Ventricular TLR4 Levels Abrogate TLR2-Knockout-Mediated Adverse Cardiac Remodeling upon Pressure Overload in Mice

Involvement of the Toll-like receptor 4 (TLR4) in maladaptive cardiac remodeling and heart failure (HF) upon pressure overload has been studied extensively, but less is known about the role of TLR2. Interplay and redundancy of TLR4 with TLR2 have been reported in other organs but were not investigated during cardiac dysfunction. We explored whether TLR2 deficiency leads to less adverse cardiac remodeling upon chronic pressure overload and whether TLR2 and TLR4 additively contribute to this. We subjected 35 male C57BL/6J mice (wildtype (WT) or TLR2 knockout (KO)) to sham or transverse aortic constriction (TAC) surgery. After 12 weeks, echocardiography and electrocardiography were performed, and hearts were extracted for molecular and histological analysis. TLR2 deficiency (n = 14) was confirmed in all KO mice by PCR and resulted in less hypertrophy (heart weight to tibia length ratio (HW/TL), smaller cross-sectional cardiomyocyte area and decreased brain natriuretic peptide (BNP) mRNA expression, p < 0.05), increased contractility (QRS and QTc, p < 0.05), and less inflammation (e.g., interleukins 6 and 1β, p < 0.05) after TAC compared to WT animals (n = 11). Even though TLR2 KO TAC animals presented with lower levels of ventricular TLR4 mRNA than WT TAC animals (13.2 ± 0.8 vs. 16.6 ± 0.7 mg/mm, p < 0.01), TLR4 mRNA expression was increased in animals with the largest ventricular mass, highest hypertrophy, and lowest ejection fraction, leading to two distinct groups of TLR2 KO TAC animals with variations in cardiac remodeling. This variation, however, was not seen in WT TAC animals even though heart weight/tibia length correlated with expression of TLR4 in these animals (r = 0.078, p = 0.005). Our data suggest that TLR2 deficiency exacerbates adverse cardiac remodeling and that ventricular TLR2 and TLR4 additively contribute to adverse cardiac remodeling during chronic pressure overload. Therefore, both TLRs may be therapeutic targets to prevent or interfere in the underlying molecular processes.

Protective Effect of Ulinastatin on Acute Lung Injury in Diabetic Sepsis Rats

ObjectiveTo establish a rat model of diabetic sepsis, to observe the protective effect of ulinastatin on acute lung injury in diabetic sepsis rats, and to explore the possible mechanism from the aspects of inflammatory response, oxidative stress, hypoxia-inducible factor-1 α expression and pulmonary microvascular permeability. MethodsA total of 50 SPF adult male SD rats were randomly selected, 38 were fed with high fat diet, and the remaining 12 were fed with ordinary diet. Feed five weeks later, the fatty group according to 30 mg/kg body weight, abdominal single injection of STZ, three days after injection, in a random blood glucose or greater tendency for 16.7 L for type 2 diabetes mellitus rats building success, take 36 ChengMo rats were randomly divided into four groups, each group of 12, namely D group of type 2 diabetes, the DS group of type 2 diabetes mellitus and sepsis, Group U ulinastatin pretreatment group. Twelve ordinary feed group were selected as C group, namely blank control group. After successful modeling of type 2 diabetic rats, DS group and U group were injected with endotoxin (LPS) at 5mg/kg via tail vein to construct diabetic sepsis lung injury rat model. Group U was injected with ulinastatin 100kU/kg caudal vein one hour before LPS. 4h later (4 rats in each group were injected with 2 mL 1% Evans blue solution through tail vein half an hour before execution), blood was collected and lung tissues were removed. HE staining was used to observe the pathological changes of lung tissues. The wet/dry ratio (W/D) of lung tissue was determined. Serum IL-1β, IL-18 and TNF-A were detected by ELISA. The contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were detected according to the kit instruction. Western blot was used to detect the hypoxia-inducible factor-1 α (HIF-1α) protein in renal tissue. The expression of TLR4mRNA in lung tissues was detected by rt-pcr. Evans blue staining was used to detect pulmonary microvascular permeability. ResultsCompared with the control group, the lung interstitium in group D was thickened to a certain extent, with a small amount of inflammatory cell infiltration and a little exudate in some alveolar cavities. Compared with D group, DS group had more serious damage, with obvious pulmonary interstitial hyperemia and edema, a large amount of exudation in alveolar cavity, significantly increased inflammatory cells, necrosis and swelling of alveolar epithelial cells. In group U, there were more inflammatory cells in the interstitium and part of alveolar cavities, epithelial cells were exfoliated occasionally in the lumen, and the interstitium widened and the loss was less than that in group DS. Compared with group C, the wet/dry weight ratio (W/D), serum IL-1β, IL-18 and TNF-A contents, hiF-1 α protein expression, TLR4 mRNA expression and pulmonary microvascular permeability of rats in other groups were significantly increased (P<0.01 or P<0.05). The content of MDA and SOD activity in serum decreased (P<0.01). Compared with group D, the wet/dry weight ratio (W/D), serum IL-1β, IL-18 and TNF-A contents, hiF-1 α protein expression, TLR4 mRNA expression and pulmonary microvascular permeability in DS group and U group were significantly increased (P<0.01 or P<0.05). The content of MDA in serum was increased (P<0.01 or P<0.05), and the activity of SOD in DS group was decreased (P<0.01). ; Compared with DS group, the wet/dry weight ratio (W/D), serum IL-1β, IL-18, TNF-A contents, hiF-1 α protein expression, TLR4 mRNA expression, and pulmonary microvascular permeability of rats in GROUP U were significantly decreased (P < 0.01 or P<0.05). The content of MDA in serum decreased (P<0.01), and the activity of SOD increased (P<0.01). ConclusionUlinastatin can effectively reduce acute lung injury induced by diabetic sepsis in rats, and its mechanism may be related to inhibiting inflammatory response, reducing oxidative stress, regulating hypoxia response pathway and improving pulmonary microvascular permeability.

