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Phagocytosis-induced apoptosis of macrophages is linked to uptake, killing and degradation of bacteria
Abstract
Phagocytosis and intracellular destruction of pathogens by phagocytes is a crucial defense mechanism of the innate immune response during infection. It has been reported a number of times that the interaction with pyogenic, extracellular bacteria leads to the apoptotic death of phagocytes. The signaling events that cause this form of cell death are largely unknown. In this study, we demonstrate a link between uptake, killing and degradation of Escherichia coli bacteria and induction of apoptosis in macrophages. Treatment of murine RAW 264.7 macrophages with bafilomycin A(1), a phagosome acidification inhibitor, reduced killing and degradation of phagocytosed bacteria and significantly decreased macrophage apoptosis. The stable overexpression of constitutively active or dominant-negative mutants of the small GTPase Rab5a increased bacterial phagocytosis and consecutively apoptosis. In these cells, relative killing and degradation were not affected, linking the increased apoptosis to enhanced uptake and suggesting that the apoptosis-inducing signal derives from the higher incidence of degradation events or an accumulation of phagosomes of a late maturation stage. These results thus provide a link between bacterial phagocytosis and degradation and the induction of apoptosis in macrophages. We propose that this form of apoptosis is the physiological conclusion of an innate immune response against pyogenic bacteria.
... Pathogens adapted for intracellular life, notably bacteria, utilise virulence factors to forcibly induce apoptosis in phagocytes as a way of suppressing the host's immune defences and promoting infection [9,10]. On the other hand, apoptosis induced by phagocytes, such as mammalian neutrophils [11] and macrophages [12], upon microbe internalisation (PICD), is host-mediated and effectively terminates the antimicrobial response [13]. PICD by immune cells serves to deter the systemic spread of microbes while advertising its compromised condition, ultimately leading to the resolution of inflammation. ...
... We have isolated amebocytes from L. polyphemus and examined their post-phagocytic behaviour. Amebocytes, like vertebrate phagocytes, undergo rapid cell death upon microbe internalisation, proceeding through the molecular and morphological programmes characteristic of apoptosis [11,[33][34][35][36]. Here, phagocytic amebocytes demonstrated reduced inner mitochondrial trans-membrane potential (DW m ) (Fig. 2), a concurrent increase in caspase-like activities (Fig. 6) and extracellularisation of PS (Fig. 3), followed by DNA fragmentation (Fig. 4) and impaired plasma-membrane potential (Fig. 3). ...
... It is now well documented that apoptosis following phagocytosis of bacteria by macrophages, neutrophils and monocytes is required for the resolution of inflammation [11,12,[33][34][35][36][40][41][42]. In contrast, PICD has received little attention in invertebrates, even though hemocytes are Inducible phenoloxidase activity of hemocyanin in the presence of amebocytes exhibiting apoptotic properties. ...
Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.
... Instilling AC in the lung prior to infection worsens bacterial clearance and lung inflammation (108). However, clearance of AC that have undergone phagocytosisinduced cell death after engulfing bacteria contributes to effective clearance of numerous lung pathogens and to lung repair following infection (163)(164)(165). Instilling AC in the lung following infection improves bacterial clearance and decreases lung inflammation (147), further evidence that the context of AC clearance determines the subsequent immune effects. ...
... An alternate possibility is that the observed increase in bacterial killing with increased miR-34a is due to increased apoptosis of Mø. Successful killing of S. pneumoniae (291)(292)(293)(294) and other select bacteria (164,259,(419)(420)(421) can involve phagocytosis-induced apoptosis of Mø. ...
Apoptotic cell clearance (efferocytosis) is the essential biological process by which dead cells, called apoptotic cells (AC), are engulfed and recycled by living cells called phagocytes. This process is regulated by many receptors that differentiate between apoptotic and viable cells by recognizing molecules expressed on apoptotic cells, particularly a phospholipid called phosphatidylserine (PS). Binding of recognition receptors to PS leads to intracellular signaling, culminating in engulfment of the bound AC. Little is known about how this process is negatively regulated; most variation in engulfment capacity has been explained by the differing repertoires of AC recognition receptors on different phagocytes.
In these chapters we describe two negative regulators of AC clearance: SIRPalpha and miR-34a. Both are highly expressed on the resident phagocyte of the alveolar space, the alveolar macrophage (AM). Both contribute to a low AM engulfment capacity. First, we show that expression of signal regulatory protein alpha (SIRPalpha), a previously described inhibitory receptor, is downregulated by treatment of AM with glucocorticoids, leading to increased efferocytosis. Second, we show that this glucocorticoid-augmented efferocytosis (GCAE) increases the susceptibility of mice to bacterial pneumonia. Third, we show miR-34a is a master-regulator of AC clearance in macrophages. We identify three target genes through which miR-34a can enhance AC uptake: Axl, Sirtuin1 (SIRT1) and GTP-ase regulator associated with focal adhesion kinase-1 (GRAF1). Finally, we show that expression of miR-34a alone increases macrophage bacterial killing. Collectively, these data suggest that limited AC uptake - rather than something to be "corrected" - is an important component of the AM identity and is integral to maintaining appropriate responses to potential lung pathogens.
... Furthermore, the phagosome related pathway hsa04145 plays a role in inflammation and degradation of biological materials and involves OmpA family of proteins [68]. Reduced levels of these proteins indicate a successful bacterial breakdown [69,70]. In our study, OmpA family proteins had the highest abundance (base mean = 8449.74), ...
Changes in the oral microbiome may contribute to oral pathologies, especially in patients undergoing cancer therapy. Interactions between oral microbiome and oral mucosa may exacerbate inflammation. We determined whether probiotic-controlled plaque formation could impact proximal oral mucosa gene expression profiles in healthy volunteers. A 3-weeks balanced sample collection design from healthy volunteers (HVs) was implemented. At Week-1 plaques samples and labial mucosa brush biopsies were obtained from HVs in the morning (N = 4) and/or in the afternoon (N = 4), and groups were flipped at Week-3. A fruit yogurt and tea diet were given 2-4hrs before sample collection. mRNA gene expression analysis was completed using RNA-Seq and DESeq2. Bacterial taxa relative abundance was determined by 16S HOMINGS. Bacterial diversity changes and metabolic pathway enrichment were determined using PRIMERv7 and LEfSe programs. Alpha- and beta-diversities did not differ morning (AM) vs. afternoon (PM). The most affected KEGG pathway was Toll-like receptor signaling in oral mucosa. Eighteen human genes and nine bacterial genes were differentially expressed in plaque samples. Increased activity for 'caries-free' health-associated calcifying Corynebacterium matruchotii and reduced activity for Aggregatibacter aphrophilus, an opportunistic pathogen, were observed. Microbial diversity was not altered after 8 hours plaque formation in healthy individuals as opposed to gene expression.
... Phagocytosis of pathogens lead to apoptosis of macrophages [25]. Hence, we investigated whether altered CYLD expression affects the survival of normal macrophages. ...
Acute myeloid leukemia (AML) is the most aggressive hematopoietic malignancy characterized by uncontrolled proliferation of myeloid progenitor cells within the bone marrow. Tumor suppressor cylindromatosis (CYLD) is a deubiquitinating enzyme, which suppresses inflammatory response in macrophages. Macrophages have a central role in the defense against foreign substances and circulating cancer cells by their professional phagocytic capacity. Little is known about contributions of CYLD to changes in biological properties of human macrophages and its involvement in AML. The present study, therefore, explored whether macrophage functions in healthy individuals and AML patients are influenced by CYLD. To this end, ninety-two newly diagnosed AML patients and 80 healthy controls were recruited. The mRNA expression levels of inflammation-related genes were evaluated by real-time PCR, cell maturation, phagocytosis and apoptosis assays by flow cytometry and secretion of inflammatory cytokines by ELISA. As a result, AML patients with the low CYLD expression were significantly higher in M4/M5 than other subtypes according to the FAB type. The low CYLD expression was also closely associated with older patients and enhanced level of LDH in AML. Moreover, treatment of normal macrophages with CYLD siRNA enhanced activation of STAT-1, leading to increases in expressions of maturation markers and IL-6 production as well as suppression in cell apoptosis and phagocytosis, while macrophage phagocytosis from AML M4/M5b was higher than that from healthy controls upon CYLD siRNA transfection through STAT1 signalling. In conclusion, the inhibitory effects of CYLD on macrophage functions are expected to affect the immune response in AML.
... Myeloid cells are the most evolutionarily ancient aspect of a specialized immune system and the cornerstone of innate immunity in vertebrates 1 . Among myeloid cells, monocytes play a pivotal role in innate immunity as phagocytes, cytokine producers and sources of antigen presenting cells that patrol the blood and assume tissue resident sites [2][3][4][5] . ...
Myeloid cell heterogeneity is known but whether it is cell-intrinsic or environmentally-directed remains unclear. Here, an inducible/reversible system pausing myeloid differentiation allowed definition of clone-specific functions that clustered monocytes into subsets with distinctive molecular features. These subsets were orthogonal to the classical/non-classical categorization and had inherent, restricted characteristics that did not shift under homeostasis, post-irradiation or with infectious stress. Rather, their functional fate was constrained by chromatin accessibility established at or before the granulocyte-monocyte or monocyte-dendritic progenitor level. Subsets of primary monocytes had differential ability to control distinct infectious agents in vivo. Therefore, monocytes are a heterogeneous population of functionally restricted subtypes defined by the epigenome of their progenitors that are differentially selected by physiologic challenges with limited plasticity to transition from one subset to another.
... The division of the cells used in this experiment can occur no sooner than after 50 min in the most optimal conditions, which is not the case in the in vivo environment. If the cells were captured by the macrophages, killing 40%-70% of biomass (depending on cell type and expression profile) would require at least 2 h with subsequent decomposition for additional 6 h (Frankenberg et al., 2008). ...
It was shown recently that bacterial strains, which can act specifically against malignant cells, can be used efficiently in cancer therapy. Many appropriate bacterial strains are either pathogenic or invasive and there is a substantial shortage of methods with which to monitor in vivo the distribution of bacteria used in this way. Here, it is proposed to use a Layer-by-Layer (LbL) approach that can encapsulate individual bacterial cells with fluorescently labeled polyelectrolytes (PE)s and magnetite nanoparticles (NP)s. The NP enable remote direction in vivo to the site in question and the labeled shells in the far-red emission spectra allow non-invasive monitoring of the distribution of bacteria in the body. The magnetic entrapment of the modified bacteria causes the local concentration of the bacteria to increase by a factor of at least 5. The PEs create a strong barrier, and it has been shown in vitro experiments that the division time of bacterial cells coated in this way can be regulated, resulting in control of their invasion into tissues. That animals used in the study survived and did not suffer septic shock, which can be attributed to PE capsules that prevent release of endotoxins from bacterial cells.
... The conversion of M2 to M1 macrophage, a prospective target in cancer immunotherapy, is likely to be aided by the indigenous gut microbiota such as B. fragilis (Deng et al., 2016). B. fragilis has been reported to enhance the phagocytic functions of macrophages of the innate immune system and polarize them to an M1 phenotype (Frankenberg et al., 2008;Deng et al., 2016). Additionally, Bifidobacterium bifidum cell surface polysaccharides can activate the TLR2/MyD88 pathway and also suppress experimental colitis through Treg induction (Verma et al., 2018). ...
The human gut microbiota are critical for preserving the health status because they are required for digestion and nutrient acquisition, the development of the immune system, and energy metabolism. The gut microbial composition is greatly influenced by the colonization of the recalcitrant pathogen Helicobacter pylori (H. pylori) and the conventional antibiotic regimens that follow. H. pylori is considered to be the main microorganism in gastric carcinogenesis, and it appears to be required for the early stages of the process. However, a non-H. pylori microbiota profile is also suggested, primarily in the later stages of tumorigenesis. On the other hand, specific groups of gut microbes may produce beneficial byproducts such as short-chain fatty acids (acetate, butyrate, and propionate) that can modulate inflammation and tumorigenesis pathways. In this review, we aim to present how H. pylori influences the population of the gut microbiota to modify the host immunity and trigger the development of gastric carcinogenesis. We will also highlight the effect of the gut microbiota on immunotherapeutic approaches such as immune checkpoint blockade in cancer treatment to present a perspective for further development of innovative therapeutic paradigms to prevent the progression of H. pylori-induced stomach cancer.
... Presence of Bafilomycin A1 drastically reduced the uptake of CNDs. Thus, phagocytosis will be a major route of uptake of the CNDs 86,87 . In case of the N-CNDs also presence of Chlorpromazine reduced uptake of the CNDs. ...
Carbon nanodots with opposite chirality possess the same major physicochemical properties such as optical features, hydrodynamic diameter, and colloidal stability. Here, a detailed analysis about the comparison of the concentration of both carbon nanodots is carried out, putting a threshold to when differences in biological behavior may be related to chirality and may exclude effects based merely on differences in exposure concentrations due to uncertainties in concentration determination. The present study approaches this comparative analysis evaluating two basic biological phenomena, the protein adsorption and cell internalization. We find how a meticulous concentration error estimation enables the evaluation of the differences in biological effects related to chirality. Chirality is known to impact the biological activity of materials but concentration differences can often lead to errors in analysis. Here, the authors report on detailed concertation analysis of different chiral carbon nanodots to accurately investigate chiral effects on the protein absorption and cell internalisation.
