Targeting CD13/aminopeptidase N as a novel therapeutic approach for scleroderma fibrosis.

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune multisystem disease with poorly understood pathogenesis and ineffective treatment options. Soluble CD13 (sCD13), generated by cleavage of cell surface CD13 via matrix metalloproteinase 14 (MMP14), signals through the bradykinin receptor B1 (B1R) to elicit pro-inflammatory, pro-arthritic, and pro-angiogenic responses. In this study we explored the anti-fibrotic potential of targeting the sCD13-B1R axis in SSc. METHODS: The expression of CD13, B1R and MMP14 was examined in SSc skin and explanted dermal fibroblasts. The efficacy of B1R antagonists in the inhibition on fibrosis was determined in vitro and in vivo. RESULTS: Expression of the genes for CD13, B1R and MMP14 was elevated in skin biopsies from patients with diffuse cutaneous (dc)SSc. Notably, single cell analysis of SSc skin biopsies revealed the highest BDKRB1 expression in COL8A1-positive myofibroblasts, a population exclusively seen in SSc. TGF-ß induced the expression of BDKRB1 and production of sCD13 by dcSSc skin fibroblasts. Treatment of dcSSc fibroblasts with sCD13 promoted fibrotic gene expression, signaling, cell proliferation, migration, and gel contraction. The profibrotic sCD13 or TGFß responses were prevented by a B1R antagonist. Mice lacking Cd13 or Bdkrb1 were resistant to bleomycin-induced skin fibrosis and inflammation. Pharmacological B1R inhibition had a comparable antifibrotic effect. CONCLUSION: These results are the first to demonstrate a key role for sCD13 in SSc skin fibrosis, and suggest that targeting the sCD13-B1R signaling axis is a promising novel therapeutic approach for SSc.

Elevated neutrophil extracellular traps in systemic sclerosis-associated vasculopathy and suppression by a synthetic prostacyclin analog.

OBJECTIVES: Neutrophils and neutrophil extracellular traps (NETs) contribute to the vascular complications of multiple diseases, but their role in systemic sclerosis (SSc) is understudied. We sought to test the hypothesis that NETs are implicated in SSc vasculopathy and that treatment with prostacyclin analogs may ameliorate SSc vasculopathy not only through vasodilation but also by inhibiting NET release. METHODS: Blood from 125 patients with SSc (87 diffuse cutaneous SSc and 38 limited cutaneous SSc) was collected at a single academic medical center. Vascular complications such as digital ulcers, pulmonary artery hypertension, and scleroderma renal crisis were recorded. The association between circulating NETs and vascular complications was determined using in vitro and ex vivo assays. The impact of the synthetic prostacyclin analog epoprostenol on NET release was determined. RESULTS: Neutrophil activation and NET release were elevated in patients with SSc-associated vascular complications compared to matched patients without vascular complications. Neutrophil activation and NETs positively correlated with soluble E-selectin and VCAM-1, circulating markers of vascular injury. Treatment of patients with digital ischemia with a synthetic prostacyclin analog boosted neutrophil cyclic AMP, which was associated with the blunting of NET release and reduced NETs in circulation. CONCLUSION: Our study demonstrates an association between NETs and vascular complications in SSc. We also identified the potential for an additional therapeutic benefit of synthetic prostacyclin analogs, namely to reduce neutrophil hyperactivity and NET release in SSc patients.

Senescence and tissue fibrosis: opportunities for therapeutic targeting.

Cellular senescence is a key hallmark of aging. It has now emerged as a key mediator in normal tissue turnover and is associated with a variety of age-related diseases, including organ-specific fibrosis and systemic sclerosis (SSc). This review discusses the recent evidence of the role of senescence in tissue fibrosis, with an emphasis on SSc, a systemic autoimmune rheumatic disease. We discuss the physiological role of these cells, their role in fibrosis, and that targeting these cells specifically could be a new therapeutic avenue in fibrotic disease. We argue that targeting senescent cells, with senolytics or senomorphs, is a viable therapeutic target in fibrotic diseases which remain largely intractable.

Siponimod Attenuates Neuronal Cell Death Triggered by Neuroinflammation via NFκB and Mitochondrial Pathways.

