JCI Insight

Renal fibrosis represents an irreversible endpoint of chronic kidney disease. Although macrophage efferocytosis regulates the response to acute renal injury, the mechanisms by which early efferocytosis confers long-term protection against chronic fibrosis remain elusive. Here, we show that phagocyte-specific deletion of the inhibitory receptor CD300a (Cd300afl/flLyz2-Cre) mitigates obstructive renal injury and subsequent fibrosis. CD300a deficiency enhanced efferocytosis by tissue-resident macrophages during the early phase of injury, thereby suppressing the release of damage-associated molecular patterns (DAMPs) and TGF-{beta} expression. This early attenuation of the inflammatory microenvironment altered the composition of recruited neutrophils, shifting them from profibrotic SiglecF+ toward non-profibrotic SiglecF- subsets. Furthermore, loss of CD300a cell-autonomously primed neutrophils for enhanced matrix metalloproteinase-9 (MMP9) production upon injury, whereas neutrophil depletion abolished the protective effect of CD300a deficiency. Importantly, administration of a neutralizing anti-CD300a antibody to wild-type mice recapitulated these protective effects, promoting the shift toward SiglecF- neutrophils and significantly attenuating renal fibrosis. Together, our findings identify CD300a as a regulator of early macrophage efferocytosis and neutrophil-derived MMP9 production, and establish pharmacological CD300a blockade as a potential therapeutic strategy for progressive renal fibrosis.

Background and Aims: Fibroblast-neutrophil interactions are implicated in ulcerative colitis (UC) treatment resistance, but whether fibroblasts modify neutrophil inflammatory activity remains unclear. We investigated responses to fibroblast exposure, stromal depletion, and 5{beta}1-directed treatment. Methods: We integrated human and mouse colonic single-cell RNA sequencing (scRNA-seq), spatial protein profiling (CODEX), fibroblast-neutrophil coculture, and dextran sulfate sodium (DSS) mouse colitis with fibroblast activation protein (FAP) ablation or 5{beta}1-directed treatment. Readouts included cellular representation and protein fluorescence via flow cytometry, neutrophil extracellular trap (NET)-associated elastase activity, RNA programs and epithelial responses using scRNA-seq. Results: Inflamed UC tissue contained higher fractions of inflammatory fibroblasts and oncostatin M (OSM)- and CXCR4-associated neutrophils. Inflammatory fibroblasts had higher FAP-5{beta}1 module scores, and proximity to FAP-high fibroblasts was associated with higher neutrophil OSM and CXCR4 fluorescence. UC fibroblasts increased neutrophil OSM, CXCR4 and myeloperoxidase (MPO) fluorescence and NET-associated elastase activity relative to control fibroblasts, while 5{beta}1-directed treatment attenuated these responses. Both mouse FAP ablation and 5{beta}1 blockade in DSS colitis reduced histologic inflammation and overall neutrophil frequency. Ablation broadly reduced recovered neutrophil representation, whereas blockade increased OSM-positive and PADI4-positive percentages within neutrophils despite lower MPO fluorescence within these subsets. Both interventions were associated with lower epithelial chemokine scores but distinct absorptive and mucus/secretory responses. Conclusions: Fibroblast exposure modifies neutrophil inflammatory phenotype and effector-associated activity, identifying candidate therapeutic pathways in UC. Evaluating these responses alongside neutrophil representation and tissue inflammation could inform pharmacodynamic assessment of stromal-directed therapies.

