Activation Of Proinflammatory Genes Research Articles

Antioxidant and Cytoprotective Effects of Different Compounds on Cyclophosphamide-induced Nephrotoxicity

Abstract: Cyclophosphamide is a drug derived from nitrogenous mustards and is mainly indicated for the anti-neoplastic clinic. When it is metabolized in the liver, it produces acrolein, a cytotoxic metabolite capable of generating damage to the body. The oxidative, nitrative and nitrosative stress are responsible for mediating this side effect since the toxicity of the drug induces the production of reactive species of oxygen (ROS) and nitrogen (RNS). These radicals are unstable and react with other molecules that can result in cellular lesions such as lipid peroxidation, enzymatic inactivation, and excessive activation of pro-inflammatory genes, producing molecules such as malonaldehyde (MDA) and nitric oxide (NO), representing oxidative stress markers. One of the consequences of these mechanisms is nephrotoxicity. In contrast, some compounds have direct or indirect antioxidante action, which are the objects of this study: gallic acid, aminoguanidine, chrysin, hesperidine, naringin, morin-5'-sulfonic acid sodium salt and spirulina. All of them are responsible for the nephroprotective activity when administered during experiments in vitro or in vivo. Thus, this observational bibliographic research is characterized as qualitative descriptive and explanatory, since it focuses on analyzing the antioxidant properties of compounds with possible potentials to inhibit the nephrotoxicity induced by cyclophosphamide and describe the results, in addition to clarifying the biochemical activities involved in the process.

MicroRNA-296-5p Expression in COVID-19 Patients and its Relationship with Inflammatory Cytokines

Objectives: The cytokine storm, triggered by the activation of certain pro-inflammatory genes in the second phase of COVID-19, is associated with severe acute respiratory disorder. Evidence suggests that microRNAs (miR) and cytokine levels can play pivotal roles in host cell antiviral defense mechanisms. This study aimed to investigate the changes in IFN-γ inducible protein 10 (IP-10) and miR-296-5p expression, as well as their relationship with some inflammatory cytokines and biochemical variables in COVID-19 patients. Methods: This retrospective single-center study was conducted on 30 COVID-19 patients and 30 controls. The expression of miR-296-5p and IP-10 genes in peripheral blood mononuclear cells (PBMCs) was evaluated using real-time PCR. Serum levels of IL-6, TNF-α, and CRP were measured by ELISA. Results: Higher expression levels of miR-296-5p and IP-10 genes were observed in COVID-19 patients compared to controls (P=0.001). Furthermore, IP-10, IL-6, and TNF-α levels were significantly higher (PP<0.01) in COVID-19 patients than in controls. The results also showed positive correlations between miR-296-5p expression and serum levels of IL-6, CRP, and TNF-α in patients with COVID-19. ROC curve analysis of miR[1]296-5p in COVID-19 patients showed an area under the curve of 0.830, 95% CI (0.658-0.815), P<0.001, with an optimal cut-off point of 0.32965. Conclusion: Our results suggest a regulatory role for miR-296-5p in cytokine secretion in COVID-19. The results indicate that the expression of miR-296-5p and IP-10 genes in PBMCs might serve as convenient novel biomarkers for the prognosis of COVID-19.

RETRACTION: Melatonin Attenuates Apoptotic Liver Damage in Fulminant Hepatic Failure Induced by the Rabbit Hemorrhagic Disease Virus.

Tuñón MJ, Miguel BS, Crespo I, Jorquera F, Santamaría E, Alvarez M, Prieto J, González-Gallego J. Melatonin attenuates apoptotic liver damage in fulminant hepatic failure induced by the rabbit hemorrhagic disease virus. J Pineal Res 2011;50:38-45. https://doi.org/10.1111/j.1600-079X.2010.00807.x The above article, published online on 22 October 2010 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Gianluca Tosini, and John Wiley and Sons Ltd. Following publication, concerns were raised by third parties that portions of Figure 4 are duplicated in Figures 4, 2A, and 1B, respectively, of three other articles by the same author group.1-3 The authors provided some of the original data, but these were not sufficient to resolve the concerns, and the authors were unable to provide a satisfactory explanation for the concerns. The retraction has been agreed because of concerns that portions of the figure overlap with the authors' previous articles, affecting the interpretation of the data and results presented. The authors disagree with this decision. 1. Kretzmann NA, Fillmann H, Mauriz JL, Marroni, CA, Marroni N, González-Gallego J, Tuñón MJ. Effects of glutamine on pro-inflammatory gene expression and activation of nuclear factor kappa B and signal transducers and activators of transcription in TNBS-induced colitis. Inflamm Bowel Dis 2008;14:1504-1513. doi:10.1002/ibd.20543 2. Crespo I, García-Mediavilla MV, Gutiérrez B, Sánchez-Campos S, Tuñón MJ, González-Gallego J. A comparison of the effects of kaempferol and quercetin on cytokine-induced pro-inflammatory status of cultured human endothelial cells. Br J Nutr 2008;100(5):968-976. doi:10.1017/S0007114508966083 3. Laliena A, Miguel BS, Crespo I, Alvarez M, González-Gallego J, Tuñón MJ. Melatonin attenuates inflammation and promotes regeneration in rabbits with fulminant hepatitis of viral origin. J Pineal Res 2012;53:270-278. doi:10.1111/j.1600-079X.2012.00995.x.

