TLR4 mRNA Expression Research Articles

Flagellin-independent effects of a Toll-like receptor 5 polymorphism in the inflammatory response to Burkholderia pseudomallei.

BackgroundToll-like receptors (TLRs) are sentinel receptors of the innate immune system. TLR4 detects bacterial lipopolysaccharide (LPS) and TLR5 detects bacterial flagellin. A common human nonsense polymorphism, TLR5:c.1174C>T, results in a non-functional TLR5 protein. Individuals carrying this variant have decreased mortality from melioidosis, infection caused by the flagellated Gram-negative bacterium Burkholderia pseudomallei. Although impaired flagellin-dependent signaling in carriers of TLR5:c.1174C>T is well established, this study tested the hypothesis that a functional effect of TLR5:c.1174C>T is flagellin-independent and involves LPS-TLR4 pathways.Methodology/Principal findingsWhole blood from two independent cohorts of individuals genotyped at TLR5:c.1174C>T was stimulated with wild type or aflagellated B. pseudomallei or purified bacterial motifs followed by plasma cytokine measurements. Blood from individuals carrying the TLR5:c.1174C>T variant produced less IL-6 and IL-10 in response to an aflagellated B. pseudomallei mutant and less IL-8 in response to purified B. pseudomallei LPS than blood from individuals without the variant. TLR5 expression in THP1 cells was silenced using siRNA; these cells were stimulated with LPS before cytokine levels in cell supernatants were quantified by ELISA. In these cells following LPS stimulation, silencing of TLR5 with siRNA reduced both TNF-α and IL-8 levels. These effects were not explained by differences in TLR4 mRNA expression or NF-κB or IRF activation.Conclusions/SignificanceThe effects of the common nonsense TLR5:c.1174C>T polymorphism on the host inflammatory response to B. pseudomallei may not be restricted to flagellin-driven pathways. Moreover, TLR5 may modulate TLR4-dependent cytokine production. While these results may have broader implications for the role of TLR5 in the innate immune response in melioidosis and other conditions, further studies of the mechanisms underlying these observations are required.

The Inhibitory Role of M2000 (β-D-Mannuronic Acid) on Expression of Toll-like Receptor 2 and 4 in HT29 Cell Line.

Background/Objectives:Anti-inflammatory agents play a crucial role in controlling inflammatory diseases such as Inflammatory Bowel Disease (IBD) but their use is restricted due to their vast side effects. M2000 (β-D-mannuronic acid) is a new immunomodulatory drug. According to the capacity of M2000 in suppressing some molecules involved in Toll Like Receptors (TLRs) signaling and reducing oxidative stress we hypothesize that, this molecule may have a potential role in decreasing inflammatory responses in IBD. The aim of this study was to evaluate the cytotoxicity of M2000 and its effect on the gene expression of TLR2 and TLR4.Methods:HEK293 cell line was grown and divided into 96-well cell plate and MTT assay was performed. HT29 cells were cultured and treated with low and high doses of M2000. Total RNA was extracted and cDNA synthesized and quantitative real-time PCR was done to quantify the TLR2 and TLR4 mRNA expression.Results:We found that M2000 at the concentration of ≤ 1000µg/ml had no obvious cytotoxicity effect on the HEK293 cells. Also, low and high doses of M2000 could significantly down-regulate both TLR2 and TLR4 mRNA expression. Moreover, a significant reduction in gene expression of TLR2 and TLR4 in an inflammatory condition resulted in high doses of M2000 in the presence of LPS.Conclusion:Our study which was conducted in colonic epithelial cell model, shows that M2000 can be considered as a new anti-inflammatory agent in IBD. However, more comprehensive experimental and clinical studies are required to recognize the molecular mechanism of M2000 and also its safety and efficacy.

