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.

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.

Dysfunctional TLR1 reduces the therapeutic efficacy of chemotherapy by attenuating HMGB1-mediated antitumor immunity in locally advanced colorectal cancer.

Regional lymph node metastasis is an important predictor for survival outcome and an indicator for postoperative adjuvant chemotherapy in patients with colorectal cancer. Even with advances in adjuvant chemotherapeutic regimens, 5-year distant metastasis and survival rates are still unsatisfactory. Here, we evaluate the clinical significance of polymorphisms in receptors for HMGB1, which is the hallmark of chemotherapy-induced immunogenic cell death, in patients with stage II-III colon carcinoma (COAD). We found that high cytosolic HMGB1 is elicited in stage III COAD patients who received adjuvant chemotherapy. Patients with the TLR1-N248S polymorphism (rs4833095), which causes loss-of-function in HMGB1-mediated TLR1-TLR2 signaling, may influence the therapeutic efficacy of adjuvant chemotherapy, leading to a high risk of distant metastasis within 5 years [HR = 1.694, 95% CI = 1.063-2.698, p = 0.027], suggesting that TLR1-N248S is an independent prognostic factor for locally advanced colon carcinoma patients. We found that defective TLR1 impaired TLR1/2 signaling during dendritic cell (DC) maturation for the antitumor immune response under immunogenic chemotherapy oxaliplatin (OXP) treatment. Defective TLR1 on DCs impaired their maturation ability by HMGB1 and reduced the secretion of IFNγ from T cells to eradicate tumor cells in vitro. Moreover, systemic inhibition of TLR1/2 dramatically reduced the tumor-infiltrating immune cells by OXP treatment, leading to poor therapeutic response to OXP. In contrast, administration of a TLR1/2 agonist synergistically increased the benefit of OXP treatment and triggered a high density of tumor-infiltrating immune cells. We also observed that fewer tumor-infiltrating cytotoxic T lymphocytes were located within the tumor microenvironment in patients bearing the TLR1-N248S polymorphism. Overall, our results suggest that dysfunctional TLR1 may reduce the therapeutic response to adjuvant chemotherapy by impairing HMGB1-mediated DC maturation and attenuating the antitumor immune response in locally advanced colon carcinoma patients.

[Xixin Decoction improves learning and memory ability of SAMP8 by enhancing neuroprotective effect and inhibiting neuroinflammation].

This study aimed to explore the possible effect of Xixin Decoction(XXD) on the learning and memory ability of Alzheimer's disease(AD) model senescence-accelerated mouse-prone 8(SAMP8) and the related mechanism in enhancing neuroprotective effect and reducing neuroinflammation. Forty SAMP8 were randomly divided into a model group(10 mL·kg~(-1)·d~(-1)), a probiotics group(0.39 g·kg~(-1)·d~(-1)), a high-dose group of XXD granules(H-XXD, 5.07 g·kg~(-1)·d~(-1)), a medium-dose group of XXD granules(M-XXD, 2.535 g·kg~(-1)·d~(-1)), and a low-dose group of XXD granules(L-XXD, 1.267 5 g·kg~(-1)·d~(-1)). Eight senescence-accelerated mouse-resistant 1(SAMR1) of the same age and strain were assigned to the control group(10 mL·kg~(-1)·d~(-1)). After ten weeks of intragastric administration, the Morris water maze was used to test the changes in spatial learning and memory ability of mice after treatment. Meanwhile, immunofluorescence staining was used to detect the positive expression of receptor for advanced glycation end products(AGER), Toll-like receptor 1(TLR1), and Toll-like receptor 2(TLR2) in the hippocampal CA1 region of mice. Western blot was employed to test the protein expression levels of silencing information regulator 2 related enzyme 1(SIRT1), AGER, TLR1, and TLR2 in the hippocampus of mice. Enzyme linked immunosorbent assay(ELISA) was applied to assess the levels of Aß_(1-42) in the hippocampus of mice and the levels of nuclear factor κB p65(NF-κB p65), NOD-like receptor protein 3(NLRP3), tumor necrosis factor-α(TNF-α), and interleukin-1ß(IL-1ß) in the serum and hippocampus of mice. Compared with the model group, XXD significantly improved the spatial learning and memory ability of SAMP8, increased the expression of neuroprotective factors in the hippocampus, decreased the levels of neuroinflammatory factors, and inhibited the expression of Aß_(1-42). In particular, H-XXD significantly increased the expression of SIRT1 in the hippocampus of mice, reduced the expression levels of NF-κB p65, NLRP3, TNF-α, and IL-1ß in the serum and hippocampus of mice, and decreased the expression of AGER, TLR1, and TLR2 in the hippocampus of mice(P<0.05 or P<0.01). XXD may improve the spatial learning and memory ability of AD model SAMP8 by enhancing the neuroprotective effect and inhibiting neuroinflammation.

