Protease-armed, Pathogenic Extracellular Vesicles Link Smoking and Chronic Obstructive Pulmonary Disease.

Rationale: Mounting evidence demonstrates a role for extracellular vesicles (EVs) in driving lung disorders, such as chronic obstructive pulmonary disease (COPD). Although cigarette smoke (CS) is the primary risk factor for COPD, a link between CS and the EVs that could lead to COPD is unknown. Objective: To ascertain whether exposure to CS elicits a proteolytic EV signature capable of driving disease pathogenesis. Methods: Protease expression and enzymatic activity were measured in EVs harvested from the BAL fluid of smoke-exposed mice and otherwise healthy human smokers. Pathogenicity of EVs was examined using pathological tissue scoring after EV transfer into naive recipient mice. Measurements and Main Results: The analyses revealed a unique EV profile defined by neutrophil- and macrophage-derived EVs. These EVs are characterized by abundant surface expression of neutrophil elastase (NE) and matrix metalloproteinase 12 (MMP12), respectively. CS-induced mouse or human-derived airway EVs had a robust capacity to elicit rapid lung damage in naive recipient mice, with an additive effect of NE- and MMP12-expressing EVs. Conclusions: These studies demonstrate the capacity of CS to drive the generation of unique EV populations containing NE and MMP12. The coordinated action of these EVs is completely sufficient to drive emphysematous disease, and their presence could operate as a prognostic indicator for COPD development. Furthermore, given the robust capacity of these EVs to elicit emphysema in naive mice, they provide a novel model to facilitate preclinical COPD research. Indeed, the development of this model has led to the discovery of a previously unrecognized CS-induced protective mechanism against EV-mediated damage.

A Novel Animal Model of Emphysema Induced by Anti-Elastin Autoimmunity.

Loss of immune tolerance to self-antigens can promote chronic inflammation and disrupt the normal function of multiple organs, including the lungs. Degradation of elastin, a highly insoluble protein and a significant component of the lung structural matrix, generates proinflammatory molecules. Elastin fragments (EFs) have been detected in the serum of smokers with emphysema, and elastin-specific T cells have also been detected in the peripheral blood of smokers with emphysema. However, an animal model that could recapitulate T cell-specific autoimmune responses by initiating and sustaining inflammation in the lungs is lacking. In this study, we report an animal model of autoimmune emphysema mediated by the loss of tolerance to elastin. Mice immunized with a combination of human EFs plus rat EFs but not mouse EFs showed increased infiltration of innate and adaptive immune cells to the lungs and developed emphysema. We cloned and expanded mouse elastin-specific CD4+ T cells from the lung and spleen of immunized mice. Finally, we identified TCR sequences from the autoreactive T cell clones, suggesting possible pathogenic TCRs that can cause loss of immune tolerance against elastin. This new autoimmune model of emphysema provides a useful tool to examine the immunological factors that promote loss of immune tolerance to self.

Selective cleavage of fibrinogen by diverse proteinases initiates innate allergic and antifungal immunity through CD11b.

Proteinases are essential drivers of allergic airway disease and innate antifungal immunity in part through their ability cleave the clotting factor fibrinogen (FBG) into fibrinogen cleavage products (FCPs) that signal through Toll-like receptor 4 (TLR4). However, the mechanism by which FCPs engage TLR4 remains unknown. Here, we show that the proteinases from Aspergillus melleus (PAM) and other allergenic organisms rapidly hydrolyze FBG to yield relatively few FCPs that drive distinct antifungal mechanisms through TLR4. Functional FCPs, termed cryptokines, were characterized by rapid loss of the FBG α chain with substantial preservation of the ß and γ chains, including a γ chain sequence (Fibγ390-396) that binds the integrin Mac-1 (CD11b/CD18). PAM-derived cryptokines could be generated from multiple FBG domains, and the ability of cryptokines to induce fungistasis in vitro and innate allergic airway disease in vivo strongly depended on both Mac-1 and the Mac-1-binding domain of FBG (Fibγ390-396). Our findings illustrate the essential concept of proteinase-activated immune responses and for the first time link Mac-1, cryptokines, and TLR4 to innate antifungal immunity and allergic airway disease.

Laryngeal inflammatory response to smoke and vape in a murine model.

PURPOSE: To build a murine model for tobacco smoke and electronic cigarette vapor exposure to characterize the inflammatory and immune responses in the larynx. MATERIALS AND METHODS: In this pilot study, twenty-four wild-type C57BL/6 mice were divided into four groups: smoke, vapor with nicotine, vapor without nicotine, and air only. Following daily exposure for 4 months, larynges were dissected and processed with cytokine detection arrays. Each laryngeal cytokine level between the four different groups was analyzed statistically by using statistical analysis software (SAS) to calculate the analysis of variance (ANOVA). RESULTS: IL-4 was the only cytokine found to achieve statistically significant different levels in this study, with elevated levels of IL-4 in the tobacco smoke and vapor with nicotine groups compared to the levels found in the vapor without nicotine and air only groups (p = 0.0418). While statistically non-significant, prominent findings revealed up-regulation of TGF-ß2 and TGF-ß3 in the smoke group, but near-normal levels of TGF-ß2 and TGF-ß3 and suppression of IL-10 in the vapor groups (p > 0.05). CONCLUSION: The potential utility of the murine model is established for studying the inflammatory and immune effects of tobacco smoke and vapor on the mammalian larynx. IL-4 levels in mice larynges were significantly elevated in the tobacco smoke and vapor with nicotine groups.

