Anti-CELA1 antibody KF4 prevents emphysema by inhibiting stretch-mediated remodeling.

There are no therapies to prevent emphysema progression. Chymotrypsin-like elastase 1 (CELA1) is a serine protease that binds and cleaves lung elastin in a stretch-dependent manner and is required for emphysema in a murine antisense oligonucleotide model of α-1 antitrypsin (AAT) deficiency. This study tested whether CELA1 is important in strain-mediated lung matrix destruction in non-AAT-deficient emphysema and the efficacy of CELA1 neutralization. Airspace simplification was quantified after administration of tracheal porcine pancreatic elastase (PPE), after 8 months of cigarette smoke (CS) exposure, and in aging. In all 3 models, Cela1-/- mice had less emphysema and preserved lung elastin despite increased lung immune cells. A CELA1-neutralizing antibody was developed (KF4), and it inhibited stretch-inducible lung elastase in ex vivo mouse and human lung and immunoprecipitated CELA1 from human lung. In mice, systemically administered KF4 penetrated lung tissue in a dose-dependent manner and 5 mg/kg weekly prevented emphysema in the PPE model with both pre- and postinjury initiation and in the CS model. KF4 did not increase lung immune cells. CELA1-mediated lung matrix remodeling in response to strain is an important contributor to postnatal airspace simplification, and we believe that KF4 could be developed as a lung matrix-stabilizing therapy in emphysema.

Chymotrypsin-like Elastase-1 Mediates Progressive Emphysema in Alpha-1 Antitrypsin Deficiency.

Chymotrypsin-like elastase 1 (CELA1) is a serine protease that is neutralized by alpha-1antitrypsin (AAT) and prevents emphysema in a murine antisense oligonucleotide model of AAT-deficient emphysema. Mice with genetic ablation of AAT do not have emphysema at baseline but develop emphysema with injury and aging. We tested the role of the CELA1 gene in emphysema development in this genetic model of AAT-deficiency following tracheal lipopolysaccharide (LPS), 10 months of cigarette smoke exposure, aging, and a low-dose tracheal porcine pancreatic elastase (LD-PPE) model we developed. In this last model, we performed proteomic analysis to understand differences in lung protein composition. We were unable to show that AAT-deficient mice developed more emphysema than wild type with escalating doses of LPS. In the LD-PPE model, AAT-deficient mice developed significant and progressive emphysema from which Cela1-/- & AAT-deficient mice were protected. Cela1-/-& AAT-deficient lungs had more matrix-associated proteins than AAT-deficientlungs but also had more leukocyte-associated proteases. With cigarette smoke exposure, Cela1-/- &AAT-deficient mice had more emphysema than AAT-deficient mice but had less myeloperoxidase activity. Cela1-/-&AAT-deficient mice had less age-related airspace simplification than AAT-deficient and were comparable to wild type. While CELA1 promotes inflammation-independent emphysema progression and its absence preserves the lung matrix in multiple models of AAT-deficient emphysema, for unclear reasons Cela1 deficiency is associated with increased emphysema with cigarette smoke. While anti-CELA1 therapies could potentially be used to prevent emphysema progression in AAT deficiency after smoking cessation, an understanding of why and how cigarette smoke exacerbates emphysema in Cela1 deficiency and whether AAT replacement therapy mitigates this effect is needed first.

Associations of Smoking, Cytomegalovirus Serostatus, and Natural Killer Cell Phenotypes in Smokers With and At Risk for COPD.

