Adenine nucleotide translocase regulates airway epithelial metabolism, surface hydration and ciliary function.

Airway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive pulmonary disease (COPD), which is impacted by cigarette smoking and has no therapeutic options. We utilized a high-copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors to cigarette smoke. Members of the mitochondrial ADP/ATP transporter family adenine nucleotide translocase (ANT) are protective against cigarette smoke in Dictyostelium and human bronchial epithelial cells. Gene expression of ANT2 is reduced in lung tissue from COPD patients and in a mouse smoking model, and overexpression of ANT1 and ANT2 resulted in enhanced oxidative respiration and ATP flux. In addition to the presence of ANT proteins in the mitochondria, they reside at the plasma membrane in airway epithelial cells and regulate airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after exposure to cigarette smoke, both of which are key functions of the airway. Our study highlights a potential for upregulation of ANT proteins and/or of their agonists in the protection from dysfunctional mitochondrial metabolism, airway hydration and ciliary motility in COPD.This article has an associated First Person interview with the first author of the paper.

Alveolar Macrophages Inherently Express Programmed Death-1 Ligand 1 for Optimal Protective Immunity and Tolerance.

Macrophages play a central role in lung physiology and pathology. In this study, we show in mice that alveolar macrophages (AMs), unlike other macrophage types (interstitial, peritoneal, and splenic macrophages), constitutively express programmed death-1 ligand 1 (PD-L1), thereby possessing a superior phagocytic ability and the capacity to repress CTLs by cis- and trans-interacting with CD80 and programmed death-1 (PD-1), respectively. This extraordinary ability of AMs assures optimal protective immunity and tolerance within the lung. These findings uncover a unique characteristic of AMs and an innate immune function of PD-L1 and CD80 and therefore help in the understanding of lung physiology, diseases, and PD-L1/PD-1-based immunotherapy.

Toll-Interacting Protein in Pulmonary Diseases. Abiding by the Goldilocks Principle.

TOLLIP (Toll-interacting protein) is an intracellular adaptor protein with diverse actions throughout the body. In a context- and cell type-specific manner, TOLLIP can function as an inhibitor of inflammation and endoplasmic-reticulum stress, an activator of autophagy, or a critical regulator of intracellular vacuole trafficking. The distinct functions of this protein have been linked to innate immune responses and lung epithelial-cell apoptosis. TOLLIP genetic variants have been associated with a variety of chronic lung diseases, including idiopathic pulmonary fibrosis, asthma, and primary graft dysfunction after lung transplantation, and with infections, such as tuberculosis, Legionella pneumonia, and respiratory viruses. TOLLIP exists in a delicate homeostatic balance, with both positive and negative effects on the trajectory of pulmonary diseases. This translational review summarizes the genetic and molecular associations that link TOLLIP to the development and progression of noninfectious and infectious pulmonary diseases. We highlight current limitations of in vitro and in vivo models in assessing the role of TOLLIP in these conditions, and we describe future approaches that will enable a more nuanced exploration of the role of TOLLIP in pulmonary conditions. There has been a surge in recent research evaluating the role of this protein in human diseases, but critical mechanistic pathways require further exploration. By understanding its biologic functions in disease-specific contexts, we will be able to determine whether TOLLIP can be therapeutically modulated to treat pulmonary diseases.

Single cell RNA sequencing identifies IGFBP5 and QKI as ciliated epithelial cell genes associated with severe COPD.

BACKGROUND: Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. METHODS: To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. RESULTS: T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. CONCLUSIONS: scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

FSTL-1 Attenuation Causes Spontaneous Smoke-Resistant Pulmonary Emphysema.