Nephrotic syndrome, the leading cause of chronic kidney disease worldwide, is associated with injury and eventual loss of podocytes, the specialized epithelial cells that form the final layer of the kidney's filtration apparatus. The progression from injury to loss is largely irreversible. However, some forms of nephrotic syndrome undergo remission, suggesting that injured podocytes may retain a capacity for repair, the structural basis of which remains unknown. Podocytes respond to injury by the stereotypical morphological change known as foot process effacement (FPE), which can be visualized using transmission electron microscopy. Using modified ultrastructure expansion microscopy (U-ExM) protocol combined with super-resolution microscopy, we discovered that injured podocytes exhibit a three-stage progression of FPE maturation, with kinetics determined by the degree of actomyosin contractility. Through longitudinal study of adriamycin-induced nephropathy in mice, we identified three sequential stages of actomyosin reorganization within the sarcomere-like structures (SLSs) that define the injury response: focal infiltration of myosin IIA into synaptopodin-rich foot processes (Stage 1), disordered intermingling of synaptopodin/myosin IIA (Stage 2), and the formation of highly organized periodic contractile arrays oriented perpendicular to the direction of capillary blood flow (Stage 3). Longitudinal quantification revealed predictable temporal dynamics, with mature Stage 3 predominating by day 7-9 post-injury. To determine whether this sequence is conserved in human disease, we analyzed kidney biopsies from patients with minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, and diabetic nephropathy. The same three stages were present across all four diseases, and their distribution tracked disease course: acute podocytopathies exhibited heterogeneous early-stage patterns, whereas chronic injuries converged on uniform, mature SLS organization. These findings demonstrate that podocyte injury is a graded, potentially reversible process rather than a binary switch. They identify structured actomyosin remodeling as a conserved wound response across mechanistically distinct kidney diseases and suggest that therapeutic strategies targeting specific stages of this progression could alter the trajectory of diverse glomerular diseases.

While absolute blood biomarkers have long guided diagnosis, their rate of change often provides greater diagnostic value. For the Limb Girdle Muscular Dystrophies studied here, increased muscle weakness is associated with abnormal sarcolemma repair. Time course analyses were made to changes in muscle-derived plasma enzymes after modest physical activity of ambulatory inpatients. Blood samples at 20 selected time points at variable intervals were obtained at rest, and before and after routine morning activities and motor function testing. After these activities, plasma enzyme levels increased in a similar way, suggesting damage and leakage from skeletal muscle. The levels then decreased in a similar way, suggesting that muscle cells stopped leaking and enzymes were subsequently cleared from the blood. The complex temporal changes were modeled using both continuous time and data features to reduce the number of parameters required to describe the irregularly sampled, interrupted, time series data. Unlike individuals with cardiac muscle ischemia or following intense exercise, there was no extended post-injury rising phase of enzyme levels. In contradistinction, levels fell once activity ceased. Time series analyses of blood biomarkers most likely provide an assay and index of sarcolemma fragility and by extension skeletal muscle damage.

Pulmonary lymphatic vessels are essential for interstitial fluid balance and macromolecular clearance, yet the endothelial mechanisms regulating lymphatic vessel function during chronic fibroproliferative lung injury remain undefined. Here, we demonstrate that the lineage-defining transcription factor ERG in lymphatic endothelial cells (LECs) governs junctional plasticity and fluid drainage in pulmonary fibrosis. In human idiopathic pulmonary fibrosis (IPF) lungs, lymphatic ERG expression is markedly downregulated. Lineage-specific inducible deletion of Erg in murine LECs (Erg-CKO) unexpectedly conferred robust protection against bleomycin-induced pulmonary fibrosis, significantly dampening acute inflammation, reducing edema, and preserving pulmonary compliance. Mechanistically, loss of ERG enhanced lymphatic drainage capacity in vivo. Transcriptomic profiling of isolated LECs revealed that ERG deficiency activates actomyosin contractile pathways and selectively upregulates Endothelin Receptor Type B (Ednrb), driving junctional remodeling from continuous zippers into discontinuous button-like configurations that facilitate interstitial fluid entry. Pharmacological blockade of EDNRB with BQ-788 abolished junctional reorganization, eliminated the enhanced lymphatic drainage, and completely reversed the anti-fibrotic protection in Erg-CKO mice. Together, our findings identify an endothelial ERG-EDNRB regulatory axis that controls lymphatic junctional architecture and demonstrate that promoting lymphatic clearance via endothelial junctional remodeling represents a viable therapeutic strategy for fibrotic vascular remodeling.