Introduction: Chronic obstructive disease (COPD) risk factors, smoking, and chronic infection (cytomegalovirus [CMV]) may mold natural killer (NK) cell populations. What is not known is the magnitude of the effect CMV seropositivity imparts on populations of smokers with and at risk for COPD. We investigate the independent influence of CMV seropositivity on NK cell populations and differential effects when stratifying by COPD and degree of smoking history. Methods: Descriptive statistics determine the relationship between cytotoxic NK cell populations and demographic and clinical variables. Multivariable linear regression and predictive modeling were performed to determine associations between positive CMV serology and proportions of CD57+ and natural killer group 2C (NKG2C)+ NK cells. We dichotomized our analysis by those with a heavy smoking history and COPD and described the effect size of CMV seropositivity on NK cell populations. Results: When controlled for age, race, sex, pack-years smoked, body mass index, and lung function, CMV+ serostatus was independently associated with a higher proportion of CD57+, NKG2C+, and NKG2C+CD57+ NK cells. CMV+ serostatus was the sole predictor of larger NKG2C+ and CD57+NKG2C+ populations. Associations are more pronounced in those with COPD and heavy smokers. Conclusions: Among Veterans who are current and former smokers, CMV+ serostatus was independently associated with larger CD57+ and NKG2C+ populations, with a larger effect in heavy smokers and those with COPD, and was the sole predictor for increased expression of NKG2C+ and CD57+NKG2C+ populations. These findings may be broadened to include the assessment of longitudinal NK cell population change, accrued inflammatory potential, and further identification of pro-inflammatory NK cell population clusters.

CELA1 Mediates Progressive Emphysema in Alpha-1 Antitrypsin Deficiency.

Chymotrypsin-like elastase 1 ( CELA1 ) is a serine protease that is neutralized by α1-antitrypsin (AAT) and prevents emphysema in a murine antisense oligonucleotide model of AAT-deficient emphysema. Mice with genetic ablation of AAT do not have emphysema at baseline but develop emphysema with injury and aging. We tested the role of CELA1 in emphysema development in this genetic model of AAT -deficiency following tracheal lipopolysacharide (LPS), 8 months of cigarette smoke (CS) exposure, aging, and a low-dose tracheal porcine pancreatic elastase (LD-PPE) model. In this last model, we performed proteomic analysis to understand differences in lung protein composition. We were unable to show that AAT -/ - mice developed more emphysema than wild type with LPS. In the LD-PPE model, AAT -/- mice developed progressive emphysema from which Cela1 -/- &AAT -/- mice were protected. In the CS model, Cela1 -/- &AAT -/- mice had worse emphysema than AAT -/- , and in the aging model, 72-75 week-old Cela1 -/- &AAT -/- mice had less emphysema than AAT -/- mice. Proteomic analysis of AAT -/- vs. wildtype lungs in the LD-PPE model showed reduced amounts of AAT proteins and increased amounts of proteins related to Rho and Rac1 GTPases and protein oxidation. Similar analysis of Cela1 -/- &AAT -/- vs. AAT -/- lungs showed differences in neutrophil degranulation, elastin fiber synthesis, and glutathione metabolism. Thus, Cela1 prevents post-injury emphysema progression in AAT -deficiency, but it has no effect and potentially worsens emphysema in response to chronic inflammation and injury. Prior to developing anti-CELA1 therapies for AAT-deficient emphysema, an understanding of why and how CS exacerbates emphysema in Cela1 deficiency is needed.

Cytomegalovirus Seropositivity is Associated with Airflow Limitation in a Cohort of Veterans with a High Prevalence of Smoking.

BACKGROUND: Cytomegalovirus (CMV) represents an understudied chronic infection, usually contracted early in life, that causes chronic immune system alterations which may contribute to airflow limitations in a cohort of veterans with a high prevalence of smoking. We studied 172 participants at-risk for and with airflow limitation with available CMV serology to assess the relationship between CMV infection and chronic obstructive pulmonary disease (COPD)-related outcomes. METHODS: The study cohort includes 172 veterans who are smokers with or at risk for the development of COPD. Clinical data were obtained by chart abstraction at enrollment. CMV affinity (ever-exposure) and avidity testing (length of exposure) were performed on plasma samples collected at enrollment. Bivariable and multivariable logistic regression was used to determine the relationship between both cytomegalovirus affinity and avidity and odds of prevalent airflow limitation (post-bronchodilator forced expiratory volume in 1 second to forced vital capacity ratio <0.70) at enrollment. In those with airflow limitation (n=84), bivariable and multivariable logistic regression was used to determine relationships between CMV serostatus and reported exacerbations of COPD over 2 years prior to enrollment. RESULTS: Positive CMV serostatus was independently associated with a 136% higher odds of airflow limitation (95% confidence interval 1.11-5.06, P=0.03) at enrollment. Neither CMV affinity nor avidity was associated with COPD exacerbations in the 2 years prior to enrollment. CONCLUSIONS: CMV serostatus is independently associated with airflow limitation in a cohort of veterans who smoke. Investigation into the timing of infection and alterations in cellular immunity caused by chronic CMV infection and smoking-related airways disease-related outcomes is warranted.

