miRNAs: possible regulators of toll like receptors and inflammatory tumor microenvironment in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is ranked as the third most commonly diagnosed cancer and the third cause of cancer related deaths. CRC is greatly attributed to genetic and epigenetic mutations and immune dysregulation. Tumor aberrant expression of Toll-like Receptors (TLRs) can contribute to tumorigenesis. Recent studies suggested that microRNAs act as direct ligands of TLRs altering their expression and signaling pathways. AIM: To prove our concept that specific miRNA mimics may act as antagonists of their specific toll like receptors inhibiting their expression that could limit the release of pro-inflammatory and pro-tumorigenic cytokines leading to apoptosis of tumor cells. METHODS: From public microarray databases, we retrieved TLRs and miRNAs related to CRC followed by in silico docking of the selected miRNA ligands into the TLRs. Clinical validation after co-immunoprecipitation of TLRs and their interacting miRNA ligands was done. Expression of TLRs 1, 7,8 was determined by ELISA while miRNAs was measured by RT-qPCR. In addition, microRNA mimics of the down regulated miRNAs were transfected into human CRC cell lines. RESULTS: Our data demonstrate that TLRs 1, 7, 8 are up regulated in CRC compared to controls. Further, three miRNAs (-122, -29b and -15b) are relatively downregulated, while 4 miRNAs (-202, miRNA-98, -21 and -let7i) are upregulated in CRC patients compared to those with benign tumor and healthy controls. Transfection of down regulated miRNA mimics into CRC cell lines resulted in a significant reduction of the number and viability of cells as well as down regulating the expression of TLRs 1, 7 and 8 with ultimate reduction of downstream effector IL6 protein, suggesting that these miRNAs are negative regulators of carcinogenesis. CONCLUSION: MicroRNAs could act as antagonistic ligands of TLRs limiting the inflammatory tumor microenvironment.

Diversification of Toll-like receptor 1 in swamp eel (Monopterus albus).

Toll-like receptor 1 (TLR1) is a pattern recognition receptor that plays critical roles in triggering immune activation via detecting bacterial lipoproteins and lipopeptides. In this study, the genetic characteristic of TLR1 was studied for an important aquaculture fish, swamp eel Monopterus albus. The eel has been seriously threatened by infectious diseases. However, a low level of genetic heterogeneity in the fish that has resulted from a demographic bottleneck presents further challenges in breeding for disease resistance. A comparison with the homologue of closely related species M. javanensis revealed that amino acid replacement (nonsynonymous) but not silent (synonymous) differences have accumulated nonrandomly over the coding sequences of the receptors at the early stage of their phylogenetic split. The combined results from comparative analyses of nonsynonymous-to-synonymous polymorphisms showed that the receptor has undergone significant diversification in M. albus driven by adaptive selection likely after the genetic bottleneck. Some of the changes reported here have taken place in the structures mediating heterodimerization with co-receptor TLR2, ligand recognition, and/or formation of active signaling complex with adaptor, which highlighted key structural elements and strategies of TLR1 in arms race against exogenous challenges. The findings of this study will add to the knowledge base of genetic engineering and breeding for disease resistance in the eel.

Genetic polymorphisms of TLR1, TLR2, TLR3 and TLR4 in patients with recurrent or severe infections.

Toll-like receptors (TLRs) play an important role in innate immunity. Previous studies have shown that single nucleotide polymorphisms (SNPs) in the genes coding for these innate immune molecules can affect susceptibility to and the outcome of certain diseases. The aim of the present study was to examine the clinical relevance of well-studied TLR1-4 SNPs in individuals who are prone to infections. Four functional SNPs, TLR1 rs5743618 (1805C > A, Ser602Ile), TLR2 rs5743708 (2258G > A, Arg753Gln), TLR3 rs3775291 (1234C > T, Leu412Phe) and TLR4 rs4986790 (896A > G, Asp299Gly), were analysed in 155 patients with recurrent respiratory infections (n = 84), severe infections (n = 15) or common variable immunodeficiency (n = 56), and in 262 healthy controls, using the High Resolution Melting Analysis method. Polymorphisms of TLR2 rs5743708 (odds ratio [OR] 3.16; 95% confidence interval [CI] 1.45-6.83, p = .004, ap = .016) and TLR4 rs4986790 (OR 1.8; 95% CI 1.05-3.12, p = .028, ap = .112) were more frequent in patients with recurrent or severe infections than in controls. Interestingly, seven patients were found to carry both variant genotypes of TLR2 and TLR4, whereas none of the control group carried such genotypes (p  ≤ .0001). Moreover, TLR2 polymorphism was associated with increased risk for acute otitis media episodes (OR, 3.02; 95% CI 1.41-6.47; p = .012). This study indicates that children and adults who are more prone to recurrent or severe respiratory infections carry one or both variant types of TLR2 and TLR4 more often than control subjects. Genetic variations of TLRs help explain why some children are more susceptible to respiratory infections.