Zinc source modulates intestinal inflammation and intestinal integrity of broiler chickens challenged with coccidia and Clostridium perfringens

Two dietary sources of zinc (ZnSO4 or organic Zn) were tested in chickens challenged with coccidiosis (Co) or coccidiosis plus Clostridium perfringens (CoCPF). On day 14, the chickens were orally gavaged with ∼5,000 Eimeria maxima sporulated oocysts. On day 19, 20, and 21 chickens challenged with C. perfringens were given a broth culture containing 108 cfu of this bacterium. Productive performance parameters were determined at d 14, 21, and 28. On day 21, necrotic enteritis (NE) lesions were scored, and intestinal permeability was evaluated. Jejunum and cecal tonsils were collected for morphology and gene expression analysis. On day 21, organic Zn improved BW gain by 18.6% (P = 0.07), and FCR by 12% (P = 0.09) in CoCPF challenged chickens vs. birds fed ZnSO4. From 1 to 28, organic Zn increased BW gain (P = 0.02), and improved FCR (P = 0.03) vs. birds fed ZnSO4. At 21 d, NE lesions were only observed in CoCPF birds (P < 0.001), and mortality due to NE was only observed when CoCPF birds were fed ZnSO4 (P = 0.001). Organic Zn fed birds had increased villus height in the jejunum (P = 0.005) and decreased intestinal permeability (P = 0.01) vs. ZnSO4. In the jejunum, organic Zn fed birds showed a downregulation of expression of IL-8 (P = 0.02), and upregulation of IL-10 (P = 0.05) in CoCPF birds vs. ZnSO4- CoCPF birds. As main effect, birds supplemented with organic Zn had higher mRNA expression of TLR-2 (P = 0.02) and IgA (P = 0.01). In the cecal tonsils, organic Zn fed birds showed upregulation of iNOS (P = 0.008) in CoCPF birds vs. ZnSO4-CoCPF birds. Organic Zn supplementation reduced intestinal permeability and attenuated intestinal inflammation of broilers co-challenged with coccidia and C. perfringens.

TLR2 and TLR4 mediate an activation of adipose tissue renin-angiotensin system induced by uric acid

Both hyperuricemia and adipose tissue renin–angiotensin system (RAS) are closely associated with multiple metabolic and cardiovascular diseases. We previously reported that uric acid could upregulate tissue RAS in adipocytes. In the present study, we aimed to reveal the involvement of toll-like receptors (TLRs) in uric acid-induced RAS activation in adipose tissue. A hyperuricemia rat model fed with a high-fructose diet and rat primary adipocytes were used in this study. Here, we inhibited TLR2 and TLR4 expression in adipose tissue and cultured adipocytes using small interfering RNA (siRNA). We found that high fructose-fed rats had hyperuricemia, higher body weight and greater adipose tissue content. We also found that hyperuricemia rats had raising blood pressure, higher expression levels of inflammatory cytokines and RAS components in adipose tissue, which could be prevented by TLR2/4-siRNA infection. In vitro study, uric acid caused a dose- and time-dependent increase in the mRNA expression of TLR2 and TLR4 in rat adipocytes. Uric acid could increase inflammatory cytokines and upregulate tissue RAS in rat adipocytes, which were both blocked with TLR2/4-siRNA infection. TNF-α and IL-6 could also result in an activation of tissue RAS expression in adipocytes. In conclusion, TLR2/4 mediated adipose inflammation plays a key role in RAS activation induced by uric acid in adipose tissue.

Effects of restrictions on maternal feed intake on the immune indexes of umbilical cord blood and liver Toll-like receptor signaling pathways in fetal goats during pregnancy

BackgroundLiver has important immune function during fetal development and after birth. However, the effect of maternal malnutrition on immune function of the fetal liver is rarely reported. In this study, twelve pregnant goats (Xiangdong black goat, at d 45 of gestation) were assigned to the control group (fed 100% of nutritional requirements) and the restriction group (fed 60% of the intake of the control group) during gestation from d 55 to 100. Fetal goats were harvested at d 100 of gestation and immune indexes and amino acid profiles of the umbilical cord blood and liver Toll-like receptors (TLRs) signaling pathways were measured.ResultsMaternal body weight in the restriction group was lower than the control group (P < 0.05). Maternal feed intake restriction decreased (P < 0.05) heart weight, heart index, alkaline phosphatase and serum amyloid protein A in the umbilical cord blood (UCB). Moreover, only histidine was decreased in the restricted group (P = 0.084), and there were no differences in other amino acids contents in the UCB between the two groups (P > 0.05). The TLR2 and TLR4 mRNA expression in the fetal liver in the restriction group was greater (P < 0.05) than that in the control group. Furthermore, the mRNA expression levels of myeloid differentiation primary response 88 (MyD88), TNF receptor associated factor 6, nuclear factor kappa B subunit 1, NFKB inhibitor alpha, IFN-β, TGF-β, TNF-α and IL-1β in the restricted group were upregulated (P < 0.05), and the expression of TLR3 (P = 0.099) tended to be higher in the restricted group. However, protein levels of TLR2, TLR4, IκBα, phosphorylated IκBα, phosphorylated IκBα/total IκBα, TRIF and MyD88 were not affected (P > 0.05) by maternal intake restriction.ConclusionsThese results revealed that the restriction of maternal feed intake influenced the development of heart and hepatic protein synthesis at the acute phase of fetal goats and upregulated the mRNA expression of genes involved in MyD88-dependent signaling pathways and of target cytokines.

