ObjectivesCD19-targeting chimeric antigen receptor (CAR) T-cell therapy can induce long-term drug-free remission in patients with autoimmune diseases (AIDs). The efficacy of CD19-CAR T-cell therapy is presumably based on deep tissue depletion of B cells; however, such effect has not been proven in humans in vivo.MethodsSequential ultrasound-guided inguinal lymph node biopsies were performed at baseline and after CD19-CAR T-cell therapy in patients with AIDs. Results were compared with lymph node biopsies from rituximab (RTX)-treated AID patients with absence of peripheral B cells. Conventional and immunohistochemistry staining were performed on lymph node tissue to assess architecture as well the number of B cells, follicular dendritic cells (FDCs), plasma cells, T cells and macrophages.ResultsSequential lymph node biopsies were analysed from five patients with AID before and after CD19-CAR T-cell therapy and from five patients with AID after RTX treatment. In addition, non-lymphoid organ biopsies (colon, kidney and gallbladder) from three additional patients with AID after CD19-CAR T-cell therapy were analysed. CD19+and CD20+B cells were completely depleted in the lymph nodes after CD19-CAR T-cell therapy, but not after RTX treatment. Plasma cells, T cells and macrophages in the lymph nodes remained unchanged. Follicular structures were disrupted and FDCs were depleted in the lymph nodes after CD19-CAR T-cell therapy, but not after RTX. Non-lymphoid organs were completely depleted of B cells.DiscussionThis study demonstrates complete B-cell depletion in secondary lymphoid tissues of patients with AIDs following CD19-CAR T-cell therapy combined with standard lymphodepleting therapy.

Fibroblasts are important regulators of inflammation, but whether fibroblasts change phenotype during resolution of inflammation is not clear. Here we use positron emission tomography to detect fibroblast activation protein (FAP) as a means to visualize fibroblast activation in vivo during inflammation in humans. While tracer accumulation is high in active arthritis, it decreases after tumor necrosis factor and interleukin-17A inhibition. Biopsy-based single-cell RNA-sequencing analyses in experimental arthritis show that FAP signal reduction reflects a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalization) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalization). Spatial transcriptomics of human joints indicates that pro-resolving niches of CD200+DKK3+ fibroblasts cluster with type 2 innate lymphoid cells, whereas MMP3+/IL6+ fibroblasts colocalize with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilized the type 2 innate lymphoid cell phenotype and induced resolution of arthritis via CD200-CD200R1 signaling. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

OBJECTIVE:Deregulation of the cJUN/AP-1 and hedgehog/GLI2 signaling pathways has been implicated in fibroblast activation in systemic sclerosis (SSc). However, the consequences of their concomitant up-regulation are unknown. Here, we tested the hypothesis that mutual amplification of both pathways might drive persistent fibroblast activation. METHODS:Cultured fibroblasts and skin sections of patients with diffuse SSc and healthy volunteers were analyzed. cJUN/AP-1 signaling and hedgehog/GLI2 signaling were inhibited using knockdown and pharmacologic approaches. Hedgehog signaling was activated in mice by fibroblast-specific overexpression of constitutively active Smoothened. RESULTS:cJUN and GLI2 are concomitantly up-regulated and colocalize in fibroblasts of patients with SSc compared to healthy controls. Activation of hedgehog/GLI2 signaling induces the expression of cJUN in vitro and in vivo, whereas inactivation of GLI2 inhibits cJUN expression. Likewise, inactivation of cJUN impairs the expression of GLI2. This mutual regulation occurs at the level of transcription with binding of cJUN and GLI2 to specific binding motifs. Interference with this mutual amplification of cJUN signaling and GLI2 signaling inhibits fibroblast activation and collagen release: Inhibition of cJUN/AP-1 signaling ameliorates hedgehog-induced fibroblast activation and skin fibrosis in SmoACT mice with a reduction of skin thickness of 103% (P = 0.0043) in the treatment group compared to the fibrotic control group. Moreover, combined pharmacologic inhibition of cJUN/AP-1 and hedgehog/GLI2 exerts additive antifibrotic effects in a model of TGFβ-driven experimental fibrosis (TBRACT mice). CONCLUSION:The transcription factors cJUN and GLI2 reinforce each other's activity to promote fibroblast activation in SSc. Interruption of this crosstalk by combined inhibition of both pathways exerts additive antifibrotic effects at well-tolerated doses.

