Activation Of Inflammatory Genes Research Articles

Trauma-associated extracellular histones mediate inflammation via a MYD88-IRAK1-ERK signaling axis and induce lytic cell death in human adipocytes

Despite advances in the treatment and care of severe physical injuries, trauma remains one of the main reasons for disability-adjusted life years worldwide. Trauma patients often suffer from disturbances in energy utilization and metabolic dysfunction, including hyperglycemia and increased insulin resistance. White adipose tissue plays an essential role in the regulation of energy homeostasis and is frequently implicated in traumatic injury due to its ubiquitous body distribution but remains poorly studied. Initial triggers of the trauma response are mainly damage-associated molecular patterns (DAMPs) such as histones. We hypothesized that DAMP-induced adipose tissue inflammation contributes to metabolic dysfunction in trauma patients. Therefore, we investigated whether histone release during traumatic injury affects adipose tissue. Making use of a murine polytrauma model with hemorrhagic shock, we found increased serum levels of histones accompanied by an inflammatory response in white adipose tissue. In vitro, extracellular histones induced an inflammatory response in human adipocytes. On the molecular level, this inflammatory response was mediated via a MYD88-IRAK1-ERK signaling axis as demonstrated by pharmacological and genetic inhibition. Histones also induced lytic cell death executed independently of caspases and RIPK1 activity. Importantly, we detected increased histone levels in the bloodstream of patients after polytrauma. Such patients might benefit from a therapy consisting of activated protein C and the FDA-approved ERK inhibitor trametinib, as this combination effectively prevented histone-mediated effects on both, inflammatory gene activation and cell death in adipocytes. Preventing adipose tissue inflammation and adipocyte death in patients with polytrauma could help minimize posttraumatic metabolic dysfunction.

Strain-Specific Benefits of Bacillus on Growth, Intestinal Health, Immune Modulation, and Ammonia-Nitrogen Stress Resilience in Hybrid Grouper.

In the dynamic field of intensive aquaculture, the strategic application of probiotics has become increasingly crucial, particularly for enhancing resistance to environmental stressors such as ammonia-nitrogen. Over a 42-day period, this study investigated the effects of different probiotic strains-Bacillus subtilis (BS, 6-3-1, and HAINUP40)-on the health and resilience of hybrid groupers. Each strain, distinct in its origin, was assessed for its influence on growth performance, antioxidant capacity, immune gene expressions, and ammonia-nitrogen stress response in the hybrid grouper. The experimental design included a control group and three experimental groups, each supplemented with 1 × 108 CFU/g of the respective probiotic strains, respectively. Our results demonstrated notable differences in growth parameters, including final body weight (FBW) and feed conversion ratio (FCR). The 6-3-1 strain, originating from grouper, exhibited significant improvements in growth, oxidative capacity, and intestinal health. Conversely, the BS strain achieved the highest survival rates under ammonia-nitrogen stress, indicating its superior ability to regulate inflammatory responses despite its less pronounced growth-promoting effects. The HAINUP40 strain was distinguished for its growth enhancement and improvements in intestinal health, though it also showed significant activation of inflammatory genes and decreased resistance to ammonia-nitrogen stress after extended feeding. The uniqueness of this study lies in its detailed examination of the strain-specific effects of probiotics on fish in the context of ammonia-nitrogen stress, a significant challenge in contemporary aquaculture. The research revealed that host-derived probiotics, particularly the 6-3-1 strain, provided more comprehensive benefits for growth performance and stress resilience. In contrast, the BS and HAINUP40 strains exhibited varying efficiencies, with BS excelling in stress resistance and HAINUP40 promoting growth and gut health. In conclusion, this study underscores the complex roles of different probiotic strains in aquaculture, contributing to the understanding of probiotic applications and presenting new approaches to address the challenges of intensive farming.

Characterization of the cachexia pathway in pancreatic ductal adenocarcinoma.

