Dermal Fibrosis Research Articles

A comparative analysis of deferoxamine treatment modalities for dermal radiation-induced fibrosis.

The iron chelator, deferoxamine (DFO), has been shown to potentially improve dermal radiation‐induced fibrosis (RIF) in mice through increased angiogenesis and reduced oxidative damage. This preclinical study evaluated the efficacy of two DFO administration modalities, transdermal delivery and direct injection, as well as temporal treatment strategies in relation to radiation therapy to address collateral soft tissue fibrosis. The dorsum of CD‐1 nude mice received 30 Gy radiation, and DFO (3 mg) was administered daily via patch or injection. Treatment regimens were prophylactic, during acute recovery, post‐recovery, or continuously throughout the experiment (n = 5 per condition). Measures included ROS‐detection, histology, biomechanics and vascularity changes. Compared with irradiated control skin, DFO treatment decreased oxidative damage, dermal thickness and collagen content, and increased skin elasticity and vascularity. Metrics of improvement in irradiated skin were most pronounced with continuous transdermal delivery of DFO. In summary, DFO administration reduces dermal fibrosis induced by radiation. Although both treatment modalities were efficacious, the transdermal delivery showed greater effect than injection for each temporal treatment strategy. Interestingly, the continuous patch group was more similar to normal skin than to irradiated control skin by most measures, highlighting a promising approach to address detrimental collateral soft tissue injury following radiation therapy.

Insight into the role of dermal white adipose tissue loss in dermal fibrosis.

The loss of dermal white adipose tissue (dWAT) is vital to the formation of dermal fibrosis (DF), but the specific mechanism is not well understood. A few studies are reviewed to explore the role of dWAT in the formation of DF. Recent findings indicated that the adipocytes-to-myofibroblasts transition in dWAT reflects the direct contribution to the DF formation. While adipose-derived stem cells (ADSCs) contained in dWAT express antifibrotic cytokines, the loss of ADSCs leads to skin protection decreased, which indirectly exacerbates DF and tissue damage. Therefore, blocking or reversing the adipocytes-to-myofibroblasts transition or improving the survival of ADSCs in dWAT and the expression of antifibrotic cytokines may be an effective strategy for the treatment of DF.

Reconstruction of Distal Nasal Defects With a Large Postauricular Skin-Fat-Fascia Composite Graft.

Composite grafts have previously been reported to achieve a good outcome for nasal defect repair, but composite grafts have greater metabolic needs than simple skin. Therefore, the traditionally recommended size of a composite graft for nasal reconstruction is less than 1.5 cm in diameter. However, the distal nose is generally well supplied with blood vessels, which might support the use of larger composite grafts in such a highly vascularized recipient site. The aim of the article is to investigate whether a large skin-fat-fascia composite graft (larger than 2.0 cm) is viable for the repair of partial-thickness nasal defects. From October 2017 to December 2019, 13 patients with partial-thickness nasal defects underwent nasal reconstruction using a large postauricular skin-fat-fascia composite graft. Cases were followed up for 3 to 14 months postoperatively. The aesthetic outcome was evaluated in comparison with preoperative digital images. Skin-fat-fascia composite grafts survived without graft necrosis, dermal fibrosis, or skin contraction in all cases. Favorable aesthetic outcomes were obtained in all patients, and no further revision surgery was need. A postauricular composite graft larger than 2.0 cm is a safe and effective reconstruction approach for partial-thickness nasal defects. This technique offers significant advantages in terms of no additional facial scar, no visible asymmetry on the face, no additional surgery for revision, and with mild scar in the donor site of the postauricular region.

Metabolic perturbations in fibrosis disease.

Metabolic changes occur in all forms of disease but their impact on fibrosis is a relatively recent area of interest. This review provides an overview of the major metabolic pathways, glycolysis, amino acid metabolism and lipid metabolism, and highlights how they influence fibrosis at a cellular and tissue level, drawing on key discoveries in dermal, renal, pulmonary and hepatic fibrosis. The emerging influence of adipose tissue-derived cytokines is discussed and brings a link between fibrosis and systemic metabolism. To close, the concept of targeting metabolism for fibrotic therapy is reviewed, drawing on lessons from the more established field of cancer metabolism, with an emphasis on important considerations for clinical translation.

Mesenchymal Stem Cell-Derived Exosomes Ameliorate Dermal Fibrosis in a Murine Model of Bleomycin-Induced Scleroderma.