Fibronectin Extra Domain A (FN-EDA) Causes Glaucomatous Trabecular Meshwork, Retina, and Optic Nerve Damage in Mice

Background: Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage. Here, we investigate the role of an endogenous Toll-like receptor 4 (TLR4) ligand, FN-EDA, in the development of glaucoma utilizing a transgenic mouse strain (B6.EDA +/+ ) that constitutively expresses only FN containing the EDA isoform. Methods: Eyes from C57BL6/J (wild-type), B6.EDA +/+ (constitutively active EDA), B6.EDA -/- (EDA null) mice were processed for electron microscopy and consecutive images of the entire length of the TM and Schlemm’s canal (SC) from anterior to posterior were collected and montaged into a single image. ECM accumulation, basement membrane length, and size and number of giant vacuoles were quantified by ImageJ analysis. Tlr4 and Iba1 expression in the TM and ONH cells was conducted using RNAscope in situ hybridization and immunohistochemistry protocols. IOP was measured using a rebound tonometer, ON damage assessed by PPD stain, and RGC loss quantified in RBPMS labeled retina flat mounts. Results: Ultrastructure analyses show the TM of B6.EDA +/+ mice have significantly increased accumulation of ECM between TM beams with few empty spaces compared to C57BL/6J mice (P<0.05). SC basement membrane is thicker and more continuous in B6.EDA +/+ mice compared to C57BL/6J. No significant structural differences are detected in the TM of EDA null mice. Tlr4 and Iba1 expression is increased in the TM of B6.EDA +/+ mice compared to C57BL/6J eyes (p<0.05). IOP is significantly higher in B6.EDA +/+ mice compared to C57BL/6J eyes (p<0.001), and significant ON damage (p<0.001) and RGC loss (p<0.05) detected at one year of age. Tlr4 mRNA is expressed in mouse ONH cells, and is present in ganglion cell axons, microglia, and astrocytes. There is a significant increase in the area occupied by Iba-1 positive microglia cells in the ONH of B6.EDA +/+ mice compared to C57BL/6J control eyes (p<0.01). Conclusions: B6.EDA +/+ mice have increased ECM accumulation in the TM, elevated IOP, enhanced proinflammatory changes in the ONH, loss of RGCs, and ONH damage. These data suggest B6.EDA +/+ mice recapitulate many aspects of glaucomatous damage.