699 Background: Cancer cachexia is characterized by progressive weight loss and skeletal muscle degradation, contributing to 33% of pancreatic ductal adenocarcinoma (PDAC) deaths. Targeting myostatin in the myostatin-activin pathway has been shown to reverse cachexia. Here, we present comprehensive clinical and molecular characterization of the myostatin-activin pathway in PDAC. Methods: 9,607 samples of PDAC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Cachexia gene scores (GS) were calculated by averaging the positive z scores of activators and negative z scores of repressors in the myostatin-activin pathway. Activators were ACVR1B, ACVR1C, ACVR2A, ACVR2B, SMAD2, SMAD3, SMAD4, and TGFBR1, while repressors were SMAD1, SMAD5, SMAD6, SMAD7, SMURF1, and SMURF2. The top quartile (Q4) and bottom quartile (Q1) of GS were compared using chi-squared and Fisher-Exact tests. RNA deconvolution analysis with QuantiSEQ estimated cell infiltration in the tumor microenvironment (TME). Differences in overall survival (OS) were analyzed from insurance claims data and calculated from time of tissue collection using Kaplan-Meier estimates. Statistical significance was determined as a P-value adjusted for multiple comparisons ( q<0.05). Results: GS were higher in primary tumors compared to metastases (median: -0.71 vs -0.86, q<0.05). GS was associated with increased PD-L1 IHC expression (Q1 21.2% vs Q4 10.3%, q<0.001), but not TMB-high (1.9% vs 2.1%, q=1) or MSI-H status (1.1% vs 1.3%, q=1). GS correlated with increased expression of immune related genes ( CD274, CD80, IDO1, CD86, PDCD1, LAG3, CTLA4, HAVCR2, and IFNG, q<0.05). However, TME immune cell infiltration did not vary based on cachexia GS. Mutation rates of TP53 (Q1 79.4% vs Q4 73.7%), ARID1A (Q1 11.8% vs Q4 6.9%) and KRAS (Q1 92.7% vs Q4 86.4%) were associated with Q1-GS (all q<0.01), while STK11 mutations (1.1% vs 3.0%, q=0.001) were associated with Q4-GS. Spearman correlation linked cachexia GS with the lipid metabolizing genes UCP2 (rho=0.49) and UCP3 (rho=0.30), as well as the inflammatory markers CCL2/MCP-1(rho=0.34) and IL1B (rho=0.33). Decreased OS was seen with higher tumor expression of the myostatin-activin pathway activator, SMAD3 (6.6 vs 8.5 mo, HR=1.22, CI 1.12-1.33, P<0.001), and lower expression of the repressor SMAD7 (6.9 vs 8.9 mo, HR=0.91, CI 0.79-0.90, P=0.034) in metastatic PDAC. Conclusions: This is the largest molecular and clinical characterization of the myostatin-activin cachexia pathway in PDAC. Our data show that increased activation of the myostatin-activin pathway is associated with immune mediators, lipid metabolism, and inflammatory gene activation. Activators and repressors are significant predictors of survival in PDAC, suggesting possible novel therapeutic targets.

A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activation.

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1β complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1β coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1β complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.

Single-Cell RNA Sequencing Reveals Metabolic Stress-Dependent Activation of Cardiac Macrophages in a Model of Dyslipidemia-Induced Diastolic Dysfunction.

