Cytokine Response In Macrophages Research Articles

Erythropoietin and obesity-induced white adipose tissue inflammation: redefining the boundaries of the immunometabolism territory

The adipose tissue represents a critical and predominant site for the interaction between metabolic and inflammatory responses during health and disease. In the white adipose tissue microenvironment, macrophages/adipocytes cross-talk have been shown to influence the metabolic and inflammatory states of both cell types, and contribute to the development of systemic insulin resistance during obesity. Indeed, the existence of paracrine loops between mature adipocytes and macrophages, especially during obesity-induced stress, involving the release of, and response to, an array of cytokines and regulatory factors, have been extensively studied using several in vitro and in vivo model systems. Published evidence together with recent observations, brought to light the unexpected role of erythropoietin and its receptor in the regulation of white adipose tissue mass, energy homeostasis, and inflammation as demonstrated by erythropoietin effects on adipocyte development and metabolic profile, and macrophage infiltration, cytokine responses, and activation state during diet-induced obesity. In this commentary, we discuss the newly added elements and perspectives to our understanding of the erythropoietin/erythropoietin-receptor axis as a regulator of obesity-induced white adipose tissue inflammation, providing insight into its effects on cytokine responses of macrophages and adipocytes, and possible links to glucose metabolism and insulin resistance.

STING agonists induce an innate antiviral immune response against hepatitis B virus.

Chronicity of hepatitis B virus (HBV) infection is due to the failure of a host to mount a sufficient immune response to clear the virus. The aim of this study was to identify small-molecular agonists of the pattern recognition receptor (PRR)-mediated innate immune response to control HBV infection. To achieve this goal, a coupled mouse macrophage and hepatocyte culture system mimicking the intrahepatic environment was established and used to screen small-molecular compounds that activate macrophages to produce cytokines, which in turn suppress HBV replication in a hepatocyte-derived stable cell line supporting HBV replication in a tetracycline-inducible manner. An agonist of the mouse stimulator of interferon (IFN) genes (STING), 5,6-dimethylxanthenone-4-acetic acid (DMXAA), was found to induce a robust cytokine response in macrophages that efficiently suppressed HBV replication in mouse hepatocytes by reducing the amount of cytoplasmic viral nucleocapsids. Profiling of cytokines induced by DMXAA and agonists of representative Toll-like receptors (TLRs) in mouse macrophages revealed that, unlike TLR agonists that induced a predominant inflammatory cytokine/chemokine response, the STING agonist induced a cytokine response dominated by type I IFNs. Moreover, as demonstrated in an HBV hydrodynamic mouse model, intraperitoneal administration of DMXAA significantly induced the expression of IFN-stimulated genes and reduced HBV DNA replication intermediates in the livers of mice. This study thus proves the concept that activation of the STING pathway induces an antiviral cytokine response against HBV and that the development of small-molecular human STING agonists as immunotherapeutic agents for treatment of chronic hepatitis B is warranted.

Cytotoxicity and Cytokine Release in Rat Hepatocytes, C3A Cells and Macrophages Exposed to Gold Nanoparticles—Effect of Biological Dispersion Media or Corona

The study aim was to investigate how gold nanoparticles (NPs) of different sizes (20 and 100 nm) influence primary hepatocytes, the hepatocyte cell line C3A and macrophage cytokine responses when dispersed in lung or blood relevant fluids. Gold Au NPs induced cytotoxicity in primary hepatocytes at the highest dose of 66 μg/cm2, this effect was modified by the dispersant, the effect was greater with lung lining fluid (LLF). Release of interleukin (IL)-6, Monocyte chemoattractant protein-1 (MCP-1) and IL-1β was enhanced by the Au NPs and the effects were influenced by the particle size and dispersant. In medium, the smallest particle size was most effective at inducing IL-6 release, while in LLF the largest particles were most effective at inducing IL-6 release. Both 20 nm and 100 nm particles enhanced MCP-1 and IL-1β in the presence of LLF. The Au particles had no cytotoxic effects nor did they stimulate the release of cytokines in the C3A hepatoma cell line. The Au NPs had no significant impact on macrophage viability. Particles induced IL-6 and TNF-α release. LLF and serum reduced the IL-6 response while albumin enhanced the TNF-α response compared to medium dispersed Au NPs. The Au NPs did not impact on MCP-1 release, but this cytokine was enhanced by albumin and serum, while it was depressed by LLF. The macrophage responses were lower than those evoked in primary hepatocytes. In conclusion, when assessing the cytotoxic and pro-inflammatory responses induced by Au NPs, the response is influenced by the dispersant, with different dispersants having different effects in different cell types.