The Immunomodulatory Role of G2013 (α-L-Guluronic Acid) on the Expression of TLR2 and TLR4 in HT29 cell line.

The non-steroidal anti-inflammatory drugs (NSAIDs) play crucial role in the controlling of inflammatory diseases. Due to the vast side effects of NSAIDs, its use is limited. G2013 or α-L-Guluronic Acid is a new NSAID with immunomodulatory features. Considering the leading role of TLRs in inflammatory responses, in this study, we aimed to evaluate G2013 cytotoxicity and its effect on the expression of TLR2 and TLR4 molecules. HEK293-TLR2 and HEK293-TLR4 cells were cultured and seeded on 96-well cell plate, and MTT assay was performed for detecting the viability of the cells after treatment with different concentrations of G2013. HT29 cells were grown and treated with low and high doses of G2013. After total RNA extraction and cDNA synthesis, quantitative real-time PCR were performed to assess the TLR2 and TLR4 mRNA synthesis. We found that concentrations of ≤125 µg/ml of G2013 had no apparent cytotoxicity effect on the HEK293-TLR2 and -TLR4 cells. Our results indicated that after G2013 treatment (5 µg/ml) in HT29 cells, TLR2 and TLR4 mRNA expression decreased significantly compared with the untreated control group (p=0.02 and p=0.001 respectively). The results of this study revealed that G2013 can down regulate the TLR2 and TLR4 gene expression and exerts its inhibitory effect. Our findings are parallel to our previous finding which showed G2013 ability to down regulate the signaling pathway of TLRs. However, further studies are needed to identify the molecular mechanism of G2013.

Interferon-Beta Treatment Differentially Alters TLR2 and TLR4-Dependent Cytokine Production in Multiple Sclerosis Patients

Objective: Multiple sclerosis (MS) is a multifactorial chronic disease that affects the central nervous system (CNS). Toll-like receptors (TLRs) play a central role in cytokine production after pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) and contribute to CNS damage in MS patients. Here, we evaluated the effects of interferon (IFN)-β treatment in TLR2 and TLR4-dependent cytokine production and mRNA expression in whole-blood cell cultures from MS patients. Methods: We evaluated cytokine production by ELISA from whole-blood cell culture supernatants and mRNA expression by real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMCs). Results: In patients treated with IFN-β, tumor necrosis factor (TNF)-α production after exposure to TLR2 agonist (Pam₃Cys) was lower than in healthy controls and untreated MS patients. However, IFN-β treatment had no significant effect on TNF-α production after TLR4 agonist (LPS) stimulation. On the other hand, interleukin (IL)-10 production was increased in TLR4- but not in TLR2-stimulated whole-blood cell culture from MS patients under IFN-β treatment when compared to the controls. No differences in TNF-α or IL-10 mRNA expression in PBMCs from healthy controls and untreated or treated MS patients were detected, although PBMCs from treated patients presented higher levels of IL-32γ mRNA than those from controls. Conclusions: Our data suggest that IFN-β treatment alters the TLR-dependent immune response of PBMCs from MS patients. This may contribute to the beneficial effects of IFN-β treatment.

Kinetic studies on clinical and immunological modulations by intramuscular injection of Escherichia coli LPS in laying hens.

