The role of RAGE in host pathology and crosstalk between RAGE and TLR4 in innate immune signal transduction pathways.

Although the innate immune receptor protein, Receptor for Advanced Glycation End products (RAGE), has been extensively studied, there has been renewed interest in RAGE for its potential role in sepsis, along with a host of other inflammatory diseases of chronic, noninfectious origin. In contrast to other innate immune receptors, for example, Toll-like receptors (TLRs), that recognize ligands derived from pathogenic organisms that are collectively known as "pathogen-associated molecular patterns" (PAMPs) or host-derived "damage-associated molecular patterns" (DAMPs), RAGE has been shown to recognize a broad collection of DAMPs exclusively. Historically, these DAMPs have been shown to be pro-inflammatory in nature. Early studies indicated that the adaptor molecule, MyD88, might be important for this change. More recent studies have explored further the mechanisms underlying this inflammatory change. Overall, the newer results have shown that there is extensive crosstalk between RAGE and TLRs. The three canonical RAGE ligands, Advanced Glycation End products (AGEs), HMGB1, and S100 proteins, have all been shown to activate both TLRs and RAGE to varying degrees in order to induce inflammation in in vitro models. As with any field that delves deeply into innate signaling, obstacles of reagent purity may be a cause of some of the discrepancies in the literature, and we have found that commercial antibodies that have been widely used exhibit a high degree of nonspecificity. Nonetheless, the weight of published evidence has led us to speculate that RAGE may be physically interacting with TLRs on the cell surface to elicit inflammation via MyD88-dependent signaling.

AMP-activated Kinase (AMPK) Promotes Innate Immunity and Antiviral Defense through Modulation of Stimulator of Interferon Genes (STING) Signaling.

The host protein Stimulator of Interferon Genes (STING) has been shown to be essential for recognition of both viral and intracellular bacterial pathogens, but its regulation remains unclear. Previously, we reported that mitochondrial membrane potential regulates STING-dependent IFN-ß induction independently of ATP synthesis. Because mitochondrial membrane potential controls calcium homeostasis, and AMP-activated protein kinase (AMPK) is regulated, in part, by intracellular calcium, we postulated that AMPK participates in STING activation; however, its role has yet to be been defined. Addition of an intracellular calcium chelator or an AMPK inhibitor to either mouse macrophages or mouse embryonic fibroblasts (MEFs) suppressed IFN-ß and TNF-α induction following stimulation with the STING-dependent ligand 5,6-dimethyl xanthnone-4-acetic acid (DMXAA). These pharmacological findings were corroborated by showing that MEFs lacking AMPK activity also failed to up-regulate IFN-ß and TNF-α after treatment with DMXAA or the natural STING ligand cyclic GMP-AMP (cGAMP). As a result, AMPK-deficient MEFs exhibit impaired control of vesicular stomatitis virus (VSV), a virus sensed by STING that can cause an influenza-like illness in humans. This impairment could be overcome by pretreatment of AMPK-deficient MEFs with type I IFN, illustrating that de novo production of IFN-ß in response to VSV plays a key role in antiviral defense during infection. Loss of AMPK also led to dephosphorylation at Ser-555 of the known STING regulator, UNC-51-like kinase 1 (ULK1). However, ULK1-deficient cells responded normally to DMXAA, indicating that AMPK promotes STING-dependent signaling independent of ULK1 in mouse cells.

The DNA sensor, cyclic GMP-AMP synthase, is essential for induction of IFN-ß during Chlamydia trachomatis infection.

IFN-ß has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-ß expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-ß expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-ß expression. We determined that cGAS is required for IFN-ß expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-ß, coculture of cells depleted for either STING or cGAS rescued IFN-ß expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-ß expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-ß expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis.

CD4+ T cell expression of MyD88 is essential for normal resolution of Chlamydia muridarum genital tract infection.

Resolution of Chlamydia genital tract infection is delayed in the absence of MyD88. In these studies, we first used bone marrow chimeras to demonstrate a requirement for MyD88 expression by hematopoietic cells in the presence of a wild-type epithelium. Using mixed bone marrow chimeras we then determined that MyD88 expression was specifically required in the adaptive immune compartment. Furthermore, adoptive transfer experiments revealed that CD4(+) T cell expression of MyD88 was necessary for normal resolution of genital tract infection. This requirement was associated with a reduced ability of MyD88(-/-)CD4(+) T cells to accumulate in the draining lymph nodes and genital tract when exposed to the same inflammatory milieu as wild-type CD4(+) T cells. We also demonstrated that the impaired infection control we observed in the absence of MyD88 could not be recapitulated by deficiencies in TLR or IL-1R signaling. In vitro, we detected an increased frequency of apoptotic MyD88(-/-)CD4(+) T cells upon activation in the absence of exogenous ligands for receptors upstream of MyD88. These data reveal an intrinsic requirement for MyD88 in CD4(+) T cells during Chlamydia infection and indicate that the importance of MyD88 extends beyond innate immune responses by directly influencing adaptive immunity.

