Post-transcriptional regulation of IFI16 promotes inflammatory endothelial pathophenotypes observed in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease driven by endothelial cell inflammation and dysfunction, resulting in the pathological remodeling of the pulmonary vasculature. Innate immune activation has been linked to PAH development; however, the regulation, propagation, and reversibility of the induction of inflammation in PAH is poorly understood. Here, we demonstrate a role for interferon inducible protein 16 (IFI16), an innate immune sensor, as a modulator of endothelial inflammation in pulmonary hypertension, utilizing human pulmonary artery endothelial cells (PAECs). Inflammatory stimulus of PAECs with IL-1ß up-regulates IFI16 expression, inducing proinflammatory cytokine up-regulation and cellular apoptosis. IFI16 mRNA stability is regulated by post-transcriptional m6A modification, mediated by Wilms' tumor 1-associated protein (WTAP), a structural stabilizer of the methyltransferase complex, via regulation of m6A methylation of IFI16. Additionally, m6A levels are increased in the peripheral blood mononuclear cells of PAH patients compared to control, indicating that quantifying this epigenetic change in patients may hold potential as a biomarker for disease identification. In summary, our study demonstrates IFI16 mediates inflammatory endothelial pathophenotypes seen in pulmonary arterial hypertension.

Airway derived emphysema-specific alveolar type II cells exhibit impaired regenerative potential in COPD.

Emphysema, the progressive destruction of gas exchange surfaces in the lungs, is a hallmark of chronic obstructive pulmonary disease (COPD) that is presently incurable. This therapeutic gap is largely due to a poor understanding of potential drivers of impaired tissue regeneration, such as abnormal lung epithelial progenitor cells, including alveolar type II (ATII) and airway club cells. We discovered an emphysema-specific sub-population of ATII cells located in enlarged distal alveolar sacs, termed asATII cells. Single cell RNA-seq and in situ localisation revealed that asATII cells co-express the alveolar marker surfactant protein C (SPC) and the club cell marker secretaglobin-3A2 (SCGB3A2). A similar ATII sub-population derived from club cells was also identified in mouse COPD models using lineage labeling. Human and mouse ATII sub-populations formed 80-90% fewer alveolar organoids than healthy controls, indicating reduced progenitor function. Targeting asATII cells or their progenitor club cells could reveal novel COPD treatment strategies.

IL-10 Is Critical for Regulation of Cytotoxic CD4+NKG7+ T Cells in Lung Allograft Rejection but Is Not Required for Allograft Acceptance.

Lung transplant remains the primary therapeutic option for patients with end-stage lung disease, but long-term survival rates remain suboptimal compared with other solid organ transplants. Acute cellular rejection (ACR) is a significant challenge in lung transplant recipients, with T cell-mediated mechanisms playing a major role. IL-10 is known for its immunoregulatory function, although its specific role in lung allograft rejection remains unclear. Using the mouse orthotopic lung transplant model, we investigated the role of IL-10 in regulating alloeffector T cell responses. Unexpectedly, we found that IL-10 was not required for early costimulation blockade-induced allograft acceptance. However, IL-10 deficiency or blockade resulted in increased CD4+ T cell numbers, proliferation, graft infiltration, and alloeffector responses. In the absence of IL-10, CD4+ T cell responses predominated over CD8 responses during ACR in contrast to wild-type mice. Type 1 immunity (IFN-γ) responses along with elevated CD4+NKG7+ and CD4+CD107a+ responses predominated during ACR, highlighting a critical regulatory role for IL-10 in modulating CD4+ T cell alloimmune responses. We further demonstrated increased colocalization of NKG7 and CD107a in CD4+ T cells from IL-10-deficient allografts, suggesting coordination in cytotoxic activity. Together, our findings highlight a critical role for IL-10 in regulation of cytotoxic CD4+NKG7+ T cells, an effector population that needs further investigation to elucidate their role in lung allograft rejection.

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator correctors VX-445 and VX-121.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BKCa, KCa1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BKCa activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BKCa in the lung yet may lead to adverse events through BKCa activation elsewhere.