Tissue homeostasis requires mechanisms that preserve quiescence while allowing rapid activation after injury, but how tissues actively restrain injury responses during homeostasis remains poorly understood. Here we identify alveolar type 1 (AT1) cells as organizers of the distal lung niche through tonic expression of PDGFA, which maintains alveolar fibroblast identity and restrains myofibroblast activation. Cell type-specific ablation revealed that loss of AT1s, but not other major alveolar cell populations, was sufficient to evoke spontaneous fibroblast activation and tissue-wide responses that recapitulate injury. In silico ligand-receptor analysis, ex vivo screening, and conditional genetic models identified AT1-derived PDGFA as a key homeostatic signal. Loss of AT1-derived PDGFA caused spontaneous myofibroblast induction and tissue remodeling, whereas increasing PDGFA promoted fibroblast proliferation while suppressing myofibroblast differentiation. Integrated phosphoproteomic, transcriptomic, and chromatin accessibility analyses showed that PDGFA engages SHP2-ERK signaling, suppresses MRTFA-associated SRF programs, and promotes E2F-associated cell-cycle programs, thereby opposing the pro-fibrotic effects of TGF{beta}-driven fibroblast activation. Restoring PDGFA signaling abrogates myofibroblast features from human idiopathic pulmonary fibrotic lungs and attenuated fibrotic remodeling in vivo. These findings define a tonic AT1-derived PDGFA signal that maintains alveolar homeostasis and offers a therapeutic target to prevent or revert lung fibrosis.

Osteoarthritis (OA) and systemic sclerosis (SSc) are diseases with distinct etiologies but similar disease phenotypes, most prominently fibrosis, a process governed by the pathological activation of fibroblasts. Mechanisms underpinning fibrosis in these diseases are widely studied, though have yet to be directly compared. We employed single-cell RNA-seq and high-resolution spatial transcriptomics to describe the functional plasticity and the range of fibroblast activation states in both diseases. We identified disease-enriched fibroblast subsets conserved in both diseases, including fibrogenic myofibroblast-like cells, chemokine- and cytokine-expressing inflammatory fibroblasts, and lining-like fibroblasts. These subsets exhibited convergent fibrotic programs also observed in idiopathic pulmonary fibrosis, as well as overlapping osteochondral transcriptional programs. Xenium 5K spatial transcriptomics revealed that conserved, disease-associated fibroblasts reside in highly similar anatomic niches in OA synovium and SSc skin. These consisted of a fibrotic ECM-rich niche harboring myofibroblast-like cells and an inflammatory niche containing inflammatory fibroblasts adjacent to infiltrating immune cells. Disease-associated transcriptional remodeling converged on shared functional pathways and common putative upstream molecular regulators. These findings identify conserved cellular states, spatial niches, and regulatory programs across OA and SSc, suggesting that fibrotic disease is underpinned by shared pathogenic stromal states and candidate targets for therapeutic strategies are applicable across both diseases.

Duchenne muscular dystrophy (DMD) is an X-linked disorder mainly affecting young boys characterized by repeated cycles of myofiber injury/regeneration and chronic muscle inflammation. Although glucocorticoids can slow disease progression, their potential long-term adverse effects highlight the need for new steroid-sparing therapies for DMD. Annexin A1 (ANXA1) is a glucocorticoid-induced pro-resolving protein that signals primarily through formyl peptide receptor 2 (FPR2). We investigated whether the ANXA1 mimetic peptide Ac2-26 could reduce inflammation, stimulate tissue regeneration, and improve skeletal muscle function in muscular dystrophy. 3-week-old dystrophin-deficient male mdx mice were randomized to receive daily intraperitoneal injections of vehicle control, deflazacort (1 mg/kg/day), or Ac2-26 (1 mg/kg/day) for a duration of 5-weeks. Body composition, skeletal muscle mass, contractile function, and tissue histopathology/immune-cell infiltration were assessed at 8-weeks of age. Direct effects on myogenesis were also examined in vitro using primary D2-mdx myoblasts. ANXA1 was expressed predominantly by myeloid cells rather than resident dystrophic muscle cells. Both deflazacort and Ac2-26 markedly reduced intramuscular inflammation and increased myofiber size. However, Ac2-26 produced broader anabolic and functional benefits, increasing body-weight gain, lean body mass, hindlimb muscle weights, and absolute contractile force. Ac2-26 treatment also directly promoted myogenesis of dystrophic myoblasts in vitro, while pharmacological FPR2 blockade impaired myotube development. Overall, Ac2-26 mimicked the anti-inflammatory effects of deflazacort while providing superior benefits for body weight and composition, skeletal muscle growth, myofiber regeneration, and absolute muscle strength. These findings support therapeutic targeting of the ANXA1-FPR2 axis as a potential safe and effective steroid-sparing immunomodulatory strategy for DMD.