Significant role of IL-1 signaling, but limited role of inflammasome activation, in oviduct pathology during Chlamydia muridarum genital infection.

IL-1ß has been implicated in the development of oviduct pathology during Chlamydia muridarum genital infection in the mouse model. The goal of this study was to characterize the role of IL-1 signaling and the inflammasome-activation pathways during genital chlamydial infection. Compared with control mice, IL-1R-deficient mice displayed delayed clearance and increased chlamydial colonization. Consistent with the role for IL-1 signaling in infection clearance, mice deficient for the IL-1R antagonist cleared infection at a faster rate. Despite increased infection, IL-1R-deficient mice had significantly reduced oviduct pathology, which was associated with decreased numbers of neutrophils, but more macrophages, in the genital tract. IL-1ß secretion is dependent on caspase-1 and apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) inflammasome during in vitro infection of primed macrophages with C. muridarum. To investigate the role of inflammasome components during in vivo genital infection, mice lacking NLRP3, NLRC4, and ASC were tested and found to display no reduction in oviduct pathology compared with control mice. Mice deficient for ASC displayed a prolonged course of infection, which was associated with reduced T cell recruitment and proliferation. Further, ASC-deficient mice displayed normal levels of IL-1ß in genital secretions. However, a significant decrease in caspase-1-dependent IL-18 was observed in both ASC- and NLRP3-deficient mice. These data demonstrate a major role for IL-1 signaling, but a limited role for the inflammasome pathway, in IL-1ß secretion and development of oviduct pathology during genital chlamydial infection. The data also suggest an IL-1-independent role for ASC in adaptive immunity during genital chlamydial infection.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) activates stimulator of interferon gene (STING)-dependent innate immune pathways and is regulated by mitochondrial membrane potential.

The chemotherapeutic agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a potent inducer of type I IFNs and other cytokines. This ability is essential for its chemotherapeutic benefit in a mouse cancer model and suggests that it might also be useful as an antiviral agent. However, the mechanism underlying DMXAA-induced type I IFNs, including the host proteins involved, remains unclear. Recently, it was reported that the antioxidant N-acetylcysteine (NAC) decreased DMXAA-induced TNF-α and IL-6, suggesting that oxidative stress may play a role. The goal of this study was to identify host proteins involved in DMXAA-dependent signaling and determine how antioxidants modulate this response. We found that expression of IFN-ß in response to DMXAA in mouse macrophages requires the mitochondrial and endoplasmic reticulum resident protein STING. Addition of the antioxidant diphenylene iodonium (DPI) diminished DMXAA-induced IFN-ß, but this decrease was independent of both the NADPH oxidase, Nox2, and de novo generation of reactive oxygen species. Additionally, IFN-ß up-regulation by DMXAA was inhibited by agents that target the mitochondrial electron transport chain and, conversely, loss of mitochondrial membrane potential correlated with diminished innate immune signaling in response to DMXAA. Up-regulation of Ifnb1 gene expression mediated by cyclic dinucleotides was also impaired by DPI, whereas up-regulation of Ifnb1 mRNA due to cytosolic double-stranded DNA was not. Although both stimuli signal through STING, cyclic dinucleotides interact directly with STING, suggesting that recognition of DMXAA by STING may also be mediated by direct interaction.

M2a macrophages facilitate resolution of chemically-induced colitis in TLR4-SNP mice.

IMPORTANCE: Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, impacts millions of individuals worldwide and severely impairs the quality of life for patients. Dysregulation of innate immune signaling pathways reduces barrier function and exacerbates disease progression. Macrophage (Mφ) signaling pathways are potential targets for IBD therapies. While multiple treatments are available for IBD, (i) not all patients respond, (ii) responses may diminish over time, and (iii) treatments often have undesirable side effects. Genetic studies have shown that the inheritance of two co-segregating SNPs expressed in the innate immune receptor, TLR4, is associated with human IBD. Mice expressing homologous SNPs ("TLR4-SNP" mice) exhibited more severe colitis than WT mice in a DSS-induced colonic inflammation/repair model. We identified a critical role for M2a "tissue repair" Mφ in the resolution of colitis. Our findings provide insight into potential development of novel therapies targeting Mφ signaling pathways that aim to alleviate the debilitating symptoms experienced by individuals with IBD.

