Anti-inflammatory effects of chlorogenic acid from Taraxacum officinale on LTA-stimulated bovine mammary epithelial cells via the TLR2/NF-κB pathway.

Mastitis is an inflammatory disease caused by microbial infection. Chlorogenic acid (CGA), one of the major phenolic acids in Taraxacum officinale, has natural antioxidant and anti-inflammatory properties in various cell types; however, the effects of CGA on Lipoteichoic acid (LTA)-induced bovine mammary epithelial cells (BMECs) have not been investigated. In this study, the CGA content in T. officinale was determined by High-performance liquid chromatography (HPLC). BMECs were infected with LTA to induce the mastitis model. Different concentrations of CGA were administered after establishing the LTA infection. The results showed that the T. officinale contained CGA 1.36 mg/g. CGA significantly reduced the pro-inflammatory gene and protein expression of TNF-α, IL-6, and IL-1ß. In addition, CGA downregulated the NO, TLR2, and NF-κB signaling pathways in LTA-infected bovine mammary epithelial cells. Our results indicate that CGA reduced the expression of TNF-α, IL-6, IL-1ß, and TLR2 by inhibiting the phosphorylation of proteins in the NF-κB signaling pathways in a dose-dependent manner. This finding suggests that CGA may be a potential agent for the treatment of mastitis in dairy cows.

Simultaneous control of infection and inflammation with keratin-derived antibacterial peptides targeting TLRs and co-receptors.

Controlling infection-driven inflammation is a major clinical dilemma because of limited therapeutic options and possible adverse effects on microbial clearance. Compounding this difficulty is the continued emergence of drug-resistant bacteria, where experimental strategies aiming to augment inflammatory responses for enhanced microbial killing are not applicable treatment options for infections of vulnerable organs. As with corneal infections, severe or prolonged inflammation jeopardizes corneal transparency, leading to devastating vision loss. We hypothesized that keratin 6a-derived antimicrobial peptides (KAMPs) may be a two-pronged remedy capable of tackling bacterial infection and inflammation at once. We used murine peritoneal neutrophils and macrophages, together with an in vivo model of sterile corneal inflammation, to find that nontoxic and prohealing KAMPs with natural 10- and 18-amino acid sequences suppressed lipoteichoic acid (LTA)- and lipopolysaccharide (LPS)-induced NFκB and IRF3 activation, proinflammatory cytokine production, and phagocyte recruitment independently of their bactericidal function. Mechanistically, KAMPs not only competed with bacterial ligands for cell surface Toll-like receptor (TLR) and co-receptors (MD2, CD14, and TLR2) but also reduced cell surface availability of TLR2 and TLR4 through promotion of receptor endocytosis. Topical KAMP treatment effectively alleviated experimental bacterial keratitis, as evidenced by substantial reductions of corneal opacification, inflammatory cell infiltration, and bacterial burden. These findings reveal the TLR-targeting activities of KAMPs and demonstrate their therapeutic potential as a multifunctional drug for managing infectious inflammatory disease.

CD36 neutralisation blunts TLR2-IRF7 but not IRF3 pathway in neonatal mouse brain and immature human microglia following innate immune challenge.

Innate immune response in neonatal brain is associated with a robust microglial activation and induction of Toll-like Receptors (TLRs). To date, the role of the scavenger receptor CD36 in TLRs modulation, particularly TLR2 signaling, has been well established in adult brain. However, the crosstalk between TLR4, TLR2 and CD36 and its immunogenic influence in the neonatal brain remains unclear. In this study, using a CD36 blocking antibody (anti-CD36) at post-natal day 8, we evaluated the response of neonates to systemic endotoxin (lipopolysaccharide; LPS) challenge. We visualized the TLR2 response by bioluminescence imaging using the transgenic mouse model bearing the dual reporter system luciferase/green fluorescent protein under transcriptional control of a murine TLR2 promoter. The anti-CD36 treatment modified the LPS induced inflammatory profile in neonatal brains, causing a significant decrease in inflammatory cytokine levels and the TLR2 and TLR3 mediated signalling.The interferon regulatory factor 3 (IRF3) pathway remained unaffected. Treatment of the LPS-challenged human immature microglia with anti-CD36 induced a marked decrease in TLR2/TLR3 expression levels while TLR4 and IRF3 expression was not affected, suggesting the shared CD36 regulatory mechanisms in human and mouse microglia. Collectively, our results indicate that blocking CD36 alters LPS-induced inflammatory profile of mouse and human microglia, suggesting its role in fine-tuning of neuroinflammation.

