Disturbed Antioxidant Capacity in Patients with Systemic Sclerosis Associates with Lung and Gastrointestinal Symptoms.

The correct balance between reactive oxygen species and antioxidant defense in an organism is disturbed in oxidative stress. To assess oxidative balance in 36 SSc patients and 26 healthy controls (HCs), we measured reactive oxidative metabolites (ROMs), total antioxidant capacity (TAC), lipid peroxidation (measuring 4-HNE), and DNA oxidative damage (measuring 8-OHdG) in serum. Furthermore, DNA breaks in leukocytes of 35 SSc patients and 32 HCs were evaluated using COMET. While we report high ROMs for both SSc patients and age/sex matched HC samples, there was a significant increase in TAC in SSc patients as compared to HCs, and thus also a significantly higher oxidative stress index in SSc patients. TAC was significantly higher in SSc patients with ILD and gastrointestinal involvement, as well as in patients with anti-topoisomerase antibodies. We observe no difference in serum lipid peroxidation status or oxidative DNA damage. However, SSc patients had significantly more leukocyte DNA breaks than HCs; the most damage was observed in patients treated with immunosuppressives. Thus, our study confirms presence of oxidative stress and increased DNA damage in leukocytes of SSc patients; however, it points toward increased antioxidant capacity, which needs to be further studied.

P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma.

Hotspot mutations in the NRAS gene are causative genetic events associated with the development of melanoma. Currently, there are no FDA-approved drugs directly targeting NRAS mutations. Previously, we showed that p38 acts as a tumor suppressor in vitro and in vivo with respect to NRAS-mutant melanoma. We observed that because of p38 activation through treatment with the protein synthesis inhibitor, anisomycin leads to a transient upregulation of several targets of the cAMP pathway, representing a stressed cancer cell state that is often observed by therapeutic doses of MAPK inhibitors in melanoma patients. Meanwhile, genetically induced p38 or its stable transduction leads to a distinct cellular transcriptional state. Contrary to previous work showing an association of invasiveness with high p38 levels in BRAF-mutated melanoma, there was no correlation of p38 expression with NRAS-mutant melanoma invasion, highlighting the difference in BRAF and NRAS-driven melanomas. Although the role of p38 has been reported to be that of both tumor suppressor and oncogene, we show here that p38 specifically plays the role of a tumor suppressor in NRAS-mutant melanoma. Both the transient and stable activation of p38 elicits phosphorylation of mTOR, reported to be a master switch in regulating autophagy. Indeed, we observed a correlation between elevated levels of phosphorylated mTOR and a reduction in LC3 conversion (LCII/LCI), indicative of suppressed autophagy. Furthermore, a reduction in actin intensity in p38-high cells strongly suggests a role of mTOR in regulating actin and a remodeling in the NRAS-mutant melanoma cells. Therefore, p38 plays a tumor suppressive role in NRAS-mutant melanomas at least partially through the mechanism of mTOR upregulation, suppressed autophagy, and reduced actin polymerization. One or more combinations of MEK inhibitors with either anisomycin, rapamycin, chloroquine/bafilomycin, and cytochalasin modulate p38 activation, mTOR phosphorylation, autophagy, and actin polymerization, respectively, and they may provide an alternate route to targeting NRAS-mutant melanoma.

An Optimized Tissue Dissociation Protocol for Single-Cell RNA Sequencing Analysis of Fresh and Cultured Human Skin Biopsies.

We present an optimized dissociation protocol for preparing high-quality skin cell suspensions for in-depth single-cell RNA-sequencing (scRNA-seq) analysis of fresh and cultured human skin. Our protocol enabled the isolation of a consistently high number of highly viable skin cells from small freshly dissociated punch skin biopsies, which we use for scRNA-seq studies. We recapitulated not only the main cell populations of existing single-cell skin atlases, but also identified rare cell populations, such as mast cells. Furthermore, we effectively isolated highly viable single cells from ex vivo cultured skin biopsy fragments and generated a global single-cell map of the explanted human skin. The quality metrics of the generated scRNA-seq datasets were comparable between freshly dissociated and cultured skin. Overall, by enabling efficient cell isolation and comprehensive cell mapping, our skin dissociation-scRNA-seq workflow can greatly facilitate scRNA-seq discoveries across diverse human skin pathologies and ex vivo skin explant experimentations.

