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... Box: 91775-1793, Mashhad, Iran; Tel:/Fax: 0098-513-8805608; E-mails: haghparast@um.ac.ir & alireza.haghparast@gmail.com molecular patterns (PAMPs) and those molecular patterns expressed by endogenous danger signals are called damage associated molecular patterns (DAMPs) [4]. Upon recognition of PAMPs and DAMPs by PRRs, a cascade of intracellular signaling pathways is initiated leading to the activation and translocation of transcription factors like nuclear factor kappa B (NF-B) and interferon regulatory factors (IRFs) to the nucleous, promoting the production and secretion of pro-inflammatory cytokines and chemokines [4] (Fig. 1). ...
... molecular patterns (PAMPs) and those molecular patterns expressed by endogenous danger signals are called damage associated molecular patterns (DAMPs) [4]. Upon recognition of PAMPs and DAMPs by PRRs, a cascade of intracellular signaling pathways is initiated leading to the activation and translocation of transcription factors like nuclear factor kappa B (NF-B) and interferon regulatory factors (IRFs) to the nucleous, promoting the production and secretion of pro-inflammatory cytokines and chemokines [4] (Fig. 1). The local inflammatory responses initiated by the engagements of PRRs and their respective ligands (PAMPs, DAMPs) lead to the recruitment and maturation of sentient dendritic cells into the draining lymph nodes promoting the development of adaptive immune responses and ultimately creating an immunological memory [4]. ...
... Upon recognition of PAMPs and DAMPs by PRRs, a cascade of intracellular signaling pathways is initiated leading to the activation and translocation of transcription factors like nuclear factor kappa B (NF-B) and interferon regulatory factors (IRFs) to the nucleous, promoting the production and secretion of pro-inflammatory cytokines and chemokines [4] (Fig. 1). The local inflammatory responses initiated by the engagements of PRRs and their respective ligands (PAMPs, DAMPs) lead to the recruitment and maturation of sentient dendritic cells into the draining lymph nodes promoting the development of adaptive immune responses and ultimately creating an immunological memory [4]. ...
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Enormous progress in the field of vaccine development has moved the area from traditional vaccines using whole microorganisms to subunit vaccines containing only purified or modified antigenic proteins. The low immunogenic potentials of subunit vaccines compared to live attenuated pathogens, have prompted the research towards developing new adjuvants with the ability to mimic and enhance the innate and adaptive immune responses. Novel adjuvants formulations based on the discovery of pattern recognition receptors (PRRs), the main class of innate immunity sensors have recently gained interest. The major family of PRRs called Toll-like receptors (TLRs), which are expressed by a variety of cells and capable to induce innate immune responses and effective stimulation of transition from innate immune responses to adaptive immune responses. PRR ligands of natural and synthetic origin have been evaluated as potential adjuvants in a variety of applications including vaccine formulations. Apart from this, for generating effective and protective immune responses, targeted delivery of vaccine antigens along with PRR agonists to the desired cells is of great importance. New strategy of delivering vaccine antigens along with adjuvants using proper nano-sized materials has gained much attention during the recent years. Here we review the recent advances regarding PRR-based nano-adjuvants developments, focusing on current nano-materials based delivery system and their applications in biomedical sciences.
... It is not only the initiating factor of SAP, but also the factor of sustained and aggravated injury. Acute pancreatitis can damage the pancreas and systemic microcirculation, thus forming a vicious circle (1,2). Additionally, the immune-inflammatory response-mediated pancreatic microcirculation dysfunction is always active, which is also an important molecular biomarker to determine the outcome of pancreatitis (2). ...
... Acute pancreatitis can damage the pancreas and systemic microcirculation, thus forming a vicious circle (1,2). Additionally, the immune-inflammatory response-mediated pancreatic microcirculation dysfunction is always active, which is also an important molecular biomarker to determine the outcome of pancreatitis (2). In particular, the activation of the inflammatory immune response relies on the recruitment and migration of neutrophils in SAP (3). ...
... HMGB1, one of the endogenous molecules secreted from apoptotic and necrotic cells, has a pivotal influence on the development of SAP by activating the immune response (2). Once pancreatic cells are harmfully stimulated, HMGB1 in the nucleus will be modified by acetylation and then released to the extracellular space where it pairs with receptors (TLR-4) on the surface of macrophages to induce TLR-4-mediated immune responses (2,4). ...
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Background: Dachengqi decoction (DCQD), one of classic prescription of Chinese herbal medicine has been widely used in clinic to treat severe acute pancreatitis (SAP). The damage of pancreatic microcirculation plays key pathogenesis of SAP. However, little is known about the molecular pharmacological activity of DCQD on pancreatic microcirculation in SAP. Methods: Sodium taurodeoxycholate and cerulein were used to establish model of SAP in vitro and in vivo, respectively. The pancreatic pathological morphology, wet weight ratio, myeloperoxidase (MPO) activity, cell viability and microcirculatory function of the pancreas, as well as serum lipase and amylase expressions were evaluated. The expression levels of SIRT1, acety-HMGB1, TLR-4, HMGB1, IL-23, IL-17A, neutrophil chemokines (KC, LIX, and MIP-2), and inflammation-related factors (IL-6, IL-1β, and TNF-α), the translocation of HMGB1 and the interaction of SIRT-HMGB1 in the pancreas and serum were determined by ELISA real-time PCR, western blotting and immunoprecipitation. Results: In vivo studies showed that DCQD or neutralizing antibody (anti-23p19 or anti-IL-17A) could all significantly decrease lipase, amylase activity, down-regulate the expression of CD68, Myeloperoxidase (MPO), wet/weight, IL-1β, IL-6, TNF-α, and neutrophil chemokines (KC, LIX, MIP-2), alleviate pathological injury and improve pancreatic microcirculatory function in rats with SAP. Furthermore, DCQD remarkably increased SIRT1 expression, promoted SIRT1 and HMGB1 combination, reduced HMGB1 translocation from nuclear to cytoplasm, and alleviated the expression of acetyl-HMGB1, HMGB1, IL-17A, TLR-4, and IL-23 in vitro and in vivo with SAP. However, the intervention with EX527 (SIRT1 inhibitor) or r-HMGB1 (recombinant HMGB1) obliviously reverses the above mentioned influence mentioned above of DCQD in SAP. In vitro, we confirmed that DCQD could decrease HMGB1 acetylation, migration, and release, and improve the decline of cell viability, SIRT1 expression and SIRI-HMGB1 combination induced by cerulean with promoting macrophage to release IL-23 by relying on the HMGB1/TLR-4 way. Conclusions: DCQD treatment improves SAP-induced pancreatic microcirculatory dysfunction by inhibiting neutrophil-mediated inflammation via inactivating HMGB1-TLR-4-IL-23-IL-17A signaling by targeting SIRT1.
... The pathogenesis of SAP is complex and involves in the abnormal activation of pancreatic in ammatory mediators, microcirculation dysfunction, translocation of intestinal ora, apoptosis, and oxidative stress; additionally, the immune in ammatory response mediated pancreatic microcirculation dysfunction is always active, which is also an important molecular biomarker to determine the outcome of pancreatitis [2]. Notably, the activation of immune in ammatory response is relied on the recruitment and migration of neutrophil in SAP [3]. ...
... Therefore, it is an attractive treatment strategy to improve SAP by suppressing the in ammatory response mediated pancreatic microcirculation dysfunction through promoting the inactivation of neutrophil. HMGB1, one of endogenous molecule secreted from apoptotic and necrotic cell possess a pivotal in uence in the development of SAP by activating immune response [2]. Once the pancreatic cells are harmfully stimulated , the HMGB1 in the nucleus will be modi ed by acetylation and then released to the extracellular space where it pairs with receptors(TLR-4) on the surface of macrophages to induce the TLR-4-mediated immune responses [2,4]. ...
... HMGB1, one of endogenous molecule secreted from apoptotic and necrotic cell possess a pivotal in uence in the development of SAP by activating immune response [2]. Once the pancreatic cells are harmfully stimulated , the HMGB1 in the nucleus will be modi ed by acetylation and then released to the extracellular space where it pairs with receptors(TLR-4) on the surface of macrophages to induce the TLR-4-mediated immune responses [2,4]. This further promotes the releasing HMGB1 and ampli es the immune reaction. ...
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Background Dachengqi decoction (DCQD), one of classic prescription of Chinese herbal medicine has been widely used in clinic to treat severe acute pancreatitis (SAP). The damage of pancreatic microcirculation plays key pathogenesis of SAP. However, little is known about the molecular pharmacological activity of DCQD on pancreatic microcirculation in SAP. Therefore, the purpose of the study attempted to confirm the improvement of DCQD on pancreatic microcirculation is associated with suppressing neutrophil mediated immune-inflammatory response through promoting the inactivation of HMGB1-TLR-4-IL-23-IL-17A axis via targeting the SIRT1 signal pathway in SAP.Material and Methods Sodium taurodeoxycholate and cerulein were used to establish model of SAP in vitro and vivo, respectively. The pancreatic pathological morphology, wet weight ratio, myeloperoxidase (MPO) activity, cell viability and microcirculatory function of the pancreas, as well as serum lipase and amylase expressions were evaluated. The expression levels of SIRT1, acety-HMGB1, TLR-4, HMGB1, IL-23, IL-17A, neutrophil chemokines (KC, LIX, and MIP-2), and inflammation-related factors (IL-6, IL-1β, and TNF-α), the translocation of HMGB1 and the interaction of SIRT-HMGB1 in the pancreas and serum were determined by ELISA real-time PCR, western blotting and immunoprecipitation.ResultsIn-vivo studies showed DCQD or neutralizing antibody (anti-23p19 or anti-IL-17A) could significantly decrease the activity of lipase, amylase, down-regulate the expression of CD68, MPO, wet/weight, IL-1β, IL-6, TNF-α,neutrophil chemokines (KC, LIX, MIP-2 ), alleviate pathological injury, and improve the microcirculatory function of the pancreas in rats with SAP. Moreover, DCQD remarkably augmented SIRT1 expression, promoted SIRT1 and HMGB1 combination, reduced HMGB1 translocation from nuclear to cytoplasm, and alleviated the expression of acetyl-HMGB1, HMGB1, IL-17A, TLR-4 and IL-23 in vitro and vivo with SAP. However, the intervention with EX527 (SIRT1 inhibitor) or r-HMGB1 (recombinant HMGB1) could obliviously reverse the above-mentioned influence of DCQD in SAP. In vitro, we confirmed that DCQD could decrease the acetylation, migration and release of HMGB1, and improve the decline of cell viability, SIRT1, SIRI-HMGB1 combination induced by cerulein with promoting macrophage to release IL-23 through HMGB1/TLR-4. ConclusionDCQD treatment improves SAP-induced pancreatic microcirculatory dysfunction by inhibiting neutrophil-mediated inflammation through the inactivation of HMGB1-TLR-4-IL-23-IL-17A signaling via Targeting SIRT1.Trial registration: No. 365, 2020.