Crosstalk between Oxidative Stress and Inflammation Induced by Ionizing Radiation in Healthy and Cancerous Cells.

Radiotherapy (RT) is a unique modality in cancer treatment with no replacement in many cases and uses a tumoricidal dose of various ionizing radiation (IR) types to kill cancer cells. It causes oxidative stress through reactive oxygen species (ROS) production or the destruction of antioxidant systems. On the other hand, RT stimulates the immune system both directly and indirectly by releasing danger signals from stress-exposed and dying cells. Oxidative stress and inflammation are two reciprocal and closely related mechanisms, one induced and involved by the other. ROS regulates the intracellular signal transduction pathways, which participate in the activation and expression of pro-inflammatory genes. Reciprocally, inflammatory cells release ROS and immune system mediators during the inflammation process, which drive the induction of oxidative stress. Oxidative stress or inflammation-induced damages can result in cell death (CD) or survival mechanisms that may be destructive for normal cells or beneficial for cancerous cells. The present study has focused on the radioprotection of those agents with binary effects of antioxidant and anti-inflammatory mechanisms IR-induced CD.

Toward a disruptive, minimally invasive small finger joint implant concept: Cellular and molecular interactions with materials in vivo

Osteoarthritis (OA) poses significant therapeutic challenges, particularly OA that affects the hand. Currently available treatment strategies are often limited in terms of their efficacy in managing pain, regulating invasiveness, and restoring joint function. The APRICOTⓇ implant system developed by Aurora Medical Ltd (Chichester, UK) introduces a minimally invasive, bone-conserving approach for treating hand OA (https://apricot-project.eu/). By utilizing polycarbonate urethane (PCU), this implant incorporates a caterpillar track-inspired design to promote the restoration of natural movement to the joint. Surface modifications of PCU have been proposed for the biological fixation of the implant. This study investigated the biocompatibility of PCU alone or in combination with two surface modifications, namely dopamine-carboxymethylcellulose (dCMC) and calcium-phosphate (CaP) coatings. In a rat soft tissue model, native and CaP-coated PCU foils did not increase cellular migration or cytotoxicity at the implant–soft tissue interface after 3 d, showing gene expression of proinflammatory cytokines similar to that in non-implanted sham sites. However, dCMC induced an amplified initial inflammatory response that was characterized by increased chemotaxis and cytotoxicity, as well as pronounced gene activation of proinflammatory macrophages and neoangiogenesis. By 21 d, inflammation subsided in all the groups, allowing for implant encapsulation. In a rat bone model, 6 d and 28 d after release of the periosteum, all implant types were adapted to the bone surface with a surrounding fibrous capsule and no protracted inflammatory response was observed. These findings demonstrated the biocompatibility of native and CaP-coated PCU foils as components of APRICOTⓇ implants. Statement of significanceHand osteoarthritis treatments require materials that minimize irritation of the delicate finger joints. Differing from existing treatments, the APRICOTⓇ implant leverages polycarbonate urethane (PCU) for minimally invasive joint replacement. This interdisciplinary, preclinical study investigated the biocompatibility of thin polycarbonate urethane (PCU) foils and their surface modifications with calcium-phosphate (CaP) or dopamine-carboxymethylcellulose (dCMC). Cellular and morphological analyses revealed that both native and Ca-P coated PCU elicit transient inflammation, similar to sham sites, and a thin fibrous encapsulation in soft tissues and on bone surfaces. However, dCMC surface modification amplified initial chemotaxis and cytotoxicity, with pronounced activation of proinflammatory and neoangiogenesis genes. Therefore, native and CaP-coated PCU possess sought-for biocompatible properties, crucial for patient safety and performance of APRICOTⓇ implant.