Impact of patient characteristics on innate immune responses and inflammasome activation in ex vivo human lung tissues infected with influenza A virus.

Background: Influenza A virus (IAV) infection poses a persistent global health challenge, necessitating a nuanced grasp of host immune responses for optimal interventions. While the interplay between aging, immunosenescence, and IAV is recognized as key in severe lower respiratory tract infections, the role of specific patient attributes in shaping innate immune reactions and inflammasome activity during IAV infection remains under-investigated. In this study, we utilized an ex vivo infection model of human lung tissues with H3N2 IAV to discern relationships among patient demographics, IAV nucleoprotein (NP) expression, toll-like receptor (TLR) profiles, PD-1/PD-L1 markers, and cytokine production. Methods: Our cohort consisted of thirty adult patients who underwent video-assisted thoracoscopic surgery during 2018-2019. Post-surgical lung tissues were exposed to H3N2 IAV for ex vivo infections, and the ensuing immune responses were profiled using flow cytometry. Results: We observed pronounced IAV activity within lung cells, as indicated by marked NP upregulation in both epithelial cells (P = 0.022) and macrophages (P = 0.003) in the IAV-exposed group relative to controls. Notably, interleukin-2 levels correlated with variations in TLR1 expression on epithelial cells and PD-L1 markers on macrophages. Age emerged as a modulating factor, dampening innate immune reactions, as evidenced by reduced interleukin-2 and interferon-γ concentrations (both adjusted P < 0.05). Intriguingly, a subset of participants with pronounced tumor necrosis factor-alpha post-mock infection (Cluster 1) showed attenuated cytokine responses in contrast to their counterparts in Cluster 2 and Cluster 3 (all adjusted P < 0.05). Individuals in Cluster 2, characterized by a low post-mock infection NP expression in macrophages, exhibited reduced variations in both NP and TLR1-3 expressions on these cells and a decreased variation in interleukin-2 secretion in comparison to their Cluster 3 counterparts, who were identified by their elevated NP macrophage expression (all adjusted P < 0.05). Conclusion: Our work elucidates the multifaceted interplay of patient factors, innate immunity, and inflammasome responses in lung tissues subjected to ex vivo H3N2 IAV exposure, reflecting real-world lower respiratory tract infections. While these findings provide a foundation for tailored therapeutic strategies, supplementary studies are requisite for thorough validation and refinement.

Leishmania major-derived lipophosphoglycan influences the host's early immune response by inducing platelet activation and DKK1 production via TLR1/2.

Background: Platelets are rapidly deployed to infection sites and respond to pathogenic molecules via pattern recognition receptors (TLR, NLRP). Dickkopf1 (DKK1) is a quintessential Wnt antagonist produced by a variety of cell types including platelets, endothelial cells, and is known to modulate pro-inflammatory responses in infectious diseases and cancer. Moreover, DKK1 is critical for forming leukocyte-platelet aggregates and induction of type 2 cell-mediated immune responses. Our previous publication showed activated platelets release DKK1 following Leishmania major recognition. Results: Here we probed the role of the key surface virulence glycoconjugate lipophosphoglycan (LPG), on DKK1 production using null mutants deficient in LPG synthesis (Δlpg1- and Δlpg2-). Leishmania-induced DKK1 production was reduced to control levels in the absence of LPG in both mutants and was restored upon re-expression of the cognate LPG1 or LPG2 genes. Furthermore, the formation of leukocyte-platelet aggregates was dependent on LPG. LPG mediated platelet activation and DKK1 production occurs through TLR1/2. Conclusion: Thus, LPG is a key virulence factor that induces DKK1 production from activated platelets, and the circulating DKK1 promotes Th2 cell polarization. This suggests that LPG-activated platelets can drive innate and adaptive immune responses to Leishmania infection.