Multiple sclerosis (MS) is the most common autoimmune demyelinating disease of the central nervous system (CNS), consisting of heterogeneous clinical courses varying from relapsing-remitting MS (RRMS), in which disability is linked to bouts of inflammation, to progressive disease such as primary progressive MS (PPMS) and secondary progressive MS (SPMS), in which neurological disability is thought to be linked to neurodegeneration. As a result, successful therapeutics for progressive MS likely need to have both anti-inflammatory and direct neuroprotective properties. The modulation of sphingosine-1-phosphate (S1P) receptors has been implicated in neuroprotection in preclinical animal models. Siponimod/BAF312, the first oral treatment approved for SPMS, may have direct neuroprotective benefits mediated by its activity as a selective (S1P receptor 1) S1P1 and (S1P receptor 5) S1P5 modulator. We showed that S1P1 was mainly present in cortical neurons in lesioned areas of the MS brain. To gain a better understanding of the neuroprotective effects of siponimod in MS, we used both rat neurons and human-induced pluripotent stem cell (iPSC)-derived neurons treated with the neuroinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Cell survival/apoptotic assays using flow cytometry and IncuCyte live cell analyses showed that siponimod decreased TNF-α induced neuronal cell apoptosis in both rat and human iPSCs. Importantly, a transcriptomic analysis revealed that mitochondrial oxidative phosphorylation, NFκB and cytokine signaling pathways contributed to siponimod's neuroprotective effects. Our data suggest that the neuroprotection of siponimod/BAF312 likely involves the relief of oxidative stress in neuronal cells. Further studies are needed to explore the molecular mechanisms of such interactions to determine the relationship between mitochondrial dysfunction and neuroinflammation/neurodegeneration.

Systems-based identification of the Hippo pathway for promoting fibrotic mesenchymal differentiation in systemic sclerosis.

Systemic sclerosis (SSc) is a devastating autoimmune disease characterized by excessive production and accumulation of extracellular matrix, leading to fibrosis of skin and other internal organs. However, the main cellular participants in SSc skin fibrosis remain incompletely understood. Here using differentiation trajectories at a single cell level, we demonstrate a dual source of extracellular matrix deposition in SSc skin from both myofibroblasts and endothelial-to-mesenchymal-transitioning cells (EndoMT). We further define a central role of Hippo pathway effectors in differentiation and homeostasis of myofibroblast and EndoMT, respectively, and show that myofibroblasts and EndoMTs function as central communication hubs that drive key pro-fibrotic signaling pathways in SSc. Together, our data help characterize myofibroblast differentiation and EndoMT phenotypes in SSc skin, and hint that modulation of the Hippo pathway may contribute in reversing the pro-fibrotic phenotypes in myofibroblasts and EndoMTs.

Activation of cytotoxic lymphocytes through CD6 enhances killing of cancer cells.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

Single-cell sequencing reveals Hippo signaling as a driver of fibrosis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease characterized by abscesses, nodules, dissecting/draining tunnels, and extensive fibrosis. Here, we integrate single-cell RNA sequencing, spatial transcriptomics, and immunostaining to provide an unprecedented view of the pathogenesis of chronic HS, characterizing the main cellular players and defining their interactions. We found a striking layering of the chronic HS infiltrate and identified the contribution of 2 fibroblast subtypes (SFRP4+ and CXCL13+) in orchestrating this compartmentalized immune response. We further demonstrated the central role of the Hippo pathway in promoting extensive fibrosis in HS and provided preclinical evidence that the profibrotic fibroblast response in HS can be modulated through inhibition of this pathway. These data provide insights into key aspects of HS pathogenesis with broad therapeutic implications.

Herbal compound cepharanthine attenuates inflammatory arthritis by blocking macrophage M1 polarization.