A novel type lavandulyl flavonoid from Sophora flavescens as potential anti-hepatic injury agent that inhibit TLR2/NF-κB signaling pathway.

ETHNOPHARMACOLOGY RELEVANCE: Sophora flavescens Aiton, was a crucial source of Traditional Chinese Medicine (TCM) that has benefited human health for hundreds of years. Alkaloids and flavonoids were the major bioactive constituents from S. flavescens, which had been widely used for liver disease treatment in China. However, the liver-protective components of flavonoids from S. flavescens and their mechanism of action were not clear. AIM OF THE STUDY: This work aimed to evaluate the in vitro hepatoprotective activities of 35 flavonoids from S. flavescens and screen active compounds. Furthermore, it was conducted to demonstrate the hepatoprotective effects of a new active compound (kurarinol A, 1) was isolated by authors and the ethyl acetate (EtOAc) extract form S. flavescens against carbon tetrachloride (CCl4)-induced hepatic injury in Kunming (KM) mice, meanwhile revealed the potential mechanism. MATERIALS AND METHODS: The 35 flavonoids from S. flavescens were co-incubated with HepG2 cells and treated with 0.35% CCl4 for 6 h cell viability was measured by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay. Then, in vivo animal experiments, the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in the serum were analyzed, the degree of hepatic injury was examined using hematoxylin-eosin (H&E) staining, the mRNA expression of Superoxide Dismutase 2 (SOD2), Nuclear factor E2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), Interleukin 6 (IL-6), Tumor Necrosis Factor-α (TNF-α), interleukin-1ß (IL-1ß), and the protein levels of nuclear factor-kappa B p65/p-p65 (NF-κB p65/p-p65), toll-like receptor 2 (TLR2), IL-1ß and cyclooxygenase-2 (COX2) in hepatic tissues were detected. RESULTS: The lavandulyl flavonoid (kurarinol A, 1) and the EtOAc extract from S. flavescens showed protective effects on CCl4-injured HepG2 cells, increasing cell viability from 24.5% to 61.3% and 91.8%, respectively. What's more, we found that treatment with kurarinol A (1) and the EtOAc extract lead to a significant reduction in hepatotoxicity in response to acute CCl4 exposure. Compared with the model group, experimental results exhibited kurarinol A (10 mg/kg, i.p.) and the EtOAc extract (300 mg/kg, i.p.) could decrease the levels of AST, ALT, ALP and tissue damage. Further mechanistic investigations revealed that up-regulated the mRNA expression of SOD2, Nrf2, OH-1 and down-regulated the IL-1ß in liver tissues, respectively. Additionally, Western blot analyses elucidated that inhibition of IL-1ß, TLR2, COX-2, NF-κB (p65/p-p65) via TLR2/NF-κB signaling pathway by kurarinol A and the EtOAc extract contribute to its hepatoprotective activity. CONCLUSION: These findings demonstrated that the novel compound (kurarinol A, 1) possessed notable hepatoprotective activity against CCl4. It was confirmed that kurarinol A had a certain effect on mice with liver damage induced by CCl4, and its mechanism could be include inhibiting inflammation and reducing of oxidative stress reaction by regulating expression of related genes and proteins. Thus, kurarinol A could as a novel active agent that contributes to the hepatoprotective activity of S. flavescens for the treatment of live injury.

Dapagliflozin ameliorates diabetic renal injury through suppressing the self-perpetuating cycle of inflammation mediated by HMGB1 feedback signaling in the kidney.

