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Mechanisms of TNF induction by heat-killed Staphylococcus aureus differ upon the origin of mononuclear phagocytes
Abstract
Mononuclear phagocytes are among the first immune cells activated after pathogens invasion. Although they all derive from the same progenitor in the bone marrow, their characteristics differ on the compartment from which they are derived. In this work, we investigated the contribution of phagocytosis for tumor necrosis factor (TNF) production by murine mononuclear phagocytes (monocytes, peritoneal and alveolar macrophages) in response to heat-killed Staphylococcus aureus (HKSA). Mononuclear phagocytes behaved differently, depending on their compartment of residence. Indeed, when bacterial uptake or phagosome maturation was blocked, activation through membrane receptors was sufficient for a maximal production of TNF and interleukin-10 by peritoneal macrophages. In contrast, monocytes, and to a lesser extent alveolar macrophages, required phagocytosis for optimal cytokine production. While investigating the different actors of signalization, we found that p38 kinase and phosphatidylinositol 3-kinase were playing an important role in HKSA phagocytosis and TNF production. Furthermore, blocking the α(5)β(1)-integrin significantly decreased TNF production in response to HKSA in all three cell types. Finally, using mononuclear phagocytes from NOD2 knockout mice, we observed that TNF production in response to HKSA was dependent on NOD2 for monocytes and peritoneal macrophages. In conclusion, we demonstrate that the mechanisms of activation leading to TNF production in response to HKSA are specific for each mononuclear phagocyte population and involve different recognition processes and signaling pathways. The influence of the compartments on cell properties and behavior should be taken into account, to better understand cell physiology and host-pathogen interaction, and to define efficient strategies to fight infection.
... In our study, bone marrow-derived macrophages (BMMs) from both Camp ϩ/ϩ and Camp Ϫ/Ϫ mice secreted TNF-␣ in a dose-dependent manner after S. aureus challenge (2 h), with the highest increase present in Camp ϩ/ϩ BMMs (Fig. 7A). It has been reported that S. aureus induces TNF-␣ through various signaling pathways, including MAPK p38 (52); however, in our study, the levels of phosphorylation of p38 were similar in Camp ϩ/ϩ and Camp Ϫ/Ϫ infected BMMs (Fig. 7B). In another phagocytic cell model (murine [J774A.1] ...
... We investigated whether the observed increase in TNF-␣ was mediated via the MAPK p38 downstream signaling pathway. Mechanistically, heat-killed S. aureus (HKSA) has been shown to promote TNF-␣ secretion by macrophages via this pathway (52). The phosphorylation of p38 has been reported to be enhanced in both Camp ϩ/ϩ and Camp Ϫ/Ϫ BMMs challenged by LPS, although TNF-␣ secretion was only slightly increased in Camp ϩ/ϩ BMMs (68). ...
... This apparent discrepancy between our findings and those of others may be due to undetected earlier p38 phosphorylation when modulating TNF-␣ secretion in BMMs (before 2 h). It may also be due to the use of different macrophage cell lines that respond differently to S. aureus; in a previous study, the level of secretion of TNF-␣ by peritoneal macrophages was higher than that by pulmonary alveolar macrophages when challenged by HKSA (52). Furthermore, S. aureus has been shown to induce alternate pathways, such as NF-B, ERK (extracellular signal-regulated kinase), and JNK (c-Jun N-terminal kinase) phosphorylation, in RAW 264.7 macrophages (69). ...
Staphylococcus aureus, an important cause of mastitis in mammals, is becoming increasingly problematic due to the development of resistance to conventional antibiotics. The ability of S. aureus to invade host cells is key to its propensity to evade immune defense and antibiotics. This study focused on functions of cathelicidins, small cationic peptides secreted by epithelial cells and leukocytes, in the pathogenesis of S. aureus mastitis in mice. We determined that endogenous murine cathelicidin (CRAMP; Camp ) was important in controlling S. aureus infection, as cathelicidin knock-out mice ( Camp -/- ) intramammarily challenged with S. aureus had a higher bacterial burden and more severe mastitis than wild-type mice. Exogenous administration of both synthetic human cathelicidin (LL-37) and synthetic murine cathelicidin (CRAMP) (8 μM), reduced invasion of S. aureus into murine mammary epithelium. Additionally, this exogenous LL-37 was internalized into cultured mammary epithelial cells and impaired S. aureus growth in vitro . We concluded that cathelicidins may be potential therapeutic agents against mastitis; both endogenous and exogenous cathelicidins conferred protection against S. aureus infection by reducing bacterial internalization and potentially by directly killing this pathogen.
... Functional TLR2 is expressed by diverse cell populations within the lungs (Opitz et al. 2010), including alveolar macrophages (Hoogerwerf et al. 2010;Juarez et al. 2010;Kapetanovic et al. 2011), airway epithelial cells (Hertz et al. 2003;Armstrong et al. 2004;Muir et al. 2004;Sha et al. 2004;Soong et al. 2004;Mayer et al. 2007), and pulmonary endothelial cells (Pai et al. 2012). TLR2 has been shown to mediate pro-inflammatory cytokine responses of alveolar macrophages and airway epithelial cells to S. aureus in vitro (Muir et al. 2004;Soong et al. 2004;Kapetanovic et al. 2011), as well as pulmonary inflammatory responses to staphylococcal lipoteichoic acid in vivo (Knapp et al. 2008). ...
... Functional TLR2 is expressed by diverse cell populations within the lungs (Opitz et al. 2010), including alveolar macrophages (Hoogerwerf et al. 2010;Juarez et al. 2010;Kapetanovic et al. 2011), airway epithelial cells (Hertz et al. 2003;Armstrong et al. 2004;Muir et al. 2004;Sha et al. 2004;Soong et al. 2004;Mayer et al. 2007), and pulmonary endothelial cells (Pai et al. 2012). TLR2 has been shown to mediate pro-inflammatory cytokine responses of alveolar macrophages and airway epithelial cells to S. aureus in vitro (Muir et al. 2004;Soong et al. 2004;Kapetanovic et al. 2011), as well as pulmonary inflammatory responses to staphylococcal lipoteichoic acid in vivo (Knapp et al. 2008). Thus, we hypothesized that TLR2 would play an important role in the activation of innate immune responses to S. aureus infection of the lungs. ...
... **P = 0.001 Hashimoto et al. 2006a), and, possibly, peptidoglycan (Schwandner et al. 1999;Dziarski and Gupta 2005;Muller-Anstett et al. 2010;Fournier 2012), as well as secreted toxins such as the Panton-Valentine leukocidin (Zivkovic et al. 2011). Diverse resident cell populations in human and murine lungs express functional TLR2, including alveolar macrophages (Hoogerwerf et al. 2010;Juarez et al. 2010;Kapetanovic et al. 2011), airway epithelial cells (Hertz et al. 2003;Armstrong et al. 2004;Muir et al. 2004;Sha et al. 2004;Soong et al. 2004;Mayer et al. 2007), and pulmonary endothelial cells (Pai et al. 2012). ...
Staphylococcus aureus
is an important cause of acute bacterial pneumonia. Toll-like receptor 2 (TLR2) recognizes multiple components of the bacterial cell wall and activates innate immune responses to gram-positive bacteria. We hypothesized that TLR2 would have an important role in pulmonary host defense againstS. aureusTLR null (TLR2-/-) mice and wild type (WT) C57BL/6 controls were challenged with aerosolizedS. aureusat a range of inocula for kinetic studies of cytokine and antimicrobial peptide expression, lung inflammation, bacterial killing by alveolar macrophages, and bacterial clearance. Survival was measured after intranasal infection. Pulmonary induction of most pro-inflammatory cytokines was significantly blunted in TLR2-/-mice 4 and 24 h after infection in comparison with WT controls. Bronchoalveolar concentrations of cathelicidin-related antimicrobial peptide also were reduced in TLR2-/-mice. Lung inflammation, measured by enumeration of bronchoalveolar neutrophils and scoring of histological sections, was significantly blunted in TLR2-/-mice. Phagocytosis ofS. aureusby alveolar macrophages in vivo after low-dose infection was unimpaired, but viability of ingested bacteria was significantly greater in TLR2-/-mice. Bacterial clearance from the lungs was slightly impaired in TLR2-/-mice after low-dose infection only; bacterial elimination from the lungs was slightly accelerated in the TLR2-/-mice after high-dose infection. Survival after high-dose intranasal challenge was 50-60% in both groups. TLR2 has a significant role in early innate immune responses toS. aureusin the lungs but is not required for bacterial clearance and survival fromS. aureuspneumonia.
... It has been suggested that in systemic inflammatory response syndrome and/or infection, compartmentalized synthesis and release of inflammatory cytokines are equally important (63Y65) and that cells other than leukocytes may be responsible for morbidity and mortality in trauma (61), inflammatory shock (66), endotoxemia (67Y69), and/or infection (70). Studies have also shown that peripheral blood cells fail to reflect what occurs in the tissue fixed cells within the same or different organs (30,49,50,71,72). Furthermore, the concept of compartmentalization pertains to coexistence of differential (and often contrasting) responses that depend on the specific location of the immune-competent machinery, and this notion has been supported by numerous preclinical studies. ...
... The disparity in the posthemorrhage response also extends to the cytokine compartment: transcriptional activity of tumor necrosis factor ! (TNF-!), interleukin 1" (IL-1"), and transforming growth factor " (but not IL-6) and the release capacity of TNF-! and IL-1 were shown to be enhanced in Kupffer cells but diminished in peritoneal and splenic macrophages (73,74). Remarkably, compartmentalization of the gene and protein expression response after trauma-hemorrhage (30,75), infection (71), and no challenge (76) was also reported to occur in different cell types of the same organ (e.g., liver) and in cells of the same origin (e.g., mononuclear cells) but different locations (i.e., blood, spleen, peritoneum). Interestingly, data reported by the two aforementioned GLUE GRANT studies that compared genomic white blood cell and splenic re-sponses in the mouse injury/infection model further substantiate the notion of compartmentalization given that responses in those two compartments were highly dissimilar both at the early (50) and late phase after challenge (49). ...
To effectively improve outcomes of septic patients, we first need to elucidate the multifaceted pathogenesis of sepsis syndromes and related inflammatory conditions. In fulfillment of such needs, in February 2016, new definitions for sepsis and septic shock were published under the acronym Sepsis-3. Although aimed at the clinical area, Sepsis-3 will have an inevitable influence upon the field of translational research as well. Sepsis-3 brings a considerable shift regarding the experimental focal point: from inflammatory states (SIRS/CARS) to organ failure (single and multiple) as the decisive factor. This shift exposes several shortcomings of the current in vivo sepsis modeling including 1) lack of uniform scoring system for sepsis severity and organ dysfunction/failure; 2) high variability of organ dysfunction phenotypes among animal species/strains; 3) difficulty in reproducing severe, ICU-grade of organ dysfunction due to high resistance of experimental animals and others. It is intuitive that clinical Sepsis-3 should also serve as an incentive for developing a global standardized approach in sepsis modeling to maximize its translational potential. This could be achieved, for example, by generating consensus guidelines that would support scientists in their study design and optimal sepsis modeling decision-making. An implementation of such hypothetical 'Minimum Quality Threshold in Pre-clinical Sepsis Studies (MQTiPSS) guidelines across different species, has a strong potential for making mouse sepsis studies more reliable and transpolatable. We strongly believe that an internationally coordinated standardization effort in sepsis modeling will certainly serve the above purposes well.
... In this sense, gram-positive bacteria can be internalized more efficiently into monocytes and macrophages because these bacteria activate integrin through the action of TLR2 [19]. In addition, the treatment with cytokines members of the tumor necrosis factor (TNF) family induces an upregulation and redistribution of b1 integrin in epithelial cells [20], and the blocking of a5b1 integrin decreases TNF-a production in response to heat-killed S. aureus [21]. ...
... For the invasion assays in the presence of the blocking antibodies, one hour prior to the addition of the bacteria to the bMECs, the blocking antibodies anti-a5b1 integrin (concentrations and incubation times employed according to Kapetanovic et al. [21]) or anti-TLR2 (according to the manufacturer's instructions) were added separately to triplicate wells. Rat IgGs (purified from normal rat serum with protein A-sepharose beads (Sigma)) or mouse IgGs (purified from normal mouse serum purchased from Pierce) were used as the negative controls. ...
Staphylococcus aureus has the ability to invade mammary epithelial cells (bMECs) causing mastitis. This event depends primarily on the α5β1 integrin in the host cell. In addition, bMECs are a target for the hormone prolactin (PRL), which can regulate β1 integrin-dependent actions related to differentiation and lactation. Previously, we demonstrated that bovine PRL (bPRL, 5 ng/ml) stimulates S. aureus internalization into bMECs. TLR2 is important during S. aureus infections, but its activation by PRL has not yet been established. The objective of this study was to determine the role of α5β1 integrin and TLR2 during S. aureus internalization into bMECs stimulated with bPRL. We demonstrated that the prolactin-stimulated internalization of S. aureus decreases in response to the blockage of α5β1 integrin (∼80%) and TLR2 (∼80%). bPRL increases the membrane abundance (MA) of α5β1 integrin (∼20%) and induces TLR2 MA (∼2-fold). S. aureus reduces the α5β1 integrin MA in bMECs treated with bPRL (∼75%) but induces TLR2 MA in bMECs (∼3-fold). Bacteria and bPRL did not modify TLR2 MA compared with the hormone alone. S. aureus induces the activation of the transcription factor AP-1, which was inhibited in bMECs treated with bPRL and infected. In general, bPRL induces both pro- and anti-inflammatory responses in bMECs, which are abated in response to bacterial challenge. Interestingly, the canonical Stat-5 transcription factor was not activated in the challenged bMECs and/or treated with bPRL. Taken together, these results support novel functions of prolactin as a modulator of the innate immune response that do not involve the classical prolactin pathway.