Metabolic trade-offs in Neonatal sepsis triggered by TLR4 and TLR1/2 ligands result in unique dysfunctions in neural breathing circuits.

Neonatal sepsis remains one of the leading causes of mortality in newborns. Several brainstem-regulated physiological processes undergo disruption during neonatal sepsis. Mechanistic knowledge gaps exist at the interplay between metabolism and immune activation to brainstem neural circuits and pertinent physiological functions in neonates. To delineate this association, we induced systemic inflammation either by TLR4 (LPS) or TLR1/2 (PAM3CSK4) ligand administration in postnatal day 5 mice (PD5). Our findings show that LPS and PAM3CSK4 evoke substantial changes in respiration and metabolism. Physiological trade-offs led to hypometabolic-hypothermic responses due to LPS, but not PAM3CSK4, whereas to both TLR ligands blunted respiratory chemoreflexes. Neuroinflammatory pathways modulation in brainstem showed more robust effects in LPS than PAM3CSK4. Brainstem neurons, microglia, and astrocyte gene expression analyses showed unique responses to TLR ligands. PAM3CSK4 did not significantly modulate gene expression changes in GLAST-1 positive brainstem astrocytes. PD5 pups receiving PAM3CSK4 failed to maintain a prolonged metabolic state repression, which correlated to enhanced gasping latency and impaired autoresuscitation during anoxic chemoreflex challenges. In contrast, LPS administered pups showed no significant changes in anoxic chemoreflex. Electrophysiological studies from brainstem slices prepared from pups exposed to either TLR4 or PAM3CSK4 showed compromised transmission between preBötzinger complex and Hypoglossal as an exclusive response to the TLR1/2 ligand. Spatial gene expression analysis demonstrated a region-specific modulation of PAM3CSK4 within the raphe nucleus relative to other anatomical sites evaluated. Our findings suggest that metabolic changes due to inflammation might be a crucial tolerance mechanism for neonatal sepsis preserving neural control of breathing.

TLRs1-10 Protein Expression in Circulating Human White Blood Cells during Bacterial and COVID-19 Infections.

INTRODUCTION: Toll-like receptors play crucial roles in the sepsis-induced systemic inflammatory response. Septic shock mortality correlates with overexpression of neutrophilic TLR2 and TLR9, while the role of TLR4 overexpression remains a debate. In addition, TLRs are involved in the pathogenesis of viral infections such as COVID-19, where the single-stranded RNA of SARS-CoV-2 is recognized by TLR7 and TLR8, and the spike protein activates TLR4. METHODS: In this study, we conducted a comprehensive analysis of TLRs 1-10 expressions in white blood cells from 71 patients with bacterial and viral infections. Patients were divided into 4 groups based on disease type and severity (sepsis, septic shock, moderate, and severe COVID-19) and compared to 7 healthy volunteers. RESULTS: We observed a significant reduction in the expression of TLR4 and its co-receptor CD14 in septic shock neutrophils compared to the control group (p < 0.001). Severe COVID-19 patients exhibited a significant increase in TLR3 and TLR7 levels in neutrophils compared to controls (p < 0.05). Septic shock patients also showed a similar increase in TLR7 in neutrophils along with elevated intermediate monocytes (CD14+CD16+) compared to the control group (p < 0.005 and p < 0.001, respectively). However, TLR expression remained unchanged in lymphocytes. CONCLUSION: This study provides further insights into the mechanisms of TLR activation in various infectious conditions. Additional analysis is needed to assess their correlation with patient outcome and to evaluate the impact of TLR-pathway modulation during septic shock and severe COVID-19.