Molecular Changes Implicate Angiogenesis and Arterial Remodeling in Systemic Sclerosis-Associated and Idiopathic Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a common complication of systemic sclerosis (SSc) and a leading cause of mortality among patients with this disease. PH can also occur as an idiopathic condition (idiopathic pulmonary arterial hypertension). Investigation of transcriptomic alterations in vascular populations is critical to elucidating cellular mechanisms underlying pathobiology of SSc-associated and idiopathic PH. METHODS: We analyzed single-cell RNA sequencing profiles of endothelial and perivascular mesenchymal populations from explanted lung tissue of patients with SSc-associated PH (n=16), idiopathic pulmonary arterial hypertension (n=3), and healthy controls (n=15). Findings were validated by immunofluorescence staining of explanted human lung tissue. RESULTS: Three disease-associated endothelial populations emerged. Two angiogenic endothelial cell (EC) subtypes markedly expanded in SSc-associated PH lungs: tip ECs expressing canonical tip markers PGF and APLN and phalanx ECs expressing genes associated with vascular development, endothelial barrier integrity, and Notch signaling. Gene regulatory network analysis suggested enrichment of Smad1 (SMAD family member 1) and PPAR-γ (peroxisome proliferator-activated receptor-γ) regulon activities in these 2 populations, respectively. Mapping of potential ligand-receptor interactions highlighted Notch, apelin-APJ (apelin receptor), and angiopoietin-Tie (tyrosine kinase with immunoglobulin-like and EGF-like domains 1) signaling pathways between angiogenic ECs and perivascular cells. Transitional cells, expressing both endothelial and pericyte/smooth muscle cell markers, provided evidence for the presence of endothelial-to-mesenchymal transition. Transcriptional programs associated with arterial endothelial dysfunction implicated VEGF-A (vascular endothelial growth factor-A), TGF-ß1 (transforming growth factor beta-1), angiotensin, and TNFSF12 (tumor necrosis factor ligand superfamily member 12)/TWEAK (TNF-related weak inducer of apoptosis) in the injury/remodeling phenotype of PH arterial ECs. CONCLUSIONS: These data provide high-resolution insights into the complexity and plasticity of the pulmonary endothelium in SSc-associated PH and idiopathic pulmonary arterial hypertension and provide direct molecular insights into soluble mediators and transcription factors driving PH vasculopathy.

GCN5L1-mediated acetylation prevents Rictor degradation in cardiac cells after hypoxic stress.

Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia-reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.

Increased expression of CXCL6 in secretory cells drives fibroblast collagen synthesis and is associated with increased mortality in idiopathic pulmonary fibrosis.

RATIONALE: Recent data suggest that the localisation of airway epithelial cells in the distal lung in idiopathic pulmonary fibrosis (IPF) may drive pathology. We set out to discover whether chemokines expressed in these ectopic airway epithelial cells may contribute to the pathogenesis of IPF. METHODS: We analysed whole lung and single-cell transcriptomic data obtained from patients with IPF. In addition, we measured chemokine levels in blood, bronchoalveolar lavage (BAL) of IPF patients and air-liquid interface cultures. We employed ex vivo donor and IPF lung fibroblasts and an animal model of pulmonary fibrosis to test the effects of chemokine signalling on fibroblast function. RESULTS: By analysis of whole-lung transcriptomics, protein and BAL, we discovered that CXCL6 (a member of the interleukin-8 family) was increased in patients with IPF. Elevated CXCL6 levels in the BAL of two cohorts of patients with IPF were associated with poor survival (hazard ratio of death or progression 1.89, 95% CI 1.16-3.08; n=179, p=0.01). By immunostaining and single-cell RNA sequencing, CXCL6 was detected in secretory cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased collagen I levels in donor and IPF fibroblasts 4.4-fold and 1.7-fold, respectively. Both silencing of and chemical inhibition of CXCR1/2 blocked the effects of CXCL6 on collagen, while overexpression of CXCR2 increased collagen I levels 4.5-fold in IPF fibroblasts. CONCLUSIONS: CXCL6 is expressed in ectopic airway epithelial cells. Elevated levels of CXCL6 are associated with IPF mortality. CXCL6-driven collagen synthesis represents a functional consequence of ectopic localisation of airway epithelial cells in IPF.

Increased CD8+ tissue resident memory T cells, regulatory T cells and activated natural killer cells in systemic sclerosis lungs.

