Insight into potent TLR2 inhibitors for the treatment of disease caused by Mycoplasma pneumoniae based on machine learning approaches.

Mycoplasma pneumoniae (MP) is one of the most common pathogens that causes acute respiratory tract infections. Children experiencing MP infection often suffer severe complications, lung injury, and even death. Previous studies have demonstrated that Toll-like receptor 2 (TLR2) is a potential therapeutic target for treating the MP-induced inflammatory response. However, the screening of natural compounds has received more attention for the treatment of bacterial infections to reduce the likelihood of bacterial resistance. Herein, we screened compounds by combining molecular docking and machine learning approaches to find potential lead compounds for treating MP infection. First, all compounds were docked with the TLR2 receptor protein to screen for potential candidates. To predict drug bioactivity, a machine learning model (random forest) was trained for TLR2 inhibitors to obtain the predictive model. The model achieved significant squared correlation coefficient (R2) values for the training set (0.85) and validation set (0.84) of compounds. The developed machine learning model was then used to predict the pIC50 values of the top 50 candidates from the Traditional Chinese compounds and Discovery Diversity sets of compounds. As a result, these compounds are capable of inhibiting the inflammatory response induced by MP. However, prior to bringing these compounds to market, it is necessary to verify these results with additional biological testing, including preclinical and clinical studies. Moreover, the present study provides a theoretical basis for the use of natural compounds as potential candidates to treat pneumonia caused by MP.

Yeast-derived nanoparticles remodel the immunosuppressive microenvironment in tumor and tumor-draining lymph nodes to suppress tumor growth.

Microbe-based cancer immunotherapy has recently emerged as a hot topic for cancer treatment. However, serious limitations remain including infection associated side-effect and unsatisfactory outcomes in clinic trials. Here, we fabricate different sizes of nano-formulations derived from yeast cell wall (YCW NPs) by differential centrifugation. The induction of anticancer immunity of our formulations appears to inversely correlate with their size due to the ability to accumulate in tumor-draining lymph node (TDLN). Moreover, we use a percolation model to explain their distribution behavior toward TDLN. The abundance and functional orientation of each effector component are significantly improved not only in the microenvironment in tumor but also in the TDLN following small size YCW NPs treatment. In combination with programmed death-ligand 1 (PD-L1) blockade, we demonstrate anticancer efficiency in melanoma-challenged mice. We delineate potential strategy to target immunosuppressive microenvironment by microbe-based nanoparticles and highlight the role of size effect in microbe-based immune therapeutics.

Tetrahedral-Framework Nucleic Acids Carry Small Interfering RNA to Downregulate Toll-Like Receptor 2 Gene Expression for the Treatment of Sepsis.

Sepsis is caused by the invasion of pathogenic microorganisms, which can lead to excessive expression of toll-like receptors (TLRs) in cells and uncontrollable amplification of the inflammatory response. TLR2, as an essential part of the TLR family, has a significant feature in the identification of innate immune responses. Therefore, blocking the expression and activation of TLR2 can inhibit the synthesis and release of inflammatory factors and avoid the occurrence of excessive inflammatory reactions. Small interfering RNA (siRNA) can selectively target the silencing or downregulation of pathogenic genes and has the advantages of high specificity, a strong effect, and fewer adverse reactions. However, the application of siRNA is limited by its high molecular weight, poor biostability, and difficulty in passive uptake into cells. Tetrahedral-framework nucleic acid (tFNA) is a new kind of three-dimensional nucleic acid nanomaterial, which has the advantages of good biocompatibility, stable structure, and editability. In this study, we used tFNA as carriers to deliver siRNA-targeting downregulation of TLR2 expression for anti-inflammatory therapy. We show that siRNA can specifically reduce lipopolysaccharide (LPS)-induced TLR2 elevation and reduce release of inflammatory factors in LPS-induced experimental sepsis, which provides a new idea for the prevention and treatment of sepsis.

Blockade of TLR2 and TLR4 Attenuates Inflammatory Response and Parasite Load in Cutaneous Leishmaniasis.

Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a pronounced inflammatory response associated with ulcer development. Monocytes/macrophages, the main cells harboring parasites, are largely responsible for parasite control. Toll-like receptor (TLR) signaling leads to the transcription of inflammatory mediators, such as IL-1ß and TNF during innate immune response. TLR antagonists have been used in the treatment of inflammatory disease. The neutralization of these receptors may attenuate an exacerbated inflammatory response. We evaluated the ability of TLR2 and TLR4 antagonists to modulate host immune response in L. braziliensis-infected monocytes and cells from CL patient skin lesions. Following TLR2 and TLR4 neutralization, decreased numbers of infected cells and internalized parasites were detected in CL patient monocytes. In addition, reductions in oxidative burst, IL-1ß, TNF and CXCL9 production were observed. TNF production by cells from CL lesions also decreased after TLR2 and TLR4 neutralization. The attenuation of host inflammatory response after neutralizing these receptors suggests the potential of TLR antagonists as immunomodulators in association with antimonial therapy in human cutaneous leishmaniasis.

TLR2 Potentiates SR-Marco-Mediated Neuroinflammation by Interacting with the SRCR Domain.

Microglial activation-induced neuroinflammation is critical in the pathogenesis of neurodegenerative diseases. Activated microglia are regulated mainly by innate pattern recognition receptors (PRRs) on their surface, of which macrophage receptor with collagenous structure (Marco) is a well-characterized scavenger receptor constitutively expressed on specific subsets of macrophages, including microglia. Increasing evidence has shown that Marco is involved in the pathogenesis of a range of inflammatory processes. However, research on the role of Marco in regulating neuroinflammation has reported conflicting results. In the present study, we examined the role Marco played in triggering neuroinflammation and its underlying mechanisms. The results demonstrated that silencing the Marco gene resulted in a significantly reduced neuroinflammatory response and vice versa. α-Syn stimulation in Marco overexpressing cells induced a pronounced inflammatory response, suggesting that Marco alone could trigger an inflammatory response. We also found that TLR2 significantly promoted Marco-mediated neuroinflammation, indicating TLR2 was an important co-receptor of Marco. Knocking down the TLR2 gene in microglia and mouse substantia nigra resulted in decreased expression of Marco. Subsequent mechanistic studies showed that deleting the SRCR domain of Marco resulted in disruption of the inflammatory response and the interaction between TLR2 and Marco. This suggested that TLR2 binds directly to the SRCR domain of Marco and regulates Marco-mediated neuroinflammation. In summary, this investigation revealed that TLR2 could potentiate Marco-mediated neuroinflammation by interacting with the SRCR domain of Marco, providing a new target for inhibiting neuroinflammation in neurodegenerative diseases.

TLR2 activation promotes tumour growth and associates with patient survival and chemotherapy response in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, and is plagued by a paucity of targeted treatment options and tumour resistance to chemotherapeutics. The causal link between chronic inflammation and PDAC suggests that molecular regulators of the immune system promote disease pathogenesis and/or therapeutic resistance, yet their identity is unclear. Here, we couple endoscopic ultrasound-guided fine-needle aspiration, which captures tumour biopsies from all stages, with whole transcriptome profiling of PDAC patient primary tumours to reveal enrichment of the innate immune Toll-like receptor 2 (TLR2) molecular pathway. Augmented TLR2 expression associated with a 4-gene "TLR2 activation" signature, and was prognostic for survival and predictive for gemcitabine-based chemoresistance. Furthermore, antibody-mediated anti-TLR2 therapy suppressed the growth of human PDAC tumour xenografts, independent of a functional immune system. Our results support TLR2-based therapeutic targeting for precision medicine in PDAC, with further clinical utility that TLR2 activation is prognostic and predictive for chemoresponsiveness.

Attenuation of Doxorubicin-Induced Small Intestinal Mucositis by Pectins is Dependent on Pectin's Methyl-Ester Number and Distribution.