... bacteria or viruses) release pathogenassociated molecular patterns (PAMPs) 2 that are recognized by pattern recognition receptors (PRRs) of the immune system and thus trigger an inflammatory response (1). In contrast, sterile inflammation, an important characteristic of myocardial infarction, atherosclerosis, several autoimmune diseases, and cancer, is induced by the release of endogenous molecules called DAMPs following tissue stress or injury (2)(3)(4)(5). PAMPs and DAMPs are highly conserved motifs derived from the pathogens themselves (PAMPs) or from self-molecules that are normally hidden from the PRRs by compartmentalization (intracellular DAMPs) or sequestration in the ECM (extracellular DAMPs). DAMPs, similar to PAMPs, are recognized by PRRs. ...
... Preliminary unpublished data suggest a hierarchical mode of DAMP interactions. 3 Therefore, approaches to identify an initial DAMP trigger and the following DAMP-mediated "chains of command" should be the focus of future scientific attention. 4) How do DAMPs interact with PRRs? ...
Article
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In reply to internal or external danger stimuli, the body orchestrates an inflammatory response. The endogenous triggers of this process are the damage-associated molecular patterns (DAMPs). DAMPs represent a heterogeneous group of molecules that draw their origin either from inside the various compartments of the cell or from the extracellular space. Following interaction with pattern recognition receptors in cross-talk with various non-immune receptors, DAMPs determine the downstream signaling outcome of septic and aseptic inflammatory responses. In this review, the diverse nature, structural characteristics, and signaling pathways elicited by DAMPs will be critically evaluated.
... Extracellular HMGB1 is a cytokine that is considered a prototypic "alarmin" or "damage-associated molecular pattern" (DAMP) molecule that can be secreted by distressed, yet viable, cells [36]. Secreted HMGB1 is recognized by the innate immune system which initiates an appropriate inflammatory response. ...
... Extracellular HMGB1 can exert its effects on cells via binding to either RAGE, CXCR4, as well as several TLRs (e.g. TLR4, TLR2, and TLR9) [36,40,41]. The results presented herein indicating that HMGB1 ligation of the TLR4 recptors on fibroblasts is a prerquisite for collagen production (Fig. 5). ...
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Diabetic cardiomyopathy (DiCM) is characterized by myocardial fibrosis and dysfunction. In rodent models of diabetes myocardial HMGB1 increases while IL-33 decreases. The major cardiac cell type expressing HMGB1 is the myocyte while the primary IL-33 expressing cell is the fibroblast. The aim of this study was to delineate the extracellular communication pathway(s) between cardiomyocytes and fibroblasts that contributes to murine DiCM. The streptozotocin (STZ)-induced murine model of diabetes and a cardiomyocyte/fibroblast co-culture challenged with high glucose were used. In STZ mice, myocardial HMGB1 expression was increased while IL-33 expression decreased (immunoflourescence and western blot). In addition, STZ mice had an increased myocardial collagen deposition and myocardial dysfunction (pressure-volume loop analysis). An HMGB1 inhibitor (A-box) or exogenous IL-33 prevented the myocardial collagen deposition and dysfunction. In the cardiomyocyte/fibroblast co-culture model, HG increased cardiomyocyte HMGB1 secretion, decreased fibroblast IL-33 expression, and increased fibroblast collagen I production. Further, using A-box and HMGB1 shRNA transfected myocytes, we found that cardiomyocyte-derived HMGB1 dramatically potentiated the HG-induced down-regulation of IL-33 and the increase in collagen I expression in the fibroblasts. The potentiating effects of the cardiomyocytes was diminished when toll-like receptor 4 deficient (TLR4(-/-)) fibroblasts were co-cultured with wild-type myocytes. Finally, TLR4(-/-) mice with diabetes had increased myocardial expression of HMGB1, but failed to down-regulate IL-33. The diabetes-induced myocardial collagen deposition and cardiac dysfunction were significantly attenuated in TLR4(-/-) mice. In conclusion, our findings support a role for "cardiomyocyte HMGB1-fibroblast TLR4/IL-33 axis" in the development of myocardial fibrosis and dysfunction in a murine model of diabetes. Copyright © 2015. Published by Elsevier B.V.
... DAMPs are very heterogeneous in structure, physical, chemical, and biological properties, but they have in common the property to be normally sequestered from the PRRs and become exposed to them upon cellular and tissue damage. DAMPs can be defined as primary endogenous danger signals, as they originate directly from the damaged cells or tissues, and can include nucleic acid and heat-shock proteins (hsp) ( Broggi and Granucci, 2015;Crisan et al., 2016;Heil and Land, 2014). In addition to these primary DAMPs, which by majority represent intracellular molecules showing up on the outside of a cell/its microenvironment, secreted immune-modulators, such as chemokines/ cytokines produced by activated immune cells may act also as danger signals ( Broggi and Granucci, 2015; Gallo and Gallucci, 2013). ...
... DAMPs can be defined as primary endogenous danger signals, as they originate directly from the damaged cells or tissues, and can include nucleic acid and heat-shock proteins (hsp) ( Broggi and Granucci, 2015;Crisan et al., 2016;Heil and Land, 2014). In addition to these primary DAMPs, which by majority represent intracellular molecules showing up on the outside of a cell/its microenvironment, secreted immune-modulators, such as chemokines/ cytokines produced by activated immune cells may act also as danger signals ( Broggi and Granucci, 2015; Gallo and Gallucci, 2013). Indeed, a specific group of protein, the S100 family of EF-hand calcium (Ca2)binding proteins, bridges the secretory side of the (innate) immune response by acting as DAMPs and interacting with PRR, but also have direct antimicrobial properties through the process of metal limitation, termed "nutritional immunity" at the site of infection ( Zackular et al., 2015). ...
Article
Therapies with increasing specificity against pathogens follow the immune system's evolutionary course in maximizing host defence while minimizing self-harm. Nevertheless, even completely non-specific stressors, such as reactive molecular species, heat, nutrient and oxygen deprivation, and acidity can be used to preferentially harm pathogens. Strategic use of non-specific stressors requires exploiting differences in stress vulnerability between pathogens and hosts. Two basic vulnerabilities of pathogens are their inherent vulnerability to stress of their growth and replication and the degree of pathogen localization, permitting the host's use of locally and regionally intense stress to fight these pathogens. Each of the various types of non-specific stressors is present during severe infections at all levels of localization. One could propose that hosts, such as cows strategically usea coordinated system of non-specific stressors at local, regional and systemic levels to preferentially harm the pathogens within. With the rising concern over emergence of resistance to specific therapies, we should pay more scrutiny to strategies using less specific therapies in pathogen control. Hosts' active use of multiple non-specific stressors is likely an evolutionarily basic defence whose retention underlies and supplements the well-recognized immune defences that directly target pathogens.
... The depurination of the ribosome by the enzymatic A 1 -fragment of Stxs is a crucial event in the activation of host signal transduction pathways leading to the proinflammatory response [87,[137][138][139]. Inflammatory responses include, in part, the synthesis and secretion of cytokines and chemokines by immune cells following the detection of "danger signals", such as bacterial cell membrane or cell wall components, toxins or flagellins [140,141]. The regulated elicitation of inflammation leads to the elimination of microbes or foreign agents. ...
... Several studies support the concept that macrophages may be the source of cytokine production to promote tissue damage through increased toxin receptor expression and/or leukocyte recruitment. When monocytic THP-1 cells were differentiated into the macrophage-like state, the cells became relatively resistant to the cytotoxic action of Stxs, but also became capable of secretion of TNF-α, IL-1β, IL-8, MIP-1α, MIP-1β, MCP1 and Groβ [138][139][140]. Stx-induced cytokine expression was manifested in the presence or absence of lipopolysaccharide (LPS) [87,139,[149][150][151]. ...
Article
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Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are primary virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications, such as hemolytic uremic syndrome and central nervous system abnormalities. Current therapeutic options to treat patients infected with toxin-producing bacteria are limited. The structures of Stxs, toxin-receptor binding, intracellular transport and the mode of action of the toxins have been well defined. However, in the last decade, numerous studies have demonstrated that in addition to being potent protein synthesis inhibitors, Stxs are also multifunctional proteins capable of activating multiple cell stress signaling pathways, which may result in apoptosis, autophagy or activation of the innate immune response. Here, we briefly present the current understanding of Stx-activated signaling pathways and provide a concise review of therapeutic applications to target tumors by engineering the toxins.
... Although the low amounts proinflammatory cytokines may be protective against viral invasion, they are harmful to the host when overproduced [29]. To explore whether PEDV caused the increase of proinflammatory cytokines, we collected Vero cells infected with PEDV at 2h, 6h, 12h and 24h and measured the mRNA levels of several proinflammatory cytokines and HMGB1. ...
Article
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Porcine epidemic diarrhea is a devastating swine enteric disease, which is caused by porcine epidemic diarrhea virus (PEDV) infection. Our studies demonstrated that PEDV infection resulted in the up-regulation of proinflammatory cytokines. Meanwhile, PEDV infection and overexpression of viral nucleoprotein resulted in the acetylation and release of high mobility group box 1 proteins in vitro, an important proinflammatory response mediator, which contributes to the pathogenesis of various inflammatory diseases. Our studies also showed that SIRT1, histone acetyltransferase, and NF-κB regulated the acetylation and release of HMGB1. Chromatin immunoprecipitation, dual-luciferase reporter gene assay, and co-immunoprecipitation experiments illustrated that PEDV-N could induce HMGB1 transcription by interacting with C/EBP-β, which could bind to C/EBP motif in HMGB1 promotor region. Collectively, our data indicate PEDV-N contributes to HMGB1 transcription and the subsequent release/acetylation of HMGB1 during PEDV infection.