Nemaline myopathy is a severe inherited skeletal muscle disorder characterized by profound muscle weakness, impaired mobility, and early mortality in severe cases. Despite the devastating clinical burden, no disease-modifying therapies are currently available. Nemaline myopathy-causing genes either encode sarcomeric structural proteins or regulators of sarcomeric protein stability, resulting in primary defects in sarcomere structure and function. Recessive mutations in KLHL40 cause one of the most severe forms of nemaline myopathy, frequently resulting in perinatal lethality. Given its loss-of-function mechanism, KLHL40-associated nemaline myopathy is an attractive candidate for gene replacement therapy. We therefore evaluated the therapeutic potential of systemic AAV9-mediated KLHL40 gene replacement in a mouse model of severe nemaline myopathy. A single systemic administration of an AAV9 vector encoding human KLHL40 during early disease produced durable rescue of survival, skeletal muscle pathology, motor performance, and contractile function in KLHL40-deficient mice. Therapeutic benefit was accompanied by restoration of sarcomere organization and normalization of myofiber size without detectable long-term histopathological abnormalities in major organs. Collectively, our findings provide a strong preclinical foundation for developing AAV9-mediated KLHL40 gene replacement as a treatment for severe KLHL40-associated nemaline myopathy.

While the immune reset induced by CD19 CAR T therapies has shown striking promise in systemic lupus erythematosus (SLE), the nature and duration of benefit mediated by B cell depletion remains unclear. Loss of tolerance in SLE is driven by extrafollicular and germinal center interactions between dendritic cells and self-reactive T and B cells. We hypothesized that CD19 CAR-engineered regulatory T cells (CAR19 EngTreg), capable of suppressing multiple autoimmune effector cell types, might promote long-term tolerance without producing high-levels of inflammatory mediators. To generate CAR19 EngTreg, we combined CD19 CAR delivery with homology directed repair at the FOXP3 locus to drive constitutive, stable FOXP3 expression in CD4 T cells. CAR19 EngTreg expressed canonical Treg markers; exhibited minimal inflammatory cytokine production; and blocked human B cell differentiation into antibody secreting plasma cells. In humanized mouse models, transfer of CAR19 EngTreg led to a reduction in B cell numbers with low inflammatory cytokine production relative to CD19 CAR Teff, and reduced tissue inflammation. In a short-term, in vivo imiquimod-induced inflammatory model, murine CAR19 EngTreg reduced skin pathology, spleen size, and inflammatory cytokines. Finally, in two distinct mouse models of lupus, CAR19 EngTreg persisted for >4 months leading to reduced autoimmune T and B effector populations, sustained reduction in multiple autoantibodies and improved renal function and histopathology. Thus, CAR19 EngTreg may offer a promising and durable therapeutic avenue for B cell-mediated autoimmune disorders capable of long-term regulation of both autoreactive B and T cells.

Cancer genomics studies have implicated combined loss of VHL and TSC1 as sufficient to initiate clear cell renal cell carcinoma (ccRCC) in humans. Here, we engineered a mouse model with kidney-specific deletion of Vhl and Tsc1. VhlF/FTsc1F/FKsp-Cre+ mice developed multifocal renal cell carcinomas starting at 7 weeks of age with 100% penetrance, and the resulting Vhl-/-Tsc1-/- tumors recapitulated the histopathology of human VHL-/-TSC1-/- ccRCC. Integrated transcriptomic and metabolomic analyses of 4-week-old preneoplastic renal cortices deficient for Vhl and Tsc1 demonstrated heightened HIF and mTORC1 signaling and unexpectedly revealed activation of an antioxidant response regulated by NRF2 (NFE2L2). Biochemical, cell biological, multi-omic, and xenograft studies identified an NRF2 dependency in human VHL-/-TSC1-/- ccRCC cells. Altogether, we report a mouse model that recapitulates human VHL-/-TSC1-/- ccRCC, delineate metabolic and redox reprogramming driven by unrestrained HIF and mTORC1 activation in the renal cortex, and identify NRF2 as a potential therapeutic vulnerability.

