TLR5-deficiency controls dendritic cell subset development in an autoimmune diabetes-susceptible model.

Introduction: 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. Methods: To fill this knowledge gap, we generated a TLR5-deficient non-obese diabetic (NOD) mouse, an animal model of human T1D, for study. Results: 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. Discussion: 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.

Novel mechanism of Clostridium butyricum alleviated coprophagy prevention-induced intestinal inflammation in rabbit.

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-1ß 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.

Epithelial-derived interleukin-23 promotes oral mucosal immunopathology.

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.

Desulfovibrio vulgaris interacts with novel gut epithelial immune receptor LRRC19 and exacerbates colitis.

BACKGROUND: The overgrowth of Desulfovibrio, an inflammation promoting flagellated bacteria, has been found in ulcerative colitis (UC) patients. However, the molecular mechanism in promoting colitis remains unestablished. METHODS: The relative abundance Desulfovibrio vulgaris (D. vulgaris) in stool samples of UC patients was detected. Mice were treated with dextran sulfate sodium to induce colitis with or without administration of D. vulgaris or D. vulgaris flagellin (DVF), and the severity of colitis and the leucine-rich repeat containing 19 (LRRC19) signaling were assessed. The interaction between DVF and LRRC19 was identified by surface plasmon resonance and intestinal organoid culture. Lrrc19-/- and Tlr5-/- mice were used to investigate the indispensable role of LRRC19. Finally, the blockade of DVF-LRRC19 interaction was selected through virtual screening and the efficacy in colitis was assessed. RESULTS: D. vulgaris was enriched in fecal samples of UC patients and was correlated with the disease severity. D. vulgaris or DVF treatment significantly exacerbated colitis in germ-free mice and conventional mice. Mechanistically, DVF could interact with LRRC19 (rather than TLR5) in colitis mice and organoids, and then induce the production of pro-inflammatory cytokines. Lrrc19 knockdown blunted the severity of colitis. Furthermore, typhaneoside, a blockade of binding interfaces, blocked DVF-LRRC19 interaction and dramatically ameliorated DVF-induced colitis. CONCLUSIONS: D. vulgaris could promote colitis through DVF-LRRC19 interaction. Targeting DVF-LRRC19 interaction might be a new therapeutic strategy for UC therapy. Video Abstract.

Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

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.

Lawsonia intracellularis flagellin protein LfliC stimulates NF-κB and MAPK signaling pathways independently of TLR5 interaction.

Lawsonia intracellularis, a Gram-negative obligate intracellular bacterium and etiologic agent of porcine proliferative enteropathy, was observed to have a long, single, and unipolar flagellum. Bacterial flagellar filament comprises thousands of copies of the protein flagellin (FliC), and has been reported to be recognized by Toll-like receptor (TLR5) to activate the NF-κB and MAPK signaling pathways, thereby inducing the expression of proinflammatory genes. Recently, two L. intracellularis flagellin proteins, LfliC and LFliC, were reported to be involved in bacterial-host interaction and immune response. Here, to further explore the role of LfliC in proinflammatory response, we purified LfliC, and found that its exposure could activate NF-κB signaling pathway in both HEK293T and IPI-FX cells, as well as activate MAPK p38 and ERK1/2 in HEK293T cells but not in IPI-FX cells. However, our yeast two-hybrid and co-immunoprecipitation assay results revealed that LfliC has no interaction with the porcine TLR5 ECD domain though it harbors the conserved D1-like motif required for the interaction. Moreover, LfliC was identified as a substrate of the virulence-associated type III secretion system (T3SS) by using the heterologous Y. enterocolitica system. Transient expression of LfliC also activated the NF-κB and MAPK signaling pathway in HEK293T cells. Collectively, our results suggest that both the exposure and expression of L. intracellularis LfliC can induce the NF-κB and MAPK signaling pathway in mammalian cells. Our findings may provide important implications and resources for the development of diagnostic tools or vaccines and dissection of the pathogenesis of L. intracellularis.

Construction of the flagellin F mutant of Vibrio parahaemolyticus and its toxic effects on silver pomfret (Pampus argenteus) cells.

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.

Agonistic effect of peptides derived from a truncated HMGB1 acidic tail sequence in TLR5 from Salmo salar.

