Jeffrey H. Ruth

Objective: Systemic sclerosis (SSc) is an autoimmune multisystem disease with poorly understood pathogenesis and ineffective treatment options. Soluble CD13 (sCD13), generated by the 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 antifibrotic 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. Transforming growth factor beta (TGFβ) 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 pro‐fibrotic responses of sCD13 or TGFβ 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. image

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid arthritis (RA), compared to osteoarthritis or other arthritis patients. Several studies suggested an important role for MCP-1 in the massive inflammation at the damaged joint, in part due to its chemotactic and angiogenic effects. It is a known fact that the post-translational modifications (PTMs) of proteins have a significant impact on their properties. In mammals, arginine residues within proteins can be converted into citrulline by peptidylarginine deiminase (PAD) enzymes. Anti-citrullinated protein antibodies (ACPA), recognizing these PTMs, have become a hallmark for rheumatoid arthritis (RA) and other autoimmune diseases and are important in diagnostics and prognosis. In previous studies, we found that citrullination converts the neutrophil attracting chemokine neutrophil-activating peptide 78 (ENA-78) into a potent macrophage chemoattractant. Here we report that both commercially available and recombinant bacterially produced MCP-1/CCL2 are rapidly (partially) degraded upon in vitro citrullination. However, properly glycosylated MCP-1/CCL2 produced by mammalian cells is protected against degradation during efficient citrullination. Site-directed mutagenesis of the potential glycosylation site at the asparagine-14 residue within human MCP-1 revealed lower expression levels in mammalian expression systems. The glycosylation-mediated recombinant chemokine stabilization allows the production of citrullinated MCP-1/CCL2, which can be effectively used to calibrate crucial assays, such as modified ELISAs.

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.

Binding of the bromodomain and extraterminal domain proteins (BETs) to acetylated histone residues is critical for gene transcription. We sought to determine the antifibrotic efficacy and potential mechanisms of BET inhibition in systemic sclerosis (SSc). Blockade of BETs was done using a pan-BET inhibitor, JQ1; BRD2 inhibitor, BIC1; or BRD4 inhibitors AZD5153 or ARV825. BET inhibition, specifically BRD4 blockade, showed antifibrotic effects in an animal model of SSc and in patient-derived diffuse cutaneous SSc (dcSSc) fibroblasts. Transcriptome analysis of JQ1-treated dcSSc fibroblasts revealed differentially expressed genes related to extracellular matrix, cell cycle, and calcium (Ca ²⁺ ) signaling. The antifibrotic effect of BRD4 inhibition was mediated at least in part by downregulation of Ca ²⁺ /calmodulin–dependent protein kinase II α and reduction of intracellular Ca ²⁺ concentrations. On the basis of these results, we propose targeting Ca ²⁺ pathways or BRD4 as potentially novel therapeutic approaches for progressive tissue fibrosis.

Limitations of checkpoint inhibitor cancer immunotherapy include induction of autoimmune syndromes and resistance of many cancers. Since CD318, a novel CD6 ligand, is associated with the aggressiveness and metastatic potential of human cancers, we tested the effect of an anti-CD6 monoclonal antibody, UMCD6, on killing of cancer cells by human lymphocytes. UMCD6 augmented killing of breast, lung, and prostate cancer cells through direct effects on both CD8 ⁺ T cells and NK cells, increasing cancer cell death and lowering cancer cell survival in vitro more robustly than monoclonal antibody checkpoint inhibitors that interrupt the programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) axis. UMCD6 also augmented in vivo killing by human peripheral blood lymphocytes of a human breast cancer line xenotransplanted into immunodeficient mice. Mechanistically, UMCD6 upregulated the expression of the activating receptor NKG2D and downregulated expression of the inhibitory receptor NKG2A on both NK cells and CD8 ⁺ T cells, with concurrent increases in perforin and granzyme B production. The combined capability of an anti-CD6 monoclonal antibody to control autoimmunity through effects on CD4 ⁺ lymphocyte differentiation while enhancing killing of cancer cells through distinct effects on CD8 ⁺ and NK cells opens a potential new approach to cancer immunotherapy that would suppress rather than instigate autoimmunity.