Multiple toll-like receptors (TLRs) display differential bacterial and ligand specificity in the earthworm, Eisenia andrei.

Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.

Identification of TLRs as potential prognostic biomarkers for lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is one of the most common tumors with the highest cancer-related death rate worldwide. Early diagnosis of LUAD can improve survival. Abnormal expression of the Toll-like receptors (TLRs) is related to tumorigenesis and development, inflammation and immune infiltration. However, the role of TLRs as an immunotherapy target and prognostic marker in lung adenocarcinoma is not well understood and needs to be analyzed. Relevant data obtained from databases such as ONCOMINE, UALCAN, GEPIA, and the Kaplan-Meier plotter, GSCALite, GeneMANIA, DAVID 6.8, Metascape, LinkedOmics and TIMER, to compare transcriptional TLRs and survival data of patients with LUAD. The expression levels of TLR1/2/3/4/5/7/8 in LUAD tissues were significantly reduced while the expression levels of TLR6/9/10 were significantly elevated. LUAD patients having low expression of TLR1/2/3/5/8 and high expression of TLR9 had a poor overall survival while patients with low expression of TLR2/3/7 presented with worse first progress. TLR4, TLR7 and TLR8 are the 3 most frequently mutated genes in the TLR family. Correlation suggested a low to moderate correlation among TLR family. TLR family was also involved in the activation or inhibition of the famous cancer related pathways. Analysis of immune infiltrates analysis suggested that TLR1/2/7/8 levels significantly correlated with immune infiltration level. Enrichment analysis revealed that TLRs were involved in TLR signaling pathway, immune response, inflammatory response, primary immunodeficiency, regulation of IL-8 production and PI3K-Akt signaling pathway. Our results provided information on TLRs expression and potential regulatory networks in LUAD. Moreover, our results suggested TLR2/7/8 as a potential prognostic biomarker for lung adenocarcinoma.

Expression and Role of Toll-like Receptors in Facial Nerve Regeneration after Facial Nerve Injury.

Facial nerve palsy directly impacts the quality of life, with patients with facial nerve palsy showing increased rates of depression and limitations in social activities. Although facial nerve palsy is not life-threatening, it can devastate the emotional and social lives of affected individuals. Hence, improving the prognosis of patients with this condition is of vital importance. The prognosis of patients with facial nerve palsy is determined by the cause of the disease, the degree of damage, and the treatment provided. The facial nerve can be easily damaged by middle ear and temporal bone surgery, trauma or infection, and tumors of the peripheral facial nerve or tumors surrounding the nerve secondary to systemic disease. In addition, idiopathic, acquired immunodeficiency syndrome and autoimmune diseases may damage the facial nerve. The treatment used for facial paralysis depends on the cause. Treatment of facial nerve amputation injury varies depending on the degree of facial nerve damage, comorbidities, and duration of injury. Recently, interest has increased in Toll-like receptors (TLRs) related to innate immune responses, as these receptors are known to be related to nerve regeneration. In addition to innate immune cells, both neurons and glia of the central nervous system (CNS) and peripheral nervous system (PNS) express TLRs. A comprehensive literature review was conducted to assess the expression and role of TLRs in peripheral nerve injury and subsequent regeneration. Studies conducted on rats and mice have demonstrated the expression of TLR1-13. Among these, TLR2-5 and TLR7 have received the most research attention in relation to facial nerve degeneration and regeneration. TLR10, TLR11, and TLR13 increase during compression injury of the facial nerve, whereas during cutting injury, TLR1-5, TLR8, and TLR10-13 increase, indicating that these TLRs are involved in the degeneration and regeneration of the facial nerve following each type of injury. Inadequate TLR expression or absence of TLR responses can hinder regeneration after facial nerve damage. Animal studies suggest that TLRs play an important role in facial nerve degeneration and regeneration.