The present study investigated clinical and immunological modulations due to intramuscular injection of Escherichia coli LPS in 49-wk-old laying hens over 48 h post injection (p.i.). LPS induced characteristic sickness behavior but no significant body temperature alterations (P > 0.05). During experimental period decreases in blood albumin, calcium, phosphorus and tryptophan concentrations, hyperglycemia, increased plasma nitrite concentrations, leucopenia, decreased thrombocyte counts, lymphopenia, heterophilia and an increased heterophilic granulocyte/lymphocyte (H/L) ratio were observed after LPS administration. Time-dependent effects were shown on T and B cell subsets in caecal tonsils (CT) and on splenic CD3+/CD4+/CD8+ proportions, on IL-1β and -10 and inducible NO synthase mRNA expression in peripheral blood lymphocytes (PBL), liver, spleen and CT, and on the mRNA expression of the TLR4 in PBL, liver and spleen p.i. (P < 0.05). The main responding period of mentioned alterations due to LPS appears to include the period from 2 until 8 h p.i. According to the H/L ratio, the most stressful phase was 5 h p.i. T and B cell subsets in CT, the IL-1β and TLR4 mRNA expression in liver and plasma nitrite concentrations seemed to be affected for a longer period.

Innate immune response in patients with acute Zika virus infection.

Innate immunity receptors (Toll-like receptors/TLRs and RIG-like receptors/RLRs) are important for the initial recognition of Zika virus (ZIKV), modulation of protective immune response, and IFN-α and IFN-β production. Immunological mechanisms involved in protection or pathology during ZIKV infection have not yet been determined. In this study, we evaluated the mRNA expression of innate immune receptors (TLR3, TLR7, TLR8, TLR9, melanoma differentiation-associated protein 5/MDA-5, and retinoic acid inducible gene/RIG-1), its adapter molecules (Myeloid Differentiation Primary Response Gene 88/Myd88, Toll/IL-1 Receptor Domain-Containing Adaptor-Inducing IFN-β/TRIF), and cytokines (IL-6, IL-12, TNF-α, IFN-α, IFN-β, and IFN-γ) in the acute phase of patients infected by ZIKV using real-time PCR in peripheral blood. Patients with acute ZIKV infection had high expression of TLR3, IFN-α, IFN-β, and IFN-γ when compared to healthy controls. In addition, there was a positive correlation between TLR3 expression compared to IFN-α and IFN-β. Moreover, viral load is positively correlated with TLR8, RIG-1, MDA-5, IFN-α, and IFN-β. On the other hand, patients infected by ZIKV showed reduced expression of RIG-1, TLR8, Myd88, and TNF-α molecules, which are also involved in antiviral immunity. Similar expressions of TLR7, TLR9, MDA-5, TRIF, IL-6, and IL-12 were observed between the group of patients infected with ZIKV and control subjects. Our results indicate that acute infection (up to 5days after the onset of symptoms) by ZIKV in patients induces the high mRNA expression of TLR3 correlated to high expression of IFN-γ, IFN-α, and IFN-β, even though the high viral load is correlated to high expression of TLR8, RIG-1, MDA-5, IFN-α, and IFN-β in ZIKV patients.

Effect of silkworm peptide on inducting M1 type polarization and Th1 activation via TLR2-induced MyD88-dependent pathway.

The aim of this study was to explore immune activity and molecular mechanism of silkworm peptide. The cell subsets induced by silkworm peptides were detected by flow cytometry. The IFN‐γ and IL‐4 levels in CD4+ cells were measured by ELISA. TLR2 mRNA expression in mouse CD4+ T cells was detected by qRT‐PCR. Western blot was used to detect the protein expression levels of MyD88 and p‐IκB. The growth rate of Lewis lung cancer xenografts in mice of the medium‐dose group was significantly reduced, and the tumor volume was significantly smaller than that of the control group on the 14th day. The relative vitality values of spleen lymphocytes in the medium‐dose and high‐dose groups were higher than the control group. The IFN‐γ levels in the medium‐dose and high‐dose groups were significantly higher than the control group. The levels of IL‐4 were no significant change among different groups. Compared with the control group, different doses of silkworm peptide groups could increase the levels of NO, IL‐6, IL‐12, and IL‐1β. Compared with the control group, the protein expression levels of MyD88 and p‐IκB in 10 μg/ml group and 20 μg/ml groups were significantly increased compared with the control group. Silkworm peptide could induce Th1 activation and M1 type polarization, which was dose‐dependent and was relative to the effect of silkworm peptide on inhibiting tumor growth. Silkworm peptide could directly induce M1 type polarization and Th1 activation via TLR2‐induced MyD88‐dependent pathway in vitro.

