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.

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.

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.

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.

Tissue damage in chronic periodontal disease is driven by the host response to a dysbiotic microbiota, and not by bacteria directly. Among chronic inflammatory diseases of the oral cavity, inflammation and tissue damage around dental implants (peri-implantitis) is emerging as a major clinical challenge, since it is more severe and less responsive to treatment compared to inflammation around natural teeth. We tested whether oral fibroblasts from the periodontal ligament (PDLF), which are present around natural teeth but not around dental implants, actively regulate inflammatory responses to bacterial stimulation. We show that human PDLF down-regulate TNF-α post-transcriptionally in macrophages stimulated with the oral pathogen Porphyromonas gingivalis. Cell contact and secretion of IL-6 and IL-10 contribute to the modulation of inflammatory cytokine production. Although fibroblasts decreased TNF-α secretion, they enhanced the ability of macrophages to phagocytose bacteria. Surprisingly, donor matched oral fibroblasts from gingival tissues, or fibroblasts from peri-implant inflamed tissues were at least as active as PDLF in regulating macrophage responses to bacteria. In addition, priming fibroblasts with inflammatory mediators enhanced PDLF regulatory activity. A further understanding of the spectrum of fibroblast activities in inflammatory lesions is important in order to design ways to control inflammatory tissue damage.

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.\\ 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.\\ 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.\\ 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.

Whereas type I interferons (IFNs-I) were proposed to be elevated in human periodontitis, their role in the disease remains elusive. Using a bacterial-induced model of murine periodontitis, we revealed a prolonged elevation in IFN-I expression. This was due to the downregulation of TAM signaling, a major negative regulator of IFN-I. Further examination revealed that the expression of certain TAM components was reduced as a result of prolonged degradation of MYD88 by the infection. As a result of such prolonged IFN-I production, innate immunological functions of the gingiva were disrupted, and CD4+ T cells were constitutively primed by dendritic cells, leading to elevated RANKL expression and, subsequently, alveolar bone loss (ABL). Blocking IFN-I signaling restored proper immunological function and prevented ABL. Importantly, a loss of negative regulation on IFN-I expression by TAM signaling was also evident in periodontitis patients. These findings thus suggest a role for IFN-I in the pathogenesis of periodontitis.

Porphyromonas gingivalis is a gram-negative anaerobic periodontal pathogen that persists in dysbiotic mixed-species biofilms alongside a dense inflammatory infiltrate of neutrophils and other leukocytes in the subgingival areas of the periodontium. Toll-like receptor 2 (TLR2) mediates the inflammatory response to P. gingivalis and TLR2-deficient mice resist alveolar bone resorption following oral challenge with this organism. Although, MyD88 is an adaptor protein considered necessary for TLR2-induced inflammation, we now report for the first time that oral challenge with P. gingivalis leads to alveolar bone resorption in the absence of MyD88. Indeed, in contrast to prototypical TLR2 agonists, such as the lipopeptide Pam3CSK4 that activates TLR2 in a strictly MyD88-dependent manner, P. gingivalis strikingly induced TLR2 signaling in neutrophils and macrophages regardless of the presence or absence of MyD88. Moreover, genetic or antibody-mediated inactivation of TLR2 completely reduced cytokine production in P. gingivalis-stimulated neutrophils or macrophages, suggesting that TLR2 plays a non-redundant role in the host response to P. gingivalis. In the absence of MyD88, inflammatory TLR2 signaling in P. gingivalis-stimulated neutrophils or macrophages depended upon PI3K. Intriguingly, TLR2-PI3K signaling was also critical to P. gingivalis evasion of killing by macrophages, since their ability to phagocytose this pathogen was reduced in a TLR2 and PI3K-dependent manner. Moreover, within those cells that did phagocytose bacteria, TLR2-PI3K signaling blocked phago-lysosomal maturation, thereby revealing a novel mechanism whereby P. gingivalis can enhance its intracellular survival. Therefore, P. gingivalis uncouples inflammation from bactericidal activity by substituting TLR2-PI3K in place of TLR2-MyD88 signaling. These findings further support the role of P. gingivalis as a keystone pathogen, which manipulates the host inflammatory response in a way that promotes bone loss but not bacterial clearance. Modulation of these host response factors may lead to novel therapeutic approaches to improve outcomes in disease conditions associated with P. gingivalis.

Syndecan-1 (Sdc1) is an important member of the cell surface heparan sulfate proteoglycan family, highly expressed by epithelial cells in adult organisms. Sdc1 is involved in the regulation of cell migration, cell-cell and cell-matrix interactions, growth-factor, chemokine and integrin activity, and implicated in inflammatory responses and tumorigenesis. Gastrointestinal tract represents an important anatomic site where loss of Sdc1 expression was reported both in inflammation and malignancy. However, the biological significance of Sdc1 in chronic colitis-associated tumorigenesis has not been elucidated. To the best of our knowledge, this study is the first to test the effects of Sdc1 loss on colorectal tumor development in inflammation-driven colon tumorigenesis. Utilizing a mouse model of colitis-related colon carcinoma induced by the carcinogen azoxymethane (AOM), followed by the inflammatory agent dextran sodium sulfate (DSS), we found that Sdc1 deficiency results in increased susceptibility to colitis-associated tumorigenesis. Importantly, colitis-associated tumors developed in Sdc1-defficient mice were characterized by increased local production of IL-6, activation of STAT3, as well as induction of several STAT3 target genes that act as important effectors of colonic tumorigenesis. Altogether, our results highlight a previously unknown effect of Sdc1 loss in progression of inflammation-associated cancer and suggest that decreased levels of Sdc1 may serve as an indicator of colon carcinoma progression in the setting of chronic inflammation.

