TLR2 on CD4+ and CD8+ T cells promotes control of Mycobacterium tuberculosis infection.

Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout mice is due to its role in macrophages, T cells, or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-γ, TNF-α) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells affected control of Mtb in the lungs and spleens of infected mice. This suggests that T-cell co-stimulation by mycobacterial TLR2 ligands in vivo contributes to the control of Mtb infection in the lung and spleen.

An AP-3-dependent pathway directs phagosome fusion with Rab8 and Rab11 vesicles involved in TLR2 signaling.

Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.

Deletions of MGF110-9L and MGF360-9L from African swine fever virus are highly attenuated in swine and confer protection against homologous challenge.

African swine fever, caused by a large icosahedral DNA virus (African swine fever virus, ASFV), is a highly contagious disease in domestic and feral swine, thus posing a significant economic threat to the global swine industry. Currently, there are no effective vaccines or the available methods to control ASFV infection. Attenuated live viruses with deleted virulence factors are considered to be the most promising vaccine candidates; however, the mechanism by which these attenuated viruses confer protection is unclear. Here, we used the Chinese ASFV CN/GS/2018 as a backbone and used homologous recombination to generate a virus in which MGF110-9L and MGF360-9L, two genes antagonize host innate antiviral immune response, were deleted (ASFV-ΔMGF110/360-9L). This genetically modified virus was highly attenuated in pigs and provided effective protection of pigs against parental ASFV challenge. Importantly, we found ASFV-ΔMGF110/360-9L infection induced higher expression of Toll-like receptor 2 (TLR2) mRNA compared with parental ASFV as determined by RNA-Seq and RT-PCR analysis. Further immunoblotting results showed that parental ASFV and ASFV-ΔMGF110/360-9L infection inhibited Pam3CSK4-triggered activating phosphorylation of proinflammatory transcription factor NF-κB subunit p65 and phosphorylation of NF-κB inhibitor IκBα levels, although NF-κB activation was higher in ASFV-ΔMGF110/360-9L-infected cells compared with parental ASFV-infected cells. Additionally, we show overexpression of TLR2 inhibited ASFV replication and the expression of ASFV p72 protein, whereas knockdown of TLR2 had the opposite effect. Our findings suggest that the attenuated virulence of ASFV-ΔMGF110/360-9L might be mediated by increased NF-κB and TLR2 signaling.

GT1b functions as a novel endogenous agonist of toll-like receptor 2 inducing neuropathic pain.

Spinal cord microglia contribute to nerve injury-induced neuropathic pain. We have previously demonstrated that toll-like receptor 2 (TLR2) signaling is critical for nerve injury-induced activation of spinal cord microglia, but the responsible endogenous TLR2 agonist has not been identified. Here, we show that nerve injury-induced upregulation of sialyltransferase St3gal2 in sensory neurons leads to an increase in expression of the sialylated glycosphingolipid, GT1b. GT1b ganglioside is axonally transported to the spinal cord dorsal horn and contributes to characteristics of neuropathic pain such as mechanical and thermal hypersensitivity. Spinal cord GT1b functions as an TLR2 agonist and induces proinflammatory microglia activation and central sensitization. Pharmacological inhibition of GT1b synthesis attenuates nerve injury-induced spinal cord microglia activation and pain hypersensitivity. Thus, the St3gal2-GT1b-TLR2 axis may offer a novel therapeutic target for the treatment of neuropathic pain.

Ultrahigh field diffusion magnetic resonance imaging uncovers intriguing microstructural changes in the adult zebrafish brain caused by Toll-like receptor 2 genomic deletion.

