Abstract Introduction We have previously shown that response gene to complement-32 (RGC-32) promoted the Th17 and Th1 dependent proinflammatory and profibrotic responses in a murine model of immune complex mediated glomerulonephritis. The expression and function of RGC-32 in PBMC and kidney biopsies from patients with lupus nephritis (LN) have not yet been investigated. Methods RGC-32 expression was evaluated by RT-PCR, WB, flow cytometry and cDNA Array in PBMC from lupus patients and controls and in human proximal tubular epithelial cells (PTEC) and by immunohistochemistry in kidney biopsies from 25 patients with LN and 11 disease controls. Results RGC-32 mRNA expression was highest in CD3 cells followed by CD14 and CD19 cells and was increased in CD14 and CD19 cells from patients with vs those without renal disease. RGC-32 expression was induced by T cell stimulation under Th17 and Treg conditions and by serum from patients with high titer anti-dsDNA Ab. In kidneys, RGC-32 immunostaining was predominant in tubules and staining intensity was significantly higher in SLE than in non-SLE specimens. RGC-32 expression was also detected in glomeruli and in inflammatory cells in the interstitium, was significantly higher in LN specimens vs. controls and correlated with the activity (r = 0.4), chronicity (r = 0.5) and interstitial fibrosis scores (r = 0.5). RGC-32 colocalized with CD4+, CD68+ cells and C5b-9 deposition. In PTEC, RGC- 32 mRNA and protein expression was upregulated by IL-1, TNFα, TGFβ, IFNγ and sublytic C5b-9. TGFβ induced mRNA production of Collagen I and collagen III by PTEC was increased in RGC-32 transfected cells. Conclusion RGC-32 is upregulated in PBMC, glomeruli and tubulointerstitium of patients with LN. RGC-32 upregulation in PTEC is mediated by proinflammatory cytokines and complement activation and may play a role in organ damage by promoting manifestations of progressive renal disease such as interstitial fibrosis. Thus, RGC-32 is a potential therapeutic target in the treatment of LN. Funding Source N/A Topic Categories Immune Mechanisms of Human Disease (HUM)

Background:Effective skin wound healing is essential for restoring tissue integrity following injury. Repair proceeds through phases of hemostasis, inflammation, proliferation, and remodeling, but molecular mechanisms governing these stages remain poorly defined. Vascular niche cells (VNCs)-including endothelial cells, vascular smooth muscle cells (SMCs), and fibroblasts-are central regulators of healing, but the lack of longitudinal in vivo human data has limited identification of VNC-derived signals that distinguish effective repair from pathological healing such as ulcers. Thus, defining the regulation of VNCs in wound healing addresses a critical knowledge gap. Methods:We developed a protocol for wound healing using dermal forearm punch biopsies to track longitudinal repair in healthy volunteers. Single-cell and spatial transcriptomics were performed to identify and validate signaling activities within VNCs. Results:We spatiotemporally defined the inflammation, proliferation, and remodeling phases of human skin wound healing with a focus on VNCs. Spatial analysis localized this activity for VNCs and immune cells within a heterogenous granulation zone that later led to re-epithelializion. Angiogenesis was dominated by Vegf, Egf and Hif1α signaling. Extracellular matrix (ECM) remodeling occurred through Collagen, Laminin, Thrombospondin, and Fibronectin. SMCs emerged as dominant drivers of injury-induced remodeling including basement membrane and interstitial ECM components compared to fibroblasts. This SMC-led program was further defined by robust induction of TIMP1, an inhibitor of matrix degradation, which localized to granulation tissue and correlated with re-epithelialization and wound resolution. Lastly, we compared remodeling factors between healing and non-healing human diabetic foot ulcers (DFUs). SMCs in non-healing DFUs had deficient expression for core remodeling factors, including TIMP1 , indicating SMC activity is needed for effective healing. Conclusion:We identified an SMC-driven model of wound repair in which TIMP1-dependent activity underpins granulation zone formation. Failure of this program defined a mechanistic basis for impaired healing in ulcers, identifying SMCs and TIMP1 as therapeutic targets.

