JCI Insight

Neurodegenerative diseases are increasingly recognized to involve detrimental interactions between the immune and nervous systems, yet whether failure of peripheral immune tolerance actively drives neuronal loss frequently remains unclear. Here, using primary open-angle glaucoma as a model of chronic neurodegeneration, we demonstrate that dysregulated adaptive immunity is sufficient to promote retinal ganglion cell degeneration. Peripheral blood mononuclear cells from glaucoma patients, but not healthy donors, induced retinal ganglion cell loss following transfer into humanized immunodeficient mice without changes to the intraocular pressure, demonstrating a causal role for immune responses in the patient derived material. Comprehensive immune profiling further revealed selective changes in the regulatory T-cell compartment, indicating reduced activation, an impaired suppressive phenotype, and altered differentiation and trafficking states despite preserved overall regulatory T cell abundance. We further demonstrate that transient expansion of regulatory T cells preserves visual function, reduced optic nerve axonal degeneration, and limited retinal ganglion cell loss in an experimental glaucoma model. Together, these findings identify failure of regulatory T cell mediated immune tolerance as a mechanism that permits neurodegeneration in glaucoma and demonstrate that restoring immune regulation can ameliorate neuronal injury. Our data indicate that immune tolerance is a modifiable determinant of chronic neurodegeneration and suggest that immunoregulatory therapies may complement conventional pressure-lowering treatments to preserve vision in glaucoma.

APOL1 risk variants are the strongest genetic determinants of HIV-associated nephropathy (HIVAN), yet the mechanisms linking inflammation to APOL1-mediated podocyte injury remain poorly understood because authentic patient-derived human disease models are lacking. Using urine-derived podocytes established from children with HIVAN and endogenous APOL1 reporter cell lines derived from these cells, we identified a previously unrecognized pathway of inflammatory, cathepsin-dependent APOL1 proteolysis. Endogenous APOL1 cleavage was detected in patient-derived podocytes, whereas reporter cell lines enabled the identification and functional characterization of N-terminal and C-terminal APOL1 fragments with distinct intracellular localization and pathogenic functions. The nuclear N-terminal fragment activated inflammatory transcriptional programs and promoted podocyte injury, whereas the membrane-associated C-terminal fragment mediated membrane toxicity and remained susceptible to pharmacologic inhibition by inaxaplin. Cathepsin S directly cleaved APOL1 in vitro, linking inflammatory signaling to APOL1 fragmentation. These findings identify inflammatory APOL1 proteolysis as a mechanism that partitions APOL1 toxicity into distinct pathogenic programs and nominate APOL1 processing as a therapeutic target for HIV-associated and other APOL1-mediated kidney diseases.

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.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by irreversible destruction of the alveolar epithelium and impaired regeneration. Although current therapies slow disease progression, they do not restore functional alveoli, highlighting the need for regenerative approaches that promote endogenous lung repair. Here, we performed the first unbiased functional screen of 2,042 human microRNA mimics in primary mouse alveolar type II (ATII) cells to identify regulators of ATII-to-alveolar type I (ATI) cell transdifferentiation. The screen identified miR-124-3p as the most effective promoter of ATI differentiation. In vitro, miR-124-3p promoted ATII-to-ATI transdifferentiation in healthy and bleomycin-injured ATII cells while also increasing the ATII cell pool, consistent with activity on epithelial progenitors. Using the engineered AAV6.2FF capsid, we generated a vector encoding miR-124-3p, which efficiently transduced ATII cells, MHC-II club distal progenitor cells, and injury-induced KRT8 epithelial intermediates. Therapeutic administration after fibrosis establishment reduced lung fibrosis, restored alveolar architecture, and showed greater efficacy than nintedanib in the bleomycin mouse model. Mechanistically, we propose a context-dependent model whereby miR-124-3p regulates epithelial cell states through the EZH2-C/EBP axis while attenuating epithelial transcriptional programs associated with IPF. Together, these findings support AAV-mediated delivery of miR-124 to promote alveolar repair in pulmonary fibrosis.