Lack of Association of TLR1 and TLR5 Coding Variants with Mortality in a Large Multicenter Cohort of Melioidosis Patients.

Melioidosis, infection caused by Burkholderia pseudomallei, is characterized by robust innate immune responses. We have previously reported associations of TLR1 single nucleotide missense variant rs76600635 with mortality and of TLR5 nonsense variant rs5744168 with both bacteremia and mortality in single-center studies of patients with melioidosis in northeastern Thailand. The objective of this study was to externally validate the associations of rs76600635 and rs5744168 with bacteremia and mortality in a large multicenter cohort of melioidosis patients. We genotyped rs76600635 and rs5744168 in 1,338 melioidosis patients enrolled in a prospective parent cohort study conducted at nine hospitals in northeastern Thailand. The genotype frequencies of rs76600635 did not differ by bacteremia status (P = 0.27) or 28-day mortality (P = 0.84). The genotype frequencies of rs5744168 did not differ by either bacteremia status (P = 0.46) or 28-day mortality (P = 0.10). Assuming a dominant genetic model, there was no association of the rs76600635 variant with bacteremia (adjusted odds ratio [OR], 0.75; 95% CI, 0.54-1.04, P = 0.08) or 28-day mortality (adjusted OR, 0.96; 95% CI, 0.71-1.28, P = 0.77). There was no association of the rs5744168 variant with bacteremia (adjusted OR, 1.24; 95% CI, 0.76-2.03, P = 0.39) or 28-day mortality (adjusted OR, 1.22; 95% CI, 0.83-1.79, P = 0.21). There was also no association of either variant with 1-year mortality. We conclude that in a large multicenter cohort of patients hospitalized with melioidosis in northeastern Thailand, neither TLR1 missense variant rs76600635 nor TLR5 nonsense variant rs5744168 is associated with bacteremia or mortality.

An immune-competent human gut microphysiological system enables inflammation-modulation by Faecalibacterium prausnitzii.

Crosstalk of microbes with human gut epithelia and immune cells is crucial for gut health. However, there is no existing system for a long-term co-culture of human innate immune cells with epithelium and oxygen-intolerant commensal microbes, hindering the understanding of microbe-immune interactions in a controlled manner. Here, we established a gut epithelium-microbe-immune (GuMI) microphysiological system to maintain the long-term continuous co-culture of Faecalibacterium prausnitzii/Faecalibacterium duncaniae with colonic epithelium, antigen-presenting cells (APCs, herein dendritic cells and macrophages), and CD4+ naive T cells circulating underneath the colonic epithelium. In GuMI-APC condition, multiplex cytokine assays suggested that APCs contribute to the elevated level of cytokines and chemokines secreted into both apical and basolateral compartments compared to GuMI condition that lacks APC. In GuMI-APC with F. prausnitzii (GuMI-APC-FP), F. prausnitzii increased the transcription of pro-inflammatory genes such as toll-like receptor 1 (TLR1) and interferon alpha 1 (IFNA1) in the colonic epithelium, without a significant effect on cytokine secretion, compared to the GuMI-APC without bacteria (GuMI-APC-NB). In contrast, in the presence of CD4+ naive T cells (GuMI-APCT-FP), TLR1, IFNA1, and IDO1 transcription levels decreased with a simultaneous increase in F. prausnitzii-induced secretion of pro-inflammatory cytokines (e.g., IL8) compared to GuMI-APC-FP that lacks T cells. These results highlight the contribution of individual innate immune cells in regulating the immune response triggered by the gut commensal F. prausnitzii. The integration of defined populations of immune cells in the gut microphysiological system demonstrated the usefulness of GuMI physiomimetic platform to study microbe-epithelial-immune interactions in healthy and disease conditions.

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.

Synthesis and immunological evaluation of TLR1/2 ligand-conjugated RBDs as self-adjuvanting vaccine candidates against SARS-CoV-2.

We synthesized and evaluated Pam3CSK4-conjugated receptor binding domain (RBD)/deglycosylated RBD as potential anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidates. Our investigation revealed the critical importance of limiting the number of introduced Pam3CSK4 molecules to the RBD in order to preserve its antigenicity. We also confirmed the harmonious integration of the adjuvant-conjugation strategy with the glycan-shield removal strategy.