Based on the structural knowledge of TLR5 surface and using blind docking platforms, peptides derived from a truncated HMGB1 acidic tail from Salmo salar was designed as TLR5 agonistic. Additionally, a template peptide with the native N-terminal of the acidic tail sequence as a reference was included (SsOri). 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. The best peptides, termed 6WK and 5LWK, were selected for chemical synthesis and experimental functional assay. The agonist activity by immunoblotting and immunocytochemistry was determined following the NF-κBp65 phosphorylation (p-NF-κBp65) and the nuclear translocation of the NF-κBp65 subunit from the cytosol, respectively. HeLa cells stably expressing a S. salar TLR5 chimeric form (TLR5c7) showed increased p-NF-κBp65 levels regarding extracts from flagellin-treated cells. No statistically significant differences (p > 0.05) were found in the detected p-NF-κBp65 levels between cellular extracts treated with peptides or flagellin by one-way ANOVA. The image analysis of NF-κBp65 immunolabeled cells obtained by confocal microscopy showed increased nuclear NF-κBp65 co-localization in cells both 5LWK and flagellin stimulated, while 6WK and SsOri showed less effect on p65 nuclear translocation (p < 0.05). Also, an increased transcript expression profile of proinflammatory cytokines such as TNFα, IL-1ß, and IL-8 in HKL cells isolated from Salmo salar was evidenced in 5LWK - stimulated by RT-PCR analysis. Overall, the result indicates the usefulness of novel peptides as a potential immunostimulant in S. salar.

UNC93B1 facilitates the localization and signaling of TLR5M in Epinephelus coioides.

Toll-like receptor 5 (TLR5), serving as a sensor of bacterial flagellin, mediates the innate immune response to actively engage in the host's immune processes against pathogen invasion. However, the mechanism underlying TLR5-mediated immune response in fish remains unclear. Despite the presumed cell surface expression of TLR5 member form (TLR5M), its trafficking dynamics remain elusive. Here, we have identified Epinephelus coioides TLR5M as a crucial mediator of Vibrio flagellin-induced cytokine expression in grouper cells. EcTLR5M facilitated the activation of NF-κB signaling pathway in response to flagellin stimulation and exerted a modest influence on the mitogen-activated protein kinase (MAPK)-extracellular regulated kinase (ERK) signaling. The trafficking chaperone Unc-93 homolog B1 (EcUNC93B1) participated in EcTLR5M-mediated NF-κB signaling activation and downstream cytokine expression. In addition, EcUNC93B1 combined with EcTLR5M to mediate its exit from the endoplasmic reticulum, and also affected its post-translational maturation. Collectively, these findings first discovered that EcTLR5M mediated the flagellin-induced cytokine expression primarily by regulating the NF-κB signaling pathway, and EcUNC93B1 mediated EcTLR5M function through regulating its trafficking and post-translational maturation. This research expanded the understanding of fish innate immunity and provided a novel concept for the advancement of anti-vibrio immunity technology.

Intestinal permeability is associated with aggravated inflammation and myofibroblast accumulation in Graves' orbitopathy: the MicroGO study.

Background: Graves' disease (GD) and Graves' orbitopathy (GO) result from ongoing stimulation of the TSH receptor due to autoantibodies acting as persistent agonists. Orbital pre-adipocytes and fibroblasts also express the TSH receptor, resulting in expanded retro-orbital tissue and causing exophthalmos and limited eye movement. Recent studies have shown that GD/GO patients have a disturbed gut microbiome composition, which has been associated with increased intestinal permeability. This study hypothesizes that enhanced intestinal permeability may aggravate orbital inflammation and, thus, increase myofibroblast differentiation and the degree of fibrosis. Methods: Two distinct cohorts of GO patients were studied, one of which was a unique cohort consisting of blood, fecal, and retro-orbital tissue samples. Intestinal permeability was assessed by measuring serum lipopolysaccharide-binding protein (LBP), zonulin, TLR5, and TLR9 ligands. The influx of macrophages and accumulation of T-cells and myofibroblast were quantified in orbital connective tissue. The NanoString immune-oncology RNA targets panel was used to determine the transcriptional profile of active fibrotic areas within orbital sections. Results: GO patients displayed significantly higher LBP serum concentrations than healthy controls. Within the MicroGO cohort, patients with high serum LBP levels also showed higher levels of zonulin and TLR5 and TLR9 ligands in their circulation. The increased intestinal permeability was accompanied by augmented expression of genes marking immune cell infiltration and encoding key proteins for immune cell adhesion, antigen presentation, and cytokine signaling in the orbital tissue. Macrophage influx was positively linked to the extent of T cell influx and fibroblast activation within GO-affected orbital tissues. Moreover, serum LBP levels significantly correlated with the abundance of specific Gram-negative gut bacteria, linking the gut to local orbital inflammation. Conclusion: These results indicate that GO patients have enhanced intestinal permeability. The subsequent translocation of bacterial compounds to the systemic circulation may aggravate inflammatory processes within the orbital tissue and, as a consequence, augment the proportion of activated myofibroblasts, which actively secrete extracellular matrix leading to retro-orbital tissue expansion. These findings warrant further exploration to assess the correlation between specific inflammatory pathways in the orbital tissue and the gut microbiota composition and may pave the way for new microbiota-targeting therapies.

