Pirfenidone mitigates TGF-ß1-mediated fibrosis in an idiopathic inflammatory myositis-associated interstitial lung disease model.

Idiopathic inflammatory myositis (IIM) is a group of rare diseases of unknown etiology, with a pathognomonic muscular deficiency. Antisynthetase syndrome is a subtype of IIM with an associated interstitial lung disease (ILD), characterized by pulmonary inflammation and fibrosis mediated by TGF-ß. Pirfenidone is a new molecule with anti-inflammatory and anti-fibrotic properties, used for the treatment of idiopathic ILD, but has never been assessed in IIM. The aim of our study is to evaluate the effect of pirfenidone on IIM-associated ILD. Thirty-two BALB/c male mice were divided into three groups: Sham, IIM-untreated (IIM), and IIM pirfenidone-treated (IIM + PIR). IIM was induced by intramuscular injections of guinea pig muscle myosin extract and intraperitoneal injections of Pertussis toxin. Pirfenidone was given orally at a dose of 30 mg kg-1 day-1 for two months. Muscle force, blood and bronchoalveolar lavage fluid samples, as well as muscle and lung tissues, were analyzed. Progressive deterioration of muscle force and infiltration of the muscular tissue by inflammatory cells were observed with IIM. Auto-immune antibodies specific to the antisynthetase syndrome were also increased in IIM mice. Pirfenidone attenuated IIM-associated ILD with anti-inflammatory properties evidenced by decreased peribronchial inflammation and TGF-ß1 in bronchoalveolar lavage fluid. Likewise, pirfenidone attenuated pulmonary fibrosis by fine-tuning TGF-ß1-mediated epithelial-to-mesenchymal and fibrotic signaling pathways; pro-fibrotic SMAD3, ZEB2 and STAT1 expression and activation were decreased, whereas anti-fibrotic SMAD2 activation was increased. This study unravels for the first time that pirfenidone has the potential to fine-tune TGF-ß1 fibrotic signaling in IIM-associated ILD.

New insights in gut-liver axis in wild-type murine imiquimod-induced lupus.

BACKGROUND: Intestinal and hepatic manifestations of lupus seem to be underestimated in comparison to other major organ lesions. Although recent data point to gut-liver axis involvement in lupus, gut permeability dysfunction and liver inflammation need to be more investigated. OBJECTIVE: This study aims to assess fecal calprotectin, intestinal tight junction proteins and liver inflammation pathway in wild-type murine imiquimod- induced lupus. METHODS: C57BL/6 mice were topically treated on their right ears with 1.25 mg of 5% imiquimod cream, three times per week for six weeks. Fecal calprotectin was collected at day 0, 22 and 45. Renal, liver and intestinal pathology, as well as inflammatory markers, intestinal tight junction proteins, and E. coli protein in liver were assessed at sacrifice. RESULTS: At six weeks, lupus nephritis was confirmed on histopathology and NGAL and KIM-1 expression. Calprotectin rise started at day 22 and persists at day 45. Protein expression of Claudine, ZO-1 and occludin was significantly decreased. E. coli protein was significantly increased in liver with necro-inflammation and increased TLR4, TLR7, and pNFκB/NFκB liver expression. CONCLUSION: This study is the first to demonstrate early fecal calprotectin increase and liver activation of TLR4- NFκB pathway in wild-type murine imiquimod-induced lupus.

VEGF-C attenuates renal damage in salt-sensitive hypertension.

Salt-sensitive hypertension is a major risk factor for renal impairment leading to chronic kidney disease. High-salt diet leads to hypertonic skin interstitial volume retention enhancing the activation of the tonicity-responsive enhancer-binding protein (TonEBP) within macrophages leading to vascular endothelial growth factor C (VEGF-C) secretion and NOS3 modulation. This promotes skin lymphangiogenesis and blood pressure regulation. Whether VEGF-C administration enhances renal and skin lymphangiogenesis and attenuates renal damage in salt-sensitive hypertension remains to be elucidated. Hypertension was induced in BALB/c mice by a high-salt diet. VEGF-C was administered subcutaneously to high-salt-treated mice as well as control animals. Analyses of kidney injury, inflammation, fibrosis, and biochemical markers were performed in vivo. VEGF-C reduced plasma inflammatory markers in salt-treated mice. In addition, VEGF-C exhibited a renal anti-inflammatory effect with the induction of macrophage M2 phenotype, followed by reductions in interstitial fibrosis. Antioxidant enzymes within the kidney as well as urinary RNA/DNA damage markers were all revelatory of abolished oxidative stress under VEGF-C. Furthermore, VEGF-C decreased the urinary albumin/creatinine ratio and blood pressure as well as glomerular and tubular damages. These improvements were associated with enhanced TonEBP, NOS3, and lymphangiogenesis within the kidney and skin. Our data show that VEGF-C administration plays a major role in preserving renal histology and reducing blood pressure. VEGF-C might constitute an interesting potential therapeutic target for improving renal remodeling in salt-sensitive hypertension.