OBJECTIVE: Cepharanthine (CEP) is a drug candidate for tumor, viral infection, and some inflammatory diseases, but its effect on rheumatoid arthritis (RA) and the underlying mechanism are incompletely understood. METHODS: CEP was administered intraperitoneally to a collagen-induced arthritis (CIA) model. Joints went radiological and histological examination and serum cytokines were examined with cytometry-based analysis. M1 macrophages were induced from THP-1 cells or mouse bone marrow-derived macrophages with LPS and IFN-γ. Bulk RNA-seq was performed on macrophage undergoing M1-polarizatioin. Western blotting was applied to determine pathways involved in monocyte chemotaxis and polarization. Glycolysis metabolites were measured by chemiluminescence while glycolytic enzymes were examined by quantitative PCR. RESULTS: We found CEP significantly ameliorated synovial inflammation and joint destruction of CIA mice. It downregulated TNF-α levels in serum and in joints. The number of M1 macrophages were reduced in CEP-treated mice. In vitro, CEP inhibited monocyte chemotaxis to MCP-1 by downregulating CCR2 and reducing ERK1/2 signaling. Additionally, CEP suppressed M1 polarization of macrophages induced by LPS and IFN-γ. Genes involved in IFN-γ signaling, IL-6-JAK/STAT3 signaling, glycolysis, and oxidative phosphorylation process were downregulated by CEP. Several enzymes critically involved in glycolytic metabolism were suppressed by CEP, which resulted in reduced citrate in M1-polarizing macrophages. The inhibitory effect of CEP on macrophage polarization might be attributed to the blockage of TLRs-MyD88/IRAK4-IRF5 signaling pathway together with suppression of overactivated glycolytic metabolism in M1-polarizing macrophages. CONCLUSION: CEP attenuated joint inflammation by suppressing monocyte chemotaxis and proinflammatory differentiation. It has the potential to be developed into a complementary or alternative therapy for RA.

Activation of Cytotoxic Lymphocytes Through CD6 Enhances Killing of Cancer Cells.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8+ T cells. Tumor-infiltrating cytotoxic lymphocytes were found in higher proportions and were activated in UMCD6-treated mice compared to controls. Similar changes in gene expression were observed by RNA-seq analysis of NK cells treated with UMCD6. Particularly, UMCD6 up-regulated the NKG2D-DAP10 complex and activated PI3K. Thus, the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

Pansclerotic morphea is characterized by IFN-γ responses priming dendritic cell fibroblast crosstalk to promote fibrosis.

Pansclerotic morphea (PSM) is a rare, devastating disease characterized by extensive soft tissue fibrosis, secondary contractions, and significant morbidity. PSM pathogenesis is unknown, and aggressive immunosuppressive treatments rarely slow disease progression. We aimed to characterize molecular mechanisms driving PSM and to identify therapeutically targetable pathways by performing single-cell and spatial RNA-Seq on 7 healthy controls and on lesional and nonlesional skin biopsies of a patient with PSM 12 months apart. We then validated our findings using immunostaining and in vitro approaches. Fibrotic skin was characterized by prominent type II IFN response, accompanied by infiltrating myeloid cells, B cells, and T cells, which were the main IFN-γ source. We identified unique CXCL9+ fibroblasts enriched in PSM, characterized by increased chemokine expression, including CXCL9, CXCL10, and CCL2. CXCL9+ fibroblasts were related to profibrotic COL8A1+ myofibroblasts, which had enriched TGF-ß response. In vitro, TGF-ß and IFN-γ synergistically increased CXCL9 and CXCL10 expression, contributing to the perpetuation of IFN-γ responses. Furthermore, cell-to-cell interaction analyses revealed cDC2B DCs as a key communication hub between CXCL9+ fibroblasts and COL8A1+ myofibroblasts. These results define PSM as an inflammation-driven condition centered on type II IFN responses. This work identified key pathogenic circuits between T cells, cDC2Bs, and myofibroblasts, and it suggests that JAK1/2 inhibition is a potential therapeutic option in PSM.

Pharmacological inhibition of TAK1 prevents and induces regression of experimental organ fibrosis.

Multiorgan fibrosis in systemic sclerosis (SSc) accounts for substantial mortality and lacks effective therapies. Lying at the crossroad of TGF-ß and TLR signaling, TGF-ß-activated kinase 1 (TAK1) might have a pathogenic role in SSc. We therefore sought to evaluate the TAK1 signaling axis in patients with SSc and to investigate pharmacological TAK1 blockade using a potentially novel drug-like selective TAK1 inhibitor, HS-276. Inhibiting TAK1 abrogated TGF-ß1 stimulation of collagen synthesis and myofibroblasts differentiation in healthy skin fibroblasts, and it ameliorated constitutive activation of SSc skin fibroblasts. Moreover, treatment with HS-276 prevented dermal and pulmonary fibrosis and reduced the expression of profibrotic mediators in bleomycin-treated mice. Importantly, initiating HS-276 treatment even after fibrosis was already established prevented its progression in affected organs. Together, these findings implicate TAK1 in the pathogenesis of SSc and identify targeted TAK1 inhibition using a small molecule as a potential strategy for the treatment of SSc and other fibrotic diseases.

