Wang, Shaohua

Background: Alcohol-associated hepatitis (AH) is a severe inflammatory liver condition driven by dysregulated immune responses. CD8 T cells accumulate in the liver during AH, yet their functional role remains poorly defined. Methods: A murine NIAAA chronic-binge model with lipopolysaccharide (LPS) challenge was used. Single-cell RNA sequencing of liver and blood immune cells defined CD8 T cell subsets, followed by pathway analysis and reanalysis of human AH datasets. Spatial relationships were validated by immunohistochemistry of human AH liver tissue with CD8 T RM and liver sinusoidal endothelial cell (LSEC) co-staining. LSEC–CD8 T cell interactions were examined using in vitro co-culture, and signaling pathways were assessed by bulk RNA sequencing and phospho-protein analyses. IL15 function was evaluated by in vivo neutralization. Results: Single-cell RNA sequencing in the murine AH model revealed a distinct population of CD8 tissue-resident memory T cells (T RM ) with heightened activation and proinflammatory cytokine production. Analysis of human AH liver single-cell RNA sequencing data, along with immunohistochemistry validation, supported CD8 T RM enrichment in diseased tissue. IL15 emerged as a prominent pathway linked to T RM activation, and IL15 blockade reduced T RM abundance and attenuated EtOH/ lipopolysaccharide-induced liver injury. Mechanistically, LSECs not only provided a structural niche for T RM retention but also amplified IL15 signalling. In vitro, coculture experiments demonstrated that LSECs intensified activation in pre-stimulated CD8 T cells in a stimulus-dependent manner: under IL15 stimulation, LSECs boosted effector function without inducing cell death, whereas under T-cell receptor stimulation, LSECs drove hyperactivation and activation-induced cell death. Bulk RNA-seq and phospho-protein analysis identified the PI3K-AKT pathway as a shared pathway enhanced by LSEC coculture in activated CD8 T cells. These findings define a context-dependent mechanism in which LSECs promote IL15-driven signaling through AKT pathway amplification, promoting T RM persistence and inflammatory activity in AH. Conclusions: IL15-associated signaling within the hepatic microenvironment, shaped by LSEC-CD8 T-cell interactions, promotes activation and persistence of CD8 T RM cells in AH. Targeting the IL15-LSEC-AKT axis may disrupt pathogenic T RM niches and represent a promising therapeutic strategy for severe AH.

ABSTRACTInflammatory cell infiltration is a characteristic feature of COPD and correlates directly with the severity of the disease. Interleukin‐23 (IL‐23) is a pro‐inflammatory cytokine that regulates Th‐17 inflammation, which mediates many pathophysiological events in COPD. The primary goal of this study was to determine the role of IL‐23 as a mediator of key pathologic processes in cigarette smoke‐induced COPD. In this study, we report an increase in IL23 gene expression in the lung biopsies of COPD patients compared to controls and identified a positive correlation between IL23 gene expression and disease severity. In a cigarette smoke‐induced murine emphysema model, the suppression of IL‐23 with a monoclonal blocking antibody reduced the severity of cigarette smoke‐induced murine emphysema. Mechanistically, the suppression of IL‐23 was associated with a reduction in immune cell infiltration, oxidative stress injury, and apoptosis, suggesting a role for IL‐23 as an essential immune mediator of the inflammatory processes in the pathogenesis of CS‐induced emphysema.

Site specific targeting remains an elusive goal for stem cell and gene therapies in the cardiovascular field. One promising option involves use of modified 3D stent designs that deliver larger cell/gene payloads to specific disease sites using the versatility of percutaneous vascular access. Here, we engineered smooth muscle cells (SMCs) placed in a 3D metallic stent scaffold to deliver high local concentrations of an angiogenic gene (VEGF165) in a porcine model of chronic total occlusion involving ameroid placement on the proximal left circumflex (LCx) artery(n≥8 per group). Engineered SMCs were retained within the mesh stent structure (479 ± 47.4 SMC/mm2) and were competent for VEGF production in vitro (8.8 ± 1.8 ng/ml in media). Following implantation, micro CT analyses revealed that stents loaded with VEGF producing SMCs significantly enhanced vasa vasora microvessel density (5.43 ± 0.46 vs 2.14 ± 0.19 vessels/mm2, p<0.001) with a concomitant increase in tissue VEGF protein levels (277.9 ± 85.7 vs 18.8 ± 4.4 pg/mg protein, p<0.0001). Formation of endothelial cell colonies from peripheral blood mononuclear cells was significantly increased with the implantation of VEGF-secreting cells suggesting increased angiogenic potential (7.0 ± 2.0 versus 41.6 ± 4.5, EC:CFU p<0.0001). VEGF secreting mesh stent placement enhanced regional blood flow analyses determined by microsphere analyses as well as improving regional wall motion in the LCx-related territory (p<0.05) as well as left ventricular function assessed in terms of ejection fraction (34.2 ± 6.7% vs 61.2 ± 5.2% P<0.001) and dp/dt (p<0.001) Our data indicate robust clinically relevant angiogenesis can be achieved in a human scale porcine chronic vascular occlusion model following stent based delivery of angiogenic cells. This may have implications for percutaneous delivery of stem cells, genes and secreted therapeutic factors in cardiovascular disease in general and in creation of percutaneous microvascular bypass networks in the setting of chronic vaso-occlusive disease in particular.

