Porphyromonas gingivalis induction of TLR2 association with Vinculin enables PI3K activation and immune evasion.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that thrives in the inflamed environment of the gingival crevice, and is strongly associated with periodontal disease. The host response to P. gingivalis requires TLR2, however P. gingivalis benefits from TLR2-driven signaling via activation of PI3K. We studied TLR2 protein-protein interactions induced in response to P. gingivalis, and identified an interaction between TLR2 and the cytoskeletal protein vinculin (VCL), confirmed using a split-ubiquitin system. Computational modeling predicted critical TLR2 residues governing the physical association with VCL, and mutagenesis of interface residues W684 and F719, abrogated the TLR2-VCL interaction. In macrophages, VCL knock-down led to increased cytokine production, and enhanced PI3K signaling in response to P. gingivalis infection, effects that correlated with increased intracellular bacterial survival. Mechanistically, VCL suppressed TLR2 activation of PI3K by associating with its substrate PIP2. P. gingivalis induction of TLR2-VCL led to PIP2 release from VCL, enabling PI3K activation via TLR2. These results highlight the complexity of TLR signaling, and the importance of discovering protein-protein interactions that contribute to the outcome of infection.

Oral bacteria accelerate pancreatic cancer development in mice.

OBJECTIVE: Epidemiological studies highlight an association between pancreatic ductal adenocarcinoma (PDAC) and oral carriage of the anaerobic bacterium Porphyromonas gingivalis, a species highly linked to periodontal disease. We analysed the potential for P. gingivalis to promote pancreatic cancer development in an animal model and probed underlying mechanisms. DESIGN: We tracked P. gingivalis bacterial translocation from the oral cavity to the pancreas following administration to mice. To dissect the role of P. gingivalis in PDAC development, we administered bacteria to a genetically engineered mouse PDAC model consisting of inducible acinar cell expression of mutant Kras (Kras +/LSL-G12D; Ptf1a-CreER, iKC mice). These mice were used to study the cooperative effects of Kras mutation and P. gingivalis on the progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC. The direct effects of P. gingivalis on acinar cells and PDAC cell lines were studied in vitro. RESULTS: P. gingivalis migrated from the oral cavity to the pancreas in mice and can be detected in human PanIN lesions. Repetitive P. gingivalis administration to wild-type mice induced pancreatic acinar-to-ductal metaplasia (ADM), and altered the composition of the intrapancreatic microbiome. In iKC mice, P. gingivalis accelerated PanIN to PDAC progression. In vitro, P. gingivalis infection induced acinar cell ADM markers SOX9 and CK19, and intracellular bacteria protected PDAC cells from reactive oxygen species-mediated cell death resulting from nutrient stress. CONCLUSION: Taken together, our findings demonstrate a causal role for P. gingivalis in pancreatic cancer development in mice.

TNFα expression by Porphyromonas gingivalis-stimulated macrophages relies on Sirt1 cleavage.

OBJECTIVE: Periodontitis is one the most common chronic inflammatory conditions, resulting in destruction of tooth-supporting tissues and leading to tooth loss. Porphyromonas gingivalis activates host macrophages to secrete pro-inflammatory cytokines and elicit tissue damage, in part by inducing NF-kappa-B transactivation. Since NFκB transactivation is negatively regulated by the Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase enzyme Sirt1, we sought to assess if RAW264.7 macrophages exposed to P. gingivalis demonstrate impaired Sirt1 activity, to ultimately induce a pro-inflammatory response. METHODS: RAW264.7 macrophages were incubated with heat- killed P. gingivalis for 2, 4, 8, and 24 h. Stimulated RAW264.7 were assessed for TNFα expression via PCR, ELISA, and ChIP analysis. Following the activation of RAW264.7 macrophages, immunoblot analysis was executed to detect modifications in Sirt1 and the NFκB subunit RelA that is essential for NFκB transcriptional activity. RESULTS: TNFα expression was elevated 4 h after exposure to P. gingivalis. ChIP confirmed that RelA was enriched in the mouse TNFα promoter 4 h following stimulation, which correlated with the increased TNFα mRNA levels. Preceding TNFα expression, we detected Phosphoserine 536 and acetylated lysine 310 of RelA after 2 hours exposure with P. gingivalis. Moreover, reduced Sirt1 activity was associated with its cleavage in RAW264.7 protein extracts, after 2 hours of P. gingivalis exposure. Blocking TLR2/4 signaling prevented Sirt1 cleavage, loss of deacetylase activity, and TNFα secretion, while co-administering CA074Me (a cathepsin B inhibitor) with P. gingivalis reduced RelA promoter enrichment, resulting in impaired TNFα expression. CONCLUSIONS: Together, the results suggest that P. gingivalis induces TNFα expression, at least in part, by enhancing cleavage of Sirt1 via a TLR-dependent signaling circuit.

