Isotretinoin-induced myositis.

Retinoid-induced myositis is a phenomenon recognised in multiple case reports. We report a case of isotretinoin-induced myositis in an 18-year-old male patient. This case adds to the published literature as it demonstrates (i) myositis may occur after extended periods of isotretinoin use, (ii) should be considered as a differential diagnosis even when presenting asymmetrically and (iii) can continue to progress clinically and biochemically initially following the suspension of isotretinoin before being effectively treated with corticosteroids.

Emerging diagnostic and therapeutic challenges for skin fibrosis in systemic sclerosis.

Systemic sclerosis (also called scleroderma, SSc) is a chronic autoimmune disorder characterized by excessive collagen deposition leading to skin fibrosis and various internal organ manifestations. The emergent diagnostics and therapeutic strategies for scleroderma focus on early detection and targeted interventions to improve patient outcomes and quality of life. Diagnostics for SSc have evolved significantly in recent years, driven by advancements in serological markers and imaging techniques. Autoantibody profiling, especially antinuclear antibodies (ANA) and specific scleroderma-associated autoantibodies, aids in identifying subsets of scleroderma and predicting disease progression. Furthermore, novel imaging modalities, such as high-frequency ultrasonography and optical coherence tomography, enable early detection of skin fibrosis and internal organ involvement, enhancing the diagnostic precision and allowing for tailored management. Therapeutic strategies for SSc are multifaceted, targeting immune dysregulation, vascular abnormalities, and fibrotic processes. Emerging biologic agents have shown promise in clinical trials, including monoclonal antibodies directed against key cytokines involved in fibrosis, such as transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6). Additionally, small-molecule inhibitors that disrupt fibrotic pathways, like tyrosine kinase inhibitors, have exhibited potential in limiting collagen deposition and preventing disease progression. Stem cell therapy, cell ablation and gene editing techniques hold great potential in regenerating damaged tissue and halting fibrotic processes. Early intervention remains crucial in managing SSc, as irreversible tissue damage often occurs in advanced stages. Novel diagnostic methods, such as biomarkers and gene expression profiling, are being explored to identify individuals at high risk for developing progressive severe disease and intervene proactively. Furthermore, patient-tailored therapeutic approaches, employing a combination of immunosuppressive agents and targeted anti-fibrotic therapies, are being investigated to improve treatment efficacy while minimizing adverse effects. The emergent diagnostics and therapeutic strategies in scleroderma are transforming the management of this challenging disease. Nevertheless, ongoing research and clinical trials are needed to optimize the efficacy and safety of these novel approaches in the complex and diverse spectrum of SSc manifestations.

Autoantibodies Which Bind to and Activate Keratinocytes in Systemic Sclerosis.

Systemic sclerosis (SSc) is a multisystem connective tissue disease characterised by pathological processes involving autoimmunity, vasculopathy and resultant extensive skin and organ fibrosis. Recent studies have demonstrated activation and aberrant wound healing responses in the epithelial layer of the skin in this disease, implicating the epithelial keratinocytes as a source of pro-fibrotic and inflammatory mediators. In this paper, we investigated the role of Immunoglobulin G (IgG) autoantibodies directed against epithelial cells, as potential initiators and propagators of pathological keratocyte activation and the ensuing SSc fibrotic cascade. A keratinocyte cell-based ELISA is used to evaluate the binding of SSc IgG. SSc skin biopsies were stained by immunofluorescence for the presence of IgG in the keratinocyte layer. Moreover, IgG purified from SSc sera was evaluated for the potential to activate keratinocytes in tissue culture and to induce TLR2 and 3 signalling in reporter cell lines. We demonstrate enhanced binding of SSc IgG to keratinocytes and the activation of these cells leading to the release of IL-1α, representing a potential initiating pathway in this disease.

Back to the future: targeting the extracellular matrix to treat systemic sclerosis.

