Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6.

Helicobacter pylori (H. pylori, Hp) has been designated a class I carcinogen and is closely associated with severe gastric diseases. During colonization in the gastric mucosa, H. pylori develops immune escape by inducing host immune tolerance. The gastric epithelium acts as the first line of defense against H. pylori, with Toll-like receptors (TLRs) in gastric epithelial cells being sensitive to H. pylori components and subsequently activating the innate immune system. However, the mechanism of immune tolerance induced by H. pylori through the TLR signalling pathway has not been fully elucidated. In this research, we detected the expression of TLRs and inflammatory cytokines in GES-1 cells upon sustained exposure to H. pylori or H. pylori lysate from 1 to 30 generations and in Mongolian gerbils infected with H. pylori for 5 to 90 weeks. We found that the levels of TLR6 and inflammatory cytokines first increased and then dropped during the course of H. pylori treatment in vitro and in vivo. The restoration of TLR6 potentiated the expression of IL-1ß and IL-8 in GES-1 cells, which recruited neutrophils and reduced the colonization of H. pylori in the gastric mucosa of gerbils. Mechanistically, we found that persistent infection with H. pylori reduces the sensitivity of TLR6 to bacterial components and regulates the expression of inflammatory cytokines in GES-1 cells through TLR6/JNK signaling. The TLR6 agonist obviously alleviated inflammation in vitro and in vivo. Promising results suggest that TLR6 may be a potential candidate immunotherapy drug for H. pylori infection.

A Cardiolipin from Muribaculum intestinale Induces Antigen-Specific Cytokine Responses.

An systematic phenotypic screen of the mouse gut microbiome for metabolites with an immunomodulatory effect identified Muribaculum intestinale as one of only two members with an oversized effect on T-cell populations. Here we report the identification and characterization of a lipid, MiCL-1, as the responsible metabolite. MiCL-1 is an 18:1-16:0 cardiolipin, whose close relatives are found on concave lipid surfaces of both mammals and bacteria. MiCL-1 was synthesized to confirm the structural analysis and functionally characterized in cell-based assays. It has a highly restrictive structure-activity profile, as its chain-switched analog fails to induce responses in any of our assays. MiCL-1 robustly induces the production of pro-inflammatory cytokines like TNF-α, IL-6, and IL-23, but has no detectable effect on the anti-inflammatory cytokine IL-10. As is the case with other recently discovered immunomodulatory lipids, MiCL-1 requires functional TLR2 and TLR1 but not TLR6 in cell-based assays.

An Update on Toll-like Receptor 2, Its Function and Dimerization in Pro- and Anti-Inflammatory Processes.

While a certain level of inflammation is critical for humans to survive infection and injury, a prolonged inflammatory response can have fatal consequences. Pattern recognition Toll-like receptors (TLRs) are key players in the initiation of an inflammatory process. TLR2 is one of the most studied pattern recognition receptors (PRRs) and is known to form heterodimers with either TLR1, TLR4, TLR6, and TLR10, allowing it to recognize a wide range of pathogens. Although a large number of studies have been conducted over the past decades, there are still many unanswered questions regarding TLR2 mechanisms in health and disease. In this review, we provide an up-to-date overview of TLR2, including its homo- and heterodimers. Furthermore, we will discuss the pro- and anti-inflammatory properties of TLR2 and recent findings in prominent TLR2-associated infectious and neurodegenerative diseases.

Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response.

