Ageing and interferon gamma response drive the phenotype of neutrophils in the inflamed joint.

OBJECTIVE: Neutrophils are typically the most abundant leucocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint. METHODS: We performed RNA sequencing of neutrophils from healthy human blood, arthritic blood and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotype ex vivo in cultured healthy blood neutrophils. RESULTS: Blood neutrophils from healthy donors and patients with active arthritis showed largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFN-γ), as well as to tumour necrosis factor, interleukin-6 and hypoxia, in both humans and mice. Mass cytometry confirmed that healthy and arthritic donor blood neutrophils are largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFN-γ and prolonged culture. CONCLUSIONS: Circulating neutrophils from patients with arthritis resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFN-γ response and ageing as complementary drivers of the synovial fluid neutrophil phenotype.

Evaluation of the association between KIR polymorphisms and systemic sclerosis: a meta-analysis.

BACKGROUND: The results of investigations on the association between killer cell immunoglobulin-like receptor (KIR) gene polymorphisms and the risk of systemic sclerosis (SSc) are inconsistent. To comprehensively evaluate the influence of KIR polymorphisms on the risk of SSc, this meta-analysis was performed. METHODS: A systematic literature search was performed in electronic databases including Scopus and PubMed/MEDLINE to find all available studies involving KIR gene family polymorphisms and SSc risk prior to July 2019. Pooled odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were measured to detect associations between KIR gene family polymorphisms and SSc risk. RESULTS: Five articles, comprising 571 patients and 796 healthy participants, evaluating the KIR gene family polymorphisms were included in the final meta-analysis according to the inclusion and exclusion criteria, and 16 KIR genes were assessed. None of the KIR genes were significantly associated with the risk of SSc. CONCLUSIONS: The current meta-analysis provides evidence that KIR genes might not be potential risk factors for SSc risk.

Evaluation of the association between KIR polymorphisms and systemic sclerosis: a meta-analysis

Abstract Background: The results of investigations on the association between killer cell immunoglobulin-like receptor (KIR) gene polymorphisms and the risk of systemic sclerosis (SSc) are inconsistent. To comprehensively evaluate the influence of KIR polymorphisms on the risk of SSc, this meta-analysis was performed. Methods: A systematic literature search was performed in electronic databases including Scopus and PubMed/ MEDLINE to find all available studies involving KIR gene family polymorphisms and SSc risk prior to July 2019. Pooled odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were measured to detect associations between KIR gene family polymorphisms and SSc risk. Results: Five articles, comprising 571 patients and 796 healthy participants, evaluating the KIR gene family polymorphisms were included in the final meta-analysis according to the inclusion and exclusion criteria, and 16 KIR genes were assessed. None of the KIR genes were significantly associated with the risk of SSc. Conclusions: The current meta-analysis provides evidence that KIR genes might not be potential risk factors for SSc risk.(AU)

Analysis of gene expression profiles and protein-protein interaction networks in multiple tissues of systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc), a multi-organ disorder, is characterized by vascular abnormalities, dysregulation of the immune system, and fibrosis. The mechanisms underlying tissue pathology in SSc have not been entirely understood. This study intended to investigate the common and tissue-specific pathways involved in different tissues of SSc patients. METHODS: An integrative gene expression analysis of ten independent microarray datasets of three tissues was conducted to identify differentially expressed genes (DEGs). DEGs were mapped to the search tool for retrieval of interacting genes (STRING) to acquire protein-protein interaction (PPI) networks. Then, functional clusters in PPI networks were determined. Enrichr, a gene list enrichment analysis tool, was utilized for the functional enrichment of clusters. RESULTS: A total of 12, 2, and 4 functional clusters from 619, 52, and 119 DEGs were determined in the lung, peripheral blood mononuclear cell (PBMC), and skin tissues, respectively. Analysis revealed that the tumor necrosis factor (TNF) signaling pathway was enriched significantly in the three investigated tissues as a common pathway. In addition, clusters associated with inflammation and immunity were common in the three investigated tissues. However, clusters related to the fibrosis process were common in lung and skin tissues. CONCLUSIONS: Analysis indicated that there were common pathological clusters that contributed to the pathogenesis of SSc in different tissues. Moreover, it seems that the common pathways in distinct tissues stem from a diverse set of genes.