Mesenchymal stem cells (MSCs) have become a promising therapeutic strategy for scleroderma. Exosomes derived from MSCs (MSC-exosomes) possess functional properties similar to those of their source cells. In this study, we aimed to explore the potential role of MSC-exosomes in the treatment of scleroderma. MSC-exosomes were isolated from human umbilical cords through ultracentrifugation and characterized. An experimental fibrosis model was established in BALB/c mice by a subcutaneous injection of bleomycin, followed by treatment with MSC-exosomes or MSC infusions once a week for a total of four doses. Using hematoxylin and eosin and Masson's trichrome staining and immunohistochemistry, hydroxyproline content, and quantitative real-time polymerase chain reaction analyses, we investigated the effects of MSC-exosomes on dermal fibrosis and explored the underlying mechanism. MSC-exosome treatment restored the dermal architecture, reduced dermal thickness, and partially increased subcutaneous adipose tissue thickness. In addition, MSC-exosomes inhibited the expression of collagen (COL)-I, COL-III, and α-smooth muscle actin. The transforming growth factor (TGF)-β/Smad signaling pathway was also suppressed in MSC-exosome-treated mice. Taken together, our results suggest that MSC-exosomes can attenuate myofibroblast activation and collagen deposition in dermal fibrosis by downregulating the TGF-β/Smad signaling pathway. Therefore, the use of MSC-exosomes may be a potential therapeutic approach for the treatment of scleroderma.

TFH cells in systemic sclerosis

Systemic sclerosis is an autoimmune disease characterized by excessive dermal fibrosis with progression to internal organs, vascular impairment and immune dysregulation evidenced by the infiltration of inflammatory cells in affected tissues and the production of auto antibodies. While the pathogenesis remains unclear, several data highlight that T and B cells deregulation is implicated in the disease pathogenesis. Over the last decade, aberrant responses of circulating T follicular helper cells, a subset of CD4 T cells which are able to localise predominantly in the B cell follicles through a high level of chemokine receptor CXCR5 expression are described in pathogenesis of several autoimmune diseases and chronic graft-versus-host-disease. In the present review, we summarized the observed alteration of number and frequency of circulating T follicular helper cells in systemic sclerosis. We described their role in aberrant B cell activation and differentiation though interleukine-21 secretion. We also clarified T follicular helper-like cells involvement in fibrogenesis in both human and mouse model. Finally, because T follicular helper cells are involved in both fibrosis and autoimmune abnormalities in systemic sclerosis patients, we presented the different strategies could be used to target T follicular helper cells in systemic sclerosis, the therapeutic trials currently being carried out and the future perspectives from other auto-immune diseases and graft-versus-host-disease models.

The Role of T Lymphocytes in Cutaneous Scarring.

Significance: Cutaneous scarring affects millions of patients worldwide and results in significant financial and psychosocial burdens. Given the immune system's intricate involvement in the initiation and progression of wound healing, it is no surprise that the scarring outcome can be affected by the actions of various immune cells and the cytokines and growth factors they produce. Understanding the role of T cells in regulating immune responses and directing the action of wound mesenchymal cells is essential to developing antifibrotic therapies to reduce the burden of scarring.Recent Advances: As the immune system is intimately involved in wound healing, much work has examined the impact of T cells and their cytokines on the final wound outcome. New innovative tools for studying T cells have resulted in more sophisticated immunophenotyping capabilities and the ability to examine effects of individual cytokines in the wound environment.Critical Issues: Despite continued advances in the study of specific immune cells and their effects on dermal fibrosis, minimal progress has been made to modulate immune responses to result in improved wound cosmesis.Future Directions: The actions of T cells represent potential pharmacologic targets that could lead to novel bioengineered or immunoengineered therapies to improve the lives of people with cutaneous scarring.