RFX1 downregulation contributes to TLR4 overexpression in CD14+ monocytes via epigenetic mechanisms in coronary artery disease

BackgroundToll-like receptor 4 (TLR4) expression is increased in activated monocytes, which play a critical role in the pathogenesis of coronary artery disease (CAD). However, the mechanism remains unclear. Regulatory factor X1 (RFX1) is a critical transcription factor regulating epigenetic modifications. In this study, we investigated whether RFX1 and epigenetic modifications mediated by RFX1 contributed to the overexpression of TLR4 in activated monocytes.ResultsCompared with those of the controls, the mRNA and protein expression of RFX1 were downregulated and the mRNA expression of TLR4 was upregulated in CD14+ monocytes obtained from CAD patients and CD14+ monocytes obtained from healthy controls treated with low-density lipoprotein (LDL). The mRNA expression of RFX1 was negatively correlated with the mRNA expression of TLR4 in CD14+ monocytes. RFX1 knockdown led to the overexpression of TLR4 and the activation of CD14+ monocytes. In contrast, the overexpression of RFX1 inhibited TLR4 expression and the activation of CD14+ monocytes stimulated with LDL. Moreover, TLR4 was identified as a target gene of RFX1. The results indicated that RFX1 downregulation contributed to the decreased DNA methylation and histone H3 lysine 9 trimethylation and the increased H3 and H4 acetylation in the TLR4 promoter via the lack of recruitments of DNA methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone-lysine N-methyltransferase SUV39H1 (SUV39H1), which were observed in CD14+ monocytes of CAD patients.ConclusionsOur results show that RFX1 expression deficiency leads to the overexpression of TLR4 and the activation of CD14+ monocytes in CAD patients by regulating DNA methylation and histone modifications, which highlights the vital role of RFX1 in the pathogenesis of CAD.

Toll-like receptor 9, maternal cell-free DNA and myometrial cell response to CpG oligodeoxynucleotide stimulation.

Among mechanisms triggering onset of parturition, it has been recently postulated that Toll-Like Receptor (TLR)9 engagement by cell-free DNA (cfDNA) triggers inflammation, myometrial contractions, and labor in absence of infection. The current study evaluated whether direct (myometrial) or indirect (decidual) TLR9 engagement enhances human myometrial contractility. Toll-like receptor 9 expression and cellular localization were surveyed by immunohistochemistry of placenta, fetal membranes, and myometrium in term (gestational age [GA]: >37weeks) labor (TL, n=7) or term non-labor (TNL, n=7) tissues. Non-pregnant myometrium (n=4) served as reference. TLR9 mRNA expression relative to other TLRs was evaluated through the mining of an RNA-seq dataset and confirmed by RT-PCR. Immortalized human myometrial cells (hTERT-HM) were treated with incremental concentrations of TLR9 agonist ODN2395, TNF-α, or LPS. Secreted cytokines were quantified by multiplex immunoassay, and contractility was assessed by an in-gel cell contraction assay (n=9). Induction of hTERT-HM contractility was also evaluated indirectly following exposure to conditioned media from primary term decidual cells (n=4) previously stimulated with ODN2395. Toll-like receptor 9 immunostaining in placenta and amniochorion was strongest in decidual cells, but unrelated to labor. TLR9 staining intensity was significantly decreased in TL compared with TNL myometrium (P=0.002). Although total cfDNA in maternal circulation increased in TL (P=0.025 vs TNL), difference in cffDNA was non-significant. Myometrial TLR9 mRNA levels were unaffected by contractile status and far less abundant than other pro-inflammatory TLRs. hTERT-HM contractility was enhanced by LPS (P=0.002) and TNF-α (P=0.003), but not by ODN2395 (P=0.345) or supernatant of TLR9-stimulated decidual cells. Myometrial and decidual TLR9 are unlikely to directly regulate human parturition.