SCOPE: Intestinal mucositis is a common side effect of the chemotherapeutic agent doxorubicin, which is characterized by severe Toll-like receptor (TLR) 2-mediated inflammation. The dietary fiber pectin is shown to prevent this intestinal inflammation through direct inhibition of TLR2 in a microbiota-independent manner. Recent in vitro studies show that inhibition of TLR2 is determined by the number and distribution of methyl-esters of pectins. Therefore, it is hypothesized that the degree of methyl-esterification (DM) and the degree of blockiness (DB) of pectins determine attenuating efficacy on doxorubicin-induced intestinal mucositis. METHODS AND RESULTS: Four structurally different pectins that differed in DM and DB are tested on inhibitory effects on murine TLR2 in vitro, and on doxorubicin-induced intestinal mucositis in mice. These data demonstrate that low DM pectins or intermediate DM pectins with high DB have the strongest inhibitory impact on murine TLR2-1 and the strongest attenuating effect on TLR2-induced apoptosis and peritonitis. Intermediate DM pectin with a low DB is, however, also effective in preventing the induction of doxorubicin-induced intestinal damage. CONCLUSION: These pectin structures with stronger TLR2-inhibiting properties may prevent the development of doxorubicin-induced intestinal damage in patients undergoing chemotherapeutic treatment with doxorubicin.

A Novel Mechanism of Carvedilol Efficacy for Rosacea Treatment: Toll-Like Receptor 2 Inhibition in Macrophages.

Background: Rosacea, a chronic inflammatory skin disorder etiologically associated with immune cells and the antibacterial peptide cathelicidin LL-37, can be effectively treated by oral carvedilol administration. Objective: To investigate the molecular mechanisms underlying carvedilol efficacy in rosacea treatment. Methods: Skin samples of patients with rosacea were subjected to histopathological (hematoxylin and eosin) and immunohistochemical (CD68, Toll-like receptor 2 (TLR2), kallikrein 5, cathelicidin, TNF-α, and IL-1ß) evaluation. An in vivo murine rosacea-like inflammation model was established by LL-37 intradermal injection with or without carvedilol gavage-based pretreatment. Erythema proportion (Image J) and skin redness (L*a*b colorimetry) were quantified. Murine skin samples underwent pathological examination for inflammatory status and immunofluorescence staining. Murine skin and lipopolysaccharide-stimulated RAW 264.7 cells with or without carvedilol pretreatment were evaluated by quantitative reverse transcription-polymerase chain reaction and western blotting. Clinical facial images of patients were obtained using the VISIA skin analysis system before, 4, and 6 months following oral carvedilol administration. Results: Rosacea skin lesions exhibited more pronounced inflammatory cell infiltration than peripheral areas, with profound macrophage infiltration and inflammatory cytokines (TLR2, kallikrein 5, cathelicidin, TNF-α, and IL-1ß). In vivo, carvedilol alleviated inflammation in LL-37 mice, down-regulating TLR2, KLK5, and cathelicidin expression. In vitro, carvedilol decreased TLR2 expression in RAW 264.7 cells, further reducing KLK5 secretion and LL-37 expression and ultimately inhibiting rosacea-like inflammatory reactions. Clinical manifestations and facial redness obviously improved during 6-month follow-up with systemic carvedilol administration. Conclusion: Carvedilol is effective against rosacea, with inhibition of macrophage TLR2 expression as a novel anti-inflammatory mechanism.

Toll-Like Receptor 2 Antagonist Ameliorates Type 2 Diabetes Mellitus Associated Neuropathic Pain by Repolarizing Pro-inflammatory Macrophages.

Diabetic neuropathy is one of the common complications of type 2 diabetes mellitus (T2DM) with severe outcomes. The mechanisms of physiopathology of diabetic neuropathy are not well elucidated. Inflammation and inflammatory macrophages are recognized to be crucial in diabetic neuropathy. Toll-like receptor 2 (TLR2) is an important factor in innate immune response which could promote the polarization of inflammatory macrophages. In present study, we evaluated the effects of a TLR2 antagonist CU-CPT22 on diabetic neuropathy. We induced T2DM in mice by feeding with high fat diet (HFD). We measured the body weight, blood glucose level, paw withdrawal threshold, inflammatory cytokine production, and macrophages infiltration in T2DM mice. We evaluated the effects of CU-CPT22 on pro-inflammatory cytokines production, macrophage marker expression in lipopolysaccharides (LPS)-treated BMDMs. We administrated CU-CPT22 in T2DM mice and measured the pro-inflammatory cytokines levels, expression of macrophages markers in sciatic nerve (SCN), and paw withdrawal threshold. T2DM mice had significantly increased body weight and blood glucose, and had significantly decreased paw withdrawal threshold. Obvious increased pro-inflammatory cytokine level and infiltration of M1 phenotype macrophages was observed in SCN from T2DM mice. CU-CPT22 prevented pro-inflammatory cytokine production in LPS-treated BMDMs and re-polarized them to M2 phenotype. CU-CPT22 suppressed the inflammation and induced M2 macrophages in SCN from T2DM mice, and ameliorated the paw withdrawal threshold in T2DM mice. CU-CPT22 ameliorates neuropathic pain in T2DM by promoting M2 phenotype macrophages.

