Infiltration Of Pro-inflammatory Cells Research Articles

The Therapeutic Potential of Metformin for Attenuating Postsurgical Adhesion Band Formation

Objectives: Intra-abdominal adhesions are the most frequently occurring postoperative complication following abdominopelvic surgery. Peritoneal adhesion formation can lead to infertility, chronic pelvic pain, and intestinal obstruction. Several studies have shown the potential role of metformin in reducing inflammation. Here, we explored the therapeutic potency of Metformin against postsurgical adhesion band formation. Methods: We adopted Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for the experimental protocol. Peritoneal adhesions were examined macroscopically .Metformin (100 mg/kg) was administered intraperitoneally in male albino Wistar rats. After 9 days, macroscopic evidence and score of fibrotic bands based on scaling methods were performed and evaluated by the Nair and Leach scoring system. Moreover, the anti-inflammatory and antifibrosis effects were investigated by using hematoxylin and eosin, Masson's trichrome staining, the enzyme-linked immunosorbent assay (ELISA), and reverse transcription polymerase chain reaction (RT-PCR). Results: Metformin inhibited the stabilization of adhesion bands and appeared to elicit antiinflammatory responses by attenuating submucosal edema, suppressing proinflammatory cytokines, decreasing proinflammatory cell infiltration, and inhibiting oxidative stress at the site of peritoneal surgery. We also showed that metformin prevents fibrotic adhesion band formation by reducing excessive collagen deposition and suppression of profibrotic gene expression at the peritoneum adhesion tissues via modulation of Col 1A1/A3. Conclusion: These results supported the potential application of metformin in preventing postsurgical adhesion band formation by inhibiting key pathologic responses of inflammation and fibrosis in patients post-surgery.

Targeting Angiotensin-converting Enzyme Inhibitor via Enalapril Reduces Postsurgical Adhesion Band Formation

Background: Postoperative adhesions commonly occur after abdominal surgery and can lead to significant complications. There is increasing evidence that targeting the renin-angiotensin system (RAS) can reduce inflammation and fibrosis. This study investigates the therapeutic potential of enalapril, an RAS inhibitor, in a rat model of postsurgical adhesion band formation. Methods: A total of 12 male albino Wistar rats received intraperitoneal administration of enalapril (10 mg/kg). After 9 days, the anti-inflammatory and antifibrotic effects were evaluated using RTPCR and ELISA, alongside hematoxylin and eosin and Masson's trichrome staining. Result: The statistical analysis of findings showed that enalapril significantly reduced the frequency and stability of adhesion bands. It attenuated submucosal edema by suppressing pro-inflammatory cytokines, decreasing pro-inflammatory cell infiltration, and inhibiting oxidative stress at the peritoneal surgery site. Additionally, enalapril inhibited fibrotic adhesion band formation by reducing collagen deposition and suppressing the expression of profibrotic genes in peritoneal adhesion tissues. Conclusion: These findings demonstrate the therapeutic potential of enalapril in preventing postsurgical adhesion band formation by inhibiting key pathological responses of inflammation and fibrosis, supporting its use as a preventive treatment in postoperative adhesion management.

Acidovorax temperans skews neutrophil maturation and polarizes Th17 cells to promote lung adenocarcinoma development

Change within the intratumoral microbiome is a common feature in lung and other cancers and may influence inflammation and immunity in the tumor microenvironment, affecting growth and metastases. We previously characterized the lung cancer microbiome in patients and identified Acidovorax temperans as enriched in tumors. Here, we instilled A. temperans in an animal model driven by mutant K-ras and Tp53. This revealed A. temperans accelerates tumor development and burden through infiltration of proinflammatory cells. Neutrophils exposed to A. temperans displayed a mature, pro-tumorigenic phenotype with increased cytokine signaling, with a global shift away from IL-1β signaling. Neutrophil to monocyte and macrophage signaling upregulated MHC II to activate CD4+ T cells, polarizing them to an IL-17A+ phenotype detectable in CD4+ and γδ populations (T17). These T17 cells shared a common gene expression program predictive of poor survival in human LUAD. These data indicate bacterial exposure promotes tumor growth by modulating inflammation.