Toll-like receptor 2 (TLR2) belongs to the TLR protein family that plays an important role in the immune and inflammation response system. While TLR2 is predominantly expressed in immune cells, its expression has also been detected in the brain, specifically in microglia and astrocytes. Recent studies indicate that genomic deletion of TLR2 can result in impaired neurobehavioural function. It is currently not clear if the genomic deletion of TLR2 leads to any alterations in the microstructural features of the brain. In the current study, we noninvasively assess microstructural changes in the brain of TLR2-deficient (tlr2-/-) zebrafish using state-of-the art magnetic resonance imaging (MRI) methods at ultrahigh magnetic field strength (17.6 T). A significant increase in cortical thickness and an overall trend towards increased brain volumes were observed in young tlr2-/- zebrafish. An elevated T2 relaxation time and significantly reduced apparent diffusion coefficient (ADC) unveil brain-wide microstructural alterations, potentially indicative of cytotoxic oedema and astrogliosis in the tlr2-/- zebrafish. Multicomponent analysis of the ADC diffusivity signal by the phasor approach shows an increase in the slow ADC component associated with restricted diffusion. Diffusion tensor imaging and diffusion kurtosis imaging analysis revealed diminished diffusivity and enhanced kurtosis in various white matter tracks in tlr2-/- compared with control zebrafish, identifying the microstructural underpinnings associated with compromised white matter integrity and axonal degeneration. Taken together, our findings demonstrate that the genomic deletion of TLR2 results in severe alterations to the microstructural features of the zebrafish brain. This study also highlights the potential of ultrahigh field diffusion MRI techniques in discerning exceptionally fine microstructural details within the small zebrafish brain, offering potential for investigating microstructural changes in zebrafish models of various brain diseases.

Flavokawain B inhibits NF-κB inflammatory signaling pathway activation in inflammatory bowel disease by targeting TLR2.

Inflammatory bowel disease (IBD) is characterized by recurrent inflammatory reactions in the intestinal mucosa, including ulcerative colitis (UC) and Crohn's disease (CD). The expression of Toll-like receptor 2 (TLR2) has been observed to increase during the progression of IBD. Flavokawain B (FKB), a natural chalcone with potent anti-inflammatory activity, exerts its effects through inhibition of the NF-κB pathway. In this study, we aimed to investigate the effects and mechanisms of FKB targeting TLR2 in IBD. C57BL/6 J mice were treated with 2.5% dextran sulfate sodium (DSS) for 7 days, with administration of FKB or TLR2 inhibitor C29 starting on day 2 to establish the model of IBD. In vitro, bone marrow-derived macrophages (BMDMs) were stimulated with the TLR2 agonist Pam3CSK4 to explore the therapeutic effect of FKB and its pharmacological mechanism. Compared with the model group, the FKB-treated group showed significant reductions in colitis-related injuries in the IBD mouse model, including weight gain, increased colon length and reduced inflammation. FKB decreased the formation of TLR2-MyD88 complex by targeting TLR2, leading to suppression of downstream NF-κB signaling pathway. Similar therapeutic effects were observed in the C29-treated group. Additionally, in vitro data suggested that FKB exerted its anti-inflammatory effect by targeting TLR2 and inhibiting Pam3CSK4-induced activation of the NF-κB pathway. The anti-inflammatory effects of FKB were demonstrated through drug affinity responsive target stability assay and cellular thermal shift assay, revealing its binding affinity to TLR2. By inhibiting the activation of the TLR2/NF-κB signaling pathway, FKB effectively prevented DSS-induced IBD and exhibited promising potential as a therapeutic candidate for IBD treatment.

Effects of Toll-like receptor 1 and 2 agonist Pam3CSK4 on uveal melanocytes and relevant experimental mouse model.