Rationale: The role of FSTL-1 (follistatin-like 1) in lung homeostasis is unknown.Objectives: We aimed to define the impact of FSTL-1 attenuation on lung structure and function and to identify FSTL-1-regulated transcriptional pathways in the lung. Further, we aimed to analyze the association of FSTL-1 SNPs with lung disease.Methods: FSTL-1 hypomorphic (FSTL-1 Hypo) mice underwent lung morphometry, pulmonary function testing, and micro-computed tomography. Fstl1 expression was determined in wild-type lung cell populations from three independent research groups. RNA sequencing of wild-type and FSTL-1 Hypo mice identified FSTL-1-regulated gene expression, followed by validation and mechanistic in vitro examination. FSTL1 SNP analysis was performed in the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort.Measurements and Main Results: FSTL-1 Hypo mice developed spontaneous emphysema, independent of smoke exposure. Fstl1 is highly expressed in the lung by mesenchymal and endothelial cells but not immune cells. RNA sequencing of whole lung identified 33 FSTL-1-regulated genes, including Nr4a1, an orphan nuclear hormone receptor that negatively regulates NF-κB (nuclear factor-κB) signaling. In vitro, recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an Nr4a1-dependent manner. Within the COPDGene cohort, several SNPs in the FSTL1 region corresponded to chronic obstructive pulmonary disease and lung function.Conclusions: This work identifies a novel role for FSTL-1 protecting against emphysema development independent of smoke exposure. This FSTL-1-deficient emphysema implicates regulation of immune tolerance in lung macrophages through Nr4a1. Further study of the mechanisms involving FSTL-1 in lung homeostasis, immune regulation, and NF-κB signaling may provide additional insight into the pathophysiology of emphysema and inflammatory lung diseases.

Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain.

RATIONALE: Macrophage elastase (matrix metalloproteinase [MMP]-12) is a potent protease that contributes to the lung destruction that accompanies cigarette smoking; it simultaneously inhibits lung tumor angiogenesis and metastasis by catalyzing the formation of antiangiogenic peptides. Recent studies have revealed novel nonproteolytic functions of MMP12, including antimicrobial activity through a peptide within its C-terminal domain (CTD). OBJECTIVES: To determine whether the MMP12 CTD contributes to its antitumor activity in lung cancer. METHODS: We used recombinant MMP12 peptide fragments, including its catalytic domain, CTD, and a 20 amino acid peptide within the CTD (SR20), in an in vitro system to delineate their effects on non-small cell lung cancer cell proliferation and apoptosis. We translated our findings to two murine models of lung cancer, including orthotopic human xenograft and KrasLSL/G12D mouse models of lung cancer. MEASUREMENTS AND MAIN RESULTS: We show that SR20 triggers tumor apoptosis by up-regulation of gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4, sensitizing cells to an autocrine loop of TRAIL-mediated cell death. We then demonstrate the therapeutic efficacy of SR20 against two murine models of lung cancer. CONCLUSIONS: The MMP12 CTD initiates TRAIL-mediated tumor cell death through its conserved SR20 peptide.

Extreme Trait Whole-Genome Sequencing Identifies PTPRO as a Novel Candidate Gene in Emphysema with Severe Airflow Obstruction.

RATIONALE: Genetic association studies in chronic obstructive pulmonary disease have primarily tested for association with common variants, the results of which explain only a portion of disease heritability. Because rare variation is also likely to contribute to susceptibility, we used whole-genome sequencing of subjects with clinically extreme phenotypes to identify genomic regions enriched for rare variation contributing to chronic obstructive pulmonary disease susceptibility. OBJECTIVES: To identify regions of rare genetic variation contributing to emphysema with severe airflow obstruction. METHODS: We identified heavy smokers that were resistant (n = 65) or susceptible (n = 64) to emphysema with severe airflow obstruction in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We filtered whole-genome sequencing results to include only rare variants and conducted single variant tests, region-based tests across the genome, gene-based tests, and exome-wide tests. MEASUREMENTS AND MAIN RESULTS: We identified several suggestive associations with emphysema with severe airflow obstruction, including a suggestive association of all rare variation in a region within the gene ZNF816 (19q13.41; P = 4.5 × 10-6), and a suggestive association of nonsynonymous coding rare variation in the gene PTPRO (P = 4.0 × 10-5). Association of rs61754411, a rare nonsynonymous variant in PTPRO, with emphysema and obstruction was demonstrated in all non-Hispanic white individuals in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We found that cells containing this variant have decreased signaling in cellular pathways necessary for survival and proliferation. CONCLUSIONS: PTPRO is a novel candidate gene in emphysema with severe airflow obstruction, and rs61754411 is a previously unreported rare variant contributing to emphysema susceptibility. Other suggestive candidate genes, such as ZNF816, are of interest for future studies.

Variable Susceptibility to Cigarette Smoke-Induced Emphysema in 34 Inbred Strains of Mice Implicates Abi3bp in Emphysema Susceptibility.