... After the uptake of microbes, apoptosis is a physiological consequence connected to phagosomephagosome-lysosomelysosome-mediated killing of pathogens. It has been shown that the treatment with bafilomycin A, a phagosome acidification inhibitor, can inhibit the killing of phagocytosed Escherichia coli and concomitantly decrease macrophage apoptosis [63]. Rabaptin-5 was reported to be an important and decisive component of endosome fusion [64]. ...
Macrophages are important cells that regulate various innate functions. Macrophages after engulfment of pathogens proceed for phagosome maturation and finally fuse with lysosomes to kill pathogens. Although pathogen degradation is one of the important functions of phagosomes, various immune-effector functions of macrophages are also dependent on the phagosome maturation process. This review discusses signaling processes regulating phagosome maturation as well as various effector functions of macrophages such as apoptosis, antigen presentation, autophagy and inflammasome that are dependent on the phagosome maturation process. It also discusses strategies adopted by various intracellular pathogens to counteract these functions to evade intracellular destruction mechanisms. These studies may give direction for the development of new therapeutics to control various intracellular infections.
... This could be as a result of the production of reactive oxygen and nitrogen intermediates, and possibly acidification of phagosomes by the infected macrophages (Kyriazis et al. 2016). It could also involve macrophage apoptosis (Frankenberg et al. 2008). ...
Macrophages provide the first line of defense against Shigella flexneri infection in the gastrointestinal tract by inducing a variety of inflammatory and antimicrobial responses. Secondary metabolites of plants are used as drugs against infections that are resistant to common antibiotics. In this study, the innate effects of asiaticoside on the proinflammatory activity of mouse macrophages infected with S. flexneri were investigated. The viability of the infected mouse macrophages were examined using viability assay, while the pro-inflammatory cytokines productions were determined using the enzyme-linked immunosorbent assay (ELISA) for determination of IL-1β, IL-12 p40 and TNF-α levels. The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) protein were determined using the Griess assay and western blot, respectively. Statistical analyses were performed using the Statistical Package of Social Sciences (SPSS) software, version 20. The data obtained from independent experiments (n = 3) were presented as the mean ± standard error of mean (SEM). The results showed that, asiaticoside stimulated the infected macrophages by stimulating increased production of TNF-α, IL-12 p40 and NO as well as increased expression of iNOS in a dose-dependent manner. In contrast the viability of the cells and the production of IL-1β and were reduced also in a dose-dependent manner when compared to untreated cells. These results indicate that asiaticoside has immunomodulatory effects on the innate immune function of infected macrophages, showing the potential use of this compound to reduce the clinical symptoms of the infections.
... As the LY6G immunofluorescence results demonstrated, neutrophils were present during wound healing of hair follicle ORS, but they did not appear to play an important role in regeneration, as morphological changes suggestive of activation were not observed. However, apoptosis of neutrophils was detected after phagocytosis of bacteria and cellular debris [28,29], in agreement with numerous studies that had demonstrated that neutrophil phagocytosis of foreign pathogenic microorganisms could lead to their apoptosis and subsequent phagocytosis by macrophages [30,31]. Therefore, during the inflammatory response stage, it is possible that both neutrophils and macrophages are needed to promote regeneration and repair of wound tissue, but only neutrophils were present in our model. ...
Objective
Stem cells hold promise for treating hair loss. Here an in vitro mouse model was developed using outer root sheaths (ORSs) isolated from hair follicles for studying stem cell-mediated dermal papillary regeneration.
Methods
Under sterile conditions, structurally intact ORSs were isolated from hair follicles of 3-day-old Kunming mice and incubated in growth medium. Samples were collected daily for 5 days. Stem cell distribution, proliferation, differentiation, and migration were monitored during regeneration.
Results
Cell proliferation began at the glass membrane periphery then spread gradually toward the membrane center, with the presence of CD34 and CD200 positive stem cells involved in repair initiation. Next, CD34 positive stem cells migrated down the glass membrane, where some participated in ORS formation, while other CD34 cells and CD200 positive cells migrated to hair follicle centers. Within the hair follicle matrix, stem cells divided, grew, differentiated and caused outward expansion of the glass membrane to form a dermal papillary structure containing alpha-smooth muscle actin. Neutrophils attracted to the wound site phagocytosed bacterial and cell debris to protect regenerating tissue from infection.
Conclusion
Isolated hair follicle ORSs can regenerate new dermal papillary structures in vitro . Stem cells and neutrophils play important roles in the regeneration process.
... 虽然早在 20 世纪 80 年代就认识到哺乳动物和线 虫类的凋亡细胞是通过吞噬过程被清除的 [19,20] [25,26] . 吞噬诱导的巨噬细胞凋亡已得到充分阐 述 [27] . 单丹磺酰尸胺(MDC)可以聚集于酸性囊泡中, 是一种常用的细胞自噬标记荧光染料 [28] . ...
... Macrophages and neutrophils are essential for the innate immune system to remove and kill invading microorganisms during infection [31]. This is because both neutrophils and macrophages act as the first line of defense against invading organisms. ...
Pasteurella multocida B:2 is a Gram-negative organism causing haemorrhagic septicaemia (HS) in buffaloes. It causes severe pulmonary infection, leading to infiltration of numerous macrophages and neutrophils. Despite the inflammatory response, buffaloes succumb to HS. This study aims to evaluate the in-vitro efficacy of macrophages and neutrophils of buffalo following exposure to P. multocida B:2. In-vitro infections were done using 10 ⁷ cfu/ml of P. multocida B:2 for Group 1, Escherichia coli for Group 2 and Mannhaemia haemolytica A:2 for Group 3 cells. The inoculated cell cultures were harvested at 0, 30, 60 and 120 min post-exposure and the phagocytic, killing and cell death rates were determined. Both phagocytosis and killing rates of all bacteria increased over time. Phagocytosis involved between 71% and 73% neutrophils and between 60% and 64% macrophages at 120 min. Killing rate of all bacteria involved between 76% and 79% for neutrophils and between 70% and 74% for macrophages at 120 min. Death rate of neutrophils ranged between 67% in Group 3, and 88% in Group 1 at 120 min, significantly (p < 0.05) higher than Group 3 but insignificant (p > 0.05) than Group 2. Similar pattern was observed for death rate of macrophages. The phagocytosis and killing rates of P. multocida B:2 were similar to other bacterial species used in this study but more neutrophils and macrophages were dead following infection by P. multocida B:2 than M. haemolytica A:2.
... Surprisingly, (P)RR-deficient macrophages degraded both bacteria more efficiently than control macrophages suggesting that the absence of (P)RR and most of V-ATPases did not alter their bactericidal functions. In contrast to previous studies (Bidani et al. 1995, Frankenberg et al. 2008), alkalinization of lysosome pH by treatment with Bafilomycin did not impair the bactericidal activity of macrophages (Kissing et al. 2014). Finally, the exact mechanism by which (P)RR interferes with the protein level of V0 subunits remains an important question to be solved. ...
Vor zehn Jahren wurde der (Pro)Renin-Rezeptor [(P)RR] entdeckt und als neuer Bestandteil des Renin-Angiotensin-Systems beschrieben. Neuere Studien ergaben, dass der (P)RR mit der vakuolären H+-ATPase (V-ATPase) assoziiert sein kann, weshalb er auch V-ATPase associated protein 2 (ATP6ap2) genannt wird. In Osteoklasten befinden sich V-ATPase hauptsächlich an der zur Knochenoberfläche gerichteten Plasmamembran und transportieren Protonen in den extrazellulären Raum. Mäuse mit genetischer Deletion verschiedener V-ATPase-Untereinheiten charakterisiert durch einen Anstieg von Knochenmasse (Osteopetrose). In der vorliegenden Arbeit fanden wir heraus, dass (P)RR stark in reifen Osteoklasten in vitro und in vivo exprimiert wird. Mäuse mit genetischer Deletion des (P)RR in Osteoklasten wurden durch einen komplexen Knochen-Phänotyp mit reduzierter Knochendichte charakterisiert. (P)RR-defiziten Osteoklasten wiesen vermehrte Differenzierung und/oder Aktivität in vitro und in vivo auf. Wir postulieren deshalb, dass der (P)RR die in der Plasmamembran lokalisierten V-ATPase nicht direkt reguliert, sondern mit der physiologischen Aktivität der Osteoklasten durch andere Mechanismen interferiert. Macrophagen sind speziell auf die Immunabwehr ausgerichtete Fresszellen (Phagozyten). Phagozytose ist ein wesentlicher Zellprozess der die V-ATPase in Lysosomen braucht um die eingeschlossenen Pathogen zu zerstören. Wir generierten transgene Ratten mit konditionellen knockdown von (P)RR unter Nutzung eines Doxyzyclin-induzierten shRNA-Expressionssystems. Eine effiziente (P)RR-Depletion in Makrophagen wurde durch Behandlung mit Doxyzyclin in vivo im Trinkwasser und in vitro im Kulturmedium erreicht. Die vorliegende Arbeit zeigt, dass die Verschiebung des vesikulären pHs erst ziemlich spät nach (P)RR-Depletion auftritt. Wir fanden heraus, dass (P)RR-Depletion weder Phagozytose noch Endozytose beeinträchtigte, sondern für das Recycling des Transferrin-Rezeptors zur Plasmamembran wichtig ist.
... Phagocytosis studies benefit from the unique integration of functional and phenotypic information provided by FCM and the large availability of phagocytic cell types and targets (both natural and synthetic) that are suitable for the technical capabilities of FCM [508][509][510][511][512][513][514]. In many cases, FCM assays of phagocytosis are available as commercial kits, and may include simultaneous assessment of other functional aspects or consequences of phagocytosis, typically the oxidative burst [515] or apoptosis [515,518]. ...
... To determine the effect of acidification on ingested pathogens experimentally, most investigators use either bafilomycin A 1 or concanamycin A as potent V-ATPase blockers or the weak bases methylamine or chloroquine (Fig. 2b) to raise intra-phagosome pH or (phago)lysosome pH. An increased pH often resulted in less killing, e.g., of E. coli DH5α (Frankenberg et al., 2008), Mycobacterium smegmatis (Anes et al., 2006) or of Staphylococcus aureus (Bidani et al., 2000). Such reduced killing is usually ascribed to the sensitivity of microorganisms to low pH. ...
Many eukaryotic cells ingest extracellular particles in a process termed phagocytosis which entails the generation of a new intracellular compartment, the phagosome. Phagosomes change their composition over time and this maturation process culminates in their fusion with acidic, hydrolase-rich lysosomes. During the maturation process, degradation and, when applicable, killing of the cargo may ensue. Many of the events that are pathologically relevant depend on strong acidification of phagosomes by the 'vacuolar' ATPase (V-ATPase). This protein complex acidifies the lumen of some intracellular compartments at the expense of ATP hydrolysis. We discuss here the roles and importance of V-ATPase in intracellular trafficking, its distribution, inhibition and activities, its role in the defense against microorganisms and the counteractivities of pathogens.
... The process is controlled by chemokines, which have an important role in the pathophysiology of inflammatory diseases. Tumor necrosis factor (TNF)-α is a pivotal cytokine that acts upon both the innate and the adaptive immune systems (1)(2)(3). At the same time, cytokines and chemokines represent potential targets for therapy. ...
Purpose:
Inflammatory and rheumatic arthritis remain leading causes of disability worldwide. The arthritis therapeutic area commands the largest market for the prescription of biological and non-steroidal anti-inflammatory drugs (NSAID). Yet biotechnology and pharmaceutical companies conducting research and providing therapeutics in this area frequently face challenges in patient safety. The purpose of our study was to assess safety of anti-tumor necrosis factor therapies in arthritis patients.
Methods:
The present study systematically reviews adverse events of biologicals alone or in the presence of NSAIDs and other immunosuppressant therapeutics such as disease-modifying antirheumatic drugs (DMARD). We assessed the rheumatology literature that included clinical trials with anti-tumor necrosis factor (TNF) biologicals and case reports published between 2010 and 2014.
Results:
Currently approved anti-TNF biologicals in arthritis include the monoclonal antibodies infliximab, adalimumab, certolizumab pegol and golimumab, and the fusion protein etanercept. The most frequently-reported adverse event was infection. We grouped the adverse reactions as immune-mediated, hypersensitivity syndrome reactions including cutaneous and hepatic manifestation, neurological, hematological, and malignancy.
Discussion:
Most adverse events are due to the failure of host immunological control, which involves susceptibility to the drug itself, or de novo infection or reactivation of a latent bacterial or viral infection, often with a different expression of disease. Drug-induced liver injury associated with anti-TNF biologicals must be kept in mind when evaluating patients with increased liver enzymes.
