The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification

Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. FlaB exhibited the strongest immunostimulation effects. To explore the functions of flaB in infection, we constructed a flaB deletion mutant using a two-step recombination method, and in vitro experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the in vivo study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, flaB induced a significant increase in TLR5 expression based on microscopy analysis of fluorescently labelled TLR5, indicating interactions between the two proteins, which was confirmed by native PAGE and yeast two-hybrid assay. Molecular simulation of interactions between flaB and TLR5 was performed to identify the residues involved in binding, revealing two binding sites. Then, based on molecular dynamics simulations, we carried out thirteen site-directed mutations occurring at the amino acid sites of Q57, N83, N87, R91, D94, E122, D152, N312, R313, N320, L97, H316, I324 in binding regions of flaB protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of flaB mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between flaB and TLR5. Our comprehensive and systematic analysis of V. anguillarum flagellins establishes the groundwork for future design of flagellin-based vaccines.

Methylated (−)-epigallocatechin 3-O-gallate potentiates the effect of split vaccine accompanied with upregulation of Toll-like receptor 5

Split-virus vaccine serves as a major countermeasure against influenza virus, but its effectiveness and protective action are not complete. We previously demonstrated the effect of Benifuuki, a green tea cultivar in Japan, on enhancing the split-virus vaccine–elicited immune response. However, little is known about the detail mechanisms. Here, we show that EGCG3”Me intake significantly potentiated the vaccine-elicited hemagglutination inhibition titer increase. Flow cytometry analysis revealed the increased Toll-like receptor 5 (TLR5) expression after EGCG3”Me treatment in lamina propria dendritic cells (LPDCs) and macrophages, which play crucial roles in the humoral immune system. TLR5 expression correlated with the level of interleukin-6 (IL-6)/C–C chemokine type receptor 5, which are important mediators of the humoral immunity. Taken together, In vivo and ex vivo studies showed that EGCG3”Me potentiated the split-virus vaccine–elicited immune response accompanied with the upregulation of TLR5 in intestine and splenocyte macrophages.

The Relationship between the Tissue Expression of TLR2, TLR4, TLR5, and TLR7 and Systemic Inflammatory Responses in Colorectal Cancer Patients

<b><i>Background:</i></b> Colorectal cancer (CRC) is the third most commonly diagnosed malignancy globally. CRC patients with elevated plasma C-reactive protein (CRP) levels exhibit compromised prognoses. Toll-like receptors (TLRs), activating the innate and adaptive immune systems, may contribute to pro- and antitumorigenic inflammatory responses. We aimed to identify a possible link between local and systemic inflammatory responses in CRC patients by investigating the association between tissue TLRs and plasma CRP. <b><i>Methods:</i></b> Tissue expressions of TLR2, TLR4, TLR5, and TLR7 were assessed using immunohistochemistry of tissue microarray slides from 549 CRC patients surgically treated between 1998 and 2005. Blood samples were drawn preoperatively, centrifuged, aliquoted, and stored at −80°C until analysis. Plasma CRP was determined through high-sensitivity time-resolved immunofluorometric assay. We investigated the association of TLRs to clinicopathologic variables, plasma CRP, and survival. <b><i>Results:</i></b> High TLR2 expression (hazard ratio [HR] 0.59; 95% confidence interval [CI] 0.41–0.85; <i>p</i> = 0.005), high TLR5 expression (HR 0.60; 95% CI 0.45–0.83; <i>p</i> = 0.002), positive TLR7 expression (HR 0.49; 95% CI 0.33–0.72; <i>p</i> &#x3c; 0.001), and low CRP (HR 1.48; 95% CI 1.08–2.11; <i>p</i> = 0.017) were associated with a better prognosis. A high TLR2 immunoexpression was associated with a better prognosis among low-CRP patients (HR 0.53; 95% CI 0.35–0.80; <i>p</i> = 0.002), high TLR4 expression among high-CRP patients (HR 2.04; 95% CI 1.04–4.00; <i>p</i> = 0.038), high TLR5 expression among low-CRP patients (HR 0.059; 95% CI 0.37–0.92; <i>p</i> = 0.021), and positive TLR7 expression among low-CRP patients (HR 0.53; 95% CI 0.28–1.00; <i>p</i> = 0.049). In multivariate analyses, no biomarkers emerged as significant independent variables. <b><i>Conclusions:</i></b> High tissue TLR2, TLR5, and TLR7 levels were associated with a better prognosis. Among low-CRP patients, those with high TLR2, TLR5, and TLR7 immunoexpressions exhibited a better prognosis. Among high CRP patients, a high TLR4 immunoexpression was associated with a better prognosis.