... This phenomenon is currently explained by the concept of damage-associated molecular patterns (DAMPs) that trigger inflammatory responses against danger stimuli [34,35]. The release of DAMPs by tissue stress or injury was confirmed to play an important role in the pathophysiology of a wide range of diseases [36][37][38][39][40]. In the current study, no adverse events, such as severe inflammation or rejection, were observed in rabbit model, but the possibility of an inflammatory response caused by CECs damaged during preservation should be further investigated. ...
Article
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The corneal endothelium maintains corneal transparency by its pump and barrier functions; consequently, its decompensation due to any pathological reason causes severe vision loss due to corneal haziness. Corneal transplantation is the only therapeutic choice for treating corneal endothelial dysfunction, but associated problems, such as a shortages of donor corneas, the difficulty of the surgical procedure, and graft failure, still need to be resolved. Regenerative medicine is attractive to researchers as a means of providing innovative therapies for corneal endothelial dysfunction, as it now does for other diseases. We previously demonstrated the successful regeneration of corneal endothelium in animal models by injecting cultured corneal endothelial cells (CECs) in combination with a Rho kinase (ROCK) inhibitor. The purpose of the present study was to optimize the vehicle for clinical use in cell-based therapy. Our screening of cell culture media revealed that RELAR medium promoted CEC adhesion. We then modified RELAR medium by removing hormones, growth factors, and potentially toxic materials to generate a cell therapy vehicle (CTV) composed of amino acid, salts, glucose, and vitamins. Injection of CECs in CTV enabled efficient engraftment and regeneration of the corneal endothelium in the rabbit corneal endothelial dysfunction model, with restoration of a transparent cornea. The CECs retained >85% viability after a 24 hour preservation as a cell suspension in CTV at 4°C and maintained their potency to regenerate the corneal endothelium in vivo. The vehicle developed here is clinically applicable for cell-based therapy aimed at treating the corneal endothelium. Our strategy involves the generation of vehicle from a culture medium appropriate for a given cell type by removing materials that are not favorable for clinical use.
... Inflammatory response is a highly integrated biological process in host response against invaded pathogens [4]. It has been found that multiple pro-inflammatory cytokines could be rapidly induced during the inflammation [31,56] and be vigorously modulated afterward, maintaining homeostasis of the immune system [6,31,42]. ...
Article
Tumor necrosis factor alpha (TNF-α) mediated inflammatory response plays indispensable roles in organisms defending against the invaded bacteria, during which microRNAs have been found crucial by controlling multiple TNF-α-related genes. In the present study, cgi-miR-92d was annotated as a member of miR-17–92 family and could target the CDS region of lipopolysaccharide (LPS)-induced TNF-α factor (CgLITAF3) in oyster Crassostrea gigas. It was observed that cgi-miR-92d could be vigorously modulated by Vibrio splendidus or LPS stimulation while CgLITAF3 altered oppositely. Two putative binding sites of cgi-miR-92d were then found at CDS region of CgLITAF3. The interaction between cgi-miR-92d and CgLITAF3 was subsequently verified both in vitro and in vivo. As a result, a significant decrease of cellular luminescence was observed in CgLITAF3 luciferase reporter assay when cgi-miR-92d was overexpressed. The luminescent decrease was then recuperated when cgi-miR-92d inhibitor was co-transfected with miRNA mimics. Besides, CgLITAF3 transcripts were significantly down-regulated when cgi-miR-92d was overexpressed in vivo during V. splendidus challenge. Gain-of-function assay of CgLITAF3 was then conducted in HEK293T cells to verify its function. Consequently, a significant increase of TNF-α was observed during the assay. At the meantime, CgTNF was also down-regulated in gain-of-function assay of cgi-miR-92 in vivo, which was a member of TNF superfamily in oysters which could be robustly induced after pathogen stimulation. Together, these results verify the interaction between CgLITAF3 and cgi-miR-92d, which might dedicate crucially in the repaid activation of CgTNF expression during inflammatory response of oysters.
... The innate immune system provides formidable protection against the vast majority of invading microbes. Its chemo-sensors, termed danger recognition receptors, are detecting conserved microbial products (including key virulence factors) and tissue damage inflicted by the infection, boost antimicrobial defense and recruit phagocytic cell populations to eliminate the pathogen and cellular debris [3][4][5]. It appears that some "successful" pathogens have evolved mechanisms to evade these innate responses [6,7]. ...
Article
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Author Salmonella Typhimurium is a common cause of foodborne diarrhea. The disease symptoms arise already a few hours after infection. However, it had remained unclear how the immune system can mount the responses eliciting the disease symptoms so quickly. Earlier work in a mouse model had shown that the gut epithelium expresses a sensor, called NAIP/NLRC4/caspase-1 inflammasome that can detect the pathogen and mount a defense by 12-18h p.i. However, it has remained uncharacterized how inflammasome sensing drives the initial gut inflammation. Here, we found that the caspase-1 inflammasome triggers the production of IL-18, a pro-inflammatory cytokine that appears essential for the early onset of inflammation. IL-18 is driving the accumulation of NK cells into the infected mucosa, via the upregulation of NK cell chemoattractants and by the stimulation of their migratory capacity. Mature NK cells seem to induce mucosal inflammation via a perforin-mediated cytotoxic response. These data suggest that the inflammasome/IL-18/NK cell axis is a driver of early mucosal inflammation via a perforin-dependent cytotoxic NK cell response. Future work will have to address, if this mechanism is equally potent in the human gut and may contribute to ramping up the host's response during the first hours of infection. This may have implications for other gut infections and might provide leads for developing therapies.
... 18,[20][21][22] Regarding infectious diseases, the host immune system may control not only the substances from the infectious pathogens, including pathogen-associated molecular patterns (PAMPs), but also those from the injured host cells caused by infectious insults, including damage (or danger)-associated molecular patterns (DAMPs). 23,24 Furthermore, intact MPs are difficult to find in pulmonary lesions as well as in extrapulmonary lesions such as encephalopathy but are occasionally positive on polymerase chain reaction. 25 Therefore, the substances that induce CAs may not originate from MPs or other pathogens but rather from the same kind of host cells affected by insults from MP infection or other conditions (i.e., a kinds of DAMPs?). ...
Article
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There have been some limitations on early diagnosis of Mycoplasma pneumoniae (MP) infection because of no immunoglobulin M (IgM) responses and variable detection rates of polymerase chain reaction in the early stage of the disease. We wanted to discuss regarding early diagnostic method using short-term paired titration of MP-specific IgM and cold agglutinins (CAs) in the early stage of MP pneumonia. The participants of this study were 418 children with MP pneumonia during 2 recent epidemics (2006–2007 and 2011), and they were diagnosed by an anti-MP IgM antibody test (Serodia Myco II) examined twice during hospitalization at presentation and around discharge (mean of 3.4 ± 1.3 days apart). CA titers were simultaneously examined twice during study period. Anti-MP IgM antibody titer ≥1:40 and CA titer ≥1:4 were considered positive, respectively. The relationships between 2 IgM antibodies in the early stage were evaluated. Regarding MP-specific antibody titers, 148 patients showed a seroconversion, 245 patients exhibited increased titers, and 25 patients had unchanged higher titers (≥1:640) during hospitalization. The median MP-specific antibody titers at each examination time were 1:80 and 1:640, respectively; those of CAs were 1:8 and 1:32, respectively. Illness duration prior to admission showed a trend of association with both titers, and patients with shorter illness duration had a higher rate of negative titers or lower titers at each examination time. CAs and MP-specific antibody titers were correlated in the total patients at presentation and at 2nd examination (P < 0.001, respectively), and the diagnostic corresponding rates of CAs to IgM antibody test were 81% to 96% in patient subgroups. Short-term paired MP specific-IgM determinations in the acute stage may be used as a definitive diagnostic method for MP pneumonia. Paired CA titers showed a correlation with MP-specific antibody titers, suggesting they can be used as an adjuvant diagnostic method.
... Foci, that is, damaged and stressed, or infected cells release a large assortment of "alarmins," also known as part of DAMPs. For example, extracellular nucleotides, heat shock proteins, fragments of extracellular matrix components, and the extracellular nuclear high-mobility group protein B1 represent DAMPs that activate Toll-like receptors (TLR) like PAMPs and might act as cytokines [191][192][193]. Additionally, tissue-typical nonimmune cells are able to deliver sets of inflammatory cytokines like resident macrophages and other immune cells. ...
Article
Acute phase proteins (APPs) are highly conserved plasma proteins that are increasingly secreted by the liver in response to a variety of injuries, independently of their location and cause. APPs favour the systemic regulation of defence, coagulation, proteolysis and tissue repair. Various APPs have been applied as general diagnostic parameters for a long time. Through proteomic techniques, more and more APPs have been discovered to be differentially altered. Since they are not consistently explainable by a stereotypic hepatic expression of sets of APPs, most of these results have unfortunately been neglected or attributed to the non-specificity of the acute phase reaction. Moreover, it appears that various extra-hepatic tissues are also able to express APPs. These extra-hepatic APPs show focally specific roles in tissue homeostasis and repair and are released primarily into interstitial and distal fluids. Since these focal proteins might leak into the circulatory system, mixtures of hepatic and extra-hepatic APP species can be expected in blood. Hence, a selective alteration of parts of APPs might be expected. There are several hints on multiple molecular forms and fragments of tissue-derived APPs. These differences offer the chance for multiple selective determinations. Thus, specific proteoforms might indeed serve as tissue-specific disease indicators. This article is protected by copyright. All rights reserved.
... Neutrophils recognize DAMPs by specific PRRs, including Toll-like receptors (TLRs) and NOD-like receptors (NLRs). The wide variety of DAMP molecules includes DNA, proteins (such as highmobility group box 1 (HMGB1)), N-formyl peptides, extracellular matrix components, ATP and uric acid [15][16][17]. The exact signals that govern this early stage in mice are largely unknown but are probably sensed by GPCRs as treatment of neutrophils with pertussis toxin, which inhibits GPCR-Gα i signalling, limits this early chemotaxis towards the damage site 7 . ...