Host immune activation is critical for enterovirus 71 (EV71) clearance and immunopathogenesis. However, the mechanism of innate immune activation, especially of cell membrane-bound toll-like receptors (TLRs), against EV71 remains unknown. We previously demonstrated that TLR2 and its heterodimer inhibit EV71 replication. In this study, we systematically investigated the effects of TLR1/2/4/6 monomers and TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/TLR4) on EV71 replication and innate immune activation. We found that the overexpression of human- or mouse-derived TLR1/2/4/6 monomers and TLR2 heterodimer significantly inhibited EV71 replication and induced the production of interleukin (IL)-8 via activation of the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) pathways. Furthermore,human-mouse chimeric TLR2 heterodimer inhibited EV71 replication and activated innate immunity. Dominant-negative TIR-less (DN)-TLR1/2/4/6 did not exert any inhibitory effects, whereas DN-TLR2 heterodimer inhibited EV71 replication. Prokaryotic expression of purified recombinant EV71 capsid proteins (VP1, VP2, VP3, and VP4) or overexpression of EV71 capsid proteins induced the production of IL-6 and IL-8 via activation of the PI3K/AKT and MAPK pathways. Notably, two types of EV71 capsid proteins served as pathogen-associated molecular patterns for TLR monomers (TLR2 and TLR4) and TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/TLR4) and activated innate immunity. Collectively, our results revealed that membrane TLRs inhibited EV71 replication via activation of the antiviral innate response, providing insights into the EV71 innate immune activation mechanism.

Sperm activate TLR2/TLR1 heterodimerization to induce a weak proinflammatory response in the bovine uterus.

Toll-like receptor 2 (TLR2) signaling pathway is involved in the sperm-triggered uterine inflammatory response at insemination, but its precise mechanism at molecular-level remains unknown. According to the ligand specificity, TLR2 forms a heterodimer with TLR1 or TLR6 as an initial step to mediate intracellular signaling, leading to a specific type of immune response. Hence, the present study aimed to identify the active TLR2 heterodimer (TLR2/1 or TLR2/6) that is involved in sperm-uterine immune crosstalk in bovine using various models. First, in-vitro (bovine endometrial epithelial cells, BEECs) and ex-vivo (bovine uterine explant) models were employed to test different TLR2 dimerization pathways in endometrial epithelia after exposure to sperm or TLR2 agonists; PAM3 (TLR2/1 agonist), and PAM2 (TLR2/6 agonist). Additionally, in-silico approaches were performed to confirm the dimer stability using de novo protein structure prediction model for bovine TLRs. The in-vitro approach revealed that sperm triggered the mRNA and protein expression of TLR1 and TLR2 but not TLR6 in BEECs. Moreover, this model disclosed that activation of TLR2/6 heterodimer, triggers a much stronger inflammatory response than TLR2/1 and sperm in bovine uterine epithelia. In the ex-vivo model that mimics the intact uterine tissue at insemination, sperm also induced the protein expression of both TLR1 and TLR2, but not TLR6, in bovine endometrium, particularly in uterine glands. Importantly, PAM3 and sperm induced similar and low mRNA expression of pro-inflammatory cytokines and TNFA protein to a lesser extent than PAM2 in endometrial epithelia. This implied that sperm might trigger a weak inflammatory response via TLR2/TLR1 activation which is similar to that of PAM3. Additionally, the in-silico analyses showed that the existence of bridging ligands is essential for heterodimer stability in bovine TLR2 with either TLR1 or TLR6. Altogether, the present findings revealed that sperm utilize TLR2/1, but not TLR2/6, heterodimerization to trigger a weak physiological inflammatory response in the bovine uterus. This might be the way to remove excess dead sperm remaining in the uterine lumen without tissue damage for providing an ideal uterine environment for early embryo reception and implantation.

Bacterial TLR2/6 Ligands Block Ciliogenesis, Derepress Hedgehog Signaling, and Expand the Neocortex.