Pam3CSK4 activates Toll-like receptors 2 and 1 (TLR1/2), which recognize mainly molecules from gram-positive pathogens. The effect of Pam3CSK4 on various cytokine and chemokine expression in cultured human uveal melanocytes (UM) has not been studied systematically. The purpose of this study was to investigate the mechanistic expressions of seven cytokines and chemokines of interleukin- (IL-) 6, IL-10, MCP-1 (CCL-2), CXCL-1 (GRO-α), CXCL-8 (IL-8), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in UM. These cytokines are reported to be increased in intraocular fluids or tissues of the patients with endophthalmitis and non-infectious uveitis, as well as in various experimental animal uveitic models in the literature. Flow cytometry was used to measure the effects of Pam3CSK4 on the expression of TLR1/2 in UM. ELISA and Real-time PCR analysis were used to estimate the ability of Pam3CSK4 to elevate these cytokines and chemokines levels in conditioned media and cell lysates of UM, respectively. Flow cytometry measured and compared the phosphorylated MAPK pathway and activated NF-κB signals pathway in UM, treated with and without Pam3CSK4. ELISA analysis tested the effect of various signal inhibitors (ERK1/2, JNK1/2, p38 and NF-κB) on Pam3CSK4-induced IL-6 levels in cultured UM. The role of TLR2 in Pam3CSK4-induced acute anterior uveitis in experimental mouse model was tested in TLR2 knockout (TLR2 KO) mice and their wild-type C57Bl/6 controls. Pam3CSK4 increased the expression of TLR1/2 proteins in cultured UM. Pam3CSK4 significantly elevated the IL-6, MCP-1, CXCL-1, CXCL-8 protein, and mRNA levels in cultured UM, but not IL-10, TNF-α, or IFN-γ. Pam3CSK4 activated NF-κB, ERK, JNK, and p38 expression. Pam3CSK4-induced expression of IL-6 was decreased by NF-κB, ERK, INK, and p38 inhibitors; especially the NF-κB inhibitor, which can completely block the IL-6 stimulation. Intravitreal injection of Pam3CSK4 induced acute anterior uveitis in C57Bl/6 mice, this effect was significantly reduced in TLR2 KO mice. TLR1/2 plays an important role against invading pathogens, especially gram-positive bacteria; but an excessive reaction to molecules from gram-positive bacteria may promote non-infectious uveitis. UM can produce IL-6, MCP-1, CXCL-1, and CXCL-8, and are one of the target cells of TNF-α and IFN-γ. TLR-2 inhibitors might have a beneficial effect in the treatment of certain types of uveitis and other ocular inflammatory-related diseases and warrant further investigation.

Lipoprotein signal peptidase-deficient Streptococcus pneumoniae exhibits impaired Toll-like receptor 2-stimulatory activity.

Streptococcus pneumoniae is a causative agent of community-acquired pneumonia. Upon pneumococcal infection, innate immune cells recognize pneumococcal lipoproteins via Toll-like receptor 2 and induce inflammation. Here, we generated a strain of S. pneumoniae deficient in lipoprotein signal peptidase (LspA), a transmembrane type II signal peptidase required for lipoprotein maturation, to investigate the host immune response against this strain. Triton X-114 phase separation revealed that lipoprotein expression was lower in the LspA-deficient strain than in the wild-type strain. Additionally, the LspA-deficient strain decreased nuclear factor-κB activation and cytokine production in THP-1 cells, indicating impaired innate immune response against the strain.

Toll-like receptor 2, hyaluronan, and neutrophils play a key role in plaque erosion: the OPTICO-ACS study.

BACKGROUND AND AIMS: In one-third of patients with acute coronary syndrome (ACS), thrombosis occurs despite an intact fibrous cap (IFC) (IFC-ACS, 'plaque erosion'). Recent studies emphasize neutrophils as the immediate inflammatory response in this pathology, but their exact molecular activation patterns are still poorly understood and may represent future therapeutic targets. METHODS AND RESULTS: Thirty-two patients with IFC-ACS and matched patients with ACS with ruptured fibrous cap (RFC) (RFC-ACS) from the OPTICO-ACS study were included, and blood samples were collected from the local site of the culprit lesion and the systemic circulation. Neutrophil surface marker expression was quantified by flow cytometry. Neutrophil cytotoxicity towards endothelial cells was examined in an ex vivo co-culture assay. Secretion of active matrix metalloproteinase 9 (MMP9) by neutrophils was evaluated using zymography in supernatants and in plasma samples. Optical coherence tomography (OCT)-embedded thrombi were used for immunofluorescence analysis. Toll-like receptor 2 (TLR2) expression was higher on neutrophils from IFC-ACS than RFC-ACS patients. TLR2 stimulation increased the release of active MMP9 from local IFC-ACS-derived neutrophils, which also aggravated endothelial cell death independently of TLR2. Thrombi of IFC-ACS patients exhibited more hyaluronidase 2 with concomitant increase in local plasma levels of the TLR2 ligand: hyaluronic acid. CONCLUSION: The current study provides first in-human evidence for distinct TLR2-mediated neutrophil activation in IFC-ACS, presumably triggered by elevated soluble hyaluronic acid. Together with disturbed flow conditions, neutrophil-released MMP9 might be promoting endothelial cell loss-triggered thrombosis and therefore providing a potential future target for a phenotype-specific secondary therapeutic approach in IFC-ACS.