... It has been suggested that in systemic inflammatory response syndrome and/or infection, compartmentalized synthesis and release of inflammatory cytokines are equally important (63Y65) and that cells other than leukocytes may be responsible for morbidity and mortality in trauma (61), inflammatory shock (66), endotoxemia (67Y69), and/or infection (70). Studies have also shown that peripheral blood cells fail to reflect what occurs in the tissue fixed cells within the same or different organs (30,49,50,71,72). Furthermore, the concept of compartmentalization pertains to coexistence of differential (and often contrasting) responses that depend on the specific location of the immune-competent machinery, and this notion has been supported by numerous preclinical studies. ...
... The disparity in the posthemorrhage response also extends to the cytokine compartment: transcriptional activity of tumor necrosis factor ! (TNF-!), interleukin 1" (IL-1"), and transforming growth factor " (but not IL-6) and the release capacity of TNF-! and IL-1 were shown to be enhanced in Kupffer cells but diminished in peritoneal and splenic macrophages (73,74). Remarkably, compartmentalization of the gene and protein expression response after trauma-hemorrhage (30,75), infection (71), and no challenge (76) was also reported to occur in different cell types of the same organ (e.g., liver) and in cells of the same origin (e.g., mononuclear cells) but different locations (i.e., blood, spleen, peritoneum). Interestingly, data reported by the two aforementioned GLUE GRANT studies that compared genomic white blood cell and splenic re-sponses in the mouse injury/infection model further substantiate the notion of compartmentalization given that responses in those two compartments were highly dissimilar both at the early (50) and late phase after challenge (49). ...
Many preclinical studies in critical care medicine and related disciplines rely on hypothesis-driven research in mice. The underlying premise posits that mice sufficiently emulate numerous pathophysiological alterations produced by trauma/sepsis and can serve as an experimental platform for answering clinically relevant questions. Recently the lay press severely criticized the translational relevance of mouse models in critical care medicine. A series of provocative editorials were elicited by a highly-publicized research report in the Proceedings of the National Academy of Sciences (PNAS; February 2013), which identified an unrecognized gene expression profile mismatch between human and murine leukocytes following burn/trauma/endotoxemia. Based on their data, the authors concluded that mouse models of trauma/inflammation are unsuitable for studying corresponding human conditions. We believe this conclusion was not justified. In conjunction with resulting negative commentary in the popular press, it can seriously jeopardize future basic research in critical care medicine. We will address some limitations of that PNAS report to provide a framework for discussing its conclusions and attempt to present a balanced summary of strengths/weaknesses of use of mouse models. While many investigators agree that animal research is a central component for improved patient outcomes, it is important to acknowledge known limitations in clinical translation from mouse to man. The scientific community is responsible to discuss valid limitations without over-interpretation. Hopefully a balanced view of the strengths/weaknesses of using animals for trauma/endotoxemia/critical care research will not result in hasty discount of the clear need for using animals to advance treatment of critically ill patients.
... The cytosolic nucleotide oligomerization domain 2 (NOD2) protein permits the detection of bacterial peptidoglycan, via its minimal muramyl dipeptide moiety [9,10]. Host cell integrin a5b1 is involved in the host response to S. aureus [11][12][13] as well as other receptors belonging to the family of scavenger receptors, such as SR-A, MARCO (class A) and CD36 (class B) [14][15][16][17][18][19][20]. ...
... In addition, the cells are greatly influenced by their local microenvironment. For example, macrophages are extremely different from one compartment to another and their capacity to respond to bacteria or bacterial products differs from one site to another as shown for intestinal, peritoneal, alveolar, spleen, microglial macrophages and monocytes [12,[51][52][53]. ...
Staphylococcus aureus is a major human opportunistic pathogen responsible for a broad spectrum of infections ranging from benign skin infection to more severe life threatening disorders (e.g. pneumonia, sepsis), particularly in intensive care patients. Scavenger receptors (SR-A and CD36) are known to be involved in S. aureus recognition by immune cells in addition to MARCO, TLR2, NOD2 and α5β1 integrin. In the present study, we further deciphered the contribution of SR-A and CD36 scavenger receptors in the control of infection of mice by S. aureus. Using double SR-A/CD36 knockout mice (S/C-KO) and S. aureus strain HG001, a clinically relevant non-mutagenized strain, we showed that the absence of these two scavenger receptors was protective in peritoneal infection. In contrast, the deletion of these two receptors was detrimental in pulmonary infection following intranasal instillation. For pulmonary infection, susceptible mice (S/C-KO) had more colony-forming units (CFU) in their broncho-alveolar lavages fluids, associated with increased recruitment of macrophages and neutrophils. For peritoneal infection, susceptible mice (wild-type) had more CFU in their blood, but recruited less macrophages and neutrophils in the peritoneal cavity than resistant mice. Exacerbated cytokine levels were often observed in the susceptible mice in the infected compartment as well as in the plasma. The exception was the enhanced compartmentalized expression of IL-1β for the resistant mice (S/C-KO) after peritoneal infection. A similar mirrored susceptibility to S. aureus infection was also observed for MARCO and TLR2. Marco and tlr2 -/- mice were more resistant to peritoneal infection but more susceptible to pulmonary infection than wild type mice. In conclusion, our results show that innate immune receptors can play distinct and opposite roles depending on the site of infection. Their presence is protective for local pulmonary infection, whereas it becomes detrimental in the peritoneal infection.
... Recently, it was shown that both S. aureus and S. pneumoniae induce pro-inflammatory cytokine synthesis independent of TLR signaling pathways, via the NLRP3 inflammasome [29,30]. Kapetanovic et al. [31] demonstrated a NOD2-dependent (NLRC family) recognition of S. aureus in mouse monocytes, leading to elevated TNF. In this context PI3K and p38 MAPK play a central role in TNF production [31]. ...
... Kapetanovic et al. [31] demonstrated a NOD2-dependent (NLRC family) recognition of S. aureus in mouse monocytes, leading to elevated TNF. In this context PI3K and p38 MAPK play a central role in TNF production [31]. Similarly, NOD2 is important for the intracellular recognition of S. pneumoniae in both HEK293 and C57BL/6 mouse lung cells [32]. ...
IRAK4, a serine/threonine kinase is a central adaptor protein in TLR signaling. To better understand the clinical significance of IRAK4 deficiency we examined the impact of IRAK4 on bacterial recognition in human monocytes. We show that IRAK4 knockdown modulates monocyte-derived cytokine secretion in response to S. aureus and S. pneumoniae, resulting in decreased IL-12 and elevated IL-10 production, a finding also reproducible with ligands for TLR2 and TLR4. In contrast, silencing of MyD88 leads to a complete loss of cytokine secretion, indicating that IRAK4 acts as a differential regulator of bacteria/TLR-induced cytokine secretion downstream of MyD88. Further analysis revealed that this modulatory function results from IRAK4 suppression of protein kinase B (PKB/Akt). Release of suppression upon IRAK4 silencing (but not MyD88 knockdown) increases phosphorylation of PKB/Akt, counteracts NF-κB activation and finally results in a monocyte phenotype with tolerogenic features, thus unleashing Akt- and mTOR-dependent release of IL-10, along with concomitant phosphorylation of FOXO transcription factors. In line with these observations IRAK4-deficient monocytes failed to induce allogeneic CD8(+) and CD4(+) T-cell responses, an effect reverted by neutralization of IL-10. Taken together, our data highlight an unexpected role of IRAK4, Akt and mTOR in the regulation of tolerance in human monocytes.
... Also, the loss of a single TLR is expected to result in incomplete penetrance of the clinical phenotype due to high redundancy and compensatory mechanisms (25). Besides TLRs, peptidoglycan fragments of both Gram positive and Gram negative bacteria can be sensed via the cytosolic sensors NOD1 and NOD2 (26,27), and numerous studies have revealed that NOD-and MyD88-dependent signaling typically give synergistic responses (28). Moreover, the release of high levels of IL-1β by monocytes suggests that the bacteria may activate the NLPR3 inflammasome (29). ...
TLR8 is an endosomal sensor of RNA degradation products in human phagocytes, and is involved in the recognition of viral and bacterial pathogens. We previously showed that in human primary monocytes and monocyte derived macrophages, TLR8 senses entire Staphylococcus aureus and Streptococcus agalactiae (group B streptococcus, GBS), resulting in the activation of IRF5 and production of IFNβ, IL-12p70, and TNF. However, the quantitative and qualitative impact of TLR8 for the sensing of bacteria have remained unclear because selective inhibitors have been unavailable. Moreover, while we have shown that TLR2 activation attenuates TLR8-IRF5 signaling, the molecular mechanism of this crosstalk is unknown. We here used a recently developed chemical antagonist of TLR8 to determine its role in human primary monocytes challenged with S. aureus, GBS, Streptococcus pneumonia, Pseudomonas aeruginosa, and E. coli. The inhibitor completely blocked cytokine production in monocytes stimulated with TLR8-agonists, but not TLR2-, and TLR4-agonists. Upon challenge with S. aureus, GBS, and S. pneumonia, the TLR8 inhibitor almost eliminated the production of IL-1β and IL-12p70, and it strongly reduced the release of IL-6, TNF, and IL-10. With P. aeruginosa infection, the TLR8 inhibitor impaired the production of IL-12p70 and IL-1β, while with E. coli infection the inhibitor had less effect that varied depending on the strain and conditions. Signaling via TLR2, TLR4, or TLR5, but not TLR8, rapidly eliminated IRAK-1 detection by immunoblotting due to IRAK-1 modifications during activation. Silencing of IRAK-1 reduced the induction of IFNβ and TNF by TLR8 activation, suggesting that IRAK-1 is required for TLR8-IRF5 signaling. The TLR-induced modifications of IRAK-1 also correlated closely with attenuation of TLR8-IRF5 activation, suggesting that sequestration and/or modification of Myddosome components by cell surface TLRs limit the function of TLR8. Accordingly, inhibition of CD14- and TLR4-activation during E. coli challenge increased the activation of IRF5 and the production of IL-1β and IL-12p70. We conclude that TLR8 is a dominating sensor of several species of pyogenic bacteria in human monocytes, while some bacteria attenuate TLR8-signaling via cell surface TLR- activation. Taken together, TLR8 appears as a more important sensor in the antibacterial defense system than previously known.
... Notably the stimulation of the release of important cytokines such as TNF-α and IL-17 (Fig. 5). This is fundamental for the clearance of S. aureus, which have already been reported in several works [64,65]. The stimulation of their release is a determining factor for the treatment, especially in infections by resistant strains. ...
... Alveolar macrophages perform different protective functions from other phagocyte during infection. The TNF-α response is different in human or murine AM with monocytes induced by Mycobacterium tuberculosis or heat-killed Staphylococcus aureus [9,10]. AM also differ from other monocytic/macrophagic populations in susceptibility to infection or the intracellular replication kinetics for virus [11,12]. ...
Brucella
is an intracellular bacterium that causes the zoonosis brucellosis worldwide. Alveolar macrophages (AM) constitute the main cell target of inhaledBrucella.Brucellathwarts immune surveillance and evokes endoplasmic reticulum (ER) stress to replicate in macrophages via virulence factors. The GntR regulators family was concentrated as an important virulence factor in controlling virulence and intracellular survival ofBrucella. However, the detailed underlying mechanism for the host-pathogen interaction is poorly understood. In this study the BSS2_II0438 mutant (ΔGntR) was constructed. The type IV secretion system (T4SS) virulence factor genes (VirB2,VirB6, andVirB8) were down-expression in ΔGntR. ΔGntR could infect and proliferate to high titers in GAMs without a significant difference compared with the parental strain. ΔGntR infection increased the expression of ER stress marker genes GRP78, ATF6, and PERK in the early stages of its intracellular cycle but decreased the expression of these genes in the late stages. ΔGntR increased greatly the number ofBrucellaCFUs in the inactive ER stress state in GAMs. Meanwhile, ΔGntR infection increased the levels of IFN-γ, IL-1β, and TNF-α, indicating ΔGntR could induce the secretion of inflammatory but not anti-inflammatory cytokines IL-10. Taken together, our results clarified the role of the GntR inB. suis. S2 virulence expression and elucidated that GntR is potentially involved in the signaling pathway of theBrucella-induced UPR and inflammatory response in GAMs.
... For example, in contrast to blood monocytes, intestinal macrophages do not release inflammatory cytokines in response to various triggering agents [48]. The signaling cascade involved in TNF production in response to heat killed S. aureus differs between alveolar macrophages, monocytes, and peritoneal macrophages [49]. In contrast to peritoneal macrophages, activation of alveolar macrophages by TLR3 and TLR4 ligands does not lead to interferon-b production [50], and alveolar macrophages do not respond to TLR9 ligands in contrast to peritoneal macrophages and bone marrow derived macrophages [51]. ...