Article
Neutrophil migration and its role during inflammation has been the focus of increased interest in the past decade. Advances in live imaging and the use of new model systems have helped to uncover the behaviour of neutrophils in injured and infected tissues. Although neutrophils were considered to be short-lived effector cells that undergo apoptosis in damaged tissues, recent evidence suggests that neutrophil behaviour is more complex and, in some settings, neutrophils might leave sites of tissue injury and migrate back into the vasculature. The role of reverse migration and its contribution to resolution of inflammation remains unclear. In this Review, we discuss the different cues within tissues that mediate neutrophil forward and reverse migration in response to injury or infection and the implications of these mechanisms to human disease.
... Low amounts of proinflammatory cytokines may be protective against viral invasion. However, overproduced cytokines will sabotage the host immune responses [44]. It is reported that host cells initiate immune responses by producing various proinflammatory cytokines during the infection of various viruses, including West Nile virus [45], SARS-CoV [46][47][48], and hepatitis (A, B, C) viruses [49]. ...
Article
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Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV) infection, leads to significant economic losses in the swine industry worldwide. In our studies, we found that glycyrrhizin, the major component of licorice root extracts, could moderately inhibit PEDV infection in Vero cells, when analyzed by western blot, qRT-PCR and a plaque formation assay. We also revealed that glycyrrhizin inhibited the entry and replication of PEDV. In addition, we demonstrated that glycyrrhizin decreased the mRNA levels of proinflammatory cytokines. Since glycyrrhizin is a competitive inhibitor of high mobility group box-1 (HMGB1), we confirmed that TLR4 and RAGE (£ associated with PEDV pathogenesis during the infection in Vero cells. In summary, our studies provide a molecular basis for developing novel therapeutic methods to control PEDV infection, based on glycyrrhizin and its derivatives.
... Sterile inflammation is a major component of reperfusion injury. Tissue-released danger-associated molecular patterns (DAMPs) that activate cellular receptors triggering inflammasome formation [6,11,46], massive release of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [13], neutrophilic infiltrates releasing damaging proteases [22] are among the causes which concur to the progression of cardiac injury upon reoxygenation [22,30,41,43,57,70]. Cardioprotective pharmacological interventions essentially represent an attempt to evoke defensive and anti-apoptotic signals by reperfusion conditioning with b blockers, angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists, statins and antiplatelet drug [31,32,36,37], as well as antidiabetics [20] and anti-inflammatory agents [50]. ...
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The injury caused by myocardial reperfusion after ischemia can be contained by interventions aimed at reducing the inflammation and the oxidative stress that underlie exacerbation of tissue damage. Sphingolipids are a class of structural and signaling lipid molecules; among them, the inflammation mediator ceramide accumulates in the myocardium upon ischemia/reperfusion. Here, we show that, after transient coronary occlusion in mice, an increased de novo ceramide synthesis takes place at reperfusion in the ischemic area surrounding necrosis (area at risk). This correlates with the enhanced expression of the first and rate-limiting enzyme of the de novo pathway, serine palmitoyltransferase (SPT). The intraventricular administration at reperfusion of myriocin, an inhibitor of SPT, significantly protected the area at risk from damage, reducing the infarcted area by 40.9 % relative to controls not treated with the drug. In the area at risk, myriocin downregulated ceramide, reduced the content in other mediators of inflammation and reactive oxygen species, and activated the Nrf2–HO1 cytoprotective response. We conclude that an enhanced ceramide synthesis takes part in ischemia/reperfusion injury and that myriocin treatment can be proposed as a strategy for myocardial pharmacological postconditioning.
... This action can be explained by the lower leukocyte migration and consequent lower cytokine release. IL-1β and TNF-α are potent proinflammatory cytokines that are produced primarily by macrophages and neutrophils and may act on the endothelium inducing integrin expression and stimulating the proinflammatory cytokine release by other immune cells (Bradley 2008;Broggi and Granucci 2015). ...
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Ferulic acid ethyl ester (FAEE) is a derivate from ferulic acid which reportedly has antioxidant effect; however, its role on inflammation was unknown. In this study, we investigated the orally administered FAEE anti-inflammatory activity on experimental inflammation models and Complete Freund's Adjuvant (CFA)-induced arthritis in rats. CFA-induced arthritis has been evaluated by incapacitation model and radiographic knee joint records at different observation time. FAEE (po) reduced carrageenan-induced paw edema (p < 0.001) within the 1st to 5th hours at 50 and 100 mg/kg doses. FAEE 50 and 100 mg/kg, po inhibited leukocyte migration into air pouch model (p < 0.001), and myeloperoxidase, superoxide dismutase, and catalase activities (p < 0.001) increased total thiol concentration and decreased the TNF-α and IL-1β concentrations, NO, and thiobarbituric acid reactive species. In the CFA-induced arthritis, FAEE 50 and 100 mg/kg significantly reduced the edema and the elevation paw time, a joint disability parameter, since second hour after arthritis induction (p < 0.001). FAEE presented rat joint protective activity in radiographic records (p < 0.001). The data suggest that the FAEE exerts anti-inflammatory activity by inhibiting leukocyte migration, oxidative stress reduction, and pro-inflammatory cytokines.
... 24 The involvement of TLRs in diseases associated with chronic inflammation can also be mediated by their ability to recognize dangerassociated molecular patterns (DAMP), including endogenous proteins and components of apoptotic cells. 25 TLR ligands are also currently being explored as immunomodulators in oncology. 26,27 Nevertheless, emerging evidence suggests that TLR may also exert a direct effect on tumor cells and may regulate tumor progression. ...
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Recent studies on splenic marginal zone lymphoma identified distinct mutations in genes belonging to the B-cell receptor and Toll-like receptor signaling pathways, thus pointing to their potential implication in the biology of the disease. However, limited data is available regarding the exact role of TLR. We aimed at characterizing the expression pattern of TLR in splenic marginal zone lymphoma cells and their functional impact on the activation, proliferation and viability of malignant cells in vitro. Cells expressed significant levels of TLR1, TLR6, TLR7, TLR8, TLR9 and TLR10 mRNA; TLR2 and TLR4 showed a low, variable pattern of expression among patients whereas TLR3 and TLR5 mRNAs were undetectable; mRNA specific for TLR signaling molecules and adapters was also expressed. At the protein level, TLR1, TLR6, TLR7, TLR9 and TLR10 were detected. Stimulation of TLR1/2, TLR6/2 and TLR9 with their respective ligands triggered the activation of IRAK kinases, MAPK and NF-κB signaling pathways, and the induction of CD86 and CD25 activation molecules, although in a heterogeneous manner among different patient samples. TLR-induced activation and cell viability were also inhibited by a specific IRAK1/4 inhibitor, thus strongly supporting the specific role of TLR signaling in these processes. Furthermore, TLR2/6 and TLR9 stimulation also significantly increased cell proliferation. In conclusion, we demonstrate that splenic marginal zone lymphoma cells are equipped with functional TLR and signaling molecules and that the stimulation of TLR1/2, TLR2/6 and TLR9 may play a role in regulating disease pathobiology, likely promoting the expansion of the neoplastic clone. Copyright © 2015, Ferrata Storti Foundation.
... In cultured rat VSMCs stably producing RAGE, S100B protein was shown to induce production of several inflammatory mediators such as NF-jB, MCP-1, IL-6 and ICAM-1 (Hayakawa et al. 2012). HMGB1, which is released by necrotic cells, acts as a powerful proinflammatory inducer (Broggi & Granucci 2015). In VSMCs, HMGB1 could bind to several receptors such as RAGE, TLR2 or TLR4 and stimulate IL-1b expression (Jaulmes et al. 2006). ...
Article
Vascular smooth muscle cells (VSMCs) exhibit phenotypic and functional plasticity in order to respond to vascular injury. In case of the vessel damage, VSMCs are able to switch from the quiescent 'contractile' phenotype to the 'proinflammatory' phenotype. This change is accompanied by decrease in expression of smooth muscle (SM)-specific markers responsible for SM contraction and production of proinflammatory mediators that modulate induction of proliferation and chemotaxis. Indeed, activated VSMCs could efficiently proliferate and migrate contributing to the vascular wall repair. However, in chronic inflammation that occurs in atherosclerosis, arterial VSMCs become aberrantly regulated and this leads to increased VSMC dedifferentiation and extracellular matrix formation in plaque areas. Proatherosclerotic switch in VSMC phenotype is a complex and multistep mechanism that may be induced by a variety of proinflammatory stimuli and hemodynamic alterations. Disturbances in hemodynamic forces could initiate the proinflammatory switch in VSMC phenotype even in preclinical stages of atherosclerosis. Proinflammatory signals play a crucial role in further dedifferentiation of VSMCs in affected vessels and propagation of pathological vascular remodeling. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
... In addition to the antigen mimicry theory of the induction of autoreactive immunity, PAMPs function as adjuvants in the inflammatory response through the activation of innate immunity, which causes disruption of barriers and induction of cell damage. To date, many experimental autoimmune animal models involving induction by PAMPs have been developed for the study of autoimmune diseases [2,4,5]. ...
... Human HMGB1 is released by dying cells or it can be actively secreted from immune cells like macrophages or monocytes after stimulation with lipopolysaccharide (LPS), cytokines or nitric oxide through a non-classical secretory pathway. Outside the cell, it can promote inflammation in different ways: (1) it can act as a chemotactic agent recruiting leukocytes to the site of danger, (2) it can act as a DAMP stimulating innate immune cells via pattern recognition receptors (PRR), like the Receptor for Advanced Glycation End products (RAGE) and Toll-like receptor 4 (TLR4), and (3) it can also act in association to cytokines and other molecules (LPS, lipoteichoic acid, IL-1β, chemokine CXC receptor 4 (CXCR4), RNA and DNA) binding to its major transmembrane receptors and amplifying the response to PAMPs [32,34,35].The pro-inflammatory activity of Human HMGB1 has been mapped to a highly conserved 20-aminoacid-long sequence (residues 89 to 108) located in the B-box which corresponds to the TLR4 binding site (S2 Fig, blue-box) [34,36]. Concerning trypanosomatid HMGBs, most residues in this region are either identical or conservative changes relative to metazoan HMGBs, but there are some important substitutions in this region that may produce structural changes in the protein like the three proline residues 91, 97 and 98 from human HMGB1. ...