Regulatory effect of interleukin-4 in the innate inflammatory response to Rhodococcus aurantiacus infection in mice.

Interleukin (IL)-4 promotes the regression of granulomas during the late phase of Rhodococcus aurantiacus infection. In this study, the contribution of IL-4 to the initial response against this bacterium was investigated using IL-4-deficient mice. Compared with wild-type (WT) mice, IL-4-deficient mice displayed remarkably lower tumor necrosis factor (TNF)-α and IL-6 secretion in the liver, spleen, and blood at the initial phase of infection, along with improved survival. IL-4-deficient mice also showed diminished IL-10 secretion in the spleen and blood; however, hepatic IL-10 levels were similar to those observed in WT animals, and were concomitant with augmented interferon (IFN)-γ production and decreased bacterial burden in the liver at the early infection phase. Histological examination revealed reduced hepatic granuloma formation in infected IL-4-deficient mice. On challenge with heat-killed R. aurantiacus, IL-4-deficient mouse macrophages showed reduced expression of TNF-α, IL-6, and IL-10 at both the gene and protein levels compared with WT mouse cells. These findings indicate that during the initiation of R. aurantiacus-induced inflammation, IL-4 deficiency attenuates cytokine responses in macrophages, which contributes to amelioration in mouse survival and reduction of granulomatous inflammation, and augments a hepatic IFN-γ response which transiently accelerates bacterial elimination.

204: Regulatory effect of interleukin-4 in the innate immune and inflammatory responses to Rhodococcus aurantiacus infection in mice

Inoculation of wild-type (WT) mice with gram-positive bacterium Rhodococcus aurantiacus induces Th1-type granulomatous inflammation, in which interleukin (IL)-4 promotes the regression of granulomas during the late phase of infection. As IL-4 plays diverse roles in disease pathogenesis, we investigated the effect of IL-4 in the innate response to this bacterium infection by using IL-4-deficient mice. After intravenous inoculation with 1 × 10 8 CFU of bacteria, mouse survival rates, bacteria load in organs, local and systemic cytokine production, and morphological changes in liver were assessed. Following stimulation with heat-killed R. aurantiacus , cytokine production in mouse peritoneal macrophages was also examined. Compared to WT mice, IL-4-deficient mice showed the decreased production of TNF- α and IL-6 in the spleen, liver and blood during the early phase of infection, as well as low bacterial load in the liver and improvement in survival rate. IL-4-deficient mice also showed diminished IL-10 secretion in the spleen and blood, whereas their hepatic IL-10 levels were similar to those observed in WT mice, which were concomitant with the augmented hepatic IFN-γ production. Moreover, histological studies revealed reduction in hepatic granuloma formation at day 14 post-infection in IL-4-deficient mice. Upon stimulation with heat-killed R. aurantiacus , macrophages from IL-4-deficient mice showed lower expression of TNF-α, IL-6, and IL-10 at both the gene and protein levels than WT mouse cells. These findings indicate that IL-4 deficit attenuates cytokine responses in macrophages but augments the IFN-γ response during the initial stage of R. aurantiacus infection, which together contribute to amelioration of mouse survival, rapid bacterial elimination in liver at the early phase, and reduction of granulomatous inflammation. We previously demonstrated that the early IFN-γ response to R. aurantiacus is induced in mouse natural killer cells. This study thus identifies the essential effect of IL-4 on regulating the activation and function of immunocompetent cells, including macrophages and natural killer cells, during the initial phase of systemic R. aurantiacus infection.

Infliximab therapy increases the frequency of circulating CD16(+) monocytes and modifies macrophage cytokine response to bacterial infection.