Type I interferon-dependent IFIT3 signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections including COVID-19-related ARDS, although its contribution to disease pathogenesis remains poorly understood. Here, we identified two neutrophil subpopulations (A1 and A2) in the airway compartment of 52 severe COVID-19 subjects, where loss of the A2 subset correlated with increased viral burden and reduced 30-days survival. A2 neutrophils showcased a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation with consequent reduced viral catabolism, providing the first discrete mechanism of type I interferon signaling in neutrophils. The identification of this novel neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Interferon-dependent signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections, including COVID-19-related acute respiratory distress syndrome, although its contribution to disease pathogenesis remains poorly understood. Blood and airway immune cells from 52 patients with severe COVID-19 were phenotyped by flow cytometry. Samples and clinical data were collected at 2 separate time points to assess changes during ICU stay. Blockade of type I interferon and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) signaling was performed in vitro to determine their contribution to viral clearance in A2 neutrophils. We identified 2 neutrophil subpopulations (A1 and A2) in the airway compartment, where loss of the A2 subset correlated with increased viral burden and reduced 30-day survival. A2 neutrophils exhibited a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation, with consequent reduced viral catabolism, providing the first discrete mechanism to our knowledge of type I interferon signaling in neutrophils. The identification of this neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

An in vivo model for extracellular vesicle-induced emphysema.

Chronic obstructive pulmonary disease (COPD) is a debilitating chronic disease and the third-leading cause of mortality worldwide. It is characterized by airway neutrophilia, promoting tissue injury through release of toxic mediators and proteases. Recently, it has been shown that neutrophil-derived extracellular vesicles (EVs) from lungs of patients with COPD can cause a neutrophil elastase-dependent (NE-dependent) COPD-like disease upon transfer to mouse airways. However, in vivo preclinical models elucidating the impact of EVs on disease are lacking, delaying opportunities for therapeutic testing. Here, we developed an in vivo preclinical mouse model of lung EV-induced COPD. EVs from in vivo LPS-activated mouse neutrophils induced COPD-like disease in naive recipients through an α-1 antitrypsin-resistant, NE-dependent mechanism. Together, these results show a key pathogenic and mechanistic role for neutrophil-derived EVs in a mouse model of COPD. Broadly, the in vivo model described herein could be leveraged to develop targeted therapies for severe lung disease.

Chronic exposure to carbon black ultrafine particles reprograms macrophage metabolism and accelerates lung cancer.

Chronic exposure to airborne carbon black ultrafine (nCB) particles generated from incomplete combustion of organic matter drives IL-17A-dependent emphysema. However, whether and how they alter the immune responses to lung cancer remains unknown. Here, we show that exposure to nCB particles increased PD-L1+ PD-L2+ CD206+ antigen-presenting cells (APCs), exhausted T cells, and Treg cells. Lung macrophages that harbored nCB particles showed selective mitochondrial structure damage and decreased oxidative respiration. Lung macrophages sustained the HIF1α axis that increased glycolysis and lactate production, culminating in an immunosuppressive microenvironment in multiple mouse models of non-small cell lung cancers. Adoptive transfer of lung APCs from nCB-exposed wild type to susceptible mice increased tumor incidence and caused early metastasis. Our findings show that nCB exposure metabolically rewires lung macrophages to promote immunosuppression and accelerates the development of lung cancer.

Spatial mapping of SARS-CoV-2 and H1N1 lung injury identifies differential transcriptional signatures.

Severe SARS-CoV-2 infection often leads to the development of acute respiratory distress syndrome (ARDS), with profound pulmonary patho-histological changes post-mortem. It is not clear whether ARDS from SARS-CoV-2 is similar to that observed in influenza H1N1, another common viral cause of lung injury. Here, we analyze specific ARDS regions of interest utilizing a spatial transcriptomic platform on autopsy-derived lung tissue from patients with SARS-CoV-2 (n = 3), H1N1 (n = 3), and a dual infected individual (n = 1). Enhanced gene signatures in alveolar epithelium, vascular tissue, and lung macrophages identify not only increased regional coagulopathy but also increased extracellular remodeling, alternative macrophage activation, and squamous metaplasia of type II pneumocytes in SARS-CoV-2. Both the H1N1 and dual-infected transcriptome demonstrated an enhanced antiviral response compared to SARS-CoV-2. Our results uncover regional transcriptional changes related to tissue damage/remodeling, altered cellular phenotype, and vascular injury active in SARS-CoV-2 and present therapeutic targets for COVID-19-related ARDS.

