TLR4 Molecules Research Articles

Alterations in expression of TLR2 and TLR4 during successful and pathological aging

The generally accepted theory of aging is the theory of inflammaging. The leading role in its development is played by the mechanisms of innate immunity. Key receptors of innate immunity are TLRs. The patterns of age-related changes in the functioning of the TLR system remain the subject of study. The purpose of the work is to study the expression of TLR2 and TLR4 receptors in peripheral blood cells of centenarians and elderly people at the level of genes and surface molecules. The study analyzed the expression of genes and molecules TLR2 and TLR4 in young donors (n = 50), elderly people (n = 50) and centenarians (n = 100). Analysis of TLR2 and TLR4 gene expression was carried out using RT-PCR. Determination of surface expression of TLR2 and TLR4 was carried out using flow cytofluorimetry. This study is the first to analyze the age-related dynamics of expression of genes and molecules TLR2 and TLR4. It was revealed that in groups of elderly patients and long-livers, TLR2 expression is increased both at the gene and on the cell surface, compared to young donors. In contrast, TLR4 expression decreased with age. Studied groups of patients were divided depending on the aging phenotype into two subgroups: successful and pathological aging. In elderly individuals, TLR2 is increased in both phenotypes, while TLR4 is decreased in the pathological aging. In the group of centenarians with a pathological aging, a decrease in both TLR2 and TLR4 is observed, compared with young. In long-livers with successful aging, an increase in TLR2 expression was observed at the gene and protein level and TLR4 expression was comparable to the group of young individuals. Identified changes in the expression of TLR2 and TLR4, observed in different age groups, can be considered as markers of various aging phenotypes.

Effects of Aeromonas infection on the immune system, physical barriers and microflora structure in the intestine of juvenile grass carp (Ctenopharyngodon idella)

Grass carp (Ctenopharyngodon idella) is an intensively cultured and economically important herbivorous fish species in China, but its culture is often impacted by Aeromonas pathogens such as Aeromonas hydrophila and Aeromonas veronii. In this study, healthy grass carp were separately infected with A. hydrophila or A. veronii for 12, 24, 48 or 72 h. The results showed that the mRNA expression levels of intestinal inflammatory factors (tnf-α, il-1β and il-8), complement factors (c3 and c4), antimicrobial peptides (hepcidin, nk-lysin and β-defensin-1), immunoglobulins (igm and igt), and immune pathway-related signaling molecules (tlr1, tlr2, tlr4, myd88, irak4, irak1, traf6, nf-κb p65 and ap-1) were differentially upregulated in response to A. hydrophila and A. veronii challenge. Additionally, the expression levels of the intestinal pro-apoptotic genes tnfr1, tnfr2, tradd, caspase-8, caspase-3 and bax were significantly increased, whereas the expression of the inhibitory factor bcl-2 was significantly downregulated, indicating that Aeromonas infection significantly induced apoptosis in the intestine of grass carp. Moreover, the expression of intestinal tight junction proteins (occludin, zo-1, claudin b and claudin c) was significantly decreased after infection with Aeromonas. Histopathological analysis indicated the Aeromonas challenge caused severe damage to the intestinal villi with adhesions and detachment of intestinal villi accompanied by severe inflammatory cell infiltration at 12 h and 72 h. The 16S rRNA sequencing results showed that Aeromonas infection significantly altered the structure of the intestinal microflora of the grass carp at the phylum (Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes) and genus (Proteus, Cetobacterium, Bacteroides, and Aeromonas) levels. Take together, the findings of this study revealed that Aeromonas infection induces an intestinal immune response, triggers cell apoptosis, destroys physical barriers and alters microflora structure in the intestine of juvenile grass carp; the results will help to reveal the pathogenesis of intestinal bacterial diseases in grass carp.

Electroacupuncture Reduces Fibromyalgia Pain via Neuronal/Microglial Inactivation and Toll-like Receptor 4 in the Mouse Brain: Precise Interpretation of Chemogenetics.

