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Objective: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease in which activated CD4+ T cells participate in the disease process by inducing inflammation. We aimed to investigate the role of Toll-like receptor 2 (TLR2) on CD4+ T cells in RA patients, and to elucidate the underlying mechanisms by which TLR2 contributes to the pathogenesis of RA. Methods: Serum samples were collected from RA patients and healthy controls. Soluble TLR2 levels were quantified using an enzyme-linked immunosorbent assay (ELISA). Flow cytometry was employed to assess the TLR2 expression level, activation status, cytokine production, reactive oxygen species (ROS) levels, and glucose uptake capacity of CD4+ T cells. Quantitative polymerase chain reaction (qPCR) was used to measure the expression of enzymes associated with glucose and lipid metabolism. The concentration of lactic acid in the culture supernatant was determined using a dedicated detection kit. Results: RA patients had higher levels of TLR2 in their serum, which positively correlated with C-reactive protein and rheumatoid factor. The expression level of TLR2 in CD4+ T cells of RA patients was increased, and TLR2+ cells showed higher activation levels than TLR2- cells. Activation of TLR2 in CD4+ T cells of RA patients promoted their activation, TNF-α secretion, and increased production of ROS. Furthermore, TLR2 activation led to changes in enzymes related to glucose metabolism, causing a shift in glucose metabolism towards the pentose phosphate pathway. Blocking oxidative phosphorylation and the pentose phosphate pathway had varying effects on CD4+ T cell function. Conclusion: TLR2 reprograms the glucose metabolism of CD4+ T cells in RA patients, contributing to the development of RA through ROS-mediated cell hyperactivation and TNF-α secretion. Key Points • TLR2 is upregulated in CD4+T cells of RA patients and correlates with disease severity markers such as CRP and RF. • Activation of TLR2 in CD4+T cells promotes cell activation, TNF-α secretion, and increased ROS production, contributing to the pathogenesis of RA. • TLR2 activates glucose metabolism in CD4+T cells, shifting towards the pentose phosphate pathway, which may be a novel therapeutic target for RA treatment. • Blocking glucose metabolism and ROS production can reduce CD4 + T cell hyperactivation and TNF-α secretion, indicating potential therapeutic strategies for RA management. [ABSTRACT FROM AUTHOR]

Allergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. We sought to show the molecular details and the effects of 3 nonspecific lipid transfer proteins (nsLTPs; Mal d 3 [allergenic nsLTP1 from apple], Cor a 8 [allergenic nsLTP1 from hazelnut], and Pru p 3 [allergenic nsLTP1 from peach]) on epithelial cell uptake and transport. We used fluorescent imaging, flow cytometry, and proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. nsLTPs are transported across the epithelium without affecting cell membrane stability or viability, and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis. Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent on these ligands in the protein complex. nsLTPs were found to initiate cellular signaling via Toll-like receptor 2 but not the cluster of differentiation 1 protein receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the 3 allergens induced intracellular stress signaling through activation of the NOD2 pathway. Our work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. In addition, we have developed a versatile toolbox to extend these investigations to other allergens and cell types. [ABSTRACT FROM AUTHOR]

By providing innate immune modulatory stimuli, the early-life immune system can be enhanced to increase resistance to infections. Activation of innate cell surface receptors called pattern recognition receptors by Toll-like receptor (TLR) ligands is one promising approach that can help to control infections as described for listeriosis and cryptosporidiosis. In this study, the effect of TLR2/TLR1 and TLR2/TLR6 agonists was compared when injected into neonatal mice. Surprisingly, the stimulation of TLR2/TLR6 led to the death of the neonatal mice, which was not observed in adult mice. The TLR2/TLR6 agonist administration induced higher systemic and intestinal inflammation in both adult and neonatal mice when compared with TLR2/TLR1 agonist. The mortality of neonatal mice was interferon γ dependent and involved the intestinal production of interleukin-22 and interleukin-17A. This study clearly demonstrates that targeting TLRs as new control strategy of neonatal infections has to be used with caution. Depending on its heterodimeric form, TLR2 stimulation can induce more or less severe adverse effects relying on the age-related immune functions of the host. [ABSTRACT FROM AUTHOR]

Neutrophils are essential cells involved in inflammation. However, the specific mechanism of neutrophil chemotaxis induced by Treponema pallidum remains unknown. In this study, human umbilical vein endothelial cells (HUVECs) were utilized as target cells to investigate the expression levels of chemokines when stimulated with different concentrations of Tp0768 (also known as TpN44.5 or TmpA, a T. pallidum infection dependent antigen). The results indicated that Tp0768 treatment enhanced neutrophil chemotaxis in HUVECs, which was closely associated with the expression levels of CXCL1, CXCL2, and CXCL8 (also known as interleukin-8). At the same time, the results show that the Toll-like receptor 2 (TLR2) signaling pathway is activated and that endoplasmic reticulum (ER) stress occurs. Furthermore, the findings revealed that the use of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and immunoglobulin-regulated enhancer 1 (IRE1) inhibitors reduced the expression levels of CXCL1, CXCL2, and CXCL8. Additionally, inhibiting TLR2 significantly decreased the expression levels of ER stress–related proteins (PERK and IRE1), CXCL1, CXCL2, and CXCL8. Consequently, neutrophil chemotaxis was significantly inhibited after treatment with TLR2, PERK, and IRE1 inhibitors. These findings shed light on the role of Tp0768 in enhancing neutrophil chemotaxis in endothelial cells, providing a foundation for further exploration of syphilis pathogenesis and offering a new direction for the diagnosis and treatment of T. pallidum infection. [ABSTRACT FROM AUTHOR]

Introduction: Streptococcus uberis is a member of the pyogenic cluster of Streptococcus commonly associated with intramammary infection and mastitis in dairy cattle. It is a poorly controlled globally endemic pathogen responsible for a significant cause of the disease worldwide. The ruminant mammary gland provides an atypical body niche in which immune cell surveillance occurs on both sides of the epithelial tissue. S. uberis does not cause disease in nonruminant species and is an asymptomatic commensal in other body niches. S. uberis exploits the unusual niche of the mammary gland to initiate an innate response from bovine mammary macrophage (BMMO) present in the secretion (milk) in which it can resist the host immune responses. As a result - and unexpectedly - the host inflammatory response is a key step in the pathogenesis of S.uberis, without which colonisation is impaired. In contrast to other bacteria pathogenic to the bovine mammary gland, S. uberis does not elicit innate responses from epithelial tissues; initial recognition of infection is via macrophages within milk. Methods: We dissected the role of the bacterial protein SUB1154 in the inflammasome pathway using ex vivo bovine mammary macrophages isolated from milk, recombinant protein expression, and a panel of inhibitors, agonists, and antagonists. We combine this with reverse-transcription quantitative realtime PCR to investigate the mechanisms underlying SUB1154-mediated priming of the immune response. Results: Here, we show that SUB1154 is responsible for priming the NLRP3 inflammasome in macrophages found in the mammary gland. Without SUB1154, IL-1b is not produced, and we were able to restore IL-1b responses to a sub1154 deletion S. uberis mutant using recombinant SUB1154. Surprisingly, only by blocking internalisation, or the cytoplasmic TIR domain of TLR2 were we able to block SUB1154-mediated priming. Discussion: Together, our data unifies several contrasting past studies and provides new mechanistic understanding of potential early interactions between pyogenic streptococci and the host. [ABSTRACT FROM AUTHOR]

As a famous prescription in China, AnGong NiuHuang (AGNH) pill exerts good neuroprotection for ischaemic stroke (IS), but its mechanism is still unclear. In this study, the neuroprotection of AGNH was evaluated in the rat IS model which were established with the surgery of middle cerebral artery occlusion (MCAO), and the potential mechanism was elucidated by transcriptomic analysis and metabolomic analysis. AGNH treatment obviously decreased the infarct volume and Zea-Longa 5-point neurological deficit scores, improved the survival percentage of rats, regional cerebral blood flow (rCBF), and rat activity distance and activity time. Transcriptomics showed that AGNH exerted its anti-inflammatory effects by affecting the regulatory network including Tyrobp, Syk, Tlr2, Myd88 and Ccl2 as the core. Integrating transcriptomics and metabolomics identified 8 key metabolites regulated by AGNH, including L-histidine, L-serine, L-alanine, fumaric acid, malic acid, and N-(L-arginino) succinate, 1-pyrroline-4-hydroxy-2-carboxylate and 1-methylhistamine in the rats with IS. Additionally, AGNH obviously reduced Tyrobp, Syk, Tlr2, Myd88 and Ccl2 at both the mRNA and protein levels, decreased IL-1β, KC-GRO, IL-13, TNF-α, cleaved caspase 3 and p65 nucleus translocation, but increased IκBα expression. Network pharmacology analysis showed that quercetin, beta-sitosterol, baicalein, naringenin, acacetin, berberine and palmatine may play an important role in protecting against IS. Taken together, this study reveals that AGNH reduced neuroinflammation and protected against IS by inhibiting Tyrobp/Syk and Tlr2/Myd88, as well as NF-κB signalling pathway and regulating multiple metabolites. [ABSTRACT FROM AUTHOR]