Comprehensive analysis of diel rhythmic expression of the medaka toll-like receptor gene family.

Several immune-related genes, including Toll-like receptors (TLR), are associated with circadian rhythms in mammals. However, information on the circadian rhythmic expression of TLRs in fish is limited. In this study, we aimed to analyze the regulation of diel oscillations in the expression of TLR genes in Japanese medaka (Oryzias latipes). The expression analysis revealed diel expression patterns of tlr1, tlr5m, tlr21, and clock genes (bmal1 and clock1) under a 12 h light:12 h dark cycle. The clock gene response element (E-box) was identified in the transcriptional regulatory regions of tlr1, tlr5m, and tlr21. Moreover, overexpressed bmal1 and clock1 enhanced expression levels of tlr1, tlr5m, and tlr21 in medaka embryo (OLHdrR-e3) cells. The expression of tlr1, tlr5m, and tlr21 was significantly decreased in OLHdrR-e3 after generating a bmal1 knockdown using a morpholino oligo. These results indicate the regulation of the diel rhythmic expression of several fish TLRs by clock genes.

Homology Modeling, Molecular Dynamics Simulation, and Prediction of Bovine TLR2 Heterodimerization.

Toll-like receptor 2 (TLR2) is a major membrane-bound receptor with ligand and species specificity that activates the host immune response. Heterodimerization of TLR2 with TLR1 (TLR2/1) or TLR6 (TLR2/6), triggered by ligand binding, is essential to initiating the signaling pathway. Bovine TLR2 (bTLR2) heterodimerization has not been defined yet compared with human and mouse TLR2s (hTLR2 and mTLR2). The aim of the present study was to model bovine TLRs (TLRs 1, 2 and 6) and create the heterodimeric forms of the bovine TLR2 using molecular dynamics (MD) simulations. We compared the intermolecular interactions in bTLR2/1-PAM3 and bTLR2/6-PAM2 with the hTLR2 and mTLR2 complexes through docking simulations and subsequent MD analyses. The present computational findings showed that bTLR2 dimerization could have a biological function and activate the immune response, similar to hTLR2 and mTLR2. Agonists and antagonists that are designed for hTLR2 and mTLR2 can target bTLR2. However, the experimental approaches to comparing the functional immune response of TLR2 across species were missing in the present study. This computational study provides a structural analysis of the bTLR2 interaction with bTLR1 and bTLR6 in the presence of an agonist/antagonist and reveals the three-dimensional structure of bTLR2 dimerization. The present findings could guide future experimental studies targeting bTLR2 with different ligands and lipopeptides.

Effects of aging and lifelong aerobic exercise on expression of innate immune components in skeletal muscle of women.

This study examined the effects of aging and lifelong aerobic exercise on innate immune system components in the skeletal muscle of healthy women in the basal state and after an unaccustomed resistance exercise (RE) challenge. We also made exploratory between-sex comparisons with our previous report on men. Three groups of women were studied: young exercisers (YE, n = 10, 25 ± 1 yr, V̇o2max: 44 ± 2 mL/kg/min), lifelong aerobic exercisers with a 48 ± 2 yr training history (LLE, n = 7, 72 ± 2 yr, V̇o2max: 26 ± 2 mL/kg/min), and old healthy nonexercisers (OH, n = 10, 75 ± 1 yr, V̇o2max: 18 ± 1 mL/kg/min). Ten Toll-like receptors (TLRs)1-10, TLR adaptors (Myd88, TRIF), and NF-κB pathway components (IκBα, IKKß) were assessed at the mRNA level in vastus lateralis biopsies before and 4 h after RE [3×10 repetitions, 70% 1-repetition maximum (1RM)]. Basal TLR1-10 expression was minimally influenced by age or LLE in women (TLR9 only; OH > YE, +43%, P < 0.05; OH > LLE, +30%, P < 0.10) and was on average 24% higher in women versus men. Similarly, basal adaptor expression was not influenced (P > 0.05) by age or LLE in women but was on average 26% higher (myeloid differentiation primary response 88, Myd88) and 23% lower [Toll interleukin (IL)-1 receptor-containing adaptor-inducing interferon-γ, TRIF] in women versus men. RE-induced changes in women, independent of the group, in TLR3, TLR4, TLR6 (∼2.1-fold, P < 0.05), Myd88 (∼1.2-fold, P < 0.10), and IκBα (∼0.3-fold, P < 0.05). Although there were some similar RE responses in men (TLR4: 2.1-fold, Myd88: 1.2-fold, IκBα: 0.4-fold), several components responded only in men to RE (TLR1, TLR8, TRIF, and IKKß). Our findings support the sexual dimorphism of immunity, with women having greater basal skeletal muscle TLR expression and differential response to unaccustomed exercise than men.NEW & NOTEWORTHY We recently reported that aging increases basal expression of many Toll-like receptors (TLRs) in men and lifelong aerobic exercise does not prevent this effect. In addition, a resistance exercise (RE) challenge increased the expression of many TLRs. Here we show that basal TLR expression is minimally influenced by aging in women and findings support the sexual dimorphism of immunity, with women having greater basal skeletal muscle TLR expression and a differential response to unaccustomed exercise than men.