Tea polyphenols, astaxanthin, and melittin can significantly enhance the immune response of juvenile spotted knifejaw (Oplegnathus punctatus).

The frequent occurrence of diseases seriously hampers the sustainable development of the spotted knifejaw (Oplegnathus punctatus) breeding industry. Our previous genome-wide scan and cross-species comparative genomic analysis revealed that the immune gene family (Toll-like receptors, TLR) members of O. punctatus underwent a significant contraction event (tlr1, tlr2, tlr14, tlr5, and tlr23). To address immune genetic contraction may result in reduced immunity, we investigated whether adding different doses (0, 200, 400, 600, and 800 mg/kg) of immune enhancers (tea polyphenols, astaxanthin, and melittin) to the bait after 30 days of continuous feeding could stimulate the immune response of O. punctatus. We found that the expression of tlr1, tlr14, tlr23 genes in immune organs (spleen and head kidney) was stimulated when tea polyphenols were added at 600 mg/kg. The tlr2 (400 mg/kg), tlr14 (200 mg/kg), tlr5 (200 mg/kg), and tlr23 (200 mg/kg) genes expression of intestine were elevated in the tea polyphenol group. When the addition of astaxanthin is 600 mg/kg, it can effectively stimulate the expression of tlr14 gene in immune organs (liver, spleen and head kidney). In the astaxanthin group, the expression of the genes tlr1 (400 mg/kg), tlr14 (600 mg/kg), tlr5 (400 mg/kg) and tlr23 (400 mg/kg) reached their highest expression in the intestine. Besides, the addition of 400 mg/kg of melittin can effectively induce the expression of tlr genes in the liver, spleen and head kidney, except the tlr5 gene. The tlr-related genes expression in the intestine was not significantly elevated in the melittin group. We hypothesize that the immune enhancers could enhance the immunity of O. punctatus by increasing the expression of tlr genes, and thereby leading to increased resistance to diseases. Meanwhile, our findings further demonstrated that significant increases in weight gain rate (WGR), visceral index (VSI), and feed conversion rate (FCR) were observed at 400 mg/kg, 200 mg/kg and 200 mg/kg of tea polyphenols, astaxanthin and melittin in the diet, respectively. Overall, our study provided valuable insights for future immunity enhancement and viral infection prevention in O. punctatus, as well as offered guidance for the healthy development of the O. punctatus breeding industry.

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

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

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

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

Nasopharyngeal epithelial cells from patients with coronavirus disease 2019 express abnormal levels of Toll-like receptors.

Aberrant activation of the immune system has been attributed with etiology and pathogenesis of coronavirus disease 2019 (COVID-19). Here, the transcript levels of toll-like receptors (TLRs) were measured in the nasopharyngeal epithelial cells obtained from COVID-19 patients to assess the involvement of these molecules in the clinical outcome of COVID-19 patients. Nasopharyngeal swab samples were used to obtain epithelial cells from 120 COVID-19 patients and 100 healthy controls. COVID-19 cases were classified into those having clinical symptoms/needing for hospitalization, having clinical symptoms/not needing for hospitalization, and those without clinical symptoms|. The mRNA expression levels of TLRs were measured in the nasopharyngeal epithelial cells. Overall, mRNA expression of TLR1, TLR2, TLR4, and TLR6 was significantly higher in COVID-19 cases compared to controls. The mRNA expression of TLRs were all higher significantly in the samples from COVID-19 patients having clinical symptoms and needing hospitalization as well as in those with clinical symptoms/not needing for hospitalization in comparison to controls. TLR expression was significantly higher in those with clinical symptoms/needing for hospitalization and those with clinical symptoms/not needing for hospitalization compared to COVID-19 cases without clinical symptoms. In cases with clinical symptoms/needing for hospitalization and those with clinical symptoms/not needing for hospitalization, there was a correlation between TLR expression and clinicopathological findings. In conclusion, aberrant expression of TLRs in the nasopharyngeal epithelial cells from COVID-19 cases may predict the severity of the diseases and necessity for supportive cares in the hospital.