Crohn's disease (CD) has been correlated with altered macrophage response to microorganisms. Considering the efficacy of infliximab treatment on CD remission, we investigated infliximab effects on circulating monocyte subsets and on macrophage cytokine response to bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, treated or not with infliximab. Macrophages were infected with Escherichia coli, Enterococcus faecalis, Mycobacterium avium subsp. paratuberculosis (MAP) or M. avium subsp avium, and cytokine levels [tumour necrosis factor (TNF) and interleukin (IL)-10] were evaluated at different time-points. To evaluate infliximab-dependent effects on monocyte subsets, we studied CD14 and CD16 expression by peripheral blood monocytes before and after different infliximab administrations. We also investigated TNF secretion by macrophages obtained from CD16(+) and CD16(-) monocytes and the frequency of TNF(+) cells among CD16(+) and CD16(-) monocyte-derived macrophages from CD patients. Infliximab treatment resulted in elevated TNF and IL-10 macrophage response to bacteria. An infliximab-dependent increase in the frequency of circulating CD16(+) monocytes (particularly the CD14(++) CD16(+) subset) was also observed (before infliximab: 4·65 ± 0·58%; after three administrations: 10·68 ± 2·23%). In response to MAP infection, macrophages obtained from CD16(+) monocytes were higher TNF producers and CD16(+) macrophages from infliximab-treated CD patients showed increased frequency of TNF(+) cells. In conclusion, infliximab treatment increased the TNF production of CD macrophages in response to bacteria, which seemed to depend upon enrichment of CD16(+) circulating monocytes, particularly of the CD14(++) CD16(+) subset. Infliximab treatment of CD patients also resulted in increased macrophage IL-10 production in response to bacteria, suggesting an infliximab-induced shift to M2 macrophages.

Francisella tularensis LVS induction of prostaglandin biosynthesis by infected macrophages requires specific host phospholipases and lipid phosphatases.

Francisella tularensis induces the synthesis of prostaglandin E(2) (PGE(2)) by infected macrophages to alter host immune responses, thus providing a survival advantage to the bacterium. We previously demonstrated that PGE(2) synthesis by F. tularensis-infected macrophages requires cytosolic phospholipase A2 (cPLA(2)), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES1). During inducible PGE(2) synthesis, cPLA(2) hydrolyzes arachidonic acid (AA) from cellular phospholipids to be converted to PGE(2). However, in F. tularensis-infected macrophages we observed a temporal disconnect between Ser505-cPLA(2) phosphorylation (a marker of activation) and PGE(2) synthesis. These results suggested to us that cPLA(2) is not responsible for the liberation of AA to be converted into PGE(2) by F. tularensis-infected macrophages. Utilizing small-molecule inhibitors, we demonstrated that phospholipase D and diacylglycerol lipase were required for providing AA for PGE(2) biosynthesis. cPLA(2), on the other hand, was required for macrophage cytokine responses to F. tularensis. We also demonstrated for the first time that lipin-1 and PAP2a contribute to macrophage inflammation in response to F. tularensis. Our results identify both an alternative pathway for inducible PGE(2) synthesis and a role for lipid-modifying enzymes in the regulation of macrophage inflammatory function.

Mycobacterium tuberculosis Hip1 modulates macrophage responses through proteolysis of GroEL2.

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.

SARM Regulates CCL5 Production in Macrophages by Promoting the Recruitment of Transcription Factors and RNA Polymerase II to the Ccl5 Promoter

The four Toll/IL-1R domain-containing adaptor proteins MyD88, MAL, TRIF, and TRAM are well established as essential mediators of TLR signaling and gene induction following microbial detection. In contrast, the function of the fifth, most evolutionarily conserved Toll/IL-1R adaptor, sterile α and HEAT/Armadillo motif-containing protein (SARM), has remained more elusive. Recent studies of Sarm(-/-) mice have highlighted a role for SARM in stress-induced neuronal cell death and immune responses in the CNS. However, whether SARM has a role in immune responses in peripheral myeloid immune cells is less clear. Thus, we characterized TLR-induced cytokine responses in SARM-deficient murine macrophages and discovered a requirement for SARM in CCL5 production, whereas gene induction of TNF, IL-1β, CCL2, and CXCL10 were SARM-independent. SARM was not required for TLR-induced activation of MAPKs or of transcription factors implicated in CCL5 induction, namely NF-κB and IFN regulatory factors, nor for Ccl5 mRNA stability or splicing. However, SARM was critical for the recruitment of transcription factors and of RNA polymerase II to the Ccl5 promoter. Strikingly, the requirement of SARM for CCL5 induction was not restricted to TLR pathways, as it was also apparent in cytosolic RNA and DNA responses. Thus, this study identifies a new role for SARM in CCL5 expression in macrophages.