Antifibrotic effects and mechanisms of mesenchymal stem cell-derived exosomes in a systemic sclerosis mouse model: Possible contribution of miR-196b-5p

BackgroundSystemic sclerosis (SSc) is a connective tissue disorder characterized by the development of fibrosis in the skin and internal organs. Increasing evidence suggests that mesenchymal stem cells (MSCs) can be used to a treatment for fibrotic diseases. Recent studies have demonstrated that some of the biological effects of MSCs are due to the secretion of exosomes. However, the precise mechanisms underlying MSCs-derived exosomes in skin fibrosis are not well understood. ObjectiveWe aimed to elucidate the effect of MSCs-derived exosomes on skin fibrosis in SSc and the mechanism underlying their inhibitory action on fibrosis. MethodsExosome was collected from MSCs by ultracentrifugation method. We examined the suppressive effect of MSCs-derived exosome on skin fibrosis in bleomycin-induced SSc mouse model. Skin samples from the injected site were collected for further examination, and micro-RNA analysis of MSCs-derived exosome was performed. ResultsInjection of MSCs-derived exosomes significantly inhibited bleomycin-induced dermal fibrosis in mice. MSCs-derived exosomes significantly reduced the amount of collagen and the number of α-SMA+ myofibroblasts and CD68+ macrophages in lesional skin. They also reduced the expression of type I collagen and TGF-β receptor 1 in fibroblasts in vitro. Moreover, micro-RNA analysis revealed that several microRNAs in MSCs-derived exosomes have antifibrotic potential. We confirmed that overexpression of miR-196b-5p in fibroblasts significantly suppressed collagen type I alpha 2 expression. ConclusionThis study demonstrated that inhibition of collagen type I expression by miR-196b-5p in exosomes might be one of the mechanisms by which MSCs suppress skin fibrosis in an SSc mouse model.

Regulatory T cells promote innate inflammation after skin barrier breach via TGF-β activation.

Regulatory T cells (Tregs) use multiple mechanisms to attenuate inflammation and prevent autoimmunity. Tregs residing in peripheral (i.e., nonlymphoid) tissues have specialized functions; specifically, skin Tregs promote wound healing, suppress dermal fibrosis, facilitate epidermal regeneration, and augment hair follicle cycling. Here, we demonstrated that skin Tregs were transcriptionally attuned to interact with their tissue environment through increased expression of integrin and TGF-β pathway genes that influence epithelial cell biology. We identified a molecular pathway where skin Tregs license keratinocytes to promote innate inflammation after skin barrier breach. Using a single-cell discovery approach, we identified preferential expression of the integrin αvβ8 on skin Tregs Upon skin injury, Tregs used this integrin to activate latent TGF-β, which acted directly on epithelial cells to promote CXCL5 production and neutrophil recruitment. Induction of this circuit delayed epidermal regeneration but provided protection from Staphylococcus aureus infection across a compromised barrier. Thus, αvβ8-expressing Tregs in the skin, somewhat paradoxical to their canonical immunosuppressive functions, facilitated inflammation acutely after loss of barrier integrity to promote host defense against infection.

Skin atrophy caused by topical glucocorticoids is less common in patients with atopic dermatitis than in those with psoriasis.

Although the long-term use of topical glucocorticoids (TGC) may induce skin atrophy including striae distensae (SD), patients with atopic dermatitis (AD) appear to have lesser degree of skin atrophy than those with psoriasis (PSO). Periostin, encoded by POSTN, is involved in tissue remodelling processes of chronic AD lesions. This study was designed to investigate the difference in the occurrence of skin atrophy in patients with AD or PSO when treated with TGC and to elucidate the association between skin atrophy and periostin. Big data analysis using Korean Health Claims Database was performed to determine the prevalence of SD in AD and PSO patients. Blood and skin eosinophils count and dermal fibrosis between AD and PSO patients were compared, and immunohistochemistry for periostin and mRNA sequencing in the dermis were performed. Animal experiments using AD and PSO murine model were conducted. Big data analysis revealed that patients with AD have significantly lesser degree of SD than patients with PSO. The ratio of the dermal fibrous tissues and eosinophil counts were significantly higher in AD patients. In AD skin, periostin was more widely distributed in the entire dermis and POSTN mRNAs were significantly upregulated. Dermal thickness and fibrosis were significantly higher in AD mice even after TGC treatment. A significant positive correlation was observed between dermal fibrosis and tissue eosinophil counts. Lesser skin atrophy in AD patients even after long-term TGC application could be resulted from skin fibrosis caused by increased tissue eosinophils and periostin deposition.

