Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis.

BACKGROUND: Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that a Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. METHODS: We used two cell culture models of THP-1 macrophages and Caco-2 monolayers to establish the effects of MAP infection on the vitamin D metabolic axis. We also tested the effects of Calcifediol, Calcitriol, and SB203580 treatments on the relative expression of the vitamin D metabolic genes, oxidative stress biomarkers, and inflammatory cytokines profile. RESULTS: In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 h post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78 ± 0.88 and 13.90 ± 3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93 ± 0.28, 1.86 ± 0.27, and 6.34 ± 0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. CONCLUSIONS: This study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1ß, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1ß, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Pathogenic and Apathogenic Strains of Lymphocytic Choriomeningitis Virus Have Distinct Entry and Innate Immune Activation Pathways.

Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.

Bacterial surface lipoproteins mediate epithelial microinvasion by Streptococcus pneumoniae.

Streptococcus pneumoniae, a common colonizer of the upper respiratory tract, invades nasopharyngeal epithelial cells without causing disease in healthy participants of controlled human infection studies. We hypothesized that surface expression of pneumococcal lipoproteins, recognized by the innate immune receptor TLR2, mediates epithelial microinvasion. Mutation of lgt in serotype 4 (TIGR4) and serotype 6B (BHN418) pneumococcal strains abolishes the ability of the mutants to activate TLR2 signaling. Loss of lgt also led to the concomitant decrease in interferon signaling triggered by the bacterium. However, only BHN418 lgt::cm but not TIGR4 lgt::cm was significantly attenuated in epithelial adherence and microinvasion compared to their respective wild-type strains. To test the hypothesis that differential lipoprotein repertoires in TIGR4 and BHN418 lead to the intraspecies variation in epithelial microinvasion, we employed a motif-based genome analysis and identified an additional 525 a.a. lipoprotein (pneumococcal accessory lipoprotein A; palA) encoded by BHN418 that is absent in TIGR4. The gene encoding palA sits within a putative genetic island present in ~10% of global pneumococcal isolates. While palA was enriched in the carriage and otitis media pneumococcal strains, neither mutation nor overexpression of the gene encoding this lipoprotein significantly changed microinvasion patterns. In conclusion, mutation of lgt attenuates epithelial inflammatory responses during pneumococcal-epithelial interactions, with intraspecies variation in the effect on microinvasion. Differential lipoprotein repertoires encoded by the different strains do not explain these differences in microinvasion. Rather, we postulate that post-translational modifications of lipoproteins may account for the differences in microinvasion.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is an important mucosal pathogen, estimated to cause over 500,000 deaths annually. Nasopharyngeal colonization is considered a necessary prerequisite for disease, yet many people are transiently and asymptomatically colonized by pneumococci without becoming unwell. It is therefore important to better understand how the colonization process is controlled at the epithelial surface. Controlled human infection studies revealed the presence of pneumococci within the epithelium of healthy volunteers (microinvasion). In this study, we focused on the regulation of epithelial microinvasion by pneumococcal lipoproteins. We found that pneumococcal lipoproteins induce epithelial inflammation but that differing lipoprotein repertoires do not significantly impact the magnitude of microinvasion. Targeting mucosal innate immunity and epithelial microinvasion alongside the induction of an adaptive immune response may be effective in preventing pneumococcal colonization and disease.

A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron.

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.

Hazards of microplastics exposure to liver function in fishes: A systematic review and meta-analysis.