Immunological health has been challenging to characterize but could be defined as the absence of immune pathology. While shared features of some immune diseases and the concept of immunologic resilience based on age-independent adaptation to antigenic stimulation have been developed, general metrics of immune health and its utility for assessing clinically healthy individuals remain ill defined. Here we integrated transcriptomics, serum protein, peripheral immune cell frequency and clinical data from 228 patients with 22 monogenic conditions impacting key immunological pathways together with 42 age- and sex-matched healthy controls. Despite the high penetrance of monogenic lesions, differences between individuals in diverse immune parameters tended to dominate over those attributable to disease conditions or medication use. Unsupervised or supervised machine learning independently identified a score that distinguished healthy participants from patients with monogenic diseases, thus suggesting a quantitative immune health metric (IHM). In ten independent datasets, the IHM discriminated healthy from polygenic autoimmune and inflammatory disease states, marked aging in clinically healthy individuals, tracked disease activities and treatment responses in both immunological and nonimmunological diseases, and predicted age-dependent antibody responses to immunizations with different vaccines. This discriminatory power goes beyond that of the classical inflammatory biomarkers C-reactive protein and interleukin-6. Thus, deviations from health in diverse conditions, including aging, have shared systemic immune consequences, and we provide a web platform for calculating the IHM for other datasets, which could empower precision medicine. A multimodal analysis of patients with 22 different immune-mediated monogenic diseases versus matched healthy controls leads to the development of the immune health metric, which could be implemented broadly to predict responses to aging, vaccination and other immune perturbations.

Background: Arterial calcification due to deficiency of CD73 (ACDC; OMIM 211800) is a rare genetic disease resulting in calcium deposits in arteries and small joints causing claudication, resting pain, severe joint pain, and deformities. Currently, there are no standard treatments for ACDC. Our previous work identified etidronate as a potential targeted ACDC treatment, using in vitro and in vivo disease models with patient-derived cells. In this study, we test the safety and effectiveness of etidronate in attenuating the progression of lower-extremity arterial calcification and vascular blood flow based on the computed tomography (CT) calcium score and ankle–brachial index (ABI). Methods: Seven adult patients with a confirmed genetic diagnosis of ACDC were enrolled in an open-label, nonrandomized, single-arm pilot study for etidronate treatment. They took etidronate daily for 14 days every 3 months and were examined at the NIH Clinical Center bi-annually for 3 years. They received a baseline evaluation as well as yearly follow up after treatment. Study visits included imaging studies, exercise tolerance tests with ABIs, clinical blood and urine testing, and full dental exams. Results: Etidronate treatment appeared to have slowed the progression of further vascular calcification in lower extremities as measured by CT but did not have an effect in reversing vascular and/or periarticular joint calcifications in our small ACDC cohort. Conclusions: Etidronate was found to be safe and well tolerated by our patients and, despite the small sample size, appeared to show an effect in slowing the progression of calcification in our ACDC patient cohort. (ClinicalTrials.gov Identifier NCT01585402)

Response Gene to Complement (RGC)-32 modulates TGF-β-induced extracellular matrix secretion and the ability of astrocytes to undergo reactive changes in vivo during experimental autoimmune encephalomyelitis (EAE). However, the molecular pathways underlying these effects are still not well understood. In this study, we investigated how lack of RGC-32 affects the transcriptomic profile and the expression of axonal guidance molecules (AGM) in astrocytes during EAE. We performed next-generation RNA sequencing on brain neonatal astrocytes isolated from wild type (WT) and RGC-32 knock-out (KO) mice, either unstimulated or stimulated with TGF-β. Results were then validated by using Real-Time PCR. Spinal cords from WT and RGC-32 KO mice with EAE (at days 0 and 14) were stained by immunohistochemistry for the astrocyte marker GFAP, AGM, and for nuclear factor IA (NFIA), a gliogenic factor and transcriptional regulator of AGM. Lack of RGC-32 had a significant impact on the transcriptomic programs normally associated with brain development whose re-expression is usually seen in reactive astrocytes. Connectivity analysis revealed that genes coding for AGM were particularly affected. We found lower transcript levels of ephrin receptor A type 7 (Epha7), plexin A1 and Slit guidance ligand 2 in RGC-32 KO astrocytes. Moreover, our results showed that NFIA and EPHA7 are expressed by astrocytes during EAE. We found a lower number of astrocytes expressing EPHA7 and NFIA in RGC-32 KO mice with acute EAE when compared with WT mice. These results suggest that RGC-32 might facilitate reactive astrogliosis during acute EAE through regulating the expression of AGM and NFIA. Veterans Administration Merit Award I01BX001458 (to HR)

Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of response gene to complement 32 (RGC-32) alters astrocyte morphology in spinal cords during experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In addition, we found that RGC-32 regulates TGF-β-induced extracellular matrix production and growth factors expression. We investigated if the lack of RGC-32 alters the expression of the glial fibrillary acidic protein (GFAP) and of astrocytes progenitor markers vimentin and fatty acid binding protein 7 (FABP7) during EAE. Immunohistochemical analysis of spinal cords during the acute phase of EAE (day 14 post EAE induction) showed that the elongated, radial glial cell-like astrocytes from RGC-32 knock-out (KO) mice expressed higher levels of vimentin and FABP7 as compared to wild type (WT) mice, confirming their immature phenotype. In addition, we found that the density of white matter GFAP+ astrocytes was higher in WT as compared to KO mice (p=0.02). Using double staining immunohistochemistry for GFAP and connective tissue growth factor (CTGF), known to be involved in astrocyte differentiation, we found a lower number of CTGF+ astrocytes during EAE in spinal cords of RGC-32 KO when compared with WT mice (p=0.002). We next purified neonatal astrocytes from WT and RGC-32 KO mice, and we found significantly lower levels of GFAP and CTGF mRNA and protein and higher levels of vimentin in RGC-32 KO astrocytes when compared with WT astrocytes. The expression pattern of astrocytic progenitor markers and that of CTGF suggests that RGC-32 is an important regulator of astrocyte differentiation during EAE.

Monogenic diseases are often studied in isolation due to their rarity. Here we utilize multiomics to assess 22 monogenic immune-mediated conditions with age- and sex-matched healthy controls. Despite clearly detectable disease-specific and "pan-disease" signatures, individuals possess stable personal immune states over time. Temporally stable differences among subjects tend to dominate over differences attributable to disease conditions or medication use. Unsupervised principal variation analysis of personal immune states and machine learning classification distinguishing between healthy controls and patients converge to a metric of immune health (IHM). The IHM discriminates healthy from multiple polygenic autoimmune and inflammatory disease states in independent cohorts, marks healthy aging, and is a pre-vaccination predictor of antibody responses to influenza vaccination in the elderly. We identified easy-to-measure circulating protein biomarker surrogates of the IHM that capture immune health variations beyond age. Our work provides a conceptual framework and biomarkers for defining and measuring human immune health.