Conclusion:
Risk assessment in individuals undergoing treatment with biologicals represents a step towards achieving a personalized medicine approach to identify those patients that will safely benefit from this therapeutic approach. Patients and physicians must be alert of anti-TNF agents as potential causes of drug-induced liver injury and monitor the therapies. Personalizing therapeutic pharmacovigilance promises to optimize benefits while minimizing side effects.
... These observations Three types of programmed cell death, autophagy, apoptosis and oncosis, have been well described among other forms of cell death, while necrosis is defined as the sum of changes occurring in the cells after they have died regardless of the prelethal process [25,26]. Phagocytosis-induced apoptosis has been well described in the setting of a bacterial infection [27]. MDC, a well-known specific fluorescent dye that accumulates in acidic vacuoles, is often used as a marker for autophagosomes [28]. ...
Clearance of apoptotic neutrophils by macrophages is important for both the successful resolution of acute inflammation and homeostasis. However, the dynamic process of phagocytosis of apoptotic neutrophils by macrophages and the fate of macrophages after the ingestion of apoptotic neutrophils has not been well documented. In the present study, we staged the recognition and tethering, internalization, digestion and exocytosis steps of phagocytosis of apoptotic neutrophils. Furthermore, we found that after the ingestion of apoptotic cells, a subset of macrophages underwent cell death by autophagy, apoptosis or oncosis as revealed by transmission electron microscopy and confocal microscopy combined with specific dyes. The percentage of autophagic, apoptotic and oncotic macrophages were 8.00%±2.00%, 12.33%±2.08%, and 3.66%±1.50%, respectively. These results indicated that after ingestion of apoptotic neutrophils, a subset of macrophages undergoes autophagy and apoptosis. We propose that autophagy of macrophages after the ingestion of apoptotic cells may be a new mechanism present in the resolution of inflammation.
... The induction of apoptosis in intestinal cells after the ingestion of pathogenic bacteria is thought to be an important defense mechanism against infections [24,25]. LPS and IFN-γ stimulate macrophages to produce excess NO, activate stress signaling cascades, and eventually trigger an apoptotic cascade, resulting in cell death [3]. ...
Nitric oxide (NO) is a multifunctional mediator that is involved in a variety of pathologic and physiologic processes. Few studies have addressed the effect of lactic acid bacteria (LAB), especially Lactococcus lactis strains used in dairy products, on inducible nitric oxide synthase (iNOS) induction as a component of the host's gastrointestinal immune response. We investigated the ability of L. lactis strains to induce NO synthesis in the murine macrophage-like cell line J774.1 and in peritoneal macrophages from mice. The degree of NO induction was specific to the L. lactis strain used. Compared with the no-treatment control, heat treatment of L. lactis cells decreased NO and TNF-α levels but further stimulated interleukin (IL)-12 production. Adding L. lactis cells to peritoneal macrophages dose-dependently increased the production of NO and IL-10 but decreased that of IL-12p70. Adding L. lactis cells to interferon-γ-stimulated J774.1 cells enhanced cell death and the production of NO and IL-12p40, whereas addition of 1400W, a specific inhibitor of iNOS, decreased NO production and cell death. Conversely, adding 1400W to J774.1 cells further enhanced IL-12p40 production, suggesting that IL-12 production is perturbed by excess endogenous NO. IL-12 production is thought to be a marker of improved immunostimulation. Our results suggest that IL-12 production could be increased by limiting endogenous NO production.
... S. suis serotype 2 is considered an important zoonotic pathogen causing infections in humans, pigs and so on, therefore cells originates from different hosts (Lalonde et al., 2000;Pan et al., 2009;Li et al., 2013) were applied to test if NisKR contributed to the virulence of S. suis 2. Bacterial attachments to epithelial cells, colonization of mucosal surfaces, and interaction with respiratory epithelial cells are prerequisites for the induction of streptococcal infection (Lalonde et al., 2000). Polymorphonuclear leukocytes play an important role in host defense system's response to bacterial infectious (Chabot-Roy et al., 2006), and macrophages could combine potent anti-microbial functions with the ability to induce local inflammation and a systemic immune response (Frankenberg et al., 2008). Pathogens are mainly eliminated through phagocytosis, followed by intracellular digestion. ...
Two-component signal-transduction systems (TCSTSs) may regulate some virulence factors in response to external stimuli, and thus allowing Streptococcus suis serotype 2 to interact with the host, promote survival, and cause disease. Here, a mutant of the NisKR TCSTS had attenuated virulence in vitro, as exemplified by lowered hemolytic activity, reduced adherence to epithelial cells, increased elimination by macrophages, and decreased resistance to killing by neutrophils. Results also showed that this system is important for the ability of Streptococcus suis serotype 2 to survive and proliferate in an in vivo mouse model. Thus, the NisKR system plays a significant role in pathogenesis, both in colonization and invasive disease
... With regard to the role of the phagocytic progress in PICD, results are conflicting: Whereas phagocytosis and transition to the phagolysosome were found essential for induction of monocyte apoptosis [25], related to maturation and acidification of this compartment with an activation of apoptosis-modulating factors [26], [27], our finding (Fig. 2) confirms results from other infection models using N. meningitides, K. pneumoniae, Y. enterocolitica, E. coli and fungi, where apoptosis was induced independently of phagocytosis [28], [29], [30]. ...
Phagocytosis induced cell death (PICD) is crucial for controlling phagocyte effector cells, such as monocytes, at sites of infection, and essentially contributes to termination of inflammation. Here we tested the hypothesis, that during PICD bystander apoptosis of non-phagocyting monocytes occurs, that apoptosis induction is mediated via tumor necrosis factor-alpha (TNF-α and that TNF-α secretion and -signalling is causal. Monocytes were infected with Escherichia coli (E. coli), expressing green fluorescent protein (GFP), or a pH-sensitive Eos-fluorescent protein (EOS-FP). Monocyte phenotype, phagocytic activity, apoptosis, TNF-receptor (TNFR)-1, -2-expression and TNF-α production were analyzed. Apoptosis occured in phagocyting and non-phagocyting, bystander monocytes. Bacterial transport to the phagolysosome was no prerequisite for apoptosis induction, and desensitized monocytes from PICD, as confirmed by EOS-FP expressing E. coli. Co-cultivation with non-infected carboxyfluorescein-succinimidyl-ester- (CFSE-) labelled monocytes resulted in significant apoptotic cell death of non-infected bystander monocytes. This process required protein de-novo synthesis and still occurred in a diminished way in the absence of cell-cell contact. E. coli induced a robust TNF-α production, leading to TNF-mediated apoptosis in monocytes. Neutralization with an anti-TNF-α antibody reduced monocyte bystander apoptosis significantly. In contrast to TNFR2, the pro-apoptotic TNFR1 was down-regulated on the monocyte surface, internalized 30 min. p.i. and led to apoptosis predominantly in monocytes without phagocyting bacteria by themselves. Our results suggest, that apoptosis of bystander monocytes occurs after infection with E. coli via internalization of TNFR1, and indicate a relevant role for TNF-α. Modifying monocyte apoptosis in sepsis may be a future therapeutic option.
... This observation is different than those reported by He et al. who observed that cathepsin activity and lysosomal acidification both play a role in CP-induced IL-1b secretion. However, the inhibitors used in that study, CA- 074Me (a cathepsin B and L inhibitor) and bafilomycin A (a lysosomal acidification inhibitor), both have off-target effects (as do most pharmacological inhibitors) [43,44]. Additionally, CP is a small infectious elementary body (EB); 300–600 nm diameter compared to other intracellular bacteria, and so internalization of the EB is unlikely to exceed the capacity of the phagolysosome. ...
Chlamydia pneumoniae (CP) is an important human pathogen that causes atypical pneumonia and is associated with various chronic inflammatory disorders. Caspase-1 is a key component of the 'inflammasome', and is required to cleave pro-IL-1β to bioactive IL-1β. Here we demonstrate for the first time a critical requirement for IL-1β in response to CP infection. Caspase-1⁻/⁻ mice exhibit delayed cytokine production, defective clearance of pulmonary bacteria and higher mortality in response to CP infection. Alveolar macrophages harbored increased bacterial numbers due to reduced iNOS levels in Caspase-1⁻/⁻ mice. Pharmacological blockade of the IL-1 receptor in CP infected wild-type mice phenocopies Caspase-1-deficient mice, and administration of recombinant IL-1β rescues CP infected Caspase-1⁻/⁻ mice from mortality, indicating that IL-1β secretion is crucial for host immune defense against CP lung infection. In vitro investigation reveals that CP-induced IL-1β secretion by macrophages requires TLR2/MyD88 and NLRP3/ASC/Caspase-1 signaling. Entry into the cell by CP and new protein synthesis by CP are required for inflammasome activation. Neither ROS nor cathepsin was required for CP infection induced inflammasome activation. Interestingly, Caspase-1 activation during CP infection occurs with mitochondrial dysfunction indicating a possible mechanism involving the mitochondria for CP-induced inflammasome activation.
... Supporting this hypothesis, inhibition of killing and degradation of F. tularensis in the phagosome using the phagosomal acidification inhibitors bafilomycin or NH 4 Cl completely inhibited these processes. Because phagosomal acidification inhibitors prevent killing and degradation of phagocytosed bacteria [33,34] but do not prevent phagosomal permeabilization and escape of F. tularensis into the cytosol [35], these observations strongly suggest that phagosomal acidification is a critical event essential for providing a source for cytosolic DNA that subsequently activates both the AIM2 inflammasome and production of type I interferons. ...
Introduction:
The innate immune system depends on molecules collectively known as pattern recognition receptors (PRRs) to survey the extracellular space and the cytoplasm for the presence of dangerous pathogens, pathogen-derived molecules, or even self-derived molecular danger signals, which arise from tissue damage. Absent in melanoma 2 (AIM2) is a newly discovered PRR involved in the sensing of dangerous cytosolic DNA produced by infection with DNA viruses.
Discussion:
Remarkably, recent studies in AIM2-deficient mice showed that AIM2 is uniquely involved in sensing infection with the intracellular bacteria Francisella tularensis and subsequently triggering caspase-1-mediated pro-inflammatory cytokine production and macrophage cell death, which activate other components of the immune system and eliminate the infected macrophages. Here, we provide an overview of our current understanding of the role of AIM2 in innate immunity against F. tularensis in particular, and how infection of macrophages with this pathogen is thought to activate AIM2.
... Our findings showed no difference between cord blood monocytes (CBMO) and peripheral blood monocytes (PBMO) in terms of phagocytic activity and intracellular degradation of bacteria. In contrast to the earlier gold standard of plating experiments (19,20), our method allows the characterization of phagocytosisrelated phenomena on the single cell level; however, it had yet to be compared directly with culture methods. In studies of E. coli, CBMO were significantly less sensitive toward PICD than PBMO (21). ...
Neonatal sepsis is characterized by an excessive inflammatory response induced by immune cells (monocytes). We investigated the initial stage of monocyte-pathogen interaction, i.e. bacterial ingestion and degradation at the single-cell level, by comparing a new flow cytometric procedure with culture methods. We also examined the hypothesis that, in terms of phagocytosis-induced cell death (PICD), phenotype, or cytokine production, cord blood monocytes (CBMO) differ from monocytes derived from adults (peripheral blood monocytes, PBMO).
Phagocytosis and intracellular degradation were assessed by means of flow cytometry and bacterial cultures of green fluorescent protein-labeled group B Streptococci (GBS) and Escherichia coli. The production of reactive oxygen species (ROS) was measured through luminol-enhanced chemiluminescence. Apoptosis, phenotype, and cytokine production were assessed through flow cytometry.
Flow cytometry and bacterial cultures showed no difference between phagocytosis and degradation of GBS and E. coli by PBMO and CBMO. A high correlation between both methods was observed. No difference in ROS production was evident. In comparison with PBMO, CBMO apoptosis was lower after exposure to GBS and E. coli. Similarities were found between nonapoptotic monocytes and pro-inflammatory monocytes.
PICD is lower in CBMO during the early stages of monocyte-pathogen interaction. Our results emphasize that monocyte apoptosis has a potential role in tailoring the immune response in neonatal sepsis.
Polymicrobial sepsis still has a high mortality rate despite the development of antimicrobial agents, elaborate strategies to protect major organs, and the investment of numerous medical resources. Mitochondrial dysfunction, which acts as the center of energy metabolism, is clearly the basis of pathogenesis. Drugs that act on PGC1α, the master regulator of mitochondrial biosynthesis, have shown useful effects in the treatment of sepsis; therefore, we investigated the efficacy of ZLN005, a PGC1α agonist, and found significant improvement in overall survival in an animal model. The mode of action of this effect was examined, and it was shown that the respiratory capacity of mitochondria was enhanced immediately after administration and that the function of TFEB, a transcriptional regulator that promotes lysosome biosynthesis and mutually enhances PGC1α, was enhanced, as was the physical contact between mitochondria and lysosomes. ZLN005 strongly supported immune defense in early sepsis by increasing lysosome volume and acidity and enhancing cargo degradation, resulting in a significant reduction in bacterial load. ZLN005 rapidly acted on two organelles, mitochondria and lysosomes, against sepsis and interactively linked the two to improve the pathogenesis. This is the first demonstration that acidification of lysosomes by a small molecule is a mechanism of action in the therapeutic strategy for sepsis, which will have a significant impact on future drug discovery.