Investigation of the association between the Toll-like receptor 1 rs4833095 variation and gastric adenocarcinoma recurrence.

BACKGROUND: Toll-like receptors (TLRs) are a family of transmembrane receptors that play key roles in identifying invading pathogens and activating innate immunity. TLR1 has been reported to be associated with the risk of gastric cancer (GC) but that was based on only a simple statistical analysis. METHODS: We genotyped the TLR1 in 526 GC patients to investigate the association between the variation and gastric cancer survival by the multiplex polymerase chain reaction and sequencing method. The rs4833095 variation (chr4:38798089 [GRCh38. p14], T > C) in the TLR1 gene was genotyped in 526 patients who underwent GC resection. The associations between genotype, survival, and recurrence were investigated. The potential role of TLR1 in stomach cancer was investigated using clinical data from formalin-fixed, paraffin-embedded tissue samples. RESULTS: Patients with the T/C and C/C genotypes of rs4833095 had a lower risk of recurrence than those with the T/T genotype. Recurrence-free periods were substantially longer in patients with the T/C or C/C genotypes (22.6 and 22.3 months, respectively) than in those with the T/T genotype (20.7 months). Patients with the T/C or C/C genotype, low expression levels of VEGF1, high expression levels of ERBB2 and ERCC1, the absence of cancer nodules, a tumor size of less than 5 cm, and poor differentiation had a considerably reduced risk of recurrence. CONCLUSIONS: TLR1 rs4833095 was correlated with the postresection prognosis of patients with gastric cancer, suggesting that TLR1 may have a role in the onset or progression of gastric cancer.

Correlation of polymorphism in Toll-Like Receptor (TLR1 and TLR2) genes with susceptibility of pulmonary tuberculosis in Doda region of India.

BACKGROUND: Pulmonary tuberculosis has emerged as one of the leading causes of deaths across the globe. The prevalence of Mycobacterium tuberculosis has also shown an increasing trend over the time which may be attributed to the increase in multidrug resistant strains and HIV epidemics. There are several factors like change in the gene structure and cellular activities of the host and the bacterium which may have changed the host response towards tuberculosis. Additionally, the recent reports have suggested that Toll-Like Receptors (TLRs) play an important role in the activation of immune responses against various pathogens. Therefore, this study has been designed to investigate the possible correlation of TLR gene polymorphism and prevalence of pulmonary tuberculosis. METHOD: This study investigates 300 samples collected from patients with pulmonary tuberculosis (150) and healthy controls (150) from the Doda region of Jammu, India. For analysis purpose, DNA from the collected samples were isolated and subjected to sequence specific PCR amplification of TLR-1 and TLR-2 genes. The amplicons of TLR-1 and TLR-2 were further digested with restriction enzymes PvuII and Xbal, respectively, and visualized on agarose gel, subsequently. RESULT: The results suggest that frequency of TLR2 gene polymorphism (73.9%) is high in the patients below the age of 50 years, whereas, frequency of TLR-1 gene polymorphism is high (71%) in the patients above 50 years of age (p = 0.005). Further, the restriction digestion analysis of TLR1 genes has shown that nearly 78% of the confirmed normal cases exhibit homozygous normal conditions followed by 12% cases with heterozygous conditions and 10% cases of homozygous mutants. Similarly for TLR2 genes, nearly 78.6% of the confirmed normal cases have shown homozygous normal conditions followed heterozygous conditions (12.6%) and homozygous mutants (8.6%). CONCLUSION: This study establishes a preliminary correlation between TLR polymorphism and tuberculosis.