How might endotyping guide chronic obstructive pulmonary disease treatment? Current understanding, knowledge gaps and future research needs.

PURPOSE OF REVIEW: This review discusses emerging therapies directed at chronic obstructive pulmonary disease (COPD) endotypes and pathobiological processes that manifest as the disease. RECENT FINDINGS: Specific endotypes have been targeted in COPD. These include eosinophilic inflammation, overproduction of interleukin-17, chronic bronchitis and altered nature of mucous, and chronic infection. Therapies exactly directed at the cause of these endotypes or their resultant clinical findings have been assessed. Although some intermediate outcomes have seemed promising, there have been no findings that shift the paradigm of COPD therapy. SUMMARY: Basic and clinical scientists continue to define endotypes that may be directly addressed with therapeutics. As of the time of this up-to-date review, there is yet to be an endotype-directed therapy to demonstrate great clinical effect.

Imbalance in zinc homeostasis enhances lung Tissue Loss following cigarette smoke exposure.

Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease. Cadmium is a leading toxic component of cigarette smoke. Cadmium and zinc are highly related metals. Whereas, zinc is an essential metal required for normal health, cadmium is highly toxic. Zrt- and Irt-like protein 8 (ZIP8) is an avid transporter of both zinc and cadmium into cells and is abundantly expressed in the lung of smokers compared to nonsmokers. Our objective was to determine whether disturbed zinc homeostasis through diet or the zinc transporter ZIP8 increase susceptibility to lung damage following prolonged cigarette smoke exposure. METHODS: Cigarette smoke exposure was evaluated in the lungs of mice subject to insufficient and sufficient zinc intakes, in transgenic ZIP8 overexpressing mice, and a novel myeloid-specific ZIP8 knockout strain. RESULTS: Moderate depletion of zinc intakes in adult mice resulted in a significant increase in lung cadmium burden and permanent lung tissue loss following prolonged smoke exposure. Overexpression of ZIP8 resulted in increased lung cadmium burden and more extensive lung damage, whereas cigarette smoke exposure in ZIP8 knockout mice resulted in increased lung tissue loss without a change in lung cadmium content, but a decrease in zinc. CONCLUSIONS: Overall, findings were consistent with past human studies. Imbalance in Zn homeostasis increases susceptibility to permanent lung injury following prolonged cigarette smoke exposure. Based on animal studies, both increased and decreased ZIP8 expression enhanced irreversible tissue damage in response to prolonged tobacco smoke exposure. We believe these findings represent an important advancement in our understanding of how imbalance in zinc homeostasis and cadmium exposure via tobacco smoke may increase susceptibility to smoking-induced lung disease.

Exendin-4 restores airway mucus homeostasis through the GLP1R-PKA-PPARγ-FOXA2-phosphatase signaling.

Goblet cell hyperplasia and metaplasia and excessive mucus are prominent pathologies of chronic airway diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and chronic bronchitis. Chronic infection by respiratory pathogens, including Pseudomonas aeruginosa, exacerbates cyclical proinflammatory responses and mucus hypersecretion. P. aeruginosa and its virulence factor pyocyanin contribute to these pathologies by inhibiting FOXA2, a key transcriptional regulator of mucus homeostasis, through activation of antagonistic signaling pathways EGFR-AKT/ERK1/2 and IL-4/IL-13-STAT6-SPDEF. However, FOXA2-targeted therapy has not been previously explored. Here, we examined the feasibility of repurposing the incretin mimetic Exendin-4 to restore FOXA2-mediated airway mucus homeostasis. We have found that Exendin-4 restored FOXA2 expression, attenuated mucin production in COPD and CF-diseased airway cells, and reduced mucin and P. aeruginosa burden in mouse lungs. Mechanistically, Exendin-4 activated the GLP1R-PKA-PPAR-γ-dependent phosphatases PTEN and PTP1B, which inhibited key kinases within both EGFR and STAT6 signaling cascades. Our results may lead to the repurposing of Exendin-4 and other incretin mimetics to restore FOXA2 function and ultimately regulate excessive mucus in diseased airways.