GAS6 is a key homeostatic immunological regulator of host-commensal interactions in the oral mucosa.

The oral epithelium contributes to innate immunity and oral mucosal homeostasis, which is critical for preventing local inflammation and the associated adverse systemic conditions. Nevertheless, the mechanisms by which the oral epithelium maintains homeostasis are poorly understood. Here, we studied the role of growth arrest specific 6 (GAS6), a ligand of the TYRO3-AXL-MERTK (TAM) receptor family, in regulating oral mucosal homeostasis. Expression of GAS6 was restricted to the outer layers of the oral epithelium. In contrast to protein S, the other TAM ligand, which was constitutively expressed postnatally, expression of GAS6 initiated only 3-4 wk after birth. Further analysis revealed that GAS6 expression was induced by the oral microbiota in a myeloid differentiation primary response gene 88 (MyD88)-dependent fashion. Mice lacking GAS6 presented higher levels of inflammatory cytokines, elevated frequencies of neutrophils, and up-regulated activity of enzymes, generating reactive nitrogen species. We also found an imbalance in Th17/Treg ratio known to control tissue homeostasis, as Gas6-deficient dendritic cells preferentially secreted IL-6 and induced Th17 cells. As a result of this immunological shift, a significant microbial dysbiosis was observed in Gas6-/- mice, because anaerobic bacteria largely expanded by using inflammatory byproducts for anaerobic respiration. Using chimeric mice, we found a critical role for GAS6 in epithelial cells in maintaining oral homeostasis, whereas its absence in hematopoietic cells synergized the level of dysbiosis. We thus propose GAS6 as a key immunological regulator of host-commensal interactions in the oral epithelium.

Epithelial cells detect functional type III secretion system of enteropathogenic Escherichia coli through a novel NF-κB signaling pathway.

Enteropathogenic Escherichia coli (EPEC), a common cause of infant diarrhea, is associated with high risk of mortality in developing countries. The primary niche of infecting EPEC is the apical surface of intestinal epithelial cells. EPEC employs a type three secretion system (TTSS) to inject the host cells with dozens of effector proteins, which facilitate attachment to these cells and successful colonization. Here we show that EPEC elicit strong NF-κB activation in infected host cells. Furthermore, the data indicate that active, pore-forming TTSS per se is necessary and sufficient for this NF-κB activation, regardless of any specific effector or protein translocation. Importantly, upon infection with wild type EPEC this NF-κB activation is antagonized by anti-NF-κB effectors, including NleB, NleC and NleE. Accordingly, this NF-κB activation is evident only in cells infected with EPEC mutants deleted of nleB, nleC, and nleE. The TTSS-dependent NF-κB activation involves a unique pathway, which is independent of TLRs and Nod1/2 and converges with other pathways at the level of TAK1 activation. Taken together, our results imply that epithelial cells have the capacity to sense the EPEC TTSS and activate NF-κB in response. Notably, EPEC antagonizes this capacity by delivering anti-NF-κB effectors into the infected cells.

Myristoylation Confers Oral Bioavailability and Improves the Bioactivity of c(MyD 4-4), a Cyclic Peptide Inhibitor of MyD88.

Myeloid differentiation primary response 88 (MyD88) is an intracellular adaptor protein central to the signaling of multiple receptors involved in inflammation. Since innate immune inflammation promotes autoimmunity, MyD88 is an attractive target in autoimmune disease. We previously developed c(MyD 4-4), a novel cyclic peptide competitive inhibitor of MyD88 dimerization that is metabolically stable. Parenteral administration of c(MyD 4-4) reduces disease severity in a mouse model of the human autoimmune disease multiple sclerosis. We now show that N-terminal myristoylation of c(MyD 4-4) enhances the competitive inhibition of MyD88 dimerization in living cells, leading to improved inhibition of the Toll-like receptor and IL-1 receptor signaling. Importantly, myristoylation converts c(MyD 4-4) to an orally bioavailable inhibitor of MyD88. Oral administration of c(MyD 4-4) significantly lowered the inflammatory cytokines secreted by peripheral autoimmune T cells in mice immunized with myelin antigens and ameliorated disease severity in the mouse model of multiple sclerosis. Taken together, we show the conversion of a protein active region to a metabolically stable, selective cyclic peptide that is orally bioavailable.