OBJECTIVES:Metabolic changes are crucially involved in osteoclast development and may contribute to bone degradation in rheumatoid arthritis (RA). The enzyme aconitate decarboxylase 1 (Acod1) is known to link the cellular function of monocyte-derived macrophages to their metabolic status. As osteoclasts derive from the monocyte lineage, we hypothesised a role for Acod1 and its metabolite itaconate in osteoclast differentiation and arthritis-associated bone loss. METHODS:Itaconate levels were measured in human peripheral blood mononuclear cells (PBMCs) of patients with RA and healthy controls by mass spectrometry. Human and murine osteoclasts were treated with the itaconate derivative 4-octyl-itaconate (4-OI) in vitro. We examined the impact of Acod1-deficiency and 4-OI treatment on bone erosion in mice using K/BxN serum-induced arthritis and human TNF transgenic (hTNFtg) mice. SCENITH and extracellular flux analyses were used to evaluate the metabolic activity of osteoclasts and osteoclast progenitors. Acod1-dependent and itaconate-dependent changes in the osteoclast transcriptome were identified by RNA sequencing. CRISPR/Cas9 gene editing was used to investigate the role of hypoxia-inducible factor (Hif)-1α in Acod1-mediated regulation of osteoclast development. RESULTS:Itaconate levels in PBMCs from patients with RA were inversely correlated with disease activity. Acod1-deficient mice exhibited increased osteoclast numbers and bone erosion in experimental arthritis while 4-OI treatment alleviated inflammatory bone loss in vivo and inhibited human and murine osteoclast differentiation in vitro. Mechanistically, Acod1 suppressed osteoclast differentiation by inhibiting succinate dehydrogenase-dependent production of reactive oxygen species and Hif1α-mediated induction of aerobic glycolysis. CONCLUSION:Acod1 and itaconate are crucial regulators of osteoclast differentiation and bone loss in inflammatory arthritis.

Analysis of gene expression at the single-cell level could help predict the effectiveness of therapies in the field of chronic inflammatory diseases such as arthritis. Here, we demonstrate an adopted approach for processing images from the Slide-seq method. Using a puck, which consists of about 50,000 DNA barcode beads, an RNA sequence of a cell is to be read. The pucks are repeatedly brought into contact with liquids and then recorded with a conventional epifluorescence microscope. The image analysis initially consists of stitching the partial images of a sequence recording, registering images from different sequences, and finally reading out the bases. The new method enables the use of an inexpensive epifluorescence microscope instead of a confocal microscope.

Background: In heterogeneous conditions with insufficient predictable disease course, like systemic sclerosis (SSc), appropriate measurement of disease activity, as well as predictors of disease progression are necessary for optimizing individual outcome. Systemic sclerosis is associated, despite emerging new therapies with a variable unpredictable course of disease with a high morbidity and mortality. Notably, innate lymphoid cells (ILCs), specifically ILC2, are gaining recognition as significant contributors to cytokines-driven fibrotic tissue remodelling. Elevated levels of ILC2s have been observed in both skin sections and the circulation of SSc patients commpared to healthy controls. Objectives: The study aims to assess the predictive value of type 2 ILCs over a period of 5-year follow-up in patients with systemic sclerosis. Methods: Fifty-two consecutive patients meeting the 2013 ACR/EULAR classification criteria for SSc were included in the study. Clinical parameters were assessed according to EUSTAR recommendations by experienced rheumatologists. Blood samples were analysed using flow cytometry. Annual pulmonary function test (PFT), echocardiography and EKG were conducted. High-resolution computed tomography (HRCT) scans of the lungs were evaluated by experienced radiologists, who were blinded with regard to the clinical data and the study question and some patients underwent positron emission tomography (PET) scanning with tracers specific for fibroblast activation protein (FAP, 68Ga-FAP inhibitor (FAPI)-04). Disease worsening was defined as decrease in pulmonary function tests (FVC or DLCOcSB decline ≥ 10%), progression of fibrotic lesions in HRCT, switch or new begin of