IL-33 promotes type 1 cytokine expression via p38 MAPK in human NK cells.

This study tests the hypothesis that activation of MAPK by physiologically relevant concentrations of IL-33 contributes to enhanced cytokine expression by IL-12 stimulated human NK cells. While IL-33 canonically triggers type 2 cytokine responses, this cytokine can also synergize with type 1 cytokines like IL-12 to provoke IFN-γ. We show that picogram concentrations of IL-12 and IL-33 are sufficient to promote robust secretion of IFN-γ by human NK cells that greatly exceeds resposes to either cytokine alone. Nanogram doses of IL-33, potentially consistent with levels in tissue microenvironments, synergize with IL-12 to induce secretion of additional cytokines, including TNF and GM-CSF. IL-33-induced activation of the p38 MAPK pathway in human NK cells is crucial for enhanced release of IFN-γ and TNF in response to IL-12. Mechanistically, IL-33-induced p38 MAPK signaling enhances stability of IFNG transcripts and triggers A disintegrin and metalloproteinase domain 17 (ADAM17) mediated cleavage of TNF from the cell surface. These data support our hypothesis and suggest that altered sensitivity of NK cells to IL-12 in the presence of IL-33 may have important consequences in diseases associated with mixed cytokine milieus, like asthma and chronic obstructive pulmonary disease.

Unique natural killer cell subpopulations are associated with exacerbation risk in chronic obstructive pulmonary disease.

Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of death worldwide. COPD is frequently punctuated by acute exacerbations that are precipitated primarily by infections, which increase both morbidity and mortality and inflates healthcare costs. Despite the significance of exacerbations, little understanding of immune function in COPD exacerbations exists. Natural killer (NK) cells are important effectors of innate and adaptive immune responses to pathogens and NK cell function is altered in smokers and COPD. Using high-dimensional flow cytometry, we phenotyped peripheral blood NK cells from never smokers, smokers, and COPD patients and employed a non-supervised clustering algorithm to define and detect changes in NK cell populations. We identified greater than 1,000 unique NK cell subpopulations across patient groups and describe 13 altered NK populations in patients who experienced prior exacerbations. Based upon cluster sizes and associated fluorescence data, we generated a logistic regression model to predict patients with a history of exacerbations with high sensitivity and specificity. Moreover, highly enriched NK cell subpopulations implicated in the regression model exhibited enhanced effector functions as defined by in vitro cytotoxicity assays. These novel data reflect the effects of smoking and disease on peripheral blood NK cell phenotypes, provide insight into the potential immune pathophysiology of COPD exacerbations, and indicate that NK cell phenotyping may be a useful and biologically relevant marker to predict COPD exacerbations.

MAPK mutations and cigarette smoke promote the pathogenesis of pulmonary Langerhans cell histiocytosis.

Pulmonary Langerhans cell histiocytosis (PLCH) is a rare smoking-related lung disease characterized by dendritic cell (DC) accumulation, bronchiolocentric nodule formation, and cystic lung remodeling. Approximately 50% of patients with PLCH harbor somatic BRAF-V600E mutations in cells of the myeloid/monocyte lineage. However, the rarity of the disease and lack of animal models have impeded the study of PLCH pathogenesis. Here, we establish a cigarette smoke-exposed (CS-exposed) BRAF-V600E-mutant mouse model that recapitulates many hallmark characteristics of PLCH. We show that CD11c-targeted expression of BRAF-V600E increases DC responsiveness to stimuli, including the chemokine CCL20, and that mutant cell accumulation in the lungs of CS-exposed mice is due to both increased cellular viability and enhanced recruitment. Moreover, we report that the chemokine CCL7 is secreted from DCs and human peripheral blood monocytes in a BRAF-V600E-dependent manner, suggesting a possible mechanism for recruitment of cells known to dominate PLCH lesions. Inflammatory lesions and airspace dilation in BRAF-V600E mice in response to CS are attenuated by transitioning animals to filtered air and treatment with a BRAF-V600E inhibitor, PLX4720. Collectively, this model provides mechanistic insights into the role of myelomonocytic cells and the BRAF-V600E mutation and CS exposure in PLCH pathogenesis and provides a platform to develop biomarkers and therapeutic targets.