Myd88 plays a major role in the keratinocyte response to infection with Porphyromonas gingivalis.

AIM: To explore the role of keratinocyte myeloid differentiation primary response 88 (MyD88) expression in the adhesion of Porphyromonas gingivalis to the cells and its subsequent invasion and intracellular survival. MATERIALS AND METHODS: Primary mouse keratinocytes from wild-type (WT) or Myd88-/- mice were infected with P gingivalis alone or co-infected with Fusobacterium nucleatum. Bacterial adhesion and invasion were measured using fluorescent microscopy and flow cytometry, and intracellular survival in keratinocytes was quantified by an antibiotic protection assay. Keratinocyte expression of antimicrobial peptides was measured by real-time PCR. RESULTS: In the absence of MyD88, P gingivalis adherence, invasion, and intracellular survival were enhanced compared with WT keratinocytes. The presence of F nucleatum during infection increased the adhesion of P gingivalis to WT keratinocytes but reduced the adhesion to Myd88-/- keratinocytes. Fusobacterium nucleatum improved mildly the invasion and survival of P gingivalis in both cell types. Baseline expression of beta-defensin 2, 3, 4 and RegIII-γ was elevated in Myd88-/- keratinocytes compared to WT cells; however, following infection beta-defensin expression was strongly induced in WT cells but decreased dramatically in the MyD88 deficient cells. CONCLUSION: In the absence of MyD88 expression, P gingivalis adhesion to keratinocytes is improved, and invasion and intracellular survival are increased. Furthermore, keratinocyte infection by P gingivalis induces antimicrobial peptide expression in a MyD88-dependent manner. Thus, MyD88 plays a key role in the interaction between P gingivalis and keratinocytes.

Oral infection with Porphyromonas gingivalis induces peri-implantitis in a murine model: Evaluation of bone loss and the local inflammatory response.

AIM: Peri-implantitis is a major health concern, with unclear pathogenesis, and with no accessible animal models. Our aim was to establish a mouse model for peri-implantitis and to investigate mediators of inflammation. MATERIALS AND METHODS: Mice were divided into implanted versus non-implanted groups. Implants were inserted immediately following the extraction of the upper first molar. Four weeks following implantation, implanted and non-implanted mice were challenged with either Porphyromonas gingivalis or vehicle (eight mice in each subgroup, 32 mice in total). Alveolar bone loss and expression of inflammatory mediators in the soft tissue were assessed 42 days following infection. RESULTS: Porphyromonas gingivalis infection induced greater bone loss around implants than around teeth. In non-infected animals, the presence of the implant correlated with elevated expression of Il-10, Foxp3 and Rankl/Opg ratio, while Tnf-α levels were decreased relative to tissue around teeth. Six weeks following infection, Tnf-α increased significantly while the expression of Foxp3 decreased in the tissue around the implants. No significant differences in anti- or pro-inflammatory mediators were found around teeth of infected, relative to non-infected mice. CONCLUSIONS: Oral infection with P. gingivalis of mice with implants induced bone loss and a shift in gingival cytokine expression. This mouse model enables exploration of the pathogenesis of peri-implantitis and testing of novel treatments.

CX3CR1hi Monocyte/Macrophages Support Bacterial Survival and Experimental Infection-Driven Bone Resorption.

Porphyromonas gingivalis,an anaerobic bacterium strongly linked to infection-driven inflammatory bone erosion, thrives within a highly inflamed milieu and disseminates to distant sites, such as atherosclerotic plaque. We examined the role of monocyte/macrophages in determining the outcome of infection with P. gingivalis. Surprisingly, transient monocyte/macrophage depletion led to greatly improved clearance of P. gingivalis. The chemokine receptors CCR2 and CX3CR1 play a major role in monocyte recruitment and differentiation to Ly6C(hi) vs CX3CR1(hi) subsets, respectively. To determine the contribution of particular monocyte/macrophage subsets to bacterial survival, we challenged chemokine receptor knockout mice and found that P. gingivalis clearance is significantly improved in the absence of CX3CR1. CX3CR1(hi) monocyte/macrophages promote P. gingivalis survival by downregulating neutrophil phagocytosis. Furthermore, CX3CR1 knockout mice resist bone resorption in the oral cavity following challenge with P. gingivalis Our findings provide an explanation for bacterial coexistence alongside an activate neutrophil infiltrate.