Understanding the hidden relations between pro- and anti-inflammatory cytokine genes in bovine oviduct epithelium using a multilayer response surface method

An understanding gene-gene interaction helps users to design the next experiments efficiently and (if applicable) to make a better decision of drugs application based on the different biological conditions of the patients. This study aimed to identify changes in the hidden relationships between pro- and anti-inflammatory cytokine genes in the bovine oviduct epithelial cells (BOECs) under various experimental conditions using a multilayer response surface method. It was noted that under physiological conditions (BOECs with sperm or sex hormones, such as ovarian sex steroids and LH), the mRNA expressions of IL10, IL1B, TNFA, TLR4, and TNFA were associated with IL1B, TNFA, TLR4, IL4, and IL10, respectively. Under pathophysiological + physiological conditions (BOECs with lipopolysaccharide + hormones, alpha-1-acid glycoprotein + hormones, zearalenone + hormones, or urea + hormones), the relationship among genes was changed. For example, the expression of IL10 and TNFA was associated with (IL1B, TNFA, or IL4) and TLR4 expression, respectively. Furthermore, under physiological conditions, the co-expression of IL10 + TNFA, TLR4 + IL4, TNFA + IL4, TNFA + IL4, or IL10 + IL1B and under pathophysiological + physiological conditions, the co-expression of IL10 + IL4, IL4 + IL10, TNFA + IL10, TNFA + TLR4, or IL10 + IL1B were associated with IL1B, TNFA, TLR4, IL10, or IL4 expression, respectively. Collectively, the relationships between pro- and anti-inflammatory cytokine genes can be changed with respect to the presence/absence of toxins, sex hormones, sperm, and co-expression of other gene pairs in BOECs, suggesting that considerable cautions are needed in interpreting the results obtained from such narrowly focused in vitro studies.

Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients – A pilot study

The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.

The functional impact of G protein-coupled receptor 142 (Gpr142) on pancreatic \u03b2-cell in rodent

We have recently shown that the G protein-coupled receptor 142 (GPR142) is expressed in both rodent and human pancreatic β-cells. Herein, we investigated the cellular distribution of GPR142 within islets and the effects of selective agonists of GPR142 on glucose-stimulated insulin secretion (GSIS) in the mouse islets and INS-1832/13 cells. Double-immunostaining revealed that GPR142 immunoreactivity in islets mainly occurs in insulin-positive cells. Potentiation of GSIS by GPR142 activation was accompanied by increased cAMP content in INS-1832/13 cells. PKA/Epac inhibition markedly suppressed the effect of GPR142 activation on insulin release. Gpr142 knockdown (Gpr142-KD) in islets was accompanied by elevated release of MCP-1, IFNγ, and TNFα during culture period and abolished the modulatory effect of GPR142 activation on the GSIS. Gpr142-KD had no effect on Ffar1, Ffar2, or Ffar3 mRNA while reducing Gpr56 and increasing Tlr5 and Tlr7 mRNA expression. Gpr142-KD was associated with an increased expression of Chrebp, Txnip, RhoA, and mitochondrial Vdac1 concomitant with a reduced Pdx1, Pax6, and mitochondrial Vdac2 mRNA levels. Long-term exposure of INS-1832/13 cells to hyperglycemia reduced Gpr142 and Vdac2 while increased Chrebp, Txnip, and Vdac1 mRNA expression. GPR142 agonists or Bt2-cAMP counteracted this effect. Glucotoxicity-induced decrease of cell viability in Gpr142-KD INS-1 cells was not affected by GPR142-agonists while Bt2-cAMP prevented it. The results show the importance of Gpr142 in the maintenance of pancreatic β-cell function in rodents and that GPR142 agonists potentiate GSIS by an action, which most likely is due to increased cellular generation of second messenger molecule cAMP.

Induced expression of Toll-like receptor 9 in peritubular capillary endothelium correlates with the progression of tubulointerstitial lesions in autoimmune disease-prone mice.