Effect of Royal Jelly on Gene Expression of Toll-like Receptors 1-9 in Patients with Hepatitis B.

BACKGROUND: By damaging the liver, hepatitis B can result in acute and chronic diseases, such as cirrhosis or hepatocellular carcinoma. Viable treatments for such diseases using natural products and determinative biomarkers have been proposed but require evaluation to improve their effects. Therefore, this study aims to examine how effectively a specific natural product (namely, royal jelly) protects the body from the copy number of the virus, as well as TLR1 to TLR9 gene expressions. METHODS: The effectiveness of royal jelly was tested by giving it (orally) to 30 hepatitis B patients for one month. HBV copy number and mRNA levels of TLRs were explored using Real Time PCR technique, and liver enzymes were evaluated too. RESULTS: Orally treatment with royal jelly led to a significant decrease in HBV-DNA copy number, down-regulation of TLR2 and TLR8, and up-regulation of TLR3. However, mRNA levels of the TLRs were not altered in the female, while TLR1, TLR2, and TLR5 were significantly decreased in the male participants. CONCLUSIONS: It seems that royal jelly has anti-viral and anti-inflammatory roles in the in vivo conditions in a dependent manner in TLR3, TLR2, and TLR8. Therefore, it can be suggested as a safe complementary agent for patients with hepatitis B.

The differential expression of toll like receptors and RIG-1 in the placenta of neonates with in utero infections.

Novel biomarkers of in utero infections are needed to help guide early therapy. The toll like receptors (TLRs) and retinoic acid-inducible gene 1 (RIG-1) are proteins involved in the initial reaction of the innate immune system to infectious diseases. This study tested the hypothesis that a panel of TLRs and RIG-1 in the placenta could serve as an early biomarker of in utero infections. The TLRs and RIG-1 expression as determined by immunohistochemistry was scored in 10 control placentas (normal delivery or neonatal damage from known non-infectious cause), 8 placentas from documented in utero bacterial infection, and 7 placentas from documented in utero viral infections blinded to the clinical information. The non-infected placentas showed the following profile: no expression (TLR1, TLR3, TLR4, TLR7, TLR8), moderate expression (TLR2), and strong expression (RIG-1). The bacterial and viral infection cases shared the following profile: no to mild expression (TLR 2, TLR7, and RIG1), moderate expression (TLR4), and strong expression (TLR1, TLR3, and TLR8). The histologic findings in the chorionic villi were equivalent in the infected cases and controls, underscoring the need for molecular testing by the surgical pathologist when in utero infection is suspected. The results suggest that a panel of TLRs/RIG-1 analyses can allow the pathologist and/or clinician to diagnose in utero infections soon after birth. Also, treatments to antagonize the effects of TLR1, 3, and 8 may help abrogate in utero neonatal damage.

Pmorf0222, a Virulence Factor in Pasteurella multocida, Activates Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase via Toll-Like Receptor 1/2.

Pasteurella multocida primarily causes hemorrhagic septicemia and pneumonia in poultry and livestock. Identification of the relevant virulence factors is therefore essential for understanding its pathogenicity. Pmorf0222, encoding the PM0222 protein, is located on a specific prophage island of the pathogenic strain C48-1 of P. multocida. Its role in the pathogenesis of P. multocida infection is still unknown. The proinflammatory cytokine plays an important role in P. multocida infection; therefore, murine peritoneal exudate macrophages were treated with the purified recombinant PM0222, which induced the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) via the Toll-like receptor 1/2 (TLR1/2)-nuclear factor kappa B (NF-κB)/mitogen-activated protein kinase (MAPK) signaling and inflammasome activation. Additionally, the mutant strain and complemented strain were evaluated in the mouse model with P. multocida infection, and PM0222 was identified as a virulence factor, which was secreted by outer membrane vesicles of P. multocida. Further results revealed that Pmorf0222 affected the synthesis of the capsule, adhesion, serum sensitivity, and biofilm formation. Thus, we identified Pmorf0222 as a novel virulence factor in the C48-1 strain of P. multocida, explaining the high pathogenicity of this pathogenic strain.