Protective Effect of Turmeric against Aluminium-induced Prostate Damage in Rats

This study investigates the potential protective properties of turmeric (Curcuma longa L.) against aluminium (Al) toxicity-induced prostate damage in male rats. The administration of AlCl3 to rats resulted in decreased body weight, increased oxidative stress markers, hormonal depletion, apoptosis induction, and pro-inflammatory cell infiltration in the prostate tissue. Additionally, histopathological examination revealed various abnormalities. However, co-treatment with turmeric extract improved body weight, increased testosterone levels, reduced apoptosis, and mast cell infiltration. C. longa L. exhibited strong antioxidant and anti-inflammatory effects, highlighting its potential as a phytomedicine against reproductive disorders like prostatitis induced by Al exposure.

Multiple Sclerosis: Current Knowledge of the Pathology and Use of Monoclonal Antibodies as a Promising Therapy

Multiple sclerosis is an autoimmune condition characterized by an inflammatory condition and neuron demyelination, leading to a significant deterioration in the patient's quality of life as the disease progresses. The immune system reactivity in this pathology is mainly mediated by reactive T lymphocytes against myelin. The harmful substances production and proinflammatory cell infiltration occur. Currently, there is no cure, so treatment focuses on reducing the development of the individual's long-term disability by addressing symptoms, acute exacerbations, and slowing progress. The traditional treatment includes immunosuppressive substances such as corticosteroids and interferons. However, an approach to more specific, highly effective therapies such as monoclonal antibodies is currently being sought. Ofatumumab, ocrelizumab, alemtuzumab, and rituximab are commercialized monoclonal antibodies. Likewise, therapies in the research phase, such as ublituximab, inebilizumab, GNbAC1, and elezanumab, can be found. Therefore, research must continue to have more information to increase the availability of therapeutic options for patients.

Proteopathy Linked to Exon-Skipping Isoform of RGR-Opsin Contributes to the Pathogenesis of Age-Related Macular Degeneration.

Proteopathy is believed to contribute to age-related macular degeneration (AMD). Much research indicates that AMD begins in the retinal pigment epithelium (RPE), which is associated with formation of extracellular drusen, a clinical hallmark of AMD. Human RPE produces a drusen-associated abnormal protein, the exon Ⅵ-skipping splice isoform of retinal G protein-coupled receptor (RGR-d). In this study, we investigate the detrimental effects of RGR-d on cultured cells and mouse retina. ARPE-19 cells were stably infected by lentivirus overexpressing RGR or RGR-d and were treated with MG132, sometimes combined with or without endoplasmic reticulum (ER) stress inducer, tunicamycin. RGR and RGR-d protein expression, degeneration pathway, and potential cytotoxicity were explored. Homozygous RGR-d mice aged 8 or 14months were fed with a high-fat diet for 3months and then subjected to ocular examination and histopathology experiments. We confirm that RGR-d is proteotoxic under various conditions. In ARPE-19 cells, RGR-d is misfolded and almost completely degraded via the ubiquitin-proteasome system. Unlike normal RGR, RGR-d increases ER stress, triggers the unfolded protein response, and exerts potent cytotoxicity. Aged RGR-d mice manifest disrupted RPE cell integrity, apoptotic photoreceptors, choroidal deposition of complement C3, and CD86+CD32+ proinflammatory cell infiltration into retina and RPE-choroid. Furthermore, the AMD-like phenotype of RGR-d mice can be aggravated by a high-fat diet. Our study confirmed the pathogenicity of the RGR splice isoform and corroborated a significant role of proteopathy in AMD. These findings may contribute to greater comprehension of the multifactorial causes of AMD.