Abstract CD8+ tissue-resident memory T (TRM) cells provide frontline immunity in mucosal tissues. The mechanisms regulating CD8+ TRM maintenance, heterogeneity, and protective and pathological functions are largely elusive. Here, we identify a population of CD8+ TRM cells that is maintained by major histocompatibility complex class I (MHC-I) signaling, and CD80 and CD86 co-stimulation after acute influenza infection. These TRM cells have both “exhausted-like” phenotypes and memory features, and provide heterologous immunity against secondary infection. Using recombinant influenza virus, we demonstrate that the development of these PD-1hi “exhausted-like” TRM cells is dependent on the doses of the antigen during primary infection. Furthermore, we showed that lung CD11c+ antigen presenting cells are required for sustaining these TRM cells at the memory stage. Our data suggest that continuous antigen presentation in situ is required for the development and maintenance of these “exhausted-like” TRM cells in the lung. Strikingly, PD-L1 blockade after the resolution of primary infection promotes the rejuvenation of these “exhausted-like” TRM cells, restoring protective immunity at the cost of promoting post-infection inflammatory and fibrotic sequelae. Thus, PD-1 serves to limit the pathogenic capacity of “exhausted-like” TRM cells at the memory phase under specific tissue environment with chronic low-levels of antigen presentation following viral clearance. Our data indicate that TRM cell “exhaustion” is the result of a tissue-specific cellular adaptation that balances fibrotic sequelae with protective immunity.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited survival. Janus kinases (JAKs), tyrosine kinases that transduce cytokine-mediated signals, are known to be involved, but their specific roles in lung fibrosis are not well defined. In this study, the interactions between JAK1/signal transducers and activators of transcription (STAT)3 signaling and transforming growth factor-beta (TGF-β)-induced fibroblast responses were investigated using both pharmacological and siRNA approaches in human normal and IPF-derived lung fibroblasts. We found that JAK1 directly interacts with the TGF-β receptor I subunit (TβRI), and silencing JAK1 promotes myofibroblast transdifferentiation. However, the suppression of JAK1 signaling in vitro and in vivo using an inhibitor (upadacitinib) did not alter lung fibroblast activation or fibrosis development. STAT3 was constitutively active in cultured primary lung fibroblasts; this STAT3 activation required JAK1 and repressed myofibroblast transdifferentiation. Loss of phosphorylated STAT3 following transcriptional JAK1 silencing promoted myofibroblast transdifferentiation. In contrast, transcriptional silencing of unphosphorylated STAT3 suppressed TGF-β signaling, decreased SMAD3 activation, and reduced myofibroblast transdifferentiation and ECM production. Taken together, these observations support a role for JAK1/STAT3 as a direct regulator of TGF-β signaling in lung fibroblasts. Modulation of JAK1/STAT3 signaling in lung fibroblasts represents a noncanonical approach to regulating TGF-β-induced fibrosis and suggests the potential for a novel approach to treat pulmonary fibrosis.

Abstract Idiopathic pulmonary fibrosis (IPF) and rheumatoid arthritis‐associated interstitial lung disease (RA‐ILD) are two fibrotic interstitial lung diseases that share the usual interstitial pneumonia (UIP) injury pattern. Here, we report that RNA sequencing of lung biopsies from patients with RA‐ILD and IPF revealed shared and distinct disease‐causing pathways. Analysis of transcriptomic data identified a JAK2 related JAK/STAT signaling pathway gene signature that distinguishes RA‐UIP from idiopathic UIP. This was further confirmed by immunohistostaining, which identified JAK2 phosphorylation with two distinct forms of activation: a cytoplasmic form of JAK2 activation in most IPF cases (13/20) and a nuclear form of p‐JAK2 in RA‐UIP (5/5) and a minority of IPF (6/20) cases. Further immunohistostaining identified STAT5A&B as the downstream transcriptional activator for JAK2‐mediated canonical signal transduction and phosphorylation of Tyr41 on histone H3 (H3Y41ph) as the downstream epigenetic regulation site for JAK2‐mediated noncanonical signal transduction. Gene Set Enrichment Analysis (GSEA) of the RNA‐Seq data further supported this shared pathogenic mechanism for the two diseases with the enrichment of STAT5A&B target gene sets as well as the JAK2 regulated H3Y41ph target gene set. This regulatory role of JAK2 in the pathogenesis of pulmonary fibrosis was further demonstrated by the attenuation of bleomycin‐induced murine pulmonary fibrosis using a JAK2‐selective pharmacological inhibitor CEP33779. In vitro studies with normal and IPF derived lung fibroblasts revealed a central role for JAK2 as an essential intermediary molecule in TGF‐β‐mediated myofibroblast trans‐differentiation, proliferation, and extracellular matrix protein production. These observations support a crucial role for JAK2 as an intermediary molecule in fibrotic lung disease development.