Microplastics (5 mm - 1 µm) have become one of the major pollutants in the environment. Numerous studies have shown that microplastics can have negative impacts on aquatic organisms, affecting their liver function levels. However, the extent of these effects and their potential toxicological mechanisms are largely unknown. In this study, a meta-analysis and systematic review were conducted to assess the effects of microplastics on fish liver function and summarize the potential toxicological mechanisms of microplastic-induced liver toxicity. The meta-analysis results indicate that compared to the control group, exposure to microplastics significantly affects fish liver indicators: aspartate aminotransferase (AST) (p < 0.001), alanine aminotransferase (ALT) (p < 0.001), alkaline phosphatase (ALP) (p < 0.001), total protein (TP) (p < 0.001), and lactate dehydrogenase (LDH) (p < 0.001), including oxidative stress indicators: superoxide dismutase (SOD) (p < 0.001), glutathione S-transferase (GST) (p < 0.001), glutathione (GSH) (p < 0.001), and malondialdehyde (MDA) (p < 0.001) in fish liver. For fish living in different environments, the potential toxicological mechanisms of microplastics exposure on fish liver may exhibit some differences. For freshwater fish, the mechanism may be that microplastics exposure causes overproduction of reactive oxygen species (ROS) in fish hepatocyte mitochondria. ROS promotes the expression of toll-like receptor 2 (TLR2) and activates downstream molecules myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) of the TLR2 signaling pathway, leading to phosphorylation of NF-κB p65. This leads to the release of inflammatory factors and oxidative stress and inflammation in fish liver. In addition, for seawater fish, the mechanism may be that microplastics exposure can cause damage or death of fish hepatocytes, leading to continuous pathological changes, inflammation, lipid and energy metabolism disorders, thereby causing significant changes in liver function indexes.

Design and Synthesis of 3-(2H-Chromen-3-yl)-5-aryl-1,2,4-oxadiazole Derivatives as Novel Toll-like Receptor 2/1 Agonists That Inhibit Lung Cancer In Vitro and In Vivo.

Toll-like receptor (TLR) 2 is a transmembrane receptor that participates in the innate immune response by forming a heterodimer with TLR1 or TLR6. TLR2 agonists play an important role in tumor therapy. Herein, we synthesized a series of 3-(2H-chromen-3-yl)-5-aryl-1,2,4-oxadiazole derivatives and identified WYJ-2 as a potent small and selective molecule agonist of TLR2/1, with an EC50 of 18.57 ± 0.98 nM in human TLR2 and TLR1 transient-cotransfected HEK 293T cells. WYJ-2 promoted the formation of TLR2/1 heterodimers and activated the nuclear factor kappa B (NF-κB) signaling pathway. Moreover, our study indicated that WYJ-2 could induce pyroptosis in cancer cells, mediated by activating the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome. WYJ-2 exhibited effective anti-non-small cell lung cancer (NSCLC) activity in vitro and in vivo. The discovery that activating TLR2/1 induces pyroptosis in cancer cells may highlight the prospects of TLR2/1 agonists in cancer treatment in the future.

Validation and application of caged Z-DEVD-aminoluciferin bioluminescence for assessment of apoptosis of wild type and TLR2-deficient mice after ischemic stroke.

Programmed cell death or apoptosis is a critically important mechanism of tissue remodeling and regulates conditions such as cancer, neurodegeneration or stroke. The aim of this research article was to assess the caged Z-DEVD-aminoluciferin substrate for in vivo monitoring of apoptosis after ischemic stroke in TLR2-deficient mice and their TLR2-expressing counterparts. Postischemic inflammation is a significant contributor to ischemic injury development and apoptosis, and it is modified by the TLR2 receptor. Caged Z-DEVD-aminoluciferin is made available for bioluminescence enzymatic reaction by cleavage with activated caspase-3, and therefore it is assumed to be capable of reporting and measuring apoptosis. Apoptosis was investigated for 28 days after stroke in mice which ubiquitously expressed the firefly luciferase transgene. Middle cerebral artery occlusion was performed to achieve ischemic injury, which was followed with magnetic resonance imaging. The scope of apoptosis was determined by bioluminescence with caged Z-DEVD-aminoluciferin, immunofluorescence with activated caspase-3, flow cytometry with annexin-V and TUNEL assay. The linearity of Z-DEVD-aminoluciferin substrate dose effect was shown in the murine brain. Z-DEVD-aminoluciferin was validated as a good tool for monitoring apoptosis following adequate adjustment. By utilizing bioluminescence of Z-DEVD-aminoluciferin after ischemic stroke it was shown that TLR2-deficient mice had lower post-stroke apoptosis than TLR2-expressing wild type mice. In conclusion, Z-DEVD-aminoluciferin could be a valuable tool for apoptosis measurement in living mice.