Abstract 2003: Epigenetic And Phenotypic Modulation Of Adventitial Fibroblasts In Atherosclerosis By Coronary Artery Risk Gene Tcf21

The vascular adventitia has long been postulated to play a key role in atherosclerosis, but the relevant adventitial cell populations and the mechanisms by which they influence atherosclerosis have remained elusive. To characterize relevant adventitial cells and their regulators, we have combined single cell multi-omic murine and human data with human genetics. Single cell transcriptomic and epigenetic analysis of both murine and human arterial tissue revealed a unique population of adventitial fibroblasts (AdvFib). Transcriptome and epigenome analysis revealed disproportionate enrichment for coronary artery disease (CAD) GWAS risk loci and genes in AdvFib, suggesting that AdvFib actively contributes to disease. Leveraging this rich information, we created a novel AdvFib specific CreERT2 murine model to enable AdvFib specific genetic manipulation. Murine lineage tracing using this novel AdvFib-specific model of atherosclerosis revealed early expansion and transcriptomic alteration of AdvFib during plaque development, akin to phenotypic modulation of SMC. Cell-cell communication analysis demonstrated an increase in AdvFib-driven crosstalk with other cell types during early phases of atherosclerosis, including activation of pro-inflammatory (Il6, Ccl2/7, Cxcl12) genes. Computational enrichment analysis of regulatory region of genes associated with AdvFib modulation points to a prominent role for TCF21 , a CAD GWAS risk gene. Single cell time course data of atherosclerosis demonstrates that Tcf21 is expressed primarily in AdvFib with dynamic alteration of its expression during atherosclerosis. AdvFib-specific Tcf21 loss in murine model of atherosclerosis resulted in altered expression of ECM and inflammatory genes, with corresponding changes in recruitment of inflammatory cells. Mechanistically, through histone modification cut-and-tag in human coronary AdvFib, we showed that TCF21 regulates AdvFib activation through epigenetic reprograming of key cytokine and cell fate regulators by binding and altering H3K27Acetylation. Collectively, our results begin to establish the role of AdvFib in atherosclerosis and highlight the importance of TCF21 in this process through a precise epigenetic mechanism.

HSP90, a Common Therapeutic Target for Suppressing Skin Injury Caused by Exposure to Chemically Diverse Classes of Blistering Agents.

Vesicants such as arsenicals and mustards produce highly painful cutaneous inflammatory and blistering responses, hence developed as chemical weapons during World War I/II. Here, using lewisite and sulfur mustard surrogates, namely phenylarsine oxide (PAO) and 2-chloroethyl ethyl sulfide (CEES), respectively, we defined a common underlying mechanism of toxic action by these two distinct classes of vesicants. Murine skin exposure to these chemicals causes tissue destruction characterized by increase in skin bifold thickness, Draize score, infiltration of inflammatory cells, and apoptosis of epidermal and dermal cells. RNA sequencing analysis identified ∼346 inflammatory genes that were commonly altered by both PAO and CEES, along with the identification of cytokine signaling activation as the top canonical pathway. Activation of several proinflammatory genes and pathways is associated with phosphorylation-dependent activation of heat shock protein 90α (p-HSP90α). Topical treatment with known HSP90 inhibitors SNX-5422 and IPI-504 post PAO or CEES skin challenge significantly attenuated skin damage including reduction in overall skin injury and clinical scores. In addition, highly upregulated inflammatory genes Saa3, Cxcl1, Ccl7, IL-6, Nlrp3, Csf3, Chil3, etc. by both PAO and CEES were significantly diminished by treatment with HSP90 inhibitors. These drugs not only reduced PAO- or CEES-induced p-HSP90α expression but also its client proteins NLRP3 and pP38 and the expression of their target inflammatory genes. Our data confirm a critical role of HSP90 as a shared underlying molecular target of toxicity by these two distinct vesicants and provide an effective and novel medical countermeasure to suppress vesicant-induced skin injury. SIGNIFICANCE STATEMENT: Development of effective and novel mechanism-based antidotes that can simultaneously block cutaneous toxic manifestations of distinct vesicants is important and urgently needed. Due to difficulties in determining the exact nature of onsite chemical exposure, a potent drug that can suppress widespread cutaneous damage may find great utility. Thus, this study identified HSP90 as a common molecular regulator of cutaneous inflammation and injury by two distinct warfare vesicants, arsenicals and mustards, and HSP90 inhibitors afford significant protection against skin damage.