Circadian Rhythm Disruption Aggravates DSS-Induced Colitis in Mice with Fecal Calprotectin as a Marker of Colitis Severity.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic immunologically mediated pathology that remains a major health burden. Circadian rhythm disruption leads to a deregulation in the immune system which is a major risk factor for IBD. AIMS: Since fecal calprotectin (FC) has been a useful tool for monitoring IBD, we aimed to evaluate the effect of circadian rhythm alteration on gut inflammation status and whether FC is associated with the severity of colitis. METHODS: C57BL/6J mice were exposed to circadian shifts for 3 months, and then colitis was induced by 2% dextran sulfate sodium (DSS). Colitis was evaluated according to clinical symptoms and histological scoring. Plasma and intestinal inflammatory and permeability markers as well as fecal and intestinal calprotectin were assessed. RESULTS: Circadian shifts aggravated DSS-induced colitis with increased diarrhea, flatulence, and fecal blood associated with decreased colon length. In addition, intestinal cryptic architecture was lost with the presence of increased inflammation, mucosal muscle thickening, and cryptic abscesses. Plasma tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, and C-reactive protein upregulations were paralleled by the deterioration of intestinal permeability. Calprotectin expression and distribution increased in the intestines and feces of shifted animals, and levels highly correlated with the increases in intestinal inflammation and permeability. CONCLUSIONS: Circadian rhythm disruption aggravates DSS-induced colitis, whereas fecal and intestinal calprotectin associates with the severity of disease. Calprotectin might be a useful marker and tool for assessing patients at risk of IBD due to lifestyles with disruptive sleep patterns.

Basic Signaling in Cardiac Fibroblasts.

Cardiac fibroblasts are commonly known as supporting cells of the cardiac network and exert many essential functions that are fundamental for normal cardiac growth as well as for cardiac remodeling process during pathological conditions. This review focuses on the roles of cardiac fibroblasts in the formation and regulation of the extracellular matrix components, and in maintaining structural, biochemical and mechanical properties of the heart. Additionally, though considered as non-excitable cells, we review the functional expression in cardiac fibroblasts of a wide variety of transmembrane ion channels which activity may contribute to key regulation of cardiac physiological processes. All together, cardiac fibroblasts which actively participate to fundamental regulation of cardiac physiology and physiopathology processes may represent pertinent targets for pharmacological approaches of cardiac diseases and lead to new tracks of therapeutic strategies. J. Cell. Physiol. 232: 725-730, 2017. © 2016 Wiley Periodicals, Inc.

An optimized protocol for purification of functional islets of Langerhans.

Islets of Langerhans and ß-cell isolation constitute routinely used cell models for diabetic research, and refining islet isolation protocols and cell quality assessment is a high priority. Numerous protocols have been published describing isolate of islets, but often rigorous and systematic assessment of their integrity is lacking. Herein, we propose a new protocol for optimal generation of islets. Pancreases from mice and rats were excised and digested using a low-activity collagenase solution and islets were then purified by a series of sedimentations and a Percoll gradient. Islets were maintained in culture for 5 days, during which viability, pro/antiapoptotic, and islet-specific genes, glucose-stimulated calcium entry, glucose uptake, and insulin secretion were assessed. The commonly used islet isolation technique by collagenase injection through the common bile duct (CBD) was also performed and compared with the present approach. This new protocol produced islets that retained a healthy status as demonstrated by the yield of stable living cells. Furthermore, calcium oscillation, glucose uptake, and insulin secretion remained intact in the islet cultures. This was reproducible when many rodent species were used, and neither sex nor age affected the cells behavior. When compared with the CBD technique, islet physiology was similar. Finally, this approach was used to uncover new ion channel candidates implicated in insulin secretion. In conclusion, this study outlines an efficient protocol for islet preparation that may support research into new therapeutic targets in diabetes research.

Fish oil attenuates neurologic severity of antiphospholipid syndrome in a mice experimental model.

INTRODUCTION: Murine experimental models of antiphospholipid syndrome (eAPLS) showed neurologic dysfunction and therapeutic effect of the anticoagulant enoxaparin is well established. Omega-3 fatty acids and curcumin, tested in neuroinflammation and auto-immunity diseases, might be interesting therapeutic candidates. The aim of this study was to evaluate the effects of these candidates on neurologic severity in eAPLS. METHODS: One month after immunization of BALB/c mice with beta-2-glycoprotein I, daily treatments were initiated with enoxaparin (1 mg/kg), omega-3 fatty acids (0.5 g/kg), and curcumin (200 mg/kg) for 3 months. RESULTS: Mortality was significantly decreased by enoxaparin and omega-3 treatments. Fish oil and curcumin group exhibited the highest mean of swimming behavior in forced swim test in surviving mice. Mice under omega-3 fatty acids or curcumin presented low anxiety-like behavior in the elevated plus-maze test. Cerebral histopathology revealed heavy inflammatory infiltrates in cortical and subcortical regions with vacuolization, swelling, and degeneration of astrocytes in the control group, with aggravation under curcumin; no infiltrate was retrieved in enoxaparin and omega-3 groups. CONCLUSION: Our study is the first to demonstrate a potential therapeutic effect of omega-3 fatty acids in eAPLS. Enoxaparin and omega-3 fatty acids combination would be interesting for further investigation.