Design, synthesis, biological evaluation and in silico studies of novel quinoline derivatives as potential radioprotective molecules targeting the TLR2 and p53 pathways.

Ionizing radiation in space, radiation devices or nuclear disasters are major threats to human health and public security. In this paper, in order to find the potential novel compounds decreasing the radiation-induced damage by targeting p53 apoptosis pathway and TLR2 passway, a series of novel quinoline derivatives were designed, synthesized, and evaluated their biological activities. Most of the synthesized compounds showed significant radioprotective effects in vitro, and the compound 5 has the best performance. Therefore, we verified its radioprotective activity in vivo and investigated the mechanism of its excellent activity. The results in vivo indicated that compound 5 not only markedly enhanced the survival rate (80 %) of mice 30 days after lethal exposure to irradiation, but also significantly reduced the radiation-induced damage to haematopoietic system and intestinal tissue of mice. The mechanistic studies indicated that compound 5 acted on the p53 pathway to reduce radiation-induced cell apoptosis and at the same time stimulated TLR2 to up-regulate the expressions of radiation protection factors. Molecular dynamics study shows that compound 5 would effectively bind to the TLR2 protein and further revealed the binding mechanism. Taken together, all the findings of our study demonstrate the quinoline derivative 5 is a potent radioprotective compound, which holds a great therapeutic potential for further development.

P. gingivalis-Induced TLR2 Interactome Analysis Reveals Association with PARP9.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium strongly associated with periodontal disease. Toll-like receptor 2 (TLR2) is indispensable for the host response to P. gingivalis, but P. gingivalis escapes from immune clearance via TLR2-dependent activation of phosphoinositide-3-kinase (PI3K). To probe the TLR2-dependent escape pathway of P. gingivalis, we analyzed the TLR2 interactome induced following P. gingivalis infection or activation by a synthetic lipopeptide TLR2/1 agonist on human macrophages overexpressing TLR2. Interacting proteins were stabilized by cross-linking and then immunoprecipitated and analyzed by mass spectrometry. In total, 792 proteins were recovered and network analysis enabled mapping of the TLR2 interactome at baseline and in response to infection. The P. gingivalis infection-induced TLR2 interactome included the poly (ADP-ribose) polymerase family member mono-ADP-ribosyltransferase protein 9 (PARP9) and additional members of the PARP9 complex (DTX3L and NMI). PARP9 and its complex members are highly upregulated in macrophages exposed to P. gingivalis or to the synthetic TLR2/1 ligand Pam3Cys-Ser-(Lys)4 (PAM). Consistent with its known role in virally induced interferon production, PARP9 knockdown blocked type I interferon (IFN-I) production in response to P. gingivalis and reduced inflammatory cytokine production. We found that P. gingivalis drives signal transducer and activation of transcription (STAT) 1 (S727) phosphorylation through TLR2-PARP9, explaining PARP9's role in the induction of IFN-I downstream of TLR2. Furthermore, PARP9 knockdown reduced PI3K activation by P. gingivalis, leading to improved macrophage bactericidal activity. In summary, PARP9 is a novel TLR2 interacting partner that enables IFN-I induction and P. gingivalis immune escape in macrophages downstream of TLR2 sensing.

Analysis of TLR2 in Primary Endocrine Resistant of Breast Cancer.