Taurodeoxycholate ameliorates DSS-induced colitis in mice

BackgroundInflammatory bowel disease (IBD) is typically managed using medications such as 5-aminosalicylic acid (5-ASA), glucocorticoids, anti-TNFα Ab, or anti-IL-12/23 Ab. However, some patients do not respond well to these treatments or frequently experience relapses. Therefore, alternative therapeutic options are needed. Since the activation of the inflammasome is crucial to the pathogenesis of IBD, inhibiting the inflammasome may be beneficial for patients. Materials and methodsWe tested the efficacy of taurodeoxycholate (TDCA), which is a known G-protein coupled receptor 19 (GPCR19) agonist, in a mouse colitis model induced by dextran sodium sulfate (DSS). ResultsIn the mouse colitis model, TDCA prevented loss of body weight, shortening of the colon, production of pro-inflammatory cytokines, infiltration of pro-inflammatory cells, and mucosal ulceration in the colon. In vitro, TDCA inhibited the activation of NF-κB in bone marrow-derived macrophages (BMDMs) by activating the cAMP-PKA axis. TDCA downregulated the expression of purinergic receptor P2X7 (P2X7R) and enhanced the colocalization of P2X7R with GPCR19, and inhibited the Ca2+ mobilization of BMDMs when stimulated with ATP or BzATP, which plays a pivotal role in activating the NLRP3 inflammasome (N3I) via P2X7R. TDCA inhibited the oligomerization of NLRP3-ASC and downregulated the expression of NLRP3 and ASC, as well as suppressed the maturation of pro-caspase-1 and pro-IL-1β. TDCA also increased the percentage of M2 macrophages while decreasing the number of M1 macrophages, Th1, Th2, and Th17 cells in the colon. ConclusionTDCA ameliorated DSS-induced colitis in mice, possibly by inhibiting both the priming phase (via the GPCR19-cAMP-PKA-NF-κB axis) and the activation phase (via the GPCR19-P2X7R-NLRP3-Caspase 1-IL-1β axis) of N3I signaling.

Unsymmetrical Phosphodiesters as GPR84 Antagonists with High Blood Exposure for the Treatment of Lung Inflammation.

GPR84 is a proinflammatory G protein-coupled receptor that mediates myeloid immune cell functions. Blocking GPR84 with antagonists is a promising approach for treating inflammatory and fibrotic diseases. Previously, a GPR84 antagonist 604c, with a symmetrical phosphodiester structure, has displayed promising efficacy in a mouse model of ulcerative colitis. However, the low blood exposure resulting from physicochemical properties prevented its uses in other inflammatory diseases. In this study, a series of unsymmetrical phosphodiesters with lower lipophilicity were designed and tested. The representative compound 37 exhibited a 100-fold increase in mouse blood exposure compared to 604c while maintaining in vitro activity. In a mouse model of acute lung injury, 37 (30 mg/kg, po) significantly reduced the infiltration of proinflammatory cells and the release of inflammatory cytokines and ameliorated pathological changes equally or more effectively than N-acetylcysteine (100 mg/kg, po). These findings suggest that 37 is a promising candidate for treating lung inflammation.

OP31 High fat diet exacerbates colitis through microbial metabolite deoxycholic acids induced-ferroptosis

Abstract Background High-fat diet (HFD) is closely related to the occurrence and development of IBD, which increases the level of deoxycholic acid (DCA). DCA-treated mice showed pro-inflammatory cell infiltration and colonic inflammation. Ferroptosis, a newly recognized form of cell death characterized by iron overload and the generation of reactive oxygen species (ROS) has recently been observed in colitis. Whether DCA affects ferroptosis and whether it contributes to colitis has not been explored. Methods Twenty-four Female C57BL/6 mice were randomly divided into 4 groups: control group, DCA group, DSS group, and DSS plus DCA group. Real-time PCR and Western blotting were used to detect the transcription and protein expression of ferroptosis-related genes. RNA sequencing was used to analyze the changes of differential genes in the intestinal tissue of mice treated with DCA. Transmission electron microscope was employed to observed the changes of intestinal epithelial cell morphology and intracellular mitochondria. Finally, the binding force between DCA and HIF-2α was calculated by molecular dynamics to explore the structural biological basis of the interaction between DCA and HIF-2α, and screen innovative drugs. Results We found that DCA exacerbated the disease activity index, histological damage and cell death in mice with colitis. In vivo, ferroptosis KEGG pathway was enriched by RNA sequencing in colonic tissues. DCA treatment significantly induced ferroptosis in DSS-induced mice, as evidenced by iron overload, ROS and MDA production, accompanied by decreased expression of GPX4 and increased expression of ACSL4. Transmission electron microscopy also confirmed the occurrence of ferroptosis in colonic tissues. In vitro, DCA induced ferroptosis were also observed in IEC-6 cells and Caco-2 cells under the combined treatment of Lipopolysaccharide (LPS). Interestingly, RNA sequencing revealed that the expression levels of the apical iron divalent metal transporter-1 (DMT1) were significantly upregulated in DCA treated-mice with colitis. DMT1 is a direct target gene of hypoxia inducible factor (HIF)-2α. We also found that DCA could increase the level of DMT1 and HIF-2α in the intestinal epithelial cell lines. Pharmacological inhibition of HIF-2α significantly inhibited the expression of DMT1 and blocked the promotion of ferroptosis by DCA. These data provide a mechanistic basis for the widely reported link between ferroptosis and colitis risk. Conclusion Taken together, these findings suggest that DCA aggravates colitis through the induction of ferroptosis in the HIF2α-DMT1 signaling pathway and provides a new perspective for UC prevention in the future.