Twelve toll-like receptor (TLR) genes in the family Equidae - comparative genomics, selection and evolution.

Toll-like receptors (TLRs) represent an important part of the innate immune system. While human and murine TLRs have been intensively studied, little is known about TLRs in non-model species. The order Perissodactyla comprises a variety of free-living and domesticated species exposed to different pathogens in different habitats and is therefore suitable for analyzing the diversity and evolution of immunity-related genes. We analyzed TLR genes in the order Perissodactyla with a focus on the family Equidae. Twelve TLRs were identified by bioinformatic analyses of online genomic resources; their sequences were confirmed in equids by genomic DNA re-sequencing of a panel of nine species. The expression of TLR11 and TLR12 was confirmed in the domestic horse by cDNA sequencing. Phylogenetic reconstruction of the TLR gene family in Perissodactyla identified six sub-families. TLR4 clustered together with TLR5; the TLR1-6-10 subfamily showed a high degree of sequence identity. The average estimated evolutionary divergence of all twelve TLRs studied was 0.3% among the Equidae; the most divergent CDS were those of Equus caballus and Equus hemionus kulan (1.34%) in the TLR3, and Equus africanus somaliensis and Equus quagga antiquorum (2.1%) in the TLR1 protein. In each TLR gene, there were haplotypes shared between equid species, most extensively in TLR3 and TLR9 CDS, and TLR6 amino acid sequence. All twelve TLR genes were under strong negative overall selection. Signatures of diversifying selection in specific codon sites were detected in all TLRs except TLR8. Differences in the selection patterns between virus-sensing and non-viral TLRs were observed.

Delineating the functional role of the PPE50 (Rv3135) - PPE51 (Rv3136) gene cluster in the pathophysiology of Mycobacterium tuberculosis.

The extraordinary success of Mycobacterium tuberculosis (M. tb) has been attributed to its ability to modulate host immune responses, and its genome encodes multiple immunomodulatory factors, including several proteins of the multigenic PE_PPE family. To understand its role in M. tb pathophysiology we have characterised the PPE50 (Rv3135)-PPE51 (Rv3136) gene cluster, one of nine PPE-PPE clusters in the genome. We demonstrate here that this cluster is operonic, and that PPE50 and PPE51 interact - the first demonstration of PPE-PPE interaction. THP-1 macrophages infected with recombinant Mycobacterium smegmatis strains expressing PPE50 and PPE51 showed lower intracellular viability than the control, which correlated with an increase in transcript levels of iNOS2. Infected macrophages also exhibited an upregulation in levels of IL-10, indicating an immunomodulatory role for these proteins. Using pull-downs and signalling assays, we identified TLR1 to be the cognate receptor for PPE50 - all the phenotypes observed on infection of THP-1 macrophages were reversed on pre-treatment with an anti-TLR1 antibody, validating the functional outcome of PPE50-TLR1 interaction. Our data reveals a TLR1 dependent role for the PPE50-PPE51 cluster in promoting bacillary persistence, via CFU reduction and concomitant upregulation of the anti-inflammatory response - a two-pronged strategy to circumvent host immune surveillance.

Validation of a Toll-like Receptor (TLR)2/TLR1 Activation Assay for Biological Standardization of Arthrospira/Limnospira Immune-Enhancing Potency.