High Mobility Group Box 1 in Pig Amniotic Membrane Experimentally Infected with E. coli O55

Intra-amniotic infections (IAI) are one of the reasons for preterm birth. High mobility group box 1 (HMGB1) is a nuclear protein with various physiological functions, including tissue healing. Its excessive extracellular release potentiates inflammatory reaction and can revert its action from beneficial to detrimental. We infected the amniotic fluid of a pig on the 80th day of gestation with 1 × 104 colony forming units (CFUs) of E. coli O55 for 10 h, and evaluated the appearance of HMGB1, receptor for glycation endproducts (RAGE), and Toll-like receptor (TLR) 4 in the amniotic membrane and fluid. Sham-infected amniotic fluid served as a control. The expression and release of HMGB1 were evaluated by Real-Time PCR, immunofluorescence, immunohistochemistry, and ELISA. The infection downregulated HMGB1 mRNA expression in the amniotic membrane, changed the distribution of HMGB1 protein in the amniotic membrane, and increased its level in amniotic fluid. All RAGE mRNA, protein expression in the amniotic membrane, and soluble RAGE level in the amniotic fluid were downregulated. TLR4 mRNA and protein expression and soluble TLR4 were all upregulated. HMGB1 is a potential target for therapy to suppress the exaggerated inflammatory response. This controlled expression and release can, in some cases, prevent the preterm birth of vulnerable infants. Studies on suitable animal models can contribute to the development of appropriate therapy.

H3K4me3 Histone ChIP-Seq Analysis Reveals Molecular Mechanisms Responsible for Neutrophil Dysfunction in HIV-Infected Individuals

Peripheral neutrophils in HIV-infected individuals are characterized by impairment of chemotaxis, phagocytosis, bactericidal activity, and oxidative burst ability regardless of whether patients are receiving antiretroviral therapy or not. Neutrophil dysfunction leads not only to increased susceptibility to opportunistic infections but also to tissue damage through the release of reactive oxygen species (ROS), proteases, and other potentially harmful effector molecules contributing to AIDS progression. In this study, we demonstrated high levels of histone H3 lysine K4 trimethylated (H3K4me3) and dysregulation of DNA transcription in circulating neutrophils of HIV-infected subjects. This dysregulation was accompanied by a deficient response of neutrophils to LPS, impaired cytokine/chemokine/growth factor synthesis, and increased apoptosis. Chromatin immunoprecipitation sequencing (ChIPseq) H3K4me3 histone analysis revealed that the most spectacular abnormalities were observed in the exons, introns, and promoter-TSS regions. Bioinformatic analysis of Gene Ontology, including biological processes, molecular function, and cellular components, demonstrated that the main changes were related to the genes responsible for cell activation, cytokine production, adhesive molecule expression, histone remodeling via upregulation of methyltransferase process, and downregulation of NF-κB transcription factor in canonical pathways. Abnormalities within H3K4me3 implicated LPS-mediated NF-κB canonical activation pathway that was a result of low amounts of κB DNA sites within histone H3K4me3, low NF-κB (p65 RelA) and TLR4 mRNA expression, and reduced free NF-κB (p65 RelA) accumulation in the nucleus. Genome-wide survey of H3K4me3 provided evidence that chromatin modifications lead to an impairment within the canonical NF-κB cell activation pathway causing the neutrophil dysfunction observed in HIV-infected individuals.

Paroxetine effect on expression of TLR2 and TLR4 genes in PBMCs of mentally normal individuals

Background: Paroxetine is one of the well-known antidepressants. Recent studies have focused on paroxetine’s probable immuno-modulatory effects, since findings have indicated inflammation’s role in the pathophysiology of depression. Therefore, in the present study, TLR2 and TLR4 mRNA genes expression was assessed in paroxetine-treated peripheral blood mononuclear cells (PBMCs). Methods: Venous blood samples were drawn from five healthy men (20-40 years old). Peripheral blood mononuclear cells (PBMCs) were isolated from samples and were cultured. After the first incubation for 24h, phytohemagglutinin plus lipopolysaccharide were added to the cells and then were incubated for 24h. Thereafter, cells were treated with different concentrations of paroxetine in the presence or absence of inhibitors of 5-HT2 and 5-HT7 receptors. After incubation for 48h, RNA was extracted and cDNA was synthesized. Using the real-Time PCR technique, TLR2 and TLR4 genes mRNA expression were evaluated. Statistical analysis of data were carried out using GraphPad Prism 7. Results: TLR2 and TLR4 mRNA expression were significantly increased in response to paroxetine at all concentrations. Furthermore, the co-culture of cells with the drug and the 5-HT2R and 5HT7R inhibitor simultaneously revealed that paroxetine’s immuno-modulatory effects viaTLR2 are dependent on serotonin, while it is independent of serotonin in the case of TLR4. Conclusion: Considering paroxetine’s effect in modulating immune responses via increasing TLR2 and TLR4 expression, paroxetine could have therapeutic potentials in diseases with a deficiency in these receptors.