Fibromyalgia (FM) is a complex, chronic, widespread pain syndrome that can cause significant health and economic burden. Emerging evidence has shown that neuroinflammation is an underlying pathological mechanism in FM. Toll-like receptors (TLRs) are key mediators of the immune system. TLR4 is expressed primarily in microglia and regulates downstream signaling pathways, such as MyD88/NF-κB and TRIF/IRF3. It remains unknown whether electroacupuncture (EA) has therapeutic benefit in attenuating FM pain and what role the TLR4 pathway may play in this effect. We compared EA with sham EA to eliminate the placebo effect due to acupuncture. We demonstrated that intermittent cold stress significantly induced an increase in mechanical and thermal FM pain in mice (mechanical: 2.48 ± 0.53 g; thermal: 5.64 ± 0.32 s). EA but not sham EA has an analgesic effect on FM mice. TLR4 and inflammatory mediator-related molecules were increased in the thalamus, medial prefrontal cortex, somatosensory cortex (SSC), and amygdala of FM mice, indicating neuroinflammation and microglial activation. These molecules were reduced by EA but not sham EA. Furthermore, a new chemogenetics method was used to precisely inhibit SSC activity that displayed an anti-nociceptive effect through the TLR4 pathway. Our results imply that the analgesic effect of EA is associated with TLR4 downregulation. We provide novel evidence that EA modulates the TLR4 signaling pathway, revealing potential therapeutic targets for FM pain.

Sex differences in the biomechanical and biochemical responses of caudal rat intervertebral discs to injury.

Intervertebral disc degeneration (IDD) is a major cause of low back pain (LBP) worldwide. Sexual dimorphism, or sex-based differences, appear to exist in the severity of LBP. However, it is unknown if there are sex-based differences in the inflammatory, biomechanical, biochemical, and histological responses of intervertebral discs (IVDs). Caudal (Coccygeal/Co) bone-disc-bone motion segments were isolated from multiple spinal levels (Co8 to Co14) of male and female Sprague-Dawley rats. Changes in motion segment biomechanics and extracellular matrix (ECM) biochemistry (glycosaminoglycan [GAG], collagen [COL], water, and DNA content) were evaluated at baseline and in response to chemical insult (lipopolysaccharide [LPS]) or puncture injury ex vivo. We also investigated the contributions of Toll-like receptor (TLR4) signaling on responses to LPS or puncture injury ex vivo, using a small molecule TLR4 inhibitor, TAK-242. Findings indicate that IVD motion segments from female donors had greater nitric oxide (NO) release in LPS groups compared to male donors. HMGB1 release was increased in punctured discs, but not LPS injured discs, with no sex effect. Although both male and female discs exhibited reductions in dynamic moduli in response to LPS and puncture injuries, dynamic moduli from female donors were higher than male donors across all groups. In uninjured (baseline) samples, a significant sex effect was observed in nucleus pulposus (NP) DNA and water content. Female annulus fibrosus (AF) also had higher DNA, GAG, and COL content (normalized bydry weight), but lower water content than male AF. Additional injury- and sex-dependent effects were observed in AF GAG/DNA and COL/DNA content. Finally, TAK-242 improved the dynamic modulus of female but not male punctured discs. Our findings demonstrate that there are differences in rat IVD motion segments based on sex, and that the response to injury in inflammatory, biomechanical, biochemical, and histological outcomes also exhibit sex differences. TLR4 inhibition protected against loss of mechanical integrity of puncture-injured IVD motion segments, with differences responses based on donor sex.

The role of regulatory T cells in the pathogenesis of acute kidney injury.

The incidence of acute kidney injury (AKI) is on the rise and is associated with high mortality; however, there are currently few effective treatments. Moreover, the relationship between Tregs and other components of the immune microenvironment (IME) in the pathogenesis of AKI remains unclear. We downloaded four publicly accessible AKI datasets, GSE61739, GSE67401, GSE19130, GSE81741, GSE19288 and GSE106993 from the gene expression omnibus (GEO) database. Additionally, we gathered two kidney single-cell sequencing (scRNA-seq) samples from the Department of Organ Transplantation at Zhujiang Hospital of Southern Medical University to investigate chronic kidney transplant rejection (CKTR). Moreover, we also collected three samples of normal kidney tissue from GSE131685. By analysing the differences in immune cells between the AKI and Non-AKI groups, we discovered that the Non-AKI group contained a significantly greater number of Tregs than the AKI group. Additionally, the activation of signalling pathways, such as inflammatory molecules secretion, immune response, glycolytic metabolism, NOTCH, FGF, NF-κB and TLR4, was significantly greater in the AKI group than in the Non-AKI group. Additionally, analysis of single-cell sequencing data revealed that Tregs in patients with chronic kidney rejection and in normal kidney tissue have distinct biology, including immune activation, cytokine production, and activation fractions of signalling pathways such as NOTCH and TLR4. In this study, we found significant differences in the IME between AKI and Non-AKI, including differences in Tregs cells and activation levels of biologically significant signalling pathways. Tregs were associated with lower activity of signalling pathways such as inflammatory response, inflammatory molecule secretion, immune activation, glycolysis.