Pathogens or endogenous molecules can reprogram innate immunity. This process can take the form of priming or tolerance depending on the activating signal, and favors enhanced resistance of infection and other insults, by modulating inflammation. Similarly to their organ-specific properties, reprogramming of macrophages and NK cells, is also compartmentalized.
... Interestingly, whereas heat-killed P. aeruginosa induced a significant pro-inflammatory cytokine response at our 6 hour timepoint following instillation, administration of heat-killed S. aureus did not lead to this increased inflammatory cytokine production, despite a significant increase in total cells and neutrophils in the lavage fluid. These results appear to contradict other studies which demonstrated a significant increase in TNF-α production after exposure of alveolar macrophages to heat-killed S. aureus in vitro 42 . It is possible that different methods of heat-killing may have altered the immunogenic by-products of S. aureus, which led to a reduction in cytokine induction. ...
Cystic fibrosis (CF) is characterized by recurrent airway infections with antibiotic-resistant bacteria and chronic inflammation. Chicken cathelicin-2 (CATH-2) has been shown to exhibit antimicrobial activity against antibiotic-resistant bacteria and to reduce inflammation. In addition, exogenous pulmonary surfactant has been suggested to enhance pulmonary drug delivery. It was hypothesized that CATH-2 when combined with an exogenous surfactant delivery vehicle, bovine lipid extract surfactant (BLES), would exhibit antimicrobial activity against CF-derived bacteria and downregulate inflammation. Twelve strains of CF-pathogens were exposed to BLES+CATH-2 in vitro and killing curves were obtained to determine bactericidal activity. Secondly, heat-killed bacteria were administered in vivo to elicit a pro-inflammatory response with either a co-administration or delayed administration of BLES+CATH-2 to assess the antimicrobial-independent, anti-inflammatory properties of BLES+CATH-2. CATH-2 alone exhibited potent antimicrobial activity against all clinical strains of antibiotic-resistant bacteria, while BLES+CATH-2 demonstrated a reduction, but significant antimicrobial activity against bacterial isolates. Furthermore, BLES+CATH-2 reduced inflammation in vivo when either co-administered with killed bacteria or after delayed administration. The use of a host-defense peptide combined with an exogenous surfactant compound, BLES+CATH-2, is shown to exhibit antimicrobial activity against antibiotic-resistant CF bacterial isolates and reduce inflammation.
... Intestinal macrophages despite possessing normal phagocytic and bactericidal activity are unable to produce cytokines upon activation (Smythies et al. 2005 ). The mechanisms involved in TNF production by monocytes, peritoneal macrophages, and alveolar macrophages upon activation by S. aureus are different (Kapetanovic et al. 2011 ). In contrast to monocytes and peritoneal macrophages, alveolar macrophages do not develop endotoxin tolerance (Smith et al. 1994 ;Fitting et al. 2004 ;Hoogerwerf et al. 2010 ). ...
Sepsis is a complex syndrome characterized by a dysregulated inflammatory response to systemic infection. It is a multifarious pathology that is one of chief causes of death in intensive care units (ICUs) worldwide (Hotchkiss et al. 2009). In the past, sepsis mainly occurred due to infection developing from wounds received on the battlefield. Another frequent occurrence of sepsis was puerperal fever in women, following delivery. By the end of the eighteenth century, Alexander Gordon of Aberdeen claimed that puerperal fever was an infectious and contagious disease. In 1847, Ignaz Semmelweis, a Hungarian doctor working in Vienna successfully demonstrated this was indeed the case, and succeeded, by antiseptic methods, in reducing the mortality due to puerperal sepsis from 16 % to less than 1 % in his clinic. But he was not trusted till the work of Louis Pasteur who also advocated antiseptic methods and hygiene in 1879. In the meantime, Victor Feltz and León Coze, two Alsatian doctors had discovered that there were bacteria within the blood of a woman who died of puerperal fever. Patients with puerperal fever were the first humans in whom antibiotics (prontosil) were tested and shown to be successful in 1936. © 2014 Springer Science+Business Media New York. All rights reserved.
... Also, the role of p38 in S. aureus internalization has been demonstrated in phagocytic cells (e.g. macrophages and monocytes) by pharmacological inhibition (Kapetanovic et al., 2011). However, in NPPCs (ca. ...
Staphylococcus aureus is an etiological agent of human and animal diseases, and it is able to internalize into non-professional phagocytic cells (i.e. bovine mammary epithelial cells, bMECs), which is an event that is related to chronic and recurrent infections. bMECs contribute to host innate immune responses (IIR) through TLR pathogen recognition, whereby TLR2 is the most relevant for S. aureus. In a previous report, we showed that sodium butyrate (NaB, 0.5mM), which is a short chain fatty acid (SCFA), reduced S. aureus internalization into bMECs by modulating their IIR. However, the molecular mechanism of this process has not been described, which was the aim of this study. The results showed that the TLR2 membrane abundance (MA) and mRNA expression were induced by 0.5mM NaB ∼1.6-fold and ∼1.7-fold, respectively. Additionally, 0.5mM NaB induced p38 phosphorylation, but not JNK1/2 or ERK1/2 phosphorylation in bMECs, which reached the baseline when the bMECs were S. aureus-challenged. Additionally, bMECs that were treated with 0.5mM NaB (24h) showed activation of 8 transcriptional factors (AP-1, E2F-1, FAST-1, MEF-1, EGR, PPAR, ER and CBF), which were partially reverted when the bMECs were S. aureus-challenged. Additionally, 0.5mM NaB (24h) up-regulated mRNA expression of the antimicrobial peptides, TAP (∼4.8-fold), BNBD5 (∼3.2-fold) and BNBD10 (∼2.6-fold). Notably, NaB-treated and S. aureus-challenged bMECs increased the mRNA expression of all of the antimicrobial peptides that were evaluated, and this was evident for LAP and BNBD5. In the NaB-treated bMECs, we did not detect significant expression changes for IL-1β and IL-6 and only TNF-α, IL-10 and IL-8 were induced. Interestingly, the NaB-treated and S. aureus-challenged bMECs maintained the anti-inflammatory response that was induced by this SCFA. In conclusion, our results suggest that 0.5mM NaB activates bMECs via TLR2/p38, which leads to improved antimicrobial defense before/after pathogen invasion, and NaB may exert anti-inflammatory effects during infection.
... Next, we attempted to investigate the mechanism by which adenosine inhibited S. aureus phagocytosis. It has been reported that MAPK modulate S. aureus phagocytosis (35). In this work, we showed that, compared with the WT strain, the S. aureus ΔadsA mutant increased p38 phosphorylation, but had no effect on ERK phosphorylation (Fig. 5A). ...
Staphylococcus aureus is a common cause of bacterial infections in respiratory diseases. It secretes molecules to dampen host immunity, and the recently identified adenosine is one of these molecules. The type IIA secretory phospholipase A2 (sPLA2-IIA) is a host protein endowed with antibacterial properties, especially against Gram-positive bacteria such as S. aureus. However, the role of adenosine in sPLA2-IIA-mediated S. aureus killing by host is still unknown. The present studies showed that the S. aureus mutant lacking adenosine production (∆adsA strain) increased sPLA2-IIA expression in guinea pig airways and was cleared more efficiently, compared with the wild-type strain. S. aureus ∆adsA strain induced sPLA2-IIA expression by alveolar macrophages after phagocytic process via NOD2-NF-κB-dependent mechanism. However, S. aureus adenosine (wild-type and adsA-complemented strains) and exogenous adenosine downregulated S. aureus phagocytosis by alveolar macrophages, leading to inhibition of sPLA2-IIA expression. This occurred through inhibition of p38 phosphorylation via adenosine receptors A2a-, A2b-, and protein kinase A-dependent pathways. Taken together, our studies suggest that, in the airway, S. aureus escapes sPLA2-IIA-mediated killing through adenosine-mediated inhibition of phagocytosis and sPLA2-IIA expression.
... Although TLR2, TLR2/4, or TLR9 deficiency did not modify the response of peritoneal macrophages to S. aureus there was a reduced response in MyD88-deficient cells with reduced expression of cytokines such as TNFα (Kapetanovic et al., 2007). The same group also found a role for Nod2 in S. aureus recognition, though this response was seen in monocytes and peritoneal macrophages but not in alveolar macrophages (Kapetanovic et al., 2011). Degradation of S. aureus in the phagolysosome by lysozyme is required for optimal TLR stimulation (Wolf et al., 2011). ...
Macrophages are critical mediators of innate immune responses against bacteria. The Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus express a range of virulence factors, which challenge macrophages' immune competence. We review how macrophages respond to this challenge. Macrophages employ a range of strategies to phagocytose and kill each pathogen. When the macrophages capacity to clear bacteria is overwhelmed macrophages play important roles in orchestrating the inflammatory response through pattern recognition receptor-mediated responses. Macrophages also ensure the inflammatory response is tightly constrained, to avoid tissue damage, and play an important role in downregulating the inflammatory response once initial bacterial replication is controlled.
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... While the SSAg predominantly activate T cells of the adaptive immune system through TCR, certain other staphylococcal components such as the cell wall peptidoglycans, lipoteichoic acid, lipoproteins, unmethylated bacterial DNA containing the CpG motifs and even the intact bacteria, collectively called pathogen-associated molecular patterns (PAMPs), can activate the immune system through pathogen recognition receptors (PRR) comprising the tolllike receptors (TLR) and the NOD-like receptors (NLR), expressed by cells of both innate and adaptive arms of the immune system [9][10][11]. This leads to production of several proinflammatory cytokines, chemokines, and other inflammatory mediators [6,[12][13][14][15][16]. Some studies have shown that staphylococcal PAMPs can even directly activate T cells to a proinflammatory phenotype in a TLR2-Myd88 dependent manner [17], while others have demonstrated that S. aureus and staphylococcal PAMPs are able to override the immune regulatory functions of T regulatory cells, thereby indirectly promoting inflammation [18]. ...
Staphylococcus aureus is capable of causing a spectrum of human illnesses. During serious S. aureus infections, the staphylococcal pathogen-associated molecular patterns (PAMPs) such as peptidoglycan, lipoteichoic acid, and lipoproteins and even intact S. aureus, are believed to act in conjunction with the staphylococcal superantigens (SSAg) to activate the innate and adaptive immune system, respectively, and cause immunopathology. However, recent studies have shown that staphylococcal PAMPs could suppress inflammation by several mechanisms and protect from staphylococcal toxic shock syndrome, a life-threatening systemic disease caused by toxigenic S. aureus. Given the contradictory pro- and anti-inflammatory roles of staphylococcal PAMPs, we examined the effects of S. aureus-derived molecular patterns on immune responses driven by SSAg in vivo using HLA-DR3 and HLA-DQ8 transgenic mice. Our study showed that neither S. aureus-derived peptidoglycans (PGN), lipoteichoic acid (LTA), nor heat-killed Staphylococcus aureus (HKSA) inhibited SSAg-induced T cell proliferation in vitro. They failed to antagonize the immunostimulatory effects of SSAg in vivo as determined by their inability to attenuate systemic cytokine/chemokine response and reduce SSAg-induced T cell expansion. These staphylococcal PAMPs also failed to protect HLA-DR3 as well as HLA-DQ8 transgenic mice from either SSAg-induced toxic shock or pneumonia induced by a SSAg-producing strain of S. aureus.
... S. aureus is an important nosocomial and community-acquired pathogen and a leading cause of human infections worldwide [1,2]. HISA is a common model for studying phagocytosis, phagosome acidification, bacterial recognition, and cytokine production in response to S. aureus in phagocytes [29][30][31][32]. Here, we report that HISA induced production of NFκB1, IL1α, IL1β and TLR2 and that inhibition of NF-κB decreased NFκB1, IL1α, IL1β and TLR2 expression. ...
Nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor which controls the expression of various genes involved in immune responses; however, it is not clear whether NF-κB activation is critical for phagocytosis when Staphylococcus aureus is the pathogen. Using oligonucleotide microarrays, we investigated whether NF-κB cascade genes are altered in a mouse leukemic monocyte macrophage cell line (RAW 264.7) when the cells were stimulated to activate a host innate immune response against live S. aureus or heat-inactivated S. aureus (HISA). NF-κB cascade genes such as Nfκb1, Nfκbiz, Nfκbie, Rel, Traf1, and Tnfaip3 were up-regulated by all treatments at one hour after incubation. NF-κB is important in activating phagocytosis in RAW 264.7 cells infected with S. aureus. Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled HISA and decreased the expression of NFκB1, IL1α, IL1β and TLR2 in this cell line. Our results demonstrate that S. aureus may activate the NF-κB pathway and that NF-κB activation is required for phagocytosis of S. aureus by macrophages in a RAW 246.7 cell line.
... No studies have been performed using isolated AM. This lack of knowledge is not trivial, since several studies have shown that AM differ from other monocytic/macrophagic populations in several ways, including the cytokine response to infection or to stimulation with microbial antigens (18)(19)(20)(21)(22)(23), the susceptibility to infection or the intracellular replication kinetics for certain pathogens (22,23), the signaling pathways or innate immunity receptors involved in responses to antigenic stimuli (24,25), and several other aspects (26). Notably, a recent study showed that AM can constitutively carry pathogens from the lung to the draining lymph nodes even before dendritic cells (27). ...