β-Caryophyllene Ameliorates 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis through the Downregulation of Mitogen-Activated Protein Kinase/EGR1/TSLP Signaling Axis

Atopic dermatitis (AD) is one of the most common inflammatory skin diseases accompanied by severe itching. β-caryophyllene (BCP), which displays anti-inflammatory activity, is a natural agonist of cannabinoid receptor 2. However, the therapeutic effects of BCP on atopic dermatitis (AD) remain poorly understood. The current study aimed to evaluate the topical therapeutic efficacy of BCP in an AD-like mouse model. Thymic Stromal Lymphopoietin (TSLP) is a keratinocyte-derived cytokine that drives AD pathogenesis. This study also investigated the effect of BCP on the interleukin 4 (IL-4)-induced expression of TSLP in HaCaT keratinocytes. We found that the topical application of BCP alleviated AD-like skin inflammation and inhibited the infiltration of proinflammatory cells into skin lesions. Moreover, the topical application of BCP reduced EGR1 (Early Growth Response 1) and TSLP expression in AD-like skin lesions. We also found that BCP inhibited IL-4-induced TSLP expression by downregulating mitogen-activated protein kinase (MAPK)-mediated EGR1 expression in HaCaT keratinocytes. These findings demonstrate that BCP ameliorates DNCB-induced AD-like skin lesions through the downregulation of the MAPK/EGR1/TSLP signaling axis. BCP may be applicable for developing topical therapeutic agents for chronic skin inflammatory diseases, such as AD.

A biochemical and histology experimental approach to investigate the adverse effect of chronic lead acetate and dietary furan on rat lungs.

Despite lead widespread environmental pollution, its effect on humans and livestock's respiratory systems remains inadequately investigated. Similarly, furan is industrially relevant with enormous environmental presence. Lead and furan can be ingested -via lead pipes contaminated water and heat-treated food respectively. Thus, humans are inadvertently exposed continuously. Lead toxicity is well studied, and furan have earned a position on the IARC's list of carcinogens. Here, we evaluate the effect of co-exposure to lead and furan on rat lungs. Thirty Wistar rats were grouped randomly into six cohorts (n = 6) consisting of a control group, furan alone group, lead acetate (PbAc) alone group and three other groups co-exposure to graded PbAc (1, 10 & 100µg/L) alongside a constant furan (8mg/kg) dose. After twenty-eight days, enzymatic and non-enzymatic antioxidant, oxidative stress and inflammatory biomarkers were biochemically evaluated. The ELISA-based technique was used to measure oxidative-DNA damage (8-OHG), tumour protein 53 (TP53) expressed and tumour necrotic factor-alpha (TNF-α) level. Dose-dependent increases (p < 0.05) in reactive oxygen and nitrogen species, malondialdehyde, nitric oxide, myeloperoxidase, TNF-α and TP53 level, with an associated decrease (p < 0.05) in enzymatic and non-enzymatic antioxidants were observed in the furan, PbAc and the co-treated rats relative to the control. In addition, PbAc and furan treatment impaired the histoarchitectural structures of rat lungs, exemplified by pro-inflammatory cell infiltration and trafficking into the bronchioles and alveolar spaces. Co-exposure to furan and PbAc may contribute to lung dysfunction via loss of redox balance, genomic damage/instability, inflammation and disrupted histoarchitectural features.