Constructing and Validating a Network of Potential Olfactory Sheathing Cell Transplants Regulating Spinal Cord Injury Progression.

The pathology of spinal cord injury (SCI), including primary and secondary injuries, primarily involves hemorrhage, ischemia, edema, and inflammatory responses. Cell transplantation has been the most promising treatment for SCI in recent years; however, its specific molecular mechanism remains unclear. In this study, bioinformatics analysis verified by experiment was used to elucidate the hub genes associated with SCI and to discover the underlying molecular mechanisms of cell intervention. GSE46988 data were downloaded from the Gene Expression Omnibus dataset. In our study, differentially expressed genes (DEGs) were reanalyzed using the "R" software (R v4.2.1). Functional enrichment and protein-protein interaction network analyses were performed, and key modules and hub genes were identified. Network construction was performed for the hub genes and their associated miRNAs. Finally, a semi-quantitative analysis of hub genes and pathways was performed using quantitative real-time polymerase chain reaction. In total, 718 DEGs were identified, mainly enriched in immune and inflammation-related functions. We found that Cd4, Tp53, Rac2, and Akt3 differed between vehicle and transplanted groups, suggesting that these genes may play an essential role in the transplantation of olfactory ensheathing cells, while a toll-like receptor signaling pathway was significantly enriched in Gene set enrichment analysis, and then, the differences were statistically significant by experimentally verifying the expression of their associated molecules (Tlr4, Nf-κb, Ikkβ, Cxcl2, and Tnf-α). In addition, we searched for upstream regulatory molecules of these four central genes and constructed a regulatory network. This study is the first to construct a regulatory network for olfactory ensheathing cell transplantation in treating SCI, providing a new idea for SCI cell therapy.

TLR4, MyD88, and TNF-α Expression in the Lungs of Akkaraman and Romanov Lambs in Response to LPS and LTA.

Toll-like receptors are involved in the recognition of bacterial toxins, which cause infection in the respiratory system. This study aimed to evaluate microanatomical and histological alterations in the lungs of 24 healthy Akkaraman and Romanov lambs after the administration of lipoteichoic acid (LTA), lipopolysaccharide (LPS), and LTA + LPS and investigate the gene, protein, and immune expression levels of TLR4, MyD88, and TNF-α molecules, known to have immune functions. Microanatomical examinations showed thickened peribronchial and alveolar walls in the lungs of groups LTA, LPS, and LTA + LPS of both breeds due to immune cell infiltration. TLR4, MyD88, and TNF-α immunoexpressions were positive to varying degrees in the cytoplasm and nucleus of the bronchial and bronchiolar luminal epithelial cells, alveolar epithelial cells, and alveolar macrophages. TLR4 and TNF-α protein expressions were statistically different in the LPS-treated Romanov lambs, compared to the other groups. Among the Akkaraman lambs, TLR4 gene expression was significantly higher in group LPS, and among the Romanov lambs, TLR4, MyD88, and TNF-α gene expressions were significantly higher in group LTA + LPS. Therefore, TLR4, MyD88, and TNF-α molecules, involved in the immune response, were found to be expressed at different levels against LTA and LPS in the lungs of two different sheep breeds.

Lipopolysaccharide induces skin scarring through the TLR4/Myd88 inflammatory signaling pathway in dermal fibroblasts

Skin scarring is a frequent complication of the wound healing process. Bacterial contamination and prolonged inflammation in wounds are thought to play significant roles during scar formation, but little is known about their specific mechanisms of action. In this study, hypertrophic scar derived fibroblasts (HSFs) and paired normal skin derived fibroblasts (NSFs) were used to evaluate the effects of lipopolysaccharide (LPS) on inflammation-induced skin scarring and explore the inflammation-mediated mechanism of activity of LPS on dermal fibroblasts. LPS was found to significantly upregulate the expression of the proinflammatory molecules TLR4, Myd88, TRAF6, and p65, and the fibrosis-related proteins Col I, Col III, and α-SMA, in NSFs. Blocking Myd88 expression with T6167923 downregulated the expression of Col I, Col III, and α-SMA, whereas activating Myd88 expression with CL075 significantly upregulated their expression in LPS-treated NSFs. LPS was found to delay wound healing and increase skin scarring in cell and mouse models. These results showed that LPS could induce scar formation through the TLR4/Myd88 signaling pathway in dermal fibroblasts, suggesting that the downregulation of excessive inflammation in wound tissues inhibits skin scarring and improves scar appearance.