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.

Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterized by muscle fiber degeneration, immune infiltration, and protein aggregation. Despite the prominent immune infiltrates that characterizes IBM muscle, the factors driving immune infiltration remain unknown, and the repertoire and spatial organization of infiltrating immune populations remain poorly defined. Here, we used high-resolution spatial transcriptomic profiling to define the cellular and spatial architecture of IBM muscle. Immune profiling revealed a complex inflammatory landscape dominated by interferon-responsive CD8+ T cells and interferon-stimulated antigen-presenting macrophages, which organized into spatially localized immune hubs surrounding myofibers. Myofibers within these immune-rich microenvironments exhibited increased expression of interferon-responsive genes and HLA class I and II antigen presentation machinery components across fiber subtypes. In addition, we identified muscle-intrinsic remodeling and regenerative programs that may precede or contribute to immune recruitment, characterized by focal spatial activation of genes involved in proteostasis, cytoskeletal organization, and myofiber repair. Together, these findings define the spatial immune landscape of IBM muscle and reveal coordinated immune and muscle-intrinsic programs that shape disease pathology.

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.

Chronic lung allograft dysfunction (CLAD) is a major cause of death after lung transplantation and manifests principally as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Although pulmonary macrophages are key regulators of lung injury and repair, their contributions to CLAD pathogenesis remain under-examined. We performed single-cell RNA sequencing-based transcriptomic analysis of CLAD (7 BOS, 7 RAS) and control lung tissue (n = 6), complemented by comparative and functional analyses. Two distinct macrophage populations were identified in CLAD: inflammatory macrophages expressing leukocyte immunoglobulin-like receptors (LILR), and fibrotic macrophages characterized by expression of osteopontin (SPP1). LILR macrophages were present in both BOS and RAS, whereas SPP1 macrophages were selectively enriched in RAS. Both populations were identified in idiopathic pulmonary fibrosis and cross-tissue comparisons. Surface marker-based sorting strategies were developed to isolate both populations. Functional studies demonstrated that LILR macrophages exhibited enhanced phagocytic activity, promoted T cell chemotaxis and secreted inflammatory mediators, whereas SPP1 macrophages produced soluble factors that drove fibroblast activation and contraction. These findings identify distinct inflammatory and fibrotic macrophage programs associated with CLAD, link RAS to conserved fibrotic macrophage states observed in pulmonary fibrosis and highlight macrophage populations as potential therapeutic targets for inflammatory and fibrotic allograft injury.

Osteoarthritis (OA) is the most common chronic joint disease and a leading cause of disability worldwide, yet no disease-modifying therapies are available. Although OA has traditionally been viewed as a cartilage-centered degenerative disease, growing evidence suggests that synovial inflammation is a critical driver of disease progression. However, the upstream mechanisms and actionable targets that govern this inflammatory response remain poorly defined. Connexin 43 (Cx43), a key regulator of musculoskeletal homeostasis that forms both gap junctions and hemichannels (HCs), is upregulated in osteoarthritic cartilage and synovium, but the specific contribution of Cx43 channels remains unclear. Here, we show that selective inhibition of Cx43 HCs attenuates OA progression in a murine post-traumatic OA model. Using adeno-associated virus (AAV)-mediated gene deletion, we found that Cx43 knockdown in joint tissues reduced cartilage erosion, synovial hyperplasia, subchondral bone sclerosis, and OA-associated pain. To dissect HC-specific functions, we developed a monoclonal antibody, Cx43(M1), that blocks Cx43 HCs without affecting junctional communication. Cx43(M1) preserved cartilage integrity, suppressed synovial inflammation, and alleviated pain behaviors, even when treatment was initiated after established cartilage damage. Mechanistically, the antibody preferentially localized to synovial macrophages, where it inhibited HC activity, reduced ATP and PGE2 release, and suppressed pro-inflammatory gene expression. Macrophage-specific Cx43 deletion recapitulated these protective effects, and Cx43 HC blockade in primary human OA synovial macrophages similarly inhibited inflammatory and catabolic responses. These findings identify synovial macrophage Cx43 HCs as upstream drivers of OA and support a paradigm shift from a predominantly chondrocyte-centered view toward synovial macrophage directed intervention.