Background/Purpose: CD73 is a ecto-5′-nucleotidase located on the plasma membrane that generates adenosine from AMP in the extracellular space, and functions as a checkpoint against immune and vascular activation. CD73 inhibitors that eliminate this immune checkpoint blockade are being studied in phase 2/3 cancer clinical trials. We recently identified that lack of CD73 promotes vascular thromboinflammation and neutrophil extracellular trap (NET) formation in mice. The role of CD73 in human NETosis, however, remains to be studied. Arterial calcification due to deficiency of CD73 (ACDC) is a rare, autosomal recessive condition (~20 patients globally) that results in occlusive peripheral vascular disease. We hypothesized that the congenital absence of CD73 and its function as an immune checkpoint would heighten innate immune activation in patients with ACDC. Methods: To test this hypothesis, six patients with molecularly confirmed ACDC were recruited to the NIH for mechanistic studies. Using purified neutrophils from ACDC patients and healthy controls, spontaneous and LPS-induced NET formation was quantified by both Sytox extracellular DNA (eDNA) labeling and NET-associated MPO activity. Results: Absence of surface CD73 was confirmed by flow cytometry. Relative to healthy controls, four of six (4/6) patients with ACDC demonstrated increased ex vivo spontaneous NET formation, but not LPS-triggered NETosis. The four ACDC patients with increased spontaneous NETosis had a mean 1.7-fold increase in Sytox-stained eDNA and 2-fold increase in MPO activity compared with contemporary healthy controls. Conclusion: These data reveal marked, spontaneous NETosis in patients deficient in the ectoenzyme CD73. Comparison with LPS-stimulated neutrophils demonstrates near maximal NETosis at baseline in patients with ACDC, consistent with a model where CD73 tonically suppresses neutrophil activation to maintain vascular and immune homeostasis. Ongoing studies are exploring the mechanism of spontaneous NETosis, thrombin formation, and fibrinolysis in patients with ACDC. Taken together, these data will have relevance to patients with ACDC, and inform the thromboinflammatory risk of cancer patients receiving CD73 inhibitors.

Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) is a critical process in atherosclerosis. Here, we investigated the involvement of sublytic C5b-9 effector Response Gene to Complement 32 (RGC-32) in cell cycle activation, phenotypic switch, and production of extracellular matrix (ECM) in SMC. Overexpression of RGC-32 augmented C5b-9-induced cell cycle activation and proliferation of SMC in an ERK1-dependent manner and silencing of RGC-32 inhibited C5b-9-induced cell cycle activation. C5b-9-induced cell cycle activation also required phosphorylation of RGC-32 at threonine 91. We found that ECM components fibronectin and collagens I-V were expressed by SMC in human aortic atherosclerotic tissue. Silencing of RGC-32 in cultured SMC was followed by a significant reduction in TGF-β-induced expression of SMC differentiation markers myocardin, SM22 and α-SMA, and that of collagens I, IV and V. These data suggest that RGC-32 participates in both sublytic C5b-9-induced cell cycle activation and TGF-β-induced ECM production.

The deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessively inherited disease that has undergone extensive phenotypic expansion since being first described in patients with fevers, recurrent strokes, livedo racemosa, and polyarteritis nodosa in 2014. It is now recognized that patients may develop multisystem disease that spans multiple medical subspecialties. Here, we describe the findings from a large single center longitudinal cohort of 60 patients, the broad phenotypic presentation, as well as highlight the cohort’s experience with hematopoietic cell transplantation and COVID-19. Disease manifestations could be separated into three major phenotypes: inflammatory/vascular, immune dysregulatory, and hematologic, however, most patients presented with significant overlap between these three phenotype groups. The cardinal features of the inflammatory/vascular group included cutaneous manifestations and stroke. Evidence of immune dysregulation was commonly observed, including hypogammaglobulinemia, absent to low class-switched memory B cells, and inadequate response to vaccination. Despite these findings, infectious complications were exceedingly rare in this cohort. Hematologic findings including pure red cell aplasia (PRCA), immune-mediated neutropenia, and pancytopenia were observed in half of patients. We significantly extended our experience using anti-TNF agents, with no strokes observed in 2026 patient months on TNF inhibitors. Meanwhile, hematologic and immune features had a more varied response to anti-TNF therapy. Six patients received a total of 10 allogeneic hematopoietic cell transplant (HCT) procedures, with secondary graft failure necessitating repeat HCTs in three patients, as well as unplanned donor cell infusions to avoid graft rejection. All transplanted patients had been on anti-TNF agents prior to HCT and received varying degrees of reduced-intensity or non-myeloablative conditioning. All transplanted patients are still alive and have discontinued anti-TNF therapy. The long-term follow up afforded by this large single-center study underscores the clinical heterogeneity of DADA2 and the potential for phenotypes to evolve in any individual patient.