Macrophage activation by heparanase is mediated by TLR-2 and TLR-4 and associates with plaque progression.

OBJECTIVE: Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. METHODS AND RESULTS: Highly purified heparanase was added to mouse peritoneal macrophages and macrophage-like J774 cells, and the levels of tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll-like receptor-2 and Toll-like receptor-4 knockout mice were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction, stable angina, and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with stable angina or acute myocardial infarction. Addition or overexpression of heparanase variants resulted in marked increase in tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 levels. Mouse peritoneal macrophages harvested from Toll-like receptor-2 or Toll-like receptor-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute myocardial infarction, compared with patients with stable angina and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared with specimens of stable plaque and controls. CONCLUSIONS: Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression toward vulnerability.

Cells exposed to sublethal oxidative stress selectively attract monocytes/macrophages via scavenger receptors and MyD88-mediated signaling.

The innate immune system responds to endogenous molecules released during cellular stress or those that have undergone modifications normally absent in healthy tissue. These structures are detected by pattern-recognition receptors, alerting the immune system to "danger." In this study, we looked for early signals that direct immune cells to cells undergoing stress before irreversible damage takes place. To avoid detecting signals emanating from apoptotic or necrotic cells we exposed fibroblasts to sublethal oxidative stress. Our results indicate that both nonenzymatic chemical reactions and aldehyde dehydrogenase-2-mediated enzymatic activity released signals from fibroblasts that selectively attracted CD14(+) monocytes but not T, NK, and NKT cells or granulocytes. Splenocytes from MyD88(-/-) mice did not migrate, and treatment with an inhibitory peptide that blocks MyD88 dimerization abrogated human monocyte migration. Monocyte migration was accompanied by downmodulation of CD14 expression and by the phosphorylation of IL-1R-associated kinase 1, a well-known MyD88-dependent signaling molecule. The scavenger receptor inhibitors, dextran sulfate and fucoidan, attenuated monocyte migration toward stressed cells and IL-1R-associated kinase 1 phosphorylation. Surprisingly, although monocyte migration was MyD88 dependent, it was not accompanied by inflammatory cytokine secretion. Taken together, these results establish a novel link between scavenger receptors and MyD88 that together function as sensors of oxidation-associated molecular patterns and induce monocyte motility. Furthermore, the data indicate that MyD88 independently regulates monocyte activation and motility.

IL-10 mediates resistance to adoptive transfer experimental autoimmune encephalomyelitis in MyD88(-/-) mice.

MyD88 is an adaptor molecule that functions in the innate signaling induced by proinflammatory adjuvants that interact with TLRs. Mice lacking MyD88, for example, resist active experimental autoimmune encephalomyelitis (EAE) induced by immunization with an encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide in CFA. We reasoned that MyD88(-/-) mice, nevertheless, should be susceptible to EAE mediated by adoptive transfer of activated encephalitogenic T cell lines, which do not require adjuvant signaling for their effector functions. We now report, however, that mice lacking MyD88 also resist adoptive EAE mediated by an anti-MOG T cell line that is strongly encephalitogenic in wild-type (WT) mice. The transferred anti-MOG T cells proliferated, secreted INF-gamma, and migrated to the CNS in the MyD88(-/-) mice, as they did in WT mice, but inflammatory infiltrates did not progress and clinical EAE did not develop. The resistance of the MyD88(-/-) mice to adoptive EAE mediated by the otherwise encephalitogenic T cells was found to result from the secretion of IL-10 by recipient T cells of two different specificities: those specific for MOG and those responding to the T cell clone itself-both anticlonotypic and antiergotypic T regulators were detected. IL-10-producing anti-MOG T cells isolated from immunized MyD88(-/-) mice suppressed the induction of active EAE in WT recipients. Moreover, the absence of IL-10 production in MyD88/IL-10 double-knockout mice rendered the mice susceptible to adoptive transfer of EAE. Thus, MyD88 signaling appears to be a key factor in determining the cytokine phenotype of T cells involved in autoimmune inflammation and regulation.

TLR2-dependent inflammatory response to Porphyromonas gingivalis is MyD88 independent, whereas MyD88 is required to clear infection.