EHP-101 alleviates angiotensin II-induced fibrosis and inflammation in mice

Some cannabinoids showed anti-inflammatory and antifibrotic activities. EHP-101 is an oral lipidic formulation of the novel non-psychotropic cannabidiol aminoquinone VCE-004.8, which showed antifibrotic activity in murine models of systemic sclerosis induced by bleomycin. We herein examined the effect of EHP-101 on cardiac and other organ fibrosis in a mouse model induced by Angiotensin II. VCE-004.8 inhibited TGFβ- and Ang II-induced myofibroblast differentiation in cardiac fibroblasts detected by α-SMA expression. VCE-004.8 also inhibited Ang II-induced ERK 1 + 2 phosphorylation, NFAT activation and mRNA expression of IL1β, IL6, Col1A2 and CCL2 in cardiac fibroblasts. Mice infused with Ang II resulted in collagen accumulation in left ventricle, aortic, dermal, renal and pulmonary tissues; oral administration of EHP-101, Ajulemic acid and Losartan improved these phenotypes. In myocardial tissue, Ang II induced infiltration of T cells and macrophages together with the accumulation of collagen and Tenascin C; those were all reduced by either EHP-101 or Losartan treatment. Cardiac tissue RNA-Seq analyses revealed a similar transcriptomic signature for both treatments for inflammatory and fibrotic pathways. However, the gene set enrichment analysis comparing data from EHP-101 vs Losartan showed specific hallmarks modified only by EHP-101. Specifically, EHP-101 inhibited the expression of genes such as CDK1, TOP2A and MKi67 that are regulated to the E2 factor family of transcription factors. This study suggests that the oral administration of EHP-101 prevents and inhibits cardiac inflammation and fibrosis. Furthermore, EHP-101 inhibits renal, pulmonary and dermal fibrosis. EHP-101 could offer new opportunities in the treatment of cardiac fibrosis and other fibrotic diseases.

Dermoscopy-An Aid in the Diagnosis of Mycetoma.

A 45-year-old farmer consulted for a gradually increasing swelling over right leg. Examination revealed hard swelling with multiple discharging sinuses over right foot. Many pin-point hypopigmented macules arranged in a schematic pattern were seen, corresponding to the site of administration of indigenous medication [Figure 1]. Dermoscopic examination using DermLite™ DL3-3Gen under contact polarized mode from one of the active sinus revealed small black grains, surrounded by bluish white veil suggestive of deeper fungal colonies [Figure 2a]. White structureless area around the openings corresponding to dermal fibrosis was seen[Figure 2b]. Skin punch biopsy from the lesion demonstrated periodic-acid-schiff positive, pigmented fungal colonies over a background of granulation tissue [Figure 3].Figure 1: Diffuse woody hard swelling over dorsum of right foot with multiple discharging sinusesFigure 2: a. Dermoscopy of the sinus opening shows multiple small black grains. b. The sinuses are surrounded by bluish white veil (Red arrow). Crust surrounding the sinus opening can be seen (Yellow arrow) Structureless pink-white areas related to the underlying fibrosis was seen surrounding to the lesion (Star). (DL3 dermoscope, polarized mode 10x)Figure 3: Histopathology shows pigmented fungal colony comprising of radiating hyphae of eumycetoma, showing splendor hoeppli phenomenon and surrounded by dense neutrophilic infiltrate. (PAS stain, 10x)Mycetoma is a granulomatous infection that can be caused by bacteria (actinomycotic mycetoma) or fungi (eumycotic mycetoma). Grains represent the hallmark of mycetoma and color of grains is often suggestive of the probable causative agent. However, diagnostic facilities are often limited and inaccessible to many patients affected by this neglected tropical disease.[1] Dermoscopy is an easy, quick, and noninvasive tool, which can be helpful in visualizing minute grains and ascertain the type of mycetoma.[1] Black colored granules are highly suggestive of eumycotic mycetoma and its visualization by dermoscopy can help in early institution of definitive treatment. The dermoscopic images can also be stored and transferred electronically and an expert opinion sought through teledermoscopy.[2] Dermoscopy has been explored in the diagnosis of many pigmentary disorders, granulomatous infective diseases like leprosy and sporotrichosis.[34] The current report reinstitutes the importance of dermoscopy in diagnosis of dermatological disorders with clinico-dermoscopic-pathological correlation.[2] Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

OC-0286 Wnt-associated DKK3 mediates radiation-induced dermal inflammation and fibrosis

OC-0286 Wnt-associated DKK3 mediates radiation-induced dermal inflammation and fibrosis

TGF\u03b2 promotes fibrosis by MYST1-dependent epigenetic regulation of autophagy