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.

Oral squamous cell carcinoma (OSCC) is a lethal disease whose incidence is increasing. Epidemiologic studies demonstrate an association between periodontitis and oral cancer, and periodontal pathogens are implicated in the pathogenesis of numerous disorders, including rheumatoid arthritis, cardiovascular diseases, diabetes and gastrointestinal malignancies. Nevertheless, a causal role for periodontal pathogens in OSCC has not been shown, partly due to the lack of an appropriate animal model. Here, utilizing a newly-established murine model of periodontitis-associated oral tumorigenesis, we report that chronic bacterial infection promotes OSCC, and that augmented signaling along the IL-6-STAT3 axis underlies this effect. Our results indicate that periodontal pathogens P. gingivalis and F. nucleatum stimulate tumorigenesis via direct interaction with oral epithelial cells through Toll-like receptors. Furthermore, oral pathogens stimulate human OSCC proliferation and induce expression of key molecules implicated in tumorigenesis. To the best of our knowledge, these findings represent the first demonstration of a mechanistic role for oral bacteria in chemically induced OSCC tumorigenesis. These results are highly relevant for the design of effective prevention and treatment strategies for OSCC.

Cysteine proteases (gingipains) from Porphyromonas gingivalis are key virulence factors in chronic periodontitis. Innate immune receptors CD14, Toll-like receptor (TLR) 2 and TLR4 are important in P. gingivalis recognition. We examined the ability of gingipains to cleave CD14, TLR2 and TLR4, and the consequences for the cellular response to bacterial challenge. Macrophages were exposed to Arg (RgpA and RgpB)- and Lys (Kgp)-gingipains, and residual expression of TLR2, TLR4 and CD14 was determined by flow cytometry. The cellular response to live bacteria following exposure to purified gingipains was evaluated by TNFα production and bacterial phagocytosis. RgpA and Kgp decreased CD14 detection in a concentration (p = 0.0000002)- and time (p = 0.03)-dependent manner, whereas RgpB had no significant effect. TLR2 and TLR4 expression were unaffected. Reduction in CD14 expression was more efficient with Lys-gingipain than with Arg-gingipain. A reduced CD14 surface level correlated with decreased TNFα secretion and bacterial phagocytosis following challenge with live P. gingivalis, but the response to heat-killed bacteria was unaffected. Therefore, gingipains reduce CD14 expression without affecting expression of the bacterial-sensing TLRs. Reduced CD14 expression depends on the gingipain hemagglutinin/adhesion site and results in macrophage hyporesponsiveness to bacterial challenge. Further studies are needed to determine if reduced CD14 expression is linked to periodontitis induced by P. gingivalis.

Tumor microenvironment, and particularly tumor-associated macrophages (TAMs), represent a key contributing factor in pancreatic ductal adenocarcinoma (PDAC) pathogenesis. Here we report that heparanase (predominant enzyme degrading heparan sulfate, the main polysaccharide found at the cell surface and extracellular matrix) directs tumor-promoting behavior of TAM in PDAC.\\ A mouse model of heparanase-overexpressing pancreatic carcinoma (n = 5 mice/group), tumor-associated macrophages ex vivo, primary wild-type and heparanase-null macrophages, and histological specimens from PDAC patients (n = 16), were analyzed, applying immunostaining, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, cell proliferation, and heparanase activity assays. All statistical tests are two-sided.\\ We found that overexpression of heparanase is associated with increased TAM infiltration in both experimental (P = .002) and human (P = .01) PDAC. Moreover, macrophages derived from heparanase-rich tumors (which grew faster in mouse hosts), display pronounced procancerous phenotype, evidenced by overexpression of MSR-2, IL-10, CCL2, VEGF, and increased production of IL-6, an important player in PDAC pathogenesis. Furthermore, in vitro heparanase enzyme-rendered macrophages (stimulated by necrotic cells which are often present in PDAC tissue) procancerous, as exemplified by their enhanced production of key cytokines implicated in PDAC (including IL-6), as well as by their ability to induce STAT3 signaling and to augment pancreatic carcinoma cell proliferation. In agreement, we observed activation of STAT3 in experimental and clinical specimens of heparanase-overexpressing PDAC.\\ Our findings underscore a novel function of heparanase in molecular decision-making that guides cancer-promoting action of TAM and imply that heparanase expression status may become highly relevant in defining a target patient subgroup that is likely to benefit the most from treatment modalities targeting TAM/IL-6/STAT3.

Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis, can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides "bystander" protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR and can contribute to the persistence of microbial communities that drive dysbiotic diseases.