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Background: High Mobility Group B (HMGB) proteins are nuclear architectural factors involved in chromatin remodeling and important nuclear events. HMGBs also play key roles outside the cell acting as alarmins or Damage-associated Molecular Patterns (DAMPs). In response to a danger signal these proteins act as immune mediators in the extracellular milieu. Moreover, these molecules play a central role in the pathogenesis of many autoimmune and both infectious and sterile inflammatory chronic diseases. Principal findings: We have previously identified a High mobility group B protein from Trypanosoma cruzi (TcHMGB) and showed that it has architectural properties interacting with DNA like HMGBs from other eukaryotes. Here we show that TcHMGB can be translocated to the cytoplasm and secreted out of the parasite, a process that seems to be stimulated by acetylation. We report that recombinant TcHMGB is able to induce an inflammatory response in vitro and in vivo, evidenced by the production of Nitric Oxide and induction of inflammatory cytokines like TNF-α, IL-1β and IFN-γ gene expression. Also, TGF-β and IL-10, which are not inflammatory cytokines but do play key roles in Chagas disease, were induced by rTcHMGB. Conclusions: These preliminary results suggest that TcHMGB can act as an exogenous immune mediator that may be important for both the control of parasite replication as the pathogenesis of Chagas disease and can be envisioned as a pathogen associated molecular pattern (PAMP) partially overlapping in function with the host DAMPs.
... specific PRRs, including Toll-like receptors (TLRs) and NOD-like receptors (NLRs). This wide variety of DAMP molecules includes DNA, proteins (such as high-mobility group box 1; HMGB1), N-formyl peptides, extracellular matrix components, ATP and uric acid.[59][60][61] The specific signals that govern this early stage of recruitment vary among studies in part due to different injuries and different tissues. ...
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Chemotaxis is fundamental for leukocyte migration in immunity and inflammation and contributes to the pathogenesis of many human diseases. Although chemokines and various other chemoattractants were initially appreciated as important mediators of acute inflammation, in the past years they have emerged as critical mediators of cell migration during immune surveillance, organ development, and cancer progression. Such advances in our knowledge in chemokine biology have paved the way for the development of specific pharmacological targets with great therapeutic potential. Chemoattractants may belong to different classes, including a complex chemokine system of approximately 50 endogenous molecules that bind to G protein‐coupled receptors, which are expressed by a wide variety of cell types. Also, an unknown number of other chemoattractants may be generated by pathogens and damaged/dead cells. Therefore, blocking chemotaxis without causing side effects is an extremely challenging task. In this review, we focus on recent advances in understanding how the chemokine system orchestrates immune cell migration and positioning at the whole organ level in homeostasis, inflammation, and infection.
... [9] The host immune cells react with not only the pathogen-derived substances, including pathogen-associated molecular patterns (PAMPs), but also the substances from the host cells, including damage (danger)associated molecular patterns (DAMPs) such as heat shock proteins. [10] Because same kinds of immune cells and immune proteins, including Igs and complements, are seen pathologic lesions in nearly all infectious or infection-related immunemediated diseases, it is possible that the main function of the host immune cells and immune proteins may be same on the molecular level. Thus, it was proposed that immune cells and immune proteins control toxic substances against the host cells, based on their size and biochemical characteristics of the substances. ...
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Kawasaki disease (KD) may be an acute systemic immune-mediated disease which occurs after infection of unknown KD pathogen(s). The aim of this study is to evaluate the changes in platelet count and immunoglobulin (Ig) levels (IgG, IgM, IgA, and IgE) during hospitalization. Forty-three patients with complete KD who received intravenous Ig at 2 g/kg were enrolled in South Korea. The platelet count and Ig levels of the patients were examined twice at presentation and around discharge (mean 6.2 ± 2.4 days apart) and the relationships between platelet level and Ig levels were evaluated. The mean patient age was 31 ± 18 months; 28 patients were male and 15 were female. The values of all parameters measured, with the exception of IgE, were significantly increased at the second examination compared with their values at presentation. These values gradually increased over time after fever onset, over periods ranging from 2 to 16 days. The extent by which platelet levels increased over these 2 time points was correlated with the extents by which IgG (P < .01), IgM (P < .01), and IgA levels (P = .01) increased. Both the platelet count and the Ig (IgG, IgM, and IgA) levels increased with a correlation each other during the early convalescent stage of KD. This finding suggests that all Ig subtypes except IgE and platelets may be involved in the recovery from KD and that the extent of increased parameters may reflect the degree of systemic inflammation in acute KD.
... In an explanatory attempt, Polly Matzinger put forward the danger signal theory. This proposes that the immune system does not so much discriminate between endogenous and exogenous signals but increases responses to danger signals, regardless if they are exogenous pathogenic bacteria or endogenous damaged tissues [41]. However, the hazard model does not adequately explain the exquisite specificity of adaptive immune responses in autoimmune diseases. ...
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Autoinflammatory and autoimmune diseases are characterized by an oversensitive immune system with loss of the physiological endogenous regulation, involving multifactorial self-reactive pathological mechanisms of mono- or polygenic nature. Failure in regulatory mechanisms triggers a complex network of dynamic relationships between innate and adaptive immunity, leading to coexistent autoinflammatory and autoimmune processes. Sustained exposure to a trigger or a genetic alteration at the level of the receptors of the natural immune system may lead to abnormal activation of the innate immune system, adaptive system activation, loss of self-tolerance, and systemic inflammation. The IL-1 family members critically activate and regulate innate and adaptive immune responses’ diversity and plasticity in autoimmune and/or autoinflammatory conditions. The IL-23/IL-17 axis is key in the communication between innate immunity (IL-23-producing myeloid cells) and adaptive immunity (Th17- and IL-17-expressing CD8+ T cells). In psoriasis, these cytokines are decisive to the different clinical presentations, whether as plaque psoriasis (psoriasis vulgaris), generalized pustular psoriasis (pustular psoriasis), or mixed forms. These forms reflect a gradient between autoimmune pathophysiology with predominant adaptive immune response and autoinflammatory pathophysiology with predominant innate immune response. 1. Introduction Autoinflammatory and autoimmune diseases are characterized by immune system hyperactivity, typically featuring an against-self pathological process. They are systemic diseases and mono- or polygenic. The innate immune system directly causes tissue inflammation in autoinflammatory diseases. An adaptive immune dysregulation—against self—is found in autoimmune diseases. Both combined are present in mixed autoinflammatory-autoimmune pattern diseases (Figure 1).
... All these molecules behave as danger signals, thus alerting the immune system to trigger a response. Some of them are well-known contributors to the onset a chronic low-inflammatory state (Chan et al., 2012;Broggi and Granucci, 2015). ...
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The receptor of advanced glycation end products (RAGE) is a multi-ligand binding protein that was initially described as an AGEs-binding protein. Other proteins, some of them classified as danger signals or alarmins have been described as RAGE ligands. Of note, RAGE shares some of its ligands with some member of the Toll-like receptor family. Once RAGE is engaged by any of its ligands, the receptor is able to trigger robust pro-inflammatory cellular responses. At present, RAGE has emerged as a relevant actor in the onset and development of chronic inflammation, which underlies many human diseases. The receptor of advanced glycation end products is a multi-ligand receptor.RAGE variants are mainly originated by alternative splicing or proteolytic cleavage.RAGE and TLRs share common ligands and signalling pathways.RAGE activation is associated with activation of pro-inflammatory signalling.RAGE activation by AGEs play a key a role in the development and progression of diabetic nephropathy.Most of the misfolded proteins linked to neurodegeneration act as RAGE ligands.RAGE activation plays a key role in the disturbance of endothelial functions and cardiovascular homeostasis.RAGE has been regarded as the bridge association between chronic inflammation and cancer.RAGE activation in tumour cells promotes proliferation, invasion, angiogenesis and metastasis.
... In recent years, the hypothesis regarding the initiation triggers, the initiation itself, and the course of the autoimmunity process has changed. From Burnet's self-non-self theory [1], through the infection non-self theory by Janeway [2,3] (according to which the antigenpresenting cells [APSs] react with the pathogen-associated molecular patterns [PAMPs], e.g. ...
Article
The phenomenon of autoimmunity develops as a result of the triggering factor released by damaged cells. This leads to an infiltration of CD4+ cells involved in stimulating the effector cells cytotoxicity and stimulating the humoral response. One of the most common autoimmune disorders are autoimmune thyroid diseases, including Hashimoto's thyroiditis and Graves's diseases. Helper T lymphocytes, which are divided into Th1, Th2, Tregs, and the relatively new groups Th17, Th22, and Th9, are involved in the pathogenesis of AITD. CD4+ cell subtypes mature and differentiate by specific transcription factors and in a specific interleukin environment. Not only are Th1 and Th2 cells involved in the development of AITD, but also Th17, Th22, and Th9 lymphocytes and their correlation to Tregs lymphocytes. The plasticity of the CD4+ cells is very important, affecting the balance between these cells, as well the factors modulating their phenotypic variability. Patients with AITD have an increased percentage of Th17, Th22, and Th9 cells as well as defective function of Tregs lymphocytes. The balance between Th17 cells (and also other cytotoxic T cells) and Treg cells is also very important. Understanding the role of CD4 cells in the pathogenesis of AITD may be important not only for the development of the knowledge, but also for determining therapeutic targets.
... Forward migration of neutrophils refers to the general migration pattern of neutrophils, cells moving from the bloodstream to damaged interstitial areas, through transendothelial migration (TEM) [9][10][11][12][13]. DAMPs such as ATP, HMGB1 protein, N-formyl peptides (fMLP), and uric acid act as early cues for neutrophil aggregation [14][15][16][17]. After initiation, neutrophil aggregation is amplified, and neutrophil clusters are created along damaged/infected areas. ...