Metabolic distress is often associated with heart failure with preserved ejection fraction (HFpEF) and represents a therapeutic challenge. Metabolism-induced systemic inflammation links comorbidities with HFpEF. How metabolic changes affect myocardial inflammation in the context of HFpEF is not known. We found that ApoE knockout mice fed a Western diet recapitulate many features of HFpEF. Single-cell RNA sequencing was used for expression analysis of CD45+ cardiac cells to evaluate the involvement of inflammation in diastolic dysfunction. We focused bioinformatics analysis on macrophages, obtaining high-resolution identification of subsets of these cells in the heart, enabling us to study the outcomes of metabolic distress on the cardiac macrophage infiltrate and to identify a macrophage-to-cardiomyocyte regulatory axis. To test whether a clinically relevant sodium glucose cotransporter-2 inhibitor could ameliorate the cardiac immune infiltrate profile in our model, mice were randomized to receive the sodium glucose cotransporter-2 inhibitor dapagliflozin or vehicle for 8 weeks. ApoE knockout mice fed a Western diet presented with reduced diastolic function, reduced exercise tolerance, and increased pulmonary congestion associated with cardiac lipid overload and reduced polyunsaturated fatty acids. The main immune cell types infiltrating the heart included 4 subpopulations of resident and monocyte-derived macrophages, determining a proinflammatory profile exclusively in ApoE knockout- Western diet mice. Lipid overload had a direct effect on inflammatory gene activation in macrophages, mediated through endoplasmic reticulum stress pathways. Investigation of the macrophage-to-cardiomyocyte regulatory axis revealed the potential effects on cardiomyocytes of multiple inflammatory cytokines secreted by macrophages, affecting pathways such as hypertrophy, fibrosis, and autophagy. Finally, we describe an anti-inflammatory effect of sodium glucose cotransporter-2 inhibitor in this model. Using single-cell RNA sequencing , in a model of diastolic dysfunction driven by hyperlipidemia, we have determined the effects of metabolic distress on cardiac inflammatory cells, in particular on macrophages, and suggest sodium glucose cotransporter-2 inhibitors as potential therapeutic agents for the targeting of a specific phenotype of HFpEF.

Chromatin Remodeling Complex PBAF Activates and Represses Inflammatory Genes.

The PBAF chromatin remodeling complex regulates chromatin state and gene transcription in higher eukaryotes. In this work, we studied the role of PBAF in the regulation of NF-κB-and JAK/STAT-dependent activation of inflammatory genes. We performed knockdown of specific module subunit BAF200, which resulted in destruction of the entire PBAF specific module and changed the level of the genes transcription of both pathways. PBAF can be both an activator and a repressor of inflammatory genes. Thus, PBAF is an important regulator of inflammatory gene expression.

The SGLT2I Dapagliflozin reverses metabolic stress driving myocardial inflammation in a mouse model of heart failure with preserved ejection fraction: insights from single-cell RNA sequencing

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is often associated with metabolic distress and represents a therapeutic challenge. Metabolism-induced systemic inflammation links comorbidities with HFpEF. How metabolic changes impact myocardial inflammation in the context of HFpEF is not known. Methods We generated a model of metabolic HFpEF by feeding ApoE knockout (ApoE KO) mice a high-fat diet (HFD), and used single-cell expression analysis (scRNA-seq) on CD45+ cardiac cells to evaluate the involvement of inflammation. By focusing our analysis on macrophages, we obtained high-resolution identification of subsets of these cells in the heart, enabling us to study the outcomes of metabolic distress on the cardiac macrophage infiltrate and identify a macrophage-to-cardiomyocyte (CMC) regulatory axis. To test whether a clinically relevant sodium glucose cotransporter-2 inhibitor (SGLT2i) could ameliorate the cardiac immune infiltrate profile in our model, ApoE KO-HDF mice were randomized to receive the SGLT2i dapagliflozin (DAPA) or vehicle for 8 weeks. Results We describe a new metabolic HFpEF model: mice present with reduced diastolic function, reduced exercise tolerance, and increased pulmonary congestion associated with cardiac lipid overload and reduced polyunsaturated fatty acids. The main immune cell types infiltrating the heart included 4 sub-populations of resident and monocyte-derived macrophages displaying a pro-inflammatory profile exclusively in ApoE KO-HFD mice. Lipid overload had a direct effect on inflammatory gene activation in macrophages, mediated through endoplasmic-reticulum stress pathways. Investigation of the macrophage-to-CMC regulatory axis revealed the potential effects on CMCs of multiple inflammatory cytokines secreted by macrophages, impacting pathways such as hypertrophy, fibrosis, and autophagy. Finally, we describe the anti-inflammatory effect of SGLT2i. Conclusions Through a newly described model of HFpEF based on metabolic stress, and using scRNA-seq, we have determined the effects of metabolic distress on cardiac inflammatory cells, in particular on macrophages, and suggest SGLT2i as potential therapeutic agents for the targeting of a specific phenotype of HFpEF.