Streptococcus suis Serotype 2 Type IV Secretion Effector SspA-1 Induces Proinflammatory Cytokine Production via TLR2 Endosomal and Type I Interferon Signaling.

The subtilisin-like protease-1 (SspA-1) plays an important role in the pathogenesis of a highly virulent strain of Streptococcus suis 2. However, the mechanism of SspA-1-triggered excessive inflammatory response is still unknown. In this study, we demonstrated that activation of type I IFN signaling is required for SspA-1-induced excessive proinflammatory cytokine production. Further experiments showed that the TLR2 endosomal pathway mediates SspA-1-induced type I IFN signaling and the inflammatory response. Finally, we mapped the major signaling components of the related pathway and found that the TIR adaptor proteins Mal, TRAM, and MyD88 and the downstream activation of IRF1 and IRF7 were involved in this pathway. These results explain the molecular mechanism by which SspA-1 triggers an excessive inflammatory response and reveal a novel effect of type I IFN in S. suis 2 infection, possibly providing further insights into the pathogenesis of this highly virulent S. suis 2 strain.

Targeting dendritic cells with TLR-2 ligand-coated nanoparticles loaded with Mycobacterium tuberculosis epitope induce antituberculosis immunity.

Novel vaccination strategies are crucial to efficiently control tuberculosis, as proposed by the World Health Organization under its flagship program "End TB Strategy." However, the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), particularly in those coinfected with HIV-AIDS, constitutes a major impediment to achieving this goal. We report here a novel vaccination strategy that involves synthesizing a formulation of an immunodominant peptide derived from the Acr1 protein of Mtb. This nanoformulation in addition displayed on the surface a toll-like receptor-2 ligand to offer to target dendritic cells (DCs). Our results showed an efficient uptake of such a concoction by DCs in a predominantly toll-like receptor-2-dependent pathway. These DCs produced elevated levels of nitric oxide, proinflammatory cytokines interleukin-6, interleukin-12, and tumor necrosis factor-α, and upregulated the surface expression of major histocompatibility complex class II molecules as well as costimulatory molecules such as CD80 and CD86. Animals injected with such a vaccine mounted a significantly higher response of effector and memory Th1 cells and Th17 cells. Furthermore, we noticed a reduction in the bacterial load in the lungs of animals challenged with aerosolized live Mtb. Therefore, our findings indicated that the described vaccine triggered protective anti-Mtb immunity to control the tuberculosis infection.

Interplay between nuclear factor-κB, p38 MAPK, and glucocorticoid receptor signaling synergistically induces functional TLR2 in lung epithelial cells.