Dapagliflozin, the Sodium-glucose cotransporter 2 (SGLT2) inhibitor class of glucose-lowering agents, has shown the significantly nephroprotective effects to reduce the risk of kidney failure in diabetes. However, the underlying mechanisms are incompletely understood to explain the beneficial effects of dapagliflozin on kidney function. Here, we demonstrated that the administered of dapagliflozin for 12 weeks improved the proteinuria, histomorphology damage, oxidative stress, and macrophage infiltrations in the kidney of streptozotocin (STZ)-induced diabetic mice. Meanwhile, dapagliflozin attenuated the renal inflammation and fibrosis by reducing the pro-inflammatory factors interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor α (TNF-α) and anti-fiber factor fibronectin (FN) and elevating the anti-inflammatory factor IL-10. Our data revealed that dapagliflozin exerted anti-inflammatory effects by inhibiting the activation of high mobility group box 1 (HMGB1)/TLR2/4/NF-κB signaling pathway. Consistently, we found that dapagliflozin suppressed the expression of HMGB1 and downstream TLR2/4/NF-κB signaling proteins in the human proximal tubular (HK-2) stimulated by high glucose and lipids or HMGB1 and RAW264.7 cells stimulated by IL-1ß, respectively. Further experiments were performed in the indirect co-culture model of RAW264.7 and HK-2 cells induced by high glucose and lipids. The results again confirmed the effects of dapagliflozin on alleviating inflammatory response and regulating the proportions of M1/M2 macrophage. It is indicated that the feedback signaling of HMGB1 between the tubules and macrophage involves in the persistence of the inflammation. These data demonstrate that dapagliflozin suppress the self-perpetuating inflammation by blocking the feedback loop of HMGB1 in the kidney, which contribute to ameliorate the renal injury in diabetes.

Quercetin alleviates Mycoplasma gallisepticum-induced inflammatory damage and oxidative stress through inhibition of TLR2/MyD88/NF-κB pathway in vivo and in vitro.

Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) in chickens leads to enormous economic damage to the poultry industry yearly. The active components and mechanism of action of the traditional herbal remedy Ephedra houttuynia powder (EHP), which had been approved for clinical treatment against MG infection in China, remain unknown. In this study, the active components of EHP against MG were screened using a network pharmacological method, additionally, we studied the mechanism of action of the screened results (quercetin (QUE)). The findings demonstrated that QUE was an essential element of EHP against MG infection, effectively attenuating MG-induced oxidative stress and activation of the TLR2/MyD88/NF-κB pathway. Following QUE therapy, IL-1, IL-6, and TNF-α content and expression were downregulated, whereas IL-4 and IL-10 expression were upregulated, eventually suppressing the inflammatory response both in vitro and in vivo. Together, this study presents a strong rationale for using QUE as a therapeutic strategy to inhibit MG infection-induced inflammatory damage and oxidative stress.

α-Synuclein induces prodromal symptoms of Parkinson's disease via activating TLR2/MyD88/NF-κB pathway in Schwann cells of vagus nerve in a rat model.

BACKGROUND: Increasing evidence suggests that patients with Parkinson's disease (PD) present with peripheral autonomic dysfunction (AutD) that even precedes motor deficits, through which α-synuclein can spread to the central nervous system. However, the pathological mechanisms underlying AutD in prodromal PD remain unclear. Here, we investigated the role of α-synuclein and its interplay with the activation of Schwann cells (SCs) of the vagus nerve in AutD. METHODS: Rats were subjected to injection with adeno-associated viruses containing the human mutated A53T gene (AAV-A53T) or an empty vector into the left cervical vagus nerve and evaluated for gastrointestinal symptoms, locomotor functions, intestinal blood flow, and nerve electrophysiology. Further, we examined the impact of α-synucleinopathy on vagus nerves, SCs, and central nervous system neurons using electron microscopy, immunofluorescence, immunohistochemistry, and western blot. Finally, the role of Toll-like receptor 2 (TLR2) in regulating the neuroinflammation in the vagus nerve via MyD88 and NF-κB pathway was determined using genetic knockdown. RESULTS: We found that rats injected with AAV-A53T in the vagus nerve exhibited prominent signs of AutD, preceding the onset of motor deficits and central dopaminergic abnormalities by at least 3 months, which could serve as a model for prodromal PD. In addition, reduced intestinal blood flow and decreased nerve conduction velocity were identified in AAV-A53T-injected rats, accompanied by disrupted myelin sheaths and swollen SCs in the vagus nerve. Furthermore, our data demonstrated that p-α-synuclein was deposited in SCs but not in axons, activating the TLR2/MyD88/NF-κB signaling pathway and leading to neuroinflammatory responses. In contrast, silencing the TLR2 gene not only reduced inflammatory cytokine expression but also ameliorated vagal demyelination and secondary axonal loss, consequently improving autonomic function in rats. CONCLUSIONS: These observations suggest that overexpression of α-synuclein in the vagus nerve can induce symptoms of AutD in prodromal PD, and provide support for a deeper understanding of the pathological mechanisms underlying AutD and the emergence of effective therapeutic strategies for PD.

Vibrio cholerae Porin OmpU Activates Dendritic Cells via TLR2 and the NLRP3 Inflammasome.

