Objectives Diffuse cutaneous Systemic Sclerosis (dcSSc) is a life-limiting autoimmune disease with minimal treatment options. Autologous hematopoietic stem cell transplantation (ASCT) is a potent disease-modifying therapy in dcSSc; however, its effects on fibroblasts are unknown. We recently showed that dermal fibroblasts (DFs in patients with dcSSc develop a cancer-like phenotype characterized by genomic instability and increased double-stranded DNA breaks (DSBs).[1] However, little is known about the mechanisms promoting DF survival following the accumulation of genomic mutations. We hypothesized that in dcSSc DF genomic instability results in the accumulation of genomic mutations; and that this results in the activation of Protein Kinase R-like ER Kinase (PERK) and transcription of forkhead box1 (FOXO1) promoting resistance-to-apoptosis and fibrosis (Figure 1). Methods We used whole exome sequencing (WES) to characterize the mutational frequencies and their associated signatures in dcSSc patients who did not undergo ASCT (dcSSc, N=35) and those who did (post-ASCT, N=9). We also generated DFs from dcSSc, post-AHSCT (or age/sex matched healthy controls (HC), (N=8-10 patients/group), and quantified the frequency of DSBs via γ-H2AX levels (immunoblot (IB)), and DSB nuclear foci (confocal microscopy). We measured the relative ROS levels, mitochondrial membrane potential, and phospho-PERK (active) in DFs to mechanistically link DSB with PERK activation using flow cytometry and/or IB, respectively. We also determined the downstream effects of PERK activation on mitochondria (eg, mitochondrial dynamics and biogenesis). Then, we measured FOXO1 activation via nuclear translocation, IB, and expression of its downstream mRNA target SOD2. Finally, mitochondrial-dependent resistance-to-apoptosis was determined at baseline, and following treatment with cyclophosphamide, a PERK or a FOXO1-inhibitor using TUNEL and cleaved caspase 9/3 levels (IB). Results dcSSc patients’ DFs had increased genomic instability and DSBs compared to patients treated with ASCT. dcSSc DFs had increased indicators associated with PERK activation (phospho-PERK, ROS, and mitochondrial membrane potential). This was associated with increased mitochondrial remodeling, mitochondrial biogenesis, and mitochondrial fusion. Importantly, FOXO1 was exclusively activated in dcSSc, but not HC or post-ASCT DFs. Inhibition of PERK or FOXO1 resulted in increased mitochondrial-dependent apoptosis. Conclusion Our study highlights a novel mechanism whereby genotoxic signals in dcSSc promote cell survival via a PERK/FOXO1-dependent axis and associated metabolic remodeling (Figure 1). It also provides mechanistic insights related to how changes to the mutational landscape reduce pro-fibrotic signals in DF after ASCT. Future studies targeting this dysregulated pathway may provide an additional rationale for exploring it therapeutically in patients with dcSSc. References [1.] Gniadecki R. J Autoimmun 2022;131:102847.

Resting collecting lymphatic vessels (cLVs) sense edema in distal joints and initiate contractions via unknown mechanisms. Rheumatoid arthritis (RA) patients have lymphatic drainage deficiencies from affected joints, and defects in the synovial lymphatic system exacerbate inflammatory-erosive arthritis in animal models. To understand this, we generated Efhd1-CreERT2 and Myoc-CreERT2 mice for cell-specific genetic gain and loss of function studies. These mice were crossed with tdTomato reporter (Ai9) mice, and studies showed selective tamoxifen-induced transgene expression in CD31-/CD34+ telocyte-like cells in knee and ankle synovium, and in networks physically associated with mast cells proximal to popliteal lymphatic vessels (PLVs). Consistent with the known loss of CD31-/CD34+ telocyte in RA synovium, these cells were also decreased in TNF-tg knees and partially recovered by anti-TNF treatment. Ultrastructural and gene expression studies confirmed a distinct telocyte phenotype versus closely related fibroblasts. In vivo depletion studies in tamoxifen-treated Efhd1-CreERT2 and Myoc-CreERT2 mice crossed to diphtheria toxin alpha-floxed (DTAflox) mice demonstrated telocyte requirements for physiologic lymphatic drainage and resolution of joint inflammation and focal erosions from zymosan-induced arthritis in the knee. In vitro studies demonstrated increased sensitivity to osmotic shock and decreased motility versus fibroblasts, and telocyte potential to differentiate into myofibroblasts on stiff matrix. Collectively, these findings support a model of joint homeostasis in which osmotic pressure-sensing telocyte networks extend from the synovium into mast cells proximal to joint-draining cLVs, and telocyte loss is associated with defects in the synovial lymphatic system and increased susceptibility to joint inflammation and structural damage from arthritis.