The inducible secreting TLR5 agonist, CBLB502, enhances the anti-tumor activity of CAR133-NK92 cells in colorectal cancer.

OBJECTIVE: CAR-T/NK cells have had limited success in the treatment of solid tumors, such as colorectal cancer (CRC), in part because of the heterogeneous nature of tumor-associated antigens that lead to antigen-negative relapse after the initial response. This barrier might be overcome by enhancing the recruitment and durability of endogenous immune cells. METHODS: Immunohistochemistry and flow cytometry were used to assess the expression of CD133 antigen in tissue microarrays and cell lines, respectively. Retroviral vector transduction was used to generate CBLB502-secreting CAR133-NK92 cells (CAR133-i502-NK92). The tumor killing capacity of CAR133-NK92 cells in vitro and in vivo were quantified via LDH release, the RTCA assay, and the degranulation test, as well as measuring tumor bioluminescence signal intensity in mice xenografts. RESULTS: We engineered CAR133-i502-NK92 cells and demonstrated that those cells displayed enhanced proliferation (9.0 × 104 cells vs. 7.0 × 104 cells) and specific anti-tumor activities in vitro and in a xenogeneic mouse model, and were well-tolerated. Notably, CBLB502 secreted by CAR133-i502-NK92 cells effectively activated endogenous immune cells. Furthermore, in hCD133+/hCD133- mixed cancer xenograft models, CAR133-i502-NK92 cells suppressed cancer growth better than the counterparts (n = 5, P = 0.0297). Greater T-cell infiltration was associated with greater anti-tumor potency (P < 0.0001). CONCLUSIONS: Armed with a CBLB502 TLR5 agonist, CAR133-NK92 cells were shown to be capable of specifically eliminating CD133-positive colon cancer cells in a CAR133-dependent manner and indirectly eradicating CD133-negative colon cancer cells in a CBLB502-specific endogenous immune response manner. This study describes a novel technique for optimizing CAR-T/NK cells for the treatment of antigenically-diverse solid tumors.

Toll-like receptor 5 tunes hepatic and pancreatic stellate cells activation.

OBJECTIVE: Stellate cells are responsible for liver and pancreas fibrosis and strictly correlate with tumourigenesis. Although their activation is reversible, an exacerbated signalling triggers chronic fibrosis. Toll-like receptors (TLRs) modulate stellate cells transition. TLR5 transduces the signal deriving by the binding to bacterial flagellin from invading mobile bacteria. DESIGN: Human hepatic and pancreatic stellate cells were activated by the administration of transforming growth factor-beta (TGF-ß). TLR5 was transiently knocked down by short-interference RNA transfection. Reverse Transcription-quantitativePCR and western blot were performed to analyse the transcript and protein level of TLR5 and the transition players. Fluorescence microscopy was performed to identify these targets in spheroids and in the sections of murine fibrotic liver. RESULTS: TGF-ß-activated human hepatic and pancreatic stellate cells showed an increase of TLR5 expression. TLR5 knockdown blocked the activation of those stellate cells. Furthermore, TLR5 busted during murine liver fibrosis and co-localised with the inducible Collagen I. Flagellin suppressed TLR5, COL1A1 and ACTA2 expression after the administration of TGF-ß. Instead, the antagonist of TLR5 did not block the effect of TGF-ß. Wortmannin, a specific AKT inhibitor, induced TLR5 but not COL1A1 and ACTA2 transcript and protein level. CONCLUSION: TGF-ß-mediated activation of hepatic and pancreatic stellate cells requires the over-expression of TLR5. Instead, its autonomous signalling inhibits the activation of the stellate cells, thus prompting a signalling through different regulatory pathways.

TLR5S negatively regulates the TLR5M-mediated NF-κB signaling pathway in Epinephelus coioides.