An immunoinformatics approach to epitope-based vaccine design against PspA in Streptococcus pneumoniae

BackgroundStreptococcus pneumoniae (SPN) is the agent responsible for causing respiratory diseases, including pneumonia, which causes severe health hazards and child deaths globally. Antibiotics are used to treat SPN as a first-line treatment, but nowadays, SPN is showing resistance to several antibiotics. A vaccine can overcome this global problem by preventing this deadly pathogen. The conventional methods of wet-laboratory vaccine design and development are an intense, lengthy, and costly procedure. In contrast, epitope-based in silico vaccine designing can save time, money, and energy. In this study, pneumococcal surface protein A (PspA), one of the major virulence factors of SPN, is used to design a multi-epitope vaccine. MethodsFor designing the vaccine, the sequence of PspA was retrieved, and then, phylogenetic analysis was performed. Several CTL epitopes, HTL epitopes, and LBL epitopes of PspA were all predicted by using several bioinformatics tools. After checking the antigenicity, allergenicity, and toxicity scores, the best epitopes were selected for the vaccine construction, and then, physicochemical and immunological properties were analyzed. Subsequently, vaccine 3D structure prediction, refinement, and validation were performed. Molecular docking, molecular dynamic simulation, and immune simulation were performed to ensure the binding between HLA and TLR4. Finally, codon adaptation and in silico cloning were performed to transfer into a suitable vector. ResultsThe constructed multi-epitope vaccine showed a strong binding affinity with the receptor molecule TLR4. Analysis of molecular dynamic simulation, C-immune simulation, codon adaptation, and in silico cloning validated that our designed vaccine is a suitable candidate against SPN. ConclusionThe in silico analysis has proven the vaccine as an alternative medication to combat against S. pneumoniae. The designated vaccine can be further tested in the wet lab, and a novel vaccine can be developed.

The genes expression status of inflammatory determinants following the oral administration of Mannuronic acid in patients with rheumatoid arthritis

Introduction: Rheumatoid arthritis (RA) is a progressive multifactorial inflammatory disorder. According to numerous evidence, pro-inflammatory markers such as TNF-a, IL-6, IL-22, MYD88 and TLR2 play a substantial role in the pathogenesis and persistence of this disease. B-D-Mannuronic acid (M2000) is a new immunosuppressive drug whose therapeutic effects have been approved in several clinical trials and the results of the phase III clinical trial of this drug in RA patients were potent and efficient. Therefore, the present investigation was designed to evaluate its anti-inflammatory effects on the expression of mentioned factors in RA patients. Material and methods: This research was carried out on 12 healthy individuals and 12 patients with RA and M2000 was administered to the patients orally at a dose of 500 mg twice daily for 12 weeks. The peripheral blood mononuclear cells (PBMCs) were collected from the patients before and after treatment with M2000 to investigate the gene expression levels of TNF-a, IL-6, IL-22, MYD88 and TLR2 molecules in them using Real-time PCR. Results: This study data represented a higher gene expression in all target molecules in the RA patients in comparison to the healthy individuals. Furthermore, the outcomes showed that after 12 weeks of therapy with M2000, the gene expression levels of inflammatory factors TNF-a, IL-6, IL-22, MYD88 and TLR2 decreased significantly in treated patients compared to before therapy. The gene expression results were following the clinical and paraclinical assessments. Conclusion: In conclusion, M2000 as a newly approved anti-inflammatory and immunosuppressive drug, can be proposed as a therapeutic agent in RA patients.

Lipoteichoic Acid Inhibits Lipopolysaccharide-Induced TLR4 Signaling by Forming an Inactive TLR4/MD-2 Complex Dimer.

LPS interacts with TLR4, which play important roles in host-against-pathogen immune responses, by binding to MD-2 and inducing an inflammatory response. In this study, to our knowledge, we found a novel function of lipoteichoic acid (LTA), a TLR2 ligand, that involves suppression of TLR4-mediated signaling independently of TLR2 under serum-free conditions. LTA inhibited NF-κB activation induced by LPS or a synthetic lipid A in a noncompetitive manner in human embryonic kidney 293 cells expressing CD14, TLR4, and MD-2. This inhibition was abrogated by addition of serum or albumin. LTAs from different bacterial sources also inhibited NF-κB activation, although LTA from Enterococcus hirae had essentially no TLR2-mediated NF-κB activation. The TLR2 ligands tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) and macrophage-activating lipopeptide-2 (MALP-2) did not affect the TLR4-mediated NF-κB activation. In bone marrow-derived macrophages from TLR2-/- mice, LTA inhibited LPS-induced IκB-α phosphorylation and production of TNF, CXCL1/KC, RANTES, and IFN-β without affecting cell surface expression of TLR4. LTA did not suppress IL-1β-induced NF-κB activation mediated through signaling pathways shared with TLRs. LTAs including E. hirae LTA, but not LPS, induced association of TLR4/MD-2 complexes, which was suppressed by serum. LTA also increased association of MD-2, but not TLR4 molecules. These results demonstrate that, under serum-free conditions, LTA induces association of MD-2 molecules to promote formation of an inactive TLR4/MD-2 complex dimer that in turn prevents TLR4-mediated signaling. The presence of LTA that poorly induces TLR2-mediated activation but inhibits TLR4 signaling provides insight into the role of Gram-positive bacteria in suppressing inflammation induced by Gram-negative bacteria in organs such as the intestines where serum is absent.