Myocardial fibrosis is a common end point of acute and chronic cardiovascular injury, yet the spatially organized fibroblast (Fb) and immune-cell states that sustain fibrotic remodeling across etiologies remain incompletely defined. We applied cyclic in situ hybridization (cISH)-based spatial transcriptomics to myocardial biopsies from patients with acute myocardial infarction (AMI), ischemic cardiomyopathy (ICM), hypertrophic cardiomyopathy (HCM), postviral myocardial fibrosis (PVMF), and systemic sclerosis associated primary heart involvement (SSc-pHI). CODEX-based protein imaging provided orthogonal support for cell subpopulations and cellular neighborhoods. Spatially informed clustering identified six Fb subpopulations: COL1A1hi/POSTNhi, NOTCH3+, TIMP1hi, GSNhi/DCNhi, AP1hi Fbs, and myofibroblasts. COL1A1hi/POSTNhi and NOTCH3+ Fbs were enriched in AMI, whereas GSNhi/DCNhi Fbs and myofibroblasts predominated in chronic fibrotic remodeling. These subpopulations displayed distinct extracellular matrix, inflammatory, and vasoregulatory programs. Infarction-associated reparative niches were enriched in COL1A1hi/POSTNhi or NOTCH3+ Fbs and SPP1+ or APOE+ macrophages (M{Phi}), whereas chronic fibroinflammatory niches were enriched in GSNhi/DCNhi or AP1hi Fbs or myofibroblasts with C1Q+ M{Phi} and plasma cells. The GSNhi/DCNhi Fb niche marked areas of subendocardial fibrosis, whereas the AP1hi Fb niche localized to perivascular fibrosis. In SSc-pHI, the actively fibrogenic COL1A1hi/POSTNhi Fb state characteristic of infarct scar formation was present at higher frequency than in other chronic entities and was individually associated with cardiac dysfunction, whereas the chronic GSNhi/DCNhi Fb niche was associated with the extent of interstitial fibrosis and anti-topoisomerase I autoantibodies. These data show that human myocardial fibrosis is organized into spatially distinct Fb subpopulations that assemble into context-specific fibroinflammatory niches across chronic fibrotic remodeling and repair after AMI.

Understanding the shared and disease-specific mechanisms that drive glomerular injury remains a major unmet need for developing more precise and effective treatments for glomerular diseases. Here, we integrate histology-guided spatial transcriptomics with trajectory inference and cell-state deconvolution to resolve glomerular injury across Membranous Nephropathy, Focal Segmental Glomerulosclerosis, and Lupus Nephritis. Herein, we show that glomerular diseases arising from distinct initiating mechanisms converge on common pathways during progressive loss of kidney function. We used spatial transcriptomics to analyze kidney biopsies from 59 individuals with glomerular diseases and controls and identified 792 glomeruli, including structurally injured glomeruli missed by transcriptomic clustering alone. Glomeruli shared interferon- and complement- associated inflammatory programs together with suppression of oxidative and lipid metabolic pathways, while retaining disease-preferential transcriptional features. Increased pseudotime correlated with lower estimated glomerular filtration rate, and cell- state deconvolution linked progression to podocyte loss and cellular remodeling. Parallel analysis of passive Heymann nephritis, a rat model of membranous nephropathy, recapitulated complement activation and localized complement activity to multiple renal cell populations extending into the periglomerular niche. These findings define a spatially resolved, cross-disease continuum of glomerular injury and identify coordinated local complement activation as a convergent feature of progressive glomerular damage.