Stimulator of IFN gene is critical for induction of IFN-beta during Chlamydia muridarum infection.

Type I IFN signaling has recently been shown to be detrimental to the host during infection with Chlamydia muridarum in both mouse lung and female genital tract. However, the pattern recognition receptor and the signaling pathways involved in chlamydial-induced IFN-beta are unclear. Previous studies have demonstrated no role for TLR4 and a partial role for MyD88 in chlamydial-induced IFN-beta. In this study, we demonstrate that mouse macrophages lacking TLR3, TRIF, TLR7, or TLR9 individually or both TLR4 and MyD88, still induce IFN-beta equivalent to wild type controls, leading to the hypothesis that TLR-independent cytosolic pathogen receptor pathways are crucial for this response. Silencing nucleotide-binding oligomerization domain 1 in HeLa cells partially decreased chlamydial-induced IFN-beta. Independently, small interfering RNA-mediated knockdown of the stimulator of IFN gene (STING) protein in HeLa cells and mouse oviduct epithelial cells significantly decreased IFN-beta mRNA expression, suggesting a critical role for STING in chlamydial-induced IFN-beta induction. Conversely, silencing of mitochondria-associated antiviral signaling proteins and the Rig-I-like receptors, RIG-I, and melanoma differentiation associated protein 5, had no effect. In addition, induction of IFN-beta depended on the downstream transcription IFN regulatory factor 3, and on activation of NF-kappaB and MAPK p38. Finally, STING, an endoplasmic reticulum-resident protein, was found to localize in close proximity to the chlamydial inclusion membrane during infection. These results indicate that C. muridarum induces IFN-beta via stimulation of nucleotide-binding oligomerization domain 1 pathway, and TLR- and Rig-I-like receptor-independent pathways that require STING, culminating in activation of IFN regulatory factor 3, NF-kappaB, and p38 MAPK.

Interferon regulatory transcription factor 3 protects mice from uterine horn pathology during Chlamydia muridarum genital infection.

Mice with the type I interferon (IFN) receptor gene knocked out (IFNAR KO mice) or deficient for alpha/beta IFN (IFN-α/ß) signaling clear chlamydial infection earlier than control mice and develop less oviduct pathology. Initiation of host IFN-ß transcription during an in vitro chlamydial infection requires interferon regulatory transcription factor 3 (IRF3). The goal of the present study was to characterize the influence of IRF3 on chlamydial genital infection and its relationship to IFN-ß expression in the mouse model. IRF3 KO mice were able to resolve infection as well as control mice, overcoming increased chlamydial colonization and tissue burden early during infection. As previously observed for IFNAR KO mice, IRF3 KO mice generated a potent antigen-specific T cell response. However, in contrast to IFNAR KO mice, IRF3 KO mice exhibited unusually severe dilatation and pathology in the uterine horns but normal oviduct pathology after infection. Although IFN-ß expression in vivo was dependent on the presence of IRF3 early in infection (before day 4), the IFN-independent function of IRF3 was likely driving this phenotype. Specifically, early during infection, the number of apoptotic cells and the number of inflammatory cells were significantly less in uterine horns from IRF3 KO mice than in those from control mice, despite an increased chlamydial burden. To delineate the effects of IFN-ß versus IRF3, neutralizing IFN-ß antibody was administered to wild-type (WT) mice during chlamydial infection. IFN-ß depletion in WT mice mimicked that in IFNΑR KO mice but not that in IRF3 KO mice with respect to both chlamydial clearance and reduced oviduct pathology. These data suggest that IRF3 has a role in protection from uterine horn pathology that is independent of its function in IFN-ß expression.

MyD88 deficiency leads to decreased NK cell gamma interferon production and T cell recruitment during Chlamydia muridarum genital tract infection, but a predominant Th1 response and enhanced monocytic inflammation are associated with infection resolution.

