CD7 activation regulates cytotoxicity-driven pathology in systemic sclerosis, yielding a target for selective cell depletion.

OBJECTIVES: Cytotoxic T cells and natural killer (NK) cells are central effector cells in cancer and infections. Their effector response is regulated by activating and inhibitory receptors. The regulation of these cells in systemic autoimmune diseases such as systemic sclerosis (SSc) is less defined. METHODS: We conducted ex vivo analysis of affected skin and blood samples from 4 SSc patient cohorts (a total of 165 SSc vs 80 healthy individuals) using single-cell transcriptomics, flow cytometry and multiplex immunofluorescence staining. We further analysed the effects of costimulatory modulation in functional assays, and in a severely affected SSc patient who was treated on compassionate use with a novel anti-CD3/CD7 immunotoxin treatment. RESULTS: Here, we show that SSc-affected skin contains elevated numbers of proliferating T cells, cytotoxic T cells and NK cells. These cells selectively express the costimulatory molecule CD7 in association with cytotoxic, proinflammatory and profibrotic genes, especially in recent-onset and severe disease. We demonstrate that CD7 regulates the cytolytic activity of T cells and NK cells and that selective depletion of CD7+ cells prevents cytotoxic cell-induced fibroblast contraction and inhibits their profibrotic phenotype. Finally, anti-CD3/CD7 directed depletive treatment eliminated CD7+ skin cells and stabilised disease manifestations in a severely affected SSc patient. CONCLUSION: Together, the findings imply costimulatory molecules as key regulators of cytotoxicity-driven pathology in systemic autoimmune disease, yielding CD7 as a novel target for selective depletion of pathogenic cells.

Separating friend from foe: Inhibition of TGF-ß-induced detrimental SMAD1/5/9 phosphorylation while maintaining protective SMAD2/3 signaling in OA chondrocytes.

OBJECTIVE: Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial to control cartilage homeostasis. However, TGF-ß can also have detrimental effects by signaling via SMAD1/5/9 and thereby contribute to diseases like osteoarthritis (OA). In this study, we aimed to block TGF-ß-induced SMAD1/5/9 signaling in primary human OA chondrocytes, while maintaining functional SMAD2/3 signaling. DESIGN: Human OA chondrocytes were pre-incubated with different concentrations of ALK4/5/7 kinase inhibitor SB-505124 before stimulation with TGF-ß. Changes in SMAD C-terminal phosphorylation were analyzed using Western blot and response genes were measured with quantitative Polymerase Chain Reaction. To further explore the consequences of our ability to separate pathways, we investigated TGF-ß-induced chondrocyte hypertrophy. RESULTS: Pre-incubation with 0.5 µM SB-505124, maintained ±50% of C-terminal SMAD2/3 phosphorylation and induction of JUNB and SERPINE1, but blocked SMAD1/5/9-C phosphorylation and expression of ID1 and ID3. Furthermore, TGF-ß, in levels comparable to those in the synovial fluid of OA patients, resulted in regulation of hypertrophic and dedifferentiation markers in OA chondrocytes; i.e. an increase in COL10, RUNX2, COL1A1, and VEGF and a decrease in ACAN expression. Interestingly, in a subgroup of OA chondrocyte donors, blocking only SMAD1/5/9 caused stronger inhibition on TGF-ß-induced RUNX2 than blocking both SMAD pathways. CONCLUSION: Our findings indicate that using low dose of SB-505124 we maintained functional SMAD2/3 signaling that blocks RUNX2 expression in a subgroup of OA patients. We are the first to show that SMAD2/3 and SMAD1/5/9 pathways can be separately modulated using low and high doses of SB-505124 and thereby split TGF-ß's detrimental from protective function in chondrocytes.

Prediction of the Effect of the Osteoarthritic Joint Microenvironment on Cartilage Repair.