A West Nile virus NS4B P38G mutant induces a differential innate cytokine response in human cell lines. (VAC8P.1005)

Abstract West Nile virus (WNV)-induced neurological disease has become a public health concern. No vaccines have been approved for human use. We have previously shown that an attenuated WNV, the nonstructural(NS)4B-P38G mutant, induced stronger innate cytokine and T cell responses than wild-type WNV. Mice immunized with this mutant were all protected from subsequent lethal wild-type WNV infection. Furthermore, TLR7 and MyD88 expression is required for host protection from NS4B P38G infection. Here, we have examined innate cytokine response in THP-1 cells (human monocytic cells) and THP-1 derived macrophages following with NS4B P38G infection. We have found that NS4B P38G induced stronger type 1 IFN and pro-inflammatory cytokine responses in THP-1 cells than those infected by wild-type WNV. Interestingly, we noted that there was a higher production of pro-inflammatory cytokines, but not IFN-b in NS4B P38G infected THP-1 derived macrophages. Our results also showed an upregulation of TLR4, TLR7 and RIG-I expression in THP-1 cells following NS4B P38G infection, whereas TLR2 expression was significantly increased in mutant infected THP-1 derived macrophages. Similar as our findings in mice, NS4B P38G mutant had a very low replication rate in both types of human cells. Overall, these results suggest that NS4B P38G induced a differential innate cytokine response in human monocytic cells and macrophages, possibly via different pathogen recognition receptor signaling pathways in these cells.

Differential cytokine response by macrophages to ManLAM of Mycobacterium tuberculosis under various culture conditions (INC7P.419)

Abstract Lipoarabinomannan, a lipoglycan is a major virulence factor for Mycobacterium. Mannosylated form of Lipoarabinomannan (ManLAM) is commanly found in pathogenic Mycobacterium tuberculosis. It has been shown that ManLams inhibit production of TNFα and IL-12 by macrophages under in vitro culture conditions. In this study, we tested cytokine responses of macrophages stimulated with ManLAM under various culture conditions. ManLAM was isolated from M. tuberculosis strain H37Rv (ATCC 25618). Macrophages (J744.1; murine macrophage cell line) were stimulated with ManLAM in three different conditions, either alone or in co-cultures with fibroblasts (3T3; ATCC, CRL1658) in contact-culture and non-contact culture systems. Cytokines were measured using commercially available ELISA kits. In comparison to unstimulated controls, TNFα, IL-6 and IFNγ levels increased significantly in co-culture systems after stimulation with ManLAM while a slight increase occurred for IL-12 in the same cultures. However, we observed no change in TNFα, IL-6 and IL-12 for stimulated macrophages when they were cultured alone in the absence of fibroblasts. These results suggested that macrophage activity to ManLAM changed in the presence of the fibroblast in co-culture systems that might be an in vitro model for granuloma formation in tuberculosis infections.

Genetic characteristics and pathogenic mechanisms of periodontal pathogens.

Periodontal disease is caused by a group of bacteria that utilize a variety of strategies and molecular mechanisms to evade or overcome host defenses. Recent research has uncovered new evidence illuminating interesting aspects of the virulence of these bacteria and their genomic variability. This paper summarizes some of the strategies utilized by the major species - Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis - implicated in the pathogenesis of periodontal disease. Whole-genome sequencing of 14 diverse A. actinomycetemcomitans strains has revealed variations in their genetic content (ranging between 0.4% and 19.5%) and organization. Strikingly, isolates from human periodontal sites showed no genomic changes during persistent colonization. T. forsythia manipulates the cytokine responses of macrophages and monocytes through its surface glycosylation. Studies have revealed that bacterial surface-expressed O-linked glycans modulate T-cell responses during periodontal inflammation. Periodontal pathogens belonging to the "red complex" consortium express neuraminidases, which enables them to scavenge sialic acid from host glycoconjugates. Analysis of recent data has demonstrated that the cleaved sialic acid acts as an important nutrient for bacterial growth and a molecule for the decoration of bacteria surfaces to help evade the host immune attack. In addition, bacterial entry into host cells is also an important prerequisite for the lifestyle of periodontal pathogens such as P. gingivalis. Studies have shown that, after its entry into the cell, this bacterium uses multiple sorting pathways destined for autophagy, lysosomes, or recycling pathways. In addition, P. gingivalis releases outer membrane vesicles which enter cells via endocytosis and cause cellular functional impairment.