Microbial components have a range of direct effects on the fetal brain. However, little is known about the cellular targets and molecular mechanisms that mediate these effects. Neural progenitor cells (NPCs) control the size and architecture of the brain and understanding the mechanisms regulating NPCs is crucial to understanding brain developmental disorders. We identify ventricular radial glia (vRG), the primary NPC, as the target of bacterial cell wall (BCW) generated during the antibiotic treatment of maternal pneumonia. BCW enhanced proliferative potential of vRGs by shortening the cell cycle and increasing self-renewal. Expanded vRGs propagated to increase neuronal output in all cortical layers. Remarkably, Toll-like receptor 2 (TLR2), which recognizes BCW, localized at the base of primary cilia in vRGs and the BCW-TLR2 interaction suppressed ciliogenesis leading to derepression of Hedgehog (HH) signaling and expansion of vRGs. We also show that TLR6 is an essential partner of TLR2 in this process. Surprisingly, TLR6 alone was required to set the number of cortical neurons under healthy conditions. These findings suggest that an endogenous signal from TLRs suppresses cortical expansion during normal development of the neocortex and that BCW antagonizes that signal through the TLR2/cilia/HH signaling axis changing brain structure and function. IMPORTANCE Fetal brain development in early gestation can be impacted by transplacental infection, altered metabolites from the maternal microbiome, or maternal immune activation. It is less well understood how maternal microbial subcomponents that cross the placenta, such as bacterial cell wall (BCW), directly interact with fetal neural progenitors and neurons and affect development. This scenario plays out in the clinic when BCW debris released during antibiotic therapy of maternal infection traffics to the fetal brain. This study identifies the direct interaction of BCW with TLR2/6 present on the primary cilium, the signaling hub on fetal neural progenitor cells (NPCs). NPCs control the size and architecture of the brain and understanding the mechanisms regulating NPCs is crucial to understanding brain developmental disorders. Within a window of vulnerability before the appearance of fetal immune cells, the BCW-TLR2/6 interaction results in the inhibition of ciliogenesis, derepression of Sonic Hedgehog signaling, excess proliferation of neural progenitors, and abnormal cortical architecture. In the first example of TLR signaling linked to Sonic Hedgehog, BCW/TLR2/6 appears to act during fetal brain morphogenesis to play a role in setting the total cell number in the neocortex.

BacSp222 bacteriocin as a novel ligand for TLR2/TLR6 heterodimer.

OBJECTIVE AND DESIGN: BacSp222 bacteriocin is a bactericidal and proinflammatory peptide stimulating immune cells to produce selected cytokines and NO in NF-ĸB dependent manner. This study aims to identify the receptor which mediates this activity. METHODS: We applied fluorescently labeled BacSp222 and a confocal microscopy imaging to analyze the direct interaction of the bacteriocin with the cells. Reporter HEK-Blue cells overexpressing human toll-like receptors (TLR2, TLR4, TLR5 or TLR2/TLR1 and TLR2/TLR6 heterodimers) were stimulated with BacSp222, and then the activity of NF-ĸB-dependent secreted embryonic alkaline phosphatase (SEAP) was measured. In turn, formylated peptide receptor (FPR) or TLR2 antagonists were used to verify bacteriocin-stimulated TNF production by murine monocyte-macrophage cell lines. RESULTS: BacSp222 undergoes internalization into cells without disturbing the cell membrane. FPR antagonists do not affect TNF produced by BacSp222-stimulated murine macrophage-like cells. In contrast, BacSp222 stimulates NF-ĸB activation in HEK-Blue overexpressing TLR2 or TLR2/TLR6 heterodimer, but not TLR2/TLR1, TLR4 or TLR5 receptors. Moreover, TLR2-specific antagonists inhibit NF-ĸB signaling in BacSp222-stimulated HEK-Blue TLR2/TLR6 cells and reduce TNF release by BacSp222-treated RAW 264.7 and P388.D1. CONCLUSIONS: BacSp222 is a novel ligand for TLR2/TLR6 heterodimer. By binding TLR complex the bacteriocin undergoes internalization, inducing proinflammatory signaling that employs MyD88 and NF-ĸB pathways.

Gene Silencing of Toll-like Receptor 2 Gene Expression as a Tactic to Control Mycobacterium Tuberculosis and Granuloma Formation.