Alveolar macrophages (AM) seem to constitute the main cellular target of inhaled brucellae. Here, we show that Brucella abortus invades and replicates in murine AM without inducing cytotoxicity. B. abortus infection induced a statistically significant increase of tumor necrosis factor alpha (TNF-α), CXCL1 or keratinocyte chemoattractant
(KC), interleukin-1β (IL-1β), IL-6, and IL-12 in AM from C57BL/6 mice and BALB/c mice, but these responses were generally
weaker and/or delayed compared to those elicited in peritoneal macrophages. Studies using knockout mice for TLR2, TLR4, and
TLR9 revealed that TNF-α and KC responses were mediated by TLR2 recognition. Brucella infection reduced in a multiplicity of infection-dependent manner the expression of major histocompatibility complex class
II (MHC-II) molecules induced by gamma interferon (IFN-γ) in AM. The same phenomenon was induced by incubation with heat-killed
B. abortus (HKBA) or the lipidated form of the 19-kDa outer membrane protein of Brucella (L-Omp19), and it was shown to be mediated by TLR2 recognition. In contrast, no significant downregulation of MHC-II was
induced by either unlipidated Omp19 or Brucella LPS. In a functional assay, treatment of AM with either L-Omp19 or HKBA reduced the MHC-II-restricted presentation of OVA
peptides to specific T cells. One week after intratracheal infection, viable B. abortus was detected in AM from both wild-type and TLR2 KO mice, but CFU counts were higher in the latter. These results suggest
that B. abortus survives in AM after inhalatory infection in spite of a certain degree of immune control exerted by the TLR2-mediated inflammatory
response. Both the modest nature of the latter and the modulation of MHC-II expression by the bacterium may contribute to
such survival.
... Irrespective of the underlying mechanism, it is likely that this state of low LPS responsiveness may be normal for equine PMs despite having a common progenitor cell to AMs (Gorgani et al., 2008;Kapetanovic et al., 2011), consistent with significantly different functional and phenotypic properties. Similar to flow cytometry, morphological differences between AMs and PMs were also observed by light microscopy, supporting differentiation in their function and phenotype. ...
... The contribution of various signaling molecules on IFN-γ and GM-CSF production was also analyzed using specific inhibitors (Fig. 1C). The optimal concentration for each inhibitory drug was chosen according to the literature (16- 19) and our own experience (20). CpG-ODN or IL-15/IL-18 alone did not induce any IFN-γ or GM-CSF secretion. ...
Natural killer (NK) cells are important for innate immunity in particular through the production of interferon-gamma (IFN-γ) and granulocyte-macrophage colony stimulating factor (GM-CSF). Both cytokines are important in restoration of immune function of tolerized leukocytes under inflammatory events. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors, rarely seeking their protein expression. We previously showed that murine spleen NK cells express TLR9 intracellularly and respond to CpG oligodeoxynucleotide (CpG-ODN) by producing IFN-γ and GM-CSF. However, to get such production the presence of accessory cytokines (such as IL-15 and IL-18) was required, whereas CpG-ODN or accessory cytokines alone did not induce IFN-γ or GM-CSF. We show here that TLR9 overlaps with the Golgi apparatus in NK cells. Furthermore, CpG-ODN stimulation in the presence of accessory cytokines induces the phosphorylation of c-jun, STAT3 and IκBα. IFN-γ and GM-CSF production requires NF-κB and STAT3 activation as well as Erk-dependent mechanisms for IFN-γ and p38 signaling for GM-CSF. Using knockout-mice, we show that UNC93b1 and IL-12 (produced by NK cells themselves) are also necessary for IFN-γ and GM-CSF production. IFN-γ production was found to be MyD88- and TLR9-dependent, whereas GM-CSF was TLR9-independent but dependent on Stimulator of Interferon Genes (STING), a cytosolic adaptor recently described for DNA sensing. Our study thereby allows us to gain insight into the mechanisms of synergy between accessory cytokines and CpG-ODN in NK cells. It also identifies a new and alternative signaling pathway for CpG-ODN in murine NK cells.
... The pro-apoptotic pathway is activated upon endocytosis of the TNF-α TNF receptor complex [9]; [11], [13], [14], whereas binding of TNF-α to TNF receptors on the outer membrane skews to anti-apoptotic signalling [9]. Contact to a pathogen may be sufficient for monocytes to secrete TNF-α [15], [16], [17], [18]. ...
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.
... Unlike osteoblasts at other sites, mandibular osteoblasts are in an oral environment. Therefore, it may not be a mere coincidence that the production of inflammatory cytokines, such as tumor necrosis factor, TNF-α, and of antimicrobial peptides such as defensin is under the control of α 5 β 1 integrin in gingival epithelial cells and immune cells, both of which face bacterial attacks [31,32]. Indeed, alteration in mandibular osteoblast TNFα mRNA level in response to LIPUS was similar to that of RANKL mRNA (data not shown). ...
It is unclear how bone cells at different sites detect mechanical loading and how site-specific mechanotransduction affects bone homeostasis. To differentiate the anabolic mechanical responses of mandibular cells from those of calvarial and long bone cells, we isolated osteoblasts from C57B6J mouse bones, cultured them for 1week, and subjected them to therapeutic low intensity pulsed ultrasound (LIPUS). While the expression of the marker proteins of osteoblasts and osteocytes such as alkaline phosphatase and FGF23, as well as Wnt1 and β-catenin, was equally upregulated, the expression of mandibular osteoblast messages related to bone remodeling and apoptosis differed from that of messages of other osteoblasts, in that the messages encoding the pro-remodeling protein RANKL and the anti-apoptotic protein Bcl-2 were markedly upregulated from the very low baseline levels. Blockage of the PI3K and α(5)β(1) integrin pathways showed that the mandibular osteoblast required mechanotransduction downstream of α(5)β(1) integrin to upregulate expression of the proteins β-catenin, p-Akt, Bcl-2, and RANKL. Mandibular osteoblasts thus must be mechanically loaded to preserve their capability to promote remodeling and to insure osteoblast survival, both of which maintain intact mandibular bone tissue. In contrast, calvarial Bcl-2 is fully expressed, together with ILK and phosphorylated mTOR, in the absence of LIPUS. The antibody blocking α(5)β(1) integrin suppressed both the baseline expression of all calvarial proteins examined and the LIPUS-induced expression of all mandibular proteins examined. These findings indicate that the cellular environment, in addition to the tridermic origin, determines site-specific bone homeostasis through the remodeling and survival of osteoblastic cells. Differentiated cells of the osteoblastic lineage at different sites transmit signals through transmembrane integrins such as α(5)β(1) integrin in mandibular osteoblasts, whose signaling may play a major role in controlling bone homeostasis.
Sepsis has been identified by the World Health Organization (WHO) as a global health priority. There has been a tremendous effort to decipher underlying mechanisms responsible for organ failure and death, and to develop new treatments. Despite saving thousands of animals over the last three decades in multiple preclinical studies, no new effective drug has emerged that has clearly improved patient outcomes. In the present review, we analyze the reasons for this failure, focusing on the inclusion of inappropriate patients and the use of irrelevant animal models. We advocate against repeating the same mistakes and propose changes to the research paradigm. We discuss the long-term consequences of surviving sepsis and, finally, list some putative approaches-both old and new-that could help save lives and improve survivorship.
Alveolar macrophages (AM) are sentinels of the immune response to bacteria in the lung. AM development in the lung results in a unique differentiation program which distinguishes AM from other tissue macrophages and equips them to subserve their primary physiological roles in pulmonary homeostasis. The response to bacteria requires an adaption of function to ensure that host defense is achieved while limiting unwanted inflammatory responses in the airspace. AM utilize a range of receptors to phagocytose bacteria but their intracellular killing capacity is finite. With larger inocula of infection AM play critical roles in regulating the inflammatory response. Signal transduction via diverse pattern recognition receptors allows AM to coordinate the immune response of a range of different cell types and to contribute to the cytokine network during infection. A key feature of the optimal AM response to bacterial infection is that the inflammatory response is kept at the minimum required to successfully control infection. When the inflammatory response is upregulated, it is tightly controlled and promptly downsized to minimize tissue damage and prevent compromise to gas exchange in the alveolar space. Bacteria in turn subvert these responses to try and establish their own ecological niche, which can as a consequence result in pneumonia or its complications. © Springer Science+Business Media New York 2013. All rights are reserved.
The type IIA secretory phospholipase A2 (sPLA2-IIA) is a host defense protein endowed with antibacterial activity, especially against Gram positive bacteria. Cystic fibrosis (CF) is a genetic disease due to mutations of Cftr gene. In the lungs, CFTR mutation favored bacterial colonization by bacterial pathogens, of which S. aureus and P. aeruginosa are the most isolated. These two bacterial species sequentially colonized airways of CF patients: S. aureus is predominant in young patients and P. aeruginosa in adults. But the mechanisms involved in this switch of bacterial prevalence are still unknown. In this work we showed that sPLA2-IIA levels were increased in lungs of CF patients compared to lungs of non-CF patients and that sPLA2-IIA levels increased with age of patients. sPLA2-IIA recovered from CF patients expectorations was active and killed specifically S. aureus. Using animal models of lung infection, we demonstrated the selectivity of sPLA2-IIA against S. aureus and that P. aeruginosa induced sPLA2-IIA expression, the latter contributed to S . aureus elimination from the airways. Finally, we identified epithelial cells as a major source of sPLA2-IIA in CF airways. In these cells, P. aeruginosa induced sPLA2-IIA expression through injection of ExoS toxin and activation of KLF2 transcription factor. Taken together, these results indicate that i) P. aeruginosa-induced sPLA2-IIA expression in CF airways participated to S. aureus elimination and ii) a bacteria eliminate another bacteria by manipulating host innate immunity.
Knocking down methyl CpG binding protein 2 (MeCP2) enhances NF-κB activation in human peripheral blood mononuclear cells (PBMC). In this study, we examined whether this caused the expression of cytokines to be elevated. Increased levels of TNFα, IL-6, and IL-3 mRNAs were observed in human PBMC made MeCP2 deficient with a lentiviral shRNA MeCP2 vector and in splenocytes from MeCP2-null mice. TNFα neutralizing antibody attenuated expression of IL-6 and TNFα but did not affect expression of IL-3. Lipopolysaccharide-mediated increases in TNFα, IL-6, and IL-3 mRNAs were also enhanced in MeCP2-deficient PBMC. Two inhibitors of NF-κB blocked the increased levels of IL-6, TNFα, and IL-3 in MeCP2-deficient PBMC treated with lipopolysaccharide. MeCP2 deficiency also enhanced expression of IL-6 and TNFα mRNAs in the THP1 human monocyte cell line, which were also attenuated by the NF-κB inhibitors. In chromatin immunoprecipitation assays, the binding of the NF-κB family member p65 and acetylated H3 to the TNFα promoter was greater after treatment with LPS in MeCP2-deficient THP1 cells. MeCP2 did not bind to the TNFα promoter. In summary, the data indicates that MeCP2 deficiency increases expression of TNFα and other inflammatory cytokines by enhancing NF-κB signaling.
Copyright © 2015. Published by Elsevier B.V.
Macrophages respond to Gram-negative bacterial pathogens by phagocytosis and pro-inflammatory gene expression. These responses may require GTPases that have been implicated in cytoskeletal alterations and activation of NF-kappaB. To determine the role of Rac1 and Cdc42 in signal transduction events triggered by Pseudomonas aeruginosa, we expressed GTP binding-deficient alleles of Rac1 or Cdc42, or Chim-GAP, a Rac1/Cdc42-specific GTPase-activating protein domain, in a subline of RAW 264.7 cells, and challenged the transfected cells with a laboratory strain of P. aeruginosa, PAO1. Expression of Rac1 N17, Cdc42 N17, or Chim-GAP led to a marked reduction of phagocytosis. In contrast, nuclear translocation of p65 NF-kappaB was unaffected by expression of the same constructs. Incubation of macrophages with PAO1 led to NF-kappaB-dependent expression of inducible nitric-oxide synthase, COX-2, and tumor necrosis factor-alpha, which was unaffected by inhibition of Rac1 or Cdc42 function. Isogenic strains of PAO1 that lacked surface adhesins were poorly ingested; however, they induced pro-inflammatory gene expression with an efficiency equal to that of PAO1. These results indicate that the signal transduction events leading to phagocytosis and pro-inflammatory protein expression are distinct. Rac1 and Cdc42 serve as effectors of phagocytosis, but not NF-kappaB-dependent gene expression, in the macrophage response to P. aeruginosa.
Large conductance, calcium-activated K+ (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation
by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous α5β1 integrin
ligand, enhances BK channel current through both Ca2+- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293
cells expressing murine BK channel α-subunits (mSlo) are acutely potentiated following α5β1 integrin activation. The effect occurs in a Ca2+-dependent manner, 1–3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations
for mSlo are left-shifted at free Ca2+ concentrations ≥1 μm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca2+ sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the
critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced
by α5β1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel α-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca2+ sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in
response to integrin-extracellular matrix interactions.