Type I Interferon Dependent hsa-miR-145-5p Downregulation Modulates MUC1 and TLR4 Overexpression in Salivary Glands From Sjögren’s Syndrome Patients

Sjögren’s syndrome (SS) is an autoimmune disease that mainly affects salivary glands (SG) and is characterized by overactivation of the type I interferon (IFN) pathway. Type I IFNs can decrease the levels of hsa-miR-145-5p, a miRNA with anti-inflammatory roles that is downregulated in SG from SS-patients. Two relevant targets of hsa-miR-145-5p, mucin 1 (MUC1) and toll-like receptor 4 (TLR4) are overexpressed in SS-patients and contribute to SG inflammation and dysfunction. This study aimed to evaluate if hsa-miR-145-5p modulates MUC1 and TLR4 overexpression in SG from SS-patients in a type I IFN dependent manner. Labial SG (LSG) biopsies from 9 SS-patients and 6 controls were analyzed. We determined hsa-miR-145-5p levels by TaqMan assays and the mRNA levels of MUC1, TLR4, IFN-α, IFN-β, and IFN-stimulated genes (MX1, IFIT1, IFI44, and IFI44L) by real time-PCR. We also performed in vitro assays using type I IFNs and chemically synthesized hsa-miR-145-5p mimics and inhibitors. We validated the decreased hsa-miR-145-5p levels in LSG from SS-patients, which inversely correlated with the type I IFN score, mRNA levels of IFN-β, MUC1, TLR4, and clinical parameters of SS-patients (Ro/La autoantibodies and focus score). IFN-α or IFN-β stimulation downregulated hsa-miR-145-5p and increased MUC1 and TLR4 mRNA levels. Hsa-miR-145-5p overexpression decreased MUC1 and TLR4 mRNA levels, while transfection with a hsa-miR-145-5p inhibitor increased mRNA levels. Our findings show that type I IFNs decrease hsa-miR-145-5p expression leading to upregulation of MUC1 and TLR4. Together, this suggests that type I interferon-dependent hsa-miR-145-5p downregulation contributes to the perpetuation of inflammation in LSG from SS-patients.

Serum Amyloid A1/Toll-Like Receptor-4 Axis, an Important Link between Inflammation and Outcome of TBI Patients

Traumatic brain injury (TBI) is one of the leading causes of mortality and disability worldwide without any validated biomarker or set of biomarkers to help the diagnosis and evaluation of the evolution/prognosis of TBI patients. To achieve this aim, a deeper knowledge of the biochemical and pathophysiological processes triggered after the trauma is essential. Here, we identified the serum amyloid A1 protein-Toll-like receptor 4 (SAA1-TLR4) axis as an important link between inflammation and the outcome of TBI patients. Using serum and mRNA from white blood cells (WBC) of TBI patients, we found a positive correlation between serum SAA1 levels and injury severity, as well as with the 6-month outcome of TBI patients. SAA1 levels also correlate with the presence of TLR4 mRNA in WBC. In vitro, we found that SAA1 contributes to inflammation via TLR4 activation that releases inflammatory cytokines, which in turn increases SAA1 levels, establishing a positive proinflammatory loop. In vivo, post-TBI treatment with the TLR4-antagonist TAK242 reduces SAA1 levels, improves neurobehavioral outcome, and prevents blood–brain barrier disruption. Our data support further evaluation of (i) post-TBI treatment in the presence of TLR4 inhibition for limiting TBI-induced damage and (ii) SAA1-TLR4 as a biomarker of injury progression in TBI patients.