Fibrosis is the excessive deposition of a stable extracellular matrix (ECM); fibrotic tissue is composed principally of highly crosslinked type I collagen and highly contractile myofibroblasts. Systemic sclerosis (SSc) is a multisystem autoimmune connective tissue disease characterized by skin and organ fibrosis. The fibrotic process has been recognized in SSc for >40 years, but drugs with demonstrable efficacy against SSc fibrosis in ameliorating the lung involvement have only recently been identified. Unfortunately, these treatments are ineffective at improving the skin score in patients with SSc. Previous clinical trials in SSc have largely focused on the cross-purposing of anti-inflammatory drugs and the use of immunosuppressive drugs from the transplantation field, which address inflammatory and/or autoimmune processes. Limited examination has taken place of specific anti-fibrotic agents developed through their ability to directly target the ECM in SSc by, for example, alleviating the persistent matrix stiffness and mechanotransduction that might be required for both the initiation and maintenance of fibrosis, including in SSc. However, because of the importance of the ECM in the SSc phenotype, attempts have now been made to identify drugs that specifically target the ECM, including some drugs that are currently under consideration for the treatment of cancer.

Identification of autoantigens and their potential post-translational modification in EGPA and severe eosinophilic asthma.

Background: The chronic airway inflammation in severe eosinophilic asthma (SEA) suggests potential autoimmune aetiology with unidentified autoantibodies analogous to myeloperoxidase (MPO) in ANCA-positive EGPA (eosinophilic granulomatosis with polyangiitis). Previous research has shown that oxidative post-translational modification (oxPTM) of proteins is an important mechanism by which autoantibody responses may escape immune tolerance. Autoantibodies to oxPTM autoantigens in SEA have not previously been studied. Methods: Patients with EGPA and SEA were recruited as well as healthy control participants. Autoantigen agnostic approach: Participant serum was incubated with slides of unstimulated and PMA-stimulated neutrophils and eosinophils, and autoantibodies to granulocytes were identified by immunofluorescence with anti-human IgG FITC antibody. Target autoantigen approach: Candidate proteins were identified from previous literature and FANTOM5 gene set analysis for eosinophil expressed proteins. Serum IgG autoantibodies to these proteins, in native and oxPTM form, were detected by indirect ELISA. Results: Immunofluorescence studies showed that serum from patients with known ANCA stained for IgG against neutrophils as expected. In addition, serum from 9 of 17 tested SEA patients stained for IgG to PMA-stimulated neutrophils undergoing NETosis. Immunofluorescent staining of eosinophil slides was evident with serum from all participants (healthy and with eosinophilic disease) with diffuse cytoplasmic staining except for one SEA individual in whom subtle nuclear staining was evident. FANTOM5 gene set analysis identified TREM1 (triggering receptor expressed on myeloid cells 1) and IL-1 receptor 2 (IL1R2) as eosinophil-specific targets to test for autoantibody responses in addition to MPO, eosinophil peroxidase (EPX), and Collagen-V identified from previous literature. Indirect ELISAs found high concentrations of serum autoantibodies to Collagen-V, MPO, and TREM1 in a higher proportion of SEA patients than healthy controls. High concentrations of serum autoantibodies to EPX were evident in serum from both healthy and SEA participants. The proportion of patients with positive autoantibody ELISAs was not increased when examining oxPTM compared to native proteins. Discussion: Although none of the target proteins studied showed high sensitivity for SEA, the high proportion of patients positive for at least one serum autoantibody shows the potential of more research on autoantibody serology to improve diagnostic testing for severe asthma. Clinical trial registration: ClinicalTrials.gov, identifier, NCT04671446.