BACKGROUND: Previous clinical studies have suggested that Toll-like receptor (TLR)2 had predictive function for endocrine resistance in HER2-positive breast cancer (BCa). Nevertheless, it remains unclear whether TLR2 would relate to development of endocrine therapy resistance in triple-positive breast cancer (TPBC). METHODS: Bioinformatic analysis of TLR2 was carried out through a database. Ten tumor tissues were obtained from TPBC patients who underwent surgery, with five patients displaying primary resistance to tamoxifen (TAM) with the remaining 5 being sensitive. Different levels of proteins were identified through mass spectrometry analysis and confirmed through reverse transcription polymerase chain reaction (RT-PCR) and western blot. TAM-resistant cell lines (BT474-TAM) were established by continuous exposure to TAM, and TAM resistance was assessed via IC50. Additionally, TLR2 mRNA was analyzed through western blot and RT-PCR in BT474, BT474-TAM, MCF-7, and MCF10A cells. Furthermore, TLR2-specific interference sequences were utilized to downregulate TLR2 expression in BT474-TAM cells to elucidate its role in TAM resistance. RESULTS: TLR2 had a correlation with decreased relapse-free survival in BCa patients from the GSE1456-GPL96 cohort, and it was involved in cancer development predominantly mediated by MAPK and PI3K pathways. TLR2 protein expression ranked in the top 5 proteins within the TAM-resistant group, and was 1.9 times greater than that in the sensitive group. Additionally, TLR2 mRNA and protein expression increased significantly in the established TAM-resistant BT474/TAM cell lines. The sensitivity of TAM was restored upon TLR2 downregulation in BT474/TAM cells. CONCLUSIONS: TLR2 might have a therapeutic value as it was involved in the TAM resistance in TPBC, with potential to be a marker for primary endocrine resistance.

p53 suppresses the inflammatory response following respiratory syncytial virus infection by inhibiting TLR2.

-Respiratory syncytial virus (RSV) is a pivotal virus leading to acute lower respiratory tract infections in children under 5 years old. This study aimed to explore the correlation between p53 and Toll-like receptors (TLRs) post RSV infection. p53 levels exhibited a substantial decrease in nasopharyngeal aspirates (NPAs) from infants with RSV infection compared to control group. Manipulating p53 expression had no significant impact on RSV replication or interferon signaling pathway. Suppression of p53 expression led to heightened inflammation following RSV infection in A549 cells or airways of BALB/c mice. while stabilizing p53 expression using Nutlin-3a mitigated the inflammatory response in A549 cells. Additionally, Inhibiting p53 expression significantly increased Toll-like receptor 2 (TLR2) expression in RSV-infected epithelial cells and BALB/c mice. Furthermore, the TLR2 inhibitor, C29, effectively reduced inflammation mediated by p53 in A549 cells. Collectively, our results indicate that p53 modulates the inflammatory response after RSV infection through TLR2.

Methadone Requires the Co-Activation of µ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells.

Methadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates µ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cells (MCs), are not well characterized. MCs express µ-opioid and Toll-like receptors (TLRs) and constitute an important cell lineage involved in allergy and effective innate immunity responses. In the present study, murine bone-marrow-derived mast cells (BMMCs) were treated with methadone to evaluate cell viability by flow cytometry, cell morphology with immunofluorescence and scanning electron microscopy, reactive oxygen species (ROS) production, and intracellular calcium concentration ([Ca2+]i) increase. We found that exposure of BMMCs to 0.5 mM or 1 mM methadone rapidly induced cell death by forming extracellular DNA traps (ETosis). Methadone-induced cell death depended on ROS formation and [Ca2+]i. Using pharmacological approaches and TLR4-defective BMMC cultures, we found that µ-opioid receptors were necessary for both methadone-induced ROS production and intracellular calcium increase. Remarkably, TLR4 receptors were also involved in methadone-induced ROS production as it did not occur in BMMCs obtained from TLR4-deficient mice. Finally, confocal microscopy images showed a significant co-localization of µ-opioid and TLR4 receptors that increased after methadone treatment. Our results suggest that methadone produces MCETosis by a mechanism requiring a novel crosstalk pathway between µ-opioid and TLR4 receptors.

Comprehensive exploration of hub genes involved in oxidative stress in rhegmatogenous retinal detachment based on bioinformatics analysis.