Article
Neutrophils are highly motile innate immune cells; they actively migrate in response to inflammatory signals. Using two-photon intravital microscopy, we discovered that neutrophils form stable clusters upon phototoxicity at a certain threshold. Without significant damage to the collagen structure of mouse dermis, neutrophils aggregated together with nearby neutrophils. Surprisingly, this in situ neutrophil clustering resulted in rigorous changes of migratory direction. The density of residing neutrophils was also a critical factor affecting clustering. Additionally, we found that the triggering point of neutrophil aggregation was correlated with the structure of the extracellular matrix in the ear dermis, where autofluorescence was strongly observed. This swarming behavior of neutrophils may reflect an unknown communication mechanism of neutrophils during migration under sterile injury.
... IL-1β is a cytokine that is inducible through the activation of PRRs such as TLRs, by microbial products or damaged cell factors [39], once the recognition of the ligands through the receptors activates the downstream signaling pathways activating the NF-κB, activator protein (AP)-1, MAPK, and type I IFNs pathways, resulting in an upregulation of inflammatory mediators, as well as chemotactic factors [40]. IL-1β is synthesized as a precursor peptide (pro-IL-1β) that is cut to generate its mature form (mIL-1β); this process involves caspase 1, and the proenzyme (procaspase-1) requires it to be cut by the inflammasome, which is a multimeric cytosolic protein complex, composed of NLR family-pyrin domain containing 3 (NALP3) and the adapter protein containing CARD (ASC) and caspase-1; once IL-1β is cut by this complex, it binds to the IL-1R1 receptor, thus initiating the signaling that induces DOI: http://dx.doi.org ...
... In addition, periodontal disease results not from individual pathogens but rather from polymicrobial synergy and dysbiosis, which perturbs the ecologically balanced biofilm associated with periodontal tissue homeostasis 2 . Bacterial cell wall components and intracellular contents can stimulate the host immune system, leading to the production and release of pro-inflammatory mediators 3 . Matrix metalloproteinases (MMPs), proteolytic enzymes that are responsible for the degradation of the organic extracellular matrix (ECM), have been reported to be associated with periodontal tissue destruction and alveolar bone absorption 4,5 . ...
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Periodontal homeostasis is maintained by the dynamic equilibrium between cell death, differentiation and proliferation of resident cells in the periodontal microenvironment. Loss of resident periodontal ligament fibroblasts (PDLFs) has been a major challenge in the periodontal treatment. This study aimed to investigate the exact role of necroptotic cell death in periodontal diseases. Elevated levels of receptor-interacting protein serine-threonine kinases -1 (RIPK1), phosphorylated RIPK3, mixed lineage kinase domain-like protein (MLKL), phosphorylated MLKL and FLIPL were observed in gingival tissues collected from patients with untreated chronic periodontitis; whereas no difference in caspase 8 was observed between the periodontitis and healthy control group. In contrast to the high incidence of necroptotic cell death in monocytes during live P. gingivalis infection with a low multiplicity of infection (MOI), necroptosis was only observed in PDLFs with a high MOI. Priming PDLFs with frozen thawed monocytes enhanced proinflammatory responses to P. gingivalis infection; moreover, frozen thawed monocytes stimulation triggered RIPK1, RIPK3 and MLKL-mediated-necroptotic cell death in PDLFs. These results indicated that RIPK3 and MLKL-mediated-necroptotic cell death participated in the pathogenesis of periodontitis, and DAMPs released from monocytes after P. gingivalis stimulation by necroptosis triggered not only inflammatory responses, but also necroptosis of PDLFs.
... ELISA measurement of NFκB activation was carried out on whole cell lysates to confirm that C. pecorum-dependent NFκB nuclear translocation at 24 hpi was associated with the expected increase of NFκB activation. Infection with the relatively low MOI of 1 was used in order to limit potential infectioninduced cell lysis, which could trigger danger signaling due to extracellular detection of normally cytoplasmic components (Broggi and Granucci, 2014). Such signaling has been shown to strongly impact C. pecorum development in HeLa (Leonard et al., 2015). ...
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Nuclear factor kappa B (NFκB) is an inflammatory transcription factor that plays an important role in the host immune response to infection. The potential for chlamydiae to activate NFκB has been an area of interest, however most work has focused on chlamydiae impacting human health. Given that inflammation characteristic of chlamydial infection may be associated with severe disease outcomes or contribute to poor overall fitness in farmed animals, we evaluated the ability of porcine chlamydiae to induce NFκB activation in vitro. C. pecorum infection induced both NFκB nuclear translocation and activation at 2 hours post infection (hpi), an effect strongly enhanced by suppression of host de novo protein synthesis. C. suis and C. trachomatis showed less capacity for NFκB activation compared to C. pecorum, suggesting a species-specific variation in NFκB activation. At 24 hpi, C. pecorum induced significant NFκB activation, an effect not abolished by penicillin (beta lactam)-induced chlamydial stress. C. pecorum-dependent secretion of interleukin 6 was also detected in the culture supernatant of infected cells at 24 hpi, and this effect, too, was unchanged by penicillin-induced chlamydial stress. Taken together, these results suggest that NFκB participates in the early inflammatory response to C. pecorum and that stressed chlamydiae can promote inflammation.
... DNA, high mobility group protein B1, N-formyl peptides, extracellular matrix proteins, adenosine triphosphate (ATP), and uric acid are examples of DAMPs that drive early neutrophil recruitment in tissue. Their roles in this process have been previously reviewed (22)(23)(24). Tissue resident cells, such as macrophages and mast cells, also recognize DAMPs, PAMPs, and inflammatory cytokines. Upon stimulation, these resident cells produce and secrete inflammatory cytokines of their own, including interleukin-8 (IL-8), a potent neutrophil chemoattractant (25)(26)(27)(28). ...
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Neutrophils are the primary responders to infection, rapidly migrating to sites of inflammation and clearing pathogens through a variety of antimicrobial functions. This response is controlled by a complex network of signals produced by vascular cells, tissue resident cells, other immune cells, and the pathogen itself. Despite significant efforts to understand how these signals are integrated into the neutrophil response, we still do not have a complete picture of the mechanisms regulating this process. This is in part due to the inherent disadvantages of the most-used experimental systems: in vitro systems lack the complexity of the tissue microenvironment and animal models do not accurately capture the human immune response. Advanced microfluidic devices incorporating relevant tissue architectures, cell-cell interactions, and live pathogen sources have been developed to overcome these challenges. In this review, we will discuss the in vitro models currently being used to study the neutrophil response to infection, specifically in the context of cell-cell interactions, and provide an overview of their findings. We will also provide recommendations for the future direction of the field and what important aspects of the infectious microenvironment are missing from the current models.
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DNA methyltransferase, a key enzyme mediating DNA methylation, is involved in numerous processes including genomic imprinting, X chromosome inactivation, transposable element suppression, and immune defense in vertebrates. In the present study, a DNA cytosine-5-methyltransferase 3 was identified from oyster Crassostrea gigas (designed as CgDNMT3). There were a PWWP domain, a PHD domain and a DNA-methylase domain in the deduced amino acid sequences of CgDNMT3, and the conserved motifs I, IV, VI, Ⅷ, IX and X were identified in its C-terminal catalytic DNA-methylase domain. The mRNA transcripts of CgDNMT3 were detected in haemocytes, mantle, gill, adductor muscle, digestive gland and labial palp, with higher expression level in haemocytes (6.54 folds of those in gill, p < 0.01). The expression level of CgDNMT3 mRNA in haemocytes increased significantly after LPS primed (2.87 folds of that in control group, p < 0.05) in vitro or Vibrio splendidus challenging (1.94 folds of that in control group, p < 0.05) in vivo. Immunocytochemical analysis revealed that CgDNMT3 protein was distributed mainly in cytoplasm and partial in nucleus of oyster haemocytes. After CgDNMT3 was transfected and expressed in HEK293T cells, the DNA 5-methylcytosine (5-mc) level in the transfected group was significantly increased, which was 1.22 folds (p < 0.05) of the pcDNA-3.1 group. The expressions of oyster CgIL17-1, CgIL17-2 and CgIL17-5 in haemocytes increased (13.05 folds, 4.78 folds and 9.41 folds of that in control group, respectively) at 12 h after V. splendidus challenging, but the increase were significantly inhibited when the oysters were pre-treated with DNA methyltransferase inhibitor 5-Azacytidine, which were 9 folds, 1.93 folds and 3.22 folds of that in control group, respectively. These results collectively suggested that CgDNMT3 was a conserved member of DNA methyltransferase 3 family in oyster, and participated in regulating the expression of cytokines during immune response.
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Influenza A virus (IAV) is a serious global health problem worldwide due to frequent and severe outbreaks. IAV causes significant morbidity and mortality in the elderly population, due to the ineffectiveness of the vaccine and the alteration of T cell immunity with ageing. The cellular and molecular link between ageing and virus infection is unclear and it is possible that damage associated molecular patterns (DAMPs) may play a role in the raised severity and susceptibility of virus infections in the elderly. DAMPs which are released from damaged cells following activation, injury or cell death can activate the immune response through the stimulation of the inflammasome through several types of receptors found on the plasma membrane, inside endosomes after endocytosis as well as in the cytosol. In this review, the detriment in the immune system during aging and the links between influenza virus infection and ageing will be discussed. In addition, the role of DAMPs such as HMGB1 and S100/Annexin in ageing, and will in the enhanced morbidity and mortality to severe influenza infection in ageing will be highlighted. Copyright © 2015. Published by Elsevier B.V.