Meibomian gland dysfunction (MGD), a disorder of the eyelid's modified sebaceous glands, is the leading cause of dry eye disease and ocular surface morbidity, yet the immune mechanisms driving gland obstruction remain poorly defined. In a cross-sectional study of 66 patients with ocular surface inflammation, we used meibography and spectral flow cytometry of tear washes to identify a disease-associated, remodeled neutrophil state whose abundance is associated with gland atrophy. Using single-cell transcriptomics in a murine model of immune-mediated MGD, we revealed a disease-associated neutrophil state that exhibited ocular surface-enrichment, CD14 and ICAM-1 expression, and elevated IFN-{gamma} response and inflammatory signatures. Spatial transcriptomics localized IFN-{gamma} signaling and neutrophil migration signatures to the periglandular compartment. The remodeled neutrophils exhibited PAD4-dependent histone citrullination, with Padi4 deletion reducing NET-associated obstructive plugging, thus identifying PAD4-dependent NETotic activity as their disease-producing output. Inhibition of IFN-{gamma} signaling phenocopied Padi4 deficiency, yet combined disruption of these pathways provided no additive protection, indicating that IFN-{gamma} and PAD4 function as separable required inputs. Remodeled neutrophils accumulated under both conditions, uncoupling disease severity from cell abundance alone. Our findings support immune-mediated obstructive MGD as a mechanistic endotype driven by the IFN-{gamma}- and PAD4-dependent effector output of a remodeled neutrophil state.

Introduction: C3 glomerulopathy (C3G) is driven by fluid-phase alternative complement pathway dysregulation, with emerging evidence linking glomerular neutrophil infiltration to disease severity. Neutrophil extracellular traps (NETs) are implicated in other forms of glomerulonephritis. However, their participation in the pathogenesis of C3G remains undefined. Methods: Kidney biopsies from 33 patients with C3G (15 with dense deposit disease [DDD] and 18 with C3 glomerulonephritis [C3GN]) were compared with 15 anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) biopsies as a neutrophil-rich disease control in this retrospective cross-sectional study. Glomerular neutrophils and NETs were identified using immunofluorescence, staining for myeloperoxidase, citrullinated histone H3, peptidyl arginine deiminase-4, and DNA. Supervised machine learning was used to quantify glomerular NET formation, and the data were correlated with kidney function at time of biopsy using linear regression. Results: Intraglomerular NETs were abundant and detected in the majority of glomeruli in C3G biopsies. Compared with AAV, C3G showed a significantly higher fraction of neutrophils forming NETs, despite similar neutrophil counts per glomerulus. NET abundance was similar in DDD and C3GN. In exploratory analyses, a greater proportion of glomeruli containing NETs was associated with lower kidney function (estimated glomerular filtration rate) at biopsy, and this association remained significant after adjustment for age, C3G subtype, and interstitial fibrosis. Conclusions: These observations demonstrate that intraglomerular NETs are a common and prominent observation in C3G and are associated with reduced kidney function at biopsy. These findings raise the possibility that NET deposition in glomeruli is a previously unrecognized driver of glomerular injury in C3G.