Rhegmatogenous retinal detachment (RRD) is a type of ophthalmologic emergency, if left untreated, the blindness rate approaches 100 %. The RRD patient postoperative recovery of visual function is unsatisfactory, most notably due to photoreceptor death. We conducted to identify the key genes for oxidative stress (OS) in RRD through bioinformatics analysis and clinical validation, thus providing new ideas for the recovery of visual function in RRD patients after surgery. A gene database for RRD was obtained from the Gene Expression Omnibus (GEO) database (GSE28133). Then we screened differentially expressed OS genes (DEOSGs) from the database and assessed the critical pathways in RRD with Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Protein-protein interaction (PPI) networks and hub genes among the common DEOSGs were identified. In addition, we collected general information and vitreous fluid from 42 patients with RRD and 22 controls [11 each of epiretinal membrane (EM) and macular hole (MH)], examined the expression levels of proteins encoded by hub genes in vitreous fluid by enzyme-linked immunosorbent assay (ELISA) to further assess the relationship between the ELISA data and the clinical characteristics of patients with RRD. Ten hub genes (CCL2, ICAM1, STAT3, CD4, ITGAM, PTPRC, CCL5, IL18, TLR2, VCAM1) were finally screened out from the dataset. The ELISA results showed that, compared with the control group, patients with RRD: TLR2 and ICAM-1 were significantly elevated, and CCL2 had a tendency to be elevated, but no statistically significant; RRD patients and MH patients compared with EM patients: STAT3 and VCAM-1 were significantly elevated. We found affected eyes of RRD patients compared with healthy eyes: temporal and nasal retinal nerve fiber layer (RNFL) were significantly thickened. By correlation analysis, we found that: STAT3 was negatively correlated with ocular perfusion pressure (OPP); temporal RNFL was not only significantly positively correlated with CCL2, but also negatively correlated with Scotopic b-wave amplitude. These findings help us to further explore the mechanism of RRD development and provide new ideas for finding postoperative visual function recovery.

Study on anti-inflammatory effect of Shangkehuangshui in vitro and in vivo based on TLR4/TLR2-NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shangkehuangshui (SK) has been traditionally used to treat traumatic injury, soft tissue and bone injury in Foshan hospital of traditional Chinese medicine for more than 60 years, which composed of many Chinese herbs such as Coptis chinensis Franch., Gardenia jasminoides Ellis, Phellodendron chinense Schneid. and etc. SK exhibits heat-clearing and detoxifying, enhancing blood circulation to eliminate blood stasis properties, and demonstrates noteworthy clinical efficacy. Nevertheless, the underlying mechanism remains uncertain. AIM OF THE STUDY: The early study found that SK had good anti-inflammatory effects in acute soft tissue injury model. This research is to verify the anti-inflammatory properties of SK both in vitro and in vivo via TLR4/TLR2-NF-κB signaling pathway, to clarify the underlying mechanisms responsible for the curative effect of SK. METHODS: The RAW264.7 cells inflammatory model was established with lipopolysaccharide (LPS) in vitro. NO and TNF-α, IL-6, IL-1ß were determined with Griess method and ELISA method respectively. The mRNA and protein expression levels of TLR4/TLR2-NF-κB pathway were evaluated by qPCR and Western blot method. In vivo experiment, chronic soft tissue injury rat models were established by tracking gastrocnemius muscle with electrical stimulation, then local appearance and pathological changes were observed and recorded, the contents of inflammatory factors in serum and tissue were performed. Moreover, we also measured and contrasted the expression of TLR4/TLR2-NF-κB related factors. RESULTS: SK effectively inhibited the LPS-induced generation of inflammatory cytokines, including NO, TNF-α, IL-6 and IL-1ß in RAW264.7 cells, and significantly suppressed the expression of TLR4, TLR2, MyD88, IκB, and NF-κB. In vivo, SK remarkably decreased the damage appearance scores after 4 and 14 days of administration and inhibit the quantity of NO and leukocytes present in the serum. Additionally, the inflammatory infiltration in the pathological section was alleviated, myofibrillar hyperplasia and blood stasis were reduced. SK markedly downregulated NO, TNF-α, IL-6 and IL-1ß in injured tissues of rats, also declined the expression of TLR4, TLR2, MyD88, IκB, NF-κB, IL-6, TNF-α and IL-1ß. CONCLUSION: This study revealed that SK had obvious effects of anti-inflammatory actions in vivo and vitro, effectively reduced acute and chronic soft tissue injury in clinical, this might be attributed to inhibit the TLR4/TLR2-NF-κB pathway, further inhibit the expression of downstream relevant pro-inflammatory cytokines.