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Persistence, more recently termed the chlamydial stress response, is a viable but non-infectious state constituting a divergence from the characteristic chlamydial biphasic developmental cycle. Damage/danger associated molecular patterns (DAMPs) are normal intracellular components or metabolites that, when released from cells, signal cellular damage/lysis. Purine metabolite DAMPs, including extracellular ATP and adenosine, inhibit chlamydial development in a species-specific manner. Viral co-infection has been shown to reversibly abrogate Chlamydia inclusion development, suggesting persistence/chlamydial stress. Because viral infection can cause host cell DAMP release, we hypothesized DAMPs may influence chlamydial development. Therefore, we examined the effect of extracellular ATP, adenosine, and cyclic AMP exposure, at 0 and 14 hours post infection, on C. pecorum and C. trachomatis serovar E development. In the absence of de novo host protein synthesis, exposure to DAMPs immediately post or at 14 hours post infection reduced inclusion size; however, the effect was less robust upon 14 hours post infection exposure. Additionally, upon exposure to DAMPs immediately post infection, bacteria per inclusion and subsequent infectivity were reduced in both Chlamydia species. These effects were reversible, and C. pecorum exhibited more pronounced recovery from DAMP exposure. Aberrant bodies, typical in virus-induced chlamydial persistence, were absent upon DAMP exposure. In the presence of de novo host protein synthesis, exposure to DAMPs immediately post infection reduced inclusion size, but only variably modulated chlamydial infectivity. Because chlamydial infection and other infections may increase local DAMP concentrations, DAMPs may influence Chlamydia infection in vivo, particularly in the context of poly-microbial infections.
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“There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes.” We have come a long way since this declamation by Sir Ralph Bloomfield Bonington in George Bernard Shaw’s 1906 play, The Doctor’s Dilemma. Stimulation of phagocytes is even
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This chapter contains the core of the innate immune defense systems and consequently marks the most voluminous chapter of the book. It consists of an initial recognition part and a subsequent executive part. Perception of infectious or sterile injury is ensured by PRM-bearing innate immune cells which possess the property to recognize MAMPs and DAMPs. Following recognition, the various cell-bound PRMs including TLRs, NLRs, RLRs, and ALRs trigger signalling pathways which promote, via strictly regulated transcriptional and translational processes, the secretion of mediator substances to elicit efferent inflammatory responses. Intracellular formation of multiprotein complexes, the inflammasomes, plays a pivotal role in the establishment of the inflammatory milieu. Among the vital mediator substances of inflammation, several families of pro-inflammatory or anti-inflammatory cytokines such as the interleukin-1 family, interferons, tumor necrosis factor, and interleukin-17 family, as well as the chemokine family, take center stage in shaping and regulating promotion and resolution of inflammation. The efforts of all these substances in ensuring effective defense are supported and complemented by the process of phagocytosis that can be recognized as one of the critical biological events of the innate immune defense system mainly executed by the “professionals” among phagocytosing cells, the macrophages, neutrophils, and dendritic cells. In fact, dangerous foreign bodies such as bacteria or fungi as well as apoptotic and necrotic cells can be cleared from infectiously or sterilely damaged tissue by these professional phagocytes. The chapter closes with the notoriously repeated comment that all these sophisticated pathways of a robust innate immune defense may lead to pathologies and many human diseases, when they take place in an uncontrolled and dysregulated way.
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Background: Infection in patients with systemic inflammation is difficult to diagnose with a single biomarker. We aimed to clarify the time course of change in the gene expression profile of innate immune receptors in infectious or sterile inflammation and to establish an early diagnostic method using canonical discriminant analysis (CDA) of the gene expression profile. Methods: To compare infectious and sterile inflammation, we used cecal ligation and puncture (CLP) and 20% full-thickness burn injury (Burn) models. C57BL/6 mice underwent sham treatment (n=9×3 groups), CLP (n=12×3 groups), or Burn (n=12×3 groups) injury. Mice were sacrificed at 6, 12, and 24 hours after injury, and total RNA was extracted from whole blood. We used quantitative real-time PCR to investigate gene expression of innate immune receptors TLR2, TLR4, TLR9, NLRP3, and RIG-I. To evaluate all gene expression together as patterns, each value was standardized, and CDA was performed at each time point. Results: Gene expression of TLR2 and TLR4 was already significantly increased in both CLP and Burn compared to sham mice at 6 hours after injury (p<0.05). Gene expression of TLR9 was significantly decreased in CLP compared to sham and Burn mice at 12 and 24 hours after injury (p<0.05) but not at 6 hours. Gene expression of NLRP3 was significantly increased in CLP and Burn compared to sham mice at 6 and 24 hours after injury (p<0.05). In the CDA, each group showed distinctive gene expression patterns at just 6 hours after injury. Each group was clearly classified, and the classification error rates were 0% at all of the time points. Conclusions: CDA of the gene expression profile of innate immune receptors could be a novel approach for diagnosing the pathophysiology of complicated systemic inflammation from the early stage of injury.
Chapter
Most of the syndromes associated with enteritis and colitis in horses are fundamentally inflammatory in nature, with a number of shared pathophysiologic processes. An understanding of these underlying abnormalities informs the approach to diagnosis and management of enteritis and colitis. Knowledge of the specific etiologies associated with these conditions provides the clinician with the opportunity to implement specific therapies directed at these etiologies, in order to maximize the response to treatment.
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Macrophage differentiation and polarization are essential players in the success of the wound-healing process. Acute simple wounds progress from inflammation to proliferation/regeneration and, finally, to remodeling. In injured skin, macrophages either reside in the epithelium or are recruited from monocytes. Their main role is supported by their plasticity, which allows them to adopt different phenotypic states, such as the M1-inflammatory state, in which they produce TNF and NO, and the M2-reparative state, in which they resolve inflammation and exhibit a reparative function. Reparative macrophages are an essential source of growth factors such as TGF-β and VEGF and are not found in nonhealing wounds. This review discusses the differences between macrophage phenotypes in vitro and in vivo, how macrophages originate, and how they cross-communicate with other cellular components in a wound. This review also highlights the dysregulation of macrophages that occurs in nonhealing versus overhealing wounds and fibrosis. Then, the therapeutic manipulation of macrophages is presented as an attractive strategy for promoting healing through the secretion of growth factors for angiogenesis, keratinocyte migration, and collagen production. Finally, Hoxa3 overexpression is discussed as an example of the therapeutic repolarization of macrophages to the normal maturation state and phenotype with better healing outcomes.
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The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease.
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Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release large quantities of tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can precipitate tissue injury and lethal shock (endotoxemia). Antagonists of TNF and IL-1 have shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis. Here a potential late mediator of lethality is identified and characterized in a mouse model. High mobility group–1 (HMG-1) protein was found to be released by cultured macrophages more than 8 hours after stimulation with endotoxin, TNF, or IL-1. Mice showed increased serum levels of HMG-1 from 8 to 32 hours after endotoxin exposure. Delayed administration of antibodies to HMG-1 attenuated endotoxin lethality in mice, and administration of HMG-1 itself was lethal. Septic patients who succumbed to infection had increased serum HMG-1 levels, suggesting that this protein warrants investigation as a therapeutic target.
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Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release.
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HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-kB kinase (IKK)b-and IKKa-dependent NF-kB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKb-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKa non-canonical pathway was not known. In this study, we show that the IKKa-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKa KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12's G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury. The Journal of Immunology, 2012, 188: 2380–2386. H igh-mobility group box 1 (HMGB1) is a nonhistone, chromatin architectural protein that is ubiquitously expressed by all mammalian cells, but it functions out-side cells as a potent cytokine and chemoattractant. In vivo, HMGB1 is passively released by necrotic cells and actively se-creted by immune effector cells (1–4). Extracellular HMGB1 sig-nals through the receptor for advanced glycation end-products (RAGE), TLR2, and TLR4 (3–9). In this capacity HMGB1 acts as an alarmin or damage-associated molecular pattern that senses tissue damage and elicits a variety of proinflammatory responses (reviewed in 3, 4, 6, 10, 11). Moreover, the chemotactic activity of HMGB1 is an important initiating aspect of the wound healing response and how cells migrate to repair damaged tissues (12, 13). Cell migration to HMGB1 requires the action of several inter-connected signal transduction pathways. RAGE ligand-induced cell migration requires RAGE interaction with diaphanous-1, which is required for Rac-1 and Cdc42-regulated cell movement (14). We have previously shown that cellular chemotaxis toward HMGB1 in vitro requires canonical NF-kB activation in a variety of cell types (fibroblasts, mesoangioblasts, macrophages, and neutrophils) in vitro and also for the respective migration of neutrophils and mesoangioblasts in in vivo mouse models of HMGB1-elicited peritonitis and muscle damage (15, 16). HMGB1 induction of canonical NF-kB signaling and fibroblast chemotaxis requires ERK activation (15) and Src family kinases, which reorganize the cellular cytoskeleton and induce Src, FAK, and paxillin phos-phorylation (17). Time-lapse video microscopy experiments have revealed that the inhibitor of NF-kB kinase (IKK)b and IKKa signaling pathways are essential for cells to become polarized to an HMGB1 gradient, which is indicative of critical functional roles in the initial steps of directed cell movement (16). Finally, we have also reported that the activity of IKKb-dependent canonical NF-kB signaling is mechanistically essential for cells to maintain RAGE expression for their HMGB1 migratory response, whereas the IKKa-driven noncanonical NF-kB p52-RelB signaling path-way is simultaneously critical for HMGB1-elicited chemotaxis for a different reason (16). In this study, we have defined the mechanism of action of the IKKa-driven NF-kB RelB/p52 signaling pathway for HMGB1 chemotaxis. Surprisingly, for cells to migrate in response to HMGB1, the NF-kB noncanonical pathway is solely required to
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We established a protocol for the prediction of the coding sequences of unidentified human genes based on the double selection and sequence analysis of cDNA clones with inserts carrying unreported 5′-terminal sequences and with insert sizes corresponding to nearly full-length transcripts. By applying the protocol, cDNA clones with inserts longer than 2 kb were isolated from a cDNA library of human immature myeloid cell line KG-1, and the coding sequences of 40 new genes were predicted. A computer search of the sequences indicated that 20 genes contained sequences similar to known genes in the GenBank/EMBL databases. The sequences of the remaining 20 genes were entirely new, and characteristic protein motifs or domains were identified in 32 genes. Other sequence features noted were that the coding sequences of 23 genes were followed by relatively long stretches of 3′-untranslated sequences and that 5 genes contained repetitive sequences in their 3′-untranslated regions. The chromosomal location of these genes has been determined. By increasing the scale of the above analysis, the coding sequences of many unidentified genes can be predicted.