Mycobacterium tuberculosis (MT) is the causative agent of tuberculosis (TB) in humans. Tuberculosis is one of the top 10 causes of mortality worldwide, resulting in 1.8 million deaths and 10.4 million new cases in 2016. Understanding the fundamental features of MT biology is critical to the eradication of MT in the future. Due to the increasing frequency of antimicrobial treatment resistance and problems in vaccine development, the pathogenesis of TB for its survival and growth is highly dependent on host lipids and stimulated-lipid droplets formation. Toll-like receptor 2 (TLR2) forms heterophilic dimers with TLR1 and TLR6, therefore, recognizing many MT components. Both of these receptors identify the invading antigen and activate downstream protein kinases. Some studies demonstrated that the cyclooxygenase-2 (COX-2) promoter-driven gene expression includes connecting sites for transcription factors, such as nuclear factor-kappa B, CREB, NFAT, and c/EBPß. The current study aimed to investigate the role of the TLR2 receptor in positively regulating prostaglandin E2 production in M. bovis (BCG) infected macrophages in vivo using a human monocytic cell line THP-1. Our results revealed that MT infection triggers a time-dependent increase in COX-2 expression via pathways involving TLR2 receptor activation and enhances COX-2 expression, leading to an increase in lipid droplet formation and suppression of macrophage activation.

Structural and functional implications of leucine-rich repeats in toll-like receptor1 subfamily.

Leucine-rich repeats (LRRs) - the protein-protein and protein-ligand interaction motif of proteins participating in a plethora of functions in plants, vertebrates, invertebrates, and prokaryotes - are a fascinating piece of conserved yet versatile structural motif. In toll-like receptors (TLRs), this domain forms the extracellular part that is preceded by an intracellular toll/interleukin-1 receptor (TIR) domain. The extracellular part is crucial for recognizing a structurally diverse set of viral, bacterial, fungal, and parasite-derived components, while the TIR domain is recruited for activation of downstream signaling following recognition. The distinct ability of the paralogs TLR1 and TLR6 to dimerize with TLR2 and recognize different ligands intrigued and motivated us to exchange the dimerizing and ligand-binding residues between TLR1/6 and note the effect on dimer formation and ligand binding. The appreciable sequence modification brought about no significant alteration in the native scaffold of the motif, as revealed from the comparison of simulations with wild-type dimers. Moreover, docking of the exchanged ligands to the variant proteins supported favorable binding. Thus, the structural stability and the functional plasticity offered by the motif might be the reason for its extensive use across cellular functions and life forms, a feature crucial for coevolution and the knowledge essential for therapeutics.

Cross-Talk between HLA Class I and TLR4 Mediates P-Selectin Surface Expression and Monocyte Capture to Human Endothelial Cells.

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin ß4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.

Bovine colostrum promoted ileal health in newborn lambs at 24 h after birth: insight from intestinal morphology and innate immunity.

The contribution of colostrum to passive immunity transfer and intestinal protection in newborn ruminants is well known; however, it is currently unclear how colostrum intake affects intestinal innate immunity. We investigated the effects of bovine colostrum intake on ileal morphology, expression of genes involved in intestinal innate immunity, and serum concentrations of inflammatory cytokines in newborn lambs. Twenty-seven newborn male Hu lambs were used, of which 18 were bottle-fed either bovine colostrum (C24h; n = 9) or bovine mature milk (M24h; n = 9) within the first 2 h after birth at an intake of approximately 8% of BW; the remaining nine lambs did not receive any feeding (N24h). Blood and ileal tissue samples were collected after the lambs were slaughtered at 24 h after birth. Ileal villus height and villus height-to-crypt depth ratio were significantly higher in C24h than those in N24h and M24h lambs (P < 0.01). Messenger RNA (mRNA) abundance of toll-like receptor (TLR)-2, TLR3, TLR4, TLR6, TLR7, TLR8 and tumour necrosis factor alpha in the ileum was lower in C24h than that in N24h lambs (P < 0.05). Moreover, C24h lambs had a lower TLR3 mRNA abundance (P < 0.01) and a trend of lower TLR6 (P = 0.06) and interleukin 1 beta (P = 0.08) expression compared with those in M24h lambs. We also observed strong positive correlations of tumour necrosis factor alpha expression with that of TLR2 (r = 0.71; P < 0.001), TLR4 (r = 0.88; P < 0.001) and TLR8 (r = 0.83; P < 0.001). Interestingly, the expression of barrier-related molecules, including mucin-13, lysozyme, claudin (CLDN)-1, CLDN2, CLDN4, CLDN7, CLDN12, occludin, zonula occluden-1 and junctional adhesion molecule-1, was consistently lower in C24h lambs than that in N24h and M24h lambs (P < 0.05). These results indicated that the beneficial roles of colostrum intake on intestinal protection in newborn lambs were associated with low TLR expression, which was reflected by improved intestinal development and reduced inflammatory response. Further studies using fluorescence in situ hybridisation and immunohistochemical methods are needed to further explore the mechanisms underlying the lower expression of intestinal barrier-related molecules due to colostrum feeding.