Mild Hypothermia Alleviates CLP-induced Multiple Organ Dysfunction by Mitigating Pyroptosis Through the TLR4/NF-κB/NLRP3 Signaling Pathway

Multiple organ failure secondary to severe sepsis leads to increased morbidity and mortality and is often accompanied by inflammation and immune system dysfunction. Mild hypothermia has been shown to have anti-inflammatory properties, but whether it can exert a protective effect in cases of multiple organ failure remains unclear. Thus, in this study, we investigated the protective effect of mild hypothermia on septic multiple organ failure and the underlying mechanism for this effect. Sepsis was induced through the cecal ligation and puncture (CLP) method. Rats were then housed at normal (36-38°C) or mild hypothermic (32-34°C) temperature for 10 h. CLP-induced effects on inflammatory cytokines and biochemical markers in serum were reversed by mild hypothermia. The pathological injury score and the expressions of pyroptosis markers, including TLR4, MyD88 and NF-κB signaling molecules, showed a similar trend. Moreover, 3 d survival of CLP rats was improved by mild hypothermia. Mild hypothermia alleviated CLP-induced organ failure and the downstream effects on pyroptosis, probably through the TLR4/NF-κB/NLRP3 signaling pathway.

Vaccinomics to Design a Multiepitope Vaccine against Legionella pneumophila.

Legionella pneumophila is found in the natural aquatic environment and can resist a wide range of environmental conditions. There are around fifty species of Legionella, at least twenty-four of which are directly linked to infections in humans. L. pneumophila is the cause of Legionnaires' disease, a potentially lethal form of pneumonia. By blocking phagosome-lysosome fusion, L. pneumophila lives and proliferates inside macrophages. For this disease, there is presently no authorized multiepitope vaccine available. For the multi-epitope-based vaccine (MEBV), the best antigenic candidates were identified using immunoinformatics and subtractive proteomic techniques. Several immunoinformatics methods were utilized to predict B and T cell epitopes from vaccine candidate proteins. To construct an in silico vaccine, epitopes (07 CTL, 03 HTL, and 07 LBL) were carefully selected and docked with MHC molecules (MHC-I and MHC-II) and human TLR4 molecules. To increase the immunological response, the vaccine was combined with a 50S ribosomal adjuvant. To maximize vaccine protein expression, MEBV was cloned and reverse-translated in Escherichia coli. To prove the MEBV's efficacy, more experimental validation is required. After its development, the resulting vaccine is greatly hoped to aid in the prevention of L. pneumophila infections.

Mycobacterium tuberculosis dormancy regulon proteins Rv2627c and Rv2628 as Toll like receptor agonist and as potential adjuvant

During latency, DosR proteins of Mycobacterium tuberculosis (M.tb) get activated and help the bacterium to remain dormant. We have shown earlier that 2 such proteins Rv2627c and Rv2628 are immunogenic and induce a TH1 kind of immune response. In this study, through in-vitro experiments we have confirmed that Rv2627c and Rv2628 proteins act as protein Toll-Like Receptor (TLR) agonist-adjuvant. Rv2627c and Rv2628 stimulated THP-1 macrophages showed an increased expression of TLR2, TLR4 and co-stimulatory molecules CD40, CD80, CD86 and antigen presenting molecule HLA-DR. Further studies also found enhanced expression of downstream signaling molecules of TLR activation like MyD88, NF-κB-p65 and pro-inflammatory cytokines. Inhibition studies using TLR blocking antibodies decreased the expression of co-stimulatory molecules, MyD88, NF-κB-p65, and pro-inflammatory cytokines. Rv2627c and Rv2628 stimulation of HEK-TLR2 reporter cell line confirmed the interaction of these proteins with TLR2. Moreover, molecular docking and simulations of Rv2627c and Rv2628 proteins with TLR2 and TLR4 showed stable interactions. The adjuvant activity of Rv2628 was further validated by a protein adjuvanted with pre-clinically validated peptides as multi-epitope vaccine construct which showed good binding with TLR2 and TLR4 and activate dendritic cells and induce sustained pro-inflammatory cytokine response by C-ImmSim analysis. We propose that our vaccine construct will produce a better immune response than BCG and can be taken up as a post-exposure therapeutic subunit vaccine along with standard TB therapy. We also anticipate that our construct can be taken up as a protein adjuvant with other vaccine candidates as these can activate macrophages through TLR signaling.