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High mobility group box 1 (HMGB1) is a DNA-bi nding protein possesses cytokine-like, pro-inflammatory properties when released extracellularly in the C23-C45 disulfide form. HMGB1 also plays a key role as a mediator of acute and chronic inflammation in models of sterile injury. Although HMGB1 interacts with multiple pattern recognition receptors (PRRs), many of its effects in injury models occur through an interaction with Toll-like Receptor 4 (TLR4). HMGB1 interacts directly with the TLR4/MD2 complex, although the nature of this interaction remains unclear. We demonstrate that optimal HMGB1-dependent TLR4 activation in vitro requires the co-receptor, CD14. TLR4 and MD2 are recruited into CD14-containing lipid rafts of RAW246.7 macrophages following stimulation with HMGB1, and TLR4 interacts closely with the lipid raft protein GM1. Furthermore, we show that HMGB1 stimulates TNF-α release in WT but not in TLR4(-/-), CD14(-/-), TRIF(-/-) or MyD88(-/-) macrophages. HMGB1 induces the release of MCP-1, IP-10 and MIP-1α in a TLR4 and CD14-dependent manner. Thus, efficient recognition of HMGB1 by the TLR4/MD2 complex requires CD14.
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Tissue damage causes inflammation, by recruiting leukocytes and activating them to release proinflammatory mediators. We show that high-mobility group box 1 protein (HMGB1) orchestrates both processes by switching among mutually exclusive redox states. Reduced cysteines make HMGB1 a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine and further cysteine oxidation to sulfonates by reactive oxygen species abrogates both activities. We show that leukocyte recruitment and activation can be separated. A nonoxidizable HMGB1 mutant in which serines replace all cysteines (3S-HMGB1) does not promote cytokine production, but is more effective than wild-type HMGB1 in recruiting leukocytes in vivo. BoxA, a HMGB1 inhibitor, interferes with leukocyte recruitment but not with activation. We detected the different redox forms of HMGB1 ex vivo within injured muscle. HMGB1 is completely reduced at first and disulfide-bonded later. Thus, HMGB1 orchestrates both key events in sterile inflammation, leukocyte recruitment and their induction to secrete inflammatory cytokines, by adopting mutually exclusive redox states.
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The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1β, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1β, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.
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Inflammation is a multistep process triggered when innate immune cells - for example, DCs - sense a pathogen or injured cell or tissue. Edema formation is one of the first steps in the inflammatory response; it is fundamental for the local accumulation of inflammatory mediators. Injection of LPS into the skin provides a model for studying the mechanisms of inflammation and edema formation. While it is known that innate immune recognition of LPS leads to activation of numerous transcriptional activators, including nuclear factor of activated T cells (NFAT) isoforms, the molecular pathways that lead to edema formation have not been determined. As PGE2 regulates many proinflammatory processes, including swelling and pain, and it is induced by LPS, we hypothesized that PGE2 mediates the local generation of edema following LPS exposure. Here, we show that tissue-resident DCs are the main source of PGE2 and the main controllers of tissue edema formation in a mouse model of LPS-induced inflammation. LPS exposure induced expression of microsomal PGE synthase-1 (mPGES-1), a key enzyme in PGE2 biosynthesis. mPGES-1 activation, PGE2 production, and edema formation required CD14 (a component of the LPS receptor) and NFAT. Therefore, tissue edema formation induced by LPS is DC and CD14/NFAT dependent. Moreover, DCs can regulate free antigen arrival at the draining lymph nodes by controlling edema formation and interstitial fluid pressure in the presence of LPS. We therefore suggest that the CD14/NFAT/mPGES-1 pathway represents a possible target for antiinflammatory therapies.
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After tissue damage, inflammatory cells infiltrate the tissue and release proinflammatory cytokines. HMGB1 (high mobility group box 1), a nuclear protein released by necrotic and severely stressed cells, promotes cytokine release via its interaction with the TLR4 (Toll-like receptor 4) receptor and cell migration via an unknown mechanism. We show that HMGB1-induced recruitment of inflammatory cells depends on CXCL12. HMGB1 and CXCL12 form a heterocomplex, which we characterized by nuclear magnetic resonance and surface plasmon resonance, that acts exclusively through CXCR4 and not through other HMGB1 receptors. Fluorescence resonance energy transfer data show that the HMGB1-CXCL12 heterocomplex promotes different conformational rearrangements of CXCR4 from that of CXCL12 alone. Mononuclear cell recruitment in vivo into air pouches and injured muscles depends on the heterocomplex and is inhibited by AMD3100 and glycyrrhizin. Thus, inflammatory cell recruitment and activation both depend on HMGB1 via different mechanisms.
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HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-κB kinase (IKK)β- and IKKα-dependent NF-κB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKβ-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKα non-canonical pathway was not known. In this study, we show that the IKKα-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKα KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12's G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury.
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Inflammasomes are a group of protein complexes built around several proteins, including NLRP3, NLRC4, AIM2 and NLRP6. Recognition of a diverse range of microbial, stress and damage signals by inflammasomes results in direct activation of caspase-1, which subsequently induces secretion of potent pro-inflammatory cytokines and a form of cell death called pyroptosis. Inflammasome-mediated processes are important during microbial infections and also in regulating both metabolic processes and mucosal immune responses. We review the functions of the different inflammasome complexes and discuss how aberrations in them are implicated in the pathogenesis of human diseases.
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Caspase-1 activation by inflammasome scaffolds comprised of intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the adaptor ASC is believed to be essential for production of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 during the innate immune response. Here we show, with C57BL/6 Casp11 gene-targeted mice, that caspase-11 (also known as caspase-4) is critical for caspase-1 activation and IL-1β production in macrophages infected with Escherichia coli, Citrobacter rodentium or Vibrio cholerae. Strain 129 mice, like Casp11(-/-) mice, exhibited defects in IL-1β production and harboured a mutation in the Casp11 locus that attenuated caspase-11 expression. This finding is important because published targeting of the Casp1 gene was done using strain 129 embryonic stem cells. Casp1 and Casp11 are too close in the genome to be segregated by recombination; consequently, the published Casp1(-/-) mice lack both caspase-11 and caspase-1. Interestingly, Casp11(-/-) macrophages secreted IL-1β normally in response to ATP and monosodium urate, indicating that caspase-11 is engaged by a non-canonical inflammasome. Casp1(-/-)Casp11(129mt/129mt) macrophages expressing caspase-11 from a C57BL/6 bacterial artificial chromosome transgene failed to secrete IL-1β regardless of stimulus, confirming an essential role for caspase-1 in IL-1β production. Caspase-11 rather than caspase-1, however, was required for non-canonical inflammasome-triggered macrophage cell death, indicating that caspase-11 orchestrates both caspase-1-dependent and -independent outputs. Caspase-1 activation by non-canonical stimuli required NLRP3 and ASC, but caspase-11 processing and cell death did not, implying that there is a distinct activator of caspase-11. Lastly, loss of caspase-11 rather than caspase-1 protected mice from a lethal dose of lipopolysaccharide. These data highlight a unique pro-inflammatory role for caspase-11 in the innate immune response to clinically significant bacterial infections.
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The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was considered fixed, but recently aging yeast and mammalian cells were shown to contain fewer nucleosomes. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and affects the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform and can be modelled assuming that different nucleosomal sites compete for available histones. Sites with a high propensity to occupation are almost always packaged into nucleosomes both in wild type and nucleosome-depleted cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of epigenetic regulation.
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Over the past several decades, much has been revealed about the nature of the host innate immune response to microorganisms, with the identification of pattern recognition receptors (PRRs) and pathogen-associated molecular patterns, which are the conserved microbial motifs sensed by these receptors. It is now apparent that these same PRRs can also be activated by non-microbial signals, many of which are considered as damage-associated molecular patterns. The sterile inflammation that ensues either resolves the initial insult or leads to disease. Here, we review the triggers and receptor pathways that result in sterile inflammation and its impact on human health.
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Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.
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The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease.
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During infection, vertebrates develop "sickness syndrome," characterized by fever, anorexia, behavioral withdrawal, acute-phase protein responses, and inflammation. These pathophysiological responses are mediated by cytokines, including TNF and IL-1, released during the innate immune response to invasion. Even in the absence of infection, qualitatively similar physiological syndromes occur following sterile injury, ischemia reperfusion, crush injury, and autoimmune-mediated tissue damage. Recent advances implicate high-mobility group box 1 (HMGB1), a nuclear protein with inflammatory cytokine activities, in stimulating cytokine release. HMGB1 is passively released during cell injury and necrosis, or actively secreted during immune cell activation, positioning it at the intersection of sterile and infection-associated inflammation. To date, eight candidate receptors have been implicated in mediating the biological responses to HMGB1, but the mechanism of HMGB1-dependent cytokine release is unknown. Here we show that Toll-like receptor 4 (TLR4), a pivotal receptor for activation of innate immunity and cytokine release, is required for HMGB1-dependent activation of macrophage TNF release. Surface plasmon resonance studies indicate that HMGB1 binds specifically to TLR4, and that this binding requires a cysteine in position 106. A wholly synthetic 20-mer peptide containing cysteine 106 from within the cytokine-stimulating B box mediates TLR4-dependent activation of macrophage TNF release. Inhibition of TLR4 binding with neutralizing anti-HMGB1 mAb or by mutating cysteine 106 prevents HMGB1 activation of cytokine release. These results have implications for rationale, design, and development of experimental therapeutics for use in sterile and infectious inflammation.
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The IL-1 family cytokines are regulated on transcriptional and posttranscriptional levels. Pattern recognition and cytokine receptors control pro-IL-1beta transcription whereas inflammasomes regulate the proteolytic processing of pro-IL-1beta. The NLRP3 inflammasome, however, assembles in response to extracellular ATP, pore-forming toxins, or crystals only in the presence of proinflammatory stimuli. How the activation of gene transcription by signaling receptors enables NLRP3 activation remains elusive and controversial. In this study, we show that cell priming through multiple signaling receptors induces NLRP3 expression, which we identified to be a critical checkpoint for NLRP3 activation. Signals provided by NF-kappaB activators are necessary but not sufficient for NLRP3 activation, and a second stimulus such as ATP or crystal-induced damage is required for NLRP3 activation.
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