While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of many inflammatory genes, repression is not an invariable outcome. Here, we explore synergy occurring between synthetic glucocorticoids (dexamethasone and budesonide) and proinflammatory cytokines (IL1B and TNF) on the expression of the toll-like receptor 2 (TLR2). This effect is observed in epithelial cell lines and both undifferentiated and differentiated primary human bronchial epithelial cells (pHBECs). In A549 cells, IL1B-plus-glucocorticoid-induced TLR2 expression required nuclear factor (NF)-κB and GR. Likewise, in A549 cells, BEAS-2B cells, and pHBECs, chromatin immunoprecipitation identified GR- and NF-κB/p65-binding regions ∼32 kb (R1) and ∼7.3 kb (R2) upstream of the TLR2 gene. Treatment of BEAS-2B cells with TNF or/and dexamethasone followed by global run-on sequencing confirmed transcriptional activity at these regions. Furthermore, cloning R1 or R2 into luciferase reporters revealed transcriptional activation by budesonide or IL1B, respectively, while R1+R2 juxtaposition enabled synergistic activation by IL1B and budesonide. In addition, small-molecule inhibitors and siRNA knockdown showed p38α MAPK to negatively regulate both IL1B-induced TLR2 expression and R1+R2 reporter activity. Finally, agonism of IL1B-plus-dexamethasone-induced TLR2 in A549 cells and pHBECs stimulated NF-κB- and interferon regulatory factor-dependent reporter activity and chemokine release. We conclude that glucocorticoid-plus-cytokine-driven synergy at TLR2 involves GR and NF-κB acting via specific enhancer regions, which combined with the inhibition of p38α MAPK promotes TLR2 expression. Subsequent inflammatory effects that occur following TLR2 agonism may be pertinent in severe neutrophilic asthma or chronic obstructive pulmonary disease, where glucocorticoid-based therapies are less efficacious.

Essential role of Helicobacter pylori apolipoprotein N-acyltransferase (Lnt) in stomach colonization.

Bacterial lipoproteins are post-translationally modified with acyl chains, anchoring these proteins to bacterial membranes. In Gram-negative bacteria, three enzymes complete the modifications. Lgt (which adds two acyl chains) and LspA (which removes the signal peptide) are essential. Lnt (which adds a third acyl chain) is not essential in certain bacteria including Francisella tularensis, Neisseria gonorrhoeae, and Acinetobacter baumannii. Deleting lnt results in mild to severe physiologic changes. We previously showed lnt is not essential for Helicobacter pylori growth in vitro. Here, the physiologic consequences of deleting lnt in H. pylori and the role of Lnt in the host response to H. pylori were examined using in vitro and in vivo models. Comparing wild-type, Δlnt, and complemented mutant H. pylori, no changes in growth rates or sensitivity to acid or antibiotics were observed. Since deleting lnt changes the number of acyl chains on lipoproteins and the number of acyl chains on lipoproteins impacts the innate immune response through Toll-like receptor 2 (TLR2) signaling, primary human gastric epithelial cells were treated with a purified lipoprotein from wild-type or lnt mutant H. pylori. Differential gene expression analysis indicated that lipoprotein from the lnt mutant induced a more robust TLR2 response. In a complementary approach, we infected wild-type and Tlr2-/- mice and found that both the wild-type and complemented mutant strains successfully colonized the animals. However, the lnt mutant strain was unable to colonize either mouse strain. These results show that lnt is essential for H. pylori colonization and identifies lipoprotein synthesis as a target for therapeutic intervention.

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.

Myeloid cells protect corneal nerves against sterile injury through negative-feedback regulation of TLR2-IL-6 axis.

BACKGROUND: Mounting evidence suggests that the immune system plays detrimental or protective roles in nerve injury and repair. MAIN BODY: Herein we report that both CD11bhiLy6Ghi and CD11bhiLy6ChiLy6Glo myeloid cells are required to protect corneal nerves against sterile corneal injury. Selective depletion of CD11bhiLy6Ghi or CD11bhiLy6ChiLy6Glo cells resulted in aggravation of corneal nerve loss, which correlated with IL-6 upregulation. IL-6 neutralization preserved corneal nerves while reducing myeloid cell recruitment. IL-6 replenishment exacerbated corneal nerve damage while recruiting more myeloid cells. In mice lacking Toll-like receptor 2 (TLR2), the levels of IL-6 and myeloid cells were decreased and corneal nerve loss attenuated, as compared to wild-type and TLR4 knockout mice. Corneal stromal fibroblasts expressed TLR2 and produced IL-6 in response to TLR2 stimulation. CONCLUSION: Collectively, our data suggest that CD11bhiLy6Ghi and CD11bhiLy6ChiLy6Glo myeloid cells confer corneal nerve protection under sterile injury by creating a negative-feedback loop to suppress the upstream TLR2-IL-6 axis that drives corneal nerve loss.

