High grain feeding or weaning, which could compromise the rumen epithelium by increasing ruminal short-chain fatty acid (SCFA) concentrations with pH reduction, is associated with high levels of ruminal toll-like receptor 5 (TLR5). This study aimed to determine the role of TLR5 in the rumen epithelium. Immunohistochemistry revealed that TLR5 was localized in cells on the basal side (i.e., basal and spinous layers) rather than in the granular layer in the rumen epithelium, where tight junctions are most potent, in pre- and post-weaning calves (n = 9). Primary bovine rumen epithelial cells (BRECs) obtained from Holstein cows (n = 3) were cultured to investigate the factors that upregulate TLR5; however, SCFA, low pH (pH 5.6), BHBA, L-lactate, D-lactate, and LPS did not upregulate TLR5 gene expression in BREC. Primary BREC treated with flagellin (TLR5 ligand) had higher expression of interleukin-1β (IL-1β) (P < 0.05) than BREC treated with vehicle. In addition, BREC treated with IL-1β had higher expression of antimicrobial peptides and C-X-C motif chemokine ligand 8 than BREC treated with vehicle (P < 0.05). These results suggest that ruminal TLR5 may recognize epithelial disruption via flagellin and mediate the immune response via IL-1β during high-grain feeding or weaning.

Immune checkpoint inhibitors have revolutionized anti-tumor therapy, notably improving treatment responses in various tumors. However, many patients remain non-responsive and do not experience benefits. Given that Toll-like receptors (TLRs) can counteract tumor immune tolerance by stimulating both innate and adaptive immune responses, TLR agonists are being explored as potential immune adjuvants for cancer treatment. In this study, we assessed the potential of enhancing the efficacy of immune checkpoint inhibitors by activating innate immunity with a TLR5 agonist. In a mouse tumor model, combination therapy with TLR5 agonist and anti-PD-1 significantly inhibited tumor growth. The TLR5 agonist shifted the balance from M2-like to M1-like macrophages and upregulated the expression of co-stimulatory molecules in macrophages. Furthermore, TLR5 agonist promoted the activation and tumor infiltration of CD8+ T cells. As a result, the TLR5 agonist augmented the anti-tumor efficacy of anti-PD-1, suggesting its potential in modulating the tumor microenvironment to enhance the anti-tumor response. Our findings point toward the possibility of optimizing immune checkpoint inhibitor therapy using TLR5 agonists.

At mucosal surfaces, epithelial cells provide a structural barrier and an immune defense system. However, dysregulated epithelial responses can contribute to disease states. Here, we demonstrated that epithelial cell-intrinsic production of interleukin-23 (IL-23) triggers an inflammatory loop in the prevalent oral disease periodontitis. Epithelial IL-23 expression localized to areas proximal to the disease-associated microbiome and was evident in experimental models and patients with common and genetic forms of disease. Mechanistically, flagellated microbial species of the periodontitis microbiome triggered epithelial IL-23 induction in a TLR5 receptor-dependent manner. Therefore, unlike other Th17-driven diseases, non-hematopoietic-cell-derived IL-23 served as an initiator of pathogenic inflammation in periodontitis. Beyond periodontitis, analysis of publicly available datasets revealed the expression of epithelial IL-23 in settings of infection, malignancy, and autoimmunity, suggesting a broader role for epithelial-intrinsic IL-23 in human disease. Collectively, this work highlights an important role for the barrier epithelium in the induction of IL-23-mediated inflammation.

The incidence of the autoimmune disease, type 1 diabetes (T1D), has been increasing worldwide and recent studies have shown that the gut microbiota are associated with modulating susceptibility to T1D. Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and is widely expressed on many cells, including dendritic cells (DCs), which are potent antigen-presenting cells (APCs). TLR5 modulates susceptibility to obesity and alters metabolism through gut microbiota; however, little is known about the role TLR5 plays in autoimmunity, especially in T1D.
To fill this knowledge gap, we generated a TLR5-deficient non-obese diabetic (NOD) mouse, an animal model of human T1D, for study.
We found that TLR5-deficiency led to a reduction in CD11c+ DC development in utero, prior to microbial colonization, which was maintained into adulthood. This was associated with a bias in the DC populations expressing CD103, with or without CD8α co-expression, and hyper-secretion of different cytokines, both in vitro (after stimulation) and directly ex vivo. We also found that TLR5-deficient DCs were able to promote polyclonal and islet antigen-specific CD4+ T cell proliferation and proinflammatory cytokine secretion. Interestingly, only older TLR5-deficient NOD mice had a greater risk of developing spontaneous T1D compared to wild-type mice.
In summary, our data show that TLR5 modulates DC development and enhances cytokine secretion and diabetogenic CD4+ T cell responses. Further investigation into the role of TLR5 in DC development and autoimmune diabetes may give additional insights into the pathogenesis of Type 1 diabetes.