Malassezia, the most abundant fungal commensal on the mammalian skin, has been linked to several inflammatory skin diseases such as atopic dermatitis, seborrheic dermatitis and psoriasis. This study reveals that epicutaneous application with Malassezia globosa (M. globosa) triggers skin inflammation in mice. RNA-sequencing of the resulting mouse lesions indicates activation of Interleukin-17 (IL-17) signaling and T helper 17 (Th17) cells differentiation pathways by M. globosa. Furthermore, our findings demonstrate a significant upregulation of IL-23, IL-23R, IL-17A, and IL-22 expressions, along with an increase in the proportion of Th17 and pathogenic Th17 cells in mouse skin exposed to M. globosa. In vitro experiments illustrate that M. globosa prompts human primary keratinocytes to secrete IL-23 via TLR2/MyD88/NF-κB signaling. This IL-23 secretion by keratinocytes is shown to be adequate for inducing the differentiation of pathogenic Th17 cells in the skin. Overall, these results underscore the significant role of Malassezia in exacerbating skin inflammation by stimulating IL-23 secretion by keratinocytes and promoting the differentiation of pathogenic Th17 cells. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are expressed on both immune cells and tumor cells, triggering both anti-tumor and pro-tumor responses. Therefore, TLRs have potential as prognostic biomarkers and immunotherapeutic targets. The aim of this study was to investigate TLR1, TLR2, TLR4, TLR5, and TLR6 expression and association with clinicopathological variables and survival in gastric cancer. Immunohistochemical study on cancer specimens from 564 resected gastric cancer patients was performed using tissue microarrays. The association between patient survival and TLR expression was calculated with Cox regression adjusted for confounding factors. Patients with high cytoplasmic TLR2 expression had significantly poorer 5-year survival than the low cytoplasmic TLR2 expression group in multivariate analysis (adjusted HR 1.38, 95% CI 1.11–1.71), and this estimate was similar in intestinal type (adjusted HR 1.33, 95% CI 0.98–1.80) and diffuse type (adjusted HR 1.48, 95% CI 1.06–2.05) histology subgroups. Patients with high cytoplasmic TLR6 expression group had significantly better 5-year survival compared with low cytoplasmic TLR6 expression group in multivariate analysis (adjusted HR 0.74, 95% CI 0.60–0.91). In the subgroup analysis of diffuse type of histology, the 5-year survival was better in high cytoplasmic TLR6 expression group in multivariable analysis (HR 0.62, 95% CI 0.46–0.83). In the intestinal type of histology subgroup, no significant differences between the groups were present. TLR1, TLR4, and TLR5 expression were not associated with 5-year survival. In conclusion, cytoplasmic TLR2 and TLR6 expression seem to have independent prognostic impact in gastric cancer, while TLR1, TLR4, and TLR5 do not. [ABSTRACT FROM AUTHOR]

The mammalian body possesses remarkable adaptability to cold exposure, involving intricate adjustments in cellular metabolism, ultimately leading to thermogenesis. However, cold‐induced stress can impact immune response, primarily through noradrenaline‐mediated pathways. In our study, we utilized a rat model subjected to short‐term or long‐term mild cold exposure to investigate systemic immune response during the cold acclimation. To provide human relevance, we included a group of regular cold swimmers in our study. Our research revealed complex relationship between cold exposure, neural signaling, immune response, and thermogenic regulation. One‐day cold exposure triggered stress response, including cytokine production in white adipose tissue, subsequently activating brown adipose tissue, and inducing thermogenesis. We further studied systemic immune response, including the proportion of leukocytes and cytokines production. Interestingly, γδ T cells emerged as possible regulators in the broader systemic response, suggesting their possible contribution in the dynamic process of cold adaptation. We employed RNA‐seq to gain further insights into the mechanisms by which γδ T cells participate in the response to cold. Additionally, we challenged rats exposed to cold with the Toll‐like receptor 2 agonist, showing significant modulation of immune response. These findings significantly contribute to understanding of the physiological acclimation that occur in response to cold exposure. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBDs) represent multifactorial chronic inflammatory conditions of the gastrointestinal tract. The main IBDs are Crohn's disease (CD) and ulcerative colitis (UC). CD may cause perforation, stricture or transmural inflammation, which can occur discontinuously in the entire gastrointestinal tract (GIT). UC leads to mucosal inflammation as well as mucosal atrophy in the rectum and the colon. Innate immunity is considered the first line of defense against microbial invasion; among Toll-like receptors, TLR2 is the most important for defense against mycobacterial infection. TLR2 has been reported to have a lot of functions in infectious diseases and in other pathologies, such as chronic and acute inflammatory diseases. Alfa-Smooth Muscle Actin (α-SMA) is an important biomarker in IBDs. All myofibroblasts express α-SMA, which has been found to be upregulated in CD and UC. Paraformaldehyde-fixed intestinal tissues, from patients with CD and patients with UC, were analyzed by immunostaining for TLR2 and α-SMA. Our results showed that, in the samples obtained from UC patients with inflamed mucosa, TLR2-positive epithelial cells concentrated on the mucosal surface and scattered immune cells in the connective tissue; furthermore, numerous α-SMA-positive cells (subepithelial myofibroblasts) were detected in the lamina propria and around glands, while some myofibroblasts co-localizing with α-SMA and TLR2 could be inflammatory macrophages. In CD patients, TLR2-positive enterocytes and α-SMA-positive myofibroblasts in the lamina propria of the villus have been observed. In control samples, a low positivity to α-SMA and TLR2 was observed in subepithelial myofibroblasts and scattered immune cells of the lamina propria. These data showed the recall of α-SMA-positive myofibroblasts during the inflammatory state; in addition, TLR2 expression has been observed to change in the intestinal epithelium in IBDs, demonstrating that alterations in the innate system response may contribute to the pathogenesis of these diseases. [ABSTRACT FROM AUTHOR]

Introduction: Toll-like receptors (TLRs) play a vital role in the innate immune response, recognizing pathogens and initiating the inflammatory response. Research suggests that TLRs may also have a significant impact on the development and progression of cancers, including gastric cancer (GC). Understanding the role of individual TLRs in the immunopathogenesis of gastric cancer may provide new information necessary to develop more effective diagnostic and therapeutic methods. Aim of the study: This study aimed to determine the role of selected TLR-2, -3, -4, and -9 in the immunopathogenesis of patients with newly diagnosed and untreated gastric cancer. Materials and methods: The study included 60 newly diagnosed, untreated GC patients and 25 healthy volunteers. The research included analyses assessing the percentage of the tested TLRs on T and B lymphocyte subpopulations using multicolor flow cytometry and assessing their concentration in the serum of the examined patients using ELISA tests. The statistical analyses performed included a comparison of patients in individual stages of gastric cancer, an analysis of the most common clinical subtypes of gastric cancer, and a comparative analysis of differences in the gender of recruited patients. Results: Our studies showed different expression levels of TLR-2, -3, -4, and -9 on T and B lymphocyte subpopulations, as well as their different concentrations in patients' serum. Significant differences in the expression of these receptors were observed depending on the stage of gastric cancer and its clinical subtypes. These differences were also visible in the context of patient gender. Summary: The results of our studies suggest that TLR-2, -3, -4, and -9 may play an important role in the immunopathogenesis of gastric cancer. The differential expression of these receptors depending on the stage of the disease, clinical subtype, and gender of patients may have potential diagnostic and therapeutic significance. Further research is necessary to understand better the mechanisms of action of TLRs in gastric cancer and to apply this knowledge in clinical practice. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) are tissue-resident immune cells of a hematopoietic origin that play vital roles in innate and adaptive immunity. Human MCs can be isolated and differentiated from various tissue sources, including cord blood, when supplemented with cytokines such as stem cell factor, interleukin 3, and interleukin 6. Our current research study has shown significant differences in the marker expressions of human cord blood-derived mast cells (hCBMCs) based on donor dependency and the type of medium used for culturing and differentiation. These findings are particularly relevant given the challenges of obtaining specialty media influencing MC phenotypic marker expressions. We found that hCBMCs cultured in StemSpanTM-XF medium had a moderate expression of mast/stem cell growth factor receptor Kit (c-KIT) (mRNA and protein), low expressions of FcεRI (mRNA) and TLR2 (mRNA and protein) but had high levels of MRGPRX2 (mRNA and protein) expressions. In contrast, hCBMCs cultured in Stem Line II medium expressed FcεRI and TLR2 (mRNA and protein) with higher c-KIT but had lower MRGPRX2 expressions compared to the hCBMCs cultured in the StemSpanTM-XF medium. These results suggest that it is crucial to consider both donor dependency and the medium when investigating MC functions and that further research is needed to fully understand the impact of these factors on the hCBMC marker expressions.