TLR2 agonist prevents the progression of periapical lesions in mice by reducing osteoclast activity and regulating the frequency of Tregs.

AIM: To evaluate the role of regulatory T lymphocytes (Tregs) in the presence or absence of the synthetic ligand Pam3Cys during the progression of periapical lesion in wild-type (WT) and toll-like receptor 2 knockout (TLR2KO) mice. METHODOLOGY: A total of 130 C57BL/6 male WT and TLR2KO mice were allocated into control (n = 5) and experimental (periapical lesion induction) (n = 10) groups. In specific groups (WT+Pam3cys and TLR2KO+Pam3cys), the synthetic ligand Pam3cys was administered intraperitoneally every 7 days, according to the experimental period (14, 21 and 42 days). At the end of those periods, the animals were euthanized, and the mandible and the spleen were submitted to histotechnical processing. Mandible histological sections were analysed by haematoxylin and eosin, TRAP histoenzymology and immunohistochemistry (FOXP3, RANK, RANKL and OPG). Spleen sections were analysed by immunohistochemistry (FOXP3). RESULTS: The inflammatory infiltrate and bone resorption were more intense in the TLR2KO group compared to the WT group. The animals that received the Pam3cys had smaller periapical lesions when compared to the animals that did not receive the ligand (p < .05). TLR2KO animals showed a significant increase in the number of osteoclasts when compared to TLR2KO+Pam3cys group (p < .05). At 21 days, the WT+Pam3cys group had a lower number of osteoclasts when compared to the WT animals (p = .02). FOXP3 expression was more intense in the WT+Pam3cys groups when compared to the WT animals in the 42 days (p = .03). In the spleen analysis, the WT+Pam3cys group also had a higher expression of FOXP3 when compared to the WT animals at 14 and 42 days (p = .02). Concerning RANKL, there was a reduction in staining in the KOTLR2+Pam3cys groups at 21 and 42 days (p = .03) and a higher binding ratio between RANK/RANKL in animals that did not receive the ligand. CONCLUSION: Administration of the Pam3cys increased the proliferation of Tregs, showed by FOXP3 expression and prevented the progression of the periapical lesion in WT mice. On the other hand, in the TLR2KO animals, Treg expression was lower with larger areas of periapical lesions. Finally, systemic administration of the Pam3cys in KO animals was able to limit the deleterious effects of the absence of the TLR2 receptor.

LncRNA CCRR Attenuates Postmyocardial Infarction Inflammatory Response by Inhibiting the TLR Signalling Pathway.

BACKGROUND: Timely and proper suppression of inflammation can effectively reduce myocardial injury and promote the postmyocardial infarction (post-MI) wound-healing process. We have previously found that cardiac conduction regulatory RNA (CCRR), a long noncoding RNA (lncRNA) transcribed by the gene located on chromosome 9, with abundant expression in the heart, elicits antiarrhythmic effects in heart failure, and this is a continuing study on the role of CCRR in MI. METHODS: CCRR was overexpressed in CCRR transgenic mice or after injection of adeno-associated virus-9 (AAV-9). MI surgery was performed, and cardiac function was assessed in vivo by echocardiography, followed by histologic analyses. Western blot analysis and qRT-PCR were performed to investigate the effects of CCRR on macrophages, cardiomyocytes, and cardiomyocytes cocultured with macrophages. Through microarray analysis and RNA-binding protein immunoprecipitation (RIP) and other related techniques were also employed to study the effects of CCRR on Toll-like receptor (TLR)2 and TLR4. RESULTS: We found that CCRR level was significantly decreased with increases in proinflammatory cytokines and activation of the TLR signalling pathway in the heart of the 3-day MI mice. CCRR overexpression downregulated TLR2 and TLR4 in MI and effectively inhibited the inflammatory responses in primary cardiomyocytes and macrophages cultured under hypoxic conditions. Downregulation of CCRR induced excessive inflammatory responses by activating the TLR signalling pathway. CCRR acted by suppressing TLR2 and TLR4 to inhibit the secretion of proinflammatory factors to reduce infarct size, thereby improving cardiac function. CONCLUSIONS: CCRR protected cardiomyocytes against MI injury by suppressing inflammatory response through targeting the TLR signalling pathway.