Toll-like receptor (Tlr) 9 is capable of recognizing exogenous and/or endogenous nucleic acids and plays a crucial role in innate and adaptive immunity. Recently, we showed that Tlr9 is overexpressed in podocytes, a component of the blood-urine barrier (BUB), in glomeruli of autoimmune glomerulonephritis (AGN) model mice. This study investigated the activation of peritubular capillary (PTC) endothelial cells (ECs), a component of the BUB in the tubulointerstitium, through overexpressing Tlr9, and the subsequent development of tubulointerstitial lesions (TILs) in AGN model mice. Lupus-prone BXSB/MpJ-Yaa (Yaa) and BXSB/MpJ (BXSB) mice were used as an AGN model and control, respectively. In addition to histopathological and ultrastructural techniques, protein and mRNA levels were also evaluated. The relationship between Tlr9 and TIL indices was analyzed by statistical correlation analysis. Yaa mice developed TILs and showed strong Tlr9 mRNA expression in PTC ECs at 24 weeks (wks) of age. However, BXSB mice showed no TIL but faint expression of Tlr9 mRNA at 8 and 24 wks of age. Tlr9 protein localization on PTC was almost absent in BXSB mice at both ages but intense expression was found in Yaa mice only at 24 wks of age. Relative mRNA expression of Tlr9 and its putative downstream cytokines, including interleukin 1 beta ( Il1b), Il6, interferon gamma ( Ifng), and tumor necrosis factor alpha ( Tnf) was markedly increased in isolated tubulointerstitium from Yaa mice at 24 wks of age. Furthermore, electron microscopy examination revealed PTC injury and TIL in Yaa mice at 24 wks. The expression level of Tlr9 in the tubulointerstitium was correlated with inflammatory cells in TILs, injured PTC, Ilb and Tnf expression, and damaged tubules ( P < 0.05 and 0.01). Induced expression of Tlr9 in ECs correlates with PTC injury and the development of TILs in lupus-prone AGN model mice.

Metabolic syndrome mediates proinflammatory responses of inflammatory cells in preeclampsia.

To investigate whether metabolic syndrome (MetS) is associated with exacerbation of inflammatory responses in preeclamptic (PE) patients, the dynamic changes of Th17 and Treg cells, monocytes, cytokines, and transcription pattern of inflammasome-related genes were analyzed in 35 women with PE suffering from MetS in comparison to 38 PE women without MetS and healthy pregnant women. Expression of inflammasome-related genes, cytokines, and also TLR4 was measured using real-time PCR. Serum and medium supernatant cytokines levels of PBMCs and serum levels of HMGB1 and Caspase-1 were also evaluated by ELISA. Monocytes, Th17, and Treg cells frequency were also determined by flow cytometry. PE women with MetS exhibited increased percentage of non-classical and intermediate monocytes and Th17 cells (P=0.025). Furthermore, decreased Treg cells frequency was also observed in PE women with MetS compared to PE women (P=0.019). The mRNA expression of inflammasome-related genes (Caspase-1, NLRP3, HMGB1), TLR4, IL-1β, IL-6, IL-17, IL-18, and TNF-α was significantly higher in PE patients with MetS than that of the healthy pregnant individuals (P<0.0001) and PE patients (P<0.0001). Serum levels of TGF-β and TNF-α in PE patients with MetS were increased compared to other two groups, while IL-10 levels were significantly reduced. A significant sFlt (P=0.016), Caspase-1 (P=0.012), HMGB1 (P=0.016) upregulation, and VEGF (P=0.023) downregulation were also observed in the serum of PE women having MetS compared to PE women. MetS is closely related to the exacerbation of inflammatory reactions in PE. This study indicates that, in order to diminish the systemic features of PE, prior to conceive and start a pregnancy, MetS should be severely considered and managed.

Abstract # 3170 The TLR2/4 antagonist (+)-naltrexone blocks contextual long-term memory deficits in experimental autoimmune encephalitis and associated neuroinflammation in hippocampus

Approximately half of individuals suffering from multiple sclerosis (MS) express cognitive deficits in the form of memory impairments. Previously we have shown that administration of toll-like 2/4 (TLR2/4) antagonists such as (+)-naltrexone [(+)-NTX] block neuropathic pain and associated spinal inflammation in rats. In this study, we examined the effects of (+)-NTX on memory impairments and associated brain inflammation in a rat model of MS, experimental autoimmune encephalomyelitis (EAE). Male Dark Agouti rats were induced with EAE and 14 days later received subcutaneous (+)-NTX or saline for 14 days. Contextual- and auditory-fear conditioning were then conducted to assess memory impairments one week after beginning (+)-NTX administration. We found that EAE induced impairments in long-term contextual fear memory but not short-term contextual- or auditory-related fear memory. This effect was associated with increased interleukin-1 β (IL-1 β ) mRNA expression in the hippocampus but not the amygdala. Importantly, (+)-NTX blocked EAE-induced impairments in long-term contextual fear memory and the associated increased hippocampal IL-1 β expression. Moreover, (+)-NTX blocked hippocampal expression of TLR2, TLR4, NLRP3, and IL-17 mRNA, suggesting blockade of the TLR2/4, NLRP3 inflammasome and Th17 cell signaling as additional mechanisms by which (+)-NTX exerts its therapeutic effects. These findings support the role of TLR2/4 antagonists as potential treatments for MS-related cognitive deficits and encourage future research on TLR2/4 antagonists as treatments for various symptoms associated with inflammatory-related diseases, including MS.