Tripterygium wilfordii derivative celastrol, a YAP inhibitor, has antifibrotic effects in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is characterised by extensive tissue fibrosis maintained by mechanotranductive/proadhesive signalling. Drugs targeting this pathway are therefore of likely therapeutic benefit. The mechanosensitive transcriptional co-activator, yes activated protein-1 (YAP1), is activated in SSc fibroblasts. The terpenoid celastrol is a YAP1 inhibitor; however, if celastrol can alleviate SSc fibrosis is unknown. Moreover, the cell niches required for skin fibrosis are unknown. METHODS: Human dermal fibroblasts from healthy individuals and patients with diffuse cutaneous SSc were treated with or without transforming growth factor ß1 (TGFß1), with or without celastrol. Mice were subjected to the bleomycin-induced model of skin SSc, in the presence or absence of celastrol. Fibrosis was assessed using RNA Sequencing, real-time PCR, spatial transcriptomic analyses, Western blot, ELISA and histological analyses. RESULTS: In dermal fibroblasts, celastrol impaired the ability of TGFß1 to induce an SSc-like pattern of gene expression, including that of cellular communication network factor 2, collagen I and TGFß1. Celastrol alleviated the persistent fibrotic phenotype of dermal fibroblasts cultured from lesions of SSc patients. In the bleomycin-induced model of skin SSc, increased expression of genes associated with reticular fibroblast and hippo/YAP clusters was observed; conversely, celastrol inhibited these bleomycin-induced changes and blocked nuclear localisation of YAP. CONCLUSIONS: Our data clarify niches within the skin activated in fibrosis and suggest that compounds, such as celastrol, that antagonise the YAP pathway may be potential treatments for SSc skin fibrosis.

A Novel CD206 Targeting Peptide Inhibits Bleomycin-Induced Pulmonary Fibrosis in Mice.

Activated M2-polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios, including Idiopathic Pulmonary Fibrosis (IPF). In this study, we investigated the effects of targeting the CD206 receptor in M2-like macrophages with a novel synthetic analogue of a naturally occurring Host Defense Peptide (HDP), RP-832c, to decrease profibrotic cytokines. RP-832c selectively binds to CD206 on M2-polarized bone marrow-derived macrophages (BMDM) in vitro, resulting in a time-dependent decrease in CD206 expression and a transient increase in M1-macrophage marker TNF-α. To elucidate the antifibrotic effects of RP-832c, we used a murine model of bleomycin (BLM)-induced early-stage pulmonary fibrosis. RP-832c significantly reduced fibrosis in a dose-dependent manner, and decreased CD206, TGF-ß1, and α-SMA expression in mouse lungs. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased lung fibrosis and significantly decreased inflammatory cytokines TNF-α, IL-6, IL-10, IFN-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with the FDA-approved drugs Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed. In summary, our findings showed that inhibiting the profibrotic alternatively activated M2-like macrophages using a novel peptide, RP-832c, could reduce BLM-induced pulmonary fibrosis in mice, warranting the therapeutic potential of this peptide for patients with pulmonary fibrosis.

Stratification of biological therapies by pathobiology in biologic-naive patients with rheumatoid arthritis (STRAP and STRAP-EU): two parallel, open-label, biopsy-driven, randomised trials.