Arthrospira/Limnospira is a popular botanical dietary supplement throughout the world and has been consumed as a food product for hundreds of years. Ongoing efforts from our research group are focused on evaluating the utility of a Limnospira-derived oral supplement (Immulina) in promoting resilience against influenza viral infection. Like other botanical extracts, Immulina is inherently a complex matrix with variation in the levels of its chemical constituents. Therefore, to ensure therapeutic consistency for future scientific research and clinical studies, we are developing standardization technology using a bioassay and chemical markers. Braun-type lipoproteins, a class of macromolecules that activate the Toll-like receptor (TLR)2/TLR1 signaling pathway, have been identified as a major active component within Immulina. Based on the mechanism of action of the Braun-type lipoproteins, an in vitro bioassay was established using the HEK-Blue hTLR2/TLR1 cell line to quantitate the immune-enhancing potency of Immulina. The objective of the current research was to validate that bioassay for Immulina activity quantification using the U.S. FDA guidance document for botanical drug development and U.S. Pharmacopeia recommendations. System suitability, reference standards and defining potency units were established. Validation of performance parameters included precision, specificity, accuracy, linearity, and range. Validating this bioassay for Immulina activity provides a tool for ensuring product consistency and quantifying the potency of this botanical for use in future research as well as material in the consumer market.

Toll-Like Receptor mRNA Levels in Schizophrenia: Association With Complement Factors and Cingulate Gyrus Cortical Thinning.

BACKGROUND AND HYPOTHESES: Previous studies revealed innate immune system activation in people with schizophrenia (SZ), potentially mediated by endogenous pathogen recognition receptors, notably Toll-like receptors (TLR). TLRs are activated by pathogenic molecules like bacterial lipopolysaccharides (TLR1 and TLR4), viral RNA (TLR3), or both (TLR8). Furthermore, the complement system, another key component of innate immunity, has previously been linked to SZ. STUDY DESIGN: Peripheral mRNA levels of TLR1, TLR3, TLR4, and TLR8 were compared between SZ and healthy controls (HC). We investigated their relationship with immune activation through complement expression and cortical thickness of the cingulate gyrus, a region susceptible to immunological hits. TLR mRNA levels and peripheral complement receptor mRNA were extracted from 86 SZ and 77 HC white blood cells; structural MRI scans were conducted on a subset. STUDY RESULTS: We found significantly higher TLR4 and TLR8 mRNA levels and lower TLR3 mRNA levels in SZ compared to HC. TLRs and complemental factors were significantly associated in SZ and HC, with the strongest deviations of TLR mRNA levels in the SZ subgroup having elevated complement expression. Cortical thickness of the cingulate gyrus was inversely associated with TLR8 mRNA levels in SZ, and with TLR4 and TLR8 levels in HC. CONCLUSIONS: The study underscores the role of innate immune activation in schizophrenia, indicating a coordinated immune response of TLRs and the complement system. Our results suggest there could be more bacterial influence (based on TLR 4 levels) as opposed to viral influence (based on TLR3 levels) in schizophrenia. Specific TLRs were associated with brain cortical thickness reductions of limbic brain structures.

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.

Staphylococcus aureus activates NRLP3-dependent IL-1ß secretion from human conjunctival goblet cells using α toxin and toll-like receptors 2 and 1.

We used cultured human conjunctival goblet cells to determine (i) whether the toxigenic S. aureus- induced activation of the epithelial goblet cells requires two signals to activate the NLRP3 inflammasome, (ii) if one signal is mediated by TLR1, TLR2, or TLR6, and (iii) if the S. aureus toxin α toxin is another signal for the activation of the inflammasome and secretion of mature IL-1ß. Cultured cells were incubated with siRNA to knock down the different TLRs. After stimulation with toxigenic S. aureus RN6390, pro-IL-1ß synthesis, caspase-1 activity, and mature IL-1ß secretion were measured. In a separate set of experiments, the cells were incubated with toxigenic S. aureus RN6390 or mutant S. aureus ALC837 that does not express α toxin with or without exogenous α toxin. A gentamicin protection assay was used to determine if intracellular bacteria were active. We conclude that α toxin from toxigenic S. aureus triggers two separate mechanisms required for the activation of the NLRP3 inflammasome and secretion of mature IL-1ß. In the first mechanism, α toxin secreted from internalized S. aureus produces a pore, allowing the internalized bacteria and associated pathogen-associated molecular patterns to interact with intracellular TLR2 and, to a lesser extent, TLR1. In the second mechanism, α toxin forms a pore in the plasma membrane, leading to an efflux of cytosolic K+ and influx of Ca2+. We conclude that α toxin by these two different mechanisms triggers the synthesis of pro-IL-1ß and NLRP3 components, activation of capase-1, and secretion of mature IL-1ß to defend against bacterial infection.