Regnase-1 overexpression as a novel gene therapy approach for pulmonary hypertension

Abstract Introduction Pulmonary hypertension (PH) is a chronic disease characterized by persistently increases pulmonary arterial pressure, leading on the long term to right ventricular overload and ultimately right heart failure. Until now, therapy focuses on alleviating symptoms and hence the development of a novel strategy for the treatment of this disease remains highly relevant. Purpose The importance of pro-inflammatory cytokines in progression of PH has already been established. Interestingly, patients with increased plasma levels of inflammatory markers present with more severe disease. Regnase-1 is a newly identified endoribonuclease cleaving the mRNA of pro-inflammatory cytokines therefore diminishing their levels of expression. Hence, we hypothesize that regnase-1 overexpression in lung tissue can be translated into a novel therapeutic approach for PH. Methods In vitro experiments were performed in human lung endothelial cells (HUVECs). For our in vivo study, mice were subjected to hypoxic conditions (10% O2) for 3 weeks. Regnase-1 overexpression was achieved in lung endothelial cells by transduction with targeted adeno-associated virus serotype 2 (AAV2-ESGHGYF), administered systemically by tail vein injection 3 days after exposure to hypoxia (early therapy approach). AAV2-EGFP treated mice served as controls. Right ventricular function was monitored by echocardiography and right ventricular systolic pressure was measured by right heart catheterization. The degree of pro-inflammatory cell infiltration was monitored by immunohistochemistry. Induction of fetal gene programme in the right ventricle was measured by real time qPCR. Fibrosis deposition in lung tissue was determined by Sirius Red staining of frozen sections. Results Regnase-1 overexpression led to a significantly decreased level of hypoxia-induced pro-inflammatory cytokines in HUVECs. Importantly, we could show decreased regnase-1 levels in lung endothelial cells of mice subjected to hypoxia for 3 weeks. On the other hand, regnase-1 reintroduction led to a significant improvement in right ventricular function and decreased pulmonary pressure in mice placed under hypoxic conditions. Moreover, the described gene therapy approach induced normalization of fetal gene programme in the cardiac tissue and reduced pro-inflammatory cell infiltration in lungs. Additionally, we could determine mitigation of pulmonary extracellular matrix deposition and fibrosis development in mice receiving AAV2-regnase1 as compared to controls. Conclusion AAV-mediated regnase-1 overexpression in lung endothelial cells results in amelioration of pathological events leading to PH in mice subjected to hypoxia, when the therapeutic AAV is delivered 3 days after disease induction. Further experiments will determine whether this method can be successful used for reversing already established PH. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DZHK

Liang-Ge decoction ameliorates acute lung injury in septic model rats through reducing inflammatory response, oxidative stress, apoptosis, and modulating host metabolism.