Down-Regulation of Toll-Like Receptor 5 (TLR5) Increased VEGFR Expression in Triple Negative Breast Cancer (TNBC) Based on Radionuclide Imaging

In this study, GFP-tagged TNBC 4T1 cells with down-regulated TLR5 expression (TLR5− 4T1) and normal TLR5 expression (TLR5+ 4T1) were constructed, respectively. RT-PCR and Western blot studies showed that down-regulation of TLR5 obviously increased the expression of VEGFR in 4T1 cells. Highly stable radio-probes 125I-anti-TLR5 mAb/125I-VEGF/125I-IgG were obtained with labeling rates over 85% and radiochemical purities above 90%. Among these three probes, 125I−anti−TLR5 mAb and 125I-VEGF were used for specifically imaging TNBC, while 125I-IgG was used for comparison. Whole-body phosphorus autoradiography showed clear imaging at 48 h after injection of 125I-anti-TLR5 mAb and 125I-VEGF also provided clear imaging at 24 h. Biodistribution study demonstrated a higher tumor uptake of 125I-anti-TLR5 mAb in TLR5+ group compared with that in TLR5− group (P &lt; 0.05), whereas tumor uptake of 125I-VEGF in TLR5+ group was lower than that in the TLR5− group (P &lt; 0.05). Immunohistochemical staining suggested that the expression of TLR5 was lower, whereas the expression of VEGFR, CD31, and MVD (microvessel density) was higher in TLR5− tumor-bearing mice. In summary, the down-regulation of TLR5 in TNBC promoted the VEGFR expression and angiogenesis, resulting in the proliferation of TNBC cells. TLR5/VEGF might be a better indicator for monitoring the development of TNBC.

Toll-like receptor 5-mediated signaling enhances liver regeneration in mice

Background Toll-like receptor 5 (TLR5)-mediated pathways play critical roles in regulating the hepatic immune response and show hepatoprotective effects in mouse models of hepatic diseases. However, the role of TLR5 in experimental models of liver regeneration has not been reported. This study aimed to investigate the role of TLR5 in partial hepatectomy (PHx)-induced liver regeneration. Methods We performed 2/3 PHx in wild-type (WT) mice, TLR5 knockout mice, or TLR5 agonist CBLB502 treated mice, as a model of liver regeneration. Bacterial flagellin content was measured with ELISA, and hepatic TLR5 expression was determined with quantitative PCR analyses and flow cytometry. To study the effects of TLR5 on hepatocyte proliferation, we analyzed bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression with immunohistochemistry (IHC) staining. The effects of TLR5 during the priming phase of liver regeneration were examined with quantitative PCR analyses of immediate early gene mRNA levels, and with Western blotting analysis of hepatic NF-κB and STAT3 activation. Cytokine and growth factor production after PHx were detected with real-time PCR and cytometric bead array (CBA) assays. Oil Red O staining and hepatic lipid concentrations were analyzed to examine the effect of TLR5 on hepatic lipid accumulation after PHx. Results The bacterial flagellin content in the serum and liver increased, and the hepatic TLR5 expression was significantly up-regulated in WT mice after PHx. TLR5-deficient mice exhibited diminished numbers of BrdU- and PCNA-positive cells, suppressed immediate early gene expression, and decreased cytokine and growth factor production. Moreover, PHx-induced hepatic NF-κB and STAT3 activation was inhibited in Tlr5−/− mice, as compared with WT mice. Consistently, the administration of CBLB502 significantly promoted PHx-mediated hepatocyte proliferation, which was correlated with enhanced production of proinflammatory cytokines and the recruitment of macrophages and neutrophils in the liver. Furthermore, Tlr5−/− mice displayed significantly lower hepatic lipid concentrations and smaller Oil Red O positive areas than those in control mice after PHx. Conclusion We reveal that TLR5 activation contributes to the initial events of liver regeneration after PHx. Our findings demonstrate that TLR5 signaling positively regulates liver regeneration and suggest the potential of TLR5 agonist to promote liver regeneration.