Evidence of a Role for Fibroblast Transient Receptor Potential Canonical 3 Ca2+ Channel in Renal Fibrosis.

Transient receptor potential canonical (TRPC) Ca(2+)-permeant channels, especially TRPC3, are increasingly implicated in cardiorenal diseases. We studied the possible role of fibroblast TRPC3 in the development of renal fibrosis. In vitro, a macromolecular complex formed by TRPC1/TRPC3/TRPC6 existed in isolated cultured rat renal fibroblasts. However, specific blockade of TRPC3 with the pharmacologic inhibitor pyr3 was sufficient to inhibit both angiotensin II- and 1-oleoyl-2-acetyl-sn-glycerol-induced Ca(2+) entry in these cells, which was detected by fura-2 Ca(2+) imaging. TRPC3 blockade or Ca(2+) removal inhibited fibroblast proliferation and myofibroblast differentiation by suppressing the phosphorylation of extracellular signal-regulated kinase (ERK1/2). In addition, pyr3 inhibited fibrosis and inflammation-associated markers in a noncytotoxic manner. Furthermore, TRPC3 knockdown by siRNA confirmed these pharmacologic findings. In adult male Wistar rats or wild-type mice subjected to unilateral ureteral obstruction, TRPC3 expression increased in the fibroblasts of obstructed kidneys and was associated with increased Ca(2+) entry, ERK1/2 phosphorylation, and fibroblast proliferation. Both TRPC3 blockade in rats and TRPC3 knockout in mice inhibited ERK1/2 phosphorylation and fibroblast activation as well as myofibroblast differentiation and extracellular matrix remodeling in obstructed kidneys, thus ameliorating tubulointerstitial damage and renal fibrosis. In conclusion, TRPC3 channels are present in renal fibroblasts and control fibroblast proliferation, differentiation, and activation through Ca(2+)-mediated ERK signaling. TRPC3 channels might constitute important therapeutic targets for improving renal remodeling in kidney disease.

ANO1 contributes to angiotensin-II-activated Ca2+-dependent Cl- current in human atrial fibroblasts.

Cardiac fibroblasts are an integral part of the myocardial tissue and contribute to its remodelling. This study characterises for the first time the calcium-dependent chloride channels (CaCC) in the plasma membrane of primary human atrial cardiac fibroblasts by means of the iodide efflux and the patch clamp methods. The calcium ionophore A23187 and Angiotensin II (Ang II) activate a chloride conductance in cardiac fibroblasts that shares pharmacological similarities with calcium-dependent chloride channels. This chloride conductance is depressed by RNAi-mediated selective Anoctamine 1 (ANO1) but not by Anoctamine 2 (ANO2) which has been revealed as CaCC and is inhibited by the selective ANO1 inhibitor, T16inh-A01. The effect of Ang II on anion efflux is mediated through AT1 receptors (with an EC50 = 13.8 ± 1.3 nM). The decrease of anion efflux by calphostin C and bisindolylmaleimide I (BIM I) suggests that chloride conductance activation is dependent on PKC. We conclude that ANO1 contributes to CaCC current in human cardiac fibroblasts and that this is regulated by Ang II acting via the AT1 receptor pathway.

Balancing the mind: Toward a complete picture of the interplay between gut microbiota, inflammation and major depressive disorder.

The intricate interplay existing between gut microbiota and homeostasis extends to the realm of the brain, where emerging research underscores the significant impact of the microbiota on mood regulation and overall neurological well-being and vice-versa, with inflammation playing a pivotal role in mediating these complex interactions. This comprehensive review explores the complex interplay between inflammation, alterations in gut microbiota, and their impact on major depressive disorder (MDD). It provides a cohesive framework for the puzzle pieces of this triad, emphasizing recent advancements in understanding the gut microbiota and inflammatory states' contribution to the depressive features. Two directions of communication between the gut and the brain in depression are discussed, with inflammation serving as a potential modulator. Therapeutic implications were discussed as well, drawing insights from interventional studies on the effects of probiotics on gut bacterial composition and depressive symptoms. Ultimately, this review will attempt to provide a complete and valuable framework for future research and therapeutic interventions in MDD.