TLR2 is an exceptional pattern-recognizing receptor because of its ability to heterodimerise with different types of TLRs, which allows it to recognize a wide range of molecular structures on the surface of pathogens. Polymorphisms in genes involved in the TLRs signaling cascade may be a factor in host susceptibility to the development of inflammation, affecting the outcome of a number of infectious diseases and immune diseases. The variant Arg753Gln (rs5743708) in the TLR2 gene is the most characterized missense mutation of the coding region in the TIR domain, which involves the substitution of arginine for glutamine at position 753 of the protein sequence. This functionally significant substitution leads to altered signaling and is associated with inflammatory responses. In this study, we investigated the association of the Arg753Gln (rs5743708) polymorphism of the TLR2 gene with the level of its expression in nonagenarians. The study included 82 nonagenarians. Frailty was detected in 41 subjects using a short physical performance battery, with registration in the test ≤ 7 points. It was shown that carriage of the Gln allele is statistically significantly associated with an increased risk of developing frailty; patients with the Arg/Gln genotype have a 12.8-fold higher chance of developing this geriatric syndrome. The Arg allele and the Arg/Arg genotype were found to be protective factors in the development of frailty in nonagenarians. Analysis of TLR2 gene expression in nonagenarians revealed a 2.79-fold increase in TLR2 expression relative to donors. Evaluation of TLR2 gene expression level in groups of nonagenarians with the presence and absence of frailty showed a 1.4-fold increase in TLR2 gene expression in nonagenarians with this geriatric syndrome. In patients with the Arg/Gln genotype, TLR2 gene expression was 1.3 times higher than in the group with the Arg/Arg genotype and 1.6 times higher than in the group with the Gln/Gln genotype. The increased frequency of occurrence of the Arg/Gln genotype of the Arg753Gln polymorphism of the TLR2 gene in nonagenarians with frailty may be due to increased gene expression of this receptor. It is necessary to conduct further functional and molecular genetic studies.

Inflammatory bowel diseases (IBDs) represent multifactorial chronic inflammatory conditions of the gastrointestinal tract. The main IBDs are Crohn’s disease (CD) and ulcerative colitis (UC). CD may cause perforation, stricture or transmural inflammation, which can occur discontinuously in the entire gastrointestinal tract (GIT). UC leads to mucosal inflammation as well as mucosal atrophy in the rectum and the colon. Innate immunity is considered the first line of defense against microbial invasion; among Toll-like receptors, TLR2 is the most important for defense against mycobacterial infection. TLR2 has been reported to have a lot of functions in infectious diseases and in other pathologies, such as chronic and acute inflammatory diseases. Alfa-Smooth Muscle Actin (α-SMA) is an important biomarker in IBDs. All myofibroblasts express α-SMA, which has been found to be upregulated in CD and UC. Paraformaldehyde-fixed intestinal tissues, from patients with CD and patients with UC, were analyzed by immunostaining for TLR2 and α-SMA. Our results showed that, in the samples obtained from UC patients with inflamed mucosa, TLR2-positive epithelial cells concentrated on the mucosal surface and scattered immune cells in the connective tissue; furthermore, numerous α-SMA-positive cells (subepithelial myofibroblasts) were detected in the lamina propria and around glands, while some myofibroblasts co-localizing with α-SMA and TLR2 could be inflammatory macrophages. In CD patients, TLR2-positive enterocytes and α-SMA-positive myofibroblasts in the lamina propria of the villus have been observed. In control samples, a low positivity to α-SMA and TLR2 was observed in subepithelial myofibroblasts and scattered immune cells of the lamina propria. These data showed the recall of α-SMA-positive myofibroblasts during the inflammatory state; in addition, TLR2 expression has been observed to change in the intestinal epithelium in IBDs, demonstrating that alterations in the innate system response may contribute to the pathogenesis of these diseases.

Porphyromonas gingivalis, a keystone oral pathogen implicated in development and progression of periodontitis, may also contribute to the pathogenicity of diseases such as arthritis, atherosclerosis, and Alzheimer's. P. gingivalis is a master manipulator of host immune responses due to production of a large variety of virulence factors. Among these, P. gingivalis peptidilarginine deiminase (PPAD), an enzyme unique to P. gingivalis, converts C-terminal Arg residues in bacterium- and host-derived proteins and peptides into citrulline. PPAD contributes to stimulation of proinflammatory responses in host cells and is essential for activation of the prostaglandin E2 (PGE2) synthesis pathway in gingival fibroblasts. Since P. gingivalis is recognized mainly by Toll-like receptor-2 (TLR2), we investigated the effects of PPAD activity on TLR2-dependent host cell responses to P. gingivalis, as well as to outer membrane vesicles (OMVs) and fimbriae produced by this organism. Using reporter cell lines, we found that PPAD activity was required for TLR2 activation by P. gingivalis cells and OMVs. We also found that fimbriae, an established TLR2 ligand, from wild-type ATCC 33277 (but not from its isogenic PPAD mutant) enhanced the proinflammatory responses of host cells. Furthermore, only fimbriae from wild-type ATCC 33277, but not from the PPAD-deficient strains, induced cytokine production and stimulated expression of genes within the PGE2 synthesis pathway in human gingival fibroblasts via activation of the NF-κB and MAP kinase-dependent signaling pathways. Analysis of ten clinical isolates revealed that type I FimA is preferable for TLR2 signaling enhancement. In conclusion, the data strongly suggest that both PPAD activity and fimbriae are important for TLR2-dependent cell responses to P. gingivalis infection. [ABSTRACT FROM AUTHOR]

Retinopathy of prematurity (ROP) has a dual-phase disease pathology; in phase 1, hyperoxia-induced vaso-obliteration occurs in the retinal vasculature due to increased oxidative stress (OS) and inflammation, followed by phase 2, where hypoxia increases the overproduction of growth factors, inducing retinal neovascularization. Toll-like receptor 2 and -4 (TLR2 and TLR4) overactivation, hyper-inflammation, macrophages, and neutrophil infiltration contribute to the developing ROP. AVR-121 and AVR-123 are novel classes of small-molecule dual inhibitors of TLR2/4 tested in a human leukemia monocytic cell line (THP-1) and cord-blood-derived mononuclear cells (CBMCs). Both compounds inhibited TLR2/4 signaling-related inflammatory cytokines in THP-1 cells and inhibited VEGF-induced neovascularization in human retinal endothelial cells (HRECs), which are hallmarks of ROP. In an oxygen-induced retinopathy (OIR) murine model, the intraperitoneal injection of AVR-123 in the hyperoxia phase (P7–P12) or a nanosuspension eyedrop of AVR-123 in the hypoxic phase (P12–P17) significantly reduced vaso-obliteration, angiogenesis, and inflammatory cytokine profiles while not inhibiting the necessary growth factor VEGF in the juvenile mouse eyes. The results are consistent with our hypothesis that targeting the dual TLR2/4 pathway will reduce inflammation, angiogenesis, and vaso-obliteration in vitro and in vivo and reduce cytotoxic immune cells. AVR-123 has the potential to be developed as a therapy for ROP. [ABSTRACT FROM AUTHOR]

Objective To study the effect of miR-143-3p on pyroptosis of HCT-116 cells induced by LPS + ATP. Methods The targeting relationship between miR-143-3 p and TLR2 gene was detected by dual luciferase. The pyroptosis model was established after transfection of miR-143-3p mimic. Cell apoptosis was detected by flow cytometry, and the activity of Caspase-1 and LDH was detected by biochemical method. The levels of IL-1β and IL-18 were detected by ELISA. The mRNA levels of NF-κB, TLR2, NLRP3 and GSDMD were detected by q-PCR. The expression of NLRP3 and ASC was detected by immunofluorescence. The protein expressions of TLR2, GSDMD, p-NF-κB p65 and cleave Caspase-1 were detected by Western blot. Results Dual luciferase assay showed that miR-143-3p targeted TLR2 expression. The expressions of NF-κB, TLR2, NLRP3, GSDMD mRNA and TLR2, GSDMD, p-NF- κB p65, cleave Caspase-1, ASC, NLRP3 protein in miR-143-3p mimic group and si TLR2 group were lower than those in model group. The activity of Caspase-1 and the content of LDH, IL-1β and IL-18 decreased. Conclusion miR-143-3p regulates pyroptosis of ulcerative colitis cells by targeting TLR2 gene and regulating NF-κB/NLRP3/Caspase-1 signaling pathway. [ABSTRACT FROM AUTHOR]