Human rhinovirus elicits differential cytokine response and signal transduction pathways in human macrophages as a function of viral receptor stimulation (609.2)

Major‐ and minor‐group rhinoviruses enter their host by binding to the cell surface molecules ICAM‐1 and LDL‐R, respectively, which are present on both macrophages and epithelial cells. Although epithelial cells are the primary site of productive HRV infection, previous studies have implicated macrophages in establishing the cytokine dysregulation that occurs during rhinovirus‐induced asthma exacerbations. Upon closer examination in human primary macrophages, differential signaling pathway activation was observed between major‐ and minor‐group rhinovirus upon initial HRV binding, indicating a discordant receptor‐dependent response to these rhinovirus types. Differential phosphorylation of kinases (p38, JNK, ERK5) and transcription factors (ATF‐2, CREB, CEBP‐beta) was observed between the major‐ and minor‐ group HRV treatments. Furthermore, major‐ and minor‐ group HRV elicited differential production of the asthma‐relevant cytokines CCL20, CCL2, and IL‐10. This is the first report of genetically similar viruses eliciting dissimilar cytokine release, transcription factor phosphorylation, and MAPK activation from macrophages. These results suggest that receptor dependence plays a role in establishing the inflammatory microenvironment initiated in part by monocytic‐lineage cells in the human airway upon exposure to rhinovirus.Grant Funding Source: Supported by the McNair Program, NIH R15 AI065505‐01A1,NSF 0521112, Excellence in Science Fund

CD44-deficiency attenuates the immunologic responses to LPS and delays the onset of endotoxic shock-induced renal inflammation and dysfunction.

Acute kidney injury (AKI) is a common complication during systemic inflammatory response syndrome (SIRS), a potentially deadly clinical condition characterized by whole-body inflammatory state and organ dysfunction. CD44 is a ubiquitously expressed cell-surface transmembrane receptor with multiple functions in inflammatory processes, including sterile renal inflammation. The present study aimed to assess the role of CD44 in endotoxic shock-induced kidney inflammation and dysfunction by using CD44 KO and WT mice exposed intraperitoneally to LPS for 2, 4, and 24 hours . Upon LPS administration, CD44 expression in WT kidneys was augmented at all time-points. At 2 and 4 hours, CD44 KO animals showed a preserved renal function in comparison to WT mice. In absence of CD44, the pro-inflammatory cytokine levels in plasma and kidneys were lower, while renal expression of the anti-inflammatory cytokine IL-10 was higher. The cytokine levels were associated with decreased leukocyte influx and endothelial activation in CD44 KO kidneys. Furthermore, in vitro assays demonstrated a role of CD44 in enhancing macrophage cytokine responses to LPS and leukocyte migration. In conclusion, our study demonstrates that lack of CD44 impairs the early pro-inflammatory cytokine response to LPS, diminishes leukocyte migration/chemotaxis and endothelial activation, hence, delays endotoxic shock-induced AKI.

Adenovirus E1B 19-kilodalton protein modulates innate immunity through apoptotic mimicry.