CRISPR screen identifies CEBPB as contributor to dyskeratosis congenita fibroblast senescence via augmented inflammatory gene response.

Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDRs) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend life span in DC cells. Of the cellular clones isolated due to increased life span, 34% had a guide RNA (gRNA) targeting CEBPB, while gRNAs targeting WSB1, MED28, and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA sequencing to compare DC and control cells. Expression of inflammatory genes was found to be significantly elevated (P < 0.0001) in addition to a key subset of these inflammation-related genes [IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5]. which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere length-dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB's contribution in DC cells' senescence is ROS independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.

SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.

Abstract 657: IRAK1 regulated inflammatory signal from the leukemia cells modulates tumor microenvironment and promote leukemogenesis

Abstract Hematologic malignancy associated with fibroblast growth factor receptor 1 (FGFR1) translocations develops an aggressive stem cell leukemia/lymphoma (SCLL), 80% of which progress to AML and only ~15% show survival of &amp;gt; 15 months. In addition, 23.6% of de novo AML patients overexpress FGFR1, compared with healthy individuals. Therefore, investigating how tumor-intrinsic FGFR1 signaling modulates the tumor microenvironment and their contribution to leukemogenesis is not only important for understanding leukemia initiation and progression but also critical for the development of an efficient cure for AML patients. Interleukin Receptor Associated Kinase 1 (IRAK1) is a protein kinase that initiates a cascade of signaling events eventually leading to activation of proinflammatory genes, including IL-6, IL-10, TNF-α, and IFN-γ, which play critical roles in inflammation and innate immunity. The role of IRAK1 has been reported in several different subtypes of leukemia, including B cell-like diffuse large B-cell lymphoma (ABC DLBCL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and melanoma. Most previous IRAK1 studies, however, only focused on its survival and proliferation promoting roles inside the tumor cell itself. Since most of these studies engrafted human cancer cell lines into NSG mice, which lack a functional immune system, it was impossible to investigate the function of IRAK1 in modulating the tumor microenvironment. Using our well-established syngeneic SCLL mouse model, we defined IRAK1 gene as a critical target of FGFR1 in driving leukemogenesis. Our preliminary studies revealed that IRAK1 knockout (KO) in SCLL leukemia cells leads to T cell mediated clearance of the leukemia cells. In contrast, IRAK1 expressing mock control (MC) cells induce the accumulation of myeloid derived suppressor cells (MDSCs), which sequentially suppress T cell function to promote leukemia progression. These observations indicate a critical function for IRAK1 in modulating the tumor microenvironment and promoting leukemogenesis. Most importantly, pilot cytokine profiling of the MC and IRAK1 KO leukemia cells identified IFN-γ as one of the direct targets of IRAK1 in leukemia cells, and scRNA-Seq analysis revealed predominant expression of IFNGR1 and identified activation of the IFN-γ signaling pathway in leukemia induced MDSCs. Genes in this pathway include IFN-γ directly regulated genes, such as CD274 and SOCS3. Further engraftment using either IFNGR1-/- mice or IFNG KO leukemia cells revealed the involvement of this leukemia cell intrinsic, IRAK1 regulated IFN-γ in modulating the tumor microenvironment to promote leukemogenesis. In summary, our data suggests that leukemia cell-derived, IRAK1 regulated inflammatory signals act as critical modulators in establishing a tumor favorable microenvironment during leukemogenesis. Citation Format: Ting Zhang, Xiaocui Lu, Atsuko Matsunaga, Xuexiu Fang, Stephanie Fay Mori, John Cowell, Tianxiang Hu. IRAK1 regulated inflammatory signal from the leukemia cells modulates tumor microenvironment and promote leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 657.

ACAT1/SOAT1 Blockade Suppresses LPS-Mediated Neuroinflammation by Modulating the Fate of Toll-like Receptor 4 in Microglia.