Nuclear factor kappa-B (NF-κB) pathway is a key mediator of inflammation response that plays a role in host defense for pathogen elimination, but excessive activation may lead to tissue damage or pathogen transmission. The negative regulation of NF-κB in lower vertebrates is largely unknown, hindering further understanding of immune signaling evolution. Here, we provided evidence that Epinephelus coioides soluble toll-like receptor 5 (TLR5S), a member of the TLR5 subfamily, has been newly identified as a negative regulator of NF-κB signaling. EcTLR5S was a cytoplasmic protein consisting of 17 leucine-rich repeat domains, which specifically responded to Vibrio flagellin and suppressed flagellin-induced NF-κB signaling activation and cytokine expression. The amino-terminal LRR 1-5 region was necessary for its negative regulatory function. Dual-luciferase reporter assay showed that EcTLR5S significantly inhibited the NF-κB-luc activity induced by inhibitor of NF-κB kinase α (IKKα) and IKKß. Subsequently, the functional relationship between EcTLR5M and EcTLR5S was analyzed, revealing that the negative regulatory function of EcTLR5S targeted the activation of the NF-κB pathway mediated by EcTLR5M. The above results reveal that EcTLR5S negatively regulates the flagellin-induced EcTLR5M-NF-κB pathway activation, which may prevent over-activation of immune signaling and restore homeostasis.

The role of flagellin F in Vibrio Parahaemolyticus-induced intestinal immunity and functional domain identification.

The marine pathogen Vibrio parahaemolyticus has caused huge economic losses to aquaculture. Flagellin is a key bacterial virulence factor that induces an inflammatory response via activation of Toll-like receptor 5 (TLR5) signaling. Herein, to explore the inflammatory activity of V. parahaemolyticus flagellins (flaA, flaB, flaC, flaD, flaE, and flaF), we investigated their ability to induce apoptosis in a fish cell line. All six flagellins induced severe apoptosis. Moreover, treatment with V. parahaemolyticus flagellins increased TLR5 and myeloid differentiation factor 88 (MyD88) expression and the production of TNF-α and IL-8 significantly. This indicated that flagellins might induce a TLR5-meditated immune response via an MyD88-dependent pathway. FlaF exhibited the strongest immunostimulatory effect; therefore, the interaction between TLR5 and flaF was screened using the yeast two-hybrid system. A significant interaction between the two proteins was observed, indicating that flaF binds directly to TLR5. Finally, the amino acids that participate in the TLR5-flaF interaction were identified using molecular simulation, which indicated three binding sites. These results deepen our understanding of the immunogenic properties of flagellins from V. parahaemolyticus, which could be used for vaccine development in the future.

A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages.

Background: A recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1ß secretion and their contribution to the overall immune responses remains elusive. Objective: To investigate the mechanisms underlying the production of IL-1ß from rFlaA:Betv1 stimulated macrophages. Methods: Macrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1ß to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used. Results: rFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1ß secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1ß-expression in THP-1 macrophages. Finally, lack of IL-1ß positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1ß, IL-6, and TNF-α from peritoneal macrophages. Conclusion: The mechanisms contributing to rFlaA:Betv1-induced IL-1ß secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.

Deimmunization of flagellin adjuvant for clinical application.

Flagellin is the cognate ligand for host pattern recognition receptors, toll-like receptor 5 (TLR5) in the cell surface, and NAIP5/NLRC4 inflammasome in the cytosol. TLR5-binding domain is located in D1 domain, where crucial amino acid sequences are conserved among diverse bacteria. The highly conserved C-terminal 35 amino acids of flagellin were proved to be responsible for the inflammasome activation by binding to NAIP5. D2/D3 domains, located in the central region and exposed to the outside surface of flagellar filament, are heterogeneous across bacterial species and highly immunogenic. Taking advantage of TLR5- and NLRC4-stimulating activities, flagellin has been actively developed as a vaccine adjuvant and immunotherapeutic. Because of its immunogenicity, there exist worries concerning diminished efficacy and possible reactogenicity after repeated administration. Deimmunization of flagellin derivatives while preserving the TLR5/NLRC4-mediated immunomodulatory activity should be the most reasonable option for clinical application. This review describes strategies and current achievements in flagellin deimmunization.

Upregulation of TLR5 indicates a favorable prognosis in prostate cancer.