Carrier-free subunit nanovaccine amplifies immune responses against tumors and viral infections.

Codelivering subunit antigens and Toll-like receptor (TLR) molecular adjuvants via nanocarriers can stimulate potent innate and specific immune responses. Simple and effective nanovaccines fabrication is crucial for application. However, most nanovaccines were fabricated by introducing additional delivery materials, increasing safety risk, cost and processing complexity. Herein, a carrier-free nanovaccine was facilely prepared using a TLR1/TLR2 adjuvant, Diprovocim, rich in benzene rings that could interact with aromatic residues in subunit antigens through π-π stacking without additional materials. The carrier-free nanovaccines with a narrow size distribution could target lymph nodes (LNs) after intravenous injection to mice. The carrier-free nanovaccines based on ovalbumin (OVA) can stimulate strong antibody titers and CD4+ and CD8+ T cell immune responses in mice, and it synergized with anti-PD1 showing a potent tumor suppression in B16F10-OVA tumor model of mice. Furthermore, the carrier-free nanovaccine with glycoprotein E (gE), a glycoprotein of the varicella-zoster virus (VZV), also showed potent humoral and cellular immune responses. Therefore, using subunit proteins to support Diprovocim by π-π stacking provides a new approach for the preparation and application of novel vaccines for tumor therapy and prevention of infectious diseases. STATEMENT OF SIGNIFICANCE: Codelivering subunit antigens and adjuvants via nanocarriers stimulate potent innate and specific immune responses. However, existing delivery materials for fabricating nanovaccines will inevitably increase the cost of preparation, controllability, process complexity and safety assessment. Therefore, this study easily prepared carrier-free nanovaccines using the benzene ring-rich TLR1/TLR2 adjuvant Diprovocim, which can interact with aromatic residues in subunit antigens via π-π stacking without additional materials. The carrier-free nanovaccines of OVA demonstrated a potent tumor inhibition in treating melanoma in combination with anti-PD1. And the nanovaccines of gE stimulated a strong antibody titer and cellular immune response for herpes zoster. Thus, the present study provides a new approach for the preparation of subunit vaccines to combat various cancers and virus infections.

Toll-like receptor-2 in cardiomyocytes and macrophages mediates isoproterenol-induced cardiac inflammation and remodeling.

Heart failure (HF) is the leading cause of morbidity and mortality worldwide. Activation of the innate immune system initiates an inflammatory response during cardiac remodeling induced by isoproterenol (ISO). Here, we investigated whether Toll-like receptor-2 (TLR2) mediates ISO-induced inflammation, hypertrophy, and fibrosis. TLR2 was found to be increased in the heart tissues of mouse with HF under ISO challenge. Further, cardiomyocytes and macrophages were identified as the main cellular sources of the increased TLR2 levels in the model under ISO stimulation. The effect of TLR2 deficiency on ISO-induced cardiac remodeling was determined using TLR2 knockout mice and bone marrow transplantation models. In vitro studies involving ISO-treated cultured cardiomyocytes and macrophages showed that TLR2 knockdown significantly decreased ISO-induced cell inflammation and remodeling via MAPKs/NF-κB signaling. Mechanistically, ISO significantly increased the TLR2-MyD88 interaction in the above cells in a TLR1-dependent manner. Finally, DAMPs, such as HSP70 and fibronectin 1 (FN1), were found to be released from the cells under ISO stimulation, which further activated TLR1/2-Myd88 signaling and subsequently activated pro-inflammatory cytokine expression and cardiac remodeling. In summary, our findings suggest that TLR2 may be a target for the alleviation of chronic adrenergic stimulation-associated HF. In addition, this paper points out the possibility of TLR2 as a new target for heart failure under ISO stimulation.