Abstract 13461: Early Vascular Aging is Associated With Early Cardiac Aging, Upregulation of Left Ventricle Pro-Fibrotic and Inflammatory Genes and Memory Decline in Aged Dahl Salt-Sensitive Rats

Introduction: Blood pressure (BP) begins to increase early in life in Dahl salt-sensitive rats (DSS) that consume a normal salt (NS) diet. Epidemiologic studies have shown that early vascular aging (EVA) is associated with central arterial stiffening (CAS) and hypertension, which develop earlier in life than expected in general population and contribute to cognitive impairment later in life. Hypotheses: (i) DSS rats will develop EVA earlier in life on NS diet, and with advancing age will exhibit early CV aging; (ii) In DSS, CV remodeling and CAS are implicated in memory decline. We tested these hypotheses using a longitudinal repeated measure design in DSS and their parental breed Sprague-Dawley rats (SD) at 3- & 12-mo of age. Methods: Male SD and DSS were kept on NS diet (0.5% NaCl; n=8/group) for entire experiment. Repeated measures of systolic BP (SBP), pulse wave velocity (PWV; an index of CAS), echocardiography, and non-repeated measures of Morris water maze (MWM) to test spatial memory, aortic collagen, elastin (histochemistry), and left ventricle (LV) mRNA expression (qPCR) were assessed at 3- & 12-mo. Data analyses: linear mixed-effect 2-way ANOVA, t-test & linear regression (LR) modeling. A 2-sided p<0.05 was considered significant. Results: SBP, PWV, aortic weight, wall thickness and collagen/elastin ratio were higher in DSS-3 vs. SD-3, and in DSS-12 vs. DSS-3. RWT and LV expression of pro-fibrotic, inflammatory and senescence genes were higher in DSS-12 vs. DSS-3 and vs. SD-12 (Table). DSS-12 spent more time to find a hidden platform in MWM vs. SD-12; their impaired spatial hippocampal memory was positively associated with PWV and LV mass by LR analysis. Conclusions: EVA, indexed as higher BP, PWV and aortic remodeling was associated with early CV aging, manifested by activation of LV senescence, inflammatory and pro-fibrotic genes and by higher rates of age-associated changes in BP, RWT and LV mass in DSS vs. SD. In DSS, early CV aging was associated with memory decline.

CHROMATIN REMODELING COMPLEX PBAF ACTIVATES AND REPRESSES INFLAMMATORY GENES

The PBAF chromatin remodeling complex regulates chromatin state and gene transcription in higher eukaryotes. In this work, we studied the role of PBAF in the regulation of NF–κB-and JAK/STAT-dependent activation of inflammatory genes. We performed knockdown of specific module subunit BAF200 resulted in destruction of the entire PBAF specific module and changed the level of the genes transcription of both pathways. PBAF can be both an activator and a repressor of inflammatory genes. Thus, PBAF is one of the important regulators of inflammatory gene expression.

Dichotomous roles of RIPK3 in regulating the IFN response and NLRP3 inflammasome in human monocytes.

Regulation of the profile and magnitude of toll-like receptor (TLR) responses is important for effective host defense against infections while minimizing inflammatory toxicity. The chemokine CXCL4 regulates the TLR8 response to amplify inflammatory gene and inflammasome activation while attenuating the interferon (IFN) response in primary monocytes. In this study, we describe an unexpected role for the kinase RIPK3 in suppressing the CXCL4 + TLR8-induced IFN response and providing signal 2 to activate the NLRP3 inflammasome and interleukin (IL)-1 production in primary human monocytes. RIPK3 also amplifies induction of inflammatory genes such as TNF, IL6, and IL1B while suppressing IL12B. Mechanistically, RIPK3 inhibits STAT1 activation and activates PI3K-Akt-dependent and XBP1- and NRF2-mediated stress responses to regulate downstream genes in a dichotomous manner. These findings identify new functions for RIPK3 in modulating TLR responses and provide potential mechanisms by which RIPK3 plays roles in inflammatory diseases and suggest targeting RIPK3 and XBP1- and NRF2-mediated stress responses as therapeutic strategies to suppress inflammation while preserving the IFN response for host defense.