Epithelial-mesenchymal transition (EMT) creates an environment facilitating fibrosis following alveolar epithelial cell injury. IL-23 has important roles in chronic autoimmune conditions like rheumatoid arthritis (RA), but its role in the interstitial lung disease that affects patients with RA is unclear. This study aimed to determine the profibrogenic role of IL-23 on somatic alveolar type I (ATI) epithelial cells. Primary ATI cells were isolated from rats and cultured on plastic dishes for 1–3 wk. After prolonged culture (≥14 days) on rigid culture dishes, primary ATI cells gradually acquired a mesenchymal phenotype, identified by decreased expression of caveolin-1, and reorganization of F-actin cytoskeleton, indicating the initiation of EMT by matrix stiffness. To determine how IL-23 promotes EMT in vitro, transitioning ATI cells, cultured on a stiff substrate for ≥14 days were stimulated with IL-23. The EMT phenotype was significantly enhanced by IL-23, which upregulated α-smooth muscle actin (α-SMA), collagen I/III protein, and decreased caveolin-1. Furthermore, IL-23 significantly promoted cell invasion, as well as apoptotic resistance on transitioning ATI cells. Mechanistically, IL-23-induced EMT was mammalian target of rapamycin/ribosomal protein S6 (mTOR/S6) signaling dependent and reversible by rapamycin. Transcriptional sequencing analysis of human lung fibrosis biopsy tissue revealed key roles for IL-23 in rheumatoid arthritis-associated interstitial lung disease (RA-ILD). This result was further validated by significantly upregulated IL-23 expression at the mRNA level in RA-ILD lung sections. Notably, transitioning ATI epithelial cells were abundantly detected in RA-ILD tissue. Taken together, these data support a role for IL-23 in the pathogenesis of RA lung fibrosis by promoting EMT in alveolar epithelial cells through mTOR/S6 signaling.

Exhausted-like CD8 + resident memory T cells in the lung limit immune activation–driven fibrosis.

Interleukin (IL)-17 is a T helper 17 cytokine implicated in the pathogenesis of many autoimmune diseases, including rheumatoid arthritis (RA). Although IL-17A has a well-established role in murine pulmonary fibrosis models, its role in the tissue remodeling and fibrosis occurring in idiopathic pulmonary fibrosis (IPF) and RA-associated interstitial lung disease (RA-ILD) is not very well defined. To address this question, we utilized complimentary studies to determine responsiveness of human normal and pathogenic lung fibroblasts to IL-17A and used lung biopsies acquired from patients with IPF and RA-ILD to determine IL-17A receptor (IL-17RA) expression. Both normal and pathogenic IPF lung fibroblasts express functional IL-17RA and respond to IL-17A stimulation with cell proliferation, generation of extracellular matrix (ECM) proteins, and induction of myofibroblast transdifferentiation. Small interfering RNA (siRNA) silencing of IL-17RA attenuated this fibroblast response to IL-17A on ECM production. These fibroblast responses to IL-17A are dependent on NF-κB-mediated signaling. In addition, inhibiting Janus activated kinase (JAK) 2 by either siRNA or a selective pharmacological inhibitor, AZD1480—but not a JAK1/JAK3 selective inhibitor, tofacitinib—also significantly reduced this IL-17A-induced fibrogenic response. Lung biopsies of RA-ILD patients demonstrate significantly higher IL-17RA expression in areas of fibroblast accumulation and fibrosis, compared with either IPF or normal lung tissue. These observations support a direct role for IL-17A in lung fibrosis that may be particularly relevant in the context of RA-ILD.

ABSTRACT Myofibroblasts play key roles in wound healing and pathological fibrosis. Here, we used an RNAi screen to characterize myofibroblast regulatory genes, using a high-content imaging approach to quantify α-smooth muscle actin stress fibers in cultured human fibroblasts. Screen hits were validated on physiological compliance hydrogels, and selected hits tested in primary fibroblasts from patients with idiopathic pulmonary fibrosis. Our RNAi screen led to the identification of STAT3 as an essential mediator of myofibroblast activation and function. Strikingly, we found that STAT3 phosphorylation, while responsive to exogenous ligands on both soft and stiff matrices, is innately active on a stiff matrix in a ligand/receptor-independent, but ROCK- and JAK2-dependent fashion. These results demonstrate how a cytokine-inducible signal can become persistently activated by pathological matrix stiffening. Consistent with a pivotal role for this pathway in driving persistent fibrosis, a STAT3 inhibitor attenuated murine pulmonary fibrosis when administered in a therapeutic fashion after bleomycin injury. Our results identify novel genes essential for the myofibroblast phenotype, and point to STAT3 as an important target in pulmonary fibrosis and other fibrotic diseases.