ADAM9 promotes type I interferon-mediated innate immunity during encephalomyocarditis virus infection.

Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.

Loss of ADAM15 in female mice does not worsen pressure overload cardiomyopathy, independent of ovarian hormones.

Cardiac hypertrophy is a common feature in several cardiomyopathies. We previously reported that loss of ADAM15 (disintegrin and metalloproteinase 15) worsened cardiac hypertrophy and dilated cardiomyopathy following cardiac pressure overload. Here, we investigated the impact of ADAM15 loss in female mice following cardiac pressure overload induced by transverse aortic constriction (TAC). Female Adam15-/- mice developed the same degree of cardiac hypertrophy, dilation, and dysfunction as the parallel female wild-type (WT) mice at 6 wk post-TAC. To determine if this is due to the protective effects of estrogen, which could mask the negative impact of Adam15 loss, WT and Adam15-/- mice underwent ovariectomy (OVx) 2 wk before TAC. Cardiac structure and function analyses were performed at 6 wk post-TAC. OVx similarly impacted females of both genotypes post-TAC. Calcineurin (Cn) activity was increased post-OVx-TAC, and more in Adam15-/- mice; however, this increase was not reflected in the total-to-phospho-NFAT levels. Integrin-α7 expression, which was upstream of Cn activation in male Adam15-/- -TAC mice, remained unchanged in female mice. However, activation of the mitogen-activated protein kinases (ERK, JNK, P38) was greater in Adam15-/--OVx-TAC than in WT-OVx-TAC mice. In addition, ADAM15 protein levels were significantly increased post-TAC in male but not in female WT mice. Myocardial fibrosis was comparable in non-OVx WT-TAC and Adam15-/- -TAC mice. OVx increased the perivascular fibrosis more in Adam15-/- compared with WT mice post-TAC. Our data demonstrate that loss of ovarian hormones did not fully replicate the male phenotype in the female Adam15-/- mice post-TAC. As ADAM15 levels were increased in males but not in females post-TAC, it is plausible that ADAM15 does not play a prominent role in post-TAC events in female mice. Our findings highlight the significance of factors other than sex hormones in mediating cardiomyopathies in females, which require a more thorough understanding.NEW & NOTEWORTHY Loss of ADAM15 in female mice, unlike the male mice, does not worsen the cardiomyopathy following cardiac pressure overload. Ovariectomy does not worsen the post-TAC cardiomyopathy in female Adam15-/- mice compared with female WT mice. Lack of deleterious impact of Adam15 deficiency in female mice is not because of the protective effects of ovarian hormones but could be due to a less prominent role of ADAM15 in cardiac response to post-TAC remodeling in female mice.

CC16 augmentation reduces exaggerated COPD-like disease in Cc16-deficient mice.

Low Club Cell 16 kDa protein (CC16) plasma levels are linked to accelerated lung function decline in patients with chronic obstructive pulmonary disease (COPD). Cigarette smoke-exposed (CS-exposed) Cc16-/- mice have exaggerated COPD-like disease associated with increased NF-κB activation in their lungs. It is unclear whether CC16 augmentation can reverse exaggerated COPD in CS-exposed Cc16-/- mice and whether increased NF-κB activation contributes to the exaggerated COPD in CS-exposed Cc16-/- lungs. CS-exposed WT and Cc16-/- mice were treated with recombinant human CC16 (rhCC16) or an NF-κB inhibitor versus vehicle beginning at the midpoint of the exposures. COPD-like disease and NF-κB activation were measured in the lungs. RhCC16 limited the progression of emphysema, small airway fibrosis, and chronic bronchitis-like disease in WT and Cc16-/- mice partly by reducing pulmonary inflammation (reducing myeloid leukocytes and/or increasing regulatory T and/or B cells) and alveolar septal cell apoptosis, reducing NF-κB activation in CS-exposed Cc16-/- lungs, and rescuing the reduced Foxj1 expression in CS-exposed Cc16-/- lungs. IMD0354 treatment reduced exaggerated lung inflammation and rescued the reduced Foxj1 expression in CS-exposed Cc16-/- mice. RhCC16 treatment reduced NF-κB activation in luciferase reporter A549 cells. Thus, rhCC16 treatment limits COPD progression in CS-exposed Cc16-/- mice partly by inhibiting NF-κB activation and represents a potentially novel therapeutic approach for COPD.