OBJECTIVE: Multiple observations indicate a role for lymphocytes in driving autoimmunity in SSc. While T and NK cells have been studied in SSc whole blood and bronchoalveolar lavage fluid, their role remains unclear, partly because no studies have analysed these cell types in SSc-interstitial lung disease (ILD) lung tissue. This research aimed to identify and analyse the lymphoid subpopulations in SSc-ILD lung explants. METHODS: Lymphoid populations from 13 SSc-ILD and 6 healthy control (HC) lung explants were analysed using Seurat following single-cell RNA sequencing. Lymphoid clusters were identified by their differential gene expression. Absolute cell numbers and cell proportions in each cluster were compared between cohorts. Additional analyses were performed using pathway analysis, pseudotime and cell ligand-receptor interactions. RESULTS: Activated CD16+ NK cells, CD8+ tissue resident memory T cells and Treg cells were proportionately higher in SSc-ILD compared with HC lungs. Activated CD16+ NK cells in SSc-ILD showed upregulated granzyme B, IFN-γ and CD226. Amphiregulin, highly upregulated by NK cells, was predicted to interact with epidermal growth factor receptor on several bronchial epithelial cell populations. Shifts in CD8+ T cell populations indicated a transition from resting to effector to tissue resident phenotypes in SSc-ILD. CONCLUSIONS: SSc-ILD lungs show activated lymphoid populations. Activated cytotoxic NK cells suggest they may kill alveolar epithelial cells, while their expression of amphiregulin suggests they may also induce bronchial epithelial cell hyperplasia. CD8+ T cells in SSc-ILD appear to transition from resting to the tissue resident memory phenotype.

GCN5L1-mediated acetylation prevents Rictor degradation in cardiac cells after hypoxic stress.

Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia-reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.

End-Stage Idiopathic Pulmonary Fibrosis Lung Microenvironment Promotes Impaired NK Activity.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic age-related chronic lung disease characterized by the accumulation of senescent cells. Whether impaired immune response is responsible for the accumulation of senescent cells in the IPF lung remains unknown. In this study, we characterized the NK phenotype in IPF lungs via flow cytometry using 5-dodecanoylaminofluorescein di-ß-d-galactopyranoside, markers of tissue residence, and chemokine receptors. The effect of the lung microenvironment was evaluated using lung fibroblast (LF) conditioned media (CM), and the bleomycin-induced pulmonary fibrosis mouse model was used to assess the in vivo relationship between NK cells and the accumulation of senescent cells. We found that NK cells from the lower lobe of IPF patients exhibited immune-senescent and impaired CD57-NKG2A+ phenotype. We also observed that culture of NK cells from healthy donors in CM from IPF lower lobe lung fibroblasts induced a senescent-like phenotype and impaired cytotoxic capacity. There is an impaired NK recruitment by LF, and NKs presented decreased migration toward their CM. In addition, NK cell-depleted mice treated with bleomycin showed increased collagen deposition and accumulation of different populations of senescent cells compared with controls. The IPF lung microenvironment induces a dysfunctional NK phenotype limiting the clearance of lung senescent cells and the resolution of lung fibrosis. We propose that impaired NK activity could be one of the mechanisms responsible for perpetuating the accumulation of senescent cells in IPF lungs.

Impact of enzymatic digestion on single cell suspension yield from peripheral human lung tissue.

An increasing number of translational investigations of lung biology rely on analyzing single cell suspensions obtained from human lungs. To obtain these single cell suspensions, human lungs from biopsies or research-consented organ donors must be subjected to mechanical and enzymatic digestion prior to analysis with either flow cytometry or single cell RNA sequencing. A variety of enzymes have been used to perform tissue digestion, each with potential limitations. To better understand the limitations of each enzymatic digestion protocol and to establish a framework for comparing studies across protocols, we performed five commonly published protocols in parallel from identical samples obtained from 6 human lungs. Following mechanical (gentleMACS™) and enzymatic digestion, we quantified cell count and viability using a Nexcelom Cellometer and determined cell phenotype using multiparameter spectral flow cytometry (Cytek™ Aurora). We found that all protocols were superior in cellular yield and viability when compared to mechanical digestion alone. Protocols high in dispase cleaved immune markers CD4, CD8, CD69, and CD103 and contributed to an increased monocyte to macrophage yield. Similarly, dispase led to a differential epithelial cell yield, with increased TSPN8+ and ITGA6+ epithelial cells and reduced CD66e+ cells. When compared to collagenase D, collagenase P protocols yielded increased AT1 and AT2 cells and decreased endothelial cells. These results provide a framework for selecting an enzymatic digestion protocol best suited to the scientific question and allow for comparison of studies using different protocols.

Single-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity.

BACKGROUND: In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. METHODS: We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells. RESULTS: TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log2 fold change (FC) 8.909, adjusted p-value 1.82×10-35) and control fibroblasts (log2FC 8.975, adjusted p-value 3.72×10-28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log2FC 3.136, adjusted p-value 1.41×10- 24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts. CONCLUSIONS: Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

CYB5R3 in type II alveolar epithelial cells protects against lung fibrosis by suppressing TGF-ß1 signaling.

Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a deadly disease with limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECIIs. Deficiency of CYB5R3 in AECIIs led to sustained activation of the pro-fibrotic factor TGF-ß1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and the sGC/cGMP/protein kinase G axis that modulates activation of the TGF-ß1 signaling pathway. We demonstrate that sGC agonists (BAY 41-8543 and BAY 54-6544) are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECIIs. Taken together, these results show that CYB5R3 in AECIIs is required to maintain resilience after lung injury and fibrosis and that therapeutic manipulation of the sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.

Cigarette smoking is a secondary cause of folliculin loss.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is a clinical syndrome manifesting with cystic lung disease and pneumothorax. Features of BHD result from the loss-of-function mutations of the folliculin (FLCN) gene. Chronic obstructive pulmonary disease (COPD), characterised by an irreversible airflow limitation, is primarily caused by cigarette smoking. OBJECTIVE: Given that COPD often shares structural features with BHD, we investigated the link between COPD, cigarette smoke (CS) exposure and FLCN expression. METHODS: We measured the expression of FLCN in human COPD lungs and CS-exposed mouse lungs, as well as in CS extract (CSE)-exposed immortalised human airway epithelial cells by immunoblotting. RESULTS: We found that the lung FLCN protein levels in smokers with COPD and CS exposure mice exhibit a marked decrease compared with smokers without COPD and room air exposure mice, respectively. We confirmed CS induced degradation of FLCN in immortalised human bronchial epithelial Beas-2B cells via ubiquitin proteasome system. Further, siRNA targeting FLCN enhanced CSE-induced cytotoxicity. By contrast, FLCN overexpression protected cells from CSE-induced cytotoxicity. We found that FBXO23, the ubiquitin E3 ligase subunit, specifically binds to and targets FLCN for degradation. Inhibition of ATM (ataxia-telangiectasia mutated) attenuated CSE induced FLCN degradation, suggesting a role of ATM in FLCN proteolysis. We further confirmed that the mutant of major FLCN phosphorylation site serine 62A is resistant to CSE-induced degradation and cytotoxicity. CONCLUSIONS: Our study demonstrates that CS exposure is a secondary cause of FLCN deficiency due to the enhanced proteolysis, which promoted airway epithelial cell death.

Unagi: Deep Generative Model for Deciphering Cellular Dynamics and In-Silico Drug Discovery in Complex Diseases.

Human diseases are characterized by intricate cellular dynamics. Single-cell sequencing provides critical insights, yet a persistent gap remains in computational tools for detailed disease progression analysis and targeted in-silico drug interventions. Here, we introduce UNAGI, a deep generative neural network tailored to analyze time-series single-cell transcriptomic data. This tool captures the complex cellular dynamics underlying disease progression, enhancing drug perturbation modeling and discovery. When applied to a dataset from patients with Idiopathic Pulmonary Fibrosis (IPF), UNAGI learns disease-informed cell embeddings that sharpen our understanding of disease progression, leading to the identification of potential therapeutic drug candidates. Validation via proteomics reveals the accuracy of UNAGI's cellular dynamics analyses, and the use of the Fibrotic Cocktail treated human Precision-cut Lung Slices confirms UNAGI's predictions that Nifedipine, an antihypertensive drug, may have antifibrotic effects on human tissues. UNAGI's versatility extends to other diseases, including a COVID dataset, demonstrating adaptability and confirming its broader applicability in decoding complex cellular dynamics beyond IPF, amplifying its utility in the quest for therapeutic solutions across diverse pathological landscapes.

Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c → C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.

Ewing Sarcoma and Osteosarcoma Have Distinct Immune Signatures and Intercellular Communication Networks.

PURPOSE: Ewing sarcoma and osteosarcoma are primary bone sarcomas occurring most commonly in adolescents. Metastatic and relapsed disease are associated with dismal prognosis. Although effective for some soft tissue sarcomas, current immunotherapeutic approaches for the treatment of bone sarcomas have been largely ineffective, necessitating a deeper understanding of bone sarcoma immunobiology. EXPERIMENTAL DESIGN: Multiplex immunofluorescence analysis of immune infiltration in relapsed versus primary disease was conducted. To better understand immune states and drivers of immune infiltration, especially during disease progression, we performed single-cell RNA sequencing (scRNAseq) of immune populations from paired blood and bone sarcoma tumor samples. RESULTS: Our multiplex immunofluorescence analysis revealed increased immune infiltration in relapsed versus primary disease in both Ewing sarcoma and osteosarcoma. scRNAseq analyses revealed terminally exhausted CD8+ T cells expressing co-inhibitory receptors in osteosarcoma and an effector T-cell subpopulation in Ewing sarcoma. In addition, distinct subsets of CD14+CD16+ macrophages were present in Ewing sarcoma and osteosarcoma. To determine pathways driving tumor immune infiltration, we conducted intercellular communication analyses and uncovered shared mechanisms of immune infiltration driven by CD14+CD16+ macrophages and unique pathways of immune infiltration driven by CXCL10 and CXCL12 in osteosarcoma. CONCLUSIONS: Our study provides preclinical rationale for future investigation of specific immunotherapeutic targets upon relapse and provides an invaluable resource of immunologic data from bone sarcomas.