Innate immunity, bone and cartilage metabolism in children with developmental dislocation of the hip – pilot study

Objective Search for the relationship between innate immunity and bone and cartilage metabolism in patients with developmental dislocation of the hip (DDH). Material and methods The study included 27 patients with DDH who underwent reduction of the hip at pediatric orthopaedic department of the Federal State Budgetary Educational Institution of Higher Education «Privolzhsky Research Medical University» of the Ministry of Health of the Russian Federation. The patients aged 15.0 ± 1.7 months. The study enrolled the babies diagnosed with grades III, IV, V unilateral or bilateral DDH as classified by M.V. Volkov, 1978. Patients with hip dysplasia (grade I DDH) or congenital hip subluxation (grade II DDH) were excluded from the study. The control group consisted of 15 patients without musculoskeletal pathology. The mean patients' age was 24.0 ± 1.8 months. Peripheral blood monocytes, toll-like receptor (TLR2, TLR4, TLR5) expression, serum concentrations of fibroblast growth factors (FGF), vascular endothelial growth factor (VEGF), serum magnesium, type I, II collagen and aggrecan were measured in patients of major and control groups. Results DDH patients showed statistically significant differences in all the parameters measured except for the type 2 collagen with decrease in peripheral blood monocyte and increase in TLR2 and TLR5 expression, slight increase in the serum magnesium with decreased concentration of aggrecan and increased FGF level. There was a two-fold decrease in VEGF level and a two-fold increase in type I collagen concentration. There were moderate significant correlations for monocyte matches TLR2 and TLR2 – TLR5 in major group. Three main factors detected with factor analysis included (1) monocytes, TLR2 and TLR5 as most meaningful, (2) FGF and type 2 collagen and (3) aggrecan. Conclusion The findings suggested that specific factors of innate immunity can be involved in the pathogenesis of DDH. Toll receptors regulate many metabolic pathways and connective tissue metabolism, More studies are needed to further explore this topic.

Toll-like receptor 5-mediated signaling enhances liver regeneration in mice

BackgroundToll-like receptor 5 (TLR5)-mediated pathways play critical roles in regulating hepatic immune response and show hepatoprotective effect in mouse models of hepatic diseases. However, the role of TLR5 in experimental models of liver regeneration has not been reported. This study aims to investigate the role of TLR5 in partial hepatectomy (PHx)-induced liver regeneration.MethodsWe performed 2/3 PHx in wild-type (WT) mice, TLR5 knockout mice, or TLR5 agonist CBLB502 treated mice as a model of liver regeneration. Bacterial flagellin content was measured by ELISA, and hepatic TLR5 expression was determined by quantitative PCR analyses and flow cytometry. To study the effects of TLR5 on hepatocyte proliferation, bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression were analyzed by immunohistochemistry (IHC) staining. The effects of TLR5 during the priming phase of liver regeneration was examined by quantitative PCR analyses of immediate early gene mRNA levels, and western blot analysis of hepatic NF-κB and STAT3 activation. Cytokines and growth factors production after PHx were detected using real-time PCR analyses and cytometric bead array (CBA) assays. Oil Red O staining and hepatic lipid concentrations were analyzed to examine the effect of TLR5 on hepatic lipid accumulation after PHx.ResultsThe bacterial flagellin content in serum and liver was increased and the hepatic TLR5 expression was significantly up-regulated in WT mice upon PHx. TLR5-deficient mice exhibited reduced numbers of BrdU- and PCNA-positive cells, suppressed immediate early gene expression, and decreased cytokines and growth factors production. Moreover, PHx-induced hepatic NF-κB and STAT3 activation was inhibited in Tlr5−/− mice compared with WT mice. Consistently, the administration of CBLB502 significantly promoted PHx-mediated hepatocyte proliferation correlated with enhanced production of proinflammatory cytokines and the recruitment of macrophages and neutrophils in the liver. Besides, Tlr5−/− mice displayed significantly decreased hepatic lipid concentrations and Oil Red O positive areas compared to control mice after PHx.ConclusionWe reveal that TLR5 activation contributes to the initial events of liver regeneration after PHx. Our findings demonstrate that TLR5 signaling positively regulates liver regeneration and suggest a potential application of TLR5 agonist in promoting liver regeneration.