Cystic echinococcosis (CE) is a zoonotic disease caused by the parasite Echinococcus granulosus (E. granulosus), which can lead to the formation of liver lesions. Research indicates that E. granulosus releases both Toll-like receptor 2 (TLR2) and Interleukin-9 (IL-9), which can potentially impair the body's innate immune defenses and compromise the liver's ability to fight against diseases. To investigate the role of TLR2 and IL-9 in liver damage caused by E. granulosus infection, samples were initially collected from individuals diagnosed with CE. Subsequently, BALB/c mice were infected with E. granulosus at multiple time points (4 weeks, 12 weeks, 32 weeks) and the expression levels of these markers was then assessed at each of these phases. Furthermore, a BALB/c mouse model was generated and administered anti-IL-9 antibody via intraperitoneal injection. The subsequent analysis focused on the TLR2/MyD88/NF-κB signaling pathway and the expression of IL-9 in E. granulosus was examined. A co-culture experiment was conducted using mouse mononuclear macrophage cells (RAW264.7) and hepatic stellate cells (HSCs) in the presence of E. granulosus Protein (EgP). The findings indicated elevated levels of IL-9 and TLR2 in patients with CE, with the activation of the signaling pathway significantly increased as the duration of infection progressed. Administration of anti-IL-9 in mice reduced the activation of the TLR2/MyD88/NF-κB signaling pathway, exacerbating liver injury. Moreover, EgP stimulates the TLR2/MyD88/NF-κB signaling pathway, resulting in the synthesis of α-SMA and Collagen I. The data suggest that infection with E. granulosus may stimulate the production of IL-9 through the activation of the TLR2/MyD88/NF-κB signaling pathway, which is mediated by TLR2. This activation stimulates RAW264.7 and HSCs, exacerbating liver injury and fibrosis. [ABSTRACT FROM AUTHOR]

Lipid bi‐layered particles known as membrane vesicles (MVs), produced by Gram‐positive bacteria are a communication tool throughout the entire bacterial growth. However, the MVs characteristics may vary across all stages of maternal culture growth, leading to inconsistencies in MVs research. This, in turn, hinders their employment as nanocarriers, vaccines and other medical applications. In this study, we aimed to comprehensively characterize MVs derived from Lacticaseibacillus rhamnosus CCM7091 isolated at different growth stages: early exponential (6 h, MV6), late exponential (12 h, MV12) and late stationary phase (48 h, MV48). We observed significant differences in protein content between MV6 and MV48 (data are available via ProteomeXchange with identifier PXD041580), likely contributing to their different immunomodulatory capacities. In vitro analysis demonstrated that MV48 uptake rate by epithelial Caco‐2 cells is significantly higher and they stimulate an immune response in murine macrophages RAW 264.7 (elevated production of TNFα, IL‐6, IL‐10, NO). This correlated with increased expression of lipoteichoic acid (LTA) and enhanced TLR2 signalling in MV48, suggesting that LTA contributes to the immunomodulation. In conclusion, we showed that Lacticaseibacillus rhamnosus CCM7091‐derived MVs from the late stationary phase boost the immune response the most effectively, which pre‐destines them for therapeutical application as nanocarriers. [ABSTRACT FROM AUTHOR]

Background Nucleus pulposus cell (NPC) senescence in intervertebral disc (IVD) tissue is the major pathological cause of intervertebral disc degeneration (IDD). N6-methyladenosine (m6A) methylation and gut microbiota play important roles in the progression of IDD. This study investigated whether methyltransferase-like 3 (METTL3) regulates TLR2 m6A modification and gut microbiota to influence NPC senescence. Methods An IDD rat model was established by lumbar IVD puncture and NPCs were challenged with IL-1β to mimic IVD injury. IDD rats and IL-1β-exposed NPCs were treated with METTL3-interfering lentivirus and the TLR2 agonist Pam3CSK4. Compositional changes in the rat gut microbiota were analyzed and fecal microbiota transplantation procedures were used. NPC senescence, cell cycle, and the expression of senescence-associated secretory phenotype (SASP) factors were assessed. The m6A enrichment of TLR2 and the binding of IGF2BP1 to TLR2 mRNA were examined. Results METTL3 and TLR2 were highly expressed in IDD rats. METTL3 silencing attenuated senescent phenotypes and reduced secretion of SASP factors. Pam3CSK4 reversed the beneficial effects of METTL3 silencing on NPC senescence and IVD injury. METTL3 stabilized TLR2 mRNA in an IGF2BP1-dependent manner. METTL3 silencing restored specific gut microbiota levels in IDD rats, which was further reversed by administration of Pam3CSK4. Fecal microbiota from METTL3 silenced IDD rats altered the pathological phenotypes of IDD rats. Conclusions These results demonstrate the beneficial effects of METTL3 silencing on NPC senescence and amelioration of IVD injury, involving modulation of TLR2 m6A modification and gut microbiota. These findings support METTL3 silencing as a potential therapeutic target for IDD. [ABSTRACT FROM AUTHOR]

Toll‐like receptors (TLRs) play an important role in innate immunity. Previous studies have shown that single nucleotide polymorphisms (SNPs) in the genes coding for these innate immune molecules can affect susceptibility to and the outcome of certain diseases. The aim of the present study was to examine the clinical relevance of well‐studied TLR1–4 SNPs in individuals who are prone to infections. Four functional SNPs, TLR1 rs5743618 (1805C > A, Ser602Ile), TLR2 rs5743708 (2258G > A, Arg753Gln), TLR3 rs3775291 (1234C > T, Leu412Phe) and TLR4 rs4986790 (896A > G, Asp299Gly), were analysed in 155 patients with recurrent respiratory infections (n = 84), severe infections (n = 15) or common variable immunodeficiency (n = 56), and in 262 healthy controls, using the High Resolution Melting Analysis method. Polymorphisms of TLR2 rs5743708 (odds ratio [OR] 3.16; 95% confidence interval [CI] 1.45–6.83, p =.004, ap =.016) and TLR4 rs4986790 (OR 1.8; 95% CI 1.05–3.12, p =.028, ap =.112) were more frequent in patients with recurrent or severe infections than in controls. Interestingly, seven patients were found to carry both variant genotypes of TLR2 and TLR4, whereas none of the control group carried such genotypes (p ≤.0001). Moreover, TLR2 polymorphism was associated with increased risk for acute otitis media episodes (OR, 3.02; 95% CI 1.41–6.47; p =.012). This study indicates that children and adults who are more prone to recurrent or severe respiratory infections carry one or both variant types of TLR2 and TLR4 more often than control subjects. Genetic variations of TLRs help explain why some children are more susceptible to respiratory infections. [ABSTRACT FROM AUTHOR]

Background: Cell energy metabolism controls the activation and function of dendritic cells (DCs). Inflammatory dendritic epidermal cells (IDECs) in skin lesions of atopic dermatitis (AD) express high-affinity IgE receptor (FceRI) and toll-like receptor 2 (TLR2), which mediate the generation and maintenance of inflammation. However, cellular energy metabolism and effector function of IDECs mediated by FceRI and TLR2 have not been fully elucidated. Methods: IDECs in vitro were treated with TLR2 agonist Pam3CSK4 and anti-IgE alone or in combination for 24 h. Further, we analyzed the expression of cell surface activation markers, production of inflammatory factors, and cellular energy metabolism profiles of IDECs by using flow cytometry, multiplex assay, RNA sequencing, targeted energy metabolism, and seahorse assays. Results: Compared to the unstimulated or anti-IgE groups, Pam3CSK4 alone or combined with anti-IgE groups significantly increased the expression of CD80, CD83, and CD86 on IDECs, but did not affect the expression of the above markers in the anti-IgE group. The release of inflammatory cytokines increased in the Pam3CSK4 alone or combined with anti-IgE groups, while there was a weak increasing trend in the anti-IgE group. The glycolysis/gluconeogenesis pathway of carbon metabolism was affected in all treatment groups. Furthermore, compared to the control group, we found a decrease in pyruvic acid, upregulation of PFKM, downregulation of FBP1, and increase in extracellular lactate, glycolysis rate, and glycolysis capacity after all treatments, while there was no difference between each treatment group. However, there was no difference in glycolytic reserve and mitochondrial basic and maximum respiration among all groups. Conclusion: Our results indicate that glycolysis of IDECs may be activated through FceRI and TLR2 to upregulate inflammatory factors, suggesting that danger signals from bacteria or allergens might evoke an inflammatory response from AD through the glycolysis pathway [ABSTRACT FROM AUTHOR]