Cigarette Smoke Induces Intestinal Inflammation via a Th17 Cell-Neutrophil Axis.

Epidemiological evidence finds cigarette smoking is a common risk factor for a number of diseases, not only in the lung but also in other tissues, such as the gastrointestinal tract. While it is well-documented that smoking directly drives lung inflammatory disease, how it promotes disease in peripheral tissues is incompletely understood. In this study, we utilized a mouse model of short-term smoke exposure and found increased Th17 cells and neutrophilia in the lung as well as in the circulation. Following intestinal inflammatory challenge, smoke exposed mice showed increased pathology which corresponds to enhanced intestinal Th17 cells, ILC3 and neutrophils within intestinal tissue. Using cellular depletion and genetic deficiencies, we define a cellular loop by which IL-17A and downstream neutrophils drive cigarette smoke-enhanced intestinal inflammation. Collectively, cigarette smoke induced local lung Th17 responses lead to increased systemic susceptibility to inflammatory insult through enhanced circulating neutrophils. These data demonstrate a cellular pathway by which inflammatory challenge in the lung can sensitize the intestine to enhanced pathological innate and adaptive immune responses.

Cigarette smoke-induced reduction of C1q promotes emphysema.

Alteration of innate immune cells in the lungs can promote loss of peripheral tolerance that leads to autoimmune responses in cigarette smokers. Development of autoimmunity in smokers with emphysema is also strongly linked to the expansion of autoreactive T helper (Th) cells expressing interferon gamma (Th1), and interleukin 17A (Th17). However, the mechanisms responsible for enhanced self-recognition and reduced immune tolerance in smoker with emphysema remain less clear. Here we show that C1q, a component of the complement protein 1 complex (C1), is downregulated in lung CD1a+ antigen presenting cells (APCs) isolated from emphysematous human, and mouse lung APCs after chronic cigarette smoke exposure. C1q potentiated the function of APCs to differentiate CD4+ T cells to Tregs, while it inhibited Th17 cell development and proliferation. Mice deficient in C1q that were exposed to chronic smoke exhibited exaggerated lung inflammation marked by increased Th17 cells, while reconstitution of C1q in the lungs enhanced Tregs abundance, dampened smoke-induced lung inflammation, and reversed established emphysema. Our findings demonstrate that cigarette smoke-mediated loss of C1q could play a key role in reduced peripheral tolerance, which could be explored to treat emphysema.

Electronic cigarettes disrupt lung lipid homeostasis and innate immunity independent of nicotine.

Electronic nicotine delivery systems (ENDS) or e-cigarettes have emerged as a popular recreational tool among adolescents and adults. Although the use of ENDS is often promoted as a safer alternative to conventional cigarettes, few comprehensive studies have assessed the long-term effects of vaporized nicotine and its associated solvents, propylene glycol (PG) and vegetable glycerin (VG). Here, we show that compared with smoke exposure, mice receiving ENDS vapor for 4 months failed to develop pulmonary inflammation or emphysema. However, ENDS exposure, independent of nicotine, altered lung lipid homeostasis in alveolar macrophages and epithelial cells. Comprehensive lipidomic and structural analyses of the lungs revealed aberrant phospholipids in alveolar macrophages and increased surfactant-associated phospholipids in the airway. In addition to ENDS-induced lipid deposition, chronic ENDS vapor exposure downregulated innate immunity against viral pathogens in resident macrophages. Moreover, independent of nicotine, ENDS-exposed mice infected with influenza demonstrated enhanced lung inflammation and tissue damage. Together, our findings reveal that chronic e-cigarette vapor aberrantly alters the physiology of lung epithelial cells and resident immune cells and promotes poor response to infectious challenge. Notably, alterations in lipid homeostasis and immune impairment are independent of nicotine, thereby warranting more extensive investigations of the vehicle solvents used in e-cigarettes.

Matrix remodeling in chronic lung diseases.

Multicellular organisms synthesize and renew components of their subcellular and scaffolding proteins, collectively known as the extracellular matrix molecules (ECMs). In the lung, ECMs maintain tensile strength, elasticity, and dictate the specialized function of multiple cell lineages. These functions are critical in lung homeostatic processes including cellular migration and proliferation during morphogenesis or in response to repair. Alterations in lung ECMs that expose cells to new cryptic fragments, generated in response to endogenous proteinases or exogenous toxins, are associated with the development of several common respiratory diseases. How lung ECMs provide or relay vital signals to epithelial and mesenchymal cells has shed new light on development and progression of several common chronic respiratory diseases. This review will consider how ECMs regulate lung homeostasis and their reorganization under pathological conditions that can modulate the inflammatory diseases asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). Better understanding of changes in the distribution of lung ECM could provide novel therapeutic approaches to treat chronic lung diseases.