OmpU is one of the major porins of Vibrio cholerae, a Gram-negative human pathogen. Previously, we showed that OmpU stimulates host monocytes and macrophages and induces the production of proinflammatory mediators via activation of the Toll-like receptor 1/2 (TLR1/2)-MyD88-dependent pathways. In the present study, we show that OmpU activates murine dendritic cells (DCs) via activation of the TLR2-mediated pathway and the NLRP3 inflammasome, leading to the production of proinflammatory cytokines and DC maturation. Our data reveal that although TLR2 plays an important role in providing both priming and the activation signal for the NLRP3 inflammasome in OmpU-activated DCs, OmpU is capable of activating the NLRP3 inflammasome, even in the absence of TLR2, if a priming signal is given. Furthermore, we show that the OmpU-mediated interleukin-1ß (IL-1ß) production in DCs depends on calcium flux and mitochondrial reactive oxygen species (mitoROS) generation. Interestingly, both OmpU translocation to the mitochondria of DCs as well as calcium signaling contribute to mitoROS production and prompt NLRP3 inflammasome activation. We also demonstrate that OmpU induces downstream signaling via activation of phosphoinositide-3-kinase (PI3K)-AKT, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and transcription factor NF-κB. Furthermore, our data reveal that OmpU-mediated activation of TLR2 induces signaling via PKC, MAPKs p38 and extracellular signal-regulated kinase (ERK), and transcription factor NF-κB; however, PI3K and MAPK Jun N-terminal protein kinase (JNK) are activated in TLR2 independent manner.

High tissue expression of TLRs combined with high density of tumor infiltrating lymphocytes predicts a better prognosis in colorectal cancer patients.

BACKGROUND: Colorectal cancer causes 935,000 cancer deaths yearly. High local immune cell infiltration serves as a positive prognostic factor in CRC. Toll-like receptors (TLRs) induce innate immune responses and lead to adaptive immune system activation. TLRs play protumorigenic and antitumorigenic roles. We aimed to explore the relationship between TLR immunoexpressions and the infiltration densities of T-lymphocytes in CRC. METHODS: Immunohistochemical TLR2, TLR4, TLR5, and TLR7 positivity and the density of CD3- and CD8-positive cells in tumoral and stromal tissue were evaluated from the tissue microarray slides of 549 consecutive CRC surgical patients treated at Helsinki University Hospital, Finland, between 1998 and 2005. We calculated the associations and correlations using Pearson's chi-square and Spearman's correlation tests, generating survival curves using the Kaplan-Meier method. RESULTS: Positive intratumoral CD3 and CD8 densities associated with a high TLR2 expression (p < 0.001 and p = 0.001, respectively) and a high TLR4 expression (p = 0.013 and p = 0.025). A low TLR5 immunoexpression associated with negative intratumoral CD3 (p = 0.001) and CD8 (p = 0.011) and a low stromal CD3 (p = 0.001). No association or correlation emerged between TLR7 immunoexpression and CD3 or CD8 cell density. A low CD3-CD8 tumor-stroma index indicated a worse prognosis among all TLR subgroups, except the TLR7-negative subgroup. CONCLUSIONS: We detected significant associations and correlations between high tissue TLR2, TLR4, and TLR5 immunoexpressions and high densities of CD3- and CD8-positive cells. Combining these markers may improve the prognostic evaluation of CRC patients.

Clonorchis sinensis aggravates biliary fibrosis through promoting IL-6 production via toll-like receptor 2-mediated AKT and p38 signal pathways.

Clonorchis sinensis is an important food-borne zoonotic parasite which has been linked to biliary fibrosis and cholangiocarcinoma. However, the details of the pathogenesis of C. sinensis were unclear. To explore the role and regulatory mechanism of toll-like receptor 2 (TLR2) in C. sinensis-induced biliary fibrosis, we established the C. sinensis-infected C57BL/6 mouse model with TLR2-/- and wild type (WT) mice. The mortality rate, liver lesions, TLR2 and TGF-ß1 expression, phosphorylation of Smad2/3, AKT, p38, ERK and p65, and cytokine productions were analyzed. Furthermore, similar parameters were examined in mouse biliary epithelial cells (BECs) co-cultured with C. sinensis excretory/secretory proteins (ESPs). The results showed that TLR2 expression was enhanced significantly in C. sinensis-infected WT mice and mouse BECs. C. sinensis-infected TLR2-/- mice exhibited an increased weight and a decreased mortality rate; significantly alleviated liver lesions and biliary fibrosis, reduced numbers of myofibroblasts; decreased expression of TGF-ß1 and phosphorylation level of AKT, p38 and Smad2/3; significantly decreased production of IL-6, TNF-α and IL-4, while increased production of IFN-γ compared with C. sinensis-infected WT mice. Furthermore, C. sinensis ESPs could activate TLR2-mediated AKT and p38 pathways to increase the production of IL-6 in mouse BECs. In conclusion, these data indicate that C. sinensis infection activated TGF-ß1-Smad2/3 through TLR2-mediated AKT and p38 pathways to promote IL-6 production, which resulted in myofibroblast activation and aggravating biliary fibrosis in mice.