Reduced SPAG17 Expression in Systemic Sclerosis Triggers Myofibroblast Transition and Drives Fibrosis.

Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-ß pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling.

Centromere defects, chromosome instability, and cGAS-STING activation in systemic sclerosis.

Centromere defects in Systemic Sclerosis (SSc) have remained unexplored despite the fact that many centromere proteins were discovered in patients with SSc. Here we report that lesion skin fibroblasts from SSc patients show marked alterations in centromeric DNA. SSc fibroblasts also show DNA damage, abnormal chromosome segregation, aneuploidy (only in diffuse cutaneous (dcSSc)) and micronuclei (in all types of SSc), some of which lose centromere identity while retaining centromere DNA sequences. Strikingly, we find cytoplasmic "leaking" of centromere proteins in limited cutaneous SSc (lcSSc) fibroblasts. Cytoplasmic centromere proteins co-localize with antigen presenting MHC Class II molecules, which correlate precisely with the presence of anti-centromere antibodies. CENPA expression and micronuclei formation correlate highly with activation of the cGAS-STING/IFN-ß pathway as well as markers of reactive oxygen species (ROS) and fibrosis, ultimately suggesting a link between centromere alterations, chromosome instability, SSc autoimmunity, and fibrosis.

Fibroblast A20 governs fibrosis susceptibility and its repression by DREAM promotes fibrosis in multiple organs.

In addition to autoimmune and inflammatory diseases, variants of the TNFAIP3 gene encoding the ubiquitin-editing enzyme A20 are also associated with fibrosis in systemic sclerosis (SSc). However, it remains unclear how genetic factors contribute to SSc pathogenesis, and which cell types drive the disease due to SSc-specific genetic alterations. We therefore characterize the expression, function, and role of A20, and its negative transcriptional regulator DREAM, in patients with SSc and disease models. Levels of A20 are significantly reduced in SSc skin and lungs, while DREAM is elevated. In isolated fibroblasts, A20 mitigates ex vivo profibrotic responses. Mice haploinsufficient for A20, or harboring fibroblasts-specific A20 deletion, recapitulate major pathological features of SSc, whereas DREAM-null mice with elevated A20 expression are protected. In DREAM-null fibroblasts, TGF-ß induces the expression of A20, compared to wild-type fibroblasts. An anti-fibrotic small molecule targeting cellular adiponectin receptors stimulates A20 expression in vitro in wild-type but not A20-deficient fibroblasts and in bleomycin-treated mice. Thus, A20 has a novel cell-intrinsic function in restraining fibroblast activation, and together with DREAM, constitutes a critical regulatory network governing the fibrotic process in SSc. A20 and DREAM represent novel druggable targets for fibrosis therapy.

Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis.

Activation of TLR4 by its cognate damage-associated molecular patterns (DAMPs) elicits potent profibrotic effects and myofibroblast activation in systemic sclerosis (SSc), while genetic targeting of TLR4 or its DAMPs in mice accelerates fibrosis resolution. To prevent aberrant DAMP/TLR4 activity, a variety of negative regulators evolved to dampen the magnitude and duration of the signaling. These include radioprotective 105 kDa (RP105), a transmembrane TLR4 homolog that competitively inhibits DAMP recognition of TLR4, blocking TLR4 signaling in immune cells. The role of RP105 in TLR4-dependent fibrotic responses in SSc is unknown. Using unbiased transcriptome analysis of skin biopsies, we found that levels of both TLR4 and its adaptor protein MD2 were elevated in SSc skin and significantly correlated with each other. Expression of RP105 was negatively associated with myofibroblast differentiation in SSc. Importantly, RP105-TLR4 association was reduced, whereas TLR4-TLR4 showed strong association in fibroblasts from patients with SSc, as evidenced by PLA assays. Moreover, RP105 adaptor MD1 expression was significantly reduced in SSc skin biopsies and explanted SSc skin fibroblasts. Exogenous RP105-MD1 abrogated, while loss of RP105 exaggerated, fibrotic cellular responses. Importantly, ablation of RP105 in mice was associated with augmented TLR4 signaling and aggravated skin fibrosis in complementary disease models. Thus, we believe RP105-MD1 to be a novel cell-intrinsic negative regulator of TLR4-MD2-driven sustained fibroblast activation, representing a critical regulatory network governing the fibrotic process. Impaired RP105 function in SSc might contribute to persistence of progression of the disease.