We have previously shown that MyD88 knockout (KO) mice exhibit delayed clearance of Chlamydia muridarum genital infection compared to wild-type (WT) mice. A blunted Th1 response and ineffective suppression of the Th2 response were also observed in MyD88 KO mice. The goal of the present study was to investigate specific mechanisms whereby absence of MyD88 leads to these effects and address the compensatory mechanisms in the genital tract that ultimately clear infection in the absence of MyD88. It was observed that NK cells recruited to the genital tract in MyD88 KO mice failed to produce gamma interferon (IFN-γ) mRNA and protein. This defect was associated with decreased local production of interleukin-17 (IL-17), IL-18, and tumor necrosis factor alpha (TNF-α) but normal levels of IL-12p70. Additionally, recruitment of CD4 T cells to the genital tract was reduced in MyD88 KO mice compared to that in WT mice. Although chronic infection in MyD88 KO mice resulted in oviduct pathology comparable to that of WT mice, increased histiocytic inflammation was observed in the uterine horns. This was associated with increased CCL2 levels and recruitment of macrophages as a potential compensatory mechanism. Further deletion of TLR4-TRIF signaling in MyD88 KO mice, using TLR4/MyD88 double-KO mice, did not further compromise host defense against chlamydiae, suggesting that compensatory mechanisms are Toll-like receptor (TLR) independent. Despite some polarization toward a Th2 response, a Th1 response remained predominant in the absence of MyD88, and it provided equivalent protection against a secondary infection as observed in WT mice.

Classically activated mouse macrophages produce methylglyoxal that induces a TLR4- and RAGE-independent proinflammatory response.

The highly reactive compound methylglyoxal (MG) can cause direct damage to cells and tissues by reacting with cellular macromolecules. MG has been identified as a biomarker associated with increased sepsis-induced mortality. Patients undergoing septic shock have significantly elevated circulating MG levels compared to postoperative patients and healthy controls. Furthermore, MG has been implicated in the development of type II diabetes mellitus and Alzheimer's disease. Because MG is generated during glycolysis, we hypothesized that MG may be produced by classically activated (M1) macrophages, possibly contributing to the inflammatory response. LPS and IFN-γ-treated macrophages acquired an M1 phenotype (as evidenced by M1 markers and enhanced glycolysis) and formed MG adducts, MG-H1, MG-H2, and MG-H3, which were detected using antibodies specific for MG-modified proteins (methylglyoxal 5-hydro-5-methylimidazolones). MG adducts were also increased in the lungs of LPS-treated mice. Macrophages treated with LPS and IFN-γ also exhibited decreased expression of glyoxalase 1 (Glo1), an enzyme that metabolizes MG. Concentrations of exogenous, purified MG > 0.5 mM were toxic to macrophages; however, a nontoxic dose of 0.3 mM induced TNF-α and IL-1ß, albeit to a lesser extent than LPS stimulation. Despite prior evidence that MG adducts may signal through "receptor for advanced glycation endproducts" (RAGE), MG-mediated cell death and cytokine induction by exogenous MG was RAGE-independent in primary macrophages. Finally, RAGE-deficient mice did not exhibit a significant survival advantage following lethal LPS injection. Overall, our evidence suggests that MG may be produced by M1 macrophages during sepsis, following IFN-γ-dependent down-regulation of Glo1, contributing to over-exuberant inflammation.

A mouse model of human TLR4 D299G/T399I SNPs reveals mechanisms of altered LPS and pathogen responses.

Two cosegregating single-nucleotide polymorphisms (SNPs) in human TLR4, an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I), have been associated with LPS hyporesponsiveness and differential susceptibility to many infectious or inflammatory diseases. However, many studies failed to confirm these associations, and transfection experiments resulted in conflicting conclusions about the impact of these SNPs on TLR4 signaling. Using advanced protein modeling from crystallographic data of human and murine TLR4, we identified homologous substitutions of these SNPs in murine Tlr4, engineered a knock-in strain expressing the D298G and N397I TLR4 SNPs homozygously, and characterized in vivo and in vitro responses to TLR4 ligands and infections in which TLR4 is implicated. Our data provide new insights into cellular and molecular mechanisms by which these SNPs decrease the TLR4 signaling efficiency and offer an experimental approach to confirm or refute human data possibly confounded by variables unrelated to the direct effects of the SNPs on TLR4 functionality.

Role for the chlamydial type III secretion apparatus in host cytokine expression.