Chronic obstructive pulmonary disease (COPD) is caused by a complex interaction of environmental exposures, most commonly cigarette smoke, and genetic factors. Chronic cigarette smoke exposure in the mouse is a commonly used animal model of COPD. We aimed to expand our knowledge about the variable susceptibility of inbred strains to this model and test for genetic variants associated with this trait. To that end, we sought to measure differential susceptibility to cigarette smoke-induced emphysema in the mouse, identify genetic loci associated with this quantitative trait, and find homologous human genes associated with COPD. Alveolar chord length (CL) in 34 inbred strains of mice was measured after 6 months of exposure to cigarette smoke. After testing for association, we connected a murine candidate locus to a published meta-analysis of moderate-to-severe COPD. We identified deleterious mutations in a candidate gene in silico and measured gene expression in extreme strains. A/J was the most susceptible strain in our survey (Δ CL 7.0 ± 2.2 µm) and CBA/J was the least susceptible (Δ CL -0.3 ± 1.2 µm). By integrating mouse and human genome-wide scans, we identified the candidate gene Abi3bp. CBA/J mice harbor predicted deleterious variants in Abi3bp, and expression of the gene differs significantly between CBA/J and A/J mice. This is the first report of susceptibility to cigarette smoke-induced emphysema in 34 inbred strains of mice, and Abi3bp is identified as a potential contributor to this phenotype.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor.

Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high-fat diet (HFD) in some inbred mouse strains. We conducted a genome-wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD-induced PH. Mice from 36 inbred and wild-derived strains were fed with regular diet or HFD for 20 weeks beginning at 6-12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end-systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest-path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH-related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high-fat feeding. Network-based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest-path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high-fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network-based approach adds evidence for a role for Egfr in murine PH.

Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination.

BACKGROUND: Cigarette smoke suppresses innate immunity, making smokers more susceptible to infection. The NLRP3 inflammasome is a multi-protein complex that releases interleukin (IL) -1ß and IL -18. These cytokines are critical for a timely host response to pathogens. Whether cigarette smoke affects NLRP3 protein levels, and its ability to form an inflammasome, is not known. METHODS AND RESULTS: Using the human monocyte THP1 cell line and C57BL/6 mice, we show that cigarette smoke decreases NLRP3 levels in cells by increasing ubiquitin-mediated proteasomal processing. Half-life of NLRP3 is shortened with the exposure to cigarette smoke extract. Cigarette smoke extract reduces cellular NLRP3 protein abundance in the presence of lipopolysaccharide, a known inducer of NLRP3 protein, thereby decreasing the formation of NLRP3 inflammasomes. The release of IL-1ß and IL-18 by inflammasome activation is also decreased with the exposure to cigarette smoke extract both in THP1 cells and primary human peripheral blood macrophages. CONCLUSIONS: Cigarette smoke extract decreased NLRP3 protein abundance via increased ubiquitin-mediated proteasomal processing. The release of IL-1ß and IL-18 is also decreased with cigarette smoke extract. Our findings may provide mechanistic insights on immunosuppression in smokers and unique opportunities to develop a strategy to modulate immune function.

Effects of intra-abdominal sepsis on atherosclerosis in mice.