MicroRNA-21 and microRNA-29a modulate the expression of collagen in dermal fibroblasts of patients with systemic sclerosis.

MicroRNAs (miRNAs) are well-known candidates for modulating the dysregulated signaling pathways during fibrosis. In this study, we investigated the expression pattern of 16 miRNAs, which have previously been confirmed or predicted to target genes involved in extracellular matrix (ECM) homeostasis. Primary culture of dermal fibroblasts was obtained from skin biopsies of diffused cutaneous SSc (dcSSc) patients and healthy controls. Expression of let-7a, miR-1, miR-15a, miR-17, miR-19a, miR-20a, miR-21, miR-27b, miR-26a, miR-29a, miR-29b, miR29c, miR-141, miR-125a-5p, miR-193a-3p, and miR-200a were quantified by Real-time PCR. Functional analysis of microRNAs was performed using synthetic oligonucleotides. To further confirm the pro- or anti-fibrotic effects of miRNAs, normal fibroblasts were treated with 10 ng/mL of transforming growth factor (TGF)-ß to generate an in vitro model of dermal fibrosis. miR-21 and miR-29a were upregulated and downregulated, respectively, in both dcSSc and TGF-ß-treated fibroblasts. We observed that restoration of miR-29a expression or blockade of miR-21 function negatively affected collagen production. COL1A1 expression in SSc fibroblasts is more sensitive to changes of miR-29a and miR-21 expression in compare to normal fibroblasts. miR-29a alone was effective to decrease TGF-ß-induced collagen production in dermal fibroblasts. miR-21 and TGF-ß had synergistic effects on induction of collagen production. However, neither miR-21 nor miR-29a affected alpha smooth muscle actin (α-SMA) expression in the presence or absence of TGF-ß in dermal fibroblasts. miR-21 and miR-29a as pro- and anti-fibrotic miRNAs modulate collagen production in an opposing manner. Focusing on miR-21 and miR-29s as therapeutic targets would be effective in patients with SSc or other fibrotic diseases which show aberrant expression of collagen expression.

Overexpression of apoptosis-related protein, survivin, in fibroblasts from patients with systemic sclerosis.

BACKGROUND/OBJECTIVES: Recent studies suggest that, in addition to activation and hypersecretion of matrix components, fibroblasts from patients with systemic sclerosis (SSc) are resistant to apoptosis. Previous studies have shown that survivin, a member of inhibition of apoptosis (IAP) family, plays an important role in apoptosis resistance. Accordingly, we decided to study the expression of the most important members of IAP family in SSc fibroblasts, which can block apoptosis either by binding and inhibiting caspases or through caspase-independent mechanisms. METHOD: Skin biopsy samples were obtained from 19 patients with diffuse cutaneous SSc (DcSSc) and 16 healthy controls. Dermal fibroblasts were cultured and the total RNA was isolated from cells followed by cDNA synthesis. Real-time PCR was performed using SYBR Green PCR master mix and specific primers for cIAP1, cIAP2, XIAP, and Survivin mRNA quantification. RESULTS: A significantly increased expression level of Survivin was observed in fibroblasts from SSc patients compared to controls (2.26-fold, P = 0.04). However, mRNA expression of cIAP1, cIAP2, and XIAP did not change significantly between cases and controls. CONCLUSIONS: Our results showed that survivin is upregulated in SSc skin fibroblast which may lead to resistance to apoptosis. Further studies should be performed to reveal the role of survivin in apoptosis pathway of SSc fibroblasts.

Attenuation of aquaporin-3 and epidermal growth factor receptor expression and activation in systemic sclerosis dermal fibroblasts.