Low-level light therapy (LLLT) is emerging as a promising therapeutic approach to modulate the biochemical and molecular processes within living cells. LLLT is known to produce local and systemic effects; therefore, immune cells in local tissues or in the circulation are affected by light. However, this specific effect remains weakly explored. In this study, the effect of red (650 nm) and NIR (808 nm) light on phagocytosis (respiratory burst), cytokine expression, mitochondrial activity, ROS generation, Ca²⁺ influx and membrane depolarization in macrophages in vitro is investigated. Both the phagocytic capacity and adhesion of macrophages strongly (~2.5 times) increased in the first hours after exposure to light in a dose-dependent manner. The light-evoked upregulation of phagocytosis is found to be less efficient than the maximal pharmacologically induced enhancement of ~3.2 times. Also, red/NIR light reduces the production of pro-inflammatory cytokines and activates the secretion of anti-inflammatory cytokines by several times in activated macrophages. At the same time, the viability shows a biphasic dose response: it increases after irradiation with lower doses (0.3-1 J/cm²) and decreases after treatment with higher doses (18-30 J/cm²), which is apparently associated with the upregulation of ROS generation, followed by an increase in the mitochondrial activity.
Salinity is an important ecological factor that affects physiological metabolism, survival, and distribution of marine organisms. Despite changes in the osmolarity and composition of the cytosol during salinity shifts, marine mollusks are able to maintain their metabolic function. The razor clam (Sinonovacula constricta) survives the wide range of salinity in the intertidal zone via changes in behavior and physiology. To explore the stress responses and mechanisms of salinity tolerance in razor clams, we collected transcriptomic and metabolomic data from a control group (salinity 20‰, S20) and a salinity-stress group (salinity 35‰, S35). The transcriptome data showed that genes related to the immune system, cytoskeleton remodeling, and signal transduction pathways dominated in the S35 group to counteract hypersalinity stress in the gill. The metabolomic analysis showed that 142 metabolites were significantly different between the S35 and S20 groups and that amino acid and carbohydrate metabolism were affected by hypersalinity stress. Levels of amino acids and energy substances, such as l-proline, isoleucine, and fructose, were higher in the gill of the S35 group. The combination of transcriptomic and metabolomic data indicated that metabolism of amino acids, carbohydrates, and lipids was enhanced in the gill during adaptation to high salinity. These results clarified the complex physiological processes involved in the response to hyperosmotic stress and maintenance of metabolism in the gill of razor clams. These findings provide a reference for further study of the biological responses of euryhaline shellfish to hyperosmotic stress and a molecular basis for the search for populations with high salinity tolerance.
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.
This Chapter provides an overview of ancient metazoan proteins that are made to interface (detect and control) microbes and microbial artifacts (PAMPs), especially endotoxin. The intention is to prepare readers for the more complex chapters in the next two sections (Limulus and Mammalian respectively). This discussion revolves around molecules evolved by metazoans over the course of a billion years. This evolution, a parallel development of metazoan defenses (protostome and deuterostome), a struggle against prokaryotic assault, is a competition, an arms race, in which the winner thrives, and the loser is denied the progress desired by all life. The Limulus blood system is interesting not only because it gives an exquisite way to detect endotoxin, but also because it serves to represent a set of ancient mechanisms that serve as a model system, giving a window into antiquity and thus, when compared to modern immune and coagulation systems, Limulus points back toward the beginning of these highly complex systems. Evolution, even protein evolution, it seems is not content to develop structures, it is endlessly searching for potential structures. Therefore, it is not only a science of “what is” but it is a science of “what may be” (a quest for utility) and one can view the many changes that have wheeled away in every direction from an initial structure.
Bacterial cell wall component-induced tolerance represents an important protective mechanism during microbial infection. Tolerance induced by the TLR2 agonist bacterial lipoprotein (BLP) has been shown to attenuate the inflammatory response, and simultaneously to augment antimicrobial function, thereby conferring its protection against microbial sepsis. However, the underlying mechanism by which BLP tolerance augments bactericidal activity has not been fully elucidated. Here, we reported that the induction of BLP tolerance in murine macrophages upregulated the expression of Rab20, a membrane trafficking regulator, at both the mRNA and protein levels upon bacterial infection. The knockdown of Rab20 with Rab20 specific siRNA (siRab20) did not affect the phagocytosis of Escherichia coli (E. coli), but substantially impaired the intracellular killing of the ingested E. coli in BLP-tolerized macrophages. Furthermore, Rab20 was associated with GFP-E. coli containing phagosomes, and BLP tolerization resulted in the enhanced maturation of GFP-E. coli-containing phagosomes associated with Rab20 and strong lysosomal acidification. The knockdown of Rab20 substantially diminished lysosome acidification and disturbed the fusion of GFP-E. coli containing phagosomes with lysosomes in BLP-tolerized macrophages. These results demonstrate that Rab20 plays a critical role in BLP tolerization-induced augmentation of bactericidal activity via promoting phagosome maturation and the fusion of bacteria containing phagosomes with lysosomes.
The low-molecular-mass penicillin-binding proteins, involved in peptidoglycan recycling can also produce peptidoglycan fragments capable of activating an innate immune response in host. To investigate how these proteins in Enterobacteriaceae play a role to elicit/evade innate immune responses during infections, we deleted certain endopeptidases and DD-carboxypeptidases from E. coli CS109 and studied the viability of these mutants in macrophages. The ability of infected macrophages to exert oxidative killing, express surface activation markers TLR2, MHC class II and release TNFα, were assessed. Immune responses were elevated in macrophages infected with DD-carboxypeptidase mutants but reduced for endopeptidase mutants. However, the NFκB, iNOS, and TLR2 transcripts remained elevated in macrophages infected with both mutant types. Overall, we have shown, under normal conditions endopeptidases have a tendency to elicit the immune response but their effect is suppressed by the presence of DD-carboxypeptidases. Conversely, DD-carboxypeptidases, normally, tend to reduce immune responses, as their deletions enhanced the same in macrophages. Therefore, we conclude that the roles of endopeptidases and DD-carboxypeptidases are possibly counter-active in wild-type cells where either class of enzymes suppresses each other's immunogenic properties rendering overall maintenance of low immunogenicity that helps E. coli in evading the host immune responses.
The alveolate Perkinsus marinus is the most devastating parasite of the eastern oyster Crassostrea virginica. The parasite is readily phagocytosed by oyster hemocytes, but instead of intracellular killing and digestion, P. marinus can survive phagocytosis and divide in host cells. This intracellular parasitism is accompanied by a regulation of host cell apoptosis. This study was designed to gain a better understanding of the molecular mechanisms of apoptosis regulation in oyster hemocytes following exposure to P. marinus. Regulation of apoptosis-related genes in C. virginica, and apoptosis-regulatory genes in P. marinus, were investigated via qPCR to assess the possible pathways involved during these interactions. In vitro experiments were also carried out to evaluate the effect of chemical inhibitors of P. marinus antioxidant processes on hemocyte apoptosis. Results indicate the involvement of the mitochondrial pathway (Bcl-2, anamorsin) of apoptosis in C. virginica exposed to P. marinus. In parallel, the antioxidants peroxiredoxin and superoxide dismutase were regulated in P. marinus exposed to C. virginica hemocytes suggesting that apoptosis regulation in infected oysters may be mediated by anti-oxidative processes. Chemical inhibition of P. marinus superoxide dismutase resulted in a marked increase of reactive oxygen species production and apoptosis in infected hemocytes. The implication of oxygen-dependent apoptosis during P. marinus infection and disease development in C. virginica is discussed.
Transgene expression of green fluorescent protein (GFP) has facilitated the spatio‐temporal investigation of host‐pathogen interactions; however, introduction of the GFP gene remains challenging in drug‐resistant bacteria. Herein, we report a novel far‐red fluorescent nucleic acid stain, 6‐TramTO‐3, which efficiently labels bacteria through a DNA binding mode without affecting growth and viability. Exemplarily, we stained Klebsiella pneumoniae, a major threat to hospitalized patients, and deciphered for the first time, divergent interaction strategies of antibiotic‐resistant and ‐sensitive Klebsiella strains with immune cells. 6‐TramTO‐3 constitutes an off‐the‐shelf‐reagent for real‐time analysis of bacterial infection, including strains where the use of genetically‐encoded reporters is not feasible. Eventually, our approach may aid the development of strategies to combat a major worldwide health threat: multidrug‐resistant bacteria.
Transgene expression of green fluorescent protein (GFP) has facilitated the spatio‐temporal investigation of host‐pathogen interactions; however, introduction of the GFP gene remains challenging in drug‐resistant bacteria. Herein, we report a novel far‐red fluorescent nucleic acid stain, 6‐TramTO‐3, which efficiently labels bacteria through a DNA binding mode without affecting growth and viability. Exemplarily, we stained Klebsiella pneumoniae, a major threat to hospitalized patients, and deciphered for the first time, divergent interaction strategies of antibiotic‐resistant and ‐sensitive Klebsiella strains with immune cells. 6‐TramTO‐3 constitutes an off‐the‐shelf‐reagent for real‐time analysis of bacterial infection, including strains where the use of genetically‐encoded reporters is not feasible. Eventually, our approach may aid the development of strategies to combat a major worldwide health threat: multidrug‐resistant bacteria.
Commensal Bacteroides fragilis possesses immune-regulatory characteristics. Consequently, it has been proposed as a potential novel probiotic because of its therapeutic effects on immune imbalance, mental disorders and inflammatory diseases. Macrophages play a central role in the immune response, developing either a classical-M1 or an alternative-M2 phenotype after stimulation with various signals. The interactions between macrophages and B. fragilis, however, remain to be defined. Here, a new isolate of B. fragilis, ZY-312, was shown to possess admirable properties, including tolerance to simulated gastric fluid, intestinal fluid and ox bile, and good safety (MOI = 100, 200) and adherent ability (MOI = 100) to LoVo cells. Isolate ZY-312 cell lysate promoted phagocytosis of fluorescent microspheres and pathogenic bacteria in bone marrow-derived macrophage (BMDM) cells. Gene expression of IL-12, iNOS and IL-1β in BMDM cells was increased after treatment with ZY-312, indicating the induction of M1 macrophages, consistent with enhanced secretion of NO. Cell surface expression of CD80 and CD86 was also increased. This study is the first to demonstrate that B. fragilis enhances the phagocytic functions of macrophages, polarising them to an M1 phenotype. Our findings provide insight into the close relationship between B. fragilis and the innate immune system.
Indium-containing particles (ICPs) are used extensively in the microelectronics industry. Pulmonary toxicity is observed after inhalation exposure to ICPs; however, the mechanism(s) of pathogenesis is unclear. ICPs are insoluble at physiological pH and are initially engulfed by alveolar macrophages (and likely airway epithelial cells). We hypothesized that uptake of ICPs by macrophages followed by phagolysosomal acidification results in the solubilization of ICPs into cytotoxic indium ions. To address this, we characterized the in vitro cytotoxicity of indium phosphide (InP) or indium tin-oxide (ITO) particles with macrophages (RAW cells) and lung-derived epithelial (LA-4) cells at 24 hours using metabolic (MTT) and membrane integrity (LDH) assays. InP and ITO were readily phagocytosed by RAW and LA-4 cells; however, the particles were much more cytotoxic to RAW cells and cytotoxicity was dose-dependent. Treatment of RAW cells with cytochalasin D blocked particle phagocytosis and reduced cytotoxicity. Treatment of RAW cells with bafilomycin A1, a specific inhibitor of phagolysosomal acidification, also reduced cytotoxicity but did not block particle uptake. Based on direct indium measurements, the concentration of ionic indium was increased in culture medium from RAW but not LA-4 cells following 24 hour treatment with particles. Ionic indium derived from RAW cells was significantly reduced by treatment with cytochalasin D. These data implicate macrophage uptake and solubilization of InP and ITO via phagolysosomal acidification as requisite for particle-induced cytotoxicity and the release of indium ions. This may apply to other ICPs and strongly supports the notion that ICPs require solubilization in order to be toxic.
