Flagellar filament structural protein induces Sjögren's syndrome-like sialadenitis in mice.

OBJECTIVE: Sjögren's syndrome (SS) is a systemic autoimmune disease that primarily affects lacrimal and salivary glands. We previously reported that FliC derived from Escherichia coli could induce autoimmune pancreatitis-like lesions. From these results, we speculated that FliC could also induce SS-like exocrinopathy. In this study, we investigated the effects of chronic exposure to FliC on lacrimal and salivary glands and the possibility that it might lead to an autoimmune response. METHODS: C57BL/6 mice were repeatedly injected with FliC and histological changes, serum levels of cytokine/chemokines and autoantibodies were evaluated at different time points after the final injection. The presence of sialadenitis was diagnosed by histological methods. RESULTS: In FliC-treated groups, 57% of subjects developed inflammatory cell infiltrates around ducts in mandibular salivary glands, but not lacrimal glands. In addition, serum levels of total IgG, IgG1, and IgG2a were significantly higher in FliC-treated groups. Intriguingly, serum anti-SSA/Ro levels were also significantly higher in FliC-treated groups. Cytokine analysis revealed that serum levels of IL-1ß, IL-12p70, IL-13, IFN-γ, IL-15, and IL-23 seemed to be higher in FliC-treated mice. CONCLUSIONS: Our data suggest that FliC-treated mice develop an SS-like phenotype. Our model may elucidate the relationship between commensal bacteria and SS.

Airway epithelial expression of TLR5 is downregulated in healthy smokers and smokers with chronic obstructive pulmonary disease.

The TLRs are important components of the respiratory epithelium host innate defense, enabling the airway surface to recognize and respond to a variety of insults in inhaled air. On the basis of the knowledge that smokers are more susceptible to pulmonary infection and that the airway epithelium of smokers with chronic obstructive pulmonary disease (COPD) is characterized by bacterial colonization and acute exacerbation of airway infections, we assessed whether smoking alters expression of TLRs in human small airway epithelium, the primary site of smoking-induced disease. Microarrays were used to survey the TLR family gene expression in small airway (10th to 12th order) epithelium from healthy nonsmokers (n = 60), healthy smokers (n = 73), and smokers with COPD (n = 36). Using the criteria of detection call of present (P call) ≥ 50%, 6 of 10 TLRs (TLRs 1-5 and 8) were expressed. Compared with nonsmokers, the most striking change was for TLR5, which was downregulated in healthy smokers (1.4-fold, p < 10⁻¹°) and smokers with COPD (1.6-fold, p < 10⁻¹¹). TaqMan RT-PCR confirmed these observations. Bronchial biopsy immunofluorescence studies showed that TLR5 was expressed mainly on the apical side of the epithelium and was decreased in healthy smokers and smokers with COPD. In vitro, the level of TLR5 downstream genes, IL-6 and IL-8, was highly induced by flagellin in TLR5 high-expressing cells compared with TLR5 low-expressing cells. In the context that TLR5 functions to recognize pathogens and activate innate immune responses, the smoking-induced downregulation of TLR5 may contribute to smoking-related susceptibility to airway infection, at least for flagellated bacteria.

A class V chitinase from Arabidopsis thaliana: gene responses, enzymatic properties, and crystallographic analysis.

Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end of the substrates. The crystal structure of AtChiC was determined by the molecular replacement method at 2.0 Å resolution. AtChiC was found to adopt an (ß/α)(8) fold with a small insertion domain composed of an α-helix and a five-stranded ß-sheet. From docking simulation of AtChiC with pentameric substrate, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common to class V chitinases from higher plants.

NFκB- and MAP-Kinase Signaling Contribute to the Activation of Murine Myeloid Dendritic Cells by a Flagellin A:Allergen Fusion Protein.

Fusion proteins incorporating the TLR5-ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. We recently reported a flagellin:allergen fusion protein containing the TLR5-ligand flagellin A (FlaA) from Listeriamonocytogenes and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) to prevent allergic sensitization in an experimental mouse model. This study analyzes the signaling pathways contributing to rFlaA:Betv1-mediated pro- and anti-inflammatory cytokine secretion and cell metabolism in myeloid dendritic cells (mDCs) in vitro. The influence of mammalian target of rapamycin (mTOR)-, NF?B-, and MAP kinase (MAPK)-signaling on cytokine secretion and metabolic activity of bone marrow (BM)-derived mDCs stimulated with rFlaA:Betv1 were investigated by pre-treatment with either mTOR- (rapamycin), NF?B- (dexamethason, BMS-345541, TPCA-1, triptolide, or BAY-11) or MAPK- (SP600125, U0126, or SB202190) inhibitors, respectively. rFlaA:Betv1-mediated IL-10 secretion as well as activation of mDC metabolism, rather than pro-inflammatory cytokine secretion, were inhibited by rapamycin. Inhibition of NFκB-signaling suppressed rFlaA:Betv1-induced IL-12, while inhibition of MAPK-signaling dose-dependently suppressed rFlaA:Betv1-induced IL-10 as well as pro-inflammatory IL-6 and TNF-α production. Notably, with the exception of a partial JNK-dependency, rFlaA:Betv1-mediated effects on mDC metabolism were mostly NF?B- and MAPK-independent. Therefore, MAPK-mediated activation of both NFκB- and mTOR-signaling likely is a key pathway for the production of pro- and anti-inflammatory cytokines by flagellin fusion protein vaccines.