immunosuppressive or anti-fibrotic therapy, increase in mRSS defined as increase in mRSS >5 points and ≥25% from baseline, or death. Results: The baseline cohort comprised 52 patients with SSc, including 12 patients with diffuse (23.07%) cutaneous SSc (dcSSc) and 11 (21.15%) male. Mean ± SD disease duration was 7.5 ± 6.9 years. Among the 52 patients, three (5.76%) patients were lost to follow-up and eight (15.38%) died. Circulating ILC2 were significantly increased in patients with a fibrotic subtype of disease (including important skin fibrosis and lung fibrosis, adapted from DeSScipher observational studies), followed by the vascular subtype (represented by marked digital ulcers and pulmonary arterial hypertention), while musculo-skeletal (arthritis or muscular disease as leading symptoms) and normal-like subtype had similar low levels (mean ILC2 4.18 vs. 1.80 vs. 1.28 vs. 1.01/µl respectively, p<0.001). Baseline ILC2 number correlated with lung disease progression, worsening of mRSS and mortality. Patients requiring intensive immunosuppressive or anti-fibrotic therapy during the follow-up time had significantly higher ILC2 counts at baseline (3.73/µl vs 1.25/µl, p<0.001). The ILC2 cutoff value of 1.57/µl demonstrated high sensitivity (>90%) and specificity (>70%) in identifying stable patients (area under the curve 0.879, p<0.0001). Patients with ILC2 count above 1.57/µl experienced a more rapid decline in pulmonary function test, increased mRSS scores and a higher mortality. Multivariate analysis revealed that an increased ILC2 number at baseline was a significant predictor of lung disease progression (≥10% decline in FVC, ≥10% decline in DLCO, progression of lung fibrosis in HRCT scans) and death. Furthermore, high ILC2 counts correlated with increase SUVmax and SUVmean in FAPI PET-CT imaging, indicating heightened activity in these patients. Conclusion: The baseline number of ILC2s correlated with disease progression and serves as a prognostic marker even in patients with longstanding disease, providing evidence for profibrotic role of ILC2 in SSc. REFERENCES: NIL. Acknowledgements: Supported by the German Research Foundation (grant SO 1735/2-1), Novartis Pharma GmbH Disclosure of Interests: Alina M Ramming: None declared, Christian Schmidkonz: None declared, Armin Atzinger: None declared, Maria Gabriella Raimondo: None declared, Simon Rauber: None declared, Jörg Distler: None declared, Georg Schett: None declared, Andreas Ramming Novartis Pharma GmbH, Novartis Pharma GmbH.

Objectives Deregulation of the cJUN/AP1‐ and hedgehog/GLI2 signaling pathways have been implicated in fibroblast activation in Systemic Sclerosis (SSc). However, the consequences of their concomitant upregulation are unknown. Here, we tested the hypothesis that mutual amplification of both pathways might drive persistent fibroblast activation. Methods Cultured fibroblasts and skin sections of diffuse SSc‐patients and healthy volunteers were analyzed . cJUN/AP1‐ and hedgehog/GLI2‐signaling were inhibited using knockdown and pharmacologic approaches. Hedgehog signaling was activated in mice by fibroblast‐specific overexpression of constitutively‐active Smoothend. Results cJUN and GLI2 are concomitantly upregulated and colocalize in fibroblasts of SSc patients compared to healthy controls. Activation of hedgehog/GLI2 signaling induces the expression of cJUN in vitro and in vivo , whereas inactivation of GLI2 inhibits cJUN expression. Likewise, inactivation of cJUN impairs the expression of GLI2. This mutual regulation occurs at the level of transcription with binding of cJUN and GLI2 to specific binding motifs. Interference with this mutual amplification of cJUN‐ and GLI2‐signaling inhibits fibroblast activation and collagen release: Inhibition of cJUN/AP1‐signaling ameliorates hedgehog‐induced fibroblast activation and skin fibrosis in Smo ACT ‐mice with a reduction of skin thickness of 103 % (p=0.0043) in the treatment group compared to the fibrotic control group. Moreover, combined pharmacological inhibition of cJUN/AP1‐ and hedgehog/GLI2 exerts additive antifibrotic effects in a model of TGFβ‐driven experimental fibrosis (TBR ACT mice). Conclusion The transcription factors cJUN and GLI2 reinforce each other's activity to promote fibroblast activation in SSc. Interruption of this crosstalk by combined inhibition of both pathways exerts additive anti‐fibrotic effects at well tolerated doses.