The expression of Nrf2 and TLRs in ear effusion in children with different types of otitis media and their relationship with inflammatory factors.

OBJECTIVE: This study aimed to analyze the differences in the expression of Toll-like receptors (TLRs) and nuclear factor erythroid 2-related factor 2 (Nrf2) in ear effusion in children with different types of otitis media (OM), to elaborate the relationship between the expression of TLRs and Nrf2 in ear effusion and the pathogenesis of OM, and to explore the relationship between the two indicators and pro-inflammatory cytokines in children with OM, thereby laying a scientific foundation for revealing the underlying molecular mechanisms of the progression of different types of OM. METHODS: A total of 73 children with OM who were treated in our hospital from March 2019 to July 2021 were selected as the study subjects. By using the cross-sectional investigation method, participants were divided into three groups according to the different pathological types, including the secretory OM group (30 cases), the chronic suppurative OM group (27 cases), and the cystic lesional OM group (16 cases). The levels of Nrf2, TLR2, TLR4 and proinflammatory cytokines [interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), transforming growth factor-ß 1(TGF-ß1), procalcitonin (PCT) and interleukin-1ß (IL-1ß)] were detected in ear effusion of children with different types of OM. Linear regression was used to analyze the correlation between the Nrf2, TLR2 and TLR4 expression levels and pro-inflammatory cytokines in ear effusion. RESULTS: The expression levels of TNF-α and PCT in the ear effusion of the children under 3 years old were significantly higher than that of the children between 3 and 5 years old and that of the children between 6 and 8 years old (all P < 0.001). The mRNA levels of Nrf2, TLR2 and TLR4 in the ear effusion of the children from the chronic suppurative OM group were higher than these from the secretory OM group (P < 0.001, P = 0.008 and P = 0.021). The mRNA levels of Nrf2, TLR2, and TLR4 in the ear effusion of the children from the cystic lesional OM group were higher than those from the chronic suppurative OM group (P < 0.001, P = 0.029 and P = 0.018). A prominent increase in the concentrations of IFN-γ, TNF-α, TGF-ß1, PCT and IL-1ß was found in the ear effusion of children from the chronic suppurative OM group compared to these from the secretory OM group (P = 0.021, P = 0.044, P = 0.048, P = 0.004 and P = 0.001). The concentrations of IFN-γ, TNF-α, TGF-ß1, PCT and IL-1ß in the ear effusion of the children from the cystic lesional OM group were markedly increased as compared with these from the chronic suppurative OM group (P < 0.001, P = 0.004, P = 0.003, P < 0.001 and P < 0.001). Nrf2, TLR2 and TLR4 were taken as independent variables, and inflammatory indexes, including IFN-γ, TNF-α, TGF-ß1, PCT and IL-1ß were used as dependent variables for the linear regression analysis. The results showed that Nrf2, TLR2 and TLR4 were positively correlated with the secretion levels of pro-inflammatory cytokines after adjusting for age, sex, course and the OM classification (all P < 0.05). CONCLUSION: The expressions of Nrf2, TLR2 and TLR4 in the ear effusion of children with different types of OM gradually increased with the severity of the disease, these were significantly positively correlated with the pro-inflammatory cytokines of the children. Nrf2/TLR signaling pathway maintained chronic inflammation in OM, induced damage of middle ear tissue, and promoted the transition from acute OM to chronic OM.