OBJECTIVES: Impaired wound healing and skin dehydration are the mainstay of systemic sclerosis (SSc) cutaneous manifestations. Aquaporin-3 (AQP3) has a pivotal role in skin hydration and wound healing. Epidermal growth factor receptor (EGFR) activation is impaired in SSc fibroblasts. It is unclear whether AQP3 downregulation or epidermal growth factor (EGF) signaling are the primary points of dysregulation in SSc patients. METHODS: Skin punch biopsies were obtained from 10 SSc patients and 10 healthy subjects. The mRNA and/or protein expression levels of AQP3, EGFR/p-EGFR, matrix metalloproteinase-1/2/9 (MMP-1/2/9), and tissue inhibitors of metalloproteinase-1 (TIMP1) at baseline and after EGF and transforming growth factor-ß1 (TGF-ß1) treatment was evaluated in extracted fibroblasts using real-time polymerase chain reaction and western blot analysis. RESULTS: SSc fibroblasts expressed lower AQP3 and EGFR, compared with normal fibroblasts. Normal fibroblasts increased AQP3 expression in response to EGF whereas AQP3 expression had no change in EGF-treated-SSc fibroblasts. Likewise, EGFR was activated in response to EGF in the normal group but not SSc group. Baseline expression of MMP-1/2/9 and TIMP1 was not different between SSc and controls. EGF treatment did not result in alteration of any MMPs expression in either of the groups. Combination treatment resulted in a significant upregulation of MMP-1 in normal fibroblasts compared with SSc fibroblasts, while in SSc fibroblasts MMP-9 expression was upregulated in response to treatment with TGF-ß1 only. CONCLUSION: Downregulation of AQP3 expression in SSc fibroblasts may be related to reduced EGFR expression and activation. TGF-ß1 (alone or in combination with EGF) only can upregulate AQP3 expression in SSc fibroblasts so, TGF-ß1 affect MMP-1 and MMP-9 just in SSc fibroblasts.

Downregulation of Aquaporin3 in Systemic Sclerosis Dermal Fibroblasts.

Skin dryness and thickening are hallmarks of systemic sclerosis (SSc) disease. Aquaporins (AQPs) are plasma membrane proteins that transport glycerol and water, resulting in water retention and skin hydration. Expression of AQPs has been evaluated in human normal skin. However, expression of these proteins in SSc dermal fibroblasts has not yet been reported. The aim of this study was to assess the expression profile of AQPs in dermal fibroblasts of SSc patients. Fibroblast cells were extracted from SSc and healthy skin biopsies and characterized using fibroblast surface protein antibody. The SYBR Green Real-time PCR was used to evaluate the mRNA expression of AQP1, 3, 5, 7, 9, and 10 in dermal fibroblasts. Immunoblotting was performed to confirm the results of Real-time PCR. Our data demonstrated that only AQP1, AQP3, and AQP9 were expressed in human skin fibroblasts. Moreover, the expression of AQP3 mRNA and protein were significantly decreased in SSc dermal fibroblasts compare to healthy fibroblasts. AQP3, which involves in skin hydration and wound healing through water and glycerol transmission, is downregulated in SSc fibroblasts. Based on previous studies and our results, it seems that SSc manifestations like skin dryness, abnormal wound healing, and fibrotic lesions may be related to downregulation of AQP3 in SSc fibroblasts. Therefore, induction of AQP3 expression can be a potential treatment to relieve SSc skin thickness in the future.

Effects of physical form of diet on nutrient digestibility, rumen fermentation, rumination, growth performance and protozoa population of finishing lambs.