Activation of fibroblasts is essential for physiological tissue repair. Uncontrolled activation of fibroblasts, however, may lead to tissue fibrosis with organ dysfunction. Although several pathways capable of promoting fibroblast activation and tissue repair have been identified, their interplay in the context of chronic fibrotic diseases remains incompletely understood. Here, we provide evidence that transforming growth factor-β (TGFβ) activates autophagy by an epigenetic mechanism to amplify its profibrotic effects. TGFβ induces autophagy in fibrotic diseases by SMAD3-dependent downregulation of the H4K16 histone acetyltransferase MYST1, which regulates the expression of core components of the autophagy machinery such as ATG7 and BECLIN1. Activation of autophagy in fibroblasts promotes collagen release and is both, sufficient and required, to induce tissue fibrosis. Forced expression of MYST1 abrogates the stimulatory effects of TGFβ on autophagy and re-establishes the epigenetic control of autophagy in fibrotic conditions. Interference with the aberrant activation of autophagy inhibits TGFβ-induced fibroblast activation and ameliorates experimental dermal and pulmonary fibrosis. These findings link uncontrolled TGFβ signaling to aberrant autophagy and deregulated epigenetics in fibrotic diseases and may contribute to the development of therapeutic interventions in fibrotic diseases.

Evolution of Dupilumab-Associated Cutaneous Atypical Lymphoid Infiltrates.

Observations highlighting the "unmasking" of cutaneous T-cell lymphoma after treatment with dupilumab for atopic dermatitis (AD) have been recently reported. However, there remains a paucity of literature describing the evolution of clinical and histopathological features that characterizes this phenomenon. To define the clinical and histopathologic evolution of atypical lymphoid infiltrates after the administration of dupilumab for AD. A cross-sectional study of clinical and histopathologic features in 7 consecutive patients with a diagnosis of "atypical lymphoid infiltrate" or mycosis fungoides (MF) on dupilumab for AD was performed. Seven patients with atypical lymphoid infiltrates or MF in evolution after dupilumab therapy (age range 27-74 years) were reviewed. Average duration of AD before MF diagnosis was 5.7 years, and the average duration on dupilumab treatment was 9.8 months. Notable histopathologic features across predupilumab and postdupilumab biopsies included progressive increase in the densities of the atypical lymphoid infiltrates (7/7), presence of atypical epidermotropic lymphocytes (6/7), and papillary dermal fibrosis (6/7). Small retrospective cohort study. These cases highlight the transformation of lymphoid infiltrates after dupilumab treatment for AD and emphasize the importance of clinical and histopathologic evaluation before and during treatment with dupilumab for treatment-refractory presumed AD.