Role of cellular senescence in the pathogenesis of systemic sclerosis.

PURPOSE OF REVIEW: Systemic sclerosis (SSc) is a chronic rheumatic disease that is characterized by immune activation, vasculopathy and fibrosis of the skin and internal organs. It has been proposed that premature onset of ageing pathways and associated senescent changes in cells contribute to the clinical and pathological features of SSc. The aim of this review is to critically review recent insights into the involvement of cellular senescence in SSc. RECENT FINDINGS: Cellular senescence plays a critical role in SSc pathogenesis, particularly involving endothelial cells and fibroblasts. Immunosenescence could also contribute to SSc pathogenesis by direct alteration of cellular functions or indirect promotion of defective immune surveillance. Molecular studies have shed some light on how cellular senescence contributes to fibrosis. Recent and planned proof-of-concept trials using senotherapeutics showed promising results in fibrotic diseases, including SSc. SUMMARY: There is increasing evidence implicating cellular senescence in SSc. The mechanisms underlying premature cellular senescence in SSc, and its potential role in pathogenesis, merit further investigation. Emerging drugs targeting senescence-related pathways might be potential therapeutic options for SSc.

Gut microbe-derived metabolite trimethylamine N-oxide activates PERK to drive fibrogenic mesenchymal differentiation.

Intestinal dysbiosis is prominent in systemic sclerosis (SSc), but it remains unknown how it contributes to microvascular injury and fibrosis that are hallmarks of this disease. Trimethylamine (TMA) is generated by the gut microbiome and in the host converted by flavin-containing monooxygenase (FMO3) into trimethylamine N-oxide (TMAO), which has been implicated in chronic cardiovascular and metabolic diseases. Using cell culture systems and patient biopsies, we now show that TMAO reprograms skin fibroblasts, vascular endothelial cells, and adipocytic progenitor cells into myofibroblasts via the putative TMAO receptor protein R-like endoplasmic reticulum kinase (PERK). Remarkably, FMO3 was detected in skin fibroblasts and its expression stimulated by TGF-ß1. Moreover, FMO3 was elevated in SSc skin biopsies and in SSc fibroblasts. A meta-organismal pathway thus might in SSc link gut microbiome to vascular remodeling and fibrosis via stromal cell reprogramming, implicating the FMO3-TMAO-PERK axis in pathogenesis, and as a promising target for therapy.

Soluble CD13 induces inflammatory arthritis by activating the bradykinin receptor B1.

CD13, an ectoenzyme on myeloid and stromal cells, also circulates as a shed, soluble protein (sCD13) with powerful chemoattractant, angiogenic, and arthritogenic properties, which require engagement of a G protein-coupled receptor (GPCR). Here we identify the GPCR that mediates sCD13 arthritogenic actions as the bradykinin receptor B1 (B1R). Immunofluorescence and immunoblotting verified high expression of B1R in rheumatoid arthritis (RA) synovial tissue and fibroblast-like synoviocytes (FLSs), and demonstrated binding of sCD13 to B1R. Chemotaxis, and phosphorylation of Erk1/2, induced by sCD13, were inhibited by B1R antagonists. In ex vivo RA synovial tissue organ cultures, a B1R antagonist reduced secretion of inflammatory cytokines. Several mouse arthritis models, including serum transfer, antigen-induced, and local innate immune stimulation arthritis models, were attenuated in Cd13-/- and B1R-/- mice and were alleviated by B1R antagonism. These results establish a CD13/B1R axis in the pathogenesis of inflammatory arthritis and identify B1R as a compelling therapeutic target in RA and potentially other inflammatory diseases.