FAM21 is critical for TLR2/CLEC4E-mediated dendritic cell function against Candida albicans.

FAM21 (family with sequence similarity 21) is a component of the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) protein complex that mediates actin polymerization at endosomal membranes to facilitate sorting of cargo-containing vesicles out of endosomes. To study the function of FAM21 in vivo, we generated conditional knockout (cKO) mice in the C57BL/6 background in which FAM21 was specifically knocked out of CD11c-positive dendritic cells. BMDCs from those mice displayed enlarged early endosomes, and altered cell migration and morphology relative to WT cells. FAM21-cKO cells were less competent in phagocytosis and protein antigen presentation in vitro, though peptide antigen presentation was not affected. More importantly, we identified the TLR2/CLEC4E signaling pathway as being down-regulated in FAM21-cKO BMDCs when challenged with its specific ligand Candida albicans Moreover, FAM21-cKO mice were more susceptible to C. albicans infection than WT mice. Reconstitution of WT BMDCs in FAM21-cKO mice rescued them from lethal C. albicans infection. Thus, our study highlights the importance of FAM21 in a host immune response against a significant pathogen.

Exposure to polystyrene microplastics triggers lung injury via targeting toll-like receptor 2 and activation of the NF-κB signal in mice.

Microplastics are ubiquitous pollutants with a wide range of plastic applications. More recently, microplastics are in the air and can be inhaled into the lungs, causing respiratory diseases. Knowledge of the underlying mechanisms by which microplastics may induce respiratory disease is still limited. This study used intranasal instillation to develop a model of lung injury. The histopathology result showed that the mouse lung had severe inflammatory responses, apoptosis and collagen deposition with chronic exposure to different sizes (Small: 1-5 µm and Large: 10-20 µm) of polystyrene microplastics (PS-MPS), and the damage of smaller sizes was obvious. The expression levels of the Toll-like receptors (TLRs) family, evolutionarily conserved pattern recognition receptors, were detected, and the levels of TLR2 mRNA was significantly increased. In transfection experiments, PS-MPS increased the inflammatory response in HEK293 cells with TLR2 expression. Furthermore, exposure to small polystyrene microplastics promoted oxidative stress and apoptosis, and accelerated the process of fibrosis. Interestingly, inhibition of the NF-κB signal relieves inflammation and oxidative stress, reduces apoptosis, and thus controls the fibrosis process. These results suggested that PS-MPS targeted binding to TLR2 and further exacerbated fibrosis by facilitating inflammation, oxidative stress, and apoptosis with the activation of NF-κB signal.

Toll-like Receptors and Thrombopoiesis.

Platelets are the second most abundant blood component after red blood cells and can participate in a variety of physiological and pathological functions. Beyond its traditional role in hemostasis and thrombosis, it also plays an indispensable role in inflammatory diseases. However, thrombocytopenia is a common hematologic problem in the clinic, and it presents a proportional relationship with the fatality of many diseases. Therefore, the prevention and treatment of thrombocytopenia is of great importance. The expression of Toll-like receptors (TLRs) is one of the most relevant characteristics of thrombopoiesis and the platelet inflammatory function. We know that the TLR family is found on the surface or inside almost all cells, where they perform many immune functions. Of those, TLR2 and TLR4 are the main stress-inducing members and play an integral role in inflammatory diseases and platelet production and function. Therefore, the aim of this review is to present and discuss the relationship between platelets, inflammation and the TLR family and extend recent research on the influence of the TLR2 and TLR4 pathways and the regulation of platelet production and function. Reviewing the interaction between TLRs and platelets in inflammation may be a research direction or program for the treatment of thrombocytopenia-related and inflammatory-related diseases.