TLR2 senses the SARS-CoV-2 envelope protein to produce inflammatory cytokines.

The innate immune response is critical for recognizing and controlling infections through the release of cytokines and chemokines. However, severe pathology during some infections, including SARS-CoV-2, is driven by hyperactive cytokine release, or a cytokine storm. The innate sensors that activate production of proinflammatory cytokines and chemokines during COVID-19 remain poorly characterized. In the present study, we show that both TLR2 and MYD88 expression were associated with COVID-19 disease severity. Mechanistically, TLR2 and Myd88 were required for ß-coronavirus-induced inflammatory responses, and TLR2-dependent signaling induced the production of proinflammatory cytokines during coronavirus infection independent of viral entry. TLR2 sensed the SARS-CoV-2 envelope protein as its ligand. In addition, blocking TLR2 signaling in vivo provided protection against the pathogenesis of SARS-CoV-2 infection. Overall, our study provides a critical understanding of the molecular mechanism of ß-coronavirus sensing and inflammatory cytokine production, which opens new avenues for therapeutic strategies to counteract the ongoing COVID-19 pandemic.

Salvianolic acid A alleviated inflammatory response mediated by microglia through inhibiting the activation of TLR2/4 in acute cerebral ischemia-reperfusion.

BACKGROUND: Toll-like receptor 2 and Toll-like receptor 4 (TLR2/4) on microglia have been found as important regulators in the inflammatory response during cerebral ischemia/reperfusion (I/R). In China, traditional Chinese medicine Salvia miltiorrhiza (danshen) and its some components are considered to be effective in rescuing cerebral I/R injury through clinical practice. HYPOTHESIS/PURPOSE: Here we examined the effect of Salvianolic acid A (SAA), a monomer compound in the water extract of Salvia miltiorrhiza, on TLR2/4 of microglia and its mediated inflammatory injury during cerebral I/R in vivo and in vitro. STUDY DESIGN: For exploring the effect of SAA on cerebral I/R and TLR2/4, classic middle cerebral artery occlusion (MCAO) model and oxygen glucose deprivation / reoxygenation (OGD/R) model of co-culture with primary hippocampal neurons and microglia in vitro were used. Signal pathway research and gene knockout have been applied to further explain its mechanism. METHODS: The evaluation indexes of I/R injury included infarct size, edema degree and pathology as well as primary hippocampal neurons and microglia culture, ELISA, western, RT-PCR, HE staining, immunofluorescence, flow cytometry, siRNA gene knockout were also employed. RESULTS: SAA significantly improved the degree of brain edema and ischemic area in I/R rats accompanied by decreases in levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-alpha (TNF-α). Pathological staining revealed that SAA could reduce inflammatory cell infiltration and mcirogila activation after reperfusion. Both protein and gene expression of TLR2 and TLR4 in ischemic hemisphere were obviously inhibited by SAA treatment while changes were not found in the non-ischemic hemisphere. In order to further study its mechanism, OGD/R model was used to mimic inflammatory damage of ischemic tissue by co-culturing primary rat hippocampal neurons and microglial cells. It was found that SAA also inhibited the protein and gene expression of TLR2 and TLR4 after OGD/R injury in microglia. After TLR2/4 knockout, the inhibitory effect of SAA on IL-1ß and TNF-α levels in cell supernatant and neuron apoptosis were significantly weakened in each dose group. Moreover, expression levels of myeloid differentiation factor 88 (MyD88), NFκB, IL-1ß and IL-6 in TLR2/4 mediated inflammatory pathway were reduced with SAA treatment. CONCLUSION: SAA could significantly reduce the inflammatory response and injury in cerebral ischemia-reperfusion in vivo and in vitro, and its mechanism may be through the inhibition of TLR2/4 and its related signal pathway.

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

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

Toll-like receptor 2 activation and up-regulation by high mobility group box-1 contribute to post-operative neuroinflammation and cognitive dysfunction in mice.