BACKGROUND: Despite highly effective targeted therapies for rheumatoid arthritis, about 40% of patients respond poorly, and predictive biomarkers for treatment choices are lacking. We did a biopsy-driven trial to compare the response to rituximab, etanercept, and tocilizumab in biologic-naive patients with rheumatoid arthritis stratified for synovial B cell status. METHODS: STRAP and STRAP-EU were two parallel, open-label, biopsy-driven, stratified, randomised, phase 3 trials done across 26 university centres in the UK and Europe. Biologic-naive patients aged 18 years or older with rheumatoid arthritis based on American College of Rheumatology (ACR)-European League Against Rheumatism classification criteria and an inadequate response to conventional synthetic disease-modifying antirheumatic drugs (DMARDs) were included. Following ultrasound-guided synovial biopsy, patients were classified as B cell poor or B cell rich according to synovial B cell signatures and randomly assigned (1:1:1) to intravenous rituximab (1000 mg at week 0 and week 2), subcutaneous tocilizumab (162 mg per week), or subcutaneous etanercept (50 mg per week). The primary outcome was the 16-week ACR20 response in the B cell-poor, intention-to-treat population (defined as all randomly assigned patients), with data pooled from the two trials, comparing etanercept and tocilizumab (grouped) versus rituximab. Safety was assessed in all patients who received at least one dose of study drug. These trials are registered with the EU Clinical Trials Register, 2014-003529-16 (STRAP) and 2017-004079-30 (STRAP-EU). FINDINGS: Between June 8, 2015, and July 4, 2019, 226 patients were randomly assigned to etanercept (n=73), tocilizumab (n=74), and rituximab (n=79). Three patients (one in each group) were excluded after randomisation because they received parenteral steroids in the 4 weeks before recruitment. 168 (75%) of 223 patients in the intention-to-treat population were women and 170 (76%) were White. In the B cell-poor population, ACR20 response at 16 weeks (primary endpoint) showed no significant differences between etanercept and tocilizumab grouped together and rituximab (46 [60%] of 77 patients vs 26 [59%] of 44; odds ratio 1·02 [95% CI 0·47-2·17], p=0·97). No differences were observed for adverse events, including serious adverse events, which occurred in six (6%) of 102 patients in the rituximab group, nine (6%) of 108 patients in the etanercept group, and three (4%) of 73 patients in the tocilizumab group (p=0·53). INTERPRETATION: In this biologic-naive population of patients with rheumatoid arthrtitis, the dichotomic classification into synovial B cell poor versus rich did not predict treatment response to B cell depletion with rituximab compared with alternative treatment strategies. However, the lack of response to rituximab in patients with a pauci-immune pathotype and the higher risk of structural damage progression in B cell-rich patients treated with rituximab warrant further investigations into the ability of synovial tissue analyses to inform disease pathogenesis and treatment response. FUNDING: UK Medical Research Council and Versus Arthritis.

The Cell-Permeable Derivative of the Immunoregulatory Metabolite Itaconate, 4-Octyl Itaconate, Is Anti-Fibrotic in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin fibrosis. Altered metabolism has recently been described in autoimmune diseases and SSc. Itaconate is a product of the Krebs cycle intermediate cis-aconitate and is an immunomodulator. This work examines the role of the cell-permeable derivative of itaconate, 4-octyl itaconate (4-OI), in SSc. SSc and healthy dermal fibroblasts were exposed to 4-OI. The levels of collagen Nrf2-target genes and pro-inflammatory cytokines interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were determined. Levels of reactive oxygen species (ROS) as well as the gene expression of collagen and Cellular Communication Network Factor 2 (CCN2) were measured after transforming growth factor beta 1 (TGF-ß1) stimulation in the presence or absence of 4-OI. Wild-type or Nrf2-knockout (Nrf2-KO) mouse embryonic fibroblasts (MEFs) were also treated with 4-OI to determine the role of Nrf2 in 4-OI-mediated effects. 4-OI reduced the levels of collagen in SSc dermal fibroblasts. Incubation with 4-OI led to activation of Nrf2 and its target genes heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). 4-OI activated antioxidant response element (ARE)-dependent gene expression, reduced inflammatory cytokine release and reduced TGF-ß1-induced collagen and ROS production in dermal fibroblasts. The effects of 4-OI are dependent on Nrf2. The cell-permeable derivative of itaconate 4-OI is anti-fibrotic through upregulation of Nrf2 and could be a potential therapeutic option in an intractable disease.

Bone Morphogenetic Protein Antagonist Gremlin-1 Increases Myofibroblast Transition in Dermal Fibroblasts: Implications for Systemic Sclerosis.