Increased L-Selectin on Monocytes Is Linked to the Autoantibody Profile in Systemic Sclerosis.

Monocytes are known to be implicated in the pathogenesis of systemic sclerosis (SSc), as they exert prominent migratory, adhesive, and chemotactic properties. The aim of our study was to characterize the surface expression of adhesion/chemotactic molecules (CD62L, CD11b, CCR2, CCR5) on the SSc monocytes and determine correlations with the clinical presentation of SSc. We included 38 SSc patients and 36 healthy age-and sex-matched controls. Isolated monocytes, as well as in vitro serum-treated monocytes, were analyzed by flow cytometry; additionally, soluble CD62L was measured in serum. We found increased soluble CD62L in the SSc serum samples and increased CD62L on the surface of the SSc monocytes in the in the same set of patients. Among samples with determined SSc-specific autoantibodies, the surface CD62L was the lowest in patients positive for anti-PM/Scl autoantibodies and the highest in patients with anti-topoisomerase I autoantibodies (ATA). The treatment of isolated healthy monocytes with ATA-positive SSc serum resulted in increased surface CD62L expression. Moreover, surface CCR5 was reduced on the monocytes from SSc patients with interstitial lung disease but also, along with CCR2, negatively correlated with the use of analgesics/anti-inflammatory drugs and immunosuppressants. In conclusion, increased CD62L on SSc monocytes, particularly in ATA-positive patients, provides new insights into the pathogenesis of SSc and suggests CD62L as a potential therapeutic target.

Adipose tissue and adipose secretome in systemic sclerosis.

PURPOSE OF REVIEW: Adipose tissue is closely associated with systemic sclerosis (SSc)-pathology, both anatomically and functionally. This review focuses on local effects of adipocytes in the context of adipose to mesenchymal transdifferentiation (AMT), effects of the adipose stromal vascular fraction on SSc pathogenesis and systemic effects of adipose tissue secretome. RECENT FINDINGS: Novel populations of fibroblasts evolving from adipose tissue were identified- for example COL11+ cancer-associated fibroblasts differentiated from adipose-derived stromal cells. Lipofibroblasts in human lungs were described using nonconventional markers that allow more effective population identification. These findings could make an important contribution to further clarification of adipocyte involvement in SSc.Recent studies confirmed that lipolysis contributes to fibrogenesis through AMT differentiation and release of fatty acids (FA). Unbalanced metabolism of FA has been reported in several studies in SSc. Other adipose tissue secretome molecules (e.g. lysophosphatidic acid), novel adipokines and extracellular vesicles from adipose mesenchymal stem cells make important contributions to the pro-/antifibrotic balance. SUMMARY: There is a growing evidence of important contribution of adipose tissue and its secretome to SSc pathogenesis. Novel techniques such as single-cell RNA sequencing (scRNAseq) and metabolomics, albeit challenging to use in adipose tissue, will provide further evidence.

Functional genomics atlas of synovial fibroblasts defining rheumatoid arthritis heritability.

BACKGROUND: Genome-wide association studies have reported more than 100 risk loci for rheumatoid arthritis (RA). These loci are shown to be enriched in immune cell-specific enhancers, but the analysis so far has excluded stromal cells, such as synovial fibroblasts (FLS), despite their crucial involvement in the pathogenesis of RA. Here we integrate DNA architecture, 3D chromatin interactions, DNA accessibility, and gene expression in FLS, B cells, and T cells with genetic fine mapping of RA loci. RESULTS: We identify putative causal variants, enhancers, genes, and cell types for 30-60% of RA loci and demonstrate that FLS account for up to 24% of RA heritability. TNF stimulation of FLS alters the organization of topologically associating domains, chromatin state, and the expression of putative causal genes such as TNFAIP3 and IFNAR1. Several putative causal genes constitute RA-relevant functional networks in FLS with roles in cellular proliferation and activation. Finally, we demonstrate that risk variants can have joint-specific effects on target gene expression in RA FLS, which may contribute to the development of the characteristic pattern of joint involvement in RA. CONCLUSION: Overall, our research provides the first direct evidence for a causal role of FLS in the genetic susceptibility for RA accounting for up to a quarter of RA heritability.