Post-operative cognitive dysfunction (POCD) is common and is associated with poor clinical outcome. Toll-like receptor (TLR) 3 and 4 have been implied in the development of POCD. The role of TLR2, a major brain TLR, in POCD is not clear. High mobility group box-1 (HMGB1) is a delayed inflammatory mediator and may play a role in POCD. The interaction between HMGB1 and TLRs in the perioperative period is not known. We hypothesize that TLR2 contributes to the development of POCD and that HMGB1 regulates TLR2 for this effect. To test these hypotheses, 6- to 8-week old male mice were subjected to right carotid artery exposure under isoflurane anesthesia. CU-CPT22, a TLR1/TLR2 inhibitor, at 3 mg/kg was injected intraperitoneally 30 min before surgery and 1 day after surgery. Glycyrrhizin, a HMGB1 antagonist, at 200 mg/kg was injected intraperitoneally 30 min before surgery. Mice were subjected to Barnes maze and fear conditioning tests from 1 week after surgery. Hippocampus and cerebral cortex were harvested 6 hr or 12 hr after the surgery for Western blotting, ELISA, immunofluorescent staining, and chromatin immunoprecipitation. There were neuroinflammation and impairment of learning and memory in mice with surgery. Surgery increased the expression of TLR2 and TLR4 but not TLR9 in the brain of CD-1 male mice. CU-CPT22 attenuated surgery-induced neuroinflammation and cognitive impairment. Similarly, surgery induced neuroinflammation and cognitive dysfunction in C57BL/6J mice but not in TLR2-/- mice. TLR2 staining appeared in neurons and microglia. Surgery increased HMGB1 in the cell nuclei of the cerebral cortex and hippocampus. Glycyrrhizin ameliorated this increase and the increase of TLR2 in the hippocampus after surgery. Surgery also increased the amount of tlr2 DNA precipitated by an anti-HMGB1 antibody in the hippocampus. Our results suggest that TLR2 contributes to surgery-induced neuroinflammation and cognitive impairment. HMGB1 up-regulates TLR2 expression in the hippocampus after surgery to facilitate this contribution. Thus, TLR2 and HMGB1 are potential targets for reducing POCD.

Clostridioides difficile toxins enhanced the in vitro production of CXC chemokine ligand 2 and tumor necrosis factor-α via Toll-like receptors in macrophages.

Introduction. Clostridioides difficile infection (CDI) causes toxin-mediated enteropathy, such as antibiotic-associated diarrhoea and pseudomembranous colitis. Rho-glucosylating toxin A (TcdA) and toxin B (TcdB) have been clearly implicated in pathogenesis, whereas the virulence of binary toxin (CDT) is still debated.Hypothesis statement. We hypothesized that CDT is involved in the host immune response and plays a pivotal role in establishing virulence by modulating pro-inflammatory cytokine production; this is achieved through the integral Toll-like receptor (TLR) signalling pathways.Aim. The aim of the present study was to determine whether and how CDT impacts macrophages compared to TcdA or TcdB by examining the induction of CXC chemokine ligand 2 (CXCL2) and tumour necrosis factor-α (TNF-α), both of which are crucial in mediating local and systematic inflammatory responses.Methodology. RAW264.7 cells or transfected human embryonic kidney (HEK) 293 T cells were incubated with TcdA, TcdB, or CDT. In some experiments, a neutralizing antibody against TLR2 or TLR4, or myeloid differentiation 88 inhibitory peptide were added. The amount of CXCL2 and TNF-α secreted was then measured.Results. In RAW264.7 macrophages, CXCL2 and TNF-α were produced via the Toll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) pathway in a TcdA, TcdB, or CDT dose-dependent manner. Interleukin-8 secretion was induced in TLR4/MD2/CD14-transfected, but not in TLR2-transfected, HEK 293 T cells following TcdB or CDT exposure.Conclusion. Our results showed that C. difficile toxins, including CDT, enhanced macrophage-mediated CXCL2 and TNF-α production via TLR2 and TLR4, indicating that CDT affects host immune responses.

Involvement of TLR2/4­MyD88­NF­κB signaling pathway in the pathogenesis of intracranial aneurysm.