Liang-Ge decoction (LG) has been used in the treatment of early stage of spesis and can ameliorate sepsis-associated lung injury. However, the mechanism of LG on sepsis-associated lung injury remains unknown. In this study, we established a rat model of sepsis-associated lung injury using the cecal ligation and puncture (CLP) method, and investigated the therapeutic effects of LG on lung injury in rats with sepsis. In addition, the anti-inflammatory, anti-oxidative and anti-apoptotic effects of LG on sepsis-associated lung injury model rats were evaluated. Besides, untargeted metabolomics was used to investigate the regulation of metabolites in rats with sepsis-associated lung injury after LG treatment. Our results showed that LG could decrease the wet/dry (W/D) ratio in lung and the total cell count and total protein concentration in bronchoalveolar lavage fluid (BALF) in septic model rats. Hematoxylin and eosin (HE) staining showed that LG reduced the infiltration of pro-inflammatory cells in lung. In addition, LG treatmment down-regulated the gene and protein expression of pro-inflammatory cytokins in lung tissue and BALF. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were increased and the level of methane dicarboxylic aldehyde (MDA) was decreased in lung tissue homogenate in septic model rats after LG treament. Moreover, the numbers of apoptotic cells in lung were reduced and the activity of lactic dehydrogenase (LDH) in BALF was decreased in septic model rats after LG treament. Untargeted metabolomics analysis showed that LG treatment affected the levels of 23 metabolites in lung in septic model rats such as citric acid, methionine, threonine, alpha-ketoglutaric acid, and inositol, these metabolites were associated with the glycine, serine and threonine metabolism, cysteine and methionine metabolism, inositol phosphate metabolism and citrate cycle (TCA cycle) pathways. In conclusion, our study demonstrated the therapeutic effetcts of LG on sepsis-associated lung injury model rats. Moreover, LG could inhibit the inflammatory response, oxidative stress, apoptosis and regulate metabolites related to glycine, serine and threonine metabolism, cysteine and methionine metabolism, inositol phosphate metabolism and TCA cycle in lung in sepsis-associated lung injury model rats.

Blocking DCIR mitigates colitis and prevents colorectal tumors by enhancing the GM-CSF-STAT5 pathway.

Dendritic cell immunoreceptor (DCIR; Clec4a2), a member of the C-type lectin receptor family, plays important roles in homeostasis of the immune and bone systems. However, the intestinal role of this molecule is unclear. Here, we show that dextran sodium sulfate (DSS)-induced colitis and azoxymethane-DSS-induced intestinal tumors are reduced in Clec4a2-/- mice independently of intestinal microbiota. STAT5 phosphorylation and expression of Csf2 and tight junction genes are enhanced, while Il17a and Cxcl2 are suppressed in the Clec4a2-/- mouse colon, which exhibits reduced infiltration of neutrophils and myeloid-derived suppressor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) administration ameliorates DSS colitis associated with reduced Il17a and enhanced tight junction gene expression, whereas anti-GM-CSF exacerbates symptoms. Furthermore, anti-NA2, a ligand for DCIR, ameliorates colitis and prevents colorectal tumors. These observations indicate that blocking DCIR signaling ameliorates colitis and suppresses colonic tumors, suggesting DCIR as a possible target for the treatment of these diseases.

Time Series Transcriptomic Analysis by RNA Sequencing Reveals a Key Role of PI3K in Sepsis-Induced Myocardial Injury in Mice.

Septic cardiomyopathy is the main complication and cause of death of severe sepsis with limited therapeutic strategy. However, the molecular mechanism of sepsis-induced cardiac injury remains unclear. The present study was designed to investigate differentially expressed genes (DEGs) involved in the pathogenesis of septic cardiomyopathy induced by cecal ligation and puncture (CLP) in mice. Male C57BL/6J mice (8–10 weeks old) were subjected to CLP with 21-gauge needles for 24, 48, and 72 h. Myocardial function was assessed by echocardiography. The pathological changes of the heart were evaluated by hematoxylin and eosin as well as immunohistochemical staining. Time series RNA sequencing was utilized to investigate the gene expression profiles. CLP surgery resulted in a significant decrease of animal survival rate and left ventricle contractile function, and an increase in cardiac dilation and infiltration of proinflammatory cells including Mac-2+ macrophages in a time-dependent manner. RNA sequencing identified 5,607 DEGs in septic myocardium at 24, 48, and 72 h after CLP operation. Moreover, gene ontology analysis revealed that these DEGs were mainly associated with the biological processes, including cell adhesion, immune system process, inflammatory response, and positive regulation of cell migration. KEGG pathway enrichment analysis indicated that Staphylococcus aureus infection, osteoclast differentiation, leishmaniasis, and ECM-receptor interaction were significantly altered in septic hearts. Notably, Pik3r1 and Pik3r5 were localized in the center of the gene co-expression network, and were markedly upregulated in CLP-induced septic myocardium. Further, blocking PI3Kγ by the specific inhibitor CZC24832 significantly protected against sepsis-induced cardiac impairment. The present study uncovers the gene expression signatures of CLP-induced myocardial injury and sheds light on the role of Pik3r5 in septic cardiomyopathy.