Overexpression of toll-like receptors and co-stimulatory molecules on immature dendritic cells of Crohn's disease

IntroductionCrohn's disease (CD) is a chronic, relapsing inflammatory disorder of the gastrointestinal tract resulting from a loss of homeostasis between the intestinal immune system and the gut microbiome. Also, evidence shows dendritic cells (DC) are the main antigen-presenting cells (APC) in the intestinal mucosa; they can regulate innate and adaptive immunity that is pivotal for maintaining immunological tolerance in the gut. The maturation of immature DC (iDCs) regulate by their surface molecules such as Toll-Like Receptors (TLRs) and co-stimulatory molecules (CD80 and CD86). The level of TLRs on iDCs plays a critical role in developing adaptive responses to antigens. Crohn's disease has a dysfunctional immune system and low tolerance of dendritic cells. This study aimed to investigate the expression of TLR2, TLR4, TLR5, and TLR9 and co-stimulatory molecules (CD80 and CD86). MethodologyHuman peripheral blood mononuclear cells (PBMCs) were collected from 15 CD patients and 12 healthy controls. PBMCs were isolated by density gradient centrifugation, and then iDCs were derived for 6 days. Flow cytometry was performed on iDCs to characterize and distinguish the phenotype of iDC populations. iDCs were harvested for RNA extraction, and the mRNA expression level of TLRs was examined by quantitative RT-PCR using SYBR Green chemistry. ResultThe results showed that TLR2, TLR4, TLR5, and TLR9 mRNA expression significantly increased in the iDCs isolated from patients with CD compared with healthy control P ≤ 0.0001. Although, TLR2 had the lowest expression among other TLRs on iDCs in CD patients. Also, the expression level of CD80 and CD86 were significantly higher in iDCs isolated from CD patients compared to healthy controls P < 0.001 OR = 0.75 CI (−8.76 to −3.17). ConclusionTLR2, TLR4, TLR5, and TLR9 expression on iDCs may be critical in the biological pathogenesis of CD. TLR alterations in the iDCs may contribute to determining dendritic cell maturation pathway lead to increases inflammatory response.

Elevated proportion of TLR2- and TLR4-expressing Th17-like cells and activated memory B cells was associated with clinical activity of cerebral cavernous malformations

BackgroundRecent evidences have suggested the involvement of toll-like receptor (TLR)-4 in the pathogenesis of cerebral cavernous malformations (CCM). Elevated frequency of TLR+T-cells has been associated with neurological inflammatory disorders. As T-cells and B-cells are found in CCM lesions, the objective of the present study was to evaluate the cytokine profile of T-cells expressing TLR2 and TLR4, as well as B-cell subsets, in asymptomatic (CCMAsympt) and symptomatic (CCMSympt) patients.MethodsFor our study, the cytokine profile from TLR2+ and TLR4+ T-cell and B-cell subsets in CCMAsympt and CCMSympt patients was investigated using flow cytometry and ELISA. T-cells were stimulated in vitro with anti-CD3/anti-CD28 beads or TLR2 (Pam3C) and TLR4 (LPS) ligands.ResultsCCMSymptc patients presented a higher frequency of TLR4+(CD4+ and CD8+) T-cells and greater density of TLR4 expression on these cells. With regard to the cytokine profile, the percentage of TLR2+ and TLR4+ Th17 cells was higher in CCMSympt patients. In addition, an elevated proportion of TLR4+ Tc-1 cells, as well as Tc-17 and Th17.1 cells expressing TLR2 and TLR4, was observed in the symptomatic patients. By contrast, the percentage of TLR4+ IL-10+CD4+ T cells was higher in the CCMAsympt group. Both Pam3C and LPS were more able to elevate the frequency of IL-6+CD4+T cells and Th17.1 cells in CCMSympt cell cultures. Furthermore, in comparison with asymptomatic patients, purified T-cells from the CCMSympt group released higher levels of Th17-related cytokines in response to Pam3C and, mainly, LPS, as well as after activation via TCR/CD28. Concerning the B-cell subsets, a higher frequency of memory and memory activated B-cells was observed in CCMSympt patients.ConclusionsOur findings reveal an increase in circulating Th17/Tc-17 cell subsets expressing functional TLR2 and, mainly, TLR4 molecules, associated with an increase in memory B-cell subsets in CCM patients with clinical activity of the disease.