Transforming growth factor β (TGF-β) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-β remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-β in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-β. The activation of latent TGF-β requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-β, rebalanced TGF-β signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-β in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Background: Enthesitis is a hallmark feature of psoriatic arthritis (PsA) course. So far, most of the data on enthesitis in PsA are based on clinical assessment as well as MRI or ultrasound examinations, due to the challenge in retrieving good quality entheseal tissue for molecular analysis. However, pre-clinical studies as well as surgical invasive approaches on spondyloarthritis patients showed that interleukin-17 (IL-17) is a key molecule in enthesitis pathogenesis, as also confirmed by the clinical efficacy of IL-17 inhibitors in patients with enthesitis. To date, there are no molecular data assessing how IL-17 blockade ameliorates enthesitis in humans. Therefore, deep omics analysis of these ultra-specialized tissues might expand our insights on PsA pathogenesis as well as provide information on treatment response. Objectives: To investigate the immune and the non-immune compartment in active enthesitis of PsA patients and their changes upon IL-17A inhibition. Methods: Minimal-invasive ultrasound (US)-guided biopsies of the lateral epicondyle (technology described previously[1]) were performed in 10 PsA patients with active elbow enthesitis before and after 3 months of treatment with 150mg/month s.c. secukinumab. All patients were biologicDMARD naïve at time of inclusion. Only one patient did not receive the second biopsy. Once harvested, the samples were fixed and conventional histology staining as well as second harmonic generation (SHG) were performed for morphologic evaluation and entheseal region identification[1]. Further slides were used for protein expression, through Hyperion imaging mass cytometry (IMC) and for RNA expression, through GeoMx spatial transcriptomic technology. Results: 40% of the patients included into the study were female with an average age of 54 (± 9 SD). At the time of screening DAPSA, LEI and SPARCC were (mean ± SD) 23 ± 12, 2 ± 1 and 4 ± 3, respectively, decreasing to 11 ± 10, 1 ± 1, 1 ± 1, respectively, after secukinumab. Entheseal regions within the biopsies was identified by SHG. Neutrophils (CD66b+), CD4+T cells (CD3+ CD4+) and ILC (Lineage-, CD127+) significantly decreased upon IL-17 blockade (p=0.017, 0.017, 0.021, respectively), particular with respect to the IL-17A-positive fractions of each cell type. Beside quantitative reduction of IL-17-producing immune cells, specific effects on transcriptional states of resident entheseal cells were observed. Spatial deconvolution analysis revealed a significant increase in the abundance of pro-resolving CD200+ fibroblasts[2] and ILC2 cells following treatment (p=0.055, 0.049, respectively). Moreover, these cells were found to spatially colocalize together and with MERTK+ macrophages (CD200+ R=0.57 p=0.0041, ILC2 R=0.39 p=0.063), while dissociating with CD4+ T cells (CD200+ R=-0.74 p=0.000, ILC2 R=-0.64 p=0.001). Conclusion: Here we show for the first time a full picture of cellular changes in the microarchitecture of inflamed human entheses upon anti-IL-17 treatment. Resolving enthesial inflammation was not only associated with a reduction of the cellular load of IL-17-producing cells but also with tissue intrinsic modulations towards a pro-resolving microenvironment upon treatment with IL-17 inhibitors. REFERENCES: [1] Pachowsky ML, Raimondo MG, et al. Ann Rheum Dis. 2022; 81(8):1131-1135. [2] Rauber et al., Nat Immunol. 2024, in press. Acknowledgements: NIL. Disclosure of Interests: Maria Gabriella Raimondo: None declared, Hashem Mohammadian: None declared, Stefano Alivernini: None declared, Vladyslav Fedorchenko: None declared, Simon Rauber: None declared, Hannah Labinsky: None declared, Mario Angeli: None declared, Filippo Fagni: None declared, Giulia Corte: None declared, Koray Tascilar: None declared, Lars Bräuer: None declared, Maria Antonietta D' Agostino: None declared, Georg Schett: None declared, Arnd Kleyer: None declared, Milena Pachowsky: None declared, Andreas Ramming Novartis, Novartis.