Development of a Pure Small Molecule αVβ3 Antagonist

# Contributed equallyαVβ3 has been implicated in the pathophysiology of important disorders, including osteoporosis, sickle cell disease, tumor angiogenesis, metastasis, virus invasion, glomerulonephritis, dermal and hepatic fibrosis, acute myelogenous leukemia, supravalvular aortic stenosis, bowel strictures, and T-cell lymphoma, but no αVβ3 antagonists have been approved for human therapy. The RGD-based αVβ3 antagonist MK-0429 (MK429) had favorable results in osteoporosis patients in Phase 2, but was not advanced, and the RGD-based antagonist cilengitide was studied in patients with malignancies, but did not demonstrate efficacy. Since RGD-based antagonists can under certain circumstances activate αVβ3, prime it to bind ligand, induce apoptosis, increase tumor angiogenesis and growth, and increase hepatic fibrosis, we sought to develop a pure αVβ3 antagonist that does not induce high affinity ligand binding or exposure of the epitope for mAb AP5, both of which depend on the swing-out of the β3 PSI domain. We built on Arnaout's group's studies of a variant fibronectin fragment with high affinity for αVβ3 (hFN10) that did not induce the β3 swing-out motion because a Trp substitution on the ligand formed a π-π interaction with β3 Y122 on the β1-α1 loop, thus preventing the latter's movement toward the MIDAS, which is the trigger for β3 swing-out. To develop a small-molecule that mimics the effect of hFN10, we used inferences from a predicted docking pose of MK429 into the hFN10 binding site of the αVβ3 crystal structure to rationally design a compound with an aromatic ring that directly engages β3 Y122. Based on the results of molecular dynamics simulations, the aromatic group of this compound, TDI-4161, is only 4.4 Å from Y122 (and forms a π-π interaction), which compares with 4.7 Å for hFN10 and 7.0 Å for MK429 (which does not form a π-π interaction). We analyzed compounds in 2 assays: 1. Adhesion of HEK293 cells expressing human αVβ3 (HEK-αVβ3) to fibrinogen (reported as the concentration producing 50% inhibition; IC50). 2. Exposure of the epitope for mAb AP5 (the concentration producing 50% exposure; EC50). The racemate of MK429 (rMK429) has an IC50=0.0036 µM and an EC50=0.019 µM, and cilengitide has an IC50=0.050 µM and an EC50=0.034 µM, indicating that both are potent αVβ3 antagonists and potent inducers of the αVβ3 active conformation. In sharp contrast, TDI-4161 has an IC50=0.022 µM and an EC50>10 µM, indicating that it does not induce the β3 conformational change even at 400X the IC50. TDI-4161 inhibited the binding of: purified αVβ3 to adenovirus 2 penton base (IC50 = 0.028 µM) and purified αVβ5 to vitronectin (IC50 =2.09 µM), but not purified αVβ6 to TGF-β1 Latency Associated Peptide even at 10 µM. TDI-4161inhibited adhesion of mouse endothelial cells containing murine αVβ3 to fibrinogen (IC50 = 0.437 µM). We tested TDI-4161's ability to 'prime“ αVβ3 by: incubating it with HEK-αVβ3 at 10 µM, fixing the cells with paraformaldehyde, washing, incubating with fluorescent fibrinogen, and measuring bound fibrinogen by flow cytometry. The peptide RGDS (100 µM) increased fibrinogen binding from 8.7 ± 1.5 to 24.3 ± 1.4 GMFI (p <0.001; n=3), but TDI-4161 did not (7.0 ± 2.6 GMFI; p<0.001 vs RGDS). After oral administration of TDI-4161 to mice at 100 mg/kg, the Cmax was 7.5 µM at the Tmax of 0.94 hours; the T1/2 was 3.6 hours. Once-daily oral dosing of TDI-4161 for 28 days at 10, 30, and 100 mg/kg was well tolerated in male CD-1 mice (n=3). Day 1 and day 28 plasma levels 1 hour after dosing were similar. We tested the effect of TDI-4161 on osteoclast function by adding it at 10 µM (0.1% DMSO) to bone marrow-derived murine macrophages and cultured them either on plastic or bovine bone slices with RANKL and a source of M-CSF. After 5 days, cells incubated with TDI-4161 demonstrated an osteoclast phenotype similar to that of β3 null mice; osteoclast numbers were better preserved than with rMK429, but less well than with the DMSO control. Supernatants from bone slices were assayed for cross-linked collagen degradation products (CrossLaps). When compared to the DMSO control (32.7 nM), rMK429 (10 µM) produced 82% inhibition (5.9 nM) and TDI-4161 produced 74% inhibition (8.3 nM). In summary, using inferences from structural data derived from hFN10, we have synthesized an orally bioavailable pure αVβ3 antagonist that reacts with both human and murine αVβ3, does not prime αVβ3 to bind fibrinogen, and inhibits osteoclast bone resorption. DisclosuresFilizola:Icahn School of Medicine at Mount Sinai: Patents & Royalties. Foley:Tri-Institutional Therapeutics Discovery Institute: Patents & Royalties.

Inhibition of Hsp90 Counteracts the Established Experimental Dermal Fibrosis Induced by Bleomycin.

Our previous study demonstrated that heat shock protein 90 (Hsp90) is overexpressed in the involved skin of patients with systemic sclerosis (SSc) and in experimental dermal fibrosis. Pharmacological inhibition of Hsp90 prevented the stimulatory effects of transforming growth factor-beta on collagen synthesis and the development of dermal fibrosis in three preclinical models of SSc. In the next step of the preclinical analysis, herein, we aimed to evaluate the efficacy of an Hsp90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), in the treatment of established experimental dermal fibrosis induced by bleomycin. Treatment with 17-DMAG demonstrated potent antifibrotic and anti-inflammatory properties: it decreased dermal thickening, collagen content, myofibroblast count, expression of transforming growth factor beta receptors, and pSmad3-positive cell counts, as well as leukocyte infiltration and systemic levels of crucial cytokines/chemokines involved in the pathogenesis of SSc, compared to vehicle-treated mice. 17-DMAG effectively prevented further progression and may induce regression of established bleomycin-induced dermal fibrosis to an extent comparable to nintedanib. These findings provide further evidence of the vital role of Hsp90 in the pathophysiology of SSc and characterize it as a potential target for the treatment of fibrosis with translational implications due to the availability of several Hsp90 inhibitors in clinical trials for other indications.