Although much progress has been made toward the identification of innate immune receptors, far less is known about how these receptors recognize specific microbial products. Such studies have been hampered by the need to purify compounds from microbial sources and a reliance on biological assays rather than direct binding to monitor recognition. We have employed surface plasmon resonance (SPR) binding studies using a wide range of well defined synthetic muropeptides derived from Gram-positive (lysine-containing) and Gram-negative (diaminopimelic acid (DAP)-containing) bacteria to demonstrate that Toll-like receptor 2 can recognize peptidoglycan (PGN). In the case of lysine-containing muropeptides, a limited number of compounds, which were derived from PGN remodeled by bacterial autolysins, was recognized. However, a wider range of DAP-containing muropeptides was bound with high affinity, and these compounds were derived from nascent and remodeled PGN. The difference in recognition of the two classes of muropeptides is proposed to be a strategy by the host to respond appropriately to Gram-negative and -positive bacteria, which produce vastly different quantities of PGN. It was also found that certain modifications of the carboxylic acids of isoglutamine and DAP can dramatically reduce binding, and thus, bacterial strains may employ such modifications to evade innate immune detection. Cellular activation studies employing highly purified PGN from Bacillus licheniformis, Bacillus subtilis, Escherichia coli, Lactobacillus plantarum, Micrococcus luteus, and Staphylococcus aureus support the structure binding relationship. The data firmly establish Toll-like receptor 2 as an innate immune sensor for PGN and provides an understanding of host-pathogen interactions at the molecular level.
NOD2 (the nucleotide-binding oligomerization domain containing protein 2) is known to be involved in host recognition of bacteria,
although its role in the host response to Staphylococcus aureus infection is unknown. NOD2-deficient (Nod2−/−) mice and wild-type (WT) littermate controls were injected intraperitoneally with S. aureus suspension (107 bacteria/g of body weight), and their survival was monitored. Cultured bone marrow-derived neutrophils were harvested from
Nod2−/− and WT mice and tested for cytokine production and phagocytosis. Compared to WT mice, Nod2−/− mice were significantly more susceptible to S. aureus infection (median survival of 1.5 days versus >5 days; P = 0.003) and had a significantly higher bacterial tissue burden. Cultured bone marrow-derived neutrophils from Nod2−/− and WT mice had similar levels of peritoneal neutrophil recruitment and intracellular killing, but bone marrow-derived neutrophils
from Nod2−/− mice had significantly reduced ability to internalize fluorescein-labeled S. aureus. Nod2−/− mice had significantly higher levels of Th1-derived cytokines in serum (tumor necrosis factor alpha, gamma interferon, and
interleukin-2 [IL-2]) compared to WT mice, whereas the levels of Th2-derived cytokines (IL-1β, IL-4, IL-6, and IL-10) were
similar in Nod2−/− and WT mice. Thus, mice deficient in NOD2 are more susceptible to S. aureus. Increased susceptibility is due in part to defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and
a higher bacterial tissue burden.
Type I IFNs represent a major antimicrobial defense mechanism due to their property of enhancing immune responses by priming both innate and adaptive immune cells. Plasmacytoid dendritic cells (pDC) are the major source of type I IFN in the human body and represent innate immune cells involved in first-line defense against invading pathogens. Although pDC activation has been extensively studied upon stimulation with synthetic TLR ligands, viruses, and intracellular bacteria, there is only scarce information on extracellular bacteria. In this study we show that the triggering of human pDC-derived IFN-alpha secretion by Staphylococcus aureus is independent of TLR2 and specific for coagulase-positive staphylococci. Specificity of the pDC response to S. aureus is independent of the bacterial virulence factors protein A and alpha-toxin but is mediated by Ag-specific IgG and CD32. S. aureus-induced pDC activation can be blocked by inhibitory DNA oligonucleotides and chloroquine, suggesting that engagement of TLR7/9 by bacterial nucleic acids after CD32-mediated uptake of these compounds may play a central role in this process. Altogether, we propose that in marked contrast to nonselective TLR2-dependent activation of most innate immune cells, pDC activation by S. aureus represents an Ag-specific memory response since it requires the presence of class-switched immunoglobulins.
Basic substances and acidic ionophores that increase the lysosomal pH in cultured macrophages (Ohkuma, S., and B. Poole, 1978, Proc. Natl. Acad. Sci. USA., 75:3327-3331; Poole, B., and S. Ohkuma, 1981, J. Cell Biol., 90:665-669) inhibited the digestion of heat-denatured acetylated bovine serum albumin (BSA) taken up by the cells. For several substances, the shift in pH sufficed to explain the inhibition of proteolysis. Additional effects, presumably on enzyme activities, have to be postulated for tributylamine, amantadine, and chloroquine. Sodium fluoride (10 mM) had no significant effect on the breakdown of BSA by macrophages. The breakdown of endogenous macrophage proteins, whether short lived or long lived, was inhibited approximately 40% by 10 mM NaF and 30%, or sometimes less in the case of long-lived proteins, by 100 microM chloroquine. When the cells were supplied with BSA, a mixture of cell proteins, or even inert endocytosible materials, the breakdown of endogenous long-lived proteins and the inhibitory effect of chloroquine on this process were selectively reduced. Inhibition of endocytosis by cytochalasins B or D did not affect the chloroquine-sensitive breakdown of endogenous proteins, indicating that the proteins degraded by this process were truly endogenous and not taken in from the outside by cellular cannibalism. On the other hand, when macrophage proteins were supplied extracellularly, their breakdown occurred at the same rate for short-lived and long-lived proteins, and it was strongly inhibited by chloroquine and not by NaF. It is concluded from these results that the breakdown of endogenous proteins, both short-lived and long-lived, probably takes place partly (approximately 30%) in lysosomes and partly through one or more nonlysosomal mechanism(s) unaffected by chloroquine and presumably susceptible to inhibition by fluoride. A difference must exist between short-lived and long-lived proteins in the manner in which they reach lysosomes or are handled by these organelles; this difference would account for the selective effect of the supply of endocytosible materials on the lysosomal processing of long-lived proteins.
Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases, including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin, a naturally occurring bioflavinoid, was previously shown to inhibit PtdIns 3-kinase with an IC50 of 1.3 microgram/ml (3.8 microM); inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors, with the most potent ones exhibiting IC50 values in the range of 1.7-8.4 micrograms/ml. In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 > 30 micrograms/ml). Since quercetin has also been shown to inhibit other PtdIns and protein kinases, other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (also known as 2-(4-morpholinyl)-8-phenylchromone, LY294002), completely and specifically abolished PtdIns 3-kinase activity (IC50 = 0.43 microgram/ml; 1.40 microM) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity relationship, with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in fMet-Leu-Phe-stimulated human neutrophils, as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction, the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response.
Toll-like receptors (TLRs) 2 and 4 are signal transducers for lipopolysaccharide, the major proinflammatory constituent in
the outer membrane of Gram-negative bacteria. We observed that membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans activated cells heterologously expressing TLR2 but not those expressing TLR1 or TLR4. These TLR2-expressing cells were also
stimulated by living motile B. burgdorferi, suggesting that TLR2 recognition of lipoproteins is relevant to naturalBorrelia infection. Importantly, a TLR2 antibody inhibited bacterial lipoprotein/lipopeptide-induced tumor necrosis factor release
from human peripheral blood mononuclear cells, and TLR2-null Chinese hamster macrophages were insensitive to lipoprotein/lipopeptide
challenge. The data suggest a role for the native protein in cellular activation by these ligands. In addition, TLR2-dependent
responses were seen using whole Mycobacterium avium andStaphylococcus aureus, demonstrating that this receptor can function as a signal transducer for a wide spectrum of bacterial products. We conclude
that diverse pathogens activate cells through TLR2 and propose that this molecule is a central pattern recognition receptor
in host immune responses to microbial invasion.
The central role of alveolar macrophages in the establishment of lipopolysaccharide (LPS)-induced lung inflammation is well demonstrated. They produce and release numerous proinflammatory molecules, among which is tumor necrosis factor alpha (TNF-alpha), a cytokine responsible in part for the neutrophilic alveolitis. Interleukin-10 (IL-10) produced by LPS-activated mononuclear phagocytes is a major anti-inflammatory cytokine that down-regulates TNF-alpha synthesis. We studied the ability of murine alveolar macrophages to produce IL-10 in vivo and in vitro, in response to LPS. Unexpectedly, the IL-10 protein was not detected in the whole lung and airspaces after LPS intranasal instillation. In addition, no IL-10 protein was found in supernatants of isolated and LPS-stimulated alveolar macrophages. The lack of IL-10 synthesis was confirmed by the absence of specific RNA transcripts. By contrast and as expected, autologous peritoneal macrophages produced IL-10 upon LPS challenge. Drugs that usually modify the TNF-alpha/IL-10 balance in favor of IL-10 were used without success. Thus, maneuvers allowing an increase in intracellular cAMP concentrations did not reverse this unexpected phenotype. Moreover, direct activation of protein kinase C with PMA was unable to trigger IL-10 formation by alveolar, by contrast to peritoneal, macrophages. The current findings describe a specific phenotype for murine alveolar macrophages during LPS-induced inflammation.
Nod1 is an Apaf-1-like molecule composed of a caspase-recruitment domain (CARD), nucleotide-binding domain, and leucine-rich
repeats that associates with the CARD-containing kinase RICK and activates nuclear factor κB (NF-κB). We show that self-association
of Nod1 mediates proximity of RICK and the interaction of RICK with the γ subunit of the IκB kinase (IKKγ). Similarly, the
RICK-related kinase RIP associated via its intermediate region with IKKγ. A mutant form of IKKγ deficient in binding to IKKα
and IKKβ inhibited NF-κB activation induced by RICK or RIP. Enforced oligomerization of RICK or RIP as well as of IKKγ, IKKα,
or IKKβ was sufficient for induction of NF-κB activation. Thus, the proximity of RICK, RIP, and IKK complexes may play an
important role for NF-κB activation during Nod1 oligomerization or trimerization of the tumor necrosis factor α receptor.
Bacterial lipoproteins (BLP) trigger immune responses via Toll-like receptor 2 (TLR2) and their immunostimulatory properties are attributed to the presence of a lipoylated N-terminus. Most BLP are triacylated at the N-terminus cysteine residue, but mycoplasmal macrophage-activating lipopeptide-2 kD (MALP-2) is only diacylated. Here we show that TLR6-deficient (TLR6(-/-)) cells are unresponsive to MALP-2 but retain their normal responses to lipopeptides of other bacterial origins. Reconstitution experiments in TLR2(-/-) TLR6(-/-) embryonic fibroblasts reveal that co-expression of TLR2 and TLR6 is absolutely required for MALP-2 responsiveness. Taken together, these results show that TLR6 recognizes MALP-2 cooperatively with TLR2, and appears to discriminate between the N-terminal lipoylated structures of MALP-2 and lipopeptides derived from other bacteria.
Staphylococcus aureus Cowan I and a clinically isolated coagulase-negative Staphylococcus strain, S. saprophyticus 10312, were found to have two fibronectin binding proteins, FnBPA and FnBPB. While both staphylococci bound to serum fibronectin
to a similar extent, fibronectin binding significantly increased the phagocytic activity of macrophages against S. aureus (by ca. 150%) but not against S. saprophyticus. This enhancing effect of fibronectin was inhibited by an RGD sequence-containing peptide and also by anti-very late antigen
5 antibody. This suggests that the effect is mediated by very late antigen 5 expressed on macrophages. In macrophages ingesting
fibronectin-bound Cowan I, α5 and β1 chains were associated with the cytoskeleton. Cytosolic signaling factors such as paxillin, c-Src, and c-Csk were also associated
with the cytoskeleton. On the contrary, β3 integrin transiently disappeared from the cytoskeleton when macrophages ingested the fibronectin-treated S. aureus Cowan I. Furthermore, the Src kinase family tyrosine kinase Lyn dissociated from the cytoskeleton. These cellular components
did not respond in a fibronectin-dependent manner when macrophages phagocytosed S. saprophyticus. This means that only fibronectin-treated S. aureus Cowan I induces the accumulation of very late antigen 5, which in turn induces the association of paxillin and tyrosine kinases.
It is thought that the phagocytic activity of macrophages against fibronectin-treated S. aureus was increased by signaling via the activation of very late antigen 5.
Major trauma is associated with a decreased capacity of patients' leukocytes to produce proinflammatory cytokines on in vitro stimulation. We studied leukocytes from 48 patients with trauma and showed that this hyporeactivity was restricted to gram-negative bacteria, Escherichia coli endotoxin, and unmethylated bacterial DNA, whereas Leptospira interrogans endotoxin-induced tumor necrosis factor production was similar to that observed with healthy donors. As well, tumor necrosis factor and interleukin-6 production in response to gram-positive bacteria was not altered. The expression of toll-like receptor (TLR) 2 was not reduced on patients' monocytes as compared with healthy control subjects, whereas that of TLR4 was reduced. However, the hyporeactivity to gram-negative bacteria and E. coli endotoxin cannot be fully explained by the downregulation of TLR4. Indeed, unlike proinflammatory cytokines, after stimulation with these microbial products the release of antiinflammatory cytokines was increased as compared with healthy control subjects. The increased interleukin-10 production was analyzed in terms of intracellular signaling in peripheral blood mononuclear cells from patients with trauma: our results suggest the involvement of p38 mitogen-activated protein kinase, Sp-1 transcription factor, heterotrimeric Gi protein, and phosphatidylinositol-3'-kinase. In conclusion, the immunodysregulation described for patients with trauma is not a generalized phenomenon but depends on the stimulus and the signaling pathway.