The central paradigm says that during inflammation, after completing their function, granulocytes die apoptotically in periphery to avoid destruction of self-tissues. Here we aimed to investigate the kinetic aspect of inflammatory leukocyte apoptosis and verify whether apart from neutrophils also other inflammatory leukocytes numerously undergo apoptosis. We observed that in physiological conditions, less than 7% of either resident peritoneal macrophages or lymphocytes die apoptotically. The studies on a model of acute zymosan-induced peritoneal inflammation revealed that there are two waves of inflammatory leukocyte apoptosis. The first wave corresponds to the time of maximal neutrophil accumulation in peritoneum (6 h) and the apoptotic death indeed concerns mostly neutrophils (over 30% of those cells), but also more macrophages die at this time (>10%). The second wave (at 3 days) concerns mostly macrophages (20% versus 3–6% for other populations) and coincides with the resolution of inflammation and the dominant presence of macrophages. In contrast, numbers of apoptotic T (1–3%) and B (~5%) cells do not significantly change during the whole peritonitis. The two waves of apoptosis concur with an increase of caspase-8, -9 and -3 at the transcript and activity levels. The apoptosis inducer TNF-α is produced only during first hours while nitric oxide throughout all inflammation. Moreover, during the whole course of peritonitis the expression of pro-apoptotic Bax dominates over anti-apoptotic Bcl-2. In conclusion, we characterized kinetics of apoptotic death of inflammatory leukocytes during acute peritoneal inflammation and revealed that both phagocyte populations (neutrophils and macrophages) die numerously in peritoneum.
We recently demonstrated that IL-18 injections following burn restored IFN-γ production and increased mouse survival after bacterial infection. However, it has yet to be fully elucidated how the IL-18 therapy affects the function of phagocytic cells. We investigated the effect of IL-18 therapy on function and interaction of Kupffer cells and NK cells in burned mice. C57BL/6 mice received a 20% full-thickness burn, followed by multiple injections with IL-18. Although burn-injured mice had decreased expression of IL-18 receptors on the NK/NKT cells 5 days after injury, multiple IL-18 injections restored this expression. IL-18 treatment also augmented Kupffer cell phagocytosis. Although burn decreased the number of CD68(+) Kupffer cells with phagocytic activity, IL-18 treatment partially restored their proportion, and augmented phagocytosis-induced ROS production in CD68(+) Kupffer cells after the injection of heat-killed Escherichia coli. Consistently, IL-18 restored the impaired E. coli killing activity of Kupffer cells of burn-injured mice. Such Kupffer cell activation by IL-18 was abrogated by the deletion of NK cells or IFN-γ. In conclusion, IL-18 therapy in burn-injured mice enhanced function of CD68(+) Kupffer cells via the activation of liver NK cells and augmentation of their IFN-γ production, thereby improving survival after E. coli infection.
Mitochondria have evolved from endosymbiotic alpha-proteobacteria. During the endosymbiotic process early eukaryotes dumped the major component of the bacterial cell wall, the peptidoglycan layer. Peptidoglycan is synthesized and maintained by active-site serine enzymes belonging to the penicillin-binding protein and the β-lactamase superfamily. Mammals harbor a protein named LACTB that shares sequence similarity with bacterial penicillin-binding proteins and β-lactamases. Since eukaryotes lack the synthesis machinery for peptidoglycan, the physiological role of LACTB is intriguing. Recently, LACTB has been validated in vivo to be causative for obesity, suggesting that LACTB is implicated in metabolic processes. The aim of this study was to investigate the phylogeny, structure, biochemistry and cell biology of LACTB in order to elucidate its physiological function. Phylogenetic analysis revealed that LACTB has evolved from penicillin binding-proteins present in the bacterial periplasmic space. A structural model of LACTB indicates that LACTB shares characteristic features common to all penicillin-binding proteins and β-lactamases. Recombinat LACTB protein expressed in E. coli was recovered in significant quantities. Biochemical and cell biology studies showed that LACTB is a soluble protein localized in the mitochondrial intermembrane space. Further analysis showed that LACTB preprotein underwent proteolytic processing disclosing an N-terminal tetrapeptide motif also found in a set of cell death-inducing proteins. Electron microscopy structural studies revealed that LACTB can polymerize to form stable filaments with lengths ranging from twenty to several hundred nanometers. These data suggest that LACTB filaments define a distinct microdomain in the intermembrane space. A possible role of LACTB filaments is proposed in the intramitochondrial membrane organization and microcompartmentation. The implications of these findings offer novel insight into the evolution of mitochondria. Further studies of the LACTB function might provide a tool to treat mitochondria-related metabolic diseases. Djur, växter, svampar, och ett stort antal urdjur hör till gruppen eukayota organismer. Karakteristiskt för celler i eukaryota organismer är att de består av ett antal membranomslutna rum, så kallade cellkompartement. Det största av de här rummen är cellkärnan som innehåller genetiskt material. Utanför cellkärnan finns en cytosol som i sin tur innehåller ett antal mindre partiklar eller organeller. Den viktigaste organellen för cellens energihushållning är mitokondrien. Med undantag för mitokondrien är det oklart hur cellkompartementen ursprungligen har uppstått. Mitokondrien däremot härstammar från primitiva bakterier som tidigt under evolutionen bosatte sig inne i den eukaryota cellen. Mitokondrierna bär många spår av sitt bakteriella ursprung. Mitokondrierna saknar dock den för bakterier karakteristiska cellväggen av peptidoglykan. Det är troligt att mitokondriernas bakteriella föregångare tappade cellväggen i samband med att de tog bosättning inne i den eukaryota cellen. Trots att mitokondrier saknar peptidoglykan så har mitockondrierna bevarat ett bakeriellt protein som fungerade vid syntesen av peptidoglykan. Det här proteinet kallas LACTB. Frågan som inställer sig är varför det här proteinet överhuvudtaget förekommer i eukaryota celler och vilken uppgift det har. Det frågorna tas upp i den här avhandlingen. Resultaten av studierna tyder på att LACTB fungerar som ett strukturprotein i mitokondrien. Det förefaller alltså som om LACTB delvis skulle ha övertagit den funktion som peptidoglykan har i bakterier, nämligen att fungera som en mekaniskt stödstruktur. Det här fynden ger ett nytt perspektiv på mitokondriens evolution och kan leda till nya insikter rörande cellens energihushållning.
Review discusses bacterial mechanism promoting immune evasion and release of tissue-damaging phagocyte molecules by induction of macrophage and neutrophil apoptotic secondary necrosis.
Triggering of phagocyte apoptosis is a major virulence mechanism used by some successful bacterial pathogens. A central issue in the apoptotic death context is that fully developed apoptosis results in necrotic cell autolysis (secondary necrosis) with release of harmful cell components. In multicellular animals, this occurs when apoptosing cells are not removed by scavengers, mainly macrophages. Secondary necrotic lysis of neutrophils and macrophages may occur in infection when extensive phagocyte apoptosis is induced by bacterial cytotoxins and removal of apoptosing phagocytes is defective because the apoptotic process exceeds the available scavenging capacity or targets macrophages directly. Induction of phagocyte secondary necrosis is an important pathogenic mechanism, as it combines the pathogen evasion from phagocyte antimicrobial activities and the release of highly cytotoxic molecules, particularly of neutrophil origin, such as neutrophil elastase. This pathogenicity mechanism therefore promotes the unrestricted multiplication of the pathogen and contributes directly to the pathology of several necrotizing infections, where extensive apoptosis and necrosis of macrophages and neutrophils are present. Here, examples of necrotizing infectious diseases, where phagocyte secondary necrosis is implicated, are reviewed.
Alzheimer's disease (AD) is a prevalent neurodegenerative disorder manifested by memory loss, confusion and changes in mood. A principal pathology of this debilitating disorder is extracellular deposits of amyloid-beta (Abeta) protein. The "amyloid hypothesis" postulates that a build-up of Abeta protein is responsible for neuronal loss and the ensuing symptoms of AD. One possible mechanism of Abeta clearance, and hence AD therapy, is phagocytosis of Abeta protein by microglial cells. Microglia are the brain's resident immune cells and phagocytosis is one of their innate functions. We are interested in identifying molecules that augment microglial-mediated phagocytosis of Abeta protein. We used the rodent BV-2 microglial cell line which readily phagocytose fluorescent latex beads and synthetic Abeta(1-42) peptide. BV-2 cells treated with the neuroactive drug valproic acid (VPA) showed greatly enhanced phagocytic activity for both latex beads and Abeta. VPA also reduced microglial viability by inducing apoptosis, as previously reported. The relevance of these in vitro results to the treatment of AD is unclear but further investigation into the effects of VPA on the clearance of Abeta through enhanced microglial phagocytosis is warranted.
Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18. We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate here that AIM2 is required for sensing F. tularensis. AIM2-deficient mice were extremely susceptible to F. tularensis infection, with greater mortality and bacterial burden than that of wild-type mice. Caspase-1 activation, IL-1beta secretion and cell death were absent in Aim2(-/-) macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA. Our study identifies AIM2 as a crucial sensor of F. tularensis infection and provides genetic proof of its critical role in host innate immunity to intracellular pathogens.
Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.
The weak base ammonium chloride has been previously reported to inhibit lysosomal movements and phagosome-lysosome (Ph-L) fusion in cultured mouse macrophages (M phi), thus reducing delivery, to an intraphagosomal infection, of endocytosed solutes that have concentrated in secondary lysosomes. We have now addressed the question, whether NH4Cl might affect any direct interaction (if it exists) between such infection phagosomes and earlier, nonlysosomal compartments of the endocytic pathway, i.e., solute-containing endosomes. The phagosomes studied were formed after ingestion of the mouse pathogen Mycobacterium microti and the nonpathogenic yeast Saccharomyces cerevisiae; and the endosomes were formed after nonreceptor-mediated endocytosis of electronopaque and fluorescent soluble markers. By electron microscopy, survey of the cell profiles of M phi that had been treated with 10 mM NH4Cl so that Ph-L fusion was prevented, and that displayed many ferritin-labeled endosomes, revealed numerous examples of the fusion of electronlucent endosomes, revealed numerous examples of the fusion of electronlucent vesicles with phagosomes, whether containing M. microti bacilli or S. cerevisiae yeasts. Fusion was recognized by transfer of label and by morphological evidence of fusion in progress. The fusing vesicles were classed as endosomes, not NH4Cl-lysosomes, by their appearance and provenance, and because lysosome participation was excluded by the concurrent, NH4Cl-caused block of Ph-L fusion and associated lysosomal stasis. No evidence of such phagosome-endosome (Ph-E) fusion was observed in profiles from M phi treated with chloroquine, nor in those from normal, untreated M phi. NH4Cl-treated living M phi that had ingested yeasts at 37 degrees C, followed by endocytosis of lucifer yellow at 17 degrees C (to accumulate labeled endosomes and postpone label passing to lysosomes), were then restored to 37 degrees C. Fluorescence microscopy showed that as many as half of the yeast phagosomes (previously unlabeled) rapidly became colored. We inferred that this transfer was from endosomes (by Ph-E fusion) because Ph-L passage was blocked (by the NH4Cl). We conclude that NH4Cl induces Ph-E fusion at the same time as it suppressed Ph-L fusion. We discuss the mechanisms of these concurrent effects and suggest that they are independent; and we consider the implications of NH4Cl opening a direct route for endocytosed molecules to reach an intraphagosomal infection without involving lysosomes.
The mechanism underlying phagosomal acidification was studied in thioglycolate-elicited murine macrophages. The pH of the phagosomal compartment (pHp) was measured fluorimetrically in macrophage suspensions following ingestion of fluorescein isothiocyanate-labeled Staphylococcus aureus. At 37 degrees C, pHp decreased rapidly, reaching a steady state value of 5.8-6.1, while the cytoplasmic pH remained near neutrality, pH 7.1. The phagosome to cytosol pH gradient could be collapsed by addition of nigericin, monensin, or weak bases. The substrate dependence and inhibitor sensitivity profile of phagosomal acidification were investigated in intact and permeabilized cells. Phagosomal acidification was inhibited when ATP was depleted using metabolic inhibitors or permeabilizing the plasma membrane by electroporation. In permeabilized cells, acidification could be initiated by readdition of both Mg2+ and ATP. Neither adenosine 5'-(beta,gamma-imido)triphosphate nor adenosine 5'-(gamma-thio)triphosphate supported phagosomal acidification. Inhibitors of F1F0-type H(+)-ATPase such as oligomycin and azide, and the E1E2-type H(+)-ATPase inhibitor vanadate had no effect on phagosomal acidification. In contrast, the rate of phagosomal acidification was reduced by micromolar concentrations of N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. In permeabilized cells, nitrate inhibited the acidification with an apparent Ki of 25 mM. Phagosomal acidification was also effectively blocked by the macrolide antibiotic bafilomycin A1, with an apparent Ki of approximately 3 mM in both intact and electroporated cells. In this concentration range, bafilomycin A1 selectively inhibits vacuolar H(+)-ATPases. The substrate requirement and inhibitor susceptibility profile of phagosomal acidification strongly suggest that proton translocation across the phagosomal membrane is mediated by a vacuolar-type H(+)-ATPase.