Fibronectin-EDA accumulates via reduced ubiquitination downstream of Toll-like receptor 9 activation in SSc-ILD fibroblasts.

Accumulation of excessive extracellular matrix (ECM) components from lung fibroblasts is a feature of systemic sclerosis-associated interstitial lung disease (SSc-ILD), and there is increasing evidence that innate immune signaling pathways contribute to these processes. Toll-like receptors (TLRs) are innate immune sensors activated by danger signals derived from pathogens or host molecular patterns. Several damage-associated molecular pattern (DAMP) molecules are elevated in SSc-ILD plasma, including ligands that activate TLR9, an innate immune sensor recently implicated in driving profibrotic responses in fibroblasts. Fibronectin and the isoform fibronectin-extra domain A (FN-EDA) are prominent in pathological extracellular matrix accumulation, but mechanisms promoting FN-EDA accumulation are only partially understood. Here, we show that TLR9 activation increases FN-EDA accumulation in MRC5 and SSc-ILD fibroblasts, but that this effect is independent of changes in FN-EDA gene transcription. Rather, we describe a novel mechanism where TLR9 activation inhibits FN-EDA turnover via reduced FN-EDA ubiquitination. TLR9 ligand ODN2006 reduces ubiquitinated FN-EDA destined for lysosomal degradation, an effect abrogated with TLR9 knockdown or inhibition. Taken together, these results provide rationale for disrupting the TLR9 signaling axis or FN-EDA degradation pathways to reduce FN-EDA accumulation in SSc-ILD fibroblasts. More broadly, enhancing intracellular degradation of ECM components through TLR9 inhibition or enhanced ECM turnover could be a novel strategy to attenuate pathogenic ECM accumulation in SSc-ILD.

Epigenetic Regulation of Profibrotic Macrophages in Systemic Sclerosis-Associated Interstitial Lung Disease.

OBJECTIVE: Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is the leading cause of death in patients with SSc with unclear pathogenesis and limited treatment options. Evidence strongly supports an important role for profibrotic secreted phosphoprotein 1 (SPP1)-expressing macrophages in SSc-ILD. This study was undertaken to define the transcriptome and chromatin structural changes of SPP1 SSc-ILD macrophages in order to better understand their role in promoting fibrosis and to identify transcription factors associated with open chromatin driving their altered phenotype. METHODS: We performed single-cell RNA sequencing (scRNA-Seq) on 11 explanted SSc-ILD and healthy control lung samples, as well as single-cell assay for transposase-accessible chromatin sequencing on 5 lung samples to define altered chromatin accessibility of SPP1 macrophages. We predicted transcription factors regulating SPP1 macrophages using single-cell regulatory network inference and clustering (SCENIC) and determined transcription factor binding sites associated with global alterations in SPP1 chromatin accessibility using Signac/Seurat. RESULTS: We identified distinct macrophage subpopulations using scRNA-Seq analysis in healthy and SSc-ILD lungs and assessed gene expression changes during the change of healthy control macrophages into SPP1 macrophages. Analysis of open chromatin validated SCENIC predictions, indicating that microphthalmia-associated transcription factor, transcription factor EB, activating transcription factor 6, sterol regulatory element binding transcription factor 1, basic helix-loop-helix family member E40, Kruppel-like factor 6, ETS variant transcription factor 5, and/or members of the activator protein 1 family of transcription factors regulate SPP1 macrophage differentiation. CONCLUSION: Our findings shed light on the underlying changes in chromatin structure and transcription factor regulation of profibrotic SPP1 macrophages in SSc-ILD. Similar alterations in SPP1 macrophages may underpin fibrosis in other organs involved in SSc and point to novel targets for the treatment of SSc-ILD, specifically targeting profibrotic macrophages.