As bacteria synthesize nutrients primarily in the cecum, coprophagy is indispensable for supplying rabbits with essential nutrients. Recent research has demonstrated its pivotal role in maintaining intestinal microbiota homeostasis and immune regulation in rabbits, although the specific mechanism remains unknown. Here, we used coprophagy prevention (CP) to investigate the effects of coprophagy on the cecum homeostasis and microbiota in New Zealand white rabbits. Furthermore, whether supplementation of Clostridium butyricum (C. butyricum) may alleviate the cecum inflammation and apoptosis caused by CP was also explored. Four groups were randomly assigned: control (Con), sham-coprophagy prevention (SCP), coprophagy prevention (CP), and CP and C. butyricum addition (CPCB). Compared to Con and SCP, CP augmented cecum inflammation and apoptosis, as well as bacterial adhesion to the cecal epithelial mucosa, while decreasing the expression of tight junction proteins (ZO-1, occluding, and claudin-1). The relative abundance of short-chain fatty acids (SCFAs)-producing bacteria was significantly decreased in the CP group. Inversely, there was an increase in the Firmicutes/Bacteroidetes ratio and the relative abundance of Christensenellaceae_R-7_group. Additionally, CP increased the levels of Flagellin, IFN-γ, TNF-a, and IL-1β in cecum contents and promoted the expression of TLR5/MyD88/NF-κB pathway in cecum tissues. However, the CPCB group showed significant improvements in all parameters compared to the CP group. Dietary C. butyricum supplementation significantly increased the production of SCFAs, particularly butyric acid, triggering anti-inflammatory, tissue repairing, and barrier-protective responses. Notably, CPCB effectively mitigated CP-induced apoptosis and inflammation. In summary, CP disrupts the cecum epithelial barrier and induces inflammation in New Zealand white rabbits, but these effects can be alleviated by C. butyricum supplementation. This process appears to be largely associated with the TLR5/MyD88/NF-κB signaling pathway.

The growing challenge of antibiotic resistance necessitates novel approaches for combating bacterial infections. This study explores the distinctive synergy between chlorhexidine, an antiseptic and disinfectant agent, and azithromycin, a macrolide antibiotic, in their impact on bacterial growth and virulence factors using Escherichia coli strain Crooks (ATCC 8739) as a model. Our findings reveal that the chlorhexidine and azithromycin combination demonstrates enhanced anti-bacterial effects compared to individual treatments. Intriguingly, the combination induced oxidative stress, decreased flagellin expression, impaired bacterial motility, and enhanced bacterial autoaggregation. Notably, the combined treatment also demonstrated a substantial reduction in bacterial adherence to colon epithelial cells and downregulated NF-κB in the epithelial cells. In conclusion, these results shed light on the potential of the chlorhexidine and azithromycin synergy as a compelling strategy to address the rising challenge of antibiotic resistance and may pave the way for innovative therapeutic interventions in tackling bacterial infections.

