Gene editing of alveolar organoids reveals AT2 dysfunction due to endosomal SFTPC accumulation.

Targeting the S100A2-RAGE Pathway Restores AT2 Cell Identity and Mitigates Lung Fibrosis.

Lung fibrosis, including idiopathic pulmonary fibrosis (IPF), represents a spectrum of progressive interstitial lung diseases characterized by disrupted epithelial repair, fibroblast activation, and excess extracellular matrix accumulation. A central feature of fibrotic progression is the loss of alveolar type 2 epithelial (AT2) cell identity and the emergence of aberrant transitional states that fail to support normal regeneration. Based on our re-analysis of multiple publicly available scRNA-seq datasets from the IPF Cell Atlas, S100A2 expression is tightly associated with basal-like reprogramming of AT2 cells and is a top upregulated gene at both mRNA and protein levels in IPF lungs. Furthermore, scRNA-seq data from human lung organoid models reveal that AT2 cells co-cultured with fibroblasts acquire a basal-like phenotype and express high levels of S100A2. Functionally, overexpression of S100A2 in human iPSC-derived lung alveolar epithelial type 2 (iAT2) cells leads to loss of AT2 cellular identity, increased generation of reactive oxygen species, and activation of RAGE signaling pathway. We demonstrate that pharmacologic inhibition of RAGE using Azeliragon preserves AT2 cell populations, associated with reduced oxidative stress in iAT2 cells, and significantly attenuates collagen deposition in bleomycin-induced lung fibrosis models. Collectively, our results indicate that S100A2 is a key driver of epithelial dysfunction in lung fibrosis, promoting loss of AT2 identity, aberrant basal fate acquisition, and persistent epithelial injury. Targeting the unrecognized S100A2-RAGE pathway may offer a new therapeutic strategy to restore epithelial homeostasis in lung fibrosis.

Emergence of Disease-relevant Aberrant Epithelial Cell States in Human Alveolar Organoids.

Dysregulated alveolar epithelial repair is a central aspect of Idiopathic Pulmonary Fibrosis but it has proven challenging to reliably model alveolar epithelial cell biology in-vitro. We previously reported persistent activation of Hypoxia-inducible Factor 2 (HIF2) is a hallmark of aberrant epithelial cell phenotypes in IPF. We hypothesized that HIF2 activation primes AECs for aberrant differentiation. In these studies, we investigated how primary human alveolar epithelial organoids respond to a commonly-used commercial alveolar differentiation media intended to facilitate AT2->AT1 differentiation (ADM; which includes human serum and withdrawal of multiple growth factors/inhibitors) in isolation and following biased HIF activation. Alveolar organoids from donor lungs were expanded and transitioned to ADM with pharmacologic modulators to establish HIF-biased signaling. Multimodal analysis integrating scRNA-seq, quantitative label-free proteomics, and Cell Painting revealed that ADM induces expression of several AT1 markers, yet these conditions are insufficient to generate mature AT1-like cells but drives the emergence of KRT17+/KRT5- "aberrant basaloid" -like cells which demonstrate high transcriptional similarity to populations in end-stage IPF lungs (p = 3x10-286). HIF2-biased activation significantly exacerbated these aberrant transitions. Gene module co-expression analysis (hdWGCNA) linked ADM to cytoskeletal modulation and HIF2-biased signaling to metabolic shifts and cytoskeletal rearrangement. Proteomics reinforced protein-level induction of aberrant markers (e.g., GDF15) and revealed novel signatures, including increased complement production. Orthogonal transmission electron microscopy confirmed ultrastructural remodeling, including lysosomal inclusions and mitochondrial modulation. Collectively, these data establish that ADM models disease-emergent transitional states rather than homeostatic AT1 differentiation. These data further position HIF2 as a critical potentiator of aberrant alveolar epithelial cell states in PF.

Aberrant expression of the PREX2-MAGI2 axis modulates myofibroblast differentiation in pulmonary fibrosis.

Myofibroblast differentiation is a pivotal event in idiopathic pulmonary fibrosis (IPF) and is driven by TGF-β1-dependent PTEN destabilization; however, the intermediate regulators remain incompletely defined. We tested three lung fibroblast cell lines (one normal and two IPF-derived) and identified the phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 (PREX2)-membrane-associated guanylate kinase inverted 2 (MAGI2) as a critical regulator of myofibroblast differentiation. Loss-of-function analysis of MAGI2 in normal human lung fibroblasts revealed a shift toward a myofibroblast phenotype characterized by abundant α-SMA expression. In vitro experiments revealed increased PREX2 expression and decreased MAGI2 expression in IPF-derived lung fibroblasts compared with those in normal lung fibroblasts. Consistent with this finding, in vivo experiments using bleomycin-induced murine pulmonary fibrosis revealed PREX2 upregulation in contrast to MAGI2 downregulation with the progression of lesions. Notably, PREX2 depletion in IPF-derived cells enhanced MAGI2 expression, mitigating TGF-β1-induced α-SMA expression. Conversely, PREX2 overexpression promoted myofibroblast differentiation, as evidenced by MAGI2 downregulation followed by marked α-SMA expression. The PREX2-targeting suppressive miR-338-3p upregulated MAGI2 expression in vitro and ameliorated bleomycin-induced murine pulmonary fibrosis in vivo. Immunohistochemical staining of PREX2 on lung samples obtained from patients with IPF delineated that PREX2 was exclusively expressed in the fibroblastic foci of IPF-affected lungs. Fluorescence immunostaining revealed PREX2 colocalization with α-SMA in myofibroblasts within fibroblastic foci, corroborating our hypothesis. These findings suggest that MAGI2 acts as a negative regulator of myofibroblast differentiation by stabilizing PTEN, whereas PREX2 is aberrantly expressed in the fibrotic milieu, negatively modulating MAGI2, thereby accelerating pulmonary fibrosis.