Toll­like receptor (TLR) 2/4 serves an important regulatory role in nerve tissue injury. However, the downstream and potential mechanisms remain to be elucidated. The present study was designed to investigate the roles of the TLR2/4­major myeloid differentiation response gene 88 (MyD88)­NF­κB signaling pathway in the development of intracranial aneurysm. The expression of TLR2, TLR4 and MyD88 in the blood of normal controls and patients with intracranial aneurysm were detected by quantitative PCR and ELISA. Human brain vascular smooth muscle cells were treated by Angiotensin II (Ang II) to evaluate the involvement of TLR2/4­MyD88­NF­κB signaling pathway in the process. The in vitro experiment was divided into four groups: The control group, an Ang â…¡ group, an Ang â…¡ + small interfering (si)RNA control group and an Ang â…¡ + TLR2­group. Cell viability, migration, apoptosis and expression of TLR2, TLR4, MyD88, NF­κB and phosphorylated (p­)p65 expression were detected. The results demonstrated that the expression of TLR2, TLR4, MyD88 and NF­κB at mRNA and protein levels in patients with intracranial aneurysm was significantly higher compared with corresponding protein in normal controls (P<0.05). In vitro experiments demonstrated that Ang â…¡ treatment increased the cell proliferation and migration rate but reduced the apoptotic rate compared with the control (P<0.05). The expression of TLR2, TLR4, MyD88, NF­κB and p­p65 was significantly increased in the Ang II group (vs. control; P<0.05). By contrast, TLR2­short interfering RNA reduced the cell proliferation and migration rate, and reduced the expression of TLR2, TLR4, MyD88, NF­κB and p­p65 (vs. Ang â…¡ + short interfering RNA control; P<0.05). In conclusion, the data of the present study indicated that the TLR2/4­MyD88­NF­κB signaling pathway is involved in the pathogenesis of intracranial aneurysm.

Structure based design and synthesis of novel Toll-like Receptor 2 (TLR 2) lipid antagonists.

Toll-like receptors (TLRs) play key role in innate immune response to Damage Associated Molecular Patterns (DAMPs) and Pathogen Associated Molecular Patterns (PAMPs). DAMP/PAMP-mediated activation of TLRs triggers NFκB signaling resulting in pro-inflammatory cytokine release. Using TLR2-Pam2CSK4 agonist co-crystal structure information, we designed and synthesized a novel series of Toll-like Receptor 2 (TLR2) lipid antagonists and identified compounds 14, 15 and 17 with sub-micromolar potency. TLR2 antagonists that we identified are stable for > 1.0 h in both gastric juice and PBS buffer and could be used as research tools.

Leveraging systems biology for predicting modulators of inflammation in patients with COVID-19.

Dysregulations in the inflammatory response of the body to pathogens could progress toward a hyperinflammatory condition amplified by positive feedback loops and associated with increased severity and mortality. Hence, there is a need for identifying therapeutic targets to modulate this pathological immune response. Here, we propose a single cell-based computational methodology for predicting proteins to modulate the dysregulated inflammatory response based on the reconstruction and analysis of functional cell-cell communication networks of physiological and pathological conditions. We validated the proposed method in 12 human disease datasets and performed an in-depth study of patients with mild and severe symptomatology of the coronavirus disease 2019 for predicting novel therapeutic targets. As a result, we identified the extracellular matrix protein versican and Toll-like receptor 2 as potential targets for modulating the inflammatory response. In summary, the proposed method can be of great utility in systematically identifying therapeutic targets for modulating pathological immune responses.

Staphylococcus aureus enterotoxin B disrupts nasal epithelial barrier integrity.

BACKGROUND: Staphylococcus aureus colonization and release of enterotoxin B (SEB) has been associated with severe chronic rhinosinusitis with nasal polyps (CRSwNP). The pathogenic mechanism of SEB on epithelial barriers, however, is largely unexplored. OBJECTIVE: We investigated the effect of SEB on nasal epithelial barrier function. METHODS: SEB was apically administered to air-liquid interface (ALI) cultures of primary polyp and nasal epithelial cells of CRSwNP patients and healthy controls, respectively. Epithelial cell integrity and tight junction expression were evaluated. The involvement of Toll-like receptor 2 (TLR2) activation was studied in vitro with TLR2 monoclonal antibodies and in vivo in tlr2-/- knockout mice. RESULTS: SEB applied to ALI cultures of polyp epithelial cells decreased epithelial cell integrity by diminishing occludin and zonula occludens (ZO)-1 protein expression. Antagonizing TLR2 prevented SEB-induced barrier disruption. SEB applied in the nose of control mice increased mucosal permeability and decreased mRNA expression of occludin and ZO-1, whereas mucosal integrity and tight junction expression remained unaltered in tlr2-/- mice. Furthermore, in vitro SEB stimulation resulted in epithelial production of IL-6 and IL-8, which was prevented by TLR2 antagonization. CONCLUSION & CLINICAL RELEVANCE: SEB damages nasal polyp epithelial cell integrity by triggering TLR2 in CRSwNP. Our results suggest that SEB might represent a driving factor of disease exacerbation, rather than a causal factor for epithelial defects in CRSwNP. Interfering with TLR2 triggering might provide a way to avoid the pathophysiological consequences of S. aureus on inflammation in CRSwNP.