Aging-Related Accumulation of Truncated Oxidized Phospholipids Augments Infectious Lung Injury and Endothelial Dysfunction via Cluster of Differentiation 36-Dependent Mechanism.

Truncated phospholipid oxidation products (Tr-OxPL) increase in blood circulation with aging; however, their role in the severity of vascular dysfunction and bacterial lung injury in aging groups remains poorly understood. We investigated the effects of six Tr-OxPL species: KOdiA-PC, POVPC, PONPC, PGPC, Paz-PC, and Lyso-PC on endothelial dysfunction and lung inflammation caused by heat-killed Staphylococcus aureus (HKSA) in young (aged 2-4 months) and old (aged 12-18 months) mice, organotypic culture of precisely cut lung slices, and endothelial cells (mLEC) isolated from young and old mice. HKSA and Tr-OxPL combination caused a higher degree of vascular leak, the accumulation of inflammatory cells and protein in bronchoalveolar lavage, and inflammatory gene expression in old mice lungs. HKSA caused a greater magnitude of inflammatory gene activation in cell and ex vivo cultures from old mice, which was further augmented by Tr-OxPLs. L37pA peptide targeting CD36 receptor attenuated Tr-OxPL-induced endothelial cell permeability in young and old mLEC and ameliorated KOdiA-PC-induced vascular leak and lung inflammation in vivo. Finally, CD36 knockout mice showed better resistance to KOdiA-PC-induced lung injury in both age groups. These results demonstrate the aging-dependent vulnerability of pulmonary vasculature to elevated Tr-OxPL, which exacerbates bacterial lung injury. CD36 inhibition is a promising therapeutic approach for improving pneumonia outcomes in aging population.

Neonatal administration of synthetic estrogen, diethylstilbestrol to mice up-regulates inflammatory Cxclchemokines located in the 5qE1 region in the vaginal epithelium.

A synthetic estrogen, diethylstilbestrol (DES), is known to cause adult vaginal carcinoma by neonatal administration of DES to mice. However, the carcinogenic process remains unclear. By Cap Analysis of Gene Expression method, we found that neonatal DES exposure up-regulated inflammatory Cxcl chemokines 2, 3, 5, and 7 located in the 5qE1 region in the vaginal epithelium of mice 70 days after birth. When we examined the gene expressions of these genes much earlier stages, we found that neonatal DES exposure increased these Cxcl chemokine genes expression even after 17 days after birth. It implies the DES-mediated persistent activation of inflammatory genes. Intriguingly, we also detected DES-induced non-coding RNAs from a region approximately 100 kb far from the Cxcl5 gene. The non-coding RNA up-regulation by DES exposure was confirmed on the 17-day vagina and continued throughout life, which may responsible for the activation of Cxcl chemokines located in the same region, 5qE1. This study shows that neonatal administration of DES to mice causes long-lasting up-regulation of inflammatory Cxcl chemokines in the vaginal epithelium. DES-mediated inflammation may be associated with the carcinogenic process.

Madder (Rubia cordifolia L.) Alleviates Myocardial Ischemia-Reperfusion Injury by Protecting Endothelial Cells from Apoptosis and Inflammation.

Ischemia-reperfusion injury often occurs in organ transplantation, coronary heart disease, ischemic heart disease, and other diseases, which greatly reduces clinical efficacy. This study examined the effectiveness of madder as a medicine to treat ischemia-reperfusion injury. The efficacy of madder was evaluated by measuring myocardial infarction size, coronary outflow volume, myocardial contraction rate, activation of inflammatory factors, autophagy factors, apoptosis factors, and related pathway genes in mice. The results indicated that treatment with madder can effectively reduce the area of myocardial infarction and restore arterial blood flow velocity and myocardial contractility in mice. Additionally, madder treatment inhibited the expression of inflammatory factors, autophagy factors, and apoptosis factors in mice and reduced the degree of myocardial cell injury. Studies have also shown that madder treatment can alleviate myocardial ischemia-reperfusion injury in mice and inhibit the occurrence of inflammatory response by inhibiting the activity of the NF-κB pathway. The results showed that madder was effective against ischemia-reperfusion injury, thus showing potential as a clinical drug for treating ischemia-reperfusion injury.