Background: Synovial fibroblasts play a crucial role in shaping the joint microenvironment and actively contribute to the initiation and progression of inflammatory arthritis [1]. Notably, distinct subsets of fibroblast activation protein (FAP)-positive synovial fibroblasts have been implicated in inflammation and bone erosion as well as resolution of inflammation [2]. Recent studies using 68Ga-FAPI-04 PET-CT revealed elevated FAPI tracer uptake in psoriasis patients, indicating an increased risk of developing psoriatic arthritis and correlating with disease progression and joint damage [2, 3]. Despite these findings, our understanding of FAP activation during the early stages of arthritis and its underlying regulatory mechanisms remains incomplete. Objectives: This study aimed to assess the potential of FAPI imaging in investigating early stromal remodeling during inflammatory arthritis unraveling the regulatory mechanism of early FAP activation and its role in inflammatory arthritis. Methods: We utilized Alexa Fluor 647 (AF647)-conjugated FAPI-04 to evaluate specific cell uptake in vitro via flow cytometry and in vivo using light sheet microscopy (LSFM). In vivo assessments of leukocyte infiltration were conducted with antibody-conjugated fluorophores (CD45, Ly6G). Cellular behavior was monitored over time through LSFM and flow cytometry. FAP+ synovial fibroblasts were sorted before arthritis onset and at peak inflammation, followed by single-cell RNA sequencing (scRNAseq). Results: FAPI-04-AF647 showed selective uptake by FAP-expressing cells both in vitro and in vivo. Early FAP upregulation was detected following STA modeling, prior to leukocyte infiltration. FAPI-04 uptake levels were comparable to those observed at the peak of inflammation. To elucidate whether serologic or cellular components induce early stage FAPI uptake, synovial fibroblasts were incubated with fresh or heat-inactivated KBxN serum. Heat inactivation completely abolished FAPI uptake, indicating that serological components induce fibroblast transition. Subsequently, fibroblasts were stimulated with various cytokines including complement factors, IL-6, TGFβ, and TNFα. As we did not observe unifactorial FAPI-04 uptake, scRNAseq was performed with sorted fibroblasts from joints at an early stage of disease before leukocyte infiltration occurred. Here we observed multifactorially induced FAPI-04 uptake associated with upregulation of the chemokines C-X-C motif chemokine ligand 2 (CXCL2), CXCL12 and CXCL13 and significantly altered subset characteristics distinct from fibroblast subsets at the peak of inflammation. Conclusion: Our findings highlight that FAPI-04 uptake occurs in inflammatory arthritis at early stages before leukocyte infiltration, emphasizing the pivotal role of fibroblasts throughout arthritis progression. Fibroblasts may act as sentinels of inflammation during the early stages, detectable by FAPI imaging. The identification of subtype-specific characteristics suggests the potential for developing drugs targeting specific molecular markers in fibroblast subsets, allowing for the modulation of fibroblasts in a time-course-dependent manner throughout the disease progression. REFERENCES: [1] Croft, A.P., et al. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature 570, 246-251 (2019). [2] Rauber et al., Nat Immunol. 2024, in press. [3] Fagni F, et al. Fibroblast Activation in Psoriasis Patients Assessed by 68Ga-FAPI-04 PET-CT Is Associated with Progression to Psoriatic Arthritis [abstract]. Arthritis Rheumatol. 2022; 74 (suppl 9). Acknowledgements: NIL. Disclosure of Interests: None declared.