Toll-like receptor 2 (TLR2) has been shown to recognize several classes of pathogen-associated molecular patterns including peptidoglycan (PG). However, studies linking PG with TLR2 recognition have relied mainly on the use of commercial Staphylococcus aureus PG and have not addressed TLR2 recognition of other PG types. Using highly purified PGs from eight bacteria (Escherichia coli, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Helicobacter pylori, Bacillus subtilis, Listeria monocytogenes, Streptococcus pneumoniae and S. aureus), we show that these PGs are not sensed through TLR2, TLR2/1 or TLR2/6. PG sensing is lost after removal of lipoproteins or lipoteichoic acids (LTAs) from Gram-negative and Gram-positive cell walls, respectively. Accordingly, purified LTAs are sensed synergistically through TLR2/1. Finally, we show that elicited peritoneal murine macrophages do not produce tumour necrosis factor-alpha or interleukin-6 in response to purified PGs, suggesting that PG detection is more likely to occur intracellularly (through Nod1/Nod2) rather than from the extracellular compartment.
Since the ability of peptidoglycan (PGN) to activate Toll-like receptor 2 (TLR2) was recently questioned, we reevaluated activation
of TLR2 by PGN. Polymeric soluble or insoluble Staphylococcus aureus PGN, repurified by sodium dodecyl sulfate or phenol extraction, activated TLR2 at 0.1 to 1 or 10 μg/ml, respectively, and
induced tumor necrosis factor alpha production. The TLR2 activation by PGN, but not by lipoteichoic acid, was abolished by
muramidase digestion. We conclude that polymeric S. aureus PGN is a TLR2 activator.
Phagocyte recognition and clearance of bacteria play essential roles in the host response to infection. In an on-going forward genetic screen, we identify the Drosophila melanogaster scavenger receptor Croquemort as a receptor for Staphylococcus aureus, implicating for the first time the CD36 family as phagocytic receptors for bacteria. In transfection assays, the mammalian Croquemort paralogue CD36 confers binding and internalization of Gram-positive and, to a lesser extent, Gram-negative bacteria. By mutational analysis, we show that internalization of S. aureus and its component lipoteichoic acid requires the COOH-terminal cytoplasmic portion of CD36, specifically Y463 and C464, which activates Toll-like receptor (TLR) 2/6 signaling. Macrophages lacking CD36 demonstrate reduced internalization of S. aureus and its component lipoteichoic acid, accompanied by a marked defect in tumor necrosis factor-alpha and IL-12 production. As a result, Cd36-/- mice fail to efficiently clear S. aureus in vivo resulting in profound bacteraemia. Thus, response to S. aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain, which initiates TLR2/6 signaling.
Alveolar macrophages (AMs) avidly bind and ingest inhaled environmental particles and bacteria. To identify the particle binding receptor(s) on human AMs, we used functional screening of anti-human AM hybridomas and isolated a mAb, PLK-1, which inhibits AM binding of unopsonized particles (e.g., TiO2, latex beads; 63 +/- 5 and 67 +/- 4% inhibition, respectively, measured by flow cytometry; n = 11) and unopsonized bacteria ( approximately 84 and 41% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PLK-1, respectively). The PLK-1 Ag was identified as the human class A scavenger receptor (SR) MARCO (macrophage receptor with collagenous structure) by observing specific immunolabeling of COS cells transfected with human MARCO (but not SR-AI/II) cDNA and by immunoprecipitation by PLK-1 of a protein of appropriate molecular mass (approximately 70 kDa) from both normal human bronchoalveolar lavage cells (>90% AMs) and human MARCO-transfected COS cells. PLK-1 also specifically inhibited particle binding by COS cells, only after transfection with human MARCO cDNA. Immunostaining showed specific labeling of AMs within human lung tissue, bronchoalveolar lavage samples, as well as macrophages in other sites (e.g., lymph node and liver). Using COS transfectants with different truncated forms of MARCO, allowed epitope mapping for the PLK-1 Ab to MARCO domain V between amino acid residues 420 and 431. A panel of Abs to various SRs identified expression on AMs, but failed to inhibit TiO2 or S. aureus binding. The data support a dominant role for MARCO in the human AM defense against inhaled particles and pathogens.
Heterogeneity of the macrophage lineage has long been recognized and, in part, is a result of the specialization of tissue macrophages in particular microenvironments. Circulating monocytes give rise to mature macrophages and are also heterogeneous themselves, although the physiological relevance of this is not completely understood. However, as we discuss here, recent studies have shown that monocyte heterogeneity is conserved in humans and mice, allowing dissection of its functional relevance: the different monocyte subsets seem to reflect developmental stages with distinct physiological roles, such as recruitment to inflammatory lesions or entry to normal tissues. These advances in our understanding have implications for the development of therapeutic strategies that are targeted to modify particular subpopulations of monocytes.
Macrophage responses to Francisella infection have been characterized previously by subdued proinflammatory responses; however, these studies have generally focused on macrophage cell lines or monocyte-derived macrophages. Therefore, we studied the ability of fresh human blood monocytes to engulf and respond to Francisella by using the live vaccine strain variant and Francisella novicida. Because Francisella organisms have been reported to escape from the phagolysosome into the cytosol, we hypothesized that this escape may trigger the activation of caspase-1. Francisella tularensis variants were readily taken up by fresh human CD14⁺ monocytes, inducing the release of IL-1β, as well as IL-8, in a time- and dose-dependent fashion. Importantly, whereas live and dead Escherichia coli, F. novicida, and live vaccine strain, as well as the LPS of E. coli, were able to induce abundant IL-1β mRNA synthesis and intracellular pro-IL-1β production, only live Francisella induced enhanced IL-1β processing and release (51 ± 10 vs. 7.1 ± 2.1 ng/ml, for F. novicida vs. E. coli LPS; P = 0.0032). Cytochalasin D blocked the Francisella internalization and the Francisella-induced monocyte IL-1β processing and release but not that induced by the exogenous stimulus E. coli LPS. Also, killing bacteria did not block uptake but significantly diminished the IL-1β processing and release that was induced by Francisella. Blocking bacterial escape from the phagosome into the cytosol also decreased IL-1β but not IL-8 release. These findings demonstrate that Francisella organisms efficiently induce IL-1β processing and release in fresh monocytes by means of a sensing system that requires the uptake of live bacteria capable of phagosome escape.
• bacteria
• caspase-1
• cytokine
• phagocytosis
Yeast and bacteria elicit arachidonate release in macrophages, leading to the formation of leukotrienes and prostaglandins, important mediators of inflammation. Receptors recognising various microbes have been identified, but the signalling pathways are not entirely understood. Cytosolic phospholipase A2 is a major down-stream target and this enzyme is regulated by both phosphorylation and an increase in intracellular Ca2+. Potential signal components are MAP kinases, phosphatidylinositol 3-kinase and phospholipase Cgamma2. The latter can undergo tyrosine phosphorylation, and Src family kinases might carry out this phosphorylation. Btk, a Tec family kinase, could also be important. Our aim was to further elucidate the role of Src family kinases and Btk.
Arachidonate release from murine peritoneal macrophages was measured by prior radiolabeling. Furthermore, immunoprecipitation and Western blotting were used to monitor changes in activity/phosphorylation of intermediate signal components. To determine the role of Src family kinases two different inhibitors with broad specificity (PP2 and the Src kinase inhibitor 1, SKI-1) were used as well as the Btk inhibitor LFM-A13.
Arachidonate release initiated by either Staphylococcus aureus or yeast-derived zymosan beads was shown to depend on members of the Src kinase family as well as Btk. Src kinases were found to act upstream of Btk, phosphatidylinositol 3-kinase, phospholipase Cgamma2 and the MAP kinases ERK and p38, thereby affecting all branches of the signalling investigated. In contrast, Btk was not involved in the activation of the MAP-kinases. Since the cytosolic phospholipase A2 in macrophages is regulated by both phosphorylation (via ERK and p38) and an increase in intracellular Ca2+, we propose that members of the Src kinase family are involved in both types of regulation, while the role of Btk may be restricted to the latter type.
Arachidonate release induced by either Staphylococcus aureus or zymosan was found to depend on Src family kinases as well as Btk. While members of the Src kinase family were shown to act upstream of Btk and the MAP kinases, Btk plays another role independent of MAP kinases, but down-stream of the Src family kinases.
In this study, we investigated the signaling pathway involved in IL-6 production caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium, Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused concentration- and time-dependent increases in IL-6, PGE(2), and cAMP production. PGN-mediated IL-6 production was inhibited by a nonselective cyclooxygenase (COX) inhibitor (indomethacin), a selective COX-2 inhibitor (NS398), a PGE(2) (EP2) antagonist (AH6809), a PGE(4) (EP4) antagonist (AH23848), and a protein kinase A (PKA) inhibitor (KT5720), but not by a nonselective NO synthase inhibitor (N(G)-nitro-l-arginine methyl ester). Furthermore, PGE(2), an EP2 agonist (butaprost), an EP2/PGE(3) (EP3)/EP4 agonist (misoprostol), and misoprostol in the presence of AH6809 all induced IL-6 production, whereas an EP1/EP3 agonist (sulprostone) did not. PGN caused time-dependent activations of IkappaB kinase alphabeta (IKKdbeta) and p65 phosphorylation at Ser(276), and these effects were inhibited by NS398 and KT5720. Both PGE(2) and 8-bromo-cAMP also caused IKKdbeta kinase alphabeta phosphorylation. PGN resulted in two waves of the formation of NF-kappaB-specific DNA-protein complexes. The first wave of NF-kappaB activation occurred at 10-60 min of treatment, whereas the later wave occurred at 2-12 h of treatment. The PGN-induced increase in kappaB luciferase activity was inhibited by NS398, AH6809, AH23848, KT5720, a protein kinase C inhibitor (Ro31-8220), and a p38 MAPK inhibitor (SB203580). These results suggest that PGN-induced IL-6 production involves COX-2-generated PGE(2), activation of the EP2 and EP4 receptors, cAMP formation, and the activation of PKA, protein kinase C, p38 MAPK, IKKdbeta, kinase alphabeta, p65 phosphorylation, and NF-kappaB. However, PGN-induced NO release is not involved in the signaling pathway of PGN-induced IL-6 production.
Deletion of apoptotic cells from tissues involves their phagocytosis by macrophages, dendritic cells, and tissue cells. Although much attention has been focused on the participating ligands, receptors, and mechanisms of uptake, little is known of the disposition of the ingested cell within the phagosome. Here we show that uptake of apoptotic cells by macrophages or fibroblasts results in rapid phagosome maturation, whereas macrophage phagosomes containing Ig-opsonized target cells mature at a slower rate. The early maturation was shown to depend on activation of Rho acting through Rho kinase on ezrin-radixin-moesin proteins. Blockade of Rho signaling or inhibition of moesin both delayed maturation rates to those seen with opsonized targets. By contrast, phagosome maturation in dendritic cells was slower, similar between apoptotic and opsonized target cells, and unaffected by Rho inhibition. These observations have direct implications for the clearance of dying cells and the roles played by different phagocytes in antigen digestion and presentation.
Toll-like receptors (TLRs) are involved in the sensing of microbially derived compounds. We analyzed the contribution of these
receptors to cytokine production by macrophages following stimulation with whole bacteria. Using knockout mice, we confirmed
that the TLR4 and TLR2 contribution was predominant in the induction of tumor necrosis factor alpha and interleukin-10 by
gram-negative bacteria. In contrast, the absence of TLR2 and/or TLR4 or TLR9 did not affect the response to gram-positive
bacteria. In the absence of TLR2, phagocytosis was essential for cytokine production in response to heat-killed Staphylococcus aureus (HKSA). Because intracellular sensing was important in the absence of TLR2, we evaluated the contribution of Nod1 and Nod2,
intracytoplasmic sensors of peptidoglycan-derived muropeptides, to the response to HKSA. By transfecting RAW 264.7 macrophages
with dominant negative (DN) forms of Nod1 and Nod2, we showed that both molecules inhibited NF-κB activation in response to
HKSA. The unexpected interference of DN Nod1 in the response of macrophages to gram-positive bacteria was confirmed with a
Nod2 agonist (muramyl dipeptide) in transfection experiments with HEK293T cell. Taken together, these results show the contribution
of phagocytosis and Nod molecules to the response to HKSA in macrophages and also identify possible cross talk between Nod1
and Nod2.