Porphyromonas gingivalis is a gram-negative anaerobe considered to be a major periodontal pathogen. TLR2 plays a central role in the response to P. gingivalis infection in vivo. In its absence there is a weak inflammatory response; however, bacteria are cleared rapidly compared with wild-type mice. We examined the role of the TLR adaptor proteins MyD88 and TLR/IL-1R-domain-containing adaptor-inducing IFN-beta in the inflammatory response to P. gingivalis in vivo and in the ability to clear the bacterial infection. Proinflammatory cytokine production in response to P. gingivalis infection depends on TLR2, but it does not require MyD88 or TLR/IL-1R-domain-containing adaptor-inducing IFN-beta. In contrast, the generation of intracellular toxic oxygen species and the ultimate clearance of P. gingivalis infection depend critically on MyD88, independent of TLR2. Thus, robust cytokine production and bacterial clearance are independent events mediated by distinct signaling pathways following infection with P. gingivalis.

How has neutrophil research improved our understanding of periodontal pathogenesis?

BACKGROUND: Neutrophils are the predominant cells responsible for host defence against bacterial infection. Loss of neutrophil defence, due either to deficient number or function, strongly predisposes to bacterial infections such as periodontitis. Yet, the neutrophil oxidative and proteolytic arsenal has also been implicated in perpetrating periodontal tissue damage in periodontitis. AIM: In this review, we focus on recent developments that shed light on these two aspects of neutrophil function in periodontitis. METHODS: Primary search: using PubMed search for "neutophil", "periodontal", and "periodontitis". Secondary search: using references from the articles found in the first stage. RESULTS: Early histological studies showed that infiltrating neutrophils form a wall of cells abutting the junctional epithelium in periodontal inflammatory lesions. The chronic standoff between these neutrophils and the bacterial community suggests that bacterial evasion of neutrophil clearance is a major characteristic of periodontitis. Indeed, not all functional neutrophil deficiencies increase the risk of periodontitis, an observation that points the way towards identification of particular anti-bacterial pathways essential for protection against periodontal pathogens. The net result in the majority of periodontitis patients who exhibit normal neutrophil number and function, is that neutrophils accumulate in the periodontal tissue where they are available to participate in tissue destruction. Diminished neutrophil clearance further contributes to the persistence of activated neutrophils in the periodontal tissue. CONCLUSIONS: Data on the role of neutrophils in the pathogenesis of periodontitis are mixed. Neutrophils are a critical arm of the defence against periodontitis, but bacterial evasion of the neutrophil microbicidal machinery coupled with delayed neutrophil apoptosis may transform the neutrophil from defender to perpetrator. At this stage of knowledge, attempts to induce host modulation through neutrophil suppression or activation are premature.

The cationic liposome CCS/C adjuvant induces immunity to influenza independently of the adaptor protein MyD88.

Traditional non-living vaccines are often least effective in the populations that need them most, such as neonates and elderly adults. Vaccine adjuvants are one approach to boost the immunogenicity of antigens in populations with reduced immunity. Ideally, vaccine adjuvants will increase the seroconversion rates across the population, lead to stronger immune responses, and enable the administration of fewer vaccine doses. We previously demonstrated that a cationic liposomal formulation of the commercial influenza split virus vaccine (CCS/C-HA) enhanced cellular and humoral immunity to the virus, increased seroconversion rates, and improved survival after live virus challenge in a preclinical model, as compared to the commercial vaccine as is (F-HA). We now evaluated vaccine efficacy in different strains and sexes of mice and determined the role of innate immunity in the mechanism of action of the CCS/C adjuvant by testing the response of mice deficient in Toll-like receptors or the TLR/IL-1 adaptor protein MyD88 following immunization with CCS/C-HA vs. F-HA. Although TLR2- and TLR4-deficient mice responded to F-HA immunization, F-HA immunization failed to engender a significant immune response in the absence of MyD88. In contrast, immunization with the CCS/C-HA vaccine overcame the requirement for MyD88 in the response to the commercial vaccine and improved the immune responses and seroconversion rates in all strains of mice tested, including those deficient in TLR2 and TLR4.

Intracellular Porphyromonas gingivalis Promotes the Tumorigenic Behavior of Pancreatic Carcinoma Cells.