Peripheral nerves are increasingly recognized as active components of the tumor microenvironment (TME). Yet, their role in non-muscle-invasive bladder cancer (NMIBC) and response to Bacillus Calmette-Guerin (BCG) remains poorly defined. Using multispectral imaging, we characterized autonomic and sensory nerve populations within the tumor-adjacent stroma of 241 treatment-naive high-risk NMIBC tumor regions from 141 patients. Stromal tumor-associated nerve (STaN) abundance varied substantially across patients and was associated with aggressive clinicopathological features, BCG failure, and disease progression. Unsupervised clustering resolved distinct STaN phenotypic groups with differential clinical and spatial associations. Noradrenergic and a neurochemically heterogeneous STaN populations showed the strongest associations with adverse outcomes. In contrast, a mixed population of nerves with high expression of the synaptic vesicle protein synaptophysin exhibited prominent spatial remodeling associated with BCG failure, despite limited prognostic value by abundance alone. Integration with an independent single-cell atlas nominated cancer-associated fibroblasts and mast cells as candidate cellular intermediaries of neural communication within the TME. Matched transcriptomic profiling revealed coordinated neuronal, extracellular matrix, lipid metabolism, and immune programs in STaN-high tumors, whereas STaN-low tumors preferentially enriched proliferative and hypoxic stress-response pathways. Leveraging these transcriptional features, we developed a transcriptomic classifier that distinguished STaN-high from STaN-low tumors in an independent test cohort (AUC = 0.87) and stratified clinical outcomes in additional patient cohorts. Together, these findings establish tumor innervation as a heterogeneous and clinically relevant feature of the NMIBC microenvironment, with neural abundance, phenotype, and spatial organization capturing distinct aspects of tumor behavior and clinical outcome.

Sickle cell disease (SCD) causes heterogeneous mineral imbalances including variable degrees of hypocalcemia. The kidney controls systemic calcium by reabsorbing calcium from the glomerular filtrate via paracellular transport and transcellular transport in the nephron tubules, yet it is unknown whether these processes are modulated by genetic or environmental factors or disrupted in SCD. Using SCD mouse models and single-cell multiomics, we identify the distal convoluted tubule (DCT) as the nephron segment most susceptible to calcium reabsorption dysfunction in SCD, mainly via reduction of calcium buffer protein calbindin 1 (CALB1). We show that CALB1 and its encoding mRNA are decreased in DCT cells in SCD, alongside decreased Klotho (KL)-dependent fibroblast growth factor (FGF) 23 signaling and intracellular calcium signaling. Dietary iron restriction reduces CALB1, KL, and calcium exporter SLC8A1 levels in SCD kidneys. Loss of CALB1 shifts DCT cells toward energy-inefficient glycolysis with the metabolite 2,3-diphosphoglycerate impairing KL-dependent FGF23 signaling to create a feed-forward loop suppressing calcium reabsorption. Analysis of gene expression and protein quantitative trait loci data from kidneys of genetically diverse mice revealed that Calb1 expression levels are highly heritable and co-regulated with Slc8a1, identifying a genetic axis that dictates differential capacities for calcium buffering and trafficking toward blood in the kidney. Together, these findings support a model in which genetic variation, dietary iron status, and FGF23 signaling converge on DCT calcium buffering to reduce renal calcium reabsorption in the SCD kidney. This points to personalized, genotype- and iron-dependent strategies for managing mineral metabolism in SCD patients.