Background: Paracoccidioidomycosis (PCM) is a systemic endemic fungal disease prevalent in Latin America. Previous studies revealed that host immunity against PCM is tightly regulated by several suppressive mechanisms mediated by tolerogenic plasmacytoid dendritic cells, the enzyme 2,3 indoleamine dioxygenase (IDO-1), regulatory T-cells (Tregs), and through the recruitment and activation of myeloid-derived suppressor cells (MDSCs). We have recently shown that Dectin-1, TLR2, and TLR4 signaling influence the IDO-1-mediated suppression caused by MDSCs. However, the contribution of these receptors in the production of important immunosuppressive molecules used by MDSCs has not yet been explored in pulmonary PCM. Methods: We evaluated the expression of PD-L1, IL-10, as well as nitrotyrosine by MDSCs after anti-Dectin-1, anti-TLR2, and anti-TLR4 antibody treatment followed by P. brasiliensis yeasts challenge in vitro. We also investigated the influence of PD-L1, IL-10, and nitrotyrosine in the suppressive activity of lung-infiltrating MDSCs of C57BL/6-WT, Dectin-1KO, TLR2KO, and TLR4KO mice after in vivo fungal infection. The suppressive activity of MDSCs was evaluated in cocultures of isolated MDSCs with activated T-cells. Results: A reduced expression of IL-10 and nitrotyrosine was observed after in vitro anti-Dectin-1 treatment of MDSCs challenged with fungal cells. This finding was further confirmed in vitro and in vivo by using Dectin-1KO mice. Furthermore, MDSCs derived from Dectin-1KO mice showed a significantly reduced immunosuppressive activity on the proliferation of CD4+ and CD8+ T lymphocytes. Blocking of TLR2 and TLR4 by mAbs and using MDSCs from TLR2KO and TLR4KO mice also reduced the production of suppressive molecules induced by fungal challenge. In vitro, MDSCs from TLR4KO mice presented a reduced suppressive capacity over the proliferation of CD4+ T-cells. Conclusion: We showed that the pathogen recognition receptors (PRRs) Dectin-1, TLR2, and TLR4 contribute to the suppressive activity of MDSCs by inducing the expression of several immunosuppressive molecules such as PD-L1, IL-10, and nitrotyrosine. This is the first demonstration of a complex network of PRRs signaling in the induction of several suppressive molecules by MDSCs and its contribution to the immunosuppressive mechanisms that control immunity and severity of pulmonary PCM. [ABSTRACT FROM AUTHOR]

Simple Summary: This study utilizes subtractive proteomics and pangenome analysis to identify essential non-homologous pathogenic surface proteins that may serve as potential targets for vaccine candidates. The vaccine design incorporates specific B- and T-cell epitopes coupled with adjuvants to enhance the immune response. Computational methods, such as in silico gene cloning, docking studies, and immunological simulation, are employed to assess the efficacy of the vaccine design. The study emphasizes the necessity for further validation through in vitro and in vivo testing to confirm the safety and effectiveness of the vaccine, despite the positive outcomes shown. This method represents a significant advancement in utilizing immunization to proactively prevent infections caused by S. gordonii. Streptococcus gordonii is a gram-positive, mutualistic bacterium found in the human body. It is found in the oral cavity, upper respiratory tract, and intestines, and presents a serious clinical problem because it can lead to opportunistic infections in individuals with weakened immune systems. Streptococci are the most prevalent inhabitants of oral microbial communities, and are typical oral commensals found in the human oral cavity. These streptococci, along with many other oral microbes, produce multispecies biofilms that can attach to salivary pellicle components and other oral bacteria via adhesin proteins expressed on the cell surface. Antibiotics are effective against this bacterium, but resistance against antibodies is increasing. Therefore, a more effective treatment is needed. Vaccines offer a promising method for preventing this issue. This study generated a multi-epitope vaccine against Streptococcus gordonii by targeting the completely sequenced proteomes of five strains. The vaccine targets are identified using a pangenome and subtractive proteomic approach. In the present study, 13 complete strains out of 91 strains of S. gordonii are selected. The pangenomics results revealed that out of 2835 pan genes, 1225 are core genes. Out of these 1225 core genes, 643 identified as non-homologous proteins by subtractive proteomics. A total of 20 essential proteins are predicted from non-homologous proteins. Among these 20 essential proteins, only five are identified as surface proteins. The vaccine construct is designed based on selected B- and T-cell epitopes of the antigenic proteins with the help of linkers and adjuvants. The designed vaccine is docked against TLR2. The expression of the protein is determined using in silico gene cloning. Findings concluded that Vaccine I with adjuvant shows higher interactions with TLR2, suggesting that the vaccine has the ability to induce a humoral and cell-mediated response to treat and prevent infection; this makes it promising as a vaccine against infectious diseases caused by S. gordonii. Furthermore, validation of the vaccine construct is required by in vitro and in vivo trials to check its actual potency and safety for use to prevent infectious diseases caused by S. gordonii. [ABSTRACT FROM AUTHOR]

Rosacea is a chronic facial inflammatory skin disease that occurs with dysfunction of the immune system. Colchicine was reported to have anti-inflammatory properties. However, the impact of colchicine on rosacea remains unclear. In the present study, the phenotype of rosacea lesions was evaluated by the redness score, inflammatory biomarkers were analyzed by reverse transcription PCR (RT‒PCR), and the infiltration of inflammatory cells was assessed by IHC analysis and immunofluorescence in a rosacea-like mouse model. In vitro, RT‒PCR was used to identify the inflammatory factors that Toll-like receptor 2 (TLR2) agonist caused neutrophils to produce, and immunofluorescence and coimmunoprecipitation were used to identify putative signalling pathways. We found that skin erythema and histopathological alterations, as well as elevated proinflammatory factors (IL-1β, IL-6, TNFα, CXCL2) and CAMP, were significantly ameliorated by colchicine treatment in LL37-induced rosacea-like mice. In addition, colchicine reduced the colocalization of TLR2 and neutrophils and the formation of neutrophil extracellular trap networks (NET) in mouse lesions. In neutrophils, colchicine markedly reduced TLR2 agonist-induced inflammatory biomarker expression, NET formation, and ROS production. Moreover, we found that LL37 could bind to TLR2 upon activation of TLR2 in neutrophils. Importantly, colchicine could repress the combination of TLR2 and LL37 in vivo. Finally, bioinformatics methods further validated the key molecules of neutrophil-related inflammation in rosacea, which is consistent with our experimental findings. Collectively, colchicine ameliorated rosacea-like dermatitis by regulating the neutrophil immune response activated by the TLR2 pathway, indicating that it could be an effective therapeutic option for patients with rosacea. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) recognize whole cells or components of microorganisms. Alendronate (ALN) is an anti-bone-resorptive drug that has inflammatory side effects. The aim in this study was to examine whether ALN augments TLR2 ligand-induced proinflammatory cytokine production using mouse macrophage-like RAW264.7 cells transfected with murine apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) gene (hereafter, referred to as "RAW-ASC cells"). RAW-ASC cells were pretreated with or without ALN and then incubated with or without TLR2 ligands. The levels of secreted mouse IL-1α, IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in culture supernatants and the activation of activator protein-1 (AP-1) or nuclear factor-κB (NF-κB) were measured using enzyme-linked immunosorbent assay (ELISA). The expressions of myeloid differentiation factor 88 (MyD88), caspase-11, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), ASC, NF-κB p65, and actin were analyzed via Western blotting. TLR2 expression was analyzed using flow cytometry. ALN substantially upregulated the Pam 3 CSK 4 -induced release of IL-1α, IL-1β, IL-6, and TNF-α and MyD88 expression in RAW-ASC cells. ST-2825, a MyD88 inhibitor, inhibited the ALN-augmented release of these cytokines. Pretreatment with ALN augmented Pam 3 CSK 4 -induced NF-κB activation in RAW-ASC cells and upregulated AP-1 activation. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein and ALN synergically upregulated the release of IL-1α, IL-1β, IL-6 and TNF-α in RAW-ASC cells. Our findings suggest that ALN augments TLR2 ligand-induced proinflammatory cytokine production via the upregulation of MyD88 expression, and this augmentation is accompanied by the activation of NF-κB and AP-1 in RAW-ASC cells. • ALN augmented TLR2 ligand-induced proinflammatory cytokine production by RAW-ASC cells. • ALN upregulated the expression of MyD88 and caspase-11, but not TLR2 in RAW-ASC cells. • ST-2825, a MyD88 inhibitor, inhibited ALN-augmented proinflammatory cytokine production. • ALN also upregulated the activation of NF-κB and AP-1 in RAW-ASC cells. [ABSTRACT FROM AUTHOR]

Central sensitization is an important pathophysiological mechanism underlying chronic migraine (CM). Previous studies have shown that microglial activation and subsequent inflammation in the trigeminal nucleus caudalis (TNC) contribute to central sensitization. Toll-like receptor 2 (TLR2) is a receptor expressed on the membrane of microglia and participates in central sensitization in inflammatory and chronic pain; however, its role in CM is unclear. Therefore, this study investigated TLR2 involvement in CM in detail. Mice treated with recurrent nitroglycerin (NTG) were used as a CM model. Hyperalgesia was assessed using a 50% paw mechanical threshold and a 50% periorbital threshold on a Von Frey filament pain meter. Western blotting and immunofluorescence analyses were used to detect the expression of TLR2, microglia, c-fos and CGRP in TNC. The expression of inflammatory factors (IL-6, IL-1β、 IL-10、TNF-α and IFN-β1) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). A selective TLR2 antagonist (C29) was systematically administered to observe its effect on hyperalgesia, microglia activation and the expression of c-fos, CGRP and inflammatory factors. Recurrent administration of NTG resulted in acute and chronic hypersensitivity, accompanied by upregulation of TLR2 expression and microglial activation in TNC. C29 partially inhibited pain hypersensitivity. C29 suppressed microglial activation induced by NTG administration. Inhibition of TLR2 reduced the expression of c-fos and CGRP in TNC after NTG treatment. C29 inhibited the expression of inflammatory mediators in TNC. These data showed that microglial TLR2 plays a critical role in the pathogenesis of CM by regulating microglial activation in TNC. [ABSTRACT FROM AUTHOR]

Thymopentin (TP5) plays an important role in host immunomodulation, yet its bioavailability is significantly limited by its short half-life. YW12D is a peptide with strong stability but relatively weak immunoactivity. Tuning the physicochemical properties of such molecules may yield synthetic molecules displaying optimal stability, safety and enhanced immunological activity. Here, natural peptides were modified to improve their activity by hybridization strategies. A hybrid peptide YW12D-TP5 (YTP) that combines TP5 and YW12D is designed. The half-life of YTP in plasma is significantly longer than that of YW12D and TP5. YTP also displays an improved ability to protect the host from CTX-induced weight loss and thymus and spleen indices decrease than YW12D and TP5. In addition, YTP promotes dendritic cell maturation and increases the expression of cytokines IL-1β, IL-6, TNF-α and immunoglobulins IgA, IgG, and IgM. A combination of antibody-specific blocking assay, SPR, molecular dynamics simulations and western blotting suggest that the immunomodulatory effect of YTP is associated with its activation of the TLR2-NF-кB signaling axis. In sum, we demonstrate that peptide hybridization is an effective strategy for redirecting biological activity to generate novel bioactive molecules with desired properties.