The effect of different treatments of lymph after intestinal ischemia-reperfusion in rats on macrophages in vitro.

BackgroundTo observe the effects of different treatments of lymph after intestinal I/R in rats on macrophages in vitro.MethodsForty-eight healthy SPF SD rats weighing 300 ± 20 g, were randomly divided into two groups: group A, and group B. The rats in group A were drained of lymph fluid for 180 min; the rats in group B were subjected to 60 min ischemia by clamping the SMA, followed by 120 min reperfusion and 180 min of lymph drainage. The lymph fluid collected was divided into 4 sub-groups: 1. no treatment (A1, Ly, and B1, I/R Ly); 2. protein degradation (A2, Ly PD, and B2 I/R PD); 3. endotoxin removal (A3, Ly ER, and B3, I/R ER); 4. protein degradation plus endotoxin removal (A4, Ly PD+ER, and B4, I/R PD+ER), then used to stimulate a monocyte-macrophage cell line.ResultsCompared with group A1, the levels of the inflammatory cytokines, chemokines, HMGB1 concentration, protein and mRNA expression of TLR4, HMGB1 and NF-κBp65 were significantly increased in group B1. There was a significant reduction in proinflammatory cytokines and of the expression of TLR4, NF-κBp65, and chemokines in groups A2, B2, A4, and B4. However, there were no significant decrease of these factors in groups A3 and B3.ConclusionsThe lymph fluid drained after intestinal I/R can cause inflammation in vivo and in vitro. Deproteinization of lymph fluid with proteinase K significantly reduced the concentration of proinflammatory cytokines, chemokines, TLR4 and NF-κBp65 in cell culture supernatant, exerting a protective effect on inflammatory reaction caused by the intestinal I/R. Passage of lymph fluid through an endotoxin removal column did not reduce the levels of active proinflammatory factors produced by macrophages in vitro.

Pro-inflammatory cytokine expression studies of subclinical and clinical endometritis in endometrial tissues of buffaloes.

The objective of the study was to investigate the role of pro-inflammatory cytokines in the pathogenesis of endometritis in buffaloes, which can lead to early diagnosis of endometritis. The expression of CD14, TLR-4, and IL-1β encoding gene in endometrium of buffaloes with subclinical endometritis were 1.87, 2.71, and 3.48-fold, while samples with clinical endometritis showed only 2.90, 4.76, and 8.02-fold for CD14, TLR-4, and IL-1β, respectively, though numerical value of expression was higher in subclinical and clinical endometritis but it was at par with control. The expression profile for IL-6, IL-8, and TNF-α mRNA for subclinical endometritis were 11.95, 14.87, and 12.95-fold while for clinical endometritis, values were 18.17, 37.97, and 28.83-fold for IL-6, IL-8, and TNF-α mRNA, respectively, which were significantly higher in subclinical and clinical endometritis as compared to apparently normal samples at follicular phase. In the luteal group, the subclinical endometritis sample showed 2.13, 6.43, 14.52, 22.38, 18.64, and 2.82-fold respectively, for CD14, IL-1β IL-6, IL-8, TNF-α, and TLR-4 mRNA. Except TLR-4, the values of expression were significantly higher when compared with apparently normal. Whereas, the expression values of clinical endometritic uteri were 4.01, 7.15, 17.20, 45.55, 52.58, and 32.95-fold respectively, for CD14, TLR-4, IL-1β, IL-6, IL-8, and TNF-α mRNA which were significantly higher as compared to apparently normal uteri. An increase in the mRNA expression of CD14, TLR-4, and pro-inflammatory cytokines having higher diagnostic importance in uterine inflammation.