Differential Expression of Inflammarafts in Macrophage Foam Cells and in Nonfoamy Macrophages in Atherosclerotic Lesions-Brief Report.

BACKGROUND: Reprogramming of monocytes and macrophage manifests in hyperinflammatory responses and chronification of inflammation in atherosclerosis. Recent studies focused on epigenetic, transcriptional, and metabolic alterations that characterize trained immunity. However, the underlying effector mechanisms driving the hyperinflammatory response of reprogrammed macrophages remain unclear. We hypothesized that the plasma membrane of atherosclerotic lesion macrophages undergoes reprogramming to maintain inflammarafts, enlarged lipid rafts (LR) serving as a platform for assembly of inflammatory receptor complexes. METHODS: Single-cell suspensions from the aortae of Western diet-fed Ldlr-/- mice were gated for BODIPY-high foamy and BODIPY-low nonfoamy F4/80 macrophages by flow cytometry. Inflammarafts were characterized by increased levels of LR, TLR4 (toll-like receptor-4) localization to LR, TLR4 dimers, and the proximity between TLR2, TLR1, and CD36. In a cellular model of trained immunity, LR, TLR4 dimers, and the inflammatory response were measured in bone marrow-derived macrophages subjected to a 24-hour treatment with LPS (lipopolysaccharide) or OxLDL (oxidized low-density lipoprotein), followed by a 6-day wash-out period. RESULTS: Nonfoamy macrophages, which constituted ≈40% of macrophages in atherosclerotic lesions, expressed significantly higher levels of LR and TLR4 dimers, as well as proximity ligation signals for TLR4-LR, TLR2-CD36, and TLR2-TLR1 complexes, compared with foamy macrophages. These inflammaraft measures associated, to a different degree, with plasma cholesterol and inflammatory cytokines, as well as the size of the atherosclerotic lesions and necrotic cores. The bone marrow-derived macrophages trained with LPS simulated nonfoamy atherosclerotic lesion macrophages and continued to express inflammarafts and inflammatory genes for 6 days after LPS removal and displayed a hyperinflammatory response to Pam3CSK4, a TLR2/TLR1 agonist. OxLDL-exposed, lipid-laden macrophages did not express inflammarafts. CONCLUSIONS: Our data support the hypothesis that persistent inflammarafts in nonfoamy macrophages in atherosclerotic lesions serve as effectors of macrophage reprogramming into a hyperinflammatory phenotype.

High mobility group box-1 regulates expression of EGFR, VEGF, StAR and TIMP1/2 in bovine granulosa cells through a mechanism involving TLR2/NF-κB.

Innate immunity is involved in ovarian activity through aseptic inflammation and tissue repair. High-mobility group box-1 (HMGB1) is related to placental inflammation and a driver of inflammation throughout pregnancy. The aim of this study was to investigate the interaction of HMGB1 with TLR2 in bovine ovarian granulosa cells, and the effects of HMGB1 on bovine ovarian granulosa cells in vitro. The viability of granulosa cells were not affected by HMGB1 with the concentration less than 5 µg/mL. The mRNA levels of TLR2, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), the steroidogenic acute regulatory protein (StAR), tissue inhibitors of matrix (TIMP1/2) of ovarian granulosa cells were upregulated by HMGB1(P < 0.05). The protein levels of TLR2, TLR1 and phosphorylation-NF-κB (p-NF-κB) p65 in ovarian granulosa cells increased in 5 µg/mL HMGB1 group (P < 0.05), and TLR2 decreased in siRNA-2 group (P < 0.05). IL-6 of ovarian granulosa cells was increased by 1 µg/mL and 5 µg/mL HMGB1 (P < 0.05). These results implicate that HMGB1 has interaction with TLR2, TLR1 and p-NF-κB p65 in ovarian granulosa cells, which lead to nuclear translocation of NF-κB p65 and the secretion of interleukin-6 (IL-6). HMGB1 regulates the expression of EGFR, VEGF, StAR, TIMP1/2 and the secretion of IL-6 in ovarian granulosa cells of dairy cows through activating the TLR2/NF-κB signaling pathway, which may be interfere with ovarian physiological activity.