Toll-like receptor and cytokine expression throughout the bone marrow differs between patients with low- and high-risk myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders, the pathogenesis of which involves enhanced immune signaling that promotes or selects for mutant hematopoietic stem and progenitor cells (HSPCs). In particular, toll-like receptor (TLR) expression and signaling are enhanced in MDS, and their inhibition is an attractive therapeutic strategy. Although prior studies have reported increased expression of TLR2 and its binding partners TLR1 and TLR6 in the CD34+ cells of patients with MDS (especially those with low-risk disease), TLR expression in other cell types throughout the bone marrow is largely unknown. To address this, we used mass cytometry to assess the expression of TLR1, TLR2, and TLR6 and cytokines in the bone marrow hematopoietic cells of six low/intermediate-risk and six high-risk unmatched MDS bone marrow samples, as well as healthy controls, both at baseline and in response to TLR agonists. We observed several consistent differences between the groups. Most notably, TLR expression was upregulated in multiple cell populations in the low/intermediate-risk, but not high-risk, patients. In addition, many cytokines, including interleukin-6, interleukin-8, tumor necrosis factor α, transforming growth factor ß, macrophage inflammatory protein 1ß, and granzyme B, were highly expressed from various cell types in low/intermediate-risk patients. However, these same cytokines, with the exception of transforming growth factor ß, were expressed at lower levels in high-risk MDS. Together, these findings highlight the differential role of inflammation, and specifically TLR expression, in low/intermediate- versus high-risk MDS, and suggest that elevated TLR expression and cytokine production in multiple cell types likely influences the pathogenesis of MDS in lower-risk patients.

Pilus proteins from Streptococcus pyogenes stimulate innate immune responses through Toll-like receptor 2.

The group A Streptococcus (GAS) pilus is a long, flexible, hair-like structure anchored to the cell surface that facilitates the adherence of GAS to host cells, thus playing a critical role in initiating infections. Because of its important role in GAS virulence, the pilus has become an attractive target for vaccine development. While current research mainly focuses on pilus function and its potential as a vaccine component, there is a lack of knowledge on how the host immune system recognizes and responds to this abundant surface structure. Here we show that both assembled GAS pili and individual pilus proteins induce a potent release of the proinflammatory cytokines tumor necrosis factor and interleukin-8. We further show that the surface-exposed backbone pilin and ancillary pilin 1 subunits are Toll-like receptor 2 (TLR2) agonists. Using reporter cell lines coexpressing human TLR2 in combination with either TLR1 or TLR6, we determined that activation was mediated by the TLR2/TLR6 heterodimer. Finally, we used solid-phase and flow cytometry binding assays to illustrate a direct interaction between the pilus subunits and TLR2. These results provide further support for the suitability of the pilus as a vaccine component and opens potential avenues for using GAS pili as an adjuvant or immune-modulation agent.

Endoplasmic reticulum proteins Meigo and Gp93 govern dendrite targeting by regulating Toll-6 localization.