Osteoarthritis (OA) is characterized by progressive articular cartilage loss. Human mesenchymal stromal cells (MSCs) can be used for cartilage repair therapies based on their potential to differentiate into chondrocytes. However, the joint microenvironment is a major determinant of the success of MSC-based cartilage formation. Currently, there is no tool that is able to predict the effect of a patient's OA joint microenvironment on MSC-based cartilage formation. Our goal was to develop a molecular tool that can predict this effect before the start of cartilage repair therapies. Six different promoter reporters (hIL6, hIL8, hADAMTS5, hWISP1, hMMP13, and hADAM28) were generated and evaluated in an immortalized human articular chondrocyte for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm) obtained from 32 different knee OA patients. To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model, while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. We confirmed that OAs-cm of different patients had significantly different effects on sGAG production. In addition, significant correlations were obtained between the effect of the OAs-cm on cartilage formation and promoter reporter outcome. Furthermore, we validated the predictive value of measuring two promoter reporters with an independent cohort of OAs-cm and the effect of 87.5% of the OAs-cm on MSC-based cartilage formation could be predicted. Together, we developed a novel tool to predict the effect of the OA joint microenvironment on MSC-based cartilage formation. This is an important first step toward personalized cartilage repair strategies for OA patients. Impact statement We describe the development of a novel molecular tool to predict if an osteoarthritis joint microenvironment is permissive for cartilage repair or not. Such a tool is of great importance in determining the success of mesenchymal stromal cell-based cartilage repair strategies.

Cripto favors chondrocyte hypertrophy via TGF-ß SMAD1/5 signaling during development of osteoarthritis.

Chondrocytes in mice developing osteoarthritis (OA) exhibit an aberrant response to the secreted cytokine transforming growth factor (TGF)-ß, consisting in a potentiation of intracellular signaling downstream of the transmembrane type I receptor kinase activin receptor-like kinase (ALK)1 against canonical TGF-ß receptor ALK5-mediated signaling. Unfortunately, the underlying mechanisms remain elusive. In order to identify novel druggable targets for OA, we aimed to investigate novel molecules regulating the ALK1/ALK5 balance in OA chondrocytes. We performed gene expression analysis of TGF-ß signaling modulators in joints from three different mouse models of OA and found an upregulated expression of the TGF-ß co-receptor Cripto (Tdgf1), which was validated in murine and human cartilage OA samples at the protein level. In vitro and ex vivo, elevated expression of Cripto favors the hypertrophic differentiation of chondrocytes, eventually contributing to tissue calcification. Furthermore, we found that Cripto participates in a TGF-ß-ALK1-Cripto receptor complex in the plasma membrane, thereby inducing catabolic SMAD1/5 signaling in chondrocytes. In conclusion, we demonstrate that Cripto is expressed in OA and plays a functional role promoting chondrocyte hypertrophy, thereby becoming a novel potential therapeutic target in OA, for which there is no efficient cure or validated biomarker. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Osteoarthritis-Related Inflammation Blocks TGF-ß's Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region.

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-ß signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1ß and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-ß signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1ß was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.

Interleukin-37 Protects Stem Cell-Based Cartilage Formation in an Inflammatory Osteoarthritis-Like Microenvironment.

IMPACT STATEMENT: Catabolic factors present in a damaged joint inhibit chondrogenic differentiation of mesenchymal stem cells, thereby reducing the chance for successful cartilage formation. By improving stem cell-based cartilage repair with interleukin-37 (IL37), we might be able to inhibit the worsening progression of focal cartilage defects and prevent further development of joint diseases such as osteoarthritis. This will avoid chronic pain and impaired joint mobility for patients and reduce costs for society.

Unraveling SSc Pathophysiology; The Myofibroblast.