This study was conducted to compare effects of 3 physical forms of feed including mash (diet 1), pellet (diet 2) and complete feed block (CFB; diet 3) on digestion, fermentation and performance of lambs. Twenty-one lambs with an initial average body weight of 26 ± 2.5 kg and 6 ± 1.5 months of age were assigned through a completely randomized design to 3 treatments and 7 replicates. The experimental treatments had the same formulation. The results of present experiment showed that CFB significantly increased feed intake and nutrient digestibility (P < 0.05). There was no significant difference among the diets for rumen fluid pH, blood glucose, concentration of volatile fatty acids (P > 0.05), except acetic acid (P < 0.05). The rumen ammonia nitrogen (NH3-N), mixed rumen protozoa population (RPP), Entodiniums spp., Epidiniums spp., blood urea nitrogen (BUN) concentration, rumination time adjusted for dry matter (DM), neutral detergent fiber (NDF), acid detergent fiber (ADF) intake, and total body weight gain of lambs in CFB diet were the highest among all diets (P < 0.05). Feed conversion ratio at days 31 to 45 and whole experimental period were better in CFB than in other diets (P < 0.05). Overall, according to the findings of the present study, among 3 physical forms of the diets, CFB had the best efficiency due to improvement of nutrient digestibility, rumen fermentation and performance of lambs. Therefore, the CFB diet offers the best result in lambs compared with mash and pellet diets.

Inhibition of MicroRNA-21 induces apoptosis in dermal fibroblasts of patients with systemic sclerosis.

BACKGROUND: Prolonged activation of dermal fibroblasts is the main cause of progressive fibrosis in systemic sclerosis (SSc). It seems that inhibition of apoptosis in SSc fibroblasts deregulates fibrosis. MicroRNA-21 (miR-21) is a pro-fibrotic factor with high expression in lesional areas of SSc skin and fibroblasts. METHODS: The effects of miR-21 on expression of Bcl-2 and Bax, two apoptotic genes, in dermal fibroblasts of SSc patients were evaluated using real-time polymerase chain reaction and Western blot analysis. Apoptotic cells were detected using flow cytometry and Hoechst 33258 staining assays. RESULTS: Overexpression of miR-21 using synthetic miR-21 RNA increased expression of Bcl-2, an inhibitor of apoptosis, and decreased the Bax : Bcl-2 expression ratio, a cell fate determinant, in SSc fibroblasts. Antisense inhibition of miR-21 induced a high rate of apoptosis in SSc fibroblasts. We propose that this may be associated with a decrease in Bcl-2 expression and a shift in the Bax : Bcl-2 ratio. CONCLUSIONS: Although further studies are necessary to determine the underlying apoptotic pathway, we propose that inhibition of miR-21 in dermal fibroblasts from lesional skin may be useful in harnessing progressive fibrosis in SSc.

MicroRNA-29a induces apoptosis via increasing the Bax:Bcl-2 ratio in dermal fibroblasts of patients with systemic sclerosis.

The most prominent feature of systemic sclerosis (SSc) and other diseases associated with fibrosis is the prolonged activation of fibroblasts not eliminated by apoptosis, hence characterized by accumulation of more extra cellular matrix (ECM). We tend to verify if microRNA-29a (miR-29a) as an anti-fibrotic factor could induce apoptosis in SSc fibroblasts. We did not detect apoptosis in SSc fibroblasts. We found that Bcl-2 expression was upregulated in SSc fibroblasts and the ratio of Bax:Bcl-2 in these cells was significantly lower (p = 0.02) compared to normal fibroblasts. Transfection of both SSc and transforming growth factor-ß (TGF-ß) stimulated fibroblasts by miR-29a mimic, significantly decreased the expression of two anti-apoptotic members of the Bcl-2 family, Bcl-2 (p = 0.0005, p = 0.01) and Bcl-XL (p = 0.0001, p = 0.006), resulted in enhanced Bax:Bcl-2 ratio and induced a high rate of apoptosis. Recently, miR-29 has been introduced as an anti-fibrotic factor with potential therapeutic effect on SSc. Until now, it has not been proposed whether there is a relationship between miR-29a and apoptosis in SSc. According to our results, it seems that miR-29a is a potent inducer of apoptosis in SSc fibroblasts and an attenuator of ECM production in these cells. MiR-29a disrupted the expression profiling of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-XL) which is the central point of dynamic life-death rheostat in many apoptotic pathways. Furthermore, dermal fibroblasts from patients with SSc showed elevation in TNF-α mRNA levels, while restoration of miR-29a decreases TNF-α production in these cells. Although further molecular studies are necessary to investigate the underlying apoptotic pathways, the present findings suggest that anti-fibrotic and pro-apoptotic properties of miR-29a could provide novel benefits toward the development of fibroblast-specific anti-fibrotic therapies.

c-Abl silencing reduced the inhibitory effects of TGF-ß1 on apoptosis in systemic sclerosis dermal fibroblasts.