Neuronal target recognition is performed by numerous cell-surface transmembrane proteins. Correct folding of these proteins occurs in the endoplasmic reticulum (ER) lumen of the neuronal cells before being transported to the plasma membrane of axons or dendrites. Disturbance in this protein folding process in the ER leads to dysfunction of neuronal cell surface molecules, resulting in abnormal neuronal targeting. In this study, we report that the ER-resident protein Meigo in Drosophila, governs the dendrite targeting of olfactory projection neurons (PNs) along the mediolateral axis of the antennal lobe by regulating Toll-6 localization. Loss of Meigo causes Toll-6 mislocalization in the PNs and mediolateral dendrite targeting defects, which are suppressed by Toll-6 overexpression. Furthermore, we found that the ER-chaperone protein, Gp93, also regulates the mediolateral targeting of PN dendrites by localization of the Toll-6 protein. Gp93 overexpression in the PN homozygous for the meigo mutation, partially rescued the dendrite targeting defect, while meigo knockdown decreased Gp93 expression levels in cultured cells. These results indicate that the ER-proteins Meigo and Gp93 regulate dendrite targeting by attenuating the amount and localization of cell surface receptors, including Toll-6, implying the unexpected but active involvement of ER proteins in neural wiring.

Interaction of Ectodomain of Respiratory Syncytial Virus G Protein with TLR2/ TLR6 Heterodimer: An In vitro and In silico Approach to Decipher the Role of RSV G Protein in Pro-inflammatory Response against the Virus.

BACKGROUND: Human respiratory syncytial virus (RSV) has been shown to be linked with various forms of respiratory diseases, such as common cold and lower respiratory tract illnesses like pneumonia and bronchiolitis. TLRs play critical role in generating host immune response against RSV. TLRs are expressed not only on leukocytes but also on many other cell types and can recognize RSV. Previous studies have established that RSV can interact with TLR4 and initiate inflammatory cascade of cytokines. The data from a recent study indicated that TLR2/TLR6 is involved in RSV recognition and subsequent innate immune activation. However, the nature of binding and the envelope protein of RSV involved in this interaction with TLRs are not studied yet. OBJECTIVE: We hypothesized that RSV G protein can bind to TLRs and mediate the inflammatory immune response against the virus infection. Therefore, we investigated whether RSV G protein could activate innate immune response through TLR signaling. METHODS: Different TLR antagonists were used to assess the effect of the exposure of RSV and RSV G ectodomain in human primary small airway epithelial cells (HSAECs). Various inflammatory cytokines, chemokines and type I IFNs were measured by ELISA along with their mRNA expression by qPCR. In silico interaction of RSV G protein with TLR2/TLR6 was also analyzed. RESULTS: Results of ELISA and qPCR analysis have shown that TLR2/TLR6 signaling is activated in HSAECs upon RSV and RSV G protein exposure which initiates innate immune response against RSV. Moreover, RSV envelope protein G plays a crucial role in binding and activation of TLR2/TLR6 signaling. CONCLUSION: In summary, our study shows that TLR2/TLR6 play important role in the activation of innate immune response upon RSV recognition which could be helpful in promoting RSV clearance and preventing RSV-induced disease.

RAGE-mediated functional DNA methylated modification contributes to cigarette smoke-induced airway inflammation in mice.