Abstract Endothelial cell (EC) injury is central to the pathogenesis of various renal disorders, highlighting the critical importance of vascular repair for restoring organ health. However, due to limited access to human tissue during active regeneration, the exact reparative processes remain largely unknown. This study aimed to investigate regeneration mechanisms specifically within the glomerular microvasculature by utilizing a selective endothelial cell injury (ECI) model in mice.Lineage tracing identified the origin of regenerating ECs. To uncover the transcriptional dynamics driving these reparative changes, we combined single-cell RNA sequencing and Xenium spatial transcriptomics (ST), mapping distinct glomerular EC (gEC) subpopulations across different phases of injury and recovery. Apold1 emerged as a key regulatory candidate within this process. To functionally validate this gene's specific role during the regenerative phase, Apold1-deficient (Apold1-/-) mice underwent the ECI model. After showing that gECs regenerate primarily from local, pre-existing renal ECs, our transcriptomic analysis identified 10 distinct gEC subpopulations and revealed a complex temporal pattern of regeneration. This process begins with the expression of genes associated with inflammation, followed sequentially by endothelial cell (EC) migration, the activation of immediate early genes, vascular remodeling and cellular proliferation. Within this dynamic process, the EC-specific gene Apold1 was identified as a key regulatory candidate. Apold1-/- mice exhibited impaired endothelial regeneration, characterized by increased endothelial activation and greater immune cell infiltration seven days post-ECI. To our knowledge, this is the first study to elucidate the spatiotemporal mechanisms of glomerular endothelial cell regeneration. By identifying local gECs as the primary source of repair, and stablishing the change in their expression pattern during regeneration, as well as identifying Apold1 as an intrinsic regulator, we have laid the groundwork for future research on renal microvascular disorders.

Redox homeostasis requires both inducible antioxidant defense and constitutive machinery that continuously regenerates cellular reducing capacity. How these systems are altered across lung cell populations in chronic obstructive pulmonary disease (COPD) remains unclear. We integrated donor-level single-nucleus lung transcriptomes with smoke-exposure models, independent cohorts, and large-scale pharmacologic perturbation data. Both redox programs were reduced in established COPD, but constitutive redox support showed the greater loss, concentrated in fibroblasts and lymphatic endothelium. In fibroblasts, PGC-1 target-program output showed the most negative effect among 515 regulator programs despite preserved PPARGC1A expression. Smoke exposure activated inducible antioxidant defense across experimental models, whereas this adaptive program was reduced in established COPD. Pharmacologic perturbations further separated the two arms of redox homeostasis, with redox-directed interventions producing larger transcriptional responses in inducible defense and a distinct set of compounds increasing constitutive support together with PGC-1 target output and movement away from an independently defined COPD fibroblast state. These findings identify a cell-selective loss of constitutive redox support in COPD and implicate reduced PGC-1 target-program output in the fibroblast redox phenotype.

Systemic lupus erythematosus (SLE) is the prototypical systemic autoimmune disease defined by the production of autoantibodies directed against diverse self-antigens. Antinuclear antibodies (ANA) are central to SLE diagnosis, and anti-double-stranded DNA (anti-dsDNA) antibodies are clinically used to track disease activity. However, these biomarkers provide only partial insight into the full spectrum of autoimmune changes in patients. To expand the known landscape of SLE autoreactivity shared among patients and associated with disease activity, we performed an unbiased characterization of 1,268 SLE plasma samples from 695 SLE patients and 301 healthy controls using phage immunoprecipitation and sequencing (PhIP-seq). This technique preferentially displays linear epitopes, and thus provides a complementary set of autoreactivities to previously discovered autoreactive targets. Despite the marked clinical heterogeneity of SLE, machine learning applied to this data surfaced a shared, reproducible signature that robustly discriminates between SLE and healthy controls in two validation cohorts. Autoreactivities in SLE were significantly enriched for RNA-binding protein targets. Autoreactive proteins containing Basic Acidic Dipeptides (BAD) repeat domains, features associated with biomolecular condensate formation, distinguished patients with high disease activity (by SLEDAI score) from patients with no disease activity. Among all shared autoreactivities, a peptide derived from YTHDF2 emerged as the most prevalent target across SLE cohorts occurring in ~25% of SLE patients and displayed striking sequence similarity to a peptide derived from the Epstein Barr virus (EBV) lytic switch protein BZLF1. Competition assays demonstrated higher-affinity binding to the viral peptide, supporting a model in which antiviral humoral responses to EBV may drive secondary epitope spreading toward molecularly mimicked self-antigens. Remarkably, orthogonal validation using the Luminex bead platform containing a panel of only 10 peptides, including BZLF1, demonstrated strong discrimination between SLE and healthy controls (AUC = 0.95). Together, these findings identify a rich, greatly expanded landscape of autoreactivities in SLE, candidate biomarkers associated with high disease activity, and provide mechanistic insight linking EBV-driven immunity to epitope-specific autoimmunity in lupus.