RNF138 inhibits late inflammatory gene transcription through degradation of SMARCC1 of the SWI/SNF complex.

As one of the core components of the switching or sucrose non-fermentable (SWI/SNF) complex, SMARCC1 (BAF155, SRG3) plays essential roles in activation of late inflammatory genes in response to microbial challenge. However, little is known about the mechanism of how SMARCC1 regulates the inflammatory innate response. Via functional screening, we identify the nuclear E3 ubiquitin ligase RNF138 as a negative regulator in the inflammatory innate response and show that RNF138 interacts with SMARCC1 and mediates its K48-linked polyubiquitination at position Lys643 and proteasomal degradation. As a result, the catalytic activity of RNF138 fine-tunes the kinetics of late inflammatory gene transcription by inhibiting chromatin remodeling at SWI/SNF-regulated gene loci. Reduced RNF138 and increased SMARCC1 in monocytes of rheumatoid arthritis patients are observed. These results provide mechanistic insight into the interplay among nucleosome remodeling, inflammation, and ubiquitylation and underscore the important role of the E3 ubiquitin ligases in controlling the extent and duration of inflammatory responses.

β-Sitosterol Enhances Lung Epithelial Cell Permeability by Suppressing the NF-κB Signaling Pathway.

The dysregulation between pro-inflammatory and anti-inflammatory responses during sepsis is a crucial factor in driving sepsis progression. Acute lung injury (ALI) resulting from excessive production and accumulation of inflammatory mediators in the lungs contributes to impaired lung barrier function. The activation of the NF-κB signaling pathway during inflammation leads to the transcriptional activation of multiple inflammatory genes. Given the plausible impact of NF-κB signaling suppression in mitigating lung injury, substantive evidence demonstrates beta-sitosterol (BS)'s proficient ability to block NF-κB activation. Therefore, the aim of the present investigation was to delve into the impacts of BS in the context of sepsis-induced acute lung injury, employing both a mouse model and a model involving lung epithelial cells. Sepsis-induced lung injury was simulated in mice through cecum ligation and puncture (CLP). To emulate injury in murine lung epithelial (MLE-12) cells, an experiment involving lipopolysaccharide (LPS) was administered. Evaluation of alterations in lung tissue permeability encompassed techniques such as lung wet/dry (W/D) mass ratio, Evans blue staining, and quantification of total protein concentration in bronchoalveolar lavage fluid (BALF). Lung tissue histopathological shifts were ascertained via hematoxylin and eosin (HE) staining. Additionally, the concentrations of inflammatory cytokines IL-6 and TNF-α were quantified in every lung tissue and cell group by implementing enzyme-linked immunosorbent assay (ELISA). Protein quantification for signal biomarkers was carried out using Western blotting and immunofluorescence methodologies. In tandem, the assessment of MLE-12 cell permeability was conducted by evaluating fluorescein isothiocyanate (FITC)-dextran extravasation. BS mitigated lung tissue pathologies, reduced inflammatory factors, and lowered tissue and cell permeability. BS inhibited NF-κB signaling and increased claudin-4 and claudin-5 expression, enhancing septic lung epithelial cell permeability. Through suppressing the NF-κB signaling cascade, BS effectively curtails the levels of inflammatory mediators. Simultaneously, it orchestrates the modulation of claudin-4 and claudin-5 expression, culminating in the augmentation of lung epithelial cell barrier competence, thus improving sepsis-induced lung injury.

MiR-181c, a potential mediator for acute kidney injury in a burn rat model with following sepsis.