Cholesterol is stored as cholesteryl esters by the enzymes acyl-CoA:cholesterol acyltransferases/sterol O:acyltransferases (ACATs/SOATs). ACAT1 blockade (A1B) ameliorates the pro-inflammatory responses of macrophages to lipopolysaccharides (LPS) and cholesterol loading. However, the mediators involved in transmitting the effects of A1B in immune cells is unknown. Microglial Acat1/Soat1 expression is elevated in many neurodegenerative diseases and in acute neuroinflammation. We evaluated LPS-induced neuroinflammation experiments in control vs. myeloid-specific Acat1/Soat1 knockout mice. We also evaluated LPS-induced neuroinflammation in microglial N9 cells with and without pre-treatment with K-604, a selective ACAT1 inhibitor. Biochemical and microscopy assays were used to monitor the fate of Toll-Like Receptor 4 (TLR4), the receptor at the plasma membrane and the endosomal membrane that mediates pro-inflammatory signaling cascades. In the hippocampus and cortex, results revealed that Acat1/Soat1 inactivation in myeloid cell lineage markedly attenuated LPS-induced activation of pro-inflammatory response genes. Studies in microglial N9 cells showed that pre-incubation with K-604 significantly reduced the LPS-induced pro-inflammatory responses. Further studies showed that K-604 decreased the total TLR4 protein content by increasing TLR4 endocytosis, thus enhancing the trafficking of TLR4 to the lysosomes for degradation. We concluded that A1B alters the intracellular fate of TLR4 and suppresses its pro-inflammatory signaling cascade in response to LPS.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) affects 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to 1) death through pulmonary embolism, 2) the post-thrombotic syndrome, or 3) chronic pulmonary hypertension resulting in significant chronic respiratory compromise. Inflammatory pathways are intricately involved in thrombus formation and therefore in VTE mechanisms. In pathological conditions, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal by several cell types including endothelial cells. Recent data indicated that the CD39/CD73 system involved in the metabolism of ATP into AMP might protects against thrombosis by downregulating the pro-inflammatory pathway of the inflammasome. ATP can also interact with the P2X7 receptor involved in inflammation causing a wide range of responses. To determine how the endothelial P2X7 receptor contributes to venous thromboembolism. HUVECs were incubated with BzATP, thrombin or both. HUVECs were primed with TNF-alpha prior to stimulation. We used immunofluorescence, western blot and real-time quantitative PCR analyses to assess P2X7 expression by endothelial cells, activation of p38 and NFκB activation and expression of pro-inflammatory and pro-coagulant genes. We confirmed that endothelial cells expressed P2X7 in vitro in HUVECs and in vivo after induction of venous thrombosis by ligation of the inferior vena cava. Treatment of endothelial cells with BzATP and thrombin induced the activation of p38 and NFκB signaling pathways. This appears to be associated with an increased expression of IL-1β and downregulation of thrombomodulin expression. The expression of adhesion molecules, ICAM-1 and VCAM-1, tends to increase. Our data suggest that ATP released in the extracellular space following cell damage or activation induced a pro-inflammatory response in endothelial cells through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

PARG suppresses tumorigenesis and downregulates genes controlling angiogenesis, inflammatory response, and immune cell recruitment

Chemokines are highly expressed in tumor microenvironment and play a critical role in all aspects of tumorigenesis, including the recruitment of tumor-promoting immune cells, activation of cancer-associated fibroblasts, angiogenesis, metastasis, and growth. Poly (ADP-ribose) polymerase (PARP) is a multi-target transcription regulator with high levels of poly(ADP-ribose) (pADPr) being reported in a variety of cancers. Furthermore, poly (ADP-ribose) glycohydrolase (PARG), an enzyme that degrades pADPr, has been reported to be downregulated in tumor tissues with abnormally high levels of pADPr. In conjunction to this, we have recently reported that the reduction of pADPr, by either pharmacological inhibition of PARP or PARG’s overexpression, disrupts renal carcinoma cell malignancy in vitro. Here, we use 3 T3 mouse embryonic fibroblasts, a universal model for malignant transformation, to follow the effect of PARG upregulation on cells’ tumorigenicity in vivo. We found that the overexpression of PARG in mouse allografts produces significantly smaller tumors with a delay in tumor onset. As downregulation of PARG has also been implicated in promoting the activation of pro-inflammatory genes, we also followed the gene expression profile of PARG-overexpressing 3 T3 cells using RNA-seq approach and observed that chemokine transcripts are significantly reduced in those cells. Our data suggest that the upregulation of PARG may be potentially useful for the tumor growth inhibition in cancer treatment and as anti-inflammatory intervention.

Endoplasmic Reticulum Stress in Colonic Mucosa of Ulcerative Colitis Patients Is Mediated by PERK and IRE1 Pathway Activation.