Sensoryneuron-derived TAFA4 promotes macrophage tissue repair functions.

Inflammation is a defence response to tissue damage that requires tight regulation in order to prevent impaired healing. Tissue-resident macrophages have a key role in tissue repair1, but the precise molecular mechanisms that regulate the balance between inflammatory and pro-repair macrophage responses during healing remain poorly understood. Here we demonstrate a major role for sensory neurons in promoting the tissue-repair function of macrophages. In a sunburn-like model of skin damage in mice, the conditional ablation of sensory neurons expressing the Gαi-interacting protein (GINIP) results in defective tissue regeneration and in dermal fibrosis. Elucidation of the underlying molecular mechanisms revealed a crucial role for the neuropeptide TAFA4, which is produced in the skin by C-low threshold mechanoreceptors-a subset of GINIP+ neurons. TAFA4 modulates the inflammatory profile of macrophages directly in vitro. In vivo studies in Tafa4-deficient mice revealed that TAFA4 promotes the production of IL-10 by dermal macrophages after UV-induced skin damage. This TAFA4-IL-10 axis also ensures the survival and maintenance of IL-10+TIM4+ dermal macrophages, reducing skin inflammation and promoting tissue regeneration. These results reveal a neuroimmune regulatory pathway driven by the neuropeptide TAFA4 that promotes the anti-inflammatory functions of macrophages and prevents fibrosis after tissue damage, and could lead to new therapeutic perspectives for inflammatory diseases.

OP0242 EFFECTS OF THE AUTOTAXIN INHIBITOR ZIRITAXESTAT ON SKIN AND LUNG FIBROSIS IN A MURINE GRAFT-VERSUS-HOST DISEASE MODEL OF SYSTEMIC SCLEROSIS

Background:There is an unmet medical need for new drugs to treat systemic sclerosis (SSc). Autotaxin (ATX) is a widely expressed enzyme that regulates diverse cellular processes, including proliferation, differentiation and migration, and has been implicated in the pathogenesis of SSc. Targeting ATX is a promising new strategy for treating SSc. The autotaxin inhibitor ziritaxestat (GLPG1690) is a potential first-in-class disease-modifying drug for SSc that has been shown to improve skin score in the Phase 2a NOVESA (NCT03798366) trial in patients with SSc.Objectives:To investigate the effects of ziritaxestat in a chronic graft-versus-host disease (cGvHD) murine model of SSc.Methods:Effects of ziritaxestat (10 or 30 mg/kg twice daily [bid]) on disease activity were assessed in a cGvHD murine model of SSc (allogeneic bone marrow transplantation [BMT] with B10.D2 donor and BALB/c recipient; syngeneic mice as controls). Ziritaxestat or nintedanib (60 mg/kg once daily [qd]) as active comparator was administered 21 d after BMT and continued for 35 d. Effects of ziritaxestat were assessed by clinical monitoring, histologic assessment of skin and lungs (dermal thickness, Ashcroft scores and collagen-covered area), immunofluorescence staining with Trichrome and Sirius Red for myofibroblasts, and biochemical analysis of collagen content, as measured by hydroxyproline levels.Results:Ziritaxestat 30 mg/kg bid for 35 days significantly reduced the clinical cutaneous score in the murine cGvHD model by 57% (p&lt;0.05) compared with vehicle, and to a similar extent when compared with nintedanib 60 mg/kg (38%; p&lt;0.05). Dermal accumulation of collagen and dermal thickness (Figure) were reduced with ziritaxestat 10 and 30 mg/kg compared with vehicle. At 30 mg/kg, ziritaxestat reversed the increase in the allogeneic model (p&lt;0.001), returning dermal thickness to the levels in non-fibrotic control mice. Ziritaxestat also significantly reduced pulmonary fibrosis in the cGvHD model, with reductions in the fibrotic lung area (ziritaxestat 10 and 30 mg/kg; p&lt;0.001 for both) and Ashcroft scores (ziritaxestat 30 mg/kg; p&lt;0.05). Ziritaxestat was generally well tolerated.Conclusion:Ziritaxestat improved the histological, biochemical and clinical symptom readouts of dermal and pulmonary fibrosis in a murine model, consistent with a broad and rapid disease-modifying effect in SSc.Acknowledgements:This study was funded by Galapagos NV (Mechelen, Belgium). Medical writing/editorial support was provided by Ian Faulkner, PhD (Aspire Scientific, Bollington, UK) funded by Galapagos NV.Disclosure of Interests:Yun Zhang Employee of: 4D Science, Lena Summa Employee of: 4D Science, Bertrand Heckmann Shareholder of: Galapagos, Employee of: Galapagos, Jörg H.W. Distler Shareholder of: 4D Science, Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GlaxoSmithKline, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Grant/research support from: Anamar, Active Biotech, Array Biopharma, ARXX, aTyr, Bristol Myers Squibb, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GlaxoSmithKline, Inventiva, Novartis, Sanofi-Aventis, RedX and UCB