TLR2 and TLR4 molecules and antigen-presenting cell compositions in cecal tonsils of broiler chicks (Gallus gallus domesticus) in the first two weeks of the post-hatch period.

Chickens do not have lymph nodes. Gut-associated lymphoid tissue is the major immunological organization for the digestive system. Cecal tonsils are an important part of this organization. This study is a descriptive and experimental study that was conducted to determine the histological development of the cecal tonsils and the distribution of Toll-like receptor (TLR) 2, TLR4 and antigen-presenting cells during the first 2weeks of the chick's life. The tissue sections were stained using Crossmon's triple technique, Gordon and Sweet's silver impregnation, and streptavidin-biotin-peroxidase complex methods. The classical tonsil framework with fossa and tonsillar units were observed in 4days cecal tissue. The web of reticular fibres forming the stroma of the tissue had the impression that the lymphoid cells filling in time. The development of cecal tonsil was completed histologically on the day 10 and following day 14 samples. Regardless of the antigenic stimulation, TLR2, TLR4 and CD83, major histocompatibility complex (MHC) class II molecules are present in proximal cecal tissue. However, CD83-positive dendritic cells in the germinal centre were first distinguished on day 7. Furthermore, the high antigen presentation capacity of the cecum with an intense MHC class II molecule expression was determined. Histological and immunohistochemical findings in this study revealed that both innate and adaptive cecal defence mechanisms were in the learning period during the first 2weeks. The learning period of innate immunity may require more detailed research. However, the results obtained in this study will be taken into consideration in the vaccination programmes in chicks.

P.161: Small Molecule Inhibitor of Toll-like Receptor-4, TAK-242, Attenuates Allogenic Immune Responses In Vitro

Introduction: Development of innate and adaptive immune response against transplanted islets has a major impact on the poor long term survival of allogenic islet transplants. Our previous studies have shown that TAK-242, a small molecule inhibitor of toll-like receptor-4 either in a soluble form or covalently linked to the islet surface can effectively protect islets from damage inflicted by sterile inflammation in vitro and also prolongs survival of syngeneic islet transplants. Based on recent reports indicating crosstalk between TLR-4 and major histocompatibility complex molecules, we hypothesized that TAK-242 can inhibit proliferation of lymphocytes in response to allogenic stimulation. Methods: Human peripheral blood mononuclear cells labeled with carboxyfluorescein succinimidyl ester were tested for proliferation in a one-way mixed lymphocyte reaction against mitomycin treated, CellTrace Violet-labeled allogenic splenocytes as stimulators. Proliferation of CD4+ and CD8+ T cells was analyzed by flow cytometry. Concentrations of pro-inflammatory cykokines in the culture supernatant was measured by Luminex Multiplex Bead Assay. Varying concentrations (0–30 µM) of TAK242 or MAP84, an inactive structure analog of TAK242, were added to the culture medium. Results: Addition of TAK-242 to the mixed lymphocyte reaction dose dependently inhibited proliferation of both CD4+ and CD8+ T cells (Fig. 1A). The inhibition of proliferation was optimal on day 5. Proliferation of CD4+ and CD8+ T cells was not affected by the addition of MAP84 at similar concentrations. Cytokine measurements showed that IL-2 concentrations were not inhibited by TAK-242. However, proinflammatory cytokines IL-1beta, IL-6, IP-10 and TNF-alpha were significantly inhibited specifically by TAK-242 (Fig. 1B). These results mirrored our previous data showing TAK-242 inhibition of pro-inflammatory cytokines by islets stimulated with TLR-4 ligands. Conclusion: The present data has shown for the first time that blockade of toll-like receptor-4 by TAK-242 is an effective method to attenuate development of T cell response against allogenic targets in addition to the inhibition of inflammation. Crosstalk between TLR pathway with other critical receptors involved the development of immune response is likely to play a role in this inhibition. Further experiments to decipher the mechanism underlying this unique immunosuppressive property of TAK-242 are in progress.

Designing a Multi-Epitope Vaccine against Chlamydia trachomatis by Employing Integrated Core Proteomics, Immuno-Informatics and In Silico Approaches.