INTRODUCTION: Sepsis and other infections are associated with late cardiovascular events. Although persistent inflammation is implicated, a causal relationship has not been established. We tested whether sepsis causes vascular inflammation and accelerates atherosclerosis. METHODS: We performed prospective, randomized animal studies at a university research laboratory involving adult male ApoE-deficient (ApoE-/-) and young C57B/L6 wild-type (WT) mice. In the primary study conducted to determine whether sepsis accelerates atherosclerosis, we fed ApoE-/- mice (N = 46) an atherogenic diet for 4 months and then performed cecal ligation and puncture (CLP), followed by antibiotic therapy and fluid resuscitation or a sham operation. We followed mice for up to an additional 5 months and assessed atheroma in the descending aorta and root of the aorta. We also exposed 32 young WT mice to CLP or sham operation and followed them for 5 days to determine the effects of sepsis on vascular inflammation. RESULTS: ApoE-/- mice that underwent CLP had reduced activity during the first 14 days (38% reduction compared to sham; P < 0.001) and sustained weight loss compared to the sham-operated mice (-6% versus +9% change in weight after CLP or sham surgery to 5 months; P < 0.001). Despite their weight loss, CLP mice had increased atheroma (46% by 3 months and 41% increase in aortic surface area by 5 months; P = 0.03 and P = 0.004, respectively) with increased macrophage infiltration into atheroma as assessed by immunofluorescence microscopy (0.52 relative fluorescence units (rfu) versus 0.97 rfu; P = 0.04). At 5 months, peritoneal cultures were negative; however, CLP mice had elevated serum levels of interleukin 6 (IL-6) and IL-10 (each at P < 0.05). WT mice that underwent CLP had increased expression of intercellular adhesion molecule 1 in the aortic lumen versus sham at 24 hours (P = 0.01) that persisted at 120 hours (P = 0.006). Inflammatory and adhesion genes (tumor necrosis factor α, chemokine (C-C motif) ligand 2 and vascular cell adhesion molecule 1) and the adhesion assay, a functional measure of endothelial activation, were elevated at 72 hours and 120 hours in mice that underwent CLP versus sham-operations (all at P <0.05). CONCLUSIONS: Using a combination of existing murine models for atherosclerosis and sepsis, we found that CLP, a model of intra-abdominal sepsis, accelerates atheroma development. Accelerated atheroma burden was associated with prolonged systemic, endothelial and intimal inflammation and was not explained by ongoing infection. These findings support observations in humans and demonstrate the feasibility of a long-term follow-up murine model of sepsis.

Interpretable machine learning uncovers epithelial transcriptional rewiring and a role for Gelsolin in COPD.

Transcriptomic analyses have advanced the understanding of complex disease pathophysiology including chronic obstructive pulmonary disease (COPD). However, identifying relevant biologic causative factors has been limited by the integration of high dimensionality data. COPD is characterized by lung destruction and inflammation with smoke exposure being a major risk factor. To define novel biological mechanisms in COPD, we utilized unsupervised and supervised interpretable machine learning analyses of single cell-RNA sequencing data from the gold standard mouse smoke exposure model to identify significant latent factors (context-specific co-expression modules) impacting pathophysiology. The machine learning transcriptomic signatures coupled to protein networks uncovered a reduction in network complexity and novel biological alterations in actin-associated gelsolin (GSN), which was transcriptionally linked to disease state. GSN was altered in airway epithelial cells in the mouse model and in human COPD. GSN was increased in plasma from COPD patients, and smoke exposure resulted in enhanced GSN release from airway cells from COPD patients. This method provides insights into rewiring of transcriptional networks that are associated with COPD pathogenesis and provide a novel analytical platform for other diseases.

Clathrin pit-mediated endocytosis of neutrophil elastase and cathepsin G by cancer cells.

Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with broad substrate specificity. We have recently demonstrated that NE is capable of entering tumor cell endosomes and processing novel intracellular substrates. In the current study, we sought to determine the mechanism by which NE enters tumor cells. Our results show that NE enters into early endosomal antigen-1(+) endosomes in a dynamin- and clathrin-dependent but flotillin-1- and caveolin-1-independent fashion. Cathepsin G (but not proteinase-3) also enters tumor endosomes via the same mechanism. We utilized (125)I-labeled NE to demonstrate that NE binds to the surface of cancer cells. Incubation of radiolabeled NE with lung cancer cells displays a dissociation constant (K(d)) of 284 nm. Because NE is known to bind to heparan sulfate- and chondroitin sulfate-containing proteoglycans, we treated cells with glycanases to remove these confounding factors, which did not significantly diminish cell surface binding or endosomal entry. Thus, NE and CG bind to the surface of cancer cells, presumably to a cell surface receptor, and subsequently undergo clathrin pit-mediated endocytosis.

Extracellular Release of Mitochondrial DNA: Triggered by Cigarette Smoke and Detected in COPD.