Tissue factor cytoplasmic domain exacerbates post-infarct left ventricular remodeling via orchestrating cardiac inflammation and angiogenesis

Abstract Introduction The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and implicated in cardiac injuries and dysfunction, the contribution of its cytoplasmic domain in cardiac pathology remains unclear. Purpose We aimed to investigate the contribution of the cytoplasmic domain of TF to post-infarct myocardial injury and adverse left ventricular (LV) remodeling. Methods and results Myocardial infarction was induced by permanent occlusion of the left anterior descending coronary artery. Male mice with C57BL/Jax background were used for the study. Compared with wild-type mice, mice lacking the TF cytoplasmic domain (TFΔCT) had a higher survival rate (90.5% versus 70%, p=0.0298) during a 28-day follow-up after myocardial infarction. Among surviving mice, TFΔCT mice had better cardiac function and less LV remodeling (ESV: 114.5±13.1mL for WT, 67.06±10.8mL for TFΔCT, p&amp;lt;0.001; EDV: 146.6±12.4mL for WT, 99.97±11.71mL for TFΔCT, p&amp;lt;0.001) than wild-type mice. Bone marrow chimerism indicated that deletion of the TF cytoplasmic domain in either bone marrow-derived cells or cardiac resident cells could alleviate post-infarct cardiac dysfunction. Speckle-tracking strain analysis revealed that the overall improvement of post-infarct cardiac performance in TFΔCT mice was attributed to reduced myocardial deformation in the peri-infarct region (strain-%: 11.14±0.97 for WT, 15.34±1.10 for TFΔCT, p=0.007; strain rate-/s: 3.89±0.26 for WT, 5.18±0.21 for TFΔCT, p=0.0005). Histological analysis demonstrated that TFΔCT hearts had in the infarct area greater proliferation of endothelial cells and myofibroblasts accompanied with better scar formation. Compared with wild-type hearts, infarcted TFΔCT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1)-Rac1 axis. Furthermore, infarcted TFΔCT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Conclusions Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): National University Health System of Singapore

Methotrexate Ameliorates Systemic Inflammation and Septic Associated-Lung Damage in a Cecal Ligation and Puncture Septic Rat Model.

Background: Sepsis is a serious, heterogeneous clinical entity produced by a severe and systemic host inflammatory response to infection. Methotrexate (MTX) is a folate-antagonist that induces the generation of adenosine and also inhibits JAK/STAT pathway; MTX it is widely used as an anti-inflammatory drug to control the immune system. Objective: The aim of this study was to assess the beneficial effects of a single and low dose of MTX in the systemic response and acute lung injury (ALI) induced by sepsis. As in the clinics, we treated our animals with antibiotics and fluids and performed the source control to mimic the current clinic treatment. Methods and main results: Sepsis was induced in rats by a cecal ligation puncture (CLP) procedure. Six hours after induction of sepsis, we proceeded to the source control; fluids and antibiotics were administered at 6 h and 24 h after CLP. MTX (2.5 mg/Kg) was administered 6 h after the first surgery in one CLP experimental group and to one Sham group. A protective effect of MTX was observed through a significant reduction of pro-inflammatory cytokines and a decrease infiltration of inflammatory cells in the lung. In addition, we found a regulation in adenosine receptor A2aR and the metalloproteinases by MTX. Conclusion: A single, low dose of MTX attenuates sepsis lung-associated damage by decreasing pro-inflammatory response, infiltration of pro-inflammatory cells and avoiding defective tissue lung remodeling.

Reversal of alopecia areata, osteoporosis follow treatment with activation of Tgr5 in mice.