TLR5: A prognostic and monitoring indicator for triple-negative breast cancer

A novel, highly selective biomarker is urgently needed to predict and monitor triple-negative breast cancer (TNBC) because targeting molecules are not currently available. Although associated with various malignant tumors, the role of toll-like receptor 5 (TLR5) in TNBC remains uncertain. We aimed to define the effects of TLR5 in TNBC to determine whether it could serve as a prognostic and monitoring indicator for TNBC. We established TNBC cell line 4T1 with low TLR5 expression (GFP tag; TLR5− 4T1) and with normal TLR5 expression (GFP tag; TLR5+ 4T1) using lentivirus-shRNA-TLR5 knockdown transfection and negative lentivirus transfection, respectively. Detected by western blot and qPCR, we found knockdown of TLR5 resulted in decreased expression of TLR5 and E-cadherin and increased expression of N-cadherin, vimentin, fibronectin, TRAF6, SOX2, and Twist1, which were related to EMT (epithelial–mesenchymal transition). In addition, downregulation of TLR5 increased the invasion and migration of 4T1 cells in vitro, which were investigated by CCK-8 and wound healing, as well as transwell assay and colony formation. Furthermore, the metastatic ability of TLR5− 4T1 cells to the lungs was also increased compared to TLR5+ 4T1 cells in vivo. To verify the effect of TLR5 as a monitor indicator, mice bearing TLR5+ and TLR5− 4T1 tumors injected with 125I-anti-TLR5 mAb or isotype 125I-IgG were assessed by whole body phosphor-autoradiography and fluorescence imaging in vivo. Phosphor-autoradiography of model mice revealed early tumors at 6 days after inoculation with TLR5+ 4T1, but not TLR5− 4T1 cells. Intratumoral accumulation of radioactivity positively correlated with TLR5 expression, and fluorescence imaging in vivo revealed both TLR5+ and TLR5− 4T1 tumors. Our results suggested that downregulation of TLR5 in TNBC increased tumor invasiveness and EMT expression via TRAF6 and SOX2 pathway and TLR5 could serve as a prognostic and monitoring indicator for TLR5-positive tumors.

Regulation and Molecular Mechanism of TLR5 on Resistance to Escherichia coli F18 in Weaned Piglets

Toll-like receptor 5 (TLR5) plays an important role in immune system. In this study, we performed transcriptome analysis of the duodenum in E. coli F18-resistant and -sensitive Sutai weaned piglets and analyzed the differential expression of TLR5. The cellular localization of TLR5 was investigated, and the effect of TLR5 expression on E. coli invasion was evaluated after pig small intestinal epithelial cell lines (IPEC-J2) were stimulated by E. coli. The results showed that TLR5 expression level in duodenum and jejunum were significantly higher in E. coli F18-sensitive than in E. coli F18-resistant piglets. TLR5 protein was mainly expressed in the cytoplasm and cell membrane. The expression of genes associated with the TLR5 signaling pathway were significantly higher in TLR5-overexpressed cells than in control cells. Bacterial adhesion was higher in TLR5-overexpressed cells than in blank cells and lower in TLR5 interference than in blank cells. The core promoter region of TLR5 included two CpG islands and 16 acting elements. The methylation of the mC-6 site in the second CpG island of the promoter region had a regulatory effect on TLR5 expression. Therefore, TLR5 plays an important regulatory role on E. coli invasion. Low expression of TLR5 inhibited the immune response and decreased cell damage, which was conducive to the resistance to E. coli stimulation. In conclusion, this study preliminarily revealed the molecular mechanism of TLR5 gene regulating the resistance of piglets to Escherichia coli, and provided a new candidate gene for screening Escherichia coli resistance markers in pigs.