Reduced expression of the Ion channel CFTR contributes to airspace enlargement as a consequence of aging and in response to cigarette smoke in mice.

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease resulting in respiratory failure and represents the third leading cause of global death. The two classical phenotypes of COPD are chronic bronchitis and emphysema. Owing to similarities between chronic bronchitis and the autosomal-recessive disease Cystic Fibrosis (CF), a significant body of research addresses the hypothesis that dysfunctional CF Transmembrane Conductance Regulator (CFTR) is implicated in the pathogenesis of COPD. Much less attention has been given to emphysema in this context, despite similarities between the two diseases. These include early-onset cellular senescence, similar comorbidities, and the finding that CF patients develop emphysema as they age. To determine a potential role for CFTR dysfunction in the development of emphysema, Cftr+/+ (Wild-type; WT), Cftr+/- (heterozygous), and Cftr-/- (knock-out; KO) mice were aged or exposed to cigarette smoke and analyzed for airspace enlargement. Aged knockout mice demonstrated increased alveolar size compared to age-matched wild-type and heterozygous mice. Furthermore, both heterozygous and knockout mice developed enlarged alveoli compared to their wild-type counterparts following chronic smoke exposure. Taken into consideration with previous findings that cigarette smoke leads to reduced CFTR function, our findings suggest that decreased CFTR expression sensitizes the lung to the effects of cigarette smoke. These findings may caution normally asymptomatic CF carriers against exposure to cigarette smoke; as well as highlight emphysema as a future challenge for CF patients as they continue to live longer. More broadly, our data, along with clinical findings, may implicate CFTR dysfunction in a pathology resembling accelerated aging.

NKG2D Regulation of Lung Pathology and Dendritic Cell Function Following Respiratory Syncytial Virus Infection.

Background: Respiratory syncytial virus (RSV) is a common cause of respiratory tract infection in vulnerable populations. Natural killer (NK) cells and dendritic cells (DC) are important for the effector functions of both cell types following infection. Methods: Wild-type and NKG2D-deficient mice were infected with RSV. Lung pathology was assessed by histology. Dendritic cell function and phenotype were evaluated by enzyme-linked immunosorbent assay and flow cytometry. The expression of NKG2D ligands on lung and lymph node DCs was measured by immunostaining and flow cytometry. Adoptive transfer experiments were performed to assess the importance of NKG2D-dependent DC function in RSV infection. Results: NKG2D-deficient mice exhibited greater lung pathology, marked by the accumulation of DCs following RSV infection. Dendritic cells isolated from NKG2D-deficient mice had impaired responses toward Toll-like receptor ligands. Dendritic cells expressed NKG2D ligands on their surface, which was further increased in NKG2D-deficient mice and during RSV infection. Adoptive transfer of DCs isolated from wild-type mice into the airways of NKG2D-deficient mice ameliorated the enhanced inflammation in NKG2D-deficient mice after RSV infection. Conclusion: NKG2D-dependent interactions with DCs control the phenotype and function of DCs and play a critical role in pulmonary host defenses against RSV infection.

Tuberin Regulates Prostaglandin Receptor-Mediated Viability, via Rheb, in mTORC1-Hyperactive Cells.

Tuberous sclerosis complex (TSC) is a tumor-suppressor syndrome affecting multiple organs, including the brain, skin, kidneys, heart, and lungs. TSC is associated with mutations in TSC1 or TSC2, resulting in hyperactivation of mTOR complex 1 (mTORC1). Clinical trials demonstrate that mTORC1 inhibitors decrease tumor volume and stabilize lung function in TSC patients; however, mTOR inhibitors are cytostatic not cytocidal, and long-term benefits and toxicities are uncertain. Previously, we identified rapamycin-insensitive upregulation of cyclooxygenase 2 (PTGS2/COX2) and prostaglandin E2 (PGE2) production in TSC2-deficient cells and postulated that the action of excess PGE2 and its cognate receptors (EP) contributes to cell survival. In this study, we identify upregulation of EP3 (PTGER3) expression in TSC2-deficient cells, TSC renal angiomyolipomas, lymphangioleiomyomatosis lung nodules, and epileptic brain tubers. TSC2 negatively regulated EP3 expression via Rheb in a rapamycin-insensitive manner. The EP3 antagonist, L-798106, selectively suppressed the viability of TSC2-deficient cells in vitro and decreased the lung colonization of TSC2-deficient cells. Collectively, these data reveal a novel function of TSC2 and Rheb in the regulation of EP3 expression and cell viability.Implications: Therapeutic targeting of an aberrant PGE2-EP3 signaling axis may have therapeutic benefit for TSC patients and for other mTOR-hyperactive neoplasms. Mol Cancer Res; 15(10); 1318-30. ©2017 AACR.