Our previous study indicated knockout of receptor for advanced glycation end-products (RAGE) significantly attenuated cigarette smoke (CS)-induced airway inflammation in mice. In the present study, we aim to further detect the mediatory effects of RAGE in DNA methylated modification in CS-induced airway inflammation. Lung tissues from the CS-exposed mouse model of airway inflammation were collected for profiling of DNA methylation by liquid hybridization capture-based bisulfite sequencing, which were used for conjoint analysis with our previous data of gene expression by cDNA microarray to identify functional methylated genes, as well as hub genes selected by protein-protein interaction (PPI) network analysis, and functional enrichment analyses were then performed. After RAGE knockout, 90 genes were identified by intersection of the differentially methylated genes and differentially expressed genes. According to the reversed effects of methylation in promoters on gene transcription, 14 genes with functional methylated modification were further identified, among which chemokine (C-X-C motif) ligand 1 (CXCL1), Toll-like receptor 6 (TLR6) and oncostatin M (OSM) with hypomethylation in promoters, were selected as the hub genes by PPI network analysis. Moreover, functional enrichment analyses showed the 14 functional methylated genes, including the 3 hub genes, were mainly enriched in immune-inflammatory responses, especially mitogen-activated protein kinase, tumor necrosis factor, TLRs, interleukin (IL)-6 and IL-17 pathways. The present study suggests that RAGE mediates functional DNA methylated modification in a cluster of 14 targeted genes, particularly hypomethylation in promoters of CXCL1, TLR6 and OSM, which might significantly contribute to CS-induced airway inflammation via a network of signaling pathways.

Inhibitory effect of strawberry geranium (Saxifraga stolonifera) on Toll-like receptor 2-mediated inflammatory response in human skin keratinocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Strawberry geranium (Saxifraga stolonifera [L.] Meeb) has traditionally been used as a drug to treat skin disorders in Japan. However, little is known about its physiological effects on skin keratinocytes. AIM OF THE STUDY: We investigated the anti-inflammatory effects of a strawberry geranium extract (SGE) on human skin keratinocytes. MATERIALS AND METHODS: The human keratinocyte cell line, HaCaT, was treated with SGE, and then stimulated with tumor necrosis factor (TNF)-α. The expression of 207 genes related to the innate immune system was analyzed using DNA microarrays. The effect of SGE on the target proteins in primary human epidermal keratinocytes was confirmed by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The mechanisms of action and active components involved in the suppressive effect of SGE were evaluated by fractionation and a transcription assay. RESULTS: The microarray analysis revealed that SGE primarily suppressed Toll-like receptor (TLR)2 expression through procyanidin B2 3,3'-di-O-gallate, without TLR2 downregulation, in TNF-α-stimulated HaCaT cells. SGE suppressed TLR2 expression and interleukin (IL)-8 production induced by TLR2 ligands in primary human epidermal keratinocytes and HaCaT cells. Multiple components downregulating TLR2 expression suppressed the Sp1 activity. CONCLUSIONS: We identified a novel physiological function of SGE, which suppresses TLR2 expression and TLR2-mediated inflammation in human skin keratinocytes. This study provides significant insights into the anti-inflammatory effect of SGE in human skin.

Puerarin inhibits Mycoplasma gallisepticum (MG-HS)-induced inflammation and apoptosis via suppressing the TLR6/MyD88/NF-κB signal pathway in chicken.

Mycoplasma gallisepticum (MG) is the primary etiological agent of chicken chronic respiratory disease (CRD), which mainly causes inflammatory damage of the host respiratory system. Previous studies suggest that puerarin (PUE) plays a pivotal regulatory role in inflammatory diseases, whereas the impacts of PUE on MG-induced inflammation remain unclear. This study investigated the effects of PUE on MG-HS infection in vitro and in vivo and indicated its potential therapeutic and preventive value. Experimental results showed that PUE significantly suppressed pMGA1.2 expression, promoted MG-infected cell proliferation and cell cycle process by reducing apoptosis. Histopathological examination of lung tissue showed severe histopathological lesions including thickened alveolar walls, narrowed alveolar cavity, and inflammatory cell infiltration in the MG-infected chicken group. However, PUE treatment significantly ameliorated MG-induced pathological damage in lung. Compared to the MG-infected group, PUE effectively inhibited the expression of MG-induced inflammatory genes, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), cytokines interleukin-6 (IL-6), toll-like receptor 6 (TLR6), myeloid differentiation primary response gene 88 (MyD88) and nuclear factor κB (NF-κB). Moreover, PUE dose-dependently inhibited MG-induced NF-κB p65 to enter the cell nucleus. In conclusion, our findings indicate that PUE treatment can efficiently inhibit MG-induced inflammatory response and apoptosis, and protect the lung from MG infection-induced damage by inhibiting the TLR6/MyD88/NF-κB signaling pathway activation. The study suggests that PUE may be a potential anti-inflammatory agent defense againstMGinfection in chicken.