The miRNA profile is changed after burn or sepsis and is involved in regulating inflammatory reactions. However, the function and molecular mechanism of miRNAs in regulating burn sepsis-induced acute kidney injury (AKI) are still unclear. In this study, animal and cell sepsis models were established after burned rats were injected with lipopolysaccharide (LPS) or NRK-52E cells treated with LPS, respectively. Cytokine expression, inflammatory cell infiltration, serum creatinine (Scr) and kidney injury molecule-1 (KIM-1) levels were analysed after the indicated treatments. Burn sepsis increased the expression of inflammatory factors (TNF-α and IL-1β) and chemokines (MIP-1α, MIP-2 and MCP-1). Moreover, burn sepsis promoted macrophage and neutrophil infiltration into the kidney and upregulated the levels of Scr and KIM-1 in the kidney and urine. Ectopic expression of miR-181c significantly reduced LPS-induced TLR4 protein expression, suppressed KIM-1 mRNA levels and subsequently inhibited the activation of inflammatory genes (TNF-α and IL-1β) and chemokine genes (MIP-1α, MIP-2 and MCP-1). Our results demonstrated that miR-181c could suppress TLR4 expression, reduce inflammatory factor and chemokine secretion, mitigate inflammatory cell infiltration into the kidney and downregulate KIM-1 expression, which might ultimately attenuate burn sepsis-induced AKI.

Costimulation of TLR8 responses by CXCL4 in Human Monocytes Mediated by TBK1-IRF5 Signaling and Epigenomic Remodeling

Abstract CXCL4 regulates responses of immune cells to endosomal TLRs and has been implicated in the pathogenesis of inflammatory and fibrotic diseases. However, mechanisms by which CXCL4 modulates TLR responses, and its functions in monocytes/macrophages, are still unclear. Here we report that CXCL4 changes the profile of the TLR8 response in human monocytes by selectively and dramatically amplifying inflammatory gene transcription and IL-1b production while partially attenuating the IFN response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activated TBK1/IKKe and repurposed these kinases towards an inflammatory response via coupling with IRF5, and by activating the NLRP3 inflammasome without the need for a second signal. CXCL4 strongly induced chromatin remodeling in a cooperative and synergistic manner with TLR8 signaling, inducing de novo enhancers associated with inflammatory genes. These findings identify signaling and epigenomic mechanisms that underly synergistic activation of inflammatory genes by CXCL4 and TLR8, provide a new paradigm for modulation of TLR responses that is relevant for cytokine storm, and suggest targeting the TBK1/IKKe-IRF5 axis may be beneficial in inflammatory diseases.

Tailoring PEGylated nanoparticle surface modulates inflammatory response in vascular endothelial cells

Polymer nanoparticles (NPs) are extensively studied as drug delivery systems for various therapeutic indications, including drug and imaging agent delivery to the brain. Despite intensive research, their toxicological profile has yet to be fully characterized. In particular, the more subtle effects of nanomaterials on inflammatory processes have scarcely been investigated. Surface properties of NPs are amongst parameters governing interactions between living cells and NPs. They could considerably influence the toxicity and inflammatory response of the cells exposed to NPs.Polymeric NPs investigated here present a core-shell structure. The core is constituted of hydrophobic poly(lactic acid) (PLA) block and the surface is composed of a shell of hydrophilic block of polyethylene glycol (PEG). The effect of PEG chain length coating on the expression of genes involved in the inflammation response was investigated in two vascular endothelial cell lines (bEnd.3 and HUVEC) by qPCR. Moreover, ROS generation following NP uptake was evaluated.PEGylated NPs induce a mild and transient activation of inflammatory cytokine and chemokine genes. However, differences in PEG chain length did not show any significant effect on cytokine and chemokine gene expression and PEGylated NPs did not trigger ROS generation.The present results could contribute significantly to a deeper understanding of nanomaterial interactions and toxicity with vascular endothelial cells, guiding scientists in material coating choices.