Human Toll-like receptor 2 genetic polymorphisms with tuberculosis susceptibility: A systematic review and meta-analysis.

BACKGROUND: Toll-like receptor 2 (TLR2) is a crucial factor in the development of tuberculosis. However, no studies have explored the association between TLR2 polymorphisms and tuberculosis susceptibility. OBJECTIVES: This study aimed to explore the correlation between tuberculosis susceptibility and TLR2 polymorphisms (rs3804099, rs3804100, rs1898830, rs5743708, rs121917864, and (-196-174) del). METHODS: All relevant online databases including PubMed, CNKI, WANFANG DATA, and METSTR-FMRS were systematically searched. STATA17.0 (Stata Corp LP, College Station, Texas, USA) was used. RESULTS: A total of 37 studies, covering six polymorphisms and comprising 9,474 cases and 10,295 controls, were included in this analysis. rs3804099(C vs T: OR = 1.00, 95 % CI: 0.93-1.08, CC + TC vs TT: OR = 1.04, 95 % CI: 0.98-1.10), rs3804100 (C vs T: OR = 1.19, 95 % CI: 0.93-1.07, CC + TC vs TT: OR = 0.97, 95 % CI: 0.89-1.06), rs1898830(G vs A: OR = 0.90, 95 % CI: 0.81-1.00, GG + AG vs AA: OR = 0.87, 95 % CI: 0.67-1.12), (-196 ∼174) del polymorphism (Del vs Ins: OR = 0.93,95 % CI: 0.76-1.14, DD + DI vs II: OR = 0.92,95 % CI: 0.72-1.17). CONCLUSIONS: This study indicated that only the TLR2 rs5743708 polymorphism exhibited a significant association with a higher tuberculosis risk, while TLR2 rs3804099, rs3804100, rs1898830, rs121917864, and (-196-174) del polymorphisms were not associated with tuberculosis susceptibility.

Toll-like Receptor 2-Melatonin Feedback Loop Regulates the Activation of Spinal NLRP3 Inflammasome in Morphine-Tolerant Rats.

BACKGROUND AND PURPOSE: Morphine is amongst the most effective analgesics available for the management of severe pain. However, prolonged morphine treatment leads to analgesic tolerance which limits its clinical usage. Previous studies have demonstrated that melatonin ameliorates morphine tolerance by reducing neuroinflammation. However, little is known about the relationship between Toll like receptor 2 (TLR2) and neuroinflammation in morphine tolerance. The aim of this study was to explore the role of TLR2 in morphine tolerance and its connections with melatonin and Nod-like receptor protein 3 (NLRP3) inflammasome. METHODS: Sprague-Dawley rats were treated with morphine for 7 days and tail-flick latency test was performed to identify the induction of analgesic tolerance. The roles of TLR2 in microglia activation and morphine tolerance were assessed pharmacologically, and the possible interactions between melatonin, TLR2 and NLRP3 inflammasome were investigated. KEY RESULTS: Morphine tolerance was accompanied by increased TLR2 expression and NLRP3 inflammasome activation in spinal cord. whereas melatonin level was down-regulated. Chronic melatonin administration resulted in a reduced TLR2 expression and NLRP3 inflammasome activation. Moreover, the analgesic effect of morphine was partially restored. Inhibition of TLR2 suppressed the microglia and NLRP3 inflammasome activation, as well as restored the spinal melatonin level while attenuated the development of morphine tolerance. Furthermore, the inhibition of microglia activation ameliorated morphine tolerance via inhibiting TLR2-NLRP3 inflammasome signaling in spinal cord. CONCLUSION: In this study, we directly demonstrate a TLR2-melatonin negative feedback loop regulating microglia and NLRP3 inflammasome activation during the development of morphine tolerance.

Synthesis, evaluation and molecular dynamics study of human toll-like receptor 2/6 specific monoacyl lipopeptides as candidate immunostimulants.