Ulcerative colitis (UC) is characterized by a chronic overproduction of proinflammatory cytokines. During an acute phase, the endoplasmic reticulum (ER) is overloaded and the protein folding process is impaired, a condition named ER stress. This state induces a response (unfolded protein response (UPR)), initiated by the activation of IRE1/Xbp-1, PERK/eIF2α, and ATF6 pathways, which has previously been linked to intestinal inflammation in experimental models. ER stress and UPR activation trigger the activation of proinflammatory, autophagy, and apoptosis genes, in addition to promoting protein degradation. Therefore, the goal of this study was to evaluate the activation of ER stress and UPR in colonic mucosa of UC patients. Patient and Methods. Transcriptional analysis of ER stress- and UPR-related genes was performed by qPCR from intestinal mucosa of patients with UC. We also performed in situ hybridization (ISH) and immunohistochemistry (IHQ) of PERK/eIF2α and IRE1/Xbp-1 pathways and UPR-related chaperones. Results. We first evaluated inflammatory genes via qPCR, and we observed that all analyzed proinflammatory transcripts were upregulated in UC patients. ISH and IHQ images showed that ER stress is activated via PERK/eIF2α and IRE1/Xbp-1 pathways not only in intestinal epithelial cells but also in cells of the lamina propria of UC colonic mucosa. Transcriptional analysis confirmed that EIF2AK3 was upregulated in UC patients. UPR-related genes, such as ATF3, STC2, and DDIT3, along with the chaperones and cochaperones DNAJC3, CALR, HSP90B1, and HSPA5, were also upregulated in UC patients. In addition, we observed that proapoptotic and autophagy genes (Bax and ATG6L1, respectively) were also upregulated. Conclusion. Our results suggest that ER stress and UPR are indeed activated in UC patients and this may contribute to the chronic inflammatory process seen in UC. The increased apoptosis and autophagy markers further support the activation of these findings once they are activated to counterbalance tissue damage. These findings provide new insights into the molecular mechanisms that maintain UC activity and open new possibilities to attenuate intestinal inflammation.

P.124: Withaferin A Prevents Inflammatory Phenotype in Cultured Islets

Introduction: Islets undergo enormous ischemic, and mechanical stress throughout clinical isolation steps which affect the quality of processed islets available for transplantation. Isolated islets produce pro-inflammatory cytokines and chemokines that are further exacerbated during culture. Transplantation of islets with a strong pro-inflammatory phenotype may induce the activation of IBMIR and affect overall transplant outcomes. Strategies to improve the outcome of isolation and protection of islets from pro-inflammatory gene activation is an unmet requirement. Here, we sought to investigate the protective/anti-inflammatory effect of Withaferin A: a plant-derived inhibitor of Nuclear Factor Kappa B (NFκB). Methods: A bisected split lobe isolation technique was adopted where the head and tail were isolated subsequently with a control or WA-treated (2µM) medium during isolation. WA was added in the isolation solutions including preservation media, Collagenase, Recombination Media, Wash Buffer, and also in islet culture media. The islets isolated from (+/-) WA treatment during isolation was analyzed for the viability, function, and intracellular pro-inflammatory cytokine expression. Western blots were performed to study the activation of transcription factors and stress-regulated genes post-culture. Exosomes were isolated and the presence of pro-inflammatory mediators was examined. Results: Islet damage was reduced in the WA-treated group as shown by miRNA 375 release. No significant difference in the isolation outcomes such as yield, purity, and viability of islets between WA and the control group was seen. The purity of islets post-culture was better maintained in the WA treated group, compared to the control group (Figure 1). No difference in viability and insulin secretion was observed post-culture in the two groups. The western blotting of WA-cultured islets (at 24 and 48H) showed a significant reduction in the activation of p65 NFκB (P-s536) and P-IκBα (P-s32). Interestingly, WA treatment also synergistically reduced the activation of PERK (P-T982) and IRE1 mediated ER stress signals compared to the non-WA treated cultured islets. There was a significant reduction in the intracellular mRNA expression (Tissue factor, MCP1, and IL-8) and subsequent exosomal content of pro-inflammatory mediators (IP-10, IL-6, and IL-2) in the WA treated cultured islets (24 and 48H) compared to non-WA treated islets. Conclusion: Withaferin A treatment prevented islet damage during isolation and also prevented the generation of a proinflammatory phenotype in islets post-culture. The study proposes a novel protective strategy to control islet inflammation during isolation and peritransplant steps which may improve the functional outcomes of transplantation.