BACKGROUND: Toll-like receptors (TLRs) are the key sensors of innate immunity for triggering immune responses against infections. TLRs are well known to be expressed and activated in innate immune cells, such as macrophage and dendritic cells, but we and others have found that some TLRs are also functional in epithelial cells. However, the role of an epithelial TLR in prostate cancer remains elusive. METHODS: TLR5 expression in messenger RNA and protein level in prostate cancer was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). The activation of TLR5 signaling in epithelial cells was detected upon nuclear factor-κB activation by luciferase assay and western blot analysis, and proinflammatory cytokine activation by RT-qPCR. Distinguishing between the TLR5 and NLRC4 pathways, both recognizing flagellin, is determined by small interfering RNA and proinflammatory cytokine activation. The role of TLR5 in prostate cancer was analyzed by IHC and bioinformatics using a general and single-cell database. RESULTS: In the present study, we show that TLR5, among other TLRs, is exceedingly expressed in human prostate cancer cells. This cancer epithelial cell TLR5 functions to activate the TLR5 signaling pathway in human prostate cancer cells, as it does with innate immune cell TLR5. The bacterial protein flagellin induces a robust immune response in prostate cancer cells in a TLR5-dependent but NLRC4-independent manner. TLR5 is highly expressed in prostate cancer patient specimens, and high TLR5 expression in prostate cancer patients indicates a favorable prognosis. CONCLUSIONS: TLR5, as an innate immunity receptor, is a functional TLR in human prostate cancer epithelial cells. TLR5 plays an important role in prostate cancer development and is a new potential prognosis biomarker. TLR5 may represent a novel immunotherapy target against prostate cancer.

The architecture of transmembrane and cytoplasmic juxtamembrane regions of Toll-like receptors.

Toll-like receptors (TLRs) are the important participants of the innate immune response. Their spatial organization is well studied for the ligand-binding domains, while a lot of questions remain unanswered for the membrane and cytoplasmic regions of the proteins. Here we use solution NMR spectroscopy and computer simulations to investigate the spatial structures of transmembrane and cytoplasmic juxtamembrane regions of TLR2, TLR3, TLR5, and TLR9. According to our data, all the proteins reveal the presence of a previously unreported structural element, the cytoplasmic hydrophobic juxtamembrane α-helix. As indicated by the functional tests in living cells and bioinformatic analysis, this helix is important for receptor activation and plays a role, more complicated than a linker, connecting the transmembrane and cytoplasmic parts of the proteins.

LNCGM1082-mediated NLRC4 activation drives resistance to bacterial infection.

The activation of NLRC4 is a major host response against intracellular bacteria infection. However, NLRC4 activation after a host senses diverse stimuli is difficult to understand. Here, we found that the lncRNA LNCGM1082 plays a critical role in the activation of NLRC4. LNCGM1082 in macrophages affects the maturation of interleukin (IL)-1ß and pyroptotic cell death only after exposure to an NLRC4 ligand. Similar to NLRC4-/- mice, LNCGM1082-/- mice were highly sensitive to Salmonella Typhimurium (S. T) infection. LNCGM1082 deficiency in mouse or human macrophages inhibited IL-1ß maturation and pyroptosis. Mechanistically, LNCGM1082 induced the binding of PKCδ with NLRC4 in both mice and humans. In contrast, NLRC4 did not bind PKCδ in LNCGM1082-/- macrophages. The activity of the lncRNA LNCGM1082 induced by S. T may be mediated through TLR5 in the macrophages of both mice and humans. In summary, our data indicate that TLR5-mediated LNCGM1082 activity can promote the binding of PKCδ with NLRC4 to activate NLRC4 and induce resistance to bacterial infection.

Recombinant Domain of Flagellin Promotes In Vitro a Chemotactic Inflammatory Profile in Human Immune Cells Independently of a Dendritic Cell Phenotype.

Flagellin is the major component of the flagellum in gram-positive and -negative bacteria and is also the ligand for the Toll-like receptor 5 (TLR5). The activation of TLR5 promotes the expression of proinflammatory cytokines and chemokines and the subsequent activation of T cells. This study evaluated a recombinant domain from the amino-terminus D1 domain (rND1) of flagellin from Vibrio anguillarum, a fish pathogen, as an immunomodulator in human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs). We demonstrated that rND1 induced an upregulation of proinflammatory cytokines in PBMCs, characterized at the transcriptional level by an expression peak of 220-fold for IL-1ß, 20-fold for IL-8, and 65-fold for TNF-α. In addition, at the protein level, 29 cytokines and chemokines were evaluated in the supernatant and were correlated with a chemotactic signature. MoDCs treated with rND1 showed low levels of co-stimulatory and HLA-DR molecules and kept an immature phenotype with a decreased phagocytosis of dextran. We probed that rND1 from a non-human pathogen promotes modulation in human cells, and it may be considered for further studies in adjuvant therapies based on pathogen-associated patterns (PAMPs).