A144 CHRONIC DSS LEADS TO ALTERED EXPRESSION OF IRAK4/TPL2 PATHWAYS IN UC-COLONIZED MICE

BackgroundUlcerative colitis (UC) is one of two forms of inflammatory bowel disease (IBD). The exact cause of IBD is unknown but altered host-microbe interactions and genetic susceptibility are involved in its pathogenesis. Many patients with IBD do not respond to biological therapies targeting single cytokines, therefore new therapies that target common immune pathways are being developed and need to be tested in relevant preclinical models. Previously we have shown that mice colonized with UC microbiota upregulated genes related to inflammation without induction of colitis compared to healthy volunteer-colonized mice. We thus investigated whether IRAK4 and TPL2-induced pathways, new therapeutic targets in development upstream of inflammatory cytokine gene activation, are upregulated in mice colonized with UC microbiota and chronic colitis.AimsOur aim was to characterize TPL2 and IRAK4 signalling pathways and T cell phenotypes in UC-colonized mice following chronic low-dose dextran sodium sulfate (DSS) colitis.Methods10-16-week-old germ-free C57BL/6 mice were colonized with fecal microbiota from a patient with UC experiencing a flare (n=16). Mice were housed in a gnotobiotic facility during the experiment. Three weeks following colonization, colitis was induced in half of the mice by three cycles (5 d each; 2.0%, 1.5% and 1.5%, respectively) of DSS in drinking water with a 5-d wash-out period between cycles. All mice were fed a control diet (7004, Teklad). Fecal samples were collected weekly. At sacrifice, disease activity (colon length, occult blood in feces, stool consistency, and spleen weight) was measured and colon tissue was collected for histological analysis and RNA sequencing. Mesenteric lymph nodes (MLNs) were acquired for flow cytometry to analyze T cell phenotypes.ResultsIn UC-colonized mice, chronic low-dose DSS induced softer feces (p<0.0001), shorter colon length (p<0.0001), and increased spleen weight (p<0.0001), compared with water treated mice. This was paralleled by detectable blood in stool, development of moderate colitis (DSS: 1.9+/-1.4 vs H2O: 0.5+/-0.2; p<0.02) and higher proportion of IL-17 (p=0.003) and IFN-γ-producing T cells (p=0.06) in MLNs compared with water treated mice. RNA sequencing revealed that inflammatory genes, mainly related to the IRAK4/TPL2 pathway (e.g., Gadd45b, Socs3, Il1b), were significantly increased (p≤0.05) in the colon of mice treated with DSS compared with water.ConclusionsWhen challenged with a chronic low-dose chemical injury agent, mice colonized with UC microbiota develop clinical and histological signs of colitis and upregulation of genes involved in inflammation like Gadd45b, Socs3, and Il1b. Thus, this model represents a new valuable tool for preclinical testing of new drug candidates, such as those related to the modulation of IRAK4/TPL2 pathways.Funding AgenciesCCC

Assessment of Culture/Preservation Conditions of Human Islets for Transplantation

Islet culture before clinical transplantation has been adopted by various centers, but its effect on the survival and function of islets relative to the culture conditions and media needs further assessment. Human islets were cultured or preserved under four different conditions and three media options. Parameters such as recovery, viability, function, islet damage, and gene expressions for markers of hypoxia, and inflammation were assessed after 48-h culture or preservation. Preservation of islets was performed at 4°C in Connaught’s Medical Research Lab (CMRL) and University of Wisconsin (UW) media. Islets were cultured at 22°C, 37°C, and 37°C–22°C in CMRL and PRODO culture media. Islets preserved in UW solution had visually good morphology and exhibited higher recovery with less islet damage compared with the rest of the groups, whereas islets preserved in CMRL at 4°C resulted in poor morphology, recovery, viability, and function compared with the rest of the treatment conditions. Culture at 22°C and 37°C demonstrated an increase in the expression of inflammatory and hypoxia-related genes. In conclusion, islets preserved at 4°C in UW solution showed the best overall outcomes after 48 h compared with islets cultured at 22°C, 37°C, or 37°C–22°C in PRODO. Advancement in islet culture media is warranted to reduce inflammatory gene activation and improve recovery of islets for transplantation.