AB0410 S100A4 PLASMA LEVELS CORRELATE WITH DISEASE ACTIVITY, SKIN FIBROSIS AND INTERSTITIAL LUNG DISEASE IN SYSTEMIC SCLEROSIS PATIENTS

Background:In our previous study we demonstrated that S100A4 is overexpressed in scleroderma (SSc) skin, SSc fibroblasts and preclinical models of SSc in a TGF-β dependent manner. We showed that S100A4 is a new regulator of TGF-β signalling and its inhibition prevents the pro-fibrotic effects of TGF-β. Inactivation of S100A4 prevented dermal fibrosis induced by bleomycin and in Tsk-1 miceObjectives:The aim of this study was to evaluate S100A4 in the peripheral blood of SSc patients and characterize its potential association with SSc-related features.Methods:A total of 33 patients (29 females; mean age 52.8; disease duration 4.2 years; dcSSc/lcSSc = 8/25) who met the 2013 EULAR/ACR classification criteria for SSc and 20 healthy age- and sex-matched individuals were included in this study. Plasma levels of S100A4 were measured using ELISA (CUSABIO, Houston, USA). Data are presented as median (IQR).Results:S100A4 plasma levels were significantly increased in SSc patients compared to healthy controls (78.6(32.3-146.5) vs. 43.4(32.3-53.4)ng/mL,p=0.011). Patients with diffuse cutaneous (dc)SSc had significantly higher levels of S100A4 than patients with limited cutaneous (lc)SSc or healthy controls (168.5(81.5-347.5) vs. 63.4(30.9-130.6),p=0.017,p=0.001, respectively). Plasma levels of S100A4 positively correlated with mRSS (r=0.556,p=0.001). Furthermore, S100A4 negatively correlated with forced vital capacity (FVC) and peripheral oxygen saturation (SpO2) (r=- 0.362,p=0.038;r=-0.414,p=0.029, respectively). S100A4 levels positively correlated with ESSG activity score (r=0.750,p&lt;0.001). However, only correlations between S100A4 and mRSS, and ESSG activity score were approved at corrected level of statistical significance after Bonferroni’s correction (p&lt;0.01). In a prospective analysis of patients (n=40) with progressive SSc-ILD treated with 6 (n=24) or 12 (n=16) monthly i.v. pulses of cyclophosphamide (CPA, 500 mg/m2), we observed a significant decrease in plasma S100A4 levels between the baseline samples (month 0) and blood drawn after 6 months of CPA treatment (76.3(52.9–98.6) vs. 73.2(44.4–98.6)ng/mL,p=0.013). Furthermore, baseline S100A4 levels predicted the change (m0-m6) in CRP and ESR levels after 6 months of CPA therapy (r=0.472,p=0.004;r=0.528,p=0.003, respectively).Conclusion:We demonstrate that plasma S100A4 levels are significantly increased in SSc patients compared with healthy controls. Increased S100A4 is associated with the dcSSc subset, skin involvement, deteriorated parameters of interstitial lung disease and higher disease activity. In patients with progressive SSc-ILD, S100A4 declines after 6 months of cyclophosphamide therapy and predicts the systemic inflammatory response. These data further support our previous findings on the role of S100A4 as a regulator of TGF-β induced fibrosis in SSc.Acknowledgements:Supported by MHCR023728, SVV–260373.Disclosure of Interests:None declared