OBJECTIVE: This study was to explore the role of Anti-carbamylated protein (Anti-CarP) antibodies in contributing to lung fibrosis in connective tissue disease (CTD)-associated interstitial lung disease (ILD) in an autoantigen-dependent manner.
METHODS: ELISA tested serum samples, including 89 of CTD-ILD group and 170 of non-ILD CTD, for the anti-CarP levels. Male C57BL/6 mice were used for pulmonary fibrosis model and anti-CarP treatment in vivo (n = 5), and patient serum-derived or commercialized anti-CarP for cell treatment. We identified the carbamylated membrane protein via immunofluorescence (IF) and coimmunoprecipitation followed by mass spectrometry (MS) analysis. RT-qPCR, IF and western blot were performed to explore the antigen-dependent role of anti-CarP. Native electrophoretic mobility shift assay and MS analysis were used to verify direct interaction and carbamylation sites.
RESULTS: A significantly higher serum anti-CarP level was observed in CTD with ILD than without ILD. In vivo, intrapulmonary delivery of anti-CarP induces epithelial-mesenchymal transition (EMT) and micro fibrotic foci. Carbamylation was enriched in type II alveolar epithelial cells (AEC II). A novel carbamylated membrane receptor, specifically recognized by anti-CarP, was identified as toll-like receptor 5 (TLR5). We found anti-CarP induces the nuclear translocation of NF-κB and downstream events, including EMT and expression of inflammatory cytokines in AEC II, which were reversed by TLR5 blocking or TLR5 knockdown. Moreover, up to 12 lysine carbamylation sites were found in TLR5 ectodomain, allowing the interaction of anti-CarP with carbamylated TLR5.
CONCLUSIONS: Overall, we found anti-CarP drives aberrant AEC II activation by interacting with carbamylated TLR5 to promote ILD progress.

Leaky gut has been linked to autoimmune disorders including lupus. We previously reported upregulation of anti-flagellin antibodies in the blood of lupus patients and lupus-prone mice, which led to our hypothesis that a leaky gut drives lupus through bacterial flagellin-mediated activation of toll-like receptor 5 (TLR5).
We created MRL/lpr mice with global Tlr5 deletion through CRISPR/Cas9 and investigated lupus-like disease in these mice.
Contrary to our hypothesis that the deletion of Tlr5 would attenuate lupus, our results showed exacerbation of lupus with Tlr5 deficiency in female MRL/lpr mice. Remarkably higher levels of proteinuria were observed in Tlr5 -/- MRL/lpr mice suggesting aggravated glomerulonephritis. Histopathological analysis confirmed this result, and Tlr5 deletion significantly increased the deposition of IgG and complement C3 in the glomeruli. In addition, Tlr5 deficiency significantly increased renal infiltration of Th17 and activated cDC1 cells. Splenomegaly and lymphadenopathy were also aggravated in Tlr5-/- MRL/lpr mice suggesting impact on lymphoproliferation. In the spleen, significant decreased frequencies of regulatory lymphocytes and increased germinal centers were observed with Tlr5 deletion. Notably, Tlr5 deficiency did not change host metabolism or the existing leaky gut; however, it significantly reshaped the fecal microbiota.
Global deletion of Tlr5 exacerbates lupus-like disease in MRL/lpr mice. Future studies will elucidate the underlying mechanisms by which Tlr5 deficiency modulates host-microbiota interactions to exacerbate lupus.

Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

Vibrio parahaemolyticus causes diseases in aquatic organisms, leading to substantial financial losses to the aquaculture industry; its flagellin F (flaF) protein triggers severe inflammation in host cells. To enhance the understanding of the function of flaF in V. parahaemolyticus infection, in this study, a flaF-deficient mutant was constructed by employing two-step homologous recombination. The flaF-deficient mutant induced a significantly lower toll-like receptor 5 (TLR5) expression and apoptosis in fish intestinal epithelial cells than the wild-type V. parahaemolyticus. Furthermore, fluorescence labelling and microscopy analysis of TLR5 showed that V. parahaemolyticus and its mutant strain significantly enhanced TLR5 expression. Additionally, the findings suggest that flaF deletion did not significantly affect the expression of myeloid differentiation factor 88 (MyD88) and interleukin-8 (IL-8) induced by V.parahaemolyticus. In summary, V. parahaemolyticus induced a TLR5-dependent inflammatory response and apoptosis through MyD88, which was observed to be influenced by flaF deletion. In this study, we obtained stable mutants of V. parahaemolyticus via target gene deletion-which is a rapid and effective approach-and compared the induction of inflammatory response and apoptosis by V. parahaemolyticus and its mutant strain, providing novel perspectives for functional gene research in V. parahaemolyticus.