OmpA is a major protein of the outer membrane of Escherichia coli. It is made as a larger precursor, pro-OmpA, which requires a membrane potential for processing. We now show that pro-OmpA accumulates in the cytoplasm of cells treated with carbonyl cyanide m-chlorophenylhydrazone, an uncouple which lowers the membrane potential. Upon restoration of the potential, this pro-OmpA is secreted, processed, and assembled into the outer membrane. Pro-OmpA made in vitro is also recovered with the postribosomal supernatant. It is efficiently processed to OmpA by liposomes which have bacterial leader peptidase that is exclusively internally oriented. These experiments show that: (i) the insertion of pro-OmpA into the plasma membrane is not coupled to its synthesis; (ii) insertion is promoted by the transmembrane electrochemical potential; (iii) pro-OmpA can cross a bilayer spontaneously; and (iv) pro-OmpA is processed by the same leader peptidase which converts M13 procoat to coat.
Resident alveolar macrophages (m phi) possess plasmalemmal vacuolar-type H(+)-ATPase (V-ATPase) that plays a crucial role in regulation of intracellular pH (pHi). To assess the importance of this V-ATPase to m phi effector functions, resident alveolar m phi from rabbits were activated with E. coli-derived lipopolysaccharide (LPS) and exposed to bafilomycin A1, a specific inhibitor of V-ATPase. Bafilomycin caused a significant cytosolic acidification in both the absence and presence of CO2-HCO3-, and in both unstimulated and activated m phi. Superoxide production and Fc receptor-mediated phagocytosis also were reduced in bafilomycin-treated m phi. Similar effects were elicited by acidifying the cytoplasm in the absence of bafilomycin, by lowering extracellular pH (pHo) from 7.4 to 6.5-6.6. Thus, the effects of bafilomycin on phagocytosis and superoxide production probably were related to cytosolic acidification, secondary to blockade of V-ATPase-mediated H+ extrusion across the plasma membrane. Conversely, bafilomycin significantly increased TNF-alpha release. This effect cannot be explained by a bafilomycin-induced acidosis because acidic pHo significantly reduced TNF-alpha release. The results demonstrate that V-ATPase activity is an important determinant of the effector functions of LPS-activated m phi.
We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.
Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.
The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.
Phagocytosis in macrophages is often studied using inert polymer microspheres. An implicit assumption in these studies is that such particles contain little or no specific information in their structure that affects their intracellular fate. We tested that assumption by examining macrophage phagosomes containing different kinds of particles and found that although all particles progressed directly to lysosomes, their subsequent fates varied. Within 15 min of phagocytosis, >90% of phagosomes containing opsonized sheep erythrocytes, poly-e-caprolactone microspheres, polystyrene microspheres (PS), or polyethylene glycol-conjugated PS merged with the lysosomal compartment. After that point, however, the characteristics of phagolysosomes changed in several ways that indicated differing degrees of continued interaction with the lysosomal compartment. Sheep erythrocyte phagolysosomes merged together and degraded their contents quickly, poly-e-caprolactone phagolysosomes showed intermediate levels of interaction, and PS phagolysosomes became isolated within the cytoplasm. PS were relatively inaccessible to an endocytic tracer, Texas red dextran, added after phagocytosis. Moreover, immunofluorescent staining for the lysosomal protease cathepsin L decreased in PS phagolysosomes to 23% by 4 h after phagocytosis, indicating degradation of the enzyme without replacement. Finally, PS surface labeled with fluorescein-labeled albumin showed a markedly reduced rate of protein degradation in phagolysosomes, when compared to rates measured for proteins in or on other particles. Thus, particle chemistry affected both the degree of postlysosomal interactions with other organelles and, consequently, the intracellular half-life of particle-associated proteins. Such properties may affect the ability of particles to deliver macromolecules into the major histocompatibility complex class I and II antigen presentation pathways.
Survival or destruction of a pathogen following phagocytosis depends, in part, on fusion events between the phagosome and the endosomal or lysosomal compartments. Here we use an in vitro assay to show that phagosome-endosome fusion is regulated by the small GTPase rab5 and that fusion events are influenced by an internalized live organism, Listeria monocytogenes (LM). We compare the in vitro fusion of phagosomes containing heat-killed organisms (dead LM) with that of phagosomes containing a live nonhemolytic mutant (live LMhly-). Unlike the wild-type organism, LMhly- remains trapped inside the phagosome. Phagosome-endosome fusion was reconstituted using biotinylated organisms and endosomes containing horseradish peroxidase conjugated with avidin. With both live LMhly- and dead LM preparations, in vitro phagosome-endosome fusion was time-, temperature-, and cytosol-dependent. Live LMhly- phagosomes exhibited a faster rate of fusion. Fusion in both preparations was regulated by rab5 and possibly by other GTPases. Anti-rab5 antibodies and immunodepletion of cytosolic rab5 inhibited fusion. Addition of glutatione S-transferase-rab5 in the GTP form stimulated phagosome-endosome fusion, whereas addition of a dominant negative mutant of rab5 blocked fusion. Purified live LMhly- phagosomal membranes were enriched in rab5 as revealed by Western blotting, compared with dead LM phagosomes. Fusion of endosomes with dead LM containing phagosomes required ATP and was inhibited by ATP depletion and by N-ethylmaleimide (NEM) and anti-NEM-sensitive factor (NSF) antibodies. Unexpectedly, phagosome-endosome fusion with live LMhly--containing phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Western blot analysis revealed that live LMhly--containing phagosomes were enriched for membrane-bound NSF, while dead LM containing phagosomes contained low or undetectable quantities. Washing live LMhly--containing phagosomes with 0.5 M KCl removed NSF associated with the membranes and rendered them NEM, ATP, anti-NSF antibody sensitive for fusion. We conclude that rab5 regulates phagosome-endosome fusion and that live microorganisms can up-regulate this process by recruiting rab5 to the membrane.
Monocytes and granulocytes were incubated with suspensions of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or Salmonella enteritidis and, after being washed free of bacteria, cultured for up to 48 h. Every few hours, samples of cultured cells were taken for DNA isolation. Monocytes which phagocytosed bacteria showed features of apoptotic cells, as determined by light microscopy and DNA fragmentation detected by gel electrophoresis. The phenomenon was observed 2 to 4 h after phagocytosis, in contrast, control monocytes did not show signs of apoptosis until 48 h of culture. Intact control granulocytes spontaneously became apoptotic after 12 h of culture. In contrast, degradation of DNA in cells exposed to bacteria was delayed by 12 to 24 h. In conclusion, our observation suggests that granulocytes and monocytes react differently to phagocytosis of bacteria.
Mycobacterium tuberculosis and the closely related organism Mycobacterium bovis can survive and replicate inside macrophages. Intracellular survival is at least in part attributed to the failure of mycobacterial phagosomes to undergo fusion with lysosomes. The transformation of phagosomes into phagolysosomes involves gradual acquisition of markers from the endosomal compartment. Members of the rab family of small GTPases which confer fusion competence in the endocytic pathway are exchanged sequentially onto the phagosomal membranes in the course of their maturation. To identify the step at which the fusion capability of phagosomes containing mycobacteria is compromised, we purified green fluorescent protein-labeled M. bovis BCG phagosomal compartments (MPC) and compared GTP-binding protein profiles of these vesicles with latex bead phagosomal compartments (LBC). We report that the MPC do not acquire rab7, specific for late endosomes, even 7 days postinfection, whereas this GTP-binding protein is present on the LBC within hours after phagocytosis. By contrast, rab5 is retained and enriched with time on the MPC, suggesting fusion competence with an early endosomal compartment. Prior infection of macrophages with M. bovis BCG also affected the dynamics of rab5 and rab7 acquisition by subsequently formed LBC. Selective exclusion of rab7, coupled with the retention of rab5 on the mycobacterial phagosome, may allow organisms from the M. tuberculosis complex to avert the usual physiological destination of phagocytosed material.
We have reconstituted fusion between phagosomes and lysosomes in streptolysin O-permeabilized J774-E macrophages. Fusion was assessed by measuring the delivery of avidin-conjugated horseradish peroxidase
pre-internalized into lysosomes to phagosomes containing biotinylated β-glucuronidase-conjugated paramagnetic beads (1–2 μm).
Fusion was dependent on energy and exogenously supplied cytosol. Phagosome-lysosome fusion was greatly inhibited when microtubules
were depolymerized by nocodazole treatment, suggesting that fusion occurs via microtubule-dependent transport. Furthermore,
fusion was inhibited by GTPγS and Rab GDP dissociation inhibitor. These results suggest that rab proteins are involved in
the regulation of fusion. Lastly, anti-NEM-sensitive factor (NSF) antibodies inhibited fusion, and addition of recombinant
NSF wild type partially restored the fusogenic activity, indicating that NSF is required for fusion between phagosomes and
lysosomes.
We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.
Cells from the bone marrow can present peptides that are derived from tumors, transplants, and self-tissues. Here we describe how dendritic cells (DCs) process phagocytosed cell fragments onto major histocompatibility complex (MHC) class II products with unusual efficacy. This was monitored with the Y-Ae monoclonal antibody that is specific for complexes of I-Ab MHC class II presenting a peptide derived from I-Ealpha. When immature DCs from I-Ab mice were cultured for 5-20 h with activated I-E+ B blasts, either necrotic or apoptotic, the DCs produced the epitope recognized by the Y-Ae monoclonal antibody and stimulated T cells reactive with the same MHC-peptide complex. Antigen transfer was also observed with human cells, where human histocompatibility leukocyte antigen (HLA)-DRalpha includes the same peptide sequence as mouse I-Ealpha. Antigen transfer was preceded by uptake of B cell fragments into MHC class II-rich compartments. Quantitation of the amount of I-E protein in the B cell fragments revealed that phagocytosed I-E was 1-10 thousand times more efficient in generating MHC-peptide complexes than preprocessed I-E peptide. When we injected different I-E- bearing cells into C57BL/6 mice to look for a similar phenomenon in vivo, we found that short-lived migrating DCs could be processed by most of the recipient DCs in the lymph node. The consequence of antigen transfer from migratory DCs to lymph node DCs is not yet known, but we suggest that in the steady state, i.e., in the absence of stimuli for DC maturation, this transfer leads to peripheral tolerance of the T cell repertoire to self.
The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.
To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPgammaS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)-depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPgammaS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of alpha-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.
The Rab family is part of the Ras superfamily of small GTPases. There are at least 60 Rab genes in the human genome, and a number of Rab GTPases are conserved from yeast to humans. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane traffic pathways. In the GTP-bound form, the Rab GTPases recruit specific sets of effector proteins onto membranes. Through their effectors, Rab GTPases regulate vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion.
Dendritic cells (DC) are crucial for the induction of immune responses and thus an inviting target for modulation by pathogens. We have previously shown that Plasmodium falciparum-infected erythrocytes inhibit the maturation of DCs. Intact P. falciparum-infected erythrocytes can bind directly to CD36 and indirectly to CD51. It is striking that these receptors, at least in part, also mediate the phagocytosis of apoptotic cells. Here we show that antibodies against CD36 or CD51, as well as exposure to early apoptotic cells, profoundly modulate DC maturation and function in response to inflammatory signals. Although modulated DCs still secrete tumor necrosis factor-alpha, they fail to activate T cells and now secrete IL-10. We therefore propose that intact P. falciparum-infected erythrocytes and apoptotic cells engage similar pathways regulating DC function. These findings may have important consequences for the treatment of malaria and may suggest strategies for modulating pathological immune responses in autoimmune diseases.
Apoptosis of macrophages may be a pathogen-directed mechanism of immune escape or may represent appropriate host response
to infection. Human monocyte–derived macrophages (MDMs) from healthy donors (C-MDMs) exhibited low-level constitutive apoptosis,
but culture of MDMs with opsonized serotype I Streptococcus pneumoniae (I-MDMs) for 20 h resulted in significantly increased apoptosis. I-MDM apoptosis was associated with phagocytosis of bacteria
and intracellular killing that was blocked by the caspase inhibitor z-VAD-fmk but not by Fas-blocking antibody. Paraformaldehyde-fixed
I-MDMs induced apoptosis in uninfected syngeneic monocytes at levels greater than those in monocytes incubated alone or incubated
with fixed C-MDMs. Apoptosis of syngeneic monocytes was blocked by anti-Fas antibody. The immune response of macrophages to
S. pneumoniae includes a novel form of apoptosis that is associated with successful phagocytosis and bacterial killing. This response in
vivo may regulate the inflammatory response to infection during a successful host response against S. pneumoniae
HoxB8 was the first homeobox gene identified as a cause of leukemia. In murine WEHI3B acute myeloid leukemia (AML) cells, proviral integration leads to the expression of both HoxB8 and Interleukin (IL-3). Enforced expression of HoxB8 blocks differentiation of factor-dependent myeloid progenitors, while IL-3 co-expression induces autocrine proliferation and overt leukemogenicity. Previously, we demonstrated that HoxB8 binds DNA cooperatively with members of the Pbx family of transcription factors, and that HoxB8 makes contact with the Pbx homeodomain through a hexameric sequence designated the Pbx-interaction motif (PIM). E2a-Pbx1, an oncogenic derivative of Pbx1, both retains its ability to heterodimerize with Hox proteins and arrest myeloid differentiation. This observation prompts the question of whether E2a-Pbx1 and Hox oncoproteins use endogenous Hox and Pbx proteins, respectively, to target a common set of cellular genes. Here, we use four different models of neutrophil and macrophage differentiation to determine whether HoxB8 needs to bind DNA or Pbx cofactors in order to arrest myeloid differentiation. The ability of HoxB8 to bind DNA or to bind Pbx was essential (1) to block differentiation of factor-dependent myeloid progenitors from primary marrow; (2) to block IL-6-induced monocytic differentiation of M1-AML cells; and (3) to block granulocytic differentiation of GM-CSF-dependent ECoM-G cells. However, while DNA-binding was required, the HoxB8 Pbx-interaction motif was unnecessary for preventing macrophage differentiation of ECoM-M cells. We conclude that HoxB8 prevents differentiation by directly influencing cellular gene expression, and that the genetic context within a cell dictates whether the effect of HoxB8 is dependent on a physical interaction with Pbx proteins.