Dendritic Cell Trafficking and Function in Rare Lung Diseases.

Dendritic cells (DCs) are highly specialized immune cells that capture antigens and then migrate to lymphoid tissue and present antigen to T cells. This critical function of DCs is well defined, and recent studies further demonstrate that DCs are also key regulators of several innate immune responses. Studies focused on the roles of DCs in the pathogenesis of common lung diseases, such as asthma, infection, and cancer, have traditionally driven our mechanistic understanding of pulmonary DC biology. The emerging development of novel DC reagents, techniques, and genetically modified animal models has provided abundant data revealing distinct populations of DCs in the lung, and allow us to examine mechanisms of DC development, migration, and function in pulmonary disease with unprecedented detail. This enhanced understanding of DCs permits the examination of the potential role of DCs in diseases with known or suspected immunological underpinnings. Recent advances in the study of rare lung diseases, including pulmonary Langerhans cell histiocytosis, sarcoidosis, hypersensitivity pneumonitis, and pulmonary fibrosis, reveal expanding potential pathogenic roles for DCs. Here, we provide a review of DC development, trafficking, and effector functions in the lung, and discuss how alterations in these DC pathways contribute to the pathogenesis of rare lung diseases.

NK cell activating receptor ligand expression in lymphangioleiomyomatosis is associated with lung function decline.

Lymphangioleiomyomatosis (LAM) is a rare lung disease of women that leads to progressive cyst formation and accelerated loss of pulmonary function. Neoplastic smooth muscle cells from an unknown source metastasize to the lung and drive destructive remodeling. Given the role of NK cells in immune surveillance, we postulated that NK cell activating receptors and their cognate ligands are involved in LAM pathogenesis. We found that ligands for the NKG2D activating receptor UL-16 binding protein 2 (ULBP2) and ULBP3 are localized in cystic LAM lesions and pulmonary nodules. We found elevated soluble serum ULBP2 (mean = 575 pg/ml ± 142) in 50 of 100 subjects and ULBP3 in 30 of 100 (mean = 8,300 pg/ml ± 1,515) subjects. LAM patients had fewer circulating NKG2D+ NK cells and decreased NKG2D surface expression. Lung function decline was associated with soluble NKG2D ligand (sNKG2DL) detection. The greatest rate of decline forced expiratory volume in 1 second (FEV1, -124 ± 30 ml/year) in the 48 months after enrollment (NHLBI LAM Registry) occurred in patients expressing both ULBP2 and ULBP3, whereas patients with undetectable sNKG2DL levels had the lowest rate of FEV1 decline (-32.7 ± 10 ml/year). These data suggest a role for NK cells, sNKG2DL, and the innate immune system in LAM pathogenesis.

Cutting Edge: CLEC5A Mediates Macrophage Function and Chronic Obstructive Pulmonary Disease Pathologies.

Chronic obstructive pulmonary disease (COPD) is a devastating disease with no effective therapies. We investigated the role of the C-type lectin receptor, CLEC5A, in macrophage activation and pulmonary pathogenesis in a mouse model of COPD. We demonstrate that CLEC5A is expressed on alveolar macrophages in mice exposed long-term to cigarette smoke (CS), as well as in human smokers. We also show that CLEC5A-mediated activation of macrophages enhanced cytokine elaboration alone, as well as in combination with LPS or GM-CSF in CS-exposed mice. Furthermore, usingClec5a-deficient mice, we demonstrate that CS-induced macrophage responsiveness is mediated by CLEC5A, and CLEC5A is required for the development of inflammation, proinflammatory cytokine expression, and airspace enlargement. These findings suggest a novel mechanism that promotes airway inflammation and pathologies in response to CS exposure and identifies CLEC5A as a novel target for the therapeutic control of COPD pathogenesis.