Oligodendrocyte (OLG) injury and loss are increasingly recognized as major determinants of demyelination and remyelination failure in multiple sclerosis (MS), alongside conventional immune-mediated processes. However, the mechanism underlying OLG injury and remyelination failure remains incompletely understood. Astrocyte-oligodendrocyte interactions mediated by Connexin (Cx)-43-Cx47 gap junction (GJ) communication and leukemia inhibitory factor (LIF)-LIFR/gp130 signaling are critical for oligodendroglial survival, homeostasis, and myelination. Despite their established role, how these pathways are altered during neuroinflammatory diseases remains unclear. To address this, we integrated a murine {beta}-coronavirus (RSA59)-induced in vivo model with enriched primary oligodendrocyte precursor cell (OPC) and mature OLG cultures to investigate the effect of RSA59 infection on OLGs, Cx47-mediated GJ communication, and LIF-LIFR/gp130 Signaling. We observed that RSA59 directly targets oligodendroglial lineage cells, including OPCs and mature OLGs, and induces apoptosis in mature OLGs. Despite increased OPC abundance, RSA59 infection reduces the expression of mature and myelinating markers CNPase and MBP, raising the possibility of impaired oligodendrocyte maturation and myelination. RSA59 infection also differentially regulates Cx47-mediated GJ communication and LIF-LIFR/gp130 signaling, crucial for OLG survival and homeostasis. Furthermore, our studies demonstrate that extracellular signal-regulated kinase (ERK), a downstream effector of LIF signaling, phosphorylates the Cx47 C-terminal domain (Cx47CT) at Serine-372 residue, identifying a previously unreported mechanism. Collectively, these findings reveal that RSA59 infection disrupts interconnected oligodendroglial communication and survival pathways, providing mechanistic insights into virus-induced chronic demyelination pathology. The study further identifies Cx47-mediated GJ communication and LIF-LIFR/gp130 signaling as crucial therapeutic targets, and highlights ERK-dependent Cx47CT phosphorylation as a potential regulatory mechanism.

Protein misfolding in the endoplasmic reticulum (ER) of podocytes/glomerular epithelial cells (GECs) induces ER stress and contributes to the pathogenesis of glomerular disease. Activation of IRE1 and consequently the unfolded protein response (UPR) and autophagy sustains proteostasis. The present study examines the role of Surf4 and collagen IV-5 (COL4A5) trafficking in podocyte proteostasis. Glomerular Surf4 protein expression increased in IRE1-replete mice with experimental focal segmental glomerulosclerosis (FSGS) compared to podocyte-IRE1 knockout mice with FSGS and control mice. Glomerular Surf4 gene expression was greater in human FSGS and membranous nephropathy compared to healthy controls. In cultured GECs, basal Surf4 expression was greater in IRE1-replete (control) compared to IRE1 knockout GECs, although ER stress (tunicamycin treatment) did not increase Surf4 despite activating the UPR. Tunicamycin enhanced autophagy (LC3-II) in an IRE1-dependent manner, and the amount of Surf4 localized in autophagosomes. In GECs, tunicamycin increased the amount and colocalization of endogenous COL4A5 or ectopically-expressed COL4A5-wild type (WT) and Surf4 in autophagosomes. Compared to COL4A5-WT, the human COL4A5-C1567R mutant (which is misfolded) showed enhanced basal colocalization with Surf4 in autophagosomes; colocalization was similar to COL4A5-WT after tunicamycin treatment. Degradation of COL4A5-C1567R was more rapid compared to COL4A5-WT and was comparable to degradation of COL4A5-WT in the presence of tunicamycin. Thus, during ER stress, subsets of Surf4 and COL4A5 are redirected from the secretory pathway to autophagy. Surf4 may help deliver misfolded ER cargo, such as COL4A5 for autosomal degradation, thereby sustaining proteostasis in podocytes.