Systemic sclerosis (SSc) is a severe auto-immune disease, characterized by vasculopathy and fibrosis of connective tissues. SSc has a high morbidity and mortality and unfortunately no disease modifying therapy is currently available. A key cell in the pathophysiology of SSc is the myofibroblast. Myofibroblasts are fibroblasts with contractile properties that produce a large amount of pro-fibrotic extracellular matrix molecules such as collagen type I. In this narrative review we will discuss the presence, formation, and role of myofibroblasts in SSc, and how these processes are stimulated and mediated by cells of the (innate) immune system such as mast cells and T helper 2 lymphocytes. Furthermore, current novel therapeutic approaches to target myofibroblasts will be highlighted for future perspective.

IL37 dampens the IL1ß-induced catabolic status of human OA chondrocytes.

Objective: A crucial feature of OA is cartilage degradation. This process is mediated by pro-inflammatory cytokines, among other factors, via induction of matrix-degrading enzymes. Interleukin 37 (IL37) is an anti-inflammatory cytokine and is efficient in blocking the production of pro-inflammatory cytokines during innate immune responses. We hypothesize that IL37 is therapeutic in treating the inflammatory cytokine cascade in human OA chondrocytes and can act as a counter-regulatory cytokine to reduce cartilage degradation in OA. Methods: Human OA cartilage was obtained from patients undergoing total knee or hip arthroplasty. Immunohistochemistry was applied to study IL37 protein expression in cartilage biopsies from OA patients. Induction of IL37 expression by IL1ß, OA synovium-conditioned medium and TNFα was investigated in human OA chondrocytes. Adenoviral overexpression of IL37 followed by IL1ß stimulation was performed to investigate the anti-inflammatory potential of IL37. Results: IL37 expression was detected in cartilage biopsies of OA patients and induced by IL1ß. After IL1ß stimulation, increased IL1ß, IL6 and IL8 expression was observed in OA chondrocytes. Elevated IL37 levels diminished the IL1ß-induced IL1ß , IL6 and IL8 gene levels and IL1ß and IL8 protein levels. In addition to the reduction in pro-inflammatory cytokine expression, IL37 reduced MMP1 , MMP3 , MMP13 and disintegrin and metalloproteinase with thrombospondin motifs 5 gene levels and MMP3 and MMP13 protein levels. Conclusion: IL37 is induced by IL1ß, and IL37 itself reduced IL1ß, IL6 and IL8 production, indicating that IL37 is able to induce a counter-regulatory anti-inflammatory feedback loop in chondrocytes. In addition, IL37 dampens catabolic enzyme expression. This supports IL37 as a potential therapeutic target in OA.

Commercial cow milk contains physically stable extracellular vesicles expressing immunoregulatory TGF-ß.

SCOPE: Extracellular vesicles, including exosomes, have been identified in all biological fluids and rediscovered as an important part of the intercellular communication. Breast milk also contains extracellular vesicles and the proposed biological function is to enhance the antimicrobial defense in newborns. It is, however, unknown whether extracellular vesicles are still present in commercial milk and, more importantly, whether they retained their bioactivity. Here, we characterize the extracellular vesicles present in semi-skimmed cow milk available for consumers and study their effect on T cells. METHODS AND RESULTS: Extracellular vesicles from commercial milk were isolated and characterized. Milk-derived extracellular vesicles contained several immunomodulating miRNAs and membrane protein CD63, characteristics of exosomes. In contrast to RAW 267.4 derived extracellular vesicles the milk-derived extracellular vesicles were extremely stable under degrading conditions, including low pH, boiling and freezing. Milk-derived extracellular vesicles were easily taken up by murine macrophages in vitro. Furthermore, we found that they can facilitate T cell differentiation towards the pathogenic Th17 lineage. Using a (CAGA)12-luc reporter assay we showed that these extracellular vesicles carried bioactive TGF-ß, and that anti-TGF-ß antibodies blocked Th17 differentiation. CONCLUSION: Our findings show that commercial milk contains stable extracellular vesicles, including exosomes, and carry immunoregulatory cargo. These data suggest that the extracellular vesicles present in commercial cow milk remains intact in the gastrointestinal tract and exert an immunoregulatory effect.