The Flavonoid Kurarinone Regulates Macrophage Functions via Aryl Hydrocarbon Receptor and Alleviates Intestinal Inflammation in Irritable Bowel Syndrome.

BackgroundIrritable bowel syndrome (IBS) is characterized with abdominal pain, bloating, and changes in bowel habits, and dealing with IBS is still a clinical challenge. The pathogenesis of IBS has been reported to be linked to low-grade mucosal inflammation, and macrophages contribute to the pathological process of this disease. Kurarinone (KAR), a flavanoid derived from Sophora flavescens, has been found medically effective in many inflammatory conditions and cancers. KAR was previously reported to inhibit LPS-induced expression of inflammatory cytokines in macrophages, whether and how KAR regulates the functions of macrophage in IBS remains to be elusive.MethodsWe established a TNBS-induced IBS mouse model, in which KAR was administrated, and mucosal cytokine expression was measured by qRT-PCR. Additionally, mouse macrophages were generated in vitro and their responses to LPS were evaluated by flow cytometry and qRT-PCR. AhR+/+ or AhR−/- macrophages were transferred into DTx-treated CD11b-DTR transgenic mice to investigate the role of AhR in IBS. We collected colonic biopsies and peripheral blood samples from 64 patients with IBS, and analyzed AhR expression by qRT-PCR.ResultsWe found KAR effectively alleviated visceral hypersensitivity and maintained intestinal barrier functions in mice with IBS. KAR inhibited LPS-induced macrophage activation and expression of pro-inflammatory genes, while increased anti-inflammatory gene expression including IL-10 in an AhR-dependent manner. Using macrophage-depleted mice, we found that chimera mice with AhR−/- macrophages were more susceptible to TNBS-induced IBS and the therapeutic effect of KAR on IBS was significantly impaired in mice with AhR−/- macrophages. Additionally, we found AhR expression in macrophages of IBS patients was associated with the disease severity.ConclusionOur findings provide new evidences that KAR regulates IBS development via macrophage-intrinsic AhR. KAR might show promise as an immunomodulatory therapeutic agent in treating IBS.

Gene expression analysis of subchondral bone, cartilage, and synovium in naturally occurring equine palmar/plantar osteochondral disease.

Osteoarthritis (OA) is a disease of the entire joint but the relationship between pathological events in various joint tissues is poorly understood. We examined concurrent changes in bone, cartilage, and synovium in a naturally occurring equine model of joint degeneration. Joints (n = 64) were grossly assessed for palmar/plantar osteochondral disease (POD) in racehorses that required euthanasia for unrelated reasons and assigned a grade of 0 (n = 34), 1 (n = 17), 2 or 3 (n = 13) using a recognized grading scheme. Synovium, cartilage, and subchondral bone were collected for histological and gene expression analysis. Relations between POD grade, cartilage histological score, and gene expression levels were examined using one-way analysis of variance or Kruskal-Wallis test and Spearman's correlation coefficient with corrections for multiple comparisons. Cartilage histological score increased in joints with POD grade 1 (p = 0.002) and 2 or 3 (p < 0.001) compared to 0. At grade 1, expression of COL1A1, COL2A1, and MMP1 increased and BGN decreased in subchondral bone while expression of BGN and ACAN decreased in cartilage. These changes further progressed at grades 2 and 3. POD grades 2 and 3 were associated with decreased expression of osteoclast inhibitor OPG and increased markers of cartilage degeneration (MMP13, COL1A1). Expression of the vascular endothelial growth factor decreased with POD grade and negatively correlated with cartilage histological score. Synovium showed no histological or transcriptomic changes related to pathology grade. Cartilage degeneration in POD is likely to be secondary to remodeling of the subchondral bone. Limited activation of proinflammatory and catabolic genes and moderate synovial pathology suggests distinct molecular phenotype of POD compared with OA.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to: – death through pulmonary embolism; – the post-thrombotic syndrome, or; – chronic pulmonary hypertension resulting in significant chronic respiratory compromise. Inflammatory pathways are intricately involved in venous thromboembolism mechanisms. In pathological conditions, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal. Recent data indicated that the CD39/CD73 system involved in the metabolism of ATP into AMP might protect against thrombosis by downregulating the pro-inflammatory pathway of the inflammasome. ATP can also interact with the P2X7 receptor involved in inflammation causing a wide range of responses. Determine how the endothelial P2X7 receptor contribute to venous thromboembolism. HUVEC were incubated with BzATP, thrombin or both. In some experiment, HUVEC were primed with TNFa prior to stimulation. We used immunofluorescence, western blot and real-time quantitative PCR analyses to assess P2X7 expression by endothelial cells, activation of p38 and NFkB activation and expression of pro-inflammatory and procoagulant genes. We confirmed that endothelial cells expressed P2X7 in vitro in HUVECs and in vivo after induction of venous thrombosis by ligation of the inferior vena cava. Treatment of endothelial cells with BzATP and thrombin induced the activation of p38 and NFkB signaling pathways. This was associated with an increased expression of IL-1b and tissue factor and downregulation of thrombomodulin expression. Our data suggest that ATP released in the extracellular space following cell damage or activation induced the inflammasome in endothelial through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