These studies demonstrate that treatment of macrophages with lovastatin, a cholesterol-lowering drug that blocks farnesylation and geranylgeranylation of target proteins, increases LPS-induced TNF-alpha production. This is reversed by the addition of mevalonate, which bypasses the lovastatin block. Examination of membrane localization of RhoA, Cdc42, Rac1, and Ras demonstrated decreased membrane localization of the geranylgeranylated Rho family members (RhoA, Cdc42, and Rac1) with no change in the membrane localization of farnesylated Ras. LPS-induced TNF-alpha production in the presence of the Rho family-specific blocker (toxin B from Clostridium difficile) was significantly enhanced consistent with the lovastatin data. One intracellular signaling pathway that is required for TNF-alpha production by LPS is the extracellular signal-regulated kinase (ERK). Significantly, we found prolonged ERK activation after LPS stimulation of lovastatin-treated macrophages. When we inhibited ERK, we blocked the lovastatin-induced increase in TNF-alpha production. As a composite, these studies demonstrate a negative role for one or more Rho family GTPases in LPS-induced TNF-alpha production.
Monocytes acquire a dendritic cell (DC) phenotype when cultured with GM-CSF and IL-4. By contrast, CSF-1 is a potent inducer of monocyte-to-macrophage differentiation. Increasing evidence indicates that DC development is impaired in conditions characterized by CSF-1 overproduction, including pregnancy, trauma, and diverse malignancies. To study this, we have exposed newly established monocyte-derived DC cultures to conditions of CSF-1 excess. As a consequence, differentiation is skewed toward a unique intermediate phenotype, which we have termed DC-M. Such cells exhibit macrophage-like morphology with impaired allostimulatory capacity, altered cytokine production, and a distinctive cell surface immunophenotype. In light of the emerging role of caspase activation during macrophage differentiation, the activity of caspases 3, 8, and 9 was examined in DC and DC-M cultures. It is striking that DC-M cultures exhibit a delayed and progressive increase in activation of all three caspases, associated with depolarization of mitochondrial membrane potential. Furthermore, when DC-M cultures were supplemented with an inhibitor of caspase 8 or caspase 9, impairment of DC differentiation by CSF-1 was counteracted. To investigate upstream regulators of caspase activation in DC-M cultures, experiments were performed using inhibitors of proximal CSF-1 receptor signaling. These studies demonstrated that the PI-3K inhibitors, wortmannin and LY294002, antagonize the ability of CSF-1 to inhibit DC differentiation and to promote caspase activation. Together, these data identify a novel, PI-3K-dependent pathway by which CSF-1 directs delayed caspase activation in monocytes and thereby modulates DC differentiation.
We have previously demonstrated that phagocytosed Borrelia burgdorferi induces activation programs in human peripheral blood mononuclear cells that differ qualitatively and quantitatively from
those evoked by equivalent lipoprotein-rich lysates. Here we report that ingested B. burgdorferi induces significantly greater transcription of proinflammatory cytokine genes than do lysates and that live B. burgdorferi, but not B. burgdorferi lysate, is avidly internalized by monocytes, where the bacteria are completely degraded within phagolysosomes. In the course
of these experiments, we discovered that live B. burgdorferi also induced a dose-dependent decrease in monocytes but not a decrease in dendritic cells or T cells and that the monocyte
population displayed morphological and biochemical hallmarks of apoptosis. Particularly noteworthy was the finding that apoptotic
changes occurred predominantly in monocytes that had internalized spirochetes. Abrogation of phagocytosis with cytochalasin
D prevented the death response. Heat-killed B. burgdorferi, which was internalized as well as live organisms, induced a similar degree of apoptosis of monocytes but markedly less cytokine
production. Surprisingly, opsonophagocytosis of Treponema pallidum did not elicit a discernible cell death response. Our combined results demonstrate that B. burgdorferi confined to phagolysosomes is a potent inducer of cytosolic signals that result in (i) production of NF-κB-dependent cytokines,
(ii) assembly of the inflammasome and activation of caspase-1, and (iii) induction of programmed cell death. We propose that
inflammation and apoptosis represent mutually reinforcing components of the immunologic arsenal that the host mobilizes to
defend itself against infection with Lyme disease spirochetes.
The activation of p38alpha, a MAPK family member, is associated with macrophage activation by microbial pattern molecules, such as LPS. The requirement of p38alpha in inflammatory responses has been shown in a number of studies using chemical inhibitors, though the inhibitors also inhibit p38beta and perhaps some other enzymes. In this study, we used conditional knockout of p38alpha in macrophages to address the role of p38alpha in macrophage activation. We found that p38alpha deficiency causes a significant inhibition in the production of LPS-induced TNF-alpha, IL-12, and IL-18, but it has little or no effect on IL-6 or IFN-beta production. Knockout of p38alpha in macrophages did not affect LPS-induced activation of the other major signaling pathways (NF-kappaB, Jnk, and Erk), nor did it affect the transcriptional activity of NF-kappaB. It had little inhibitory effect on LPS-induced AP-1 activity, but it significantly inhibited LPS-induced C/EBP-beta and CREB activation, indicating that the role of p38alpha in cytokine production in macrophages is at least in part through its regulation of C/EBP-beta and CREB activation. In addition, we also confirmed that p38alpha is important for phagocytosis of bacteria by macrophages. Our in vivo studies with two murine models showed that p38alpha is involved in sepsis. Collectively, our data demonstrate that p38alpha is an important player in inflammatory responses.
The contribution of Toll-like receptors (TLRs) to phagocytosis of Borrelia burgdorferi has not been extensively studied. We show that bone marrow-derived macrophages (BMDM) from MyD88−/− mice or Raw cells transfected with a dominant-negative MyD88 were unable to efficiently internalize B. burgdorferi. Knockouts of TLR2 and TLR9 or knockdown of TLR5 by small interfering RNA produced no defects in phagocytosis of B. burgdorferi. Production of inflammatory cytokines was greatly diminished in MyD88−/− BMDM but only partially affected in TLR2−/− BMDM or knockdown of TLR5 and unaffected in TLR9−/− BMDM. Cytochalasin D reduced cytokine induction, but not to the level of the MyD88−/− BMDM. Addition of cytochalasin D to TLR2−/− BMDM inhibited inflammatory responses to B. burgdorferi to the level of MyD88−/− BMDM, consistent with a role for TLR2 in both recognition of extracellular products and lysosomal sampling by TLR2 after
processing of the organism. Cytochalasin D had no impact on cytokine production in cells undergoing TLR5 knockdown. These
results suggest that MyD88, but not TLR2, TLR5, and TLR9, is important for the uptake of B. burgdorferi and that MyD88 affects inflammatory responses through both its effects on phagocytosis and its role in transducing signals
from TLR2 and TLR5.
The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the FcgammaII-receptor (FcgammaII-R). Fluorescence immunocytochemistry, demonstrating FcgammaII-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the FcgammaII-R in DQ12 phagocytosis. FcgammaII-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the FcgammaII-R. Consistent with the uptake results, FcgammaII-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF-alpha release as observed after treatment with DQ12. In conclusion, our results indicate a major role of FcgammaII-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.
Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases, including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin, a naturally occurring bioflavinoid, was previously shown to inhibit PtdIns 3-kinase with an IC50 of 1.3 mu/ml (3.8 mu M); inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors, with the most potent ones exhibiting IC50, values in the range of 1.7-8.4 mu g/mI. In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 > 30 mu g/ml). Since quercetin has also been shown to inhibit other PtdIns and protein kinases, other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound, 2-(4-morpholinyl)8-phenyl-4H-1-benzopyran-4-one (also known as 2-(4-morpholinyl-8-phenylchromone, LY294002), completely and specifically abolished PtdIns 3-kinase activity (IC50 = 0.43 mu g/ml; 1.40 mu M) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity relationship, with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in fMet-Leu-Phe-stimulated human neutrophils, as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction, the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response.
Basic substances and acidic ionophores that increase the lysosomal pH in cultured macrophages (Ohkuma, S., and B. Poole, 1978, Proc. Natl. Acad. Sci. USA., 75:3327-3331; Poole, B., and S. Ohkuma, 1981, J. Cell Biol., 90:665-669) inhibited the digestion of heat-denatured acetylated bovine serum albumin (BSA) taken up by the cells. For several substances, the shift in pH sufficed to explain the inhibition of proteolysis. Additional effects, presumably on enzyme activities, have to be postulated for tributylamine, amantadine, and chloroquine. Sodium fluoride (10 mM) had no significant effect on the breakdown of BSA by macrophages. The breakdown of endogenous macrophage proteins, whether short lived or long lived, was inhibited approximately 40% by 10 mM NaF and 30%, or sometimes less in the case of long-lived proteins, by 100 microM chloroquine. When the cells were supplied with BSA, a mixture of cell proteins, or even inert endocytosible materials, the breakdown of endogenous long-lived proteins and the inhibitory effect of chloroquine on this process were selectively reduced. Inhibition of endocytosis by cytochalasins B or D did not affect the chloroquine-sensitive breakdown of endogenous proteins, indicating that the proteins degraded by this process were truly endogenous and not taken in from the outside by cellular cannibalism. On the other hand, when macrophage proteins were supplied extracellularly, their breakdown occurred at the same rate for short-lived and long-lived proteins, and it was strongly inhibited by chloroquine and not by NaF. It is concluded from these results that the breakdown of endogenous proteins, both short-lived and long-lived, probably takes place partly (approximately 30%) in lysosomes and partly through one or more nonlysosomal mechanism(s) unaffected by chloroquine and presumably susceptible to inhibition by fluoride. A difference must exist between short-lived and long-lived proteins in the manner in which they reach lysosomes or are handled by these organelles; this difference would account for the selective effect of the supply of endocytosible materials on the lysosomal processing of long-lived proteins.
DNA from bacteria has stimulatory effects on mammalian immune cells(1-3) which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immune-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response(4-7). Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseasess(8-10). Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9(-/-)) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory Cytokine production from macrophages and maturation of dendritic cells. TLR9(-/-) mice showed resistance to the lethal effect of CPG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9(-/-) mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.
To determine the characteristics of infective endocarditis in our hospital, we reviewed all patients with that diagnosis at the University of Massachusetts Medical Center, Worcester, between 1981 and 1988. Of 113 patients with infective endocarditis, 56 (50%) had staphylococcal endocarditis. Despite aggressive medical and surgical therapy, in-hospital mortality was 25%. Forty-five (80%) of the 56 cases of staphylococcal endocarditis involved Staphylococcus aureus with a mortality of 28% vs 9% in the non-S aureus group. Mortality was higher in patients with congestive heart failure (35%), atrioventricular block (45%), atrial fibrillation (42%), and prosthetic valve endocarditis (50%). Seventy-six percent of the patients with congestive heart failure required surgery. Patients with congestive heart failure and S aureus infection had a mortality of 45%. Thirty-six patients (64%) were alive at late follow-up (mean, 28.6 months). Mortality was highest (23%) during the first 3 months following diagnosis of staphylococcal endocarditis. Staphylococcal endocarditis represents an increasingly large proportion of patients with infectious endocarditis. Mortality rates remain high despite aggressive management of the patient's condition.
A class of pyridinyl imidazoles inhibit the MAP kinase homologue, termed here reactivating kinase (RK) [Lee et al. (1994) Nature 372, 739-746]. We now show that one of these compounds (SB 203580) inhibits RK in vitro (IC50 = 0.6 microM), suppresses the activation of MAPKAP kinase-2 and prevents the phosphorylation of heat shock protein (HSP) 27 in response to interleukin-1, cellular stresses and bacterial endotoxin in vivo. These results establish that MAPKAP kinase-2 is a physiological RK substrate, and that HSP27 is phosphorylated by MAPKAP kinase-2 in vivo. The specificity of SB 203580 was indicated by its failure to inhibit 12 other protein kinases in vitro, and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo. We suggest that SB 203580 will be useful for identifying other physiological roles and targets of RK and MAPKAP kinase-2.
Induction of the adaptive immune response depends on the expression of co-stimulatory molecules and cytokines by antigen-presenting cells. The mechanisms that control the initial induction of these signals upon infection are poorly understood. It has been proposed that their expression is controlled by the non-clonal, or innate, component of immunity that preceded in evolution the development of an adaptive immune system in vertebrates. We report here the cloning and characterization of a human homologue of the Drosophila toll protein (Toll) which has been shown to induce the innate immune response in adult Drosophila. Like Drosophila Toll, human Toll is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. Both Drosophila Toll and the IL-1 receptor are known to signal through the NF-kappaB pathway. We show that a constitutively active mutant of human Toll transfected into human cell lines can induce the activation of NF-kappaB and the expression of NF-kappaB-controlled genes for the inflammatory cytokines IL-1, IL-6 and IL-8, as well as the expression of the co-stimulatory molecule B7.1, which is required for the activation of naive T cells.
Members of the tumor necrosis factor receptor (TNFR) superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation and death. TNFR-1 recruits and assembles a signaling complex containing a number of death domain (DD)-containing proteins, including the adaptor protein TRADD and the serine/threonine kinase RIP, which mediates TNF-induced NF-kappa B activation. RIP also recruits caspase-2 to the TNFR-1 signaling complex via the adaptor protein RAIDD, which contains a DD and a caspase-recruiting domain (CARD). Here, we have identified a RIP-like kinase, termed CARDIAK (for CARD-containing interleukin (IL)-1 beta converting enzyme (ICE) associated kinase), which contains a serine/threonine kinase domain and a carboxy-terminal CARD. Overexpression of CARDIAK induced the activation of both NF-kappa B and Jun N-terminal kinase (JNK). CARDIAK interacted with the TNFR-associated factors TRAF-1 and TRAF-2, and a dominant-negative form of TRAF-2 inhibited CARDIAK-induced NF-kappa B activation. Interestingly, CARDIAK specifically interacted with the CARD of caspase-1 (previously known as ICE), and this interaction correlated with the processing of pro-caspase-1 and the formation of the active p20 subunit of caspase-1. Together, these data suggest that CARDIAK may be involved in NF-kappa B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta through activation of caspase-1.