Cigarette smoke (CS) is the most common risk factor for chronic obstructive pulmonary disease (COPD). The present study aimed to elucidate whether mtDNA is released upon CS exposure and is detected in the plasma of former smokers affected by COPD as a possible consequence of airway damage. We measured cell-free mtDNA (cf-mtDNA) and nuclear DNA (cf-nDNA) in COPD patient plasma and mouse serum with CS-induced emphysema. The plasma of patients with COPD and serum of mice with CS-induced emphysema showed increased cf-mtDNA levels. In cell culture, exposure to a sublethal dose of CSE decreased mitochondrial membrane potential, increased oxidative stress, dysregulated mitochondrial dynamics, and triggered mtDNA release in extracellular vesicles (EVs). Mitochondrial DNA release into EVs occurred concomitantly with increased expression of markers that associate with DNA damage responses, including DNase III, DNA-sensing receptors (cGAS and NLRP3), proinflammatory cytokines (IL-1ß, IL-6, IL-8, IL-18, and CXCL2), and markers of senescence (p16 and p21); the majority of the responses are also triggered by cytosolic DNA delivery in vitro. Exposure to a lethal CSE dose preferentially induced mtDNA and nDNA release in the cell debris. Collectively, the results of this study associate markers of mitochondrial stress, inflammation, and senescence with mtDNA release induced by CSE exposure. Because high cf-mtDNA is detected in the plasma of COPD patients and serum of mice with emphysema, our findings support the future study of cf-mtDNA as a marker of mitochondrial stress in response to CS exposure and COPD pathology.

USP13 Deficiency Aggravates Cigarette-smoke-induced Alveolar Space Enlargement.

Alveolar enlargement is a pathological feature of emphysema. Long-term exposure to cigarette smoke (CS) is a high-risk factor for the development of emphysema. Abnormal protein ubiquitination has been implicated to regulate the development of human disorders, however, the role of protein ubiquitination in emphysema has not been well-studied. In this study, we attempted to investigate if a deubiquitinase, USP13, regulates the development of emphysema. Under a mild CS exposure condition, USP13-deficient mice show significant increases in alveolar chord length, indicating that USP13-deficient mice are susceptible to CS-induced alveolar enlargement. It has been shown that USP13 knockout reduced fibronectin expression in lungs. Here, we found that collagen levels were reduced in USP13 siRNA-transfected lung fibroblast cells. This suggests that a loss of extracellular matrix in connective tissues contributes to alveolar enlargement in USP13-deficient mice in response to CS exposure. Further, we investigated the role of USP13 in the expression of oxidative stress markers TXNIP and HMOX1. An increase in HMOX1 abundance was observed in USP13 knockdown lung fibroblast and epithelial cells. Overexpression of USP13 reduced HMOX1 protein levels in lung fibroblast cells, suggesting that modulation of USP13 levels may affect oxidative stress. Knockdown of USP13 significantly reduced TXNIP levels in lungs or lung fibroblast cells. A protein stability pulse-chase assay showed that TXNIP is instable within USP13 knockdown lung fibroblast cells. Further, the reduction of TXNIP was observed in USP13 inhibitor-treated lung epithelial cells. USP13-deficient mice also show higher levels of IgG in bronchoalveolar lavage fluid. This study provides evidence showing that USP13 deficiency plays a role in alveolar space enlargement.

PDLIM2 repression by ROS in alveolar macrophages promotes lung tumorigenesis.

One of the most fundamental and challenging questions in the field of cancer is how immunity is transformed from tumor immunosurveillance to tumor-promoting inflammation. Here, we identified the tumor suppressor PDZ-LIM domain-containing protein 2 (PDLIM2) as a checkpoint of alveolar macrophages (AMs) important for lung tumor suppression. During lung tumorigenesis, PDLIM2 expression in AMs is downregulated by ROS-activated transcription repressor BTB and CNC homology 1 (BACH1). PDLIM2 downregulation leads to constitutive activation of the transcription factor STAT3, driving AM protumorigenic polarization/activation and differentiation from monocytes attracted from the circulation to suppress cytotoxic T lymphocytes and promote lung cancer. PDLIM2 downregulation also decreases AM phagocytosis. These findings establish ROS/BACH1/PDLIM2/STAT3 as a signaling pathway driving AMs for lung tumor promotion.

RIP3-dependent necroptosis contributes to the pathogenesis of chronic obstructive pulmonary disease.