Flagellin-induced NADPH oxidase 4 activation is involved in atherosclerosis.

It is widely accepted that bacterial infection-mediated inflammation facilitates development of atherosclerosis by activating toll-like receptor (TLR) signaling system. We reasoned that NADPH oxidases (Nox), required for TLR-mediated inflammatory response, are involved in atherogenesis. Here, we show that the activation of Nox4 through TLR5 regulates the inflammation of the endothelium and in atherogenesis. Flagellin-induced interaction between the COOH region of Nox4 and the TIR domain of TLR5 led to H2O2 generation, which in turn promoted the secretion of pro-inflammatory cytokines including IL-8, as well as the expression of ICAM-1 in human aortic endothelial cells (HAECs). Knockdown of the Nox4 in HAECs resulted in attenuated expressions of IL-8 and ICAM-1 leading to a reduction in the adhesion and trans-endothelial migration of monocytes. Challenge of recombinant FliC (rFliC) to the ApoE KO mice with high-fat diet (HFD) resulted in significantly increased atherosclerotic plaque sizes compared to the saline-injected mice. However, an injection of rFliC into the Nox4ApoE DKO mice with HFDs failed to generate atherosclerotic plaque, suggesting that Nox4 deficiency resulted in significant protections against rFliC-mediated atherogenesis. We conclude that TLR5-dependent Nox4 activation and subsequent H2O2 generation play critical roles for the development of atherosclerosis.

Lactobacillus rhamnosus GG reduces hepatic TNFα production and inflammation in chronic alcohol-induced liver injury.

The therapeutic effects of probiotic treatment in alcoholic liver disease (ALD) have been studied in both patients and experimental animal models. Although the precise mechanisms of the pathogenesis of ALD are not fully understood, gut-derived endotoxin has been postulated to play a crucial role in hepatic inflammation. Previous studies have demonstrated that probiotic therapy reduces circulating endotoxin derived from intestinal gram-negative bacteria in ALD. In this study, we investigated the effects of probiotics on hepatic tumor necrosis factor-α (TNFα) production and inflammation in response to chronic alcohol ingestion. Mice were fed Lieber DeCarli liquid diet containing 5% alcohol for 8weeks, and Lactobacillus rhamnosus GG (LGG) was supplemented in the last 2 weeks. Eight-week alcohol feeding caused a significant increase in hepatic inflammation as shown by histological assessment and hepatic tissue myeloperoxidase activity assay. Two weeks of LGG supplementation reduced hepatic inflammation and liver injury and markedly reduced TNFα expression. Alcohol feeding increased hepatic mRNA expression of Toll-like receptors (TLRs) and CYP2E1 and decreased nuclear factor erythroid 2-related factor 2 expression. LGG supplementation attenuated these changes. Using human peripheral blood monocytes-derived macrophages, we also demonstrated that incubation with ethanol primes both lipopolysaccharide- and flagellin-induced TNFα production, and LGG culture supernatant reduced this induction in a dose-dependent manner. In addition, LGG treatment also significantly decreased alcohol-induced phosphorylation of p38 MAP kinase. In conclusion, probiotic LGG treatment reduced alcohol-induced hepatic inflammation by attenuation of TNFα production via inhibition of TLR4- and TLR5-mediated endotoxin activation.

CD98 positive eosinophils contribute to T helper 1 pattern inflammation.

BACKGROUND AND AIMS: The pathogenesis of inflammatory bowel disease (IBD) has not been fully understood yet. Eosinophils (Eo) are one type of the major proinflammatory cells of the chronic inflammation in the intestine. CD98 is involved in the pathogenesis of a number of inflammations. This study aims to elucidate the role of CD98(+) Eos in the initiation of intestinal inflammation. METHODS: The colon biopsies were collected from 60 patients with IBD. The expression of CD98 in the biopsies was examined by immunohistochemistry. The serum levels of the flagellin (FGN) antibody and Eo-derived mediators in the culture supernatants were assessed by enzyme-linked immunosorbent assay. The role of FGN on Eo activation was examined in a cell culture model. The role of FGN in the induction of colitis was observed in a mouse model. RESULTS: Compared to normal controls, the frequency of CD98(+) Eos was markedly increased in the IBD colon mucosa. FGN were detected in the colon biopsies and in the sera of IBD patients. Exposure to FGN induced the expression of galectin 3 (the ligand of CD98) in dendritic cells. The exposure to galectin 3 activated the CD98(+) Eos. After treatment with FGN intrarectally, mice with eosinophilia showed severe inflammation in the colon. CONCLUSIONS: The interaction of galectin 3 and CD98 can induce Eos to release chemical mediators that contributes to the initiation of the intestinal inflammation.