OBJECTIVE: Systemic Sclerosis is an autoimmune connective tissue disease which results in fibrosis of the skin and lungs. The disease is characterized by activation of myofibroblasts but what governs this is unknown. Gremlin-1 is a BMP antagonist that is developmentally regulated and we sought to investigate its role in Systemic Sclerosis. METHODS: Dermal fibroblasts were transfected with Grem1pcDNA3.1 expression vectors or empty vectors. Various markers of myofibroblasts were measured at the mRNA and protein levels. Scratch wound assays were also performed. Media Transfer experiments were performed to evaluate cytokine like effects. Various inhibitors of TGF-ß signaling and MAPK signaling were used post-transfection. siRNA to Gremlin-1 in SSc dermal fibroblasts were performed to evaluate the role of Gremlin-1. Different cytokines were incubated with fibroblasts and Gremlin-1 measured. Bleomycin was used as model of fibrosis and immunohistochemistry performed. RESULTS: Overexpression of Gremlin-1 was achieved in primary dermal fibroblasts and lead to activation of quiescent cells to myofibroblasts indicated by collagen and α-Smooth muscle actin. Overexpression also led to functional effects. This was associated with increased TGF-ß1 levels and SBE luciferase activity but not increased Thrombospondin-1 expression. Inhibition of Gremlin-1 overexpression cells with antibodies to TGF-ß1 but not isotype controls led to reduced collagen and various TGF-ß pathway chemical inhibitors also led to reduced collagen levels. In SSc cells siRNA mediated reduction of Gremlin-1 reduced collagen expression and CTGF gene and protein levels in these cells. IL-13 did not lead to elevated Gremlin-1 expression nor did IL-11. Gremlin-1 was elevated in an animal model of fibrosis compared to NaCl-treated mice. CONCLUSION: Gremlin-1 is a key regulator of myofibroblast transition leading to enhanced ECM deposition. Strategies that block Gremlin-1 maybe a possible therapeutic target in fibrotic diseases such as SSc.

COVID-19 in Northeast Bosnia and Herzegovina and patient's length of hospitalization.

BACKGROUND: Since the outbreak of COVID-19 pandemic, clinical data from various parts of the world have been reported. Up till now, there has been no clinical data with regards to COVID-19 from Bosnia and Herzegovina (B&H). The aim was to report on the first cohort of patients from B&H and to analyze factors that influence COVID-19 patient's length of hospitalization (LOH). METHODS: This retrospective cohort study was conducted at Tuzla University Clinical Center (UKC), B&H. It involved 25 COVID-19 positive patients that needed hospitalisation between March 28th and April 27th 2020. The LOH was measured from the time of admission to discharge. Factors analyzed induced age, BMI, presence of known comorbidities, serum creatinine and O2 saturation upon admission. RESULTS: The mean age was 52.92 ± 19.15 years and BMI 28.80 ± 4.22. LOH for patients with BMI < 25 was 9 ± SE2.646 days (CI 95% 3.814-14.816) vs 14.182 ± SE .937 (CI 95% 12.346-16.018 p < 0.05; HR 5.148 CI95% 1.217 to 21.772 p = 0.026) for ≥25 BMI. The mean LOH of patients with normal levels of O2 ≥ 95% was 11.667 ± SE1.202 (CI95% 8.261 to 13.739; p = 0.046), while LOH for patients with < 95% was 14.625 ± SE 1.231 CI95% 12.184 to 16.757 p = 0.042; HR 3.732 CI95%1.137-12.251 p = 0.03). Patients without known comorbidities had a mean LOH of 11.700 ± SE1.075 (CI 95% 9.592-13.808), while those with comorbidities had a mean of 14.8 ± 1.303 (CI 95% 12.247-17.353; p = 0.029) with HR2.552. CONCLUSION: LOH varied among COVID-19 patients and was prolonged when analyzed for BMI ≥25, comorbidities, elevated creatinine, and O2 saturation < 95%. Furthermore, risk factors for COVID-19 patients in B&H do not deviate from those reported in other countries.

Verteporfin inhibits the persistent fibrotic phenotype of lesional scleroderma dermal fibroblasts.