Background Spreading of inflammation from skin to joint is still an unsolved key aspect of psoriatic arthritis (PsA) pathogenesis. Psoriasis (PsO) usually anticipates the joint manifestations, suggesting the existence of a disease mediated skin-joint crosstalk. However, only 30% of the patients with psoriasis develop PsA overtime.[1] To date, it is still obscure why the inflammatory process in some patients with PsO is restrained to the skin, whereas in other patients it spreads to the joints. Objectives Using a pre-clinical model of PsA and PsO, we aimed to investigate the skin-joint axis, i.e. the spreading of psoriatic inflammation from the skin to the joints, and to address its role in PsA pathogenesis. Methods The IL-23 overexpression (IL-23OE) mouse model of psoriasis was performed in different genetic backgrounds of KAEDE-transgenic mice expressing a photo-convertible fluorescent reporter to assess cell trafficking from inflamed skin to other organs. Psoriatic skin lesions were irradiated with UV light to trigger the photoswitch from KAEDEGREEN to KAEDERED. Migration of immune cells to joints was determined by light sheet fluorescence microscopy (LSFM) and flow cytometry. Imaging flow cytometry was used to determine the phenotype of cells migrating from the skin to other organs. The interactome of skin-derived migrating cells in joints was characterized by single-cell RNA-sequencing (scRNAseq) and functional analyses. Data were validated in synovial biopsies from PsO and PsA patients by imaging mass cytometry. Results IL-23 induced initiation of inflammation at the joints was dependent on the genetic background of the mice as assessed by MRI scan and histological analysis. However, migration of immune cells from psoriatic skin to the joints was observed in both strains, protected and non-protected mice from arthritis, although other organs such as spleen and lymph nodes showed no model dependent skin-derived migration. ScRNAseq and computational fate mapping with RNA velocity analysis revealed CD2+ MCHII+ monocytes as predominant cell type escaping the inflamed skin and entering the synovial tissue. In the pre-differentiated stage, those monocytes showed similar phenotypes in both, arthritis protected and non-protected animals. However, after entering the synovial tissue further differentiation into macrophages resulted into two different phenotypes, with pro-inflammatory characteristics in mice developing arthritis. Interactome analyses between local differentiated skin-derived macrophages and tissue resident synovial cells revealed major implication of synovial fibroblasts shaping the fate of skin-derived monocytes into a protective or pro-inflammatory phenotype with capacity to initiate the cascade of inflammation. Imaging mass cytometry of synovial biopsies from patients with PsO and PsA identified niches of the synovial membrane that were protected from inflammation by a similar fibroblastic fate as observed in the murine setting. Protection from arthritis was associated with significantly increased interactions of those protective fibroblasts with innate lymphoid cells (ILC) type 2, inducing pro-survival signals and resulting into activation of pro-resolving ILC2s.[2] Conclusion Spreading of inflammation from the skin to the joint can be initiated by skin-derived monocytes. However, the interaction between migrating monocytes and stromal-resident cells is essential to define the fate towards joint inflammation or joint protection in PsA. The data might provide completely new diagnostic opportunities to estimate the risk of PsO patients to develop PsA in the future. References [1]Veale DJ, Fearon U. The pathogenesis of psoriatic arthritis. Lancet. 2018 Jun 2;391(10136):2273-2284. [2]Rauber S. et al. Resolution of inflammation by interleukin-9-producing type 2 innate lymphoid cells. Nat Med. 2017 Aug;23(8):938-944. Acknowledgements: NIL. Disclosure of Interests None Declared.

Butyrophilins are surface receptors belonging to the immunoglobulin superfamily. While several members of the butyrophilin family have been implicated in the development of unconventional T cells, butyrophilin 2a2 (Btn2a2) has been shown to inhibit conventional T cell activation. Here, we demonstrate that in steady state, the primary source of Btn2a2 are thymic epithelial cells (TEC). Absence of Btn2a2 alters thymic T cell maturation and bypasses central tolerance mechanisms. Furthermore, Btn2a2-/- mice develop spontaneous autoimmunity resembling human primary Sjögren's Syndrome (pSS), including formation of tertiary lymphoid structures (TLS) in target organs. Ligation of Btn2a2 on developing thymocytes is associated with reduced TCR signaling and CD5 levels, while absence of Btn2a2 results in increased TCR signaling and CD5 levels. These results define a novel role for Btn2a2 in promoting central tolerance by modulating TCR signaling strength and indicate a potential mechanism of pSS development.