Dendritic cells (DCs) are the sentinels of the immune system, able to interact with both naive and memory T cells. The recent observation that DCs can ingest cells dying by apoptosis has raised the possibility that DCs may, in fact, present self-derived Ags, initiating both autoimmunity and tumor-specific responses, especially if associated with appropriate danger signals. Although the process of ingestion of apoptotic cells has not been shown to induce DC maturation, the exact fate of these phagocytosing DCs remains unclear. In this paper we demonstrate that DCs that ingest apoptotic cells are able to produce TNF-alpha but have a diminished ability to produce IL-12 in response to external stimuli, a property that corresponds to a failure to up-regulate CD86. By single-cell analysis we demonstrate that these inhibitory effects are restricted to those DCs that have engulfed apoptotic cells, with bystander DCs remaining unaffected. These changes were independent of the production of anti-inflammatory cytokines TGF-beta1 and IL-10 and corresponded with a diminished capacity to stimulate naive T cells. Thus, the ingestion of apoptotic cells is not an immunologically null event but is capable of modulating DC maturation. These results have important implications for our understanding of the role of clearance of dying cells by DCs not only in the normal resolution of inflammation but also in control of subsequent immune responses to apoptotic cell-derived Ags.
Foreign particles and apoptotic bodies are eliminated from the body by phagocytic leucocytes. The initial stage of the elimination process is the internalization of the particles into a plasma membrane-derived vacuole known as the phagosome. Such nascent phagosomes, however, lack the ability to kill pathogens or to degrade the ingested targets. These properties are acquired during the course of phagosomal maturation, a complex sequence of reactions that result in drastic remodelling of the phagosomal membrane and contents. The determinants and consequences of the fusion and fission reactions that underlie phagosomal maturation are the topic of this review.
Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.
We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.
Microbe–macrophage interactions play a central role in the pathogenesis of many infections. Several bacterial pathogens induce apoptosis specifically in macrophages, but the mechanisms by which it occurs differ, and the resulting pathology can take different courses. Macrophage death caused by Shigella flexneri and Salmonella spp. has been shown to result in the release of pro-inflammatory cytokines. Conversely, Yersinia spp. induce apoptosis by suppressing the signalling pathways that lead to the production of tumour necrosis factor (TNF)-α, a cytokine essential for the control of this infection. It is likely that there are a variety of reasons why macrophages are particularly susceptible to pathogen-induced apoptosis. One reason may be the expression of surface receptors that recognize highly conserved bacterial components, such as lipopolysaccharide (LPS) and bacterial lipoproteins (BLPs). These receptors have recently been shown to activate pro-apoptotic signalling pathways. The roles of macrophage apoptosis in different disease processes are discussed.
Programmed death via apoptosis is the metazoan physiologic mode of cell death. Apoptotic cells are recognized by scavenger phagocytes via a number of membrane receptors and engulfed. Thereafter, little is known of their fate, or that of phagocytes. Here, we have traced apoptotic cells upon their engulfment by macrophages. After 3 h, apoptotic cells were contained in discrete well-defined vacuoles. Upon overnight chase, several small vesicles, possibly originating from the fragmentation of original vacuoles, were evident all over the macrophage body. Furthermore, Ags were diffused in the cytosol of some cells, which raises the possibility that epitopes from engulfed apoptotic cells may associate with macrophage MHC class I molecules and be recognized by T lymphocytes. Indeed, Ag-specific CTLs recognize and specifically lyse syngeneic macrophages upon phagocytosis of MHC class I-positive or -negative apoptotic cells, provided that they contain the relevant Ags. Synthesis and membrane expression of class I molecules by macrophages, together with functional transporters associated with Ag presentation, were necessary for recognition and lysis. The indirect presentation of epitopes from engulfed apoptotic cells by scavenger Ag-presenting phagocytes may, in the absence of "danger" signals, have implications for the establishment of central and peripheral self-tolerance.
In this study we take advantage of recently developed methods using J774 macrophages to prepare enriched fractions of early endosomes, late endosomes, dense lysosomes, as well as phagosomes of different ages enclosing 1-micron latex beads to investigate the steady state distribution and trafficking of lysosomal enzyme activity between these organelles. At steady state these cells appear to possess four different cellular structures, in addition to phagolysosomes, where acid hydrolases were concentrated. The first site of hydrolase concentration was the early endosomes, which contained the bulk of the cellular cathepsin H. This enzyme was acquired by phagosomes significantly faster than the other hydrolases tested. The second distinct site of lysosomal enzyme concentration was the late endosomes which contain the bulk of cathepsin S. The third and fourth large pools of hydrolases were found in two functionally distinct types of dense lysosomes, only one of which was found to be secreted in the presence of chloroquine or bafilomycin. Among this secreted pool was soluble furin, generally considered only as a membrane-bound trans-Golgi network resident protein. Thus, the organelles usually referred to as "lysosomes" in fact encompass a growing family of highly dynamic but functionally distinct endocytic organelles.
CD8+ cytotoxic T lymphocytes (CTLs) mediate resistance to infectious agents and tumours. Classically, CTLs recognize antigens that are localized in the cytoplasm of target cells, processed and presented as peptide complexes with class I molecules of the major histocompatibility complex (MHC). However, there is evidence for an exogenous pathway whereby antigens that are not expected to gain access to the cytoplasm are presented on MHC class I molecules. The most dramatic example is the in vivo phenomenon of cross-priming: antigens from donor cells are acquired by bone-marrow-derived host antigen-presenting cells (APCs) and presented on MHC class I molecules. Two unanswered questions concern the identity of this bone-marrow-derived cell and how such antigens are acquired. Here we show that human dendritic cells, but not macrophages, efficiently present antigen derived from apoptotic cells, stimulating class I-restricted CD8+ CTLs. Our findings suggest a mechanism by which potent APCs acquire antigens from tumours, transplants, infected cells, or even self-tissue, for stimulation or tolerization of CTLs.
The gene (ompA) for the major outer membrane protein II* from Escherichia coli K-12 has been cloned on a 5-megadalton EcoRI fragment by using phage lambda as vector. The gene is expressed during the lytic cycle of the recombinant phage and the insoluble membrane-bound protein was detected in phage plaques with a simple radioimmunoassay. Transfer of the EcoRI fragment into plasmid pSC101 and expression in a host lacking protein II* led to overproduction of protein II* and decreased production of two other major outer membrane proteins. Expression of the plasmid pSC101-ompA+ in minicells derived from an ompA minicell-producing strain led to synthesis, at high rates, of this protein and massive accumulation of a second cell envelope protein most likely representing the biosynthetic precursor of protein II*.
Outer membrane materials prepared from an Escherichia coli ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA+ strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA+ strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II or B purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II or B that is difficult to solubilize is identical with the more readily solubilized fraction.
We investigated the protein composition of J774-E clone macrophage phagosomes isolated at different stages of phagolysosome biogenesis. Phagosomes formed by internalizing antibody-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifugation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analyses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D,beta-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. Decreasing within the maturing phagosome were the FcII receptor, the mannose receptor, and alpha-adaptin. These results indicate that although the macrophage phagosome's total protein composition changes little during phagolysosome formation, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and sequestered proteins.
Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification.
Effects of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase, on the synthesis and processing of cathepsin D and cathepsin H were investigated in primary cultured rat hepatocytes. Pulse-chase experiments showed that after being synthesized as procathepsin D and procathepsin H the precursors were converted into mature forms in the control cells as the chase time elapsed. However, in the presence of 5 x 10(-7) M of bafilomycin A1, both precursors were largely secreted into the medium and no mature forms were found within the cells. Thus bafilomycin A1 mimics lysosomotropic amines with regard to perturbation of the targeting of lysosomal acid hydrolases. In contrast, bafilomycin A1 was found not to inhibit processings of proalbumin and procomplement component 3, which are thought to occur at the acidic trans-Golgi, implying that the proteolytic event of the proproteins is not sensitive to an increase of intra-Golgi pH. The results suggest that bafilomycin A1 is useful as a pH-perturbant to study the role of acidity in living cells.
Toxoplasma gondii belongs to a group of highly virulent intracellular parasites that reside in host cell vacuoles which resist typical phagosome-lysosome fusion. Live Toxoplasma replicate prodigiously within modified phagocytic vacuoles formed during invagination of the host plasma membrane. In contrast, heat-killed Toxoplasma or specific antibody (heat-inactivated)-coated live Toxoplasma-containing vacuoles readily undergo lysosome fusion and digestion in normal macrophages. Of newly recognized significance to Toxoplasma survival is the microbicidal effect of phagosome acidification, which reportedly can occur independently of fusion with other acidic vesicles. We report here that modified live Toxoplasma-containing vacuoles fail to acidify in normal macrophages, as indicated by the sensitive pH probe fluorescein. In contrast, when live Toxoplasma are coated with specific antibody (heat-inactivated), they trigger phagosome acidification when entering normal macrophages. A similar acidification is observed when normal phagocytes ingest dead Toxoplasma. Extracellular Toxoplasma are highly susceptible to acidic pH conditions, indicating that the acidification block in the modified vacuoles may be important for intracellular survival.
Various membrane ATPases have been tested for their sensitivity to bafilomycin A1, a macrolide antibiotic. F1F0 ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, E1E2 ATPases--e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na+,K+-ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum--are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. From this we conclude that bafilomycin A1 is a valuable tool for distinguishing among the three different types of ATPases and represents the first relatively specific potent inhibitor of vacuolar ATPases.
Penetration and replication of Listeria monocytogenes within intestinal epithelial cells were studied by infecting the human enterocyte-like cell line Caco-2. Entry was due to directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on bacterial entry and by electron microscopy showing bacteria inside membrane-limiting vacuoles at the early stage of infection. Only bacteria from pathogenic species (L. monocytogenes and Listeria ivanovii) were able to induce their own phagocytosis by Caco-2 cells, as opposed to Listeria seeligeri, Listeria welshimeri, and Listeria innocua. L. monocytogenes multiplied readily within Caco-2 cells, with an apparent generation time of about 90 min. Listeriolysin O was found to be a major factor promoting intracellular growth of L. monocytogenes. After being internalized at the same rate as that of its hemolytic revertant strain, a nonhemolytic mutant from L. monocytogenes failed to replicate significantly within Caco-2 cells. Electron microscopic study demonstrated that bacteria from the nonhemolytic mutant remained inside phagosomes during cellular infection, whereas hemolytic bacteria from L. monocytogenes were released free within the cytoplasm. This indicates that disruption of vacuole membranes by listeriolysin O-producing strains of L. monocytogenes might be a key mechanism allowing bacteria to escape from phagosomes and to multiply unrestricted within cell cytoplasm.
The bafilomycins A1, A2, B1, B2, C1 and C2, a new type of macrolide antibiotics with a 16-membered lactone ring, were isolated from the fermentation broth of three Streptomyces griseus strains (TU 1922, TU 2437, TU 2599) by ethyl acetate extraction and column chromatography on silica gel. The bafilomycins exhibit activity against Gram-positive bacteria and fungi. Physico-chemical data, chemical structures and biological activities are reported.
We used quantitative fluorescence microscopy to measure the pH of phagosomes in human monocytes that contain virulent Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in these phagocytes. The mean pH of phagosomes that contain live L. pneumophila was 6.1 in 14 experiments. In the same experiments, the mean pH of phagosomes containing dead L. pneumophila averaged 0.8 pH units lower than the mean pH of phagosomes containing live L. pneumophila, a difference that was highly significant (P less than 0.01 in all 14 experiments). In contrast, the mean pH of phagosomes initially containing live E. coli, which were then killed by monocytes, was the same as for phagosomes initially containing dead E. coli. The mean pH of L. pneumophila phagosomes in activated monocytes, which inhibit L. pneumophila intracellular multiplication, was the same as in nonactivated monocytes. To simultaneously measure the pH of different phagosomes within the same monocyte, we digitized and analyzed fluorescence images of monocytes that contained both live L. pneumophila and sheep erythrocytes. Within the same monocyte, live L. pneumophila phagosomes had a pH of approximately 6.1 and sheep erythrocyte phagosomes had a pH of approximately 5.0 or below. This study demonstrates that L. pneumophila is capable of modifying the pH of its phagocytic vacuole. This capability may be critical to the intracellular survival and multiplication of this and other intracellular pathogens.