Necroptosis has emerged as a potential mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we found that markers of necroptosis, including high mobility group box 1 release and phosphorylation of mixed lineage kinase domain-like protein (p-MLKL), were markedly induced in the late stage of cigarette smoking-induced (CS-induced) emphysema in mouse lung tissue as well as in lung epithelial cells and organoids with higher dosage of or more prolonged exposure to cigarette smoking extract (CSE). Apoptotic signals were also detected and maximally induced in the early stage of CS-exposed mice and CSE-treated epithelial cells. Inhibition of apoptosis by Z-VAD, a pan-caspase inhibitor, switched the cellular stress to enhanced necroptosis in lung epithelial cells and organoids treated with CSE. Depletion or inhibition of receptor-interacting protein kinase 3 (RIP3) or MLKL attenuated the CSE-induced cell death, suggesting that necroptosis contributes to CSE-induced cell death. Silencing or inhibition of RIP1 had no protective effect, indicating a RIP1-independent RIP3 activation pathway. CSE-induced necroptosis released more damage-associated molecular patterns and evoked greater engulfment but slower clearance by bone marrow-derived macrophages, leading to enhanced expression of proinflammatory cytokines Tnfα and Il6. Finally, our in vivo data verified that inhibition of necroptosis by RIP3 inhibitor GSK'872 protected mice from CS-induced emphysema and suppressed the lung inflammation. In conclusion, we provide evidence that necroptosis contributes to the pathogenesis of COPD. Targeting RIP3 and its downstream pathway may be an effective therapy for COPD.

Dual but not single PD-1 or TIM-3 blockade enhances oncolytic virotherapy in refractory lung cancer.

BACKGROUND: Programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade therapy fails in the majority of patients with cancer. Oncolytic viruses represent a new class of therapeutic agents, yet the therapeutic efficacy is still disappointing. Moreover, intratumoral injection of viruses is the main approach and preclinical studies mainly employ syngeneic or xenograft models. METHODS: Use an endogenous mouse lung cancer model that faithfully recapitulates human lung cancer, and various in vivo, ex vivo and in vitro assays, to investigate the efficacy, mechanism of action and resistance of systemically administered oncolytic vaccinia virus (oVV), immunotherapy and their combination, to find an effective therapy for refractory lung cancer. RESULTS: Resembling human lung cancers, the majority of which are largely resistant to PD-1/PD-L1 blockade and with decreased PD-L1 expression and T-cell activation by our analysis, urethane-induced endogenous lung tumors in mice show reduced PD-L1 expression, low tumor-infiltrating lymphocytes and innate resistance to PD-1/PD-L1 blockade. Intravenous administration of oVV has efficacy and synergizes with simultaneous but not single blockade of PD-1 and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) in this cancer model. Besides direct tumor cell killing, oVV induces T-cell lung recruitment, tumor infiltration, along with expression of PD-1 and TIM-3 on T cells and PD-1 and TIM-3 ligands on tumor cells and tumor-associated immune cells. Blockade of PD-1 or TIM-3 also causes their mutual induction on T cells. CONCLUSIONS: While systemic administration of oVV shows efficacy in lung cancer by killing tumor cells directly and recruiting and activating T cells for indirect tumor killing, its induction of PD-1 and TIM-3 on T cells and PD-1 and TIM-3 ligands on tumors and tumor-associated immune cells as well as mutual induction of PD-1 or TIM-3 on T cells by their blockade restricts the efficacy of oVV or its combination with single PD-1 or TIM-3 blockade. The triple combination therapy is more effective for refractory lung cancer, and possibly other cold cancers as well.

Recruitment of the inflammatory subset of monocytes to sites of ischemia induces angiogenesis in a monocyte chemoattractant protein-1-dependent fashion.

There is accumulating evidence that delivery of bone marrow cells to sites of ischemia by direct local injection or mobilization into the blood can stimulate angiogenesis. This has stimulated tremendous interest in the translational potential of angiogenic cell population(s) in the bone marrow to mediate therapeutic angiogenesis. However, the mechanisms by which these cells stimulate angiogenesis are unclear. Herein, we show that the inflammatory subset of monocytes is selectively mobilized into blood after surgical induction of hindlimb ischemia in mice and is selectively recruited to ischemic muscle. Adoptive-transfer studies show that delivery of a small number of inflammatory monocytes early (within 48 h) of induction of ischemia results in a marked increase in the local production of MCP-1, which in turn, is associated with a secondary, more robust wave of monocyte recruitment. Studies of mice genetically deficient in MCP-1 or CCR2 indicate that although not required for the early recruitment of monocytes, the secondary wave of monocyte recruitment and subsequent stimulation of angiogenesis are dependent on CCR2 signaling. Collectively, these data suggest a novel role for MCP-1 in the inflammatory, angiogenic response to ischemia.