Human mesenchymal stromal cells from different tissues exhibit unique responses to different inflammatory stimuli.

BACKGROUND: Mesenchymal stromal cell (MSC) - based therapies are emerging as promising treatment of various autoimmune diseases, however the utility of different MSC tissue sources remains elusive. We aimed to characterize MSC from different origins, namely bone marrow (BM), adipose tissue (AT) and umbilical cord (UC) and determine their functional effects on normal human lung fibroblasts (NHLF). METHODS: BM-, AT- or UC-MSC were isolated each from 3 different healthy donors. The gene expression and protein secretion were analyzed at basal level, along with TNFα-, IL-1ß- and SAA- stimulated cells using real-time PCR and Luminex technology. Effect of conditioned medium (CM) from different MSC sources on migration was determined with wound scratch assay, while mitotic and apoptotic rates were studied using immunofluorescence microscopy. RESULTS: BM-MSC expressed highest basal mRNA levels of SDF1 and VCAM-1, while other genes were similarly expressed between MSC origins. TNFα priming of AT-MSC gained a prominent increase in IDO1 and CCL5 gene expression, with 928-fold and 4396-fold changes, respectively. Among all tissue sources, basal UC-MSC released highest protein levels of most measured analytes, including IL-6, IL-8, MCP-1, ICAM1, HGF, MMP1 and CH3L1. BM- and AT-MSC derived CM enhanced wound closure in NHLF, while an opposite effect was observed with UC-MSC derived CM. Our data also suggests that MSC-CM could contribute to decreased mitotic potential and increased apoptotic rate in lung fibroblasts. CONCLUSIONS: Our study highlights origin-specific MSC profile differences and emphasizes a heterogenic response of different MSC to inflammatory stimuli.

Hi-JAKi-ng Synovial Fibroblasts in Inflammatory Arthritis With JAK Inhibitors.

The Janus kinase (JAK)-Signal transducer and activator of transcription (STAT) pathway is one of the central signaling hubs in inflammatory, immune and cancer cells. Inhibiting the JAK-STAT pathway with JAK inhibitors (jakinibs) constitutes an important therapeutic strategy in cancer and chronic inflammatory diseases like rheumatoid arthritis (RA). FDA has approved different jakinibs for the treatment of RA, including tofacitinib, baricitinib and upadacitinib, and several jakinibs are being tested in clinical trials. Here, we reviewed published studies of jakinib effects on resolving synovial pathology in inflammatory arthritis. We discussed the results of jakinibs on structural joint damage in clinical trials and explored the effects of jakinibs across different in vitro, ex vivo, and in vivo synovial experimental models. We delved rigorously into experimental designs of in vitro fibroblast studies, deconvoluted jakinib efficacy in synovial fibroblasts across diverse experimental conditions and discussed their translatability in vivo. Synovial fibroblasts can readily activate the JAK-STAT signaling pathway in response to cytokine stimulation. We highlighted rather limited effects of jakinibs on the in vitro cultured synovial fibroblasts and inferred that direct and indirect (immune cell-dependent) actions of jakinibs are required to curb the fibroblast pathology in vivo. These actions have not been mimicked optimally in current in vitro experimental designs, where inflammatory stimuli do not naturally clear out with treatment as they do in vivo. While summarizing the broad knowledge of synovial jakinib effects, our review uniquely challenges future study designs to better mimick the jakinib actions in broader cell communities, as occurring in vivo in the inflamed synovium. This can deepen our understanding of collective synovial activities of jakinibs and their therapeutic limitations, thereby fostering jakinib development in arthritis.

Olive Leaf Extract Attenuates Inflammatory Activation and DNA Damage in Human Arterial Endothelial Cells.