Cells that undergo apoptosis in response to chemical or physical stimuli repress inflammatory reactions, but cells that undergo nonapoptotic death in response to such stimuli lack this activity. Whether cells dying from viral infection exhibit a cell death-type modulatory effect on inflammatory reactions is unknown. We compared the effects on macrophage inflammatory responses of cells dying an apoptotic or a nonapoptotic death as a result of adenoviral infection. The results were exactly opposite to the predictions from the conventional paradigm. Cells dying by apoptosis induced by infection with an adenovirus type 5 (Ad5) E1B 19-kilodalton (E1B 19K) gene deletion mutant did not repress macrophage NF-κB activation or cytokine responses to proinflammatory stimuli, whereas cells dying a nonapoptotic death from infection with E1B 19K-competent, wild-type Ad5 repressed these macrophage inflammatory responses as well as cells undergoing classical apoptosis in response to chemical injury. The immunorepressive, E1B 19K-related cell death activity depended upon direct contact of the virally infected corpses with responder macrophages. Replacement of the viral E1B 19K gene with the mammalian Bcl-2 gene in cis restored the nonapoptotic, immunorepressive cell death activity of virally infected cells. These results define a novel function of the antiapoptotic, adenoviral E1B 19K protein that may limit local host innate immune inflammation during accumulation of virally infected cells at sites of infection and suggest that E1B 19K-deleted, replicating adenoviral vectors might induce greater inflammatory responses to virally infected cells than E1B 19K-positive vectors, because of the net effect of their loss-of-function mutation. We observed that cells dying a nonapoptotic cell death induced by adenovirus infection repressed macrophage proinflammatory responses while cells dying by apoptosis induced by infection with an E1B 19K deletion mutant virus did not repress macrophage proinflammatory responses and enhanced some cytokine responses. Our results define a new function of the antiapoptotic, adenoviral protein E1B 19K, which we have termed "apoptotic mimicry." Our studies suggest the possibility that the presence or absence of this E1B 19K function could alter the immunological outcome of both natural and therapeutic adenoviral infections. For example, emerging, highly immunopathogenic adenovirus serotypes might induce increased host inflammatory responses as a result of altered E1B 19K function or expression. It is also possible that engineered variations in E1B 19K expression/function could be created during adenovirus vector design that would increase the therapeutic efficacy of replicating adenovirus vectors for vaccines or oncolytic viral targeting of neoplastic cells.

Designing anti-inflammatory drugs from parasitic worms: a synthetic small molecule analogue of the Acanthocheilonema viteae product ES-62 prevents development of collagen-induced arthritis.

In spite of increasing evidence that parasitic worms may protect humans from developing allergic and autoimmune diseases and the continuing identification of defined helminth-derived immunomodulatory molecules, to date no new anti-inflammatory drugs have been developed from these organisms. We have approached this matter in a novel manner by synthesizing a library of drug-like small molecules based upon phosphorylcholine, the active moiety of the anti-inflammatory Acanthocheilonema viteae product, ES-62, which as an immunogenic protein is unsuitable for use as a drug. Following preliminary in vitro screening for inhibitory effects on relevant macrophage cytokine responses, a sulfone-containing phosphorylcholine analogue (11a) was selected for testing in an in vivo model of inflammation, collagen-induced arthritis (CIA). Testing revealed that 11a was as effective as ES-62 in protecting DBA/1 mice from developing CIA and mirrored its mechanism of action in downregulating the TLR/IL-1R transducer, MyD88. 11a is thus a novel prototype for anti-inflammatory drug development.

Inflammatory Responses Induced by Lipopolysaccharide Are Amplified in Primary Human Monocytes but Suppressed in Macrophages by Complement Protein C5a

Monocytes and macrophages are important innate immune cells equipped with danger-sensing receptors, including complement and Toll-like receptors. Complement protein C5a, acting via C5aR, is shown in this study to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. Whereas C5a enhanced secretion of LPS-induced IL-6 and TNF from primary human monocytes, C5a inhibited these responses while increasing IL-10 secretion in donor-matched human monocyte-derived macrophages differentiated by GM-CSF or M-CSF. Gαi/c-Raf/MEK/ERK signaling induced by C5a was amplified in macrophages but not in monocytes by LPS. Accordingly, the Gαi inhibitor pertussis toxin and MEK inhibitor U0126 blocked C5a inhibition of LPS-induced IL-6 and TNF production from macrophages. This synergy was independent of IL-10, PI3K, p38, JNK, and the differentiating agent. Furthermore, C5a did not inhibit IL-6 production from macrophages induced by other TLR agonists that are selective for Toll/IL-1R domain-containing adapter inducing IFN-β (polyinosinic-polycytidylic acid) or MyD88 (imiquimod), demonstrating selectivity for C5a regulation of LPS responses. Finally, suppression of proinflammatory cytokines IL-6 and TNF in macrophages did not compromise antimicrobial activity; instead, C5a enhanced clearance of the Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium from macrophages. C5aR is thus a regulatory switch that modulates TLR4 signaling via the Gαi/c-Raf/MEK/ERK signaling axis in human macrophages but not monocytes. The differential effects of C5a are consistent with amplifying monocyte proinflammatory responses to systemic danger signals, but attenuating macrophage cytokine responses (without compromising microbicidal activity), thereby restraining inflammatory responses to localized infections.