Experimental autoimmune encephalopathy (EAE)-induced hippocampal neuroinflammation and memory deficits are prevented with the non-opioid TLR2/TLR4 antagonist (+)-naltrexone.

Multiple sclerosis (MS) is associated with burdensome memory impairments and preclinical literature suggests that these impairments are linked to neuroinflammation. Previously, we have shown that toll-like receptor 4 (TLR4) antagonists, such as (+)-naltrexone [(+)-NTX], block neuropathic pain and associated spinal inflammation in rats. Here we extend these findings to first demonstrate that (+)-NTX blocks TLR2 in addition to TLR4. Additionally, we examined in two rat strains whether (+)-NTX could attenuate learning and memory disturbances and associated neuroinflammation using a low-dose experimental autoimmune encephalomyelitis (EAE) model of MS. EAE is the most commonly used experimental model for the human inflammatory demyelinating disease, MS. This low-dose model avoided motor impairments that would confound learning and memory measurements. Fourteen days later, daily subcutaneous (+)-NTX or saline injections began and continued throughout the study. Contextual and auditory-fear conditioning were conducted at day 21 to assess hippocampal and amygdalar function. With this low-dose model, EAE impaired long-term, but not short-term, contextual fear memory; both long-term and short-term auditory-cued fear memory were spared. This was associated with increased mRNA for hippocampal interleukin-1ß (IL-1ß), TLR2, TLR4, NLRP3, and IL-17 and elevated expression of the microglial marker Iba1 in CA1 and DG regions of the hippocampus, confirming the neuroinflammation observed in higher-dose EAE models. Importantly, (+)-NTX completely prevented the EAE-induced memory impairments and robustly attenuated the associated proinflammatory effects. These findings suggest that (+)-NTX may exert therapeutic effects on memory function by dampening the neuroinflammatory response in the hippocampus through blockade of TLR2/TLR4. This study suggests that TLR2 and TLR4 antagonists may be effective at treating MS-related memory deficits.

Etidronate down-regulates Toll-like receptor 2 ligand-induced chemokine production by inhibiting MyD88 expression and NF-κB activation.

OBJECTIVE: Pretreatment of J774.1 cells with etidronate, a non-nitrogen-containing bisphosphonate (non-NBP) used as an antibone resorptive drug, was previously reported to inhibit Toll-like receptor (TLR) 2 agonist-induced proinflammatory cytokine production. The present study aimed to examine the effects of etidronate on chemokine production by human monocytic U937 cells incubated with Pam3Cys-Ser-(Lys)4 (Pam3CSK4, a TLR2 ligand) and lipid A (a TLR4 ligand). METHODS: U937 cells were pretreated with or without etidronate, and then incubated with or without Pam3CSK4 or lipid A. Levels of secreted human interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) in culture supernatants and activation of nuclear factor-κB (NF-κB) p65 were measured by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) activity in supernatants. Expression of intracellular adhesion molecule (ICAM)-1 and MyD88 was analyzed by flow cytometry and Western blot analysis, respectively. RESULTS: Etidronate down-regulated IL-8 and MCP-1 production and NF-κB p65 activation induced by Pam3CSK4, but not lipid A, in U937 cells. Etidronate also inhibited MyD88 expression in U937 cells incubated with Pam3CSK4. CONCLUSION: Etidronate down-regulates IL-8 and MCP-1 production in U937 cells by inhibiting both the expression of MyD88 and activation of NF-κB p65 in the TLR2, but not TLR4, pathway.