Background: Alopecia areata is an autoimmune hair loss disease with infiltration of pro-inflammatory cells into hair follicles. The role of Tgr5 in dermatitis has attracted considerable attention. The present study aimed to investigate the effect of Tgr5 in the development of Alopecia areata.Methods: The study utilized a comparison control group design with four groups of wild-type group, wild-type+INT777 group, Tgr5−/− group, and Tgr5−/−+INT777 group. The mice were treated with INT777 (30 mg/kg/day) or the carrier solution (DMSO) intraperitoneally for 7 weeks, and the back skin was collected and analyzed by histology and immunohistochemistry staining. The lumbar vertebrae 4 has also been analyzed by DXA and Micro-CT.Results: Tgr5−/− mice displayed the decreasingly significant in hair area and length, skin thickness, and the ratio of anagen and telogen, collagen, and mast cell number and loss the bone mass than WT group. After treating with INT777, the appearance of alopecia areata and bone microstructure has improved. Immunohistochemistry and qPCR analysis showed that activation of Tgr5 can down-regulate the express of JAK1, STAT3, IL-6, TNF-α, and VEGF.Conclusion: These findings indicate that activation of Tgr5 mediated amelioration of alopecia areata and osteoporosis by down-regulated JAK1-STAT3 signaling pathway.

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

FC 037NEUTROPHILS PLAY A KEY ROLE IN THE INITIATION OF GLOMERULAR HEMATURIA IN A POSTINFECTIOUS IGAN EXPERIMENTAL MODEL

Abstract Background and Aims Hematuria is a common finding in patients with IgA nephropathy (IgAN), occurring mainly after upper respiratory tract infections. Hematuria can lead to acute kidney injury and chronic loss of renal function in IgAN. However, the mechanisms involved in egression of erythrocytes from the glomerular capillaries into the urinary space are unknown. To answer this question, we developed an infection with Streptococcus pneumoniae (SP) in a humanized experimental IgAN model (α1KICD89tg mice) that resembles the pathological and clinical findings of disease (IgA1 and soluble CD89 mesangial deposits, complement activation, proteinuria and hematuria). Method α1KICD89tg mice (12 weeks old) received an intranasal instillation of SP (107 bacteria). Blood, urine and renal samples were obtained during 1 month after induction of respiratory infection. The presence of SP in lungs from these mice was confirmed by microbiological analysis. Hematuria was quantified in the urinary sediment and renal function was determined by biochemical analysis. Renal histological characteristics were evaluated by hematoxylin/eosin, masson's trichrome and PAS staining. IgA glomerular deposits, activation of complement system and infiltration of proinflammatory cells was examined by immunohistochemistry or immunofluorescence. Circulating leukocyte populations were studied on a hemocytometer. Renal inflammatory cytokines, metalloproteases, as well as markers of tubular and glomerular damage were determined in kidneys by RT-PCR and western-blot. To further validate the role of neutrophils in this pathological setting, we selective depleted these cells through a single injection of anti-Ly6G mAb (200 µg/kg i.p). Results SP-intranasal instillation in α1KICD89tg mice increased hematuria, microalbuminuria and proteinuria, peaking at 48h after induction of the respiratory infection. SP instillation caused disruption of the glomerular basement membrane, with decreased expression of the slit diaphragm proteins nephrin and synaptopodin, as well as higher glomerular accumulation of IgA and proteins of complement system (C3, MBL). Hematuria intensity was positively correlated with the presence of interstitial F4/80+ macrophages, matrix metalloproteinase 9 (MMP-9), inflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, CCL-2, CCL5 and CX3CL1/CX3CR1) as well as p65 NF-κB activation. Hematuria was negatively correlated with anti-inflammatory IL-10 mRNA expression, Factor H levels and collagen IV content. Notably, SP infection induced expression of the tubular injury markers N-GAL and KIM-1. Increased peripheral neutrophils levels were observed in the SP-infected α1KICD89tg mice. Mechanistically, anti-Ly6G-mediated neutrophil depletion reduced SP-mediated hematuria, proteinuria and albuminuria, prevented loss of synaptopodin and nephrin, decreased renal inflammation and MMP-9 expression in α1KICD89tg mice Conclusion In a humanized mouse model of IgAN, hematuria bouts following respiratory tract infections are caused by a neutrophil-mediated alteration of the glomerular filtration barrier (podocyte damage, complement deposits and loss of Collagen IV). These findings may help to unveil novel potential therapeutic approaches to combat one of the key elements in the progression of IgAN and related conditions.