Background: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disorder caused by a loss of functional ADA2 protein. TNF inhibition (TNFi) has proven to be highly effective in treating inflammatory manifestations. Objective: We sought to explore the pathophysiology and the underlying mechanisms of TNF-inhibitor response in these patients. Methods: We performed Sanger sequencing of the ADA2 gene. We used flow cytometry, intracellular cytokine staining, transcriptome analysis, immunohistochemistry, and cell differentiation experiments to define an inflammatory signature in patients with DADA2 and studied their response to TNF-inhibitor treatment. Results: We demonstrated increased inflammatory signals and overproduction of cytokines mediated by IFN and nuclear factor kappa B pathways in patients' primary cells. Treatment with TNFi led to reduction in inflammation, rescued the skewed differentiation toward the proinflammatory M1 macrophage subset, and restored integrity of endothelial cells in blood vessels. We also report 8 novel disease-associated variants in 7 patients with DADA2. Conclusions: Our data explore the cellular mechanism underlying effective treatment with TNFi therapies in DADA2. DADA2 vasculitis is strongly related to the presence of activated myeloid cells, and the endothelial cell damage is rescued with anti-TNF treatment.

OBJECTIVE:Characterize autoantibodies and autoimmune diseases in a prospective cohort of patients with Idiopathic CD4 Lymphocytopenia (ICL) a rare immunodeficiency characterized by an absolute CD4+ T count of <300 cells/μl in the absence of HIV or HTLV infection.METHODS:Single-Center prospective study of 67 patients conducted over an 11-year period. Rheumatologic evaluation and measurement of autoantibodies were systematically conducted, and flow cytometry of immune cell subsets was performed in a subset of patients.RESULTS:54% of referred patients had clinical evidence of autoimmunity, with 34% having at least one autoimmune disease, most commonly autoimmune thyroid disease. 19%, had autoantibodies or incomplete features of autoimmune disease. Patients with autoimmune disease had more elevated serum immunoglobulins, and more effector memory T cells than those without autoimmunity.CONCLUSIONS:Evidence of autoimmunity, including autoimmune diseases, is more prevalent in ICL than the general population, and should be considered part of this syndrome.

We previously showed that lack of Response Gene to Complement 32 (RGC-32) decreases the severity of clinical scores of mice with EAE and renders spinal cord astrocytes a progenitor phenotype reminiscent of radial glia. In this study, we used RNA-sequencing to investigate whether RGC-32 deferentially regulates biological processes related to astrocyte maturation and activation. We purified neonatal astrocytes from WT and RGC-32 knock-out (KO) mice, stimulated them with TGF-β and then performed RNA-seq (Genewiz, S. Plainfield, NJ). Spinal cords from WT and RGC-32 KO mice with EAE were harvested at day 0 and at the peak of disease (day 14) and subjected to immunohistochemistry for astrocytes lineage marker GFAP, as well as radial glial marker HOPX, neural stem cell marker CD133, and proliferation marker Ki-67. Pathway enrichment analysis revealed that the genes deferentially expressed in WT but not RGC-32 KO astrocytes belonged to GO categories related to brain development, axonal guidance, cell migration and extracellular matrix (ECM) regulation. Real-Time PCR confirmed that Ephrin A7, bone morphogenetic protein 1 and polycystin 1, other genes related to brain development and ECM were significantly up-regulated in WT astrocytes after TGF-β stimulation. We found that spinal cords of RGC-32 KO mice displayed a higher number of HOPX+ cells at day 14. Similarly, the number of CD133+ cells with a radial glial morphology was higher in RGC-32 KO mice. In addition, we found that RGC-32 KO astrocytes had a higher proliferative index at the peak of EAE, as shown by their increased expression of Ki-67. These data suggest that RGC-32 is an important regulator of astrocyte maturation and reactive astrocyte formation during EAE

Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-β signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32’s ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-β. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-β-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.