Complement's role in the pathology of rheumatoid arthritis and pulmonary hypertension (PH) is not fully understood. We aimed to determine whether complement deficiency is protective against joint and lung disease using the tumor necrosis factor-transgenic (TNF-Tg) mouse model characterized by chronic inflammatory-erosive arthritis and PH. TNF-Tg synovium and lungs were analyzed with bulk and single-cell RNA-sequencing. TNF-Tg mice were crossed with complement component 3 knockout (C3KO) and factor B knockout (fBKO) mice to quantify disease. Knee histology was scored, CD45 + Ly6C-C5aR1 + cells were quantified by flow cytometry, mid-hindpaw bone volumes were determined with micro-computed tomography, and lung disease was quantified with histology and right heart catheterization. TNF-Tg mice have upregulated complement-related genes, however, C3 and fB knockout did not cause significant changes in pathology. Synovium from C3KO TNF+ mice had a greater C5aR1 + cell population than TNF-Tg mice (p < 0.0001). In early disease, C3KO TNF+ mice exhibited greater bone volumes (p < 0.05, p < 0.01) than other TNF+ mice. Complement deficiency in a chronic inflammatory model does not ameliorate joint or pulmonary disease. However, C3 deficiency may delay complement-dependent bone erosions. The influence of complement on disease may change with persistent inflammation. Chronic TNF-mediated disease may represent a pathologic endotype that is differentially mediated by complement.

Kruzer, Karen MD; Goncalves Marangoni, Roberta MD, PhD; Heckler, Ilana PhD; Elhage, Aya PharmD, MBA; Varga, John MD; Hinchcliff, Monique MD; Carns, Mary MS; Aren, Kathleen MPH; Wielgosz, Amy RN; Nuzzo, Marc BS; Venkataraman, Iswariya PhD; Korman, Benjamin MD Author Information

OBJECTIVES Symptoms of people who have systemic sclerosis (SSc) are heterogeneous and difficult to address clinically. Because diverse symptoms often co-occur and may share common underlying mechanisms, identifying symptoms that cluster together may better target treatment approaches. We sought to identify and characterize patient subgroups based on symptom experience. METHODS An exploratory hierarchical agglomerative cluster analysis was conducted to identify subgroups from a large SSc cohort from a single US academic medical center. Patient-reported symptoms of pain interference, fatigue, sleep disturbance, dyspnea, depression, and anxiety were used for clustering. A multivariate analysis of variance (MANOVA) was used to examine the relative contribution of each variable across subgroups. Analyses of variance were performed to determine participant characteristics based on subgroup assignment. Presence of symptom clusters were tallied within subgroup. RESULTS Participants (N = 587; 84% female, 41% diffuse cutaneous subtype, 59% early disease) divided into three subgroups via cluster analysis based on symptom severity: (1) no/minimal; (2) mild; and (3) moderate. Participants in mild and moderate symptoms subgroups had similar disease severity, but different symptom presentation. In the mild symptoms subgroup, pain, fatigue, and sleep disturbance was the main symptom cluster. Participants in the moderate symptoms subgroup were characterized by co-occurring pain, fatigue, sleep disturbance, depression, and anxiety. CONCLUSION Identification of distinct symptom clusters, particularly among SSc patients who experience mild and moderate symptoms, suggest potential differences in treatment approach and in mechanisms underlying symptom experience that require further study.