Porphyromonas gingivalis is a member of the dysbiotic oral microbiome associated with oral inflammation and periodontal disease. Intriguingly, epidemiological studies link P. gingivalis to an increased risk of pancreatic cancer. Given that oral bacteria are detected in human pancreatic cancer, and both mouse and human pancreata harbor microbiota, we explored the involvement of P. gingivalis in pancreatic tumorigenesis using cell lines and a xenograft model. Live P. gingivalis induced proliferation of pancreatic cancer cells; however, surprisingly, this effect was independent of Toll-like receptor 2, the innate immune receptor that is engaged in response to P. gingivalis on other cancer and immune cells, and is required for P. gingivalis to induce alveolar bone resorption. Instead, we found that P. gingivalis survives inside pancreatic cancer cells, a trait that can be enhanced in vitro and is increased by hypoxia, a central characteristic of pancreatic cancer. Increased tumor cell proliferation was related to the degree of intracellular persistence, and infection of tumor cells with P. gingivalis led to enhanced growth in vivo. To the best of our knowledge, this study is the first to demonstrate the direct effect of exposure to P. gingivalis on the tumorigenic behavior of pancreatic cancer cell lines. Our findings shed light on potential mechanisms underlying the pancreatic cancer-periodontitis link.

Involvement of Toll-like receptors 2 and 4 in the innate immune response to Treponema denticola and its outer sheath components.

Treponema denticola is considered an important oral pathogen in the development and progression of periodontal diseases. In the present study, the mechanisms of recognition and activation of murine macrophages by T. denticola and its major outer sheath protein (MSP) and lipooligosaccharide (LOS or glycolipid) were investigated. T. denticola cells and the MSP induced innate immune responses through TLR2-MyD88, whereas LOS induced a macrophage response through TLR4-MyD88. The presence of gamma interferon (IFN-gamma), or of high numbers of T. denticola, circumvented the requirement for TLR2 for the macrophage response to T. denticola, although the response was still dependent on MyD88. In contrast, synergy with IFN-gamma did not alter the TLR dependence of the response to the T. denticola surface components LOS and MSP, despite enhanced sensitivity. These data suggest that although there is flexibility in the requirements for recognition of T. denticola cells (TLR2 dependent or independent), MyD88 is a requirement for the downstream signaling events that lead to inflammation. We also demonstrate that both outer sheath molecules LOS and MSP induce macrophage tolerance to further stimulation with enterobacterial lipopolysaccharide. Tolerance induced by T. denticola components during mixed infections may represent a general mechanism through which bacteria evade clearance.

Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88.

MyD88 is a cytoplasmic adaptor protein that plays a central role in signaling downstream of the TLRs and the IL1R superfamily. We previously demonstrated that MyD88 plays a critical role in EAE, the murine model of multiple sclerosis, and showed that the MyD88 BB-loop decoy peptide RDVLPGT ameliorates EAE. We now designed and screened a library of backbone cyclized peptides based on the linear BB loop peptide, to identify a metabolically stable inhibitor of MyD88 that retains the binding properties of the linear peptide. We identified a novel cyclic peptide protein mimetic that inhibits inflammatory responses to TLR ligands, and NFκB activation in response to IL-1 activation. The inhibitor, c(MyD 4-4), is metabolically stable in comparison to the linear peptide, blocks MyD88 in a specific manner, and inhibits MyD88 function by preventing MyD88 dimerization. Finally, treatment of mice with c(MyD 4-4) reduced the severity of clinical disease in the murine EAE model of multiple sclerosis. Thus, modulation of MyD88-dependent signaling using c(MyD 4-4) is a potential therapeutic strategy to lower innate immune inflammation in autoimmune CNS disease.

Inhibition of Myeloid Differentiation Factor 88 Reduces Human and Mouse T-Cell Interleukin-17 and IFNγ Production and Ameliorates Experimental Autoimmune Encephalomyelitis Induced in Mice.

Myeloid differentiation factor 88 (MyD88) recruits signaling proteins to the intracellular domain of receptors belonging to the toll-like/interleukin-1 (IL-1) receptor superfamily. Mice lacking MyD88 are highly susceptible to infectious diseases, but tend to resist experimentally induced autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and manifest diminished allograft rejection. We reasoned that inhibition of MyD88 should influence the cytokine profile of responding T cells by blocking costimulatory molecule expression by antigen-presenting cells (APCs) and by inhibiting T-cell responses to IL-18. We now report that inhibition of MyD88 in human APCs led to decreased IFNγ and IL-17 production and a shift to IL-4 production by responding T cells in a mixed lymphocyte reaction. Direct inhibition of Myd88 in mouse and human T cells also reduced their production of IFNγ in response to IL-12/IL-18 stimulation. Finally, systemic MyD88 antagonism significantly reduced the clinical manifestations of EAE in mice. Thus, MyD88 appears to be a key factor in determining T cell phenotype and represents a potential target for therapeutic intervention.