POS0180 TYPE I INTERFERON DEPENDENT HSA-MIR-145-5P DOWNREGULATION MODULATES MUC1 AND TLR4 OVEREXPRESSION IN SALIVARY GLANDS FROM PRIMARY SJÖGREN’S SYNDROME PATIENTS

Background:Primary Sjögren’s syndrome (pSS) is an autoimmune and inflammatory disease that mainly affects the salivary glands (SG) and is characterized by an overactivation of the type I interferon pathway (IFNs I). IFNs I are known to regulate the levels of microRNAs (miRNAs), non-coding RNAs whose levels are altered in pSS. IFNs I can decrease the levels of miR-145-5p, a miRNA with anti-inflammatory roles that has been reported downregulated in SG of from pSS patients [1]. This miRNA has TLR4 and MUC1 transcripts as predicted targets. MUC1 and TLR4 are two proteins overexpressed in SG of pSS patients that contribute, through various mechanisms, to the inflammatory state and glandular dysfunction [2-3]. Thus, we propose that IFNs I may contribute to a self-perpetuating inflammation loop through a hsa-miR-145-5p dependent MUC1 and TLR4 overexpression in the SG of pSS patients.Objectives:to evaluate whether mRNA levels of MUC1 and TLR4 are modulated by hsa-miR-145-5p in a IFNs I dependent manner.Methods:13 pSS patients and 9 controls SG biopsies were analyzed. hsa-miR-145-5p levels were determined by TaqMan assays and MUC1, TLR4, IFN-α and IFN-β mRNA levels by RT-qPCR. Additionally, in vitro assays using type I IFNs and chemically synthesized hsa-miR-145-5p mimics and inhibitors were performed to study its effect on MUC1 and TLR4 expression. JAK1 and STAT1 mRNA levels were also measured.Results:By Taqman assays we validated the decreased hsa-miR-145-5p levels (p=0.0001) in SG of pSS patients compared to controls. The decreased hsa-miR-145-5p levels correlated inversely with the increased mRNA levels of IFN-β (p=0.0192) in SG of pSS-patients. The hsa-miR-145-5p downregulation also correlated inversely with the overexpression of its predicted targets MUC1 (p=0.010) and TLR4 (p=0.0004). In vitro assays showed that IFN-β induces the overexpression of JAK1 (<p=0.0001) and STAT1 (p=<0.0001) leading to the downregulation of hsa-mir-145-5p (p=<0.0001) and increased MUC1 (p=<0.0001) and TLR4 mRNA levels (p=<0.0001). Functional assays suggest a regulation of hsa-miR-145-5p on MUC1 and TLR4 expression as MUC1 and TLR4 mRNA levels were decreased in HSG cells transfected with hsa-miR-513c-3p mimic and increased in HSG cells transfected with the miRNA inhibitor.Conclusion:Our findings suggest that IFNs I could induce the downregulation of hsa-miR-145-5p leading to the overexpression of MUC1 and TLR4 in SG from pSS patients. TLR4 is activated by ectopic mucins in the SG extracellular matrix from pSS patients which induces pro-inflammatory cytokines secretion [3]. Furthermore, the high levels of the MUC1-SEC and MUC1-Y isoforms observed in SG from pSS patients may favor cytokine synthesis through the immuno-enhancing peptide of MUC1-SEC or through the formation of a MUC1-SEC/MUC1-Y complex [4]. Therefore, IFNs I may contribute to the development of SS through amplification and perpetuation of inflammation due to a hsa-miR-145-5p dependent MUC1 and TLR4 overexpression.References:[1]I. Alevizos, et al,. Arthritis Rheum, 2011;63:535-44.[2]HH. Sung, et al,. Oral Dis. 2015;21(6):730-8.[3]MJ. Barrera, et al,. Rheumatology (Oxford). 2015;54(8):1518-27[4]LM. Herbert, et al,. Cancer Res. 2004;64(21):8077-84.Acknowledgements:Fondecyt 1210055, Fondecyt 1160015, Fondecyt Iniciación 11170049, Fondecyt Iniciación 11201058, CONICYT fellowship (DJ, PC)Disclosure of Interests:None declared