Advanced Glycation End Products and Activation of Toll-like Receptor-2 and -4 Induced Changes in Aquaporin-3 Expression in Mouse Keratinocytes.

Prolonged inflammation and impaired re-epithelization are major contributing factors to chronic non-healing diabetic wounds; diabetes is also characterized by xerosis. Advanced glycation end products (AGEs), and the activation of toll-like receptors (TLRs), can trigger inflammatory responses. Aquaporin-3 (AQP3) plays essential roles in keratinocyte function and skin wound re-epithelialization/re-generation and hydration. Suberanilohydroxamic acid (SAHA), a histone deacetylase inhibitor, mimics the increased acetylation observed in diabetes. We investigated the effects of TLR2/TLR4 activators and AGEs on keratinocyte AQP3 expression in the presence and absence of SAHA. Primary mouse keratinocytes were treated with or without TLR2 agonist Pam3Cys-Ser-(Lys)4 (PAM), TLR4 agonist lipopolysaccharide (LPS), or AGEs, with or without SAHA. We found that (1) PAM and LPS significantly upregulated AQP3 protein basally (without SAHA) and PAM downregulated AQP3 protein with SAHA; and (2) AGEs (100 µg/mL) increased AQP3 protein expression basally and decreased AQP3 levels with SAHA. PAM and AGEs produced similar changes in AQP3 expression, suggesting a common pathway or potential crosstalk between TLR2 and AGEs signaling. Our findings suggest that TLR2 activation and AGEs may be beneficial for wound healing and skin hydration under normal conditions via AQP3 upregulation, but that these pathways are likely deleterious in diabetes chronically through decreased AQP3 expression.

Toll-like receptor 2 mediates Acanthamoeba-induced allergic airway inflammatory response in mice.

BACKGROUND: Repeated intranasal exposure to Acanthamoeba has been revealed to induce allergic airway inflammatory responses in mice. Based on the role of toll-like receptors (TLRs) in the pathogenesis of allergic asthma, TLRs form a link between innate and adaptive immune responses, and play an important role in the activation of various cells in the innate immune system. METHODOLOGY/PRINCIPAL FINDINGS: To determine the TLRs that are related to these immune responses, we assessed the expression levels of inflammation-related genes in mouse lung epithelial (MLE)-12 cells treated with excretory-secretory proteins (ES-P) of the Acanthamoeba strain (KA/E2) with or without the TLR antagonists. The expression levels of inflammation-related genes, such as eotaxin, TARC, macrophage-derived chemokine (MDC), and TSLP, in the TLR2 and TLR9 antagonist treatment groups were decreased, compared to those in the ES-P alone or other TLR antagonist treatment groups. In particular, a greater decrease in the relevant gene expression levels was found in the TLR2 antagonist treatment group than in the TLR9 antagonist treatment group. Allergic airway inflammation was evaluated in the wild-type (WT) and TLR2 knockout (KO) groups following KA/E2 exposure. Based on the results, allergic airway inflammatory responses (airway resistance value, inflammatory cell infiltration, Th2-related cytokine expression, mucin production, and metaplasia of lung epithelial cells and goblet cells) by KA/E2 were reduced in the TLR2 KO groups. In addition, TLR2 knockout BMDCs displayed lower activation of surface markers owing to ES-P stimulation than normal BMDCs, and KA/E2 ES-P-treated TLR2-depleted BMDCs produced fewer Th2 cytokine-expressing cells from naïve T cells than WT BMDCs. When ES-P was administered after primary lung cells were obtained from WT and TLR2 KO mice, the expression levels of inflammation-related genes were found to be significantly decreased in TLR2 KO cells compared to those in WT cells. CONCLUSIONS: These results suggest that TLR2 is involved in lung inflammatory response activation in KA/E2 intranasal infection, especially in airway tissue.

Serum amyloid A regulates TLR2/4-mediated IFN-ß signaling pathway against Marek's disease virus.