Introduction: Class II lupus nephritis (LN) is considered clinically benign, yet up to 50% of patients progress to more severe disease and no molecular characterization exists. We aimed to define the single-cell landscape of Class II LN and identify cellular and transcriptional features associated with kidney outcome. Methods: We performed single-cell RNA sequencing on kidney biopsies from fifteen Class II LN patients (eight de novo, seven regressed from prior proliferative or membranous disease) and six healthy controls, integrated with 155 LN and 30 healthy-control samples from the Accelerating Medicines Partnership in SLE spanning proliferative, membranous, and mixed LN. Findings were correlated with 52-week renal response and supported by urinary proteomics. Results: Despite histologically minimal changes, Class II LN exhibited marked immune cell expansion comparable to proliferative and membranous LN, including CCL3+ CCL4+ intermediate monocytes producing TNF, FOLR2+ SIGLEC1+ macrophages producing TGF{beta} and PDGF, and CD69+ CD8+ effector memory T cells producing IFN{gamma}. Fibroblasts and myofibroblasts were significantly expanded and displayed divergent transcriptional programs: pro-fibrotic and inflammatory myofibroblast modules were associated with worse 52-week outcomes, while interferon-responsive and matrix-remodeling fibroblast programs were associated with improvement. Pathogenic and protective fibroblast populations received overlapping immune-derived signals, suggesting that fibroblast transcriptional state, rather than the identity of incoming signals, shapes the stromal response. Baseline chronicity index captured this stromal heterogeneity and was the clinical parameter most associated with outcome. Urinary proteomics aligned Class II with proliferative rather than membranous disease. Conclusions: Class II LN harbors substantial molecular activity not captured by routine histology. Fibroblast transcriptional programs and baseline chronicity index are candidate correlates of kidney outcome that warrant validation in larger, prospectively followed cohorts.

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.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for studying individual cell populations in complex tissues during injury and repair. However, it involves extensive sample manipulation, limiting its application in rare and difficult-to-dissociate cell populations. Single-nucleus RNA sequencing (snRNA-seq) bypasses several of these challenges, but its performance in lung disease models is not well characterized. In a longitudinal murine model of bleomycin-induced lung injury, we performed whole lung snRNA-seq to systematically map 46 cell populations and states with minimal tissue processing. Our approach captured the emergence of Krt8+/Cdkn1a+ transitional alveolar cells and an expansion of macrophage populations during injury. Additionally, we identified sizeable populations of rare cell types, such as pericytes, and cells sensitive to digestion protocols, including a population of activated fibroblasts. These results indicate that snRNA-seq recapitulates the dynamic changes associated with injury as previously reported using single-cell methods and outperforms these approaches in the representation of cell types that are sensitive to processing, thereby highlighting its utility for high resolution analysis of heterogenous tissues such as lung.

Elevated intraocular pressure (IOP) and aging are major risk factors for primary open-angle glaucoma (POAG), but how aging affects IOP regulation remains poorly understood. IOP remains within a narrow range despite age-associated changes predicted to increase aqueous humor outflow (AHO) resistance at the interface between the trabecular meshwork and Schlemm's canal (SC), suggesting compensatory mechanisms preserve AHO homeostasis during aging. Single-cell RNA sequencing of mouse ocular angle tissues revealed immunomodulatory transcriptional reprogramming of SC endothelial cells in older mice, while mouse and human imaging showed reduced SC size and increased peri-SC macrophage accumulation with aging. Ligand-receptor analysis predicted enhanced macrophage-to-SC VEGFA-VEGFR signaling in aged and Tie2-haploinsufficient mice, an independent model of vascular stress and glaucoma risk. Deletion of Vegfa in CX3CR1+ macrophages increased IOP and reduced AHO facility in 9-month-old wild-type mice, demonstrating that macrophage-derived VEGFA supports AHO homeostasis. Tie2 haploinsufficiency recapitulated key age-associated SC niche changes, including peri-SC macrophage accumulation, whereas gene therapy boosting TIE2 activity protected wild-type mice against age-related changes. Together, these findings identify peri-SC macrophage-derived VEGFA as a compensatory mechanism maintaining AHO homeostasis during aging and vascular stress and support TIE2 activation as a therapeutic strategy to preserve SC function and IOP regulation.

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.