Dual epithelial-mesenchymal induction of Sfrp1/Frzb constrains alveolar epithelial differentiation in neonatal hyperoxia-induced lung injury.

Characterizing the Roles of Dysferlin Deficiency in Function, Polarization, and Secretome of Human iPSC-Derived Macrophages 2251399

Limb-girdle muscular dystrophy R2/2B (LGMDR2/2B) is an untreatable and progressive late-onset skeletal muscle disease caused by the loss of a membrane-repair protein dysferlin. Even before disease symptom onset, LGMDR2 muscles are infiltrated by pro-inflammatory macrophages (MP), implicating immune cells in disease pathogenesis. While MPs express dysferlin, defining the cell-autonomous roles of dysferlin in MP function has been challenging in vivo due to complex multicellular interactions and altered microenvironment in LGMDR2 muscle. To address this, we generated human induced pluripotent stem cell (hiPSC)-derived macrophages (iMPs) from three healthy and three LGMDR2 donors to delineate cell-autonomous roles of dysferlin in macrophage: 1) polarization, 2) transcriptional profile, 3) secretome, and 4) phagocytotic and endocytic function. Despite exhibiting comparable polarization under well-characterized pro- and anti-inflammatory cues, RNAseq analyses revealed downregulation of Gene Ontology terms related to cytokine secretion, phagocytosis, and receptor-mediated endocytosis in LGMDR2 iMPs. Proteomic analysis of iMP conditioned media revealed significant differences in 72 secreted proteins, including numerous chemokines, cytokines, and growth factors, suggesting an altered secretory phenotype. Functional assays found no significant differences in the phagocytosis of E. coli bioparticles or fluorescent myotube debris. However, receptor-mediated endocytosis of AcLDL was significantly lower in both M0 and M2 LGMDR2 vs. healthy iMPs. Pharmacological screens identified clathrin-dependent endocytosis as the primary pathway for AcLDL uptake in both genotypes, with altered clathrin trafficking and reduced scavenger receptor expression likely underlying LGMDR2 endocytic deficits. Overall, dysferlin loss in iMPs results in cell-autonomously altered transcriptome, secretome, and endocytic function, which may contribute to LGMDR2 muscle pathology and disease progression. Jain Foundation grant, NIH grant 1R01AR082979-01, National Science Foundation Graduate Research Fellowship Immune Mechanisms of Human Disease (HUM)

NUFIP1 gain of function promotes fibrotic remodeling and reveals a ZNHIT3-linked metabolic vulnerability in idiopathic pulmonary fibrosis models

Fibroblast activation and extracellular matrix deposition are central features of IPF, but the molecular programs that sustain pathogenic fibroblast states remain incompletely defined. NUFIP1 has been implicated in ribonucleoprotein biology and cellular stress responses, yet its role in pulmonary fibrosis is unknown. NUFIP1 expression was examined in lung tissues from patients with IPF, bleomycin-induced pulmonary fibrosis in mice, and TGF-β1-stimulated human lung fibroblasts. Gain-of-function studies were performed in MRC-5 fibroblasts and in mice using AAV-mediated NUFIP1 overexpression. The association between NUFIP1 and ZNHIT3 was assessed by co-immunoprecipitation and surface plasmon resonance. The effect of CB-839 on NUFIP1-ZNHIT3 interaction, fibroblast activation, and pulmonary fibrosis was evaluated in vitro and in vivo . Public bulk and single-cell RNA-seq datasets were analyzed to assess the clinical relevance of NUFIP1 expression in human IPF. NUFIP1 expression was increased in IPF lung tissue and showed spatial overlap with Vimentin-positive fibrotic regions. AAV-mediated NUFIP1 gain of function was sufficient to induce fibrotic remodeling relative to vector controls, with a magnitude comparable to a separate bleomycin reference group. NUFIP1 associated with ZNHIT3, and CB-839 altered the apparent association under the tested conditions while reducing NUFIP1-associated fibrotic readouts. Public transcriptomic analyses supported a modest donor-level increase in whole-lung NUFIP1 expression and detected NUFIP1 across multiple lineages, including IPF-associated fibroblast states. These gain-of-function data support a profibrotic effect of increased NUFIP1 in experimental models. ZNHIT3 is a candidate binding partner, and CB-839 reduced NUFIP1-associated fibrotic readouts at the tested dose. Pathway specificity, endogenous necessity, and clinical relevance remain to be established.