IntroductionStreptococcus uberis is a member of the pyogenic cluster of Streptococcus commonly associated with intramammary infection and mastitis in dairy cattle. It is a poorly controlled globally endemic pathogen responsible for a significant cause of the disease worldwide. The ruminant mammary gland provides an atypical body niche in which immune cell surveillance occurs on both sides of the epithelial tissue. S. uberis does not cause disease in non-ruminant species and is an asymptomatic commensal in other body niches. S. uberis exploits the unusual niche of the mammary gland to initiate an innate response from bovine mammary macrophage (BMMO) present in the secretion (milk) in which it can resist the host immune responses. As a result – and unexpectedly - the host inflammatory response is a key step in the pathogenesis of S.uberis, without which colonisation is impaired. In contrast to other bacteria pathogenic to the bovine mammary gland, S. uberis does not elicit innate responses from epithelial tissues; initial recognition of infection is via macrophages within milk.MethodsWe dissected the role of the bacterial protein SUB1154 in the inflammasome pathway using ex vivo bovine mammary macrophages isolated from milk, recombinant protein expression, and a panel of inhibitors, agonists, and antagonists. We combine this with reverse-transcription quantitative real-time PCR to investigate the mechanisms underlying SUB1154-mediated priming of the immune response.ResultsHere, we show that SUB1154 is responsible for priming the NLRP3 inflammasome in macrophages found in the mammary gland. Without SUB1154, IL-1β is not produced, and we were able to restore IL-1β responses to a sub1154 deletion S. uberis mutant using recombinant SUB1154. Surprisingly, only by blocking internalisation, or the cytoplasmic TIR domain of TLR2 were we able to block SUB1154-mediated priming.DiscussionTogether, our data unifies several contrasting past studies and provides new mechanistic understanding of potential early interactions between pyogenic streptococci and the host.

Activation and degranulation of mast cells (MCs) is an essential aspect of innate and adaptive immunity. Skin MCs, the most exposed to the external environment, are at risk of quickly degranulating with potentially severe consequences. Here, we define how MCs assume a tolerant phenotype via crosstalk with dermal fibroblasts (dFBs) and how this phenotype reduces unnecessary inflammation when in contact with beneficial commensal bacteria. We explore the interaction of human MCs (HMCs) and dFBs in the human skin microenvironment and test how this interaction controls MC inflammatory response by inhibiting the nuclear factor κB (NF-κB) pathway. We show that the extracellular matrix hyaluronic acid, as the activator of the regulatory zinc finger (de)ubiquitinating enzyme A20/tumor necrosis factor α-induced protein 3 (TNFAIP3), is responsible for the reduced HMC response to commensal bacteria. The role of hyaluronic acid as an anti-inflammatory ligand on MCs opens new avenues for the potential treatment of inflammatory and allergic disorders.

Abstract During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of cytokines can lead to severe COVID-19. Previous studies suggest that the spike protein (S) of SARS-CoV-2 can elicit cytokine production via the transcription factor NF-κB and the toll-like receptors (TLRs). In this study, we found that: (i) S and the S2 subunit induce CXCL10, a chemokine implicated in severe COVID-19, gene expression by human macrophage cells (THP-1); (ii) a glycogen synthase kinase-3 inhibitor attenuates this induction; (iii) S and S2 do not activate NF-κB but do activate the transcription factor IRF; (iv) S and S2 do not require TLR2 to elicit CXCL10 production or activate IRF; and (v) S and S2 elicit CXCL10 production by peripheral blood mononuclear cells (PBMCs). We also discovered that the cellular response, or lack thereof, to S and S2 is a function of the recombinant S and S2 used. While such a finding raises the possibility of confounding LPS contamination, we offer evidence that potential contaminating LPS does not underly induced increases in CXCL10. Combined, these results provide insights into the complex immune response to SARS-CoV-2 and suggest possible therapeutic targets for severe COVID-19.

Objective To investigate the protective effect of Toll-like receptor (TLR) 2 /TLR9 agonists, Pam2CSK4 (Pam) and CpG ODN (CpG) on mice infected with Acinetobacter baumannii (Ab) in the lungs. Methods Female C57 mice (6~8 weeks old) were randomly divided into PBS, Pam, CpG and Pam+CpG groups. In 24 h after intranasal immunization with different doses of the corresponding agonists, the mice were given a lethal dose of Ab infection in the lungs, and the survival rates of the mice were observed. A sublethal dose lung infection model of Ab was then established, and the bacterial colonization in the blood, lungs, liver, kidneys and spleen was measured respectively in the mice after infection. HE staining was used to observe the pathological damages in the lungs and kidneys. The protective effect of the agonists in the immunized mice against Ab was examined at 1, 3 and 7 d after immunization to explore the protective time window. Pam+CpG was used to stimulate A549 cells and RAW264.7 cells to investigate the killing or phagocytic effects on Ab. Results Compared to PBS, Pam+CpG treatment significantly improved the survival rate of the mice after a lethal dose of Ab lung infection (P

Sophisticated immune evasion strategies enable Helicobacter pylori (H. pylori) to colonize the gastric mucosa of approximately half of the world’s population. Persistent infection and the resulting chronic inflammation are a major cause of gastric cancer. To understand the intricate interplay between H. pylori and host immunity, spatial profiling was used to monitor immune cells in H. pylori infected gastric tissue. Dendritic cell (DC) and T cell phenotypes were further investigated in gastric organoid/immune cell co-cultures and mechanistic insights were acquired by proteomics of human DCs. Here, we show that ADP-heptose, a bacterial metabolite originally reported to act as a bona fide PAMP, reduces H. pylori-induced DC maturation and subsequent T cell responses. Mechanistically, we report that H. pylori uptake and subsequent DC activation by an ADP-heptose deficient H. pylori strain depends on TLR2. Moreover, ADP-heptose attenuates full-fledged activation of primary human DCs in the context of H. pylori infection by impairing type I IFN signaling. This study reveals that ADP-heptose mitigates host immunity during H. pylori infection.

Doxorubicin (DOX)-mediated cardiotoxicity can impair the clinical efficacy of chemotherapy, leading to heart failure (HF). Given the importance of circRNAs and miRNAs in HF, this paper intended to delineate the mechanism of the circular RNA 0006332 (circ -0,006,332)/microRNA (miR)-143/Toll-like receptor 2 (TLR2) axis in doxorubicin (DOX)-induced HF. The binding of miR-143 to circ -0,006,332 and TLR2 was assessed with the dual-luciferase assay, and the binding between miR-143 and circ -0,006,332 was determined with FISH, RIP, and RNA pull-down assays. miR-143 and/or circ -0,006,332 were overexpressed in rats and cardiomyocytes, followed by DOX treatment. In cardiomyocytes, miR-143 and TLR2 expression, cell viability, LDH release, ATP contents, and levels of IL-1β, IL-18, TNF-α, and pyroptosis-related molecules were examined. In rats, cardiac function, serum levels of cardiac enzymes, apoptosis, myocardial fibrosis, and levels of IL-1β, IL-18, TNF-α, TLR2, and pyroptosis-related molecules were detected. miR-143 diminished TLR2 expression by binding to TLR2, and circ -0,006,332 bound to miR-143 to downregulate miR-143 expression. miR-143 expression was reduced and TLR2 expression was augmented in DOX-induced cardiomyocytes. miR-143 inhibited DOX-induced cytotoxicity by suppressing pyroptosis in H9C2 cardiomyocytes. In DOX-induced rats, miR-143 reduced cardiac dysfunction, myocardial apoptosis, myocardial fibrosis, TLR2 levels, and pyroptosis. Furthermore, overexpression of circ -0,006,332 blocked these effects of miR-143 on DOX-induced cardiomyocytes and rats. Circ -0,006,332 stimulates cardiomyocyte pyroptosis by downregulating miR-143 and upregulating TLR2, thus promoting DOX-induced cardiac injury.