Serum amyloid A (SAA), an acute response phase protein (APP), is crucial for the innate immune response during pathogenic microorganisms' invasion. Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that activates multiple innate immune molecules, including SAA, in the host during infection. However, the pathway through which SAA participates in MDV-induced host innate immunity remains unknown. The present study aimed to elucidate the pathway through which SAA exerts its anti-MDV function. We observed that MDV infection in vivo and in vitro significantly elevated SAA expression. Furthermore, through SAA overexpression and knockdown experiments, we demonstrated that SAA could inhibit MDV replication. Subsequently, we found that SAA activated Toll-Like Receptor 2/4 (TLR2/4) -mediated Interferon Beta (IFN-ß) promoter activity and IFN regulatory factor 7 (IRF7) promoter activity. During MDV infection, SAA enhanced TLR2/4-mediated IFN-ß signal transduction and messenger RNAs (mRNAs) expression of type I IFN (IFN-I) and interferon-stimulated genes (ISGs). Finally, TLR2/4 inhibitor OxPAPC inhibits the anti-MDV activity of SAA. These results demonstrated that SAA inhibits MDV replication and enhancing TLR2/4-mediated IFN-ß signal transduction to promote IFNs and ISGs expression. This finding is the first to demonstrate the signaling pathway by which SAA exerts its anti-MDV function. It also provides new insights into the control of oncogenic herpesviruses from the perspective of acute response phase proteins.

Protective effects of Bifidobacterium bifidum FL-228.1 on dextran sulfate sodium-induced intestinal damage in mice.

PURPOSE: Numerous studies have found that probiotics benefit the intestinal barrier. However, the prophylactic effects of probiotics on the intestinal barrier, i.e., if probiotics exert protective effects in healthy individuals to defend them against harmful elements, have seldomly been reported. The present study aimed to investigate the possible mechanisms of potential strains with the function of preventing intestinal barrier damage. METHODS: This study investigated nine potential probiotic strains using in vitro and in vivo models on their intestinal barrier-protecting properties. Transcriptomic was then employed to decipher the underlying mechanisms of action of the strains. RESULTS: The results showed that the strains, to varying degrees, regulated the ratio of interleukin (IL)-10 and IL-12 in peripheral blood mononuclear cells (PBMCs), increased the transepithelial electrical resistance (TEER) values, and decreased Caco-2 cell monolayers permeability. Correspondingly, the strains showed different prophylactic efficacies in protecting mice from dextran sulfate sodium (DSS)-induced intestinal barrier damage. Remarkably, Bifidobacterium bifidum FL-228.1 (FL-228.1) showed the best prophylactic efficacies in protecting mice from DSS-induced intestinal barrier damage. Further research suggested that FL-228.1 exerted its prophylactic effects by enhancing mucin 2 (Muc2) production and Claudin (Cldn)-4 in the colon. Furthermore, the transcriptomic and protein-protein interactions (PPI) analyses indicated that the inhibition of NLRP3 and the activation of PPARγ and TLR2 could be involved in protecting the intestinal barrier by FL-228.1. CONCLUSION: Bifidobacterium bifidum FL-228.1 may be developed as a promising probiotic for the prevention of intestinal barrier damage via PPARγ/NLRP3/ TLR2 pathways by enhancing Muc2 and Cldn-4.

Dietary supplementation of n-3 PUFAs ameliorates LL37-induced rosacea-like skin inflammation via inhibition of TLR2/MyD88/NF-κB pathway.

Rosacea is a facial chronic inflammatory skin disease with dysfunction of immune and neurovascular system and treatments for rosacea are challenging. N-3 polyunsaturated fatty acids (PUFAs), one of essential fatty acids, are needed for health maintenance and exert anti-inflammation and immunomodulatory effects in a series of cutaneous diseases such as atopic dermatitis and photoaging through dietary supplementation. However, the role of n-3 PUFAs on rosacea remains to be elucidated. In this study, KEGG enrichment analysis and GO analysis indicated that the biological process and signaling pathways, including chemokine signaling pathway, regulated by n-3 PUFAs highly overlapped with those in the pathogenic biological process of rosacea, especially the erythema telangiectasia type. Next, mice were randomized to fed with a customized n-3 PUFAs diet. We showed that n-3 PUFAs ameliorated skin erythema, inhibited dermal inflammatory cell infiltration (mast cells, neutrophils, and CD4 +T cells) and suppressed elevated pro-inflammatory cytokines in LL37-induced rosacea-like mice. Besides, n-3 PUFAs were also verified to repress angiogenesis in LL37-induced mice skin. Further investigation revealed that n-3 PUFAs attenuated LL37-induced inflammation via TLR2/ MyD88/ NF-κB pathway both in mice and in keratinocytes. In conclusion, our findings underscore that dietary supplementation of n-3 PUFAs have the potential to become an efficient and safe clinical therapeutic candidate for rosacea.