It is generally accepted that the apoptosis of myofibroblasts is a crucial event in the normal wound healing. Delay in myofibroblasts apoptosis results in fibrotic diseases such as systemic sclerosis (SSc). Transforming growth factor-ß1 (TGF-ß1) is an important cytokine to induce fibroblasts differentiation into myofibroblasts. Cellular Abelson (c-Abl) is known as a TGF-ß1-modulating molecule in fibrosis. The role of c-Abl, TGF-ß1, and their interaction in SSc myofibroblasts apoptosis has not yet been fully explored. The aim of this study was to evaluate whether TGF-ß1 and inhibition of c-Abl influence Bax to Bcl-2 ratio and apoptosis in SSc and healthy dermal fibroblasts. We also would like to know whether there is interaction between TGF-ß1 and c-Abl in connection with fibroblasts apoptosis or not. Bax to Bcl-2 ratio was determined using quantitative real-time polymerase chain reaction and immunoblotting. Apoptosis was detected using annexin V and nuclear staining with Hoechst dye. Our results demonstrated that inhibition of c-Abl increased SSc and healthy dermal fibroblasts susceptibility to apoptosis through increasing in Bax to Bcl-2 mRNA and protein ratios, whereas TGF-ß1 promoted healthy fibroblasts resistance to apoptosis via decreasing Bax to Bcl-2 mRNA and protein ratios. In addition, c-Abl silencing reduced the effects of TGF-ß1 on Bax to Bcl-2 mRNA and protein ratios. These results suggested that TGF-ß1 and c-Abl individually may prevent the deletion of myofibroblasts from wounds and result in fibrosis. Results also proposed that silencing of c-Abl may promote myofibroblasts elimination from wound lesions through reduction in the TGF-ß1 inhibitory effects on apoptosis.

Attenuation of fibrosis with selective inhibition of c-Abl by siRNA in systemic sclerosis dermal fibroblasts.

Cellular abelson (c-Abl), a non-receptor tyrosine kinase, is an important molecule in the pathogenesis of systemic sclerosis. There have been reports of beneficial effects of pharmacological tyrosine kinase inhibitors, such as imatinib mesylate, on fibrosis. However, these inhibitors affect multiple tyrosine kinases including c-Abl, c-kit, and platelet-derived growth factor receptor. The effects of selective inhibition of c-Abl using small interfering RNA (siRNA) on dermal fibrosis have not yet been explored. The aim of this study is to evaluate whether specific inhibition of c-Abl by siRNA can influence the transforming growth factor-ß1 (TGF-ß1)-induced fibrotic responses. Dermal fibroblasts from systemic sclerosis patients and healthy controls were transfected with c-Abl siRNA. The expression levels of collagen type I, fibronectin, connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) were measured at both the mRNA and protein levels in the absence or presence of TGF-ß1 pro-fibrotic cytokine. In healthy dermal fibroblasts, the expression of collagen type 1, fibronectin, α-SMA, and CTGF mRNAs and proteins that were upregulated after stimulation with TGF-ß1 was markedly decreased by c-Abl siRNA. Silencing of c-Abl via siRNA efficiently reduced the basal synthesis of collagen type I, fibronectin, α-SMA, and CTGF mRNAs and proteins in systemic sclerosis fibroblasts, but it had no effect on the baseline expression of these genes and proteins in healthy dermal fibroblasts. In conclusion, specific c-Abl gene silencing using siRNA effectively reduced fibrosis-related gene expression. Inhibition of c-Abl by siRNA may be a potential therapeutic approach for fibrotic diseases such as systemic sclerosis.