There is an urgent need for alternative therapies targeting human dendritic cells (DCs) that could reverse inflammatory syndromes in many autoimmune and inflammatory diseases and organ transplantations. Here, we describe a bispecific antibody (bsAb) strategy tethering two pathogen-recognition receptors at the surface of human DCs. This cross-linking switches DCs into a tolerant profile able to induce regulatory T-cell differentiation. The bsAbs, not parental Abs, induced interleukin 10 and transforming growth factor β1 secretion in monocyte-derived DCs and human peripheral blood mononuclear cells. In addition, they induced interleukin 10 secretion by synovial fluid cells in rheumatoid arthritis and gout patients. This concept of bsAb-induced tethering of surface pathogenrecognition receptors switching cell properties opens a new therapeutic avenue for controlling inflammation and restoring immune tolerance. [ABSTRACT FROM AUTHOR]

Background: The aim was to diagnose Candida in the oral cavity of subjects with type 2 diabetes mellitus (T2DM) using a genotyping technique and compare the results with those from conventional diagnosis by Papanicolaou (Pap) staining. Methods: Palatal mucosa smears were performed on 18 dental care patients diagnosed with T2DM and grade I, II, and III prosthetic stomatitis who met the inclusion criteria; 18 healthy control subjects were also included in the study. Hemoglobin A1c (HbA1c) levels were determined from total blood. Using exfoliative cytology, the Pap staining technique was used to diagnose candidiasis. Exfoliative cytology was also used for molecular diagnosis; DNA was obtained for Candida genotyping, and RNA was used for gene expression studies. Results: Clinical patterns indicated that all subjects were positive for Candida; however, Pap analysis revealed only three positive subjects, whereas end-point polymerase chain reaction (PCR) analysis revealed 15 subjects with some type of Candida. The most common Candida species found were Candida guilliermondii (38.8%), Candida krusei (33.3%), Candida tropicalis, and Candida lusitaniae (22.2%). Interestingly, the coexpression of different species of Candida was found in various patients. In all patients, HbA1c levels were increased. Gene expression analysis showed a significant decrease (p ≤ 0.05) in TLR2 expression in positive subjects, whereas TLR4 expression did not differ significantly among patients. Conclusions: The end-point PCR technique showed better sensitivity for the diagnosis of Candida when compared with the diagnosis by Pap staining. T2DM subjects showed an increased presence of C. guilliermondii that was correlated with decreased TLR2 expression. [ABSTRACT FROM AUTHOR]

The mammal retina does not have the capacity to regenerate throughout life, although some stem and progenitor cells persist in the adult retina and might retain multipotentiality, as previously described in many tissues. In this work we demonstrate the presence of a small lineage− Sca-1+ cell population in the adult mouse retina which expresses functional TLR2 receptors as in vitro challenge with the pure TLR2 agonist Pam3CSK4 increases cell number and upregulates TLR2. Therefore, this population could be of interest in neuroregeneration studies to elucidate its role in these processes. [ABSTRACT FROM AUTHOR]

Objective To investigate the influencing factors of peripheral infections of knee joint tumor prosthesis as well as the value of serum D-D and TLR2 in predicting the infection risks so as to provide a reference for early diagnosis of tumorous periprosthetic infection （PJI） of knee joint. Methods The patients who were treated and followed up in our department from January 2008 to June 2020 were selected. According to the inclusion and exclusion criteria, 136 of the patients were selected. The data including age, gender, BMI, history of diabetes, smoking history, tumor location, stage of malignant tumor, operation time, osteotomy length, intraoperative bleeding, and the percentage of neutrophils, leukocytes, serum D-dimer, and serum TLR value 3 days after operation were collected. The risk factors of PJI and the diagnostic value of serum D-dimer and serum TLR were analyzed. Results The incidence of PJI was 11.76%. Postoperative chemotherapy and operation time ≥ 180 min were the risk factors of PJI （P < 0.05）. The area under curve （AUC） of the combination of two indicators, serum D-dimer and serum TLR2 were 0.917, 0.894 and 0.778, respectively. The AUC of TLR2 was lower than that of the combination of two indicators （P < 0.05）; The sensitivity was 0.975, 0.908 and 0.708, respectively, and the specificity was 0.75, 0.75, and 0.812, respectively. Conclusion Postoperative chemotherapy and operation time ≥ 180 min are the risk factors of PJI. The combination of D-dimer and TLR2 has good diagnostic value. [ABSTRACT FROM AUTHOR]

Objective: To investigate potential mechanism of Helicobacter pylori metabolites antagonizing host innate immunity. Methods: RNA sequencing and pathway enrichment analysis were used to analyze only LPS-stimulated gastric mucosal cells GES-1, GES-1 cells co-treated with LPS and Helicobacter pylori culture supernatant, and untreated GES-1 cells. The culture supernatant of Helicobacter pylori was filtered by a 3KD ultrafiltration tube, and the filtered filtrate (metabolite part) and the retained solution (protein part) were treated with LPS-stimulated GES-1 cells to detect activity of NF-κB pathway, phosphorylation level of NF-κB, secretion levels of NF-κB pathway effectors TNF-α, IL-6 and IL-8. Identification of key metabolites by untargeted metabolic mass spectrometry. Results: Compared with GES-1 cells stimulated only by LPS, after co-treated with LPS and Helicobacter pylori culture supernatant, expression levels of various genes were regulated and tended to the level of GES-1 in untreated gastric mucosal cells, mainly in the NF-κB pathway. After co-treatment with LPS and culture supernatant of Helicobacter pylori, activity of NF-κB pathway was inhibited (P< 0.05). Helicobacter pylori metabolites could inhibit the activity of NF-κ B pathway, inhibit phosphorylation of NF-κB, and inhibit the secretion of NF-κB pathway effectors TNF-α, IL-6 and IL-8 (P<0.05). 1, 5 and 25 µmol/L of Helicobacter pylori metabolite 2-D-Glu-copyranose (2DG) treatment inhibited activity of NF-κB pathway and phosphorylation of NF-κB in GES-1 cells, and secretion of NF-κB pathway effectors TNF-α, IL-6 and IL-8 were inhibited (P<0.05). After 2DG treatment, activity of NF-κB in GES-1 cells with TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10 knockout were significantly decreased (P<0.05); while there was no significant changes in activity of NF-κB in TLR1 and TLR2 knockout GES-1 cells. Both TLR1 and TLR2 interactions were attenuated in GES-1 cells after 2DG treatment. Molecular docking showed that 2DG could bind to TLR2 amino acid disabled R321, K347 and F349, the binding energy was -12 kcal/mol. TLR2 wild-type and mutant plasmids (R321K, K347R, F349A) were constructed, and TLR2-knockout GES-1 cells were respectively transfected. It was found that 2DG treatment did not reduce NF-κB activity in GES-1 cells transfected with TLR2 mutant. Conclusion: Helicobacter pylori metabolite 2DG can interact with TLR2, reduce the formation of heterodimers between TLR2 and TLR1, and inhibit the activity of innate immune NF-κB pathway. [ABSTRACT FROM AUTHOR]

The Arg753Gln polymorphism in Toll-like receptor 2 has been associated with an increased risk of bacterial infections as well as with protection from the late stages of Lyme disease and the acquisition of syphilis. In this study, we determined the presence of this polymorphism in samples collected from men having sex with men/men with women in the Amsterdam Cohort Studies. The presence of the polymorphism was determined by nested PCR, followed by Sanger sequencing. A set of 90 syphilis-seronegative individuals was compared to 95 syphilis-diagnosed participants. A polymorphism allele frequency of 3.9% was found in the control group and 2.63% in the syphilis case group, respectively. None of the individuals showed a homozygous Arg753Gln polymorphism. The number of self-reported sexual contacts was higher in the group of syphilis patients compared to the control group (p = 0.0063). Moreover, in the syphilis case group (n = 49), participants heterozygous for the TLR2-Arg753Gln reported higher numbers of sexual contacts (p = 0.037) compared to wild-type homozygotes. Our findings suggest a possible protective effect of TLR2-Arg753Gln in the acquisition of syphilis. In addition, the determination of the number of self-reported sexual contacts can be used in an evaluation of the protective effect of polymorphism in a population with a low prevalence of it. [ABSTRACT FROM AUTHOR]

Severe fever with thrombocytopenia syndrome (SFTS) is a recently identified infectious ailment triggered by a new strain of bunyavirus. It is distinguished by elevated fatality rates, ranging from 12 % to 30 %. The mechanism underlying the development of severe illness caused by SFTS bunyavirus (SFTSV) is not yet fully understood. To evaluate the role of the TLR2 receptor pathway in regulating Treg function in the progression of SFTS disease and possible mechanisms, sequential serum samples from 29 patients with SFTS (15 mild, 14 severe cases) were examined. Flow cytometry was employed to scrutinize the phenotypic and functional characteristics of TLR2 expression on circulating CD4 T cells, CD8 T cells, and Tregs. In all admitted patients, the evaluation of correlations between the frequencies of the aforementioned cells and SFTS index (SFTSI) was conducted. For SFTS, the levels of TLR2 on CD4 T cells and Tregs were significantly heightened when compared to those in healthy subjects. Additionally, the expression of TLR2 on Tregs exhibited a positive correlation with Ki-67 expression in Tregs and the severity of disease. Additionally, compared with those in uninfected controls, the expression levels of NF-κB in Tregs were significantly increased. Collectively, Tregs may be activated and proliferate through the stimulation of the TLR2/NF-кB pathway in reaction to SFTSV infection.