Abstract Fibroblasts are key orchestrators of inflammation. Little is known whether these cells change phenotype during resolution of inflammation. We adopted a method to visualise fibroblast activation during inflammation in humans in vivo, which is based on a fibroblast activation protein (FAP) tracer detected by positron emission tomography (PET). While tracer accumulation was high in active arthritis, it decreased significantly after TNF- and IL-17A inhibition. Biopsy-based scRNA-seq analyses in experimental arthritis demonstrated that FAP signal reduction reflected a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalisation) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalisation). Spatial transcriptomics of human joints revealed that pro-resolving niches of CD200+DKK3+ fibroblasts clustered with innate lymphoid cells (ILC)2, whereas MMP3+/IL6+ fibroblasts were co-localised with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilised the ILC2 phenotype and induced resolution of arthritis via CD200/CD200R1 pathway. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

Background Persistently activated fibroblasts status leads to progressive extracellular matrix (ECM) deposition and tissue remodeling. The hallmark of systemic sclerosis (SSc) is collagen accumulation in organs, mainly in skin and lung. Despite intensive progress in understanding disease occurence, SSc remains an intriguing disease with unknown pathology and high mortality. Sorbin and SH3 domain-containing protein2, encoded by SORBS2 is a key member of the sorbin homology family of adapter and scaffold proteins. Recent studies suggest that SORBS2 plays a role in cardiac disease, however there is no available data about its role in fibrotic conditions. Objectives We aimed to investigate the role of SORBS2 in the pathogenesis of SSc. Methods To identify molecules specifically upregulated in persistently activated fibroblasts, human fibroblasts were chronically stimulated with TGF-β and analyzed by RNA-sequencing. To evaluate the functional implication of tissue elasticity on the transcriptome of fibroblasts, multiwell stiffness assays were performed. SORBS2 expression was further analyzed in skin samples of patients with SSc and murine models of fibrosis. Fibroblast specific SORBS2 knockout mice were challenged with bleomycin to induce skin and lung fibrosis. Further specific readouts like collagen content, skin thickness, myofibroblast count, CT scans were performed. Results Upon chronic TGF-β stimulation of human normal skin fibroblasts we identified SORBS2, as significantly upregulated molecule. SORBS2 is implicated in cytoskeletal organization, cell adhesion and different signaling pathways. Moreover, extracellular stiffness induced upregulation of SORBS2 mRNA level in fibroblasts. Deletion of SORBS2 in fibroblasts led to a change of the diameter of collagen fibers and modified the elastic index of the tissue. SORBS2 expression is not only elevated in different animal models of fibrosis, but also in fibrotic skin samples of SSc patients. SORBS2 knockout mice (KO) developed significantly less skin fibrosis upon bleomycin challenge in comparison to wild type mice (WT), as assessed by measurement of dermal thickness, myofibroblast counts and hydroxyproline content. Col1a1, Col1a2 and expression of αSMA were significantly lower in SORBS2 KO mice in comparison to SORBS2 WT mice. Similarly, fibroblast specific knockout of SORBS2 showed protective effects in bleomycin induced lung fibrosis. CT scans of the lungs showed statistically significant less fibrotic changes in SORBS2 KO mice in comparison to wild type mice. Conclusion SORBS2 is engaged in a vicious circle of fibrosis. Triggered by chronic TGF-β stimulation, SORBS2 is further upregulated by the increasing stiffness of the extracellular matrix. This leads to persistently high levels of SORBS2 resulting into further production of ECM products. Deleting SORBS2 has potent antifibrotic effects in animal models of skin and lung fibrosis. As most of the currently used therapeutic approaches are focussed on early stages of the disease, SORBS2 might be an interesting new therapeutic target in established stages of SSc. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests None Declared.