157 : Dynamin inhibition interferes with cytokine responses in streptococcus pyogenes-infected human macrophages

Streptococcus pyogenes (GAS) is an important human pathogen, causing a wide range of diseases from mild superficial infections to severe invasive infections. In the present study we have analysed the ability of GAS to activate cytokine responses in human monocyte-derived macrophages. We focused on analysing the role of dynamic-dependent endocytosis and major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in these responses. One of the fundamental mechanisms of immune evasion is the ability of streptococcal virulence factors to inhibit phagocytosis or phagosome formation. Streptolysins have several functions in GAS pathogenesis. They form pores in host cell membranes and are involved in the escape of GAS from the endosome-lysosome pathway. Expression of SLO is higher in GAS isolates from severe invasive clinical cases than in serotype-matched non-invasive infections. We analysed cytokine expression in macrophages after stimulation with live, inactivated, and SLO or SLS defective bacteria. IL-1β, IL-10, and TNF-α cytokines were induced after bacterial stimulation and no differences in cytokine levels between live, inactivated, or mutant bacteria were seen. The inhibition of dynamin-dependent endocytosis with dynasore attenuated the induction of IL-1β, TNF-α, IFN-β, and CXCL-10 mRNAs in macrophages stimulated with GAS. We also showed that pro-IL-1β is expressed and cleaved into mature-IL-1β via inflammasome activation after bacterial stimulation. Interestingly, streptolysin mutants induced higher levels of mature-IL-1β, which might indicate lower pathogenesis of the mutant bacteria and more efficient responses of the innate immune system. Furthermore, we demonstrated the involvement of p38 MAPK and PI3K signalling pathways in GAS induced cytokine responses in human monocyte-derived macrophages. In conclusion, dynamin-dependent endocytosis is obligatory for the induction of the immune responses in macrophages and the bacteria deficient in streptolysin genes promote the activation of inflammasome-mediated inflammatory responses.

Enhancement of chicken macrophage cytokine response toSalmonellaTyphimurium when combined with bacteriophage P22

Enhancement of chicken macrophage cytokine response toSalmonellaTyphimurium when combined with bacteriophage P22

Abstract 189: The Role of Spleen Tyrosine Kinase, Syk, in CD36-Toll Like Receptor Cooperative Signaling in Atherosclerosis

Atherosclerosis is characterized by chronic sterile inflammation of the artery wall in which cells of the monocyte lineage accumulate in response to the deposition of low density lipoprotein (LDL). We previously established that recognition of oxidized LDL (oxLDL) by CD36 triggers assembly of a novel Toll-like receptor heterodimer composed of TLR4 and TLR6. Here we set out to understand the molecular mechanisms of CD36/TLR4/TLR6 activation and establish how it triggers downstream signals that lead to the expression of the pro-inflammatory mediators that have been directly implicated in the deleterious effects of oxLDL and atherosclerosis progression. By confocal microscopy we demonstrate that oxLDL induces CD36, TLR4 and TLR6 co-localization in intracellular compartments, but not on the cell surface of macrophages. Notably, inhibition of oxLDL endocytosis (with Dynasore) or lysosomal maturation (with Bafilomycin A or NH4Cl) blocks CD36-TLR4-TLR6 complex formation and oxLDL-induced cytokine responses in macrophages. These data indicate that both ligand internalization and lysosomal acidification are required for assembly of a functional CD36/TLR4/TLR6 signaling complex. Notably, CD36 contains a hemi-ITIM motif in the C-terminus that is reported to interact with the spleen tyrosine kinase Syk through its SH2 domain. As Syk has recently been implicated in the trafficking of CD14 and TLR4 to the endosome in response to LPS, we investigated the role of this kinase in CD36/TLR4/TLR6 signaling. We find that Syk is required for CD36 internalization and TLR4/TLR6 heterodimerization. Using a pharmacological inhibitor, we show that inhibition of Syk activity blocks oxLDL-induced TLR4-TLR6 co-precipitation and abrogates macrophage expression of both MyD88- (IL-1b, CXCL1) and TRIF-dependent (CCL5) cytokines/chemokines. Together, our data are consistent with a key role for Syk in the trafficking of CD36 and oxLDL to the lysosome, where it coordinates the assembly of a functional TLR4-TLR6 heterodimer to initiate signaling. This model highlights the importance of CD36 as a co-receptor that orchestrates TLR4-TLR6 trafficking and assembly to initiate the detrimental inflammatory responses that promote the progression of atherosclerosis.