Olive leaf extract (OLE) is used in traditional medicine as a food supplement and as an over-the-counter drug for a variety of its effects, including anti-inflammatory and anti-atherosclerotic ones. Mechanisms through which OLE could modulate these pathways in human vasculature remain largely unknown. Serum amyloid A (SAA) plays a causal role in atherosclerosis and cardiovascular diseases and induces pro-inflammatory and pro-adhesive responses in human coronary artery endothelial cells (HCAEC). Within this study we explored whether OLE can attenuate SAA-driven responses in HCAEC. HCAEC were treated with SAA (1,000 nM) and/or OLE (0.5 and 1 mg/ml). The expression of adhesion molecules VCAM-1 and E-selectin, matrix metalloproteinases (MMP2 and MMP9) and microRNA 146a, let-7e, and let-7g (involved in the regulation of inflammation) was determined by qPCR. The amount of secreted IL-6, IL-8, MIF, and GRO-α in cell culture supernatants was quantified by ELISA. Phosphorylation of NF-κB was assessed by Western blot and DNA damage was measured using the COMET assay. OLE decreased significantly released protein levels of IL-6 and IL-8, as well as mRNA expression of E-selectin in SAA-stimulated HCAEC and reduced MMP2 levels in unstimulated cells. Phosphorylation of NF-κB (p65) was upregulated in the presence of SAA, with OLE significantly attenuating this SAA-induced effect. OLE stabilized SAA-induced upregulation of microRNA-146a and let-7e in HCAEC, suggesting that OLE could fine-tune the SAA-driven activity of NF-κB by changing the microRNA networks in HCAEC. SAA induced DNA damage and worsened the oxidative DNA damage in HCAEC, whereas OLE protected HCAEC from SAA- and H2O2-driven DNA damage. OLE significantly attenuated certain pro-inflammatory and pro-adhesive responses and decreased DNA damage in HCAEC upon stimulation with SAA. The reversal of SAA-driven endothelial activation by OLE might contribute to its anti-inflammatory and anti-atherogenic effects in HCAEC.

Gene and miRNA expression in giant cell arteritis-a concise systematic review of significantly modified studies.

Giant cell arteritis (GCA) is a systemic vasculitis in individuals older than 50 years, characterized by headaches, visual disturbances, painful scalp, jaw claudication, impairment of limb arteries, and systemic inflammation, among other symptoms. GCA diagnosis is confirmed by a positive temporal artery biopsy (TAB) or by imaging modalities. A prominent acute phase response with inflammation is the hallmark of the disease, predominantly targeting large- and medium-sized arteries leading to stenosis or occlusion of arterial lumen. To date, there are no reliable tissue markers specific for GCA. Scarce reports have indicated the importance of epigenetics in GCA. The current systematic review reports significantly changed candidate biomarkers in TABs of GCA patients compared to non-GCA patients using qPCR.

Utility of serological biomarkers for giant cell arteritis in a large cohort of treatment-naïve patients.

Early diagnosis and treatment of giant cell arteritis (GCA) is crucial for preventing ischemic complications. Multiple serological markers have been identified; however, there is a distinct lack of predicting markers for GCA relapse and complications. Our main objective was to identify serological parameters in a large cohort of treatment-naïve GCA patients, which could support clinicians in evaluating the course of the disease. Clinical data was gathered, along with analyte detection using Luminex technology, ELISA, and nephelometry, among others. Unsupervised hierarchical clustering and principal component analysis of analyte profiles were performed to determine delineation of GCA patients and healthy blood donors (HBDs). Highest, significantly elevated analytes in GCA patients were SAA (83-fold > HBDs median values), IL-23 (58-fold), and IL-6 (11-fold). Importantly, we show for the first time significantly changed levels of MARCO, alpha-fetoprotein, protein C, resistin, TNC, TNF RI, M-CSF, IL-18, and IL-31 in GCA versus HBDs. Changes in levels of SAA, CRP, haptoglobin, ESR, MMP-1 and MMP-2, and TNF-alpha were found associated with relapse and visual disturbances. aCL IgG was associated with limb artery involvement, even following adjustment for multiple testing. Principal component analysis revealed clear delineation between HBDs and GCA patients. Our study reveals biomarker clusters in a large cohort of patients with GCA and emphasizes the importance of using groups of serological biomarkers, such as acute phase proteins, MMPs, and cytokines (e.g. TNF-alpha) that could provide crucial insight into GCA complications and progression, leading to a more personalized disease management.