TLR2 agonists typified by the S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (Pam2CS) motif have exhibited powerful immunostimulatory activities. Based on simplified monoacyl lipopeptide (Carbamate-linked N-Ac PamCS), we describe interesting SAR investigations where modifications are done to alter the size of substituents on the cysteine amine, introduce ionizable groups to the terminal and insert aromatic substitutions to the aliphatic chain. Our structural modifications have led to a highly specific human TLR2/6 agonist 14a (EC50 = 0.424 nM), which behaves like Pam2CSK4 by inducing NF-κB activation to trigger downstream signaling pathways, such as subsequent phosphorylation of related proteins (p65, p38) and production of key inflammatory cytokines (IL-6, IL-1ß, TNF-α). Importantly, the ability to stimulate enhanced T cell response compared to Carbamate-linked N-Ac PamCS makes compound 14a a further potential candidate immunostimulant.

Toll-like receptor 2-mediated downstream cytokine levels as determinant of malaria pathogenesis.

Background & objectives: Toll-like receptors (TLRs) are transmembrane proteins that recognize specific molecular patterns and activate downstream cytokine production usually for the eradication of invading pathogens. The objective of this study was to evaluate the genetic polymorphism of TLR2 Arg753Gln (rs 5743708) and soluble cytokines and TLR2 expression levels in malaria disease cases. Methods: The study included prospectively collected 2 ml blood samples from 153 individuals clinically suspected for malaria and confirmed by microscopy and RDT from Assam. Stratification of the study groups was done as healthy control (HC, n=150), uncomplicated malaria (UC-M, n=128) and severe malaria (SM, n=25). The PCR-restriction fragment length polymorphism (RFLP) method was applied for the analysis of TLR2 Arg753Gln polymorphism and following the ELISA for soluble serum TLR2 (sTLR2) and its associated downstream cytokines, viz. tumour necrosis factor (TNF)-α and interferon (IFN)-γ levels. Results: Variation in TLR2 Arg753Gln gene showed no association with the susceptibility and the severity of malarial infection. Soluble TLR2 expression was significantly higher in uncomplicated malaria (UC-M) cases compared to healthy controls (P=0.045) and in terms of SM cases, the expression was also found to be higher in UC-M cases (P=0.078). The TNF-α expression was significantly higher in SM cases compared to both UC-M and control (P=0.003 and P=0.004). Similarly, significantly elevated expression of IFN-γ was noted in SM cases compared to both UC-M (P=0.001) and healthy controls (P<0.001). Interpretation & conclusions: The present study suggests the association of deregulated TLR2 pathway that leads to the deleterious downstream immune response in the development of malarial pathogenicity.

Lactobacillus rhamnosus inhibits osteoclast differentiation by suppressing the TLR2/NF-κB pathway.

OBJECTIVE: This study aimed to investigate the role of ultrasonicated Lactobacillus rhamnosus extract in osteoclast differentiation and its underlying mechanism, providing new strategies for the treatment of periodontitis. MATERIALS AND METHODS: Osteoclasts were induced using macrophage colony-stimulating factor and receptor activator for nuclear factor-κB ligand. Lactobacillus rhamnosus extracts were obtained via ultrasonic crushing and ultracentrifugation. The effects of the LGG extract on osteoclast differentiation were evaluated, and the related signaling pathways were examined using western blotting. A mouse periodontitis model was established, and Lactobacillus rhamnosus extract was injected into the gingival sulcus to evaluate the inhibitory effect of Lactobacillus rhamnosus extract on alveolar bone resorption. RESULTS: At 50 µg/mL, Lactobacillus rhamnosus extract inhibited osteoclast differentiation with no effect on apoptosis and proliferation. This phenomenon was achieved by deactivating the NF-κB/c-Fos/NFATc1 signaling pathway through toll-like receptor 2. The in vivo results showed that the local injection of Lactobacillus rhamnosus extract suppressed osteoclast differentiation and alveolar bone resorption. CONCLUSION: The ultrasonicated extract of Lactobacillus rhamnosus inhibited osteoclast differentiation by suppressing the activation of the NF-κB/c-Fos/NFATc1 pathway. Furthermore, it inhibited the destruction of the alveolar bone, providing a new strategy for the use of probiotics in the treatment of periodontitis.