Fibrosis is perpetuated by an autocrine, pro-adhesive signaling loop maintained by the synthetic and contractile abilities of myofibroblasts and the stiff, highly-crosslinked extracellular matrix. Transcriptional complexes that are exquisitely responsive to mechanotransduction include the co-activator YAP1, which regulates the expression of members of the CCN family of matricellular proteins such as CCN2 and CCN1. Although selective YAP1 inhibitors exist, the effect of these inhibitors on profibrotic gene expression in fibroblasts is largely unknown, and is the subject of our current study. Herein, we use genome-wide expression profiling, real-time polymerase chain reaction and Western blot analyses, cell migration and collagen gel contraction assays to assess the ability of a selective YAP inhibitor verteporfin (VP) to block fibrogenic activities in dermal fibroblasts from healthy individual human controls and those from isolated from fibrotic lesions of patients with diffuse cutaneous systemic sclerosis (dcSSc). In control fibroblasts, VP selectively reduced expression of fibrogenic genes and also blocked the ability of TGFbeta to induce actin stress fibers in dermal fibroblasts. VP also reduced the persistent profibrotic phenotype of dermal fibroblasts cultured from fibrotic lesions of patients with dcSSc. Our results are consistent with the notion that, in the future, YAP1 inhibitors may represent a novel, valuable method of treating fibrosis as seen in dcSSc.

microRNA27a-3p mediates reduction of the Wnt antagonist sFRP-1 in systemic sclerosis.

Systemic Sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin and lung fibrosis. The Wnt pathway is clearly elevated in SSc and is pro-fibrotic via activation of canonical Wnt signalling. sFRP-1 is a Wnt antagonist that acts as a negative regulator of Wnt signalling. We sought to measure the levels of serum sFRP-1 in early diffuse SSc patients compared to healthy controls and if this is regulated by microRNA27a-3p. Ten early diffuse SSc patients and healthy controls sera were taken and sFRP-1 quantified by ELISA. Skin biopsies were also taken in five SSc patients and controls. Fibroblasts were quantified for microRNA27-3p expression by Taqman qRT-PCR with an internal microRNA to normalize. 3'UTR luciferase assays were performed to confirm direct targets of microRNA27a-3p with microRNA overexpression. Fibroblasts were transfected with microRNA27a mimics or scramble controls and using ELISA sFRP-1 was quantified. Furthermore, Collagen, Axin-2, TIMP-1 and MMP-1 were measured. Serum sFRP-1 was significantly reduced in early diffuse SSc patients. We identified microRNA27a-3p-3p as regulating sFRP-1 in dermal fibroblasts. We found significantly elevated microRNA27a-3p in isolated dermal fibroblasts from SSc patients. We confirmed that sFRP-1 is a direct target of microRNA27a-3p through cloning of the 3'UTR into a luciferase vector. ECM genes were also upregulated by microRNA27a-3p-3p and the matrix-degrading enzyme MMP-1 was suppressed. Serum sFRP-1 is reduced in diffuse SSc patients and is regulated by microRNA27a-3p and this is a direct regulation. Modulation of microRNA27a-3p levels could mediate fibrosis regression.

Wnt antagonist DKK-1 levels in systemic sclerosis are lower in skin but not in blood and are regulated by microRNA33a-3p.

BACKGROUND: Systemic sclerosis is an autoimmune skin disease which is associated with inflammation and resulting skin fibrosis. Myofibroblasts are the key cell type associated with the fibrosis but how they are differentiated is not clear. DKK-1 is a Wnt antagonist that blocks Wnt-mediated fibrosis and is reduced in fibrotic conditions. Thus, DKK-1 is a clear negative regulator of fibrosis in systemic sclerosis and its regulation is unknown. The aim of this work is to determine the levels of DKK-1 in serum and tissues of SSc and its regulation. METHODS: Skin biopsies were taken from early diffuse systemic sclerosis patients and healthy controls and DKK-1 measured by ELISA; serum was also isolated and DKK-1 quantified. DKK-1 was also measured by qRT-PCR. MicroRNA33a-3p was measured by TaqMan PCR. miR mimics and controls were transfected into dermal fibroblasts. Bleomycin mouse model was employed and compared to vehicle control treated mice, and gene expression was employed for DKK-1 and various extracellular matrix genes. RESULTS: DKK-1 is reduced in SSc skin and fibroblasts but is not reduced in the circulation in patients. MicroRNA33a-3p regulates DKK-1 levels epigenetically and is significantly reduced in SSc cells and whole tissue. DKK-1 is also reduced in the bleomycin mouse model and pro-fibrotic genes elevated. CONCLUSION: DKK-1 is reduced in SSc cells and is regulated by miR33a-3p, and restoring DKK-1 levels through epigenetic means could be a therapeutic target in systemic sclerosis.