Efficient 5-OP-RU-Induced Enrichment of Mucosa-Associated Invariant T Cells in the Murine Lung Does Not Enhance Control of Aerosol Mycobacterium tuberculosis Infection.

Mucosa-associated invariant T (MAIT) cells are an innate-like T cell subset in mammals that recognize microbial vitamin B metabolites presented by the evolutionarily conserved major histocompatibility complex class I (MHC I)-related molecule, MR1. Emerging data suggest that MAIT cells may be an attractive target for vaccine-induced protection against bacterial infections because of their rapid cytotoxic responses at mucosal services to a widely conserved bacterial ligand. In this study, we tested whether a MAIT cell priming strategy could protect against aerosol Mycobacterium tuberculosis infection in mice. Intranasal costimulation with the lipopeptide Toll-like receptor (TLR)2/6 agonist, Pam2Cys (P2C), and the synthetic MR1 ligand, 5-OP-RU, resulted in robust expansion of MAIT cells in the lung. Although MAIT cell priming significantly enhanced MAIT cell activation and expansion early after M. tuberculosis challenge, these MAIT cells did not restrict M. tuberculosis bacterial load. MAIT cells were depleted by the onset of the adaptive immune response, with decreased detection of granzyme B+ and gamma interferon (IFN-γ)+ MAIT cells relative to that in uninfected P2C/5-OP-RU-treated mice. Decreasing the infectious inoculum, varying the time between priming and aerosol infection, and testing MAIT cell priming in nitric oxide synthase 2 (NOS2)-deficient mice all failed to reveal an effect of P2C/5-OP-RU-induced MAIT cells on M. tuberculosis control. We conclude that intranasal MAIT cell priming in mice induces early MAIT cell activation and expansion after M. tuberculosis exposure, without attenuating M. tuberculosis growth, suggesting that MAIT cell enrichment in the lung is not sufficient to control M. tuberculosis infection.

Polydatin attenuates Mycoplasma gallisepticum (HS strain)-induced inflammation injury via inhibiting the TLR6/ MyD88/NF-κB pathway.

Mycoplasma gallisepticum (MG) infection is the main cause of chronic respiratory disease (CRD) characterized by severe respiratory inflammation in chickens. Polydatin (PD) is a resveratrol glycoside isolated from Polygonum cuspidatum, which has prominent anti-inflammatory effect. The purpose of this study was to investigate the therapeutic effect of PD against MG-induced inflammation in chicken and its underlying mechanism. Histopathological analysis showed that PD treatment (15, 30, and 45 mg/kg) apparently alleviated MG-induced pathological changes of chicken embryonic lung. In chicken embryo fibroblast (DF-1) cells, PD treatment (15, 30, and 60 µg/mL) could effectively suppress MG propagation, promote MG-infected cell proliferation and cell cycle progress, and inhibit MG-induced cell apoptosis. ELISA and qPCR assays showed that PD treatment significantly suppressed the expression of interleukin-6 (IL-6), IL-1ß and tumor necrosis factor-α (TNF-α) induced by MG both in vivo and in vitro. Besides, molecular studies indicated that the MG-induced levels of toll-like receptor-6 TLR6, myeloid differentiation-88 (MyD88) and nuclear factor κB (NF-κB) were significantly decreased by PD treatment. Moreover, immunofluorescence analysis showed that PD treatment restrained the MG-induced NF-κB-p65 nuclear translocation. Taken together, these results indicate the protective effects of PD against MG-induced inflammation injury in chicken were mainly by inhibiting the TLR6/MyD88/NF-κB pathway.