Transforming growth factor-beta (TGF-beta), a growth regulator of fetal hepatocytes in primary culture, also regulates death of these cells. Dose-response analysis showed that the TGF-beta concentration needed to induce hepatocyte death (2.5 ng/ml) was 5 times that needed to inhibit growth in these cells (0.5 ng/ml). In response to TGF-beta, hepatocytes induced DNA fragmentation and the appearance of nuclei with a DNA content lower than 2C (diploid content), typical of a programmed cell death model. TGF-beta-induced apoptosis in fetal hepatocytes was preceded by an induction of reactive oxygen species production and a decrease in the glutathione intracellular content, indicating that this factor induces oxidative stress in fetal hepatocytes. Studies performed to analyze levels of c-fos mRNA, a gene whose expression is modulated by redox state, demonstrated that only high, apoptotic concentrations of TGF-beta (2.5 ng/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of tert-butyl hydroperoxide. Gel mobility shift assays showed that the c-fos-induced expression was coincident with an increase in AP-1 activity. Finally, cell death induced by TGF-beta in fetal hepatocytes was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-beta in these cells. In summary, the results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-beta in fetal hepatocytes.
Maturation of phagosomes is characterized by changes in their polypeptides, phosphorylated proteins and phospholipid composition. Kinetic analyses have shown that a variety of proteins associate and dissociate from latex-containing phagosomes at precise intervals during phagolysosome biogenesis. In an attempt to link these temporal biochemical modifications to functional changes, we have examined the in vivo fusion properties of aging endosomes and phagosomes. Using an in vivo fusion assay at the electron microscope, we measured the rate of exchange of bovine serum albumin-gold (5 and 16 nm particles) between endosomes and latex-bead-containing phagosomes. The results obtained indicate that the maturation of phagosomes is accompanied by changes of their fusion properties. Early phagosomes were shown to fuse preferentially with early endocytic organelles and to gradually acquire the ability to fuse with late endocytic organelles. Furthermore, the transfer of bovine serum albumin-gold from endosomes to phagosomes is size-dependent, a process also modulated by the maturation of these organelles, in agreement with the concept that transient fusion events occur between endosomes and phagosomes. Biochemical analysis showed variations in the levels of rab proteins associated with phagosomes during maturation while other 'fusion' proteins, including synaptobrevin1 and synaptobrevin2, remained constant.
GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.
Studies of host responses to infection have traditionally focused on the direct antimicrobial activity of effector molecules (antibodies, complement, defensins, reactive oxygen and nitrogen intermediates) and immunocytes (macrophages, lymphocytes, and neutrophils among others). The discovery of the systems for programmed cell death of eukaryotic cells has revealed a unique role for this process in the complex interplay between microorganisms and their cellular targets or responding immunocytes. In particular, cells of the monocyte/macrophage lineage have been demonstrated to undergo apoptosis following intracellular infection with certain pathogens that are otherwise capable of surviving within the hostile environment of the phagosome or which can escape the phagosome. Mycobacterium tuberculosis is a prototypical 'intracellular parasite' of macrophages, and the direct induction of macrophage apoptosis by this organism has recently been reported from several laboratories. This paper reviews the current understanding of the mechanism and regulation of macrophage apoptosis in response to M. tuberculosis and examines the role this process plays in protective immunity and microbial virulence.
Apoptosis is a highly regulated process of cell death that is required for the development and homeostasis of multicellular organisms. In contrast to necrosis, apoptosis eliminates individual cells without inducing an inflammatory response. Activation or prevention of cell death could be a critical factor in the outcome of an infection. Programmed cell death has been observed as a response to infection by a wide range of animal and plant pathogens and is mediated by an array of pathogen-encoded virulence determinants. Pathogen-induced modulation of the host cell-death pathway may serve to eliminate key immune cells or evade host defenses that can act to limit the infection. Alternatively, suppression of the death pathway may facilitate the proliferation of intracellular pathogens.
Bafilomycin A(1), a selective inhibitor of V-type H(+)-translocating ATPase (V-ATPase), may be a useful adjunct in cancer chemotherapy (Altan et al. [1998] J Exp Med 187:1583-1598). Therapeutic uses of the enzyme inhibitor need to consider the agent's potential effects on normal (nontumor) cells. This study determined the effects of bafilomycin A(1) on resident alveolar macrophages (mphi). Treatment of alveolar mphi with bafilomycin A(1) (10 microM, 1 h) caused a significant decrement in cytosolic pH. This was accompanied by marked alteration of mphi bactericidal capabilities. The enzyme inhibitor caused a marginal reduction in the phagocytosis of opsonized Staphylococcus aureus and significantly suppressed intracellular killing of the phagocytosed bacteria. In keeping with the effects on intracellular killing, bafilomycin A(1) significantly reduced the production of reactive oxygen species (ROS). On the other hand, cell spreading was enhanced significantly by bafilomycin A(1). Comparable changes in ROS generation and mphi spreading were produced by altering cytosolic pH through changes in extracellular pH (pH(o)) in the absence of bafilomycin A(1). These findings suggest that the agent's effects on ROS production and mphi spreading were related to the accompanying changes in cytosolic pH. The enzyme inhibitor also altered mphi morphology, leading to the shortening of microvilli and focal loss of surface ruffles. These morphologic effects differed from those produced by altering cytosolic pH by changes in pH(o). The results demonstrate that V-ATPase activity is an important determinant of mphi functioning and structure. Therapeutic use of V-ATPase inhibitors might be expected to compromise the bactericidal activity of alveolar mphi.
Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites.
The last few years have seen the accumulation of compelling evidence that apoptosis (programmed cell death) plays a major role in promoting resolution of the acute inflammatory response. Neutrophils are constitutively programmed to undergo apoptosis, which limits their pro-inflammatory potential and leads to rapid, specific, and non-phlogistic recognition by macrophages and semi-professional phagocytes. Similar mechanisms have been implicated in clearance of eosinophils, lymphocytes, and monocytes and apoptosis also plays a role in remodeling the inflamed site by deletion of myofibroblasts. A growing understanding of the mechanisms regulating leukocyte apoptosis and of the molecules mediating safe phagocytic clearance of dying cells may yield new insights into the pathogenesis and therapy of inflammatory diseases.
Fluid-phase endocytosis is stimulated by H-ras-linked growth factor receptors and this stimulation requires activation of rab5. We utilized a GFP-rab5a:wt fusion protein to monitor GFP-rab5a:wt activation in living fibroblasts and in J774 macrophages. Control CHO cells that expressed GFP-rab5a:wt were cultured in serum-free conditions and showed GFP-rab5a:wt localized to endosomal vesicles with a mean diameter of 0.3 +/- 0.1 microm. Endosome fusion, membrane ruffling, and pinosome formation were rarely detected in these cells. Coexpression of H-ras:G12V, a constitutively active H-ras mutant that activates rab5a, in cells resulted in marked enlargement of labeled endosomes (mean diameter 0.7 +/- 0.2 microm) and large numbers of giant GFP-rab5a:wt-positive endosomes were present. Time-lapse recordings showed abundant fusion among giant labeled endosomes, and membrane ruffling and pinosome formation were commonly observed. Alterations in GFP-rab5a:wt endosome structure and activity in cells expressing H-ras:G12V were linked to rab5a activation because these changes were identical to those found in cells expressing GFP-rab5a:Q79L, a constitutively activated rab5a mutant. Furthermore, cells co-expressing H-ras:G12V and GFP-rab5a:S34N, an inactive rab5a mutant, exhibited no evidence of H-ras:G12V-induced endosome enlargement. To observe changes in endosome structure and activity that directly followed activation of GFP-rab5a:wt, we performed time-lapse recordings of cells cultured overnight in serum-free media after addition of EGF. EGF caused a rapid increase in endosome fusion and in membrane ruffling activity. Membrane ruffling was often associated with GFP-rab5a:wt-positive vesicle (pinosome) formation at the base of membrane ruffles. Endosome and pinosome fusion were common in EGF-stimulated cells. Phagocytosis is also regulated by GFP-rab5a:wt. J774 macrophages that expressed GFP-rab5a:wt showed transiently activation and recruitment of GFP-rab5a:wt to newly formed phagosomes that contained rhodamine-labeled Escherichia coli. These studies show that GFP-rab5a:wt activation results in dynamic alterations in the structure and activity of the early endosomal and early phagosomal elements.
Microbe-macrophage interactions play a central role in the pathogenesis of many infections. Several bacterial pathogens induce apoptosis specifically in macrophages, but the mechanisms by which it occurs differ, and the resulting pathology can take different courses. Macrophage death caused by Shigella flexneri and Salmonella spp. has been shown to result in the release of pro-inflammatory cytokines. Conversely, Yersinia spp. induce apoptosis by suppressing the signalling pathways that lead to the production of tumour necrosis factor (TNF)-alpha, a cytokine essential for the control of this infection. It is likely that there are a variety of reasons why macrophages are particularly susceptible to pathogen-induced apoptosis. One reason may be the expression of surface receptors that recognize highly conserved bacterial components, such as lipopolysaccharide (LPS) and bacterial lipoproteins (BLPs). These receptors have recently been shown to activate pro-apoptotic signalling pathways. The roles of macrophage apoptosis in different disease processes are discussed.
The functioning of the endocytic pathway is influenced by a distinct set of rab GTPases, including rab5a, which regulates homotypic fusion of early endosomes. Expression of a dominant active, GTPase-defective rab5a accelerates endosome fusion, causing the formation of a greatly enlarged endocytic compartment. Here we present evidence that rab5a also regulates trafficking between endosomes and lysosomes and may play a role in lysosome biogenesis. The GTPase defective rab5aQ79L mutant was inducibly expressed as an EGFP fusion in HEK293 cells, and the distribution of lysosome proteins and endocytic markers then assessed by deconvolution fluorescence microscopy. During expression of EGFP-rab5aQ79L, the lysosome proteins LAMP-1, LAMP-2 and cathepsin D were found in dilated EGFP-rab5aQ79L-positive vesicles, which also rapidly labeled with transferrin Texas Red. Exogenous tracers that normally traffic to lysosomes after prolonged chase (dextran Texas Red and DiI-LDL) also accumulated in these vesicles. Dextran Texas Red preloaded into lysosomes localized with subsequently expressed EGFP-rab5a Q79L, suggesting the existence of lysosome to endosome traffic. Cells expressing EGFP-rab5a wt or the dominant negative EGFP-rab5aS34N did not exhibit these abnormalities. Despite the dramatic alterations in lysosome protein distribution caused by expression of EGFP-rab5a Q79L, there was little change in the endocytosis or recycling of a cell-surface receptor (beta2-adrenergic receptor). However, there was a deficiency of dense beta-hexosaminidase-containing lysosomes in cells expressing EGFP-rab5aQ79L, as assessed by Percoll gradient fractionation. These results suggest that expression of a GTPase-defective rab5a affects lysosome biogenesis by alteration of traffic between lysosomes and endosomes.
Biogenesis of phagolysosomes proceeds through a sequential series of interactions with endocytic organelles, a process known to be regulated by Rab and SNARE proteins. The molecular mechanisms underlying phagosome maturation in neutrophils are, however, not clearly understood. We investigated fusion between phagosomes containing the intracellular pathogen Mycobacterium tuberculosis versus the extracellular pathogen Staphylococcus aureus (designated MCP for mycobacteria-containing phagosome and SCP for S. aureus-containing phagosome) and cytoplasmic compartments in human neutrophils. Western blot analysis of phagosomes isolated after internalisation revealed that lactoferrin (a constituent of secondary granules) and LAMP-1 were incorporated into both SCP and MCP, whereas hck (marker of azurophil granules) interacted solely with SCP. The subcellular distribution of the proteins Rab5a and syntaxin-4 suggested a role in docking of granules and/or endosomes to the target membrane in the neutrophil. We observed that during phagocytosis, Rab5a in GTP-bound form interacted with syntaxin-4 on the membrane of MCP and were retained for up to 90 minutes, whereas the complex was recruited to the SCP within 5 minutes but was selectively depleted from these vacuoles after 30 minutes of phagocytosis. Downregulation of Rab5a by antisense oligonucleotides efficiently reduced the synthesis of Rab5a, the binding of syntaxin-4 to MCP and SCP and the capacity for fusion exhibited by the pathogen-containing phagosomes, but it had no effect on bacteria internalisation. These data indicate that the difference in granule fusion is correlated with a difference in the association of Rab5a and syntaxin-4 with the phagosomes. Intracellular pathogen-containing phagosomes retain Rab5a and syntaxin-4, whereas extracellular pathogen-containing phagosomes bind briefly to this complex. These results also identified Rab5a as a key regulator of phagolysosome maturation in human neutrophils.