Objective: CD6 is an important regulator of T cell function that interacts with the ligands CD166 and CD318. To further clarify the significance of CD6 in rheumatoid arthritis (RA), we examined the effects of targeting CD6 in the mouse model of collagen‐induced arthritis (CIA), using CD6‐knockout (CD6‐KO) mice and CD6‐humanized mice that express human CD6 in lieu of mouse CD6 on their T cells.Methods: We immunized wild‐type (WT) and CD6 gene–KO mice with a collagen emulsion to induce CIA. For treatment studies using CD6‐humanized mice, mice were immunized similarly and a mouse anti‐human CD6 IgG (UMCD6) or control IgG was injected on days 7, 14, and 21. Joint tissues were evaluated for tissue damage, leukocyte infiltration, and local inflammatory cytokine production. Collagen‐specific Th1, Th9, and Th17 responses and serum levels of collagen‐specific IgG subclasses were also evaluated in WT and CD6‐KO mice with CIA.Results: The absence of CD6 reduced 1) collagen‐specific Th9 and Th17, but not Th1 responses, 2) the levels of many proinflammatory joint cytokines, and 3) serum levels of collagen‐reactive total IgG and IgG1, but not IgG2a and IgG3. Joint homogenate hemoglobin content was significantly reduced in CD6‐KO mice with CIA compared to WT mice with CIA (P < 0.05) (reduced angiogenesis). Moreover, treating CD6‐humanized mice with mouse anti‐human CD6 monoclonal antibody was similarly effective in reducing joint inflammation in CIA.Conclusion: Taken together, these data suggest that interaction of CD6 with its ligands is important for the perpetuation of CIA and other inflammatory arthritides that are T cell driven.

Cancer immunotherapy is a rapidly advancing and viable approach to treating cancer along with more traditional forms of therapy. Real-time cell analysis technologies that examine the dynamic interactions between cancer cells and the cells of the immune system are becoming more important for assessment of novel therapeutics. In this report, we use the IncuCyte® imaging system to study the killing potential of various immune cells on cancer cell lines. The IncuCyte® system tracks living cells, labeled by a red fluorescent protein, and cell death, as indicated by the caspase-3/7 reagent, which generates a green fluorescent signal upon activation of apoptotic pathways. Despite the power of this approach, obtaining commercially fluorescent cancer cell lines is expensive and limited in the range of cell lines that are available. To overcome this barrier, we developed an inexpensive method using a lentiviral construct expressing nuclear localized mKate2 red fluorescent protein to stably label cancer cells. We demonstrate that this method is effective in labeling a wide variety of cell lines, allowing for analyses of different cancers as well as different cell lines of the same type of cancer.

Objective: To explore the intrinsic role of inhibitor of DNA binding 1 (ID‐1) in rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS) and to investigate whether ID‐1 is citrullinated and autoantigenic in RA.Methods: RA patient serum ID‐1 levels were measured before and after infliximab treatment. RA FLS were transfected with a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 construct targeting ID‐1 to examine the effects of ID‐1 deletion. RA synovial fluid (SF) and homogenized synovial tissue (ST) were immunoprecipitated for ID‐1 and measured for citrullinated residues using an enzyme‐linked immunosorbent assay and Western blotting. Liquid chromatography tandem mass spectrometry (LC‐MS/MS) was performed on in vitro–citrullinated recombinant human ID‐1 (cit–ID‐1) to localize the sites of citrullination. Normal and RA sera and SF were analyzed by immunodot blotting for anti–citrullinated protein antibodies (ACPAs) to cit–ID‐1.Results: RA patient serum ID‐1 levels positively correlated with several disease parameters and were reduced after infliximab treatment. RA FLS displayed reduced growth and a robust increase in interleukin‐6 (IL‐6) and IL‐8 production upon deletion of ID‐1. ID‐1 immunodepletion significantly reduced the levels of citrullinated residues in RA SF, and citrullinated ID‐1 was detected in homogenized RA ST (n = 5 samples; P < 0.05). Immunodot blot analyses revealed ACPAs to cit–ID‐1 but not to native ID‐1, in RA peripheral blood (PB) sera (n = 30 samples; P < 0.001) and SF (n = 18 samples; P < 0.05) but not in normal PB sera. Following analyses of LC‐MS/MS results for citrullination sites and corresponding reactivity in immunodot assays, we determined the critical arginines in ID‐1 for autoantigenicity: R33, R52, and R121.Conclusion: Novel roles of ID‐1 in RA include regulation of FLS proliferation and cytokine secretion as well as autoantigenicity following citrullination.