Porphyromonas gingivalis Lipopolysaccharide Damages Mucosal Barrier to Promote Gastritis-Associated Carcinogenesis.

BACKGROUND: Recent epidemiological studies suggested correlation between gastric cancer (GC) and periodontal disease. AIMS: We aim to clarify involvement of lipopolysaccharide of Porphyromonas gingivalis (Pg.), one of the red complex periodontal pathogens, in the GC development. METHODS: To evaluate barrier function of background mucosa against the stimulations, we applied biopsy samples from 76 patients with GC using a Ussing chamber system (UCs). K19-Wnt1/C2mE transgenic (Gan) mice and human GC cell-lines ± THP1-derived macrophage was applied to investigate the role of Pg. lipopolysaccharide in inflammation-associated carcinogenesis. RESULTS: In the UCs, Pg. lipopolysaccharide reduced the impedance of metaplastic and inflamed mucosa with increases in mRNA expression of toll-like receptor (TLR) 2, tumor necrosis factor (TNF) α, and apoptotic markers. In vitro, Pg. lipopolysaccharide promoted reactive oxidative stress (ROS)-related apoptosis as well as activated TLR2-ß-catenin-signaling on MKN7, and it increased the TNFα production on macrophages, respectively. TNFα alone activated TLR2-ß-catenin-signaling in MKN7, while it further increased ROS and TNFα in macrophages. Under coculture with macrophages isolated after stimulation with Pg. lipopolysaccharide, ß-catenin-signaling in MKN7 was activated with an increase in supernatant TNFα concentration, both of which were decreased by adding a TNFα neutralization antibody into the supernatant. In Gan mice with 15-week oral administration of Pg. lipopolysaccharide, tumor enlargement with ß-catenin-signaling activation were observed with an increase in TNFα with macrophage infiltration. CONCLUSIONS: Local exposure of Pg. lipopolysaccharide may increase ROS on premalignant gastric mucosa to induce apoptosis-associated barrier dysfunction and to secrete TNFα from activated macrophages, and both stimulation of Pg. lipopolysaccharide and TNFα might activate TLR2-ß-catenin-signaling in GC.

Corydalis saxicolaBunting total alkaloid eliminates Porphyromonas gingivalis strain 33277 internalized into macrophages by inhibition of TLR2.

OBJECTIVE: This study aimed to investigate the impact of Corydalis Saxicola Bunting Total Alkaloid (CSBTA) on Porphyromonas gingivalis internalization within macrophages and explore the potential role of Toll-Like Receptor 2 (TLR2) in this process. METHODS: We established a P. gingivalis internalization model in macrophages by treating P. gingivalis-infected macrophages (MOI=100:1) with 200 µg/mL metronidazole and 300 µg/mL gentamicin for 1 h. Subsequently, the model was exposed to CSBTA at concentrations of 0.02 g/L or 1 µg/mL Pam3CSK4. After a 6 h treatment, cell lysis was performed with sterile water to quantify bacterial colonies. The mRNA expressions of TLR2 and interleukin-8 (IL-8) in macrophages were analyzed using RT-qPCR, while their protein levels were assessed via Western blot and ELISA respectively. RESULTS: P. gingivalis could internalize into macrophages and enhance the expression of TLR2 and IL-8. Activation of TLR2 by Pam3CSK4 contributed to P. gingivalis survival within macrophages and increased TLR2 and IL-8 expression. Conversely, 0.02 g/L CSBTA effectively cleared intracellular P. gingivalis, achieving a 90 % clearance rate after 6 h. Moreover, it downregulated the expression of TLR2 and IL-8 induced by P. gingivalis. However, the inhibitory effect of CSBTA on the internalized P. gingivalis model was attenuated by Pam3CSK4. CONCLUSION: CSBTA exhibited the ability to reduce the presence of live intracellular P. gingivalis and lower IL-8 expression in macrophages, possibly by modulating TLR2 activity.