Mammalian Toll-like receptors (TLRs) are expressed on innate immune cells and respond to the membrane components of Gram-positive or Gram-negative bacteria. When activated, they convey signals to transcription factors that orchestrate the inflammatory response. However, the intracellular signaling events following TLR activation are largely unknown. Here we show that TLR2 stimulation by Staphylococcus aureus induces a fast and transient activation of the Rho GTPases Rac1 and Cdc42 in the human monocytic cell line THP-1 and in 293 cells expressing TLR2. Dominant-negative Rac1N17, but not dominant-negative Cdc42N17, block nuclear factor-kappa B (NF-kappa B) transactivation. S. aureus stimulation causes the recruitment of active Rac1 and phosphatidylinositol-3 kinase (PI3K) to the TLR2 cytosolic domain. Tyrosine phosphorylation of TLR2 is required for assembly of a multiprotein complex that is necessary for subsequent NF-kappa B transcriptional activity. A signaling cascade composed of Rac1, PI3K and Akt targets nuclear p65 transactivation independently of I kappa B alpha degradation. Thus Rac1 controls a second, I kappa B-independent, pathway to NF-kappa B activation and is essential in innate immune cell signaling via TLR2.
DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.
Mutations in the human genome may result in altered phenotypes. The cystic fibrosis (CF) patient, for instance, suffers from an aberrant composition of the epithelial lining of the gastrointestinal and respiratory tract. In this particular case, a single point mutation in the cystic fibrosis conductance regulator (CFTR) gene results in major physiological changes resulting in ecological changes that generate a niche particularly attractive to a selected set of microbial pathogens. We here present a review on the dynamics of the bacterial populations inhabiting the CF lung. Studies focusing on Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa will be summarised and discussed, whereas the technology used for microbial characterisation will be shortly highlighted. Emphasis, however, will be on those studies that assessed the genetic diversity among clinical isolates that were obtained over prolonged periods of time, enabling the distinction between persistent colonisation versus frequent re-infection by the selected pathogens. Evolutionary adaptation of pathogens to the CF lung is a common theme in many of these studies.
Peripheral monocyte heterogeneity is widely acknowledged in humans but until now comparable heterogeneity has not been characterized in mice. In this issue, Geissmann et al. use chemokine receptors to define two monocyte subsets and Serbina et al. highlight the importance of selective monocyte recruitment in the innate immune response to Listeria.
Lipopolysaccharide (LPS), the gram-negative bacterial cell wall component, induces tolerance to a secondary challenge of LPS in macrophages (Mphi) as evidenced by reduced inflammatory mediator production. However, it is uncertain if heat-killed (HK) gram-positive bacteria Staphylococcus aureus (Sa) can induce a similar tolerance and alter responses to LPS. We hypothesized that HKSa induces homologous tolerance and cross tolerance to LPS stimulation in human promonocytic THP-1 cells. We measured TNF-alpha, TxB2, and IFN-gamma production and the phosphorylation of p38, JNK, and ERK-1/2 in human promonocytic THP-1 cells. HKSa (10 microg/mL) significantly stimulated naive (nonpretreated) cell TNF-alpha (P<0.05) and TxB2 production (P<0.05). However, HKSa-pretreated cells challenged secondarily with HKSa (10 microg/mL) exhibited a decrease in the production of TNF-alpha (89 +/- 5%, P<0.05) and TxB2 (85 +/- 3%, P<0.05) compared with HKSa-stimulated naive cells. By contrast, secondary LPS challenge of HKSa-pretreated cells augmented TNF-alpha (41 +/- 3%, P<0.05) and TxB2 (42 +/- 6%, P<0.05) compared with LPS-stimulated naive cells. In naive cells, HKSa and LPS stimulation also significantly phosphorylated the mitogen-activated kinases (MAPKs) p38, JNK, and ERK-1/2 (P<0.005) compared with basal levels. HKSa and LPS induced homologous tolerance as evidenced by the down-regulation of the three MAPK (P<0.05), thus paralleling data on mediator production. HKSa-pretreated cells' priming responses to LPS correlated with augmented phosphorylation of JNK and p38 (P<0.05), whereas ERK-1/2 phosphorylation remained down-regulated. In contrast to TNF-alpha and TxB2 production, HKSa-induced IFN-gamma was up-regulated (26 +/- 5%) in HKSa-pretreated cells compared with HKSa-stimulated naive cells. IFN-gamma antibody exhibited reversed priming in HKSa-pretreated cells as evidenced by a reduction in TNF-alpha. Exogenous human IFN-gamma- (1 microg/mL) and HKSa-pretreated cells secondarily stimulated with HKSa did not prevent the induction of tolerance. In contrast, exogenous IFN-gamma pretreatment prevented the induction of LPS homologous tolerance resulting in an increase in TNF-alpha production. The data demonstrate that HKSa induces homologous tolerance but causes priming to LPS.
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in signaling downstream of integrins, linking bacterial detection, cell entry, and initiation of proinflammatory response through MAPKs and NF-kappaB activation. In this study, using protein I/II from Streptococcus mutans as a model activator of FAK, we investigated the potential link between FAK and TLR pathways. Using macrophages from TLR- or MyD88-deficient mice, we report that MyD88 plays a major role in FAK-dependent protein I/II-induced cytokine release. However, response to protein I/II stimulation was independent of TLR4, TLR2, and TLR6. The data suggest that there is a cross talk between FAK and MyD88 signaling pathways. Moreover, MyD88-dependent, LPS-induced IL-6 secretion by human and murine fibroblasts required the presence of FAK, confirming that MyD88 and FAK pathways are interlinked.
To better define the incidence of sepsis and the characteristics of critically ill patients in European intensive care units.
Cohort, multiple-center, observational study.
One hundred and ninety-eight intensive care units in 24 European countries.
All new adult admissions to a participating intensive care unit between May 1 and 15, 2002.
None.
Demographic data, comorbid diseases, and clinical and laboratory data were collected prospectively. Patients were followed up until death, until hospital discharge, or for 60 days. Of 3,147 adult patients, with a median age of 64 yrs, 1,177 (37.4%) had sepsis; 777 (24.7%) of these patients had sepsis on admission. In patients with sepsis, the lung was the most common site of infection (68%), followed by the abdomen (22%). Cultures were positive in 60% of the patients with sepsis. The most common organisms were Staphylococcus aureus (30%, including 14% methicillin-resistant), Pseudomonas species (14%), and Escherichia coli (13%). Pseudomonas species was the only microorganism independently associated with increased mortality rates. Patients with sepsis had more severe organ dysfunction, longer intensive care unit and hospital lengths of stay, and higher mortality rate than patients without sepsis. In patients with sepsis, age, positive fluid balance, septic shock, cancer, and medical admission were the important prognostic variables for intensive care unit mortality. There was considerable variation between countries, with a strong correlation between the frequency of sepsis and the intensive care unit mortality rates in each of these countries.
This large pan-European study documents the high frequency of sepsis in critically ill patients and shows a close relationship between the proportion of patients with sepsis and the intensive care unit mortality in the various countries. In addition to age, a positive fluid balance was among the strongest prognostic factors for death. Patients with intensive care unit acquired sepsis have a worse outcome despite similar severity scores on intensive care unit admission.
Immune status is altered during systemic inflammatory response syndrome and sepsis. Reduced ex vivo tumor necrosis factor production has been regularly reported with lipopolysaccharide-activated monocytes. In this study, we addressed the specificity of this hyporeactivity and investigated some of the possible associated mechanistic events.
Ex vivo study.
Academic research laboratory.
Healthy controls, septic patients, and resuscitated patients after cardiac arrest (RCA). This latter group presents a systemic inflammatory response syndrome of noninfectious origin.
None.
We investigated the reactivity of patients' monocytes in terms of cytokine production, after stimulation with a Toll-like receptor (TLR) 2 (Pam3CysSK4), a TLR4 (lipopolysaccharide), a Nod2 agonist (muramyl dipeptide), or heat-killed bacteria. We also investigated the contribution of phagocytosis in cytokine production, studied the expression of intracellular bacterial peptidoglycan sensors (Nod1 and Nod2), and analyzed the messenger RNA expression of inhibitors of TLR signaling: Toll interacting protein (Tollip), suppressor of cytokine signaling-1 (SOCS1), myeloid differentiation 88 short (MyD88s), and single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR). In sepsis, tumor necrosis factor production in response to lipopolysaccharide and Pam3CysSK4 was reduced, whereas interleukin-10 production was enhanced. The responsiveness to Staphylococcus aureus, Escherichia coli, and muramyl dipeptide and the expression of Nod1 and Nod2 were similar to those obtained for healthy donors. The messenger RNA expression of Tollip and SOCS1 was unchanged, whereas that of MyD88s and SIGIRR was significantly enhanced compared with healthy controls. Monocytes from RCA patients showed a reduced production of tumor necrosis factor in response to lipopolysaccharide but neither to Pam3CysSK4 nor to heat-killed bacteria. They displayed an increased expression of SIGIRR but not of MyD88s. We showed that TLR2-dependent nuclear factor-kappaB activation was inhibited by MyD88s but not by SIGIRR. This result may explain the normal tumor necrosis factor production through TLR2 observed for monocytes of RCA patients.
There is a "reprogramming" of monocyte reactivity, and not a global hyporeactivity, during systemic inflammation, which differs in septic and RCA patients.
The TLR family senses the molecular signatures of microbial pathogens, and plays a fundamental role in innate immune responses. TLRs signal via a common pathway that leads to the expression of diverse inflammatory genes. In addition, each TLR elicits specific cellular responses to pathogens owing to differential usage of intracellular adapter proteins. Recent studies have revealed the importance of the subcellular localization of TLRs in pathogen recognition and signaling. TLR signaling pathways is negatively regulated by a number of cellular proteins to attenuate inflammation. Here, we describe recent advances in our understanding of the regulation of TLR-mediated signaling.
Innate immunity is characterised by a rapid action of host effector molecules and leukocytes aimed at limiting the multiplication of invading microbial organisms and destroying them. The recognition and destruction of microorganisms involves humoral factors (e.g., the complement system and natural antibodies) and different cell types (e.g., phagocytic cells, mast cells, natural killer cells). Microbial detection by cells involves germ line-encoded pattern-recognition receptors such as Toll-like receptors and nucleotide-binding oligomerization domain-like receptors. Cellular activation by pathogens leads to the release of antimicrobial peptides (e.g., defensins and peptidoglycan recognition proteins) and cytokines that orchestrate the anti-infectious response. Cytokines enhance phagocytosis and leukocyte microbicidal activity, allow cellular recruitment into the infectious focus, boost hematopoiesis, induce fever and lead to the production of acute phase proteins.
Tissue-resident macrophages play an important role in defense against pathogens and perform key functions in organ homeostasis, innate and adaptive immunity. Tissue macrophages originate from blood monocytes that infiltrate virtually every organ in the body. Macrophages in different tissues share many characteristics, including their ability to migrate, phagocytose particles, metabolize lipids and present antigens. Morphologically they are quite heterogeneous, and some distinct functions have been reported. The gene expression profile of macrophages is reflective of both their shared and distinct biological functions. Here, we show that macrophages from murine spleen, liver and peritoneum display dramatically different expression profiles. Clusters of genes were found to represent unique biological functions related to adhesion, antigen presentation, phagocytosis, lipid metabolism and signal transduction. Some gene families, such as integrins, are differentially expressed among the macrophages resident in different tissues, suggesting that the tissue of residence influences their biological function.
The aim of this study was to investigate the influence of delayed Rho-kinase inhibition with fasudil on second ischemic injury in a rat cerebral thrombosis model. Cerebral ischemia was induced in rats by injecting 150 mug of sodium laurate into the left internal carotid artery on day 1. In the ischemic group, the regional cerebral blood flow (rCBF) was significantly decreased 6.5 h after the injection. Fasudil (3 mg/kg/30 min i.v. infusion) significantly increased rCBF. The viscosity of whole blood was significantly increased 48 h after the injection of sodium laurate. Fasudil (10 mg/kg, i.p.) significantly decreased blood viscosity. To clarify the therapeutic time window of fasudil, rats received their first i.p. administration of fasudil (10 mg/kg) 6 h after an injection of sodium laurate. Administration of fasudil twice daily was continued until day 4. Fasudil prevented the accumulation of neutrophils within the brain as seen from measurements taken on day 3, and improved neuronal functions and reduced the infarction area as seen on day 5. Fasudil and hydroxyfasudil, an active metabolite of fasudil, concentration-dependently inhibited phosphorylation of myosin binding subunit of myosin phosphatase in neutrophils. The present results indicate that inhibition of Rho-kinase activation with fasudil is effective for the treatment of ischemic brain damage with a wide therapeutic time window by improving hemodynamic function and preventing the inflammatory responses. These results suggest that fasudil will be a novel and efficacious approach for the treatment of acute ischemic stroke.