Metabolic reprogramming of glycolysis and glutamine metabolism are key events in myofibroblast transition in systemic sclerosis pathogenesis.

Systemic Sclerosis (SSc) is a rare fibrotic autoimmune disorder for which no curative treatments currently exist. Metabolic remodelling has recently been implicated in other autoimmune diseases; however, its potential role in SSc has received little attention. Here, we aimed to determine whether changes to glycolysis and glutaminolysis are important features of skin fibrosis. TGF-ß1, the quintessential pro-fibrotic stimulus, was used to activate fibrotic pathways in NHDFs in vitro. Dermal fibroblasts derived from lesions of SSc patients were also used for in vitro experiments. Parameters of glycolytic function were assessed using by measuring extracellular acidification in response to glycolytic activators/inhibitors, whilst markers of fibrosis were measured by Western blotting following the use of the glycolysis inhibitors 2-dg and 3PO and the glutaminolysis inhibitor G968. Succinate was also measured after TGF-ß1 stimulation. Itaconate was added to SSc fibroblasts and collagen examined. TGF-ß1 up-regulates glycolysis in dermal fibroblasts, and inhibition of glycolysis attenuates its pro-fibrotic effects. Furthermore, inhibition of glutamine metabolism also reverses TGF-ß1-induced fibrosis, whilst glutaminase expression is up-regulated in dermal fibroblasts derived from SSc patient skin lesions, suggesting that enhanced glutamine metabolism is another aspect of the pro-fibrotic metabolic phenotype in skin fibrosis. TGF-ß1 was also able to enhance succinate production, with increased succinate shown to be associated with increased collagen expression. Incubation of SSc cells with itaconate, an important metabolite, reduced collagen expression. TGF-ß1 activation of glycolysis is a key feature of the fibrotic phenotype induced by TGF-B1 in skin cells, whilst increased glutaminolysis is also evident in SSc fibroblasts.

A Novel CD206 Targeting Peptide Inhibits Bleomycin Induced Pulmonary Fibrosis in Mice.

Activated M2 polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios such as Acute Respiratory Disease Syndrome (ARDS) and Idiopathic Pulmonary Fibrosis (IPF), through the production of inflammatory and fibrosis-inducing cytokines. In this study, we investigated the effect of targeting the CD206 receptor with a novel fragment of a Host Defense Peptide (HDP), RP-832c to decrease cytokines that cause fibrosis. RP-832c selectively binds to CD206 on M2 polarized bone marrow derived macrophages (BMDM) in vitro , resulting in a time-dependent decrease in CD206 expression, and a transient increase in M1 marker TNFα, which resolves over a 24hr period. To elucidate the antifibrotic effect of RP-832c, we used a murine model of bleomycin (BLM) -induced early-stage pulmonary fibrosis. RP-832c significantly reduced bleomycin-induced fibrosis in a dosage dependent manner, as well as decreased CD206, TGF-ß1 and α-SMA expression in mouse lungs. Interestingly we did not observe any changes in the resident alveolar macrophage marker CD170 expression. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased fibrosis in the lung, as well as significantly decreased inflammatory cytokines TNFα, IL-6, IL-10, INF-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with FDA approved drugs, Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed on body weight or blood chemistry. In summary, RP-832c is a potential agent to mitigate the overactivity of M2 macrophages in pathogenesis several pulmonary fibrotic diseases, including SARS-CoV-2 induced lung fibrosis.