Inhibitor of DNA binding 1 (Id1) is a nuclear protein containing a basic helix-loop-helix (bHLH) domain that regulates cell growth by selective binding and prevention of gene transcription. Sources of Id1 production in rheumatoid arthritis synovial tissue (RA ST) and its range of functional effects in RA remain to be clarified. We analyzed Id1 produced from synovial fibroblasts and endothelial cells (ECs) with histology and real-time polymerase chain reaction (RT-PCR). Fibroblast supernatants subjected to differential centrifugation to isolate and purify exosomes were measured for Id1 by enzyme-linked immunosorbent assay (ELISA). Western blotting of Id1-stimulated ECs was performed to determine the kinetics of intracellular protein phosphorylation. EC intracellular signaling pathways induced by Id1 were subsequently targeted with silencing RNA (siRNA) for angiogenesis inhibition. By PCR and histologic analysis, we found that the primary source of Id1 in STs is from activated fibroblasts that correlate with inflammatory scores in human RA ST and in joints from K/BxN serum-induced mice. Normal (NL) and RA synovial fibroblasts increase Id1 production with stimulation by transforming growth factor beta (TGF-β). Most of the Id1 released by RA synovial fibroblasts is contained within exosomes. Endothelial progenitor cells (EPCs) and human dermal microvascular ECs (HMVECs) activate the Jnk signaling pathway in response to Id1, and Jnk siRNA reverses Id1-induced HMVEC vessel formation in Matrigel plugs in vivo. Id1 is a pleotropic molecule affecting angiogenesis, vasculogenesis, and fibrosis. Our data shows that Id1 is not only an important nuclear protein, but also can be released from fibroblasts via exosomes. The ability of extracellular Id1 to activate signaling pathways expands the role of Id1 in the orchestration of tissue inflammation.

Objectives Angiogenesis contributes to the pathogenesis of rheumatoid arthritis. Fucosyltransferases (Futs) are involved in angiogenesis and tumour growth. Here, we examined the role of Fut1 in angiogenesis and K/BxN serum transfer arthritis. Methods We examined Fut1 expression in human dermal microvascular endothelial cells (HMVECs) by quantitative PCR. We performed a number of angiogenesis assays to determine the role of Fut1 using HMVECs, Fut1 null (Fut1^−/−), and wild type (wt) endothelial cells (ECs) and mice. K/BxN serum transfer arthritis was performed to determine the contribution of Fut1-mediated angiogenesis in Fut1^−/− and wt mice. A static adhesion assay was implemented with RAW264.7 (mouse macrophage cell line) and mouse ECs. Quantitative PCR, immunofluorescence and flow cytometry were performed with Fut1^−/− and wt ECs for adhesion molecule expression. Results Tumour necrosis factor-α induced Fut1 mRNA and protein expression in HMVECs. HMVECs transfected with Fut1 antisense oligodeoxynucleotide and Fut1^−/− ECs formed significantly fewer tubes on Matrigel. Fut1^−/− mice had reduced angiogenesis in Matrigel plug and sponge granuloma angiogenesis assays compared with wt mice. Fut1^−/− mice were resistant to K/BxN serum transfer arthritis and had decreased angiogenesis and leucocyte ingress into inflamed joints. Adhesion of RAW264.7 cells to wt mouse ECs was significantly reduced when Fut1 was lacking. Fut1^−/− ECs had decreased intercellular adhesion molecule-1 (ICAM-1) expression at mRNA and protein levels compared with wt ECs. ICAM-1 was also decreased in Fut1^−/− arthritic ankle cryosections compared with wt ankles. Conclusions Fut1 plays an important role in regulating angiogenesis and ICAM-1 expression in inflammatory arthritis.