Background: Vascular dementia (VaD), the second most prevalent type of dementia, lacks a well-defined cause and effective treatment. Our objective was to utilize bioinformatics analysis to discover the fundamental disease-causing genes and pathological mechanisms in individuals diagnosed with VaD. Methods: To identify potential pathogenic genes associated with VaD, we conducted weighted gene co-expression network analysis (WGCNA), differential expression analysis, and protein–protein interaction (PPI) analysis. The exploration of potential biological mechanisms involved the utilization of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. Moreover, a bilateral common carotid artery stenosis (BCAS) mouse model of VaD was established, and the expression of the hub gene, its relationship with cognitive function and its potential pathogenic mechanism were verified by cognitive behavior tests, cerebral blood flow measurement, Western blotting, and immunofluorescence experiments. Results: This study identified 293 DEGs from the brain cortex of VaD patients and healthy controls, among these genes, the Toll-like receptor 2 (TLR2) gene was identified as hub gene, and it was associated with the apoptosis-related pathway PI3K/AKT.The BCAS model demonstrated that the use of TLR2 inhibitors greatly enhanced the cognitive function of the mice (p

BackgroundParacoccidioidomycosis (PCM) is a systemic endemic fungal disease prevalent in Latin America. Previous studies revealed that host immunity against PCM is tightly regulated by several suppressive mechanisms mediated by tolerogenic plasmacytoid dendritic cells, the enzyme 2,3 indoleamine dioxygenase (IDO-1), regulatory T-cells (Tregs), and through the recruitment and activation of myeloid-derived suppressor cells (MDSCs). We have recently shown that Dectin-1, TLR2, and TLR4 signaling influence the IDO-1-mediated suppression caused by MDSCs. However, the contribution of these receptors in the production of important immunosuppressive molecules used by MDSCs has not yet been explored in pulmonary PCM.MethodsWe evaluated the expression of PD-L1, IL-10, as well as nitrotyrosine by MDSCs after anti-Dectin-1, anti-TLR2, and anti-TLR4 antibody treatment followed by P. brasiliensis yeasts challenge in vitro. We also investigated the influence of PD-L1, IL-10, and nitrotyrosine in the suppressive activity of lung-infiltrating MDSCs of C57BL/6-WT, Dectin-1KO, TLR2KO, and TLR4KO mice after in vivo fungal infection. The suppressive activity of MDSCs was evaluated in cocultures of isolated MDSCs with activated T-cells.ResultsA reduced expression of IL-10 and nitrotyrosine was observed after in vitro anti-Dectin-1 treatment of MDSCs challenged with fungal cells. This finding was further confirmed in vitro and in vivo by using Dectin-1KO mice. Furthermore, MDSCs derived from Dectin-1KO mice showed a significantly reduced immunosuppressive activity on the proliferation of CD4+ and CD8+ T lymphocytes. Blocking of TLR2 and TLR4 by mAbs and using MDSCs from TLR2KO and TLR4KO mice also reduced the production of suppressive molecules induced by fungal challenge. In vitro, MDSCs from TLR4KO mice presented a reduced suppressive capacity over the proliferation of CD4+ T-cells.ConclusionWe showed that the pathogen recognition receptors (PRRs) Dectin-1, TLR2, and TLR4 contribute to the suppressive activity of MDSCs by inducing the expression of several immunosuppressive molecules such as PD-L1, IL-10, and nitrotyrosine. This is the first demonstration of a complex network of PRRs signaling in the induction of several suppressive molecules by MDSCs and its contribution to the immunosuppressive mechanisms that control immunity and severity of pulmonary PCM.

In the immune system, the detection of pathogens through various mechanisms triggers immune responses. Several types of specific programmed cell deaths play a role in the inflammatory reaction. This study emphasizes the inflammatory response induced by Actinomycetes. Actinomyces spp. are resident bacteria in human oral plaque and often serve as a bridge for pathogenic bacteria, which lack affinity to the tooth surface, aiding their colonization of the plaque. We aim to investigate the potential role of Actinomyces oris in the early stages of oral diseases from a new perspective. Actinomyces oris MG-1 (A. oris) was chosen for this research. Differentiated THP-1 (dTHP-1) cells were transiently treated with A. oris to model the inflammatory reaction. Cell viability, as well as relative gene and protein expression levels of dTHP-1 cells, were assessed using CCK-8, quantitative real-time polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot assay. The treatment decreased cell viability and increased the expression of inflammatory genes such as IL-1R1 and NLRP3. It was also observed to significantly enhance the release of IL-1β/IL-18 into the supernatant. Immunoblot analysis revealed a notable increase in the expression of N-gasdermin D persisting up to 24 h. Conversely, in models pre-treated with TLR2 inhibitors, N-gasdermin D was detectable only 12 h post-treatment and absent at 24 h. These results suggest that Actinomyces oris MG-1 induces pyroptosis in dTHP-1 cells via TLR2, but the process is not solely dependent on TLR2.

Nocardiosis in fish, caused by Nocardia seriolae (a kind of filamentous bacterium), is a severe disease in aquaculture, affecting over 40 species of marine and freshwater fish and causing great losses. To investigate the relationship between fish nocardiosis and the gut microbiota, we first artificially infected hybrid snakehead (female Channa maculata × male Channa argus) with N. seriolae (1.0 × 107 CFU/mL), then utilized techniques such as high-throughput sequencing of 16 S rRNA, histopathology, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to analyze the changes in microbial community structure, intestinal histopathological characteristics, and related immune genes expression in hybrid snakehead. Changes in gut microbial community structure and diversity were observed starting from day 2, followed by fluctuations on day 4, and significant alterations on day 6. Four major phyla (Actinomycetes, Proteobacteria, Firmicutes, and Bacteroidetes), particularly Actinomycetes, reflected the temporal variations in the gut microbiota structure. The composition of the gut microbiota also underwent significant changes, with beneficial microorganisms such as Lactobacillus decreasing and harmful microorganisms such as Aeromonas and Nocardia increasing. The pathological sections showed that after N. seriolae injection, the fish intestinal villi became distorted and widely spaced, with increasing damage over time, leading to severe shrinkage and atrophy by the sixth day. Additionally, immune-related genes, such as TLR2 and IRF2, exhibited higher mRNA expression levels (P < 0.01) in the intestinal tissues of the infected fish. Overall, this study provides preliminary insights into the dynamic changes in gut microbial community, immune response, and intestinal pathogenic mechanisms during N. seriolae infection in hybrid snakehead, at both the levels of gut microbial composition and mRNA expression. These findings are important for better understanding and managing Nocardia infection in fish.

Background: Vibrio cholerae N-acetylglucosamine-binding protein (GbpA) is a four-domain, secretory colonization factor which is essential for chitin utilization in the environment, as well as in adherence to intestinal cells. GbpA is also involved in inducing intestinal inflammation by enhancing mucin and interleukin-8 secretion. The underlying cell signaling mechanism involved in the induction of the pro-inflammatory response and IL-8 secretion has yet to be deciphered in detail. Methods: Herein, the process through which GbpA triggers the induction of IL-8 in intestinal cells was investigated by examining the role of GbpA in intestinal cell line HT 29. Results: GbpA, specifically through the fourth domain, forms a binding connection with Toll-like receptor 2 (TLR2) and additionally, recruits TLR1 along with CD14 within a lipid raft micro-domain to initiate the signaling pathway. Notably, disruption of this micro-domain complex resulted in a reduction in IL-8 secretion. The lipid raft association served as the catalyst that invoked a downstream cellular inflammatory signaling pathway. This cascade involved the activation of various MAP kinases and NFκB and assembly of the AP-1 complex. This coordinated activation of signaling molecules eventually leads to enhanced IL-8 transcription via increased promoter activity. These findings suggested that GbpA is a crucial protein in V. cholerae, capable of inciting a pro-inflammatory response during infection by orchestrating the formation of the GbpA-TLR1/2-CD14 lipid raft complex. Activation of AP-1 and NFκB in the nucleus eventually enhanced IL-8 transcription through increased promoter activity. Conclusions: Collectively, these findings indicated that GbpA plays a pivotal role within V. cholerae by triggering a pro-inflammatory response during infection. This response is instrumented by the formation of the GbpA-TLR1/2-CD14 lipid raft complex.