Treatment of diabetic peripheral neuropathy with engineered mesenchymal stromal cell-derived exosomes enriched with microRNA-146a provide amplified therapeutic efficacy

Diabetic peripheral neuropathy (DPN) is one of the most prevalent chronic complications of diabetes mellitus with no effective treatment. We recently demonstrated that mesenchymal stromal cell (MSC)-derived exosomes (exo-naïve) alleviate neurovascular dysfunction and improve functional recovery. MicroRNA (miRNA), one of the exosomal cargos, downregulates inflammation-related genes, resulting in suppression of pro-inflammatory gene activation. In the present study, we developed engineered MSC-exosomes loaded with miR-146a (exo-146a) and compared the therapeutic effects of exo-146a with exo-naïve in diabetic (db/db) mice with DPN. Exo-146a possesses a high loading capacity, robust ability to accumulate in peripheral nerve tissues upon systemic administration, and evokes substantially enhanced therapeutic efficacy on neurological recovery compared with exo-naïve. Treatment of DPN in diabetic mice with exo-146a for two weeks significantly increased and decreased nerve conduction velocity, and thermal and mechanical stimuli threshold, respectively, whereas it took four weeks of exo-naive treatment to achieve these improvements. Compared with exo-naïve, exo-146a significantly suppressed the peripheral blood inflammatory monocytes and the activation of endothelial cells via inhibiting Toll-like receptor (TLR)-4/NF-κB signaling pathway. These data provide a proof-of-concept about both the feasibility and efficacy of the exosome-based gene therapy for DPN. The translation of this approach to the clinic has the potential to improve the prospects for people who suffer from DPN.

The Cannabinoid Receptor Agonist, WIN-55212-2, Suppresses the Activation of Proinflammatory Genes Induced by Interleukin 1 Beta in Human Astrocytes.

Background: Alterations of astrocyte function play a crucial role in neuroinflammatory diseases due to either the loss of their neuroprotective role or the gain of their toxic inflammatory properties. Accumulating evidence highlights that cannabinoids and cannabinoid receptor agonists, such as WIN55,212-2 (WIN), reduce inflammation in cellular and animal models. Thus, the endocannabinoid system has become an attractive target to attenuate chronic inflammation in neurodegenerative diseases. However, the mechanism of action of WIN in astrocytes remains poorly understood. Objective: We studied the immunosuppressive property of WIN by examining gene expression patterns that were modulated by WIN in reactive astrocytes. Materials and Methods: Transcriptomic analysis by RNA-seq was carried out using primary human astrocyte cultures stimulated by the proinflammatory cytokine interleukin 1 beta (IL1β) in the presence or absence of WIN. Real-time quantitative polymerase chain reaction analysis was conducted on selected transcripts to characterize the dose-response effects of WIN, and to test the effect of selective antagonists of cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptors (PPAR). Results: Transcriptomic analysis showed that the IL1β-induced inflammatory response is robustly inhibited by WIN pretreatment. WIN treatment alone also induced substantial gene expression changes. Pathway analysis revealed that the anti-inflammatory properties of WIN were linked to the regulation of kinase pathways and gene targets of neuroprotective transcription factors, including PPAR and SMAD (mothers against decapentaplegic homolog). The inhibitory effect of WIN was dose-dependent, but it was not affected by selective antagonists of CB1 or PPAR. Conclusions: This study suggests that targeting the endocannabinoid system may be a promising strategy to disrupt inflammatory pathways in reactive astrocytes. The anti-inflammatory activity of WIN is independent of CB1, suggesting that alternative receptors mediate the effects of WIN. These results provide mechanistic insights into the anti-inflammatory activity of WIN and highlight that astrocytes are a potential therapeutic target to ameliorate neuroinflammation in the brain.