Huc-MSCs-derived exosomes attenuate neuropathic pain by inhibiting activation of the TLR2/MyD88/NF-κB signaling pathway in the spinal microglia by targeting Rsad2.

BACKGROUND: Mesenchymal stem cells (MSCs)-derived exosomes have shown promise as a cell-free therapeutic strategy for neuropathic pain. This study was conducted to explore the potential mechanisms underlying the analgesic effects of MSC-derived exosomes in treating neuropathic pain. METHODS: Human umbilical cord MSCs (huc-MSCs)-derived exosomes were isolated and identified. BV-2 microglia were stimulated with lipopolysaccharide (LPS) in the presence or absence of exosomes. Differentially expressed proteins were identified by tandem mass tag (TMT)-based proteomic analysis. The analgesic effects of huc-MSCs-derived exosomes were evaluated in a rat model of chronic constriction injury (CCI). The underlying mechanism was investigated by flow cytometry, RT-qPCR, Western blotting, immunofluorescent staining, and small interfering RNA transfection. RESULTS: In vitro, huc-MSCs-derived exosomes suppressed LPS-induced microglial activation and inhibited activation of the TLR2/MyD88/NF-κB signaling pathway. Based on the proteomic analysis, Rsad2 was identified and confirmed to be down-regulated by huc-MSCs-derived exosomes. Importantly, knockdown of Rsad2 also inhibited microglial activation and restrained activation of the TLR2/MyD88/NF-κB signaling pathway. In vivo, intrathecal injection of exosomes ameliorated CCI-induced mechanical allodynia, down-regulated Rsad2 expression and restrained TLR2/MyD88/NF-κB signaling activation in the spinal microglia. CONCLUSION: Huc-MSCs-derived exosomes exerted analgesic effects on neuropathic pain by inhibiting activation of the TLR2/MyD88/NF-κB signaling pathway in the spinal microglia. The mechanism underlying these antinociceptive effects involved exosome-mediated interference with Rsad2 expression, thereby inhibiting microglial activation.

High Mobility Group Box 1 as an Autocrine Chemoattractant for Oligodendrocyte Lineage Cells in White Matter Stroke.

BACKGROUND: The migration of oligodendrocyte precursor cells (OPC) is a key process of remyelination, which is essential for the treatment of white matter stroke. This study aimed to investigate the role of HMGB1 (high mobility group box 1), a damage-associated molecular pattern released from dying oligodendrocytes, as an autocrine chemoattractant that promotes OPC migration. METHODS: The migratory capacity of primary cultured OPCs was measured using the Boyden chamber assay. The downstream pathway of HMGB1-mediated OPC migration was specified by siRNA-induced knockdown or pharmacological blockade of TLR2 (toll-like receptor 2), RAGE (receptor for advanced glycation end product), Src, ERK1/2 (extracellular signal-regulated kinase1/2), and FAK (focal adhesion kinase). Conditioned media were collected from oxygen-glucose deprivation-treated oligodendrocytes, and the impact on OPC migration was assessed. Lesion size and number of intralesional Olig2(+) cells were analyzed in an in vivo model of white matter stroke with N5-(1-iminoethyl)-L-ornithine (L-NIO). RESULTS: HMGB1 treatment promoted OPC migration. HMGB1 antagonism reversed such effects to untreated levels. Among the candidates for the downstream signal of HMGB1-mediated migration, the knockdown of TLR2 rather than that of RAGE attenuated the migration-promoting effect of HMGB1. Further specification of the HMGB1-TLR2 axis revealed that the phosphorylation of ERK1/2 and its downstream molecule FAK, rather than of Src, was decreased in TLR2-knockdown OPCs, and pharmacological inhibition of ERK1/2 and FAK led to decreased OPC migration. Oxygen-glucose deprivation-conditioned media promoted OPC migration, suggesting the autocrine chemoattractant function of HMGB1. In vivo, TLR2(-/-)-mice showed lesser intralesional Olig2(+) cells compared to wild-type controls in response to L-NIO induced ischemic injury regardless of HMGB1 administration. CONCLUSIONS: HMGB1, through the TLR2-ERK1/2-FAK axis, functions as an autocrine chemoattractant to promote OPC migration, which is an initial and indispensable step in remyelination. Thus, a novel treatment strategy for white matter stroke based on the HMGB1-TLR2 axis in the oligodendrocyte lineage could be feasible.