In many important human pathogens, such as Shigella and Salmonella spp., the bacterial type III secretion (T3S) apparatus is required to initiate inflammation via activation of caspase-1- or NF-kappaB-dependent genes. Using an ex vivo infection model, the goal of the present study was to determine whether the chlamydial T3S apparatus also modulates the host inflammatory response. Infections of mouse peritoneal macrophages were performed with Chlamydia muridarum, and the expression of inflammatory cytokines was monitored by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. Since there is no current genetic system for Chlamydia spp., blockade of T3S was accomplished pharmacologically using a T3S inhibitor called INP0007. It has been previously shown that INP0007 also blocks chlamydial growth in vitro and that the addition of exogenous iron completely reverses this deficit. The addition of iron to INP0007-treated C. muridarum-infected macrophages not only restored chlamydial growth deficit caused by INP0007 but also led to a multi-inclusion phenotype. Overall, T3S inhibition led to decreased interleukin-6 (IL-6), IL-1beta, and CXCL10, whereas the tumor necrosis factor alpha levels were unchanged. Rescue of chlamydial growth by addition of iron sulfate did not restore cytokine production, implying that the decreased expression of many cytokines during infection was dependent on T3S and not solely on growth. In addition, the observation that the greatest effects of INP0007 were seen at late time points during infection suggests that a temporally regulated T3S effector protein(s) may be triggering the host cytokine response.

Critical role for interleukin-1beta (IL-1beta) during Chlamydia muridarum genital infection and bacterial replication-independent secretion of IL-1beta in mouse macrophages.

Recent findings have implicated interleukin-1beta (IL-1beta) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1beta is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1beta expression during a chlamydial genital infection. In the present study, IL-1beta(-/-) mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1beta both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1beta secretion in WT mice, the major producers of IL-1beta in vivo are F4/80(+) macrophages and GR-1(+) neutrophils, but not CD45(-) epithelial cells. Although elicited mouse macrophages infected with Chlamydia muridarum in vitro secrete minimal IL-1beta, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichia coli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1beta from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1beta secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1beta secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1beta secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1beta mRNA observed in vivo in this cell type.

Type I interferon signaling exacerbates Chlamydia muridarum genital infection in a murine model.

Type I interferons (IFNs) induced during in vitro chlamydial infection exert bactericidal and immunomodulatory functions. To determine the precise role of type I IFNs during in vivo chlamydial genital infection, we examined the course and outcome of Chlamydia muridarum genital infection in mice genetically deficient in the receptor for type I IFNs (IFNAR(-/-) mice). A significant reduction in chlamydial shedding and duration of lower genital tract infection was observed in IFNAR(-/-) mice in comparison to the level of chlamydial shedding and duration of infection in wild-type (WT) mice. Furthermore, IFNAR(-/-) mice developed less chronic oviduct pathology in comparison to that in WT mice. Compared to the WT, IFNAR(-/-) mice had a greater number of chlamydial-specific T cells in their iliac lymph nodes 21 days postinfection. IFNAR(-/-) mice also exhibited earlier and enhanced CD4 T-cell recruitment to the cervical tissues, which was associated with increased expression of CXCL9 in the genital secretions of IFNAR(-/-) mice, but not with expression of CXCL10, which was reduced in the genital secretions of IFNAR(-/-) mice. These data suggest that type I IFNs exacerbate C. muridarum genital infection through an inhibition of the chlamydial-specific CD4 T-cell response.

The θ-defensin retrocyclin 101 inhibits TLR4- and TLR2-dependent signaling and protects mice against influenza infection.

Despite widespread use of annual influenza vaccines, seasonal influenza-associated deaths number in the thousands each year, in part because of exacerbating bacterial superinfections. Therefore, discovering additional therapeutic options would be a valuable aid to public health. Recently, TLR4 inhibition has emerged as a possible mechanism for protection against influenza-associated lethality and acute lung injury. Based on recent data showing that rhesus macaque θ-defensins could inhibit TLR4-dependent gene expression, we tested the hypothesis that a novel θ-defensin, retrocyclin (RC)-101, could disrupt TLR4-dependent signaling and protect against viral infection. In this study, RC-101, a variant of the humanized θ-defensin RC-1, blocked TLR4-mediated gene expression in mouse and human macrophages in response to LPS, targeting both MyD88- and TRIF-dependent pathways. In a cell-free assay, RC-101 neutralized the biologic activity of LPS at doses ranging from 0.5 to 50 EU/ml, consistent with data showing that RC-101 binds biotinylated LPS. The action of RC-101 was not limited to the TLR4 pathway because RC-101 treatment of macrophages also inhibited gene expression in response to a TLR2 agonist, Pam3CSK4, but failed to bind that biotinylated agonist. Mouse macrophages infected in vitro with mouse-adapted A/PR/8/34 influenza A virus (PR8) also produced lower levels of proinflammatory cytokine gene products in a TLR4-independent fashion when treated with RC-101. Finally, RC-101 decreased both the lethality and clinical severity associated with PR8 infection in mice. Cumulatively, our data demonstrate that RC-101 exhibits therapeutic potential for the mitigation of influenza-related morbidity and mortality, potentially acting through TLR-dependent and TLR-independent mechanisms.