Simple SummaryChlamydia trachomatis is the most common cause of blindness, ectopic pregnancy, and bacterial sexually transmitted infections. These diseases affect mostly young women but can also infect men and women of all ages. It is not difficult to treat, but it can lead to more significant health problems if left untreated. There is no licensed vaccine available for this pathogen at present. Hence, a vaccine that can control and prevent C. trachomatis infections is designed in this study by using different immuno-informatics approaches. However, the designed vaccine is the result of computational approaches; therefore, experimental validation is required to prove its effectiveness.Chlamydia trachomatis, a Gram-negative bacterium that infects the rectum, urethra, congenital sites, and columnar epithelium of the cervix. It is a major cause of preventable blindness, ectopic pregnancy, and bacterial sexually transmitted infections worldwide. There is currently no licensed multi-epitope vaccination available for this pathogen. This study used core proteomics, immuno-informatics, and subtractive proteomics approaches to identify the best antigenic candidates for the development of a multi-epitope-based vaccine (MEBV). These approaches resulted in six vaccine candidates: Type III secretion system translocon subunit CopD2, SctW family type III secretion system gatekeeper subunit CopN, SycD/LcrH family type III secretion system chaperone Scc2, CT847 family type III secretion system effector, hypothetical protein CTDEC_0668, and CHLPN 76kDa-like protein. A variety of immuno-informatics tools were used to predict B and T cell epitopes from vaccine candidate proteins. An in silico vaccine was developed using carefully selected epitopes (11 CTL, 2 HTL & 10 LBL) and then docked with the MHC molecules (MHC I & MHC II) and human TLR4. The vaccine was coupled with Cholera toxin subunit B (CTB) adjuvant to boost the immune response. Molecular dynamics (MD) simulations, molecular docking, and MMGBSA analysis were carried out to analyze the molecular interactions and binding affinity of MEBV with TLR4 and MHC molecules. To achieve the highest level of vaccine protein expression, the MEBV was cloned and reverse-translated in Escherichia coli. The highest level of expression was achieved, and a CAI score of 0.97 was reported. Further experimental validation of the MEBV is required to prove its efficacy. The vaccine developed will be useful in preventing infections caused by C. trachomatis.

Imbalanced inflammatory response in subchronic arsenic-induced liver injury and the protective effects of Ginkgo biloba extract in rats: Potential role of cytokines mediated cell-cell interactions.

Arsenic is a well-known environmental toxicant and carcinogen, which has been epidemiologically proved related to the increased hepatic disorders. Researches have shown that aseptic inflammation and abnormal immune response are associated with arsenic-induced liver injury. However, the immunotoxic effects of liver have not been extensively characterized. Ginkgo biloba extract (GBE), a natural products of G. biloba leaves with proven anti-inflammatory and potential immunoregulatory activities, was used as intervention agent to explore its protective effects on arsenic-induced hepatotoxicity. Thus, the underlying mechanism of the immunotoxic effects on arsenic-induced liver injury were investigated in 2.5, 5.0, and 10.0mg/kg NaAsO2 of Wistar rats for 16 weeks. Subsequently, GBE was used as intervention agent in 50 mg/kg for 6 weeks after cessation of arsenic exposure. The ratio of Th17 to Treg cells in peripheral blood as well as the secretion of inflammatory cytokines IL-17A, IL-6, TGF-β1, and IL-10 in serum and liver were detected. Meanwhile, the notable activation of aseptic inflammation-related molecule TLR4 and its downstream targets MyD88 and NF-κB in the liver were observed. In this work, we confirmed that subchronic exposed to arsenic triggered the infiltration of inflammatory cells in rat liver, coupled with obvious histopathological changes and aberrant hepatic serum biochemical parameters. Meanwhile, imbalanced immune response was verified by the notable abnormal ratio of Th17 to Treg cells in peripheral blood as well as the secretion of inflammatory cytokines IL-17A, IL-6, TGF-β1, and IL-10 in serum and liver of arsenic exposed rats. Further, the level of TLR4, MyD88, and NF-κB in liver both transcription and translation activity were raised. Subsequently, GBE markedly mitigated arsenic-induced liver injury, most impressively, post treatment with GBE prominently suppressed the overactivated inflammatory-related TLR4-MyD88-NF-κB pathway and evidently decreased the secretion of inflammation cytokines. Meanwhile, the disturbance of pro- and anti-inflammatory response was reversed. We concluded that the disruption of pro- and anti-inflammatory T-cells balance caused by cytokines mediated cell-cell interactions may be one of the mechanisms underlying arsenic-induced liver injury and that GBE intervention exerts an evidence protective effects, which might be closely associated with the suppression of inflammatory-related TLR4 pathway.