Dysregulation of miR-103a Mediates Cigarette Smoking-induced Lipid-laden Macrophage Formation.

Cigarette smoke (CS) is considered a major risk factor for chronic obstructive pulmonary disease (COPD) that is currently the third leading cause of death in the United States. Studies have indicated that patients with COPD have elevated blood low-density lipoprotein levels, which may contribute to the dysregulation of lipid metabolism. Accumulating data show that microRNAs (miRNAs) are involved in various human diseases. However, the role of microRNAs in the pathogenesis of COPD remains poorly defined. In this study, we found that miR-103a expression was significantly reduced in alveolar macrophages from smokers and patients with COPD versus that in alveolar macrophages from nonsmokers. Our data indicated that reactive oxygen species negatively regulate miR-103a in macrophages. Functionally, miR-103a modulates the expressions of genes involved in lipid metabolism and directly targets low-density lipoprotein receptors in macrophages. Furthermore, overexpression of miR-103a suppressed the accumulation of lipid droplets and reduced the reactive oxygen species, both in vitro and in vivo. Taken together, our findings indicate that downregulation of miR-103a contributes to cigarette smoke-induced lipid-laden macrophage formation and plays a critical role in lipid homeostasis in lung macrophages in the pathogenesis of COPD.

Association of clonal hematopoiesis with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is associated with age and smoking, but other determinants of the disease are incompletely understood. Clonal hematopoiesis of indeterminate potential (CHIP) is a common, age-related state in which somatic mutations in clonal blood populations induce aberrant inflammatory responses. Patients with CHIP have an elevated risk for cardiovascular disease, but the association of CHIP with COPD remains unclear. We analyzed whole-genome sequencing and whole-exome sequencing data to detect CHIP in 48 835 patients, of whom 8444 had moderate to very severe COPD, from four separate cohorts with COPD phenotyping and smoking history. We measured emphysema in murine models in which Tet2 was deleted in hematopoietic cells. In the COPDGene cohort, individuals with CHIP had risks of moderate-to-severe, severe, or very severe COPD that were 1.6 (adjusted 95% confidence interval [CI], 1.1-2.2) and 2.2 (adjusted 95% CI, 1.5-3.2) times greater than those for noncarriers. These findings were consistently observed in three additional cohorts and meta-analyses of all patients. CHIP was also associated with decreased FEV1% predicted in the COPDGene cohort (mean between-group differences, -5.7%; adjusted 95% CI, -8.8% to -2.6%), a finding replicated in additional cohorts. Smoke exposure was associated with a small but significant increased risk of having CHIP (odds ratio, 1.03 per 10 pack-years; 95% CI, 1.01-1.05 per 10 pack-years) in the meta-analysis of all patients. Inactivation of Tet2 in mouse hematopoietic cells exacerbated the development of emphysema and inflammation in models of cigarette smoke exposure. Somatic mutations in blood cells are associated with the development and severity of COPD, independent of age and cumulative smoke exposure.

Long-term safety of lumacaftor-ivacaftor in children aged 2-5 years with cystic fibrosis homozygous for the F508del-CFTR mutation: a multicentre, phase 3, open-label, extension study.

BACKGROUND: A previous phase 3 study showed that lumacaftor-ivacaftor was generally safe and well tolerated over 24 weeks of treatment in children aged 2-5 years with cystic fibrosis homozygous for the F508del-CFTR mutation. In this study, we aimed to assess the long-term safety of lumacaftor-ivacaftor in a rollover study of children who participated in this previous phase 3 study. METHODS: In this multicentre, phase 3, open-label, extension study (study 116; VX16-809-116), we assessed safety of lumacaftor-ivacaftor in children included in a previous multicentre, phase 3, open-label study (study 115; VX15-809-115). The study was done at 20 cystic fibrosis care centres in the USA and Canada. Children aged 2-5 years with cystic fibrosis homozygous for the F508del-CFTR mutation who completed 24 weeks of lumacaftor-ivacaftor treatment in study 115 received weight-based and age-based doses of oral lumacaftor-ivacaftor: children weighing less than 14 kg and aged younger than 6 years at study 116 screening received lumacaftor 100 mg-ivacaftor 125 mg every 12 h; children weighing 14 kg or more and aged younger than 6 years at screening received lumacaftor 150 mg-ivacaftor 188 mg every 12 h; and children aged 6 years or older received lumacaftor 200 mg-ivacaftor 250 mg every 12 h. Children received treatment for up to 96 weeks, equivalent to up to 120 weeks of treatment in total from the start of study 115 to completion of study 116. The primary endpoint was the safety and tolerability of the study drug in all participants who had received lumacaftor-ivacaftor for 24 weeks in study 115 and had received at least one dose in study 116. Secondary endpoints included change from baseline in study 115 at week 96 of study 116 in sweat chloride concentration, growth parameters, markers of pancreatic function, and lung clearance index (LCI) parameters in all children who received at least one dose of lumacaftor-ivacaftor in study 116. This study is registered with ClinicalTrials.gov, NCT03125395. FINDINGS: This extension study ran from May 12, 2017, to July 17, 2019. Of 60 participants enrolled and who received lumacaftor-ivacaftor in study 115, 57 (95%) were included in study 116 and continued to receive the study drug. A total of 47 (82%) of 57 participants completed 96 weeks of treatment. Most participants (56 [98%] of 57) had at least one adverse event during study 116, most of which were mild (19 [33%] participants) or moderate (29 [51%] participants) in severity. The most common adverse events were cough (47 [82%] participants), nasal congestion (25 [44%] participants), pyrexia (23 [40%] participants), rhinorrhoea (18 [32%] participants), and vomiting (17 [30%] participants). A total of 15 (26%) participants had at least one serious adverse event; most were consistent with underlying cystic fibrosis or common childhood illnesses. Respiratory adverse events occurred in five (9%) participants, none of which were serious or led to treatment discontinuation. Elevated aminotransferase concentrations, most of which were mild or moderate in severity, occurred in ten (18%) participants. Three (5%) participants discontinued treatment due to adverse events (two due to increased aminotransferase concentrations [one of whom had concurrent pancreatitis], considered as possibly related to study drug; and one due to gastritis and metabolic acidosis, considered unlikely to be related to study drug). No clinically significant abnormalities or changes were seen in electrocardiograms, vital signs, pulse oximetry, ophthalmological examinations, or spirometry assessments. Improvements in secondary endpoints observed in study 115 were generally maintained up to week 96 of study 116, including improvements in sweat chloride concentration (mean absolute change from study 115 baseline at week 96 of study 116 -29·6 mmol/L [95% CI -33·7 to -25·5]), an increase in growth parameters and pancreatic function, and stable lung function relative to baseline, as measured by the LCI. INTERPRETATION: Lumacaftor-ivacaftor was generally safe and well tolerated, and treatment effects were generally maintained for the duration of the extension study. These findings support the use of lumacaftor-ivacaftor for up to 120 weeks in young children with cystic fibrosis aged 2 years and older homozygous for the F508del-CFTR mutation. FUNDING: Vertex Pharmaceuticals Incorporated.

Metformin: Experimental and Clinical Evidence for a Potential Role in Emphysema Treatment.

Rationale: Cigarette smoke (CS) inhalation triggers oxidative stress and inflammation, leading to accelerated lung aging, apoptosis, and emphysema, as well as systemic pathologies. Metformin is beneficial for protecting against aging-related diseases. Objectives: We sought to investigate whether metformin may ameliorate CS-induced pathologies of emphysematous chronic obstructive pulmonary disease (COPD). Methods: Mice were exposed chronically to CS and fed metformin-enriched chow for the second half of exposure. Lung, kidney, and muscle pathologies, lung proteostasis, endoplasmic reticulum (ER) stress, mitochondrial function, and mediators of metformin effects in vivo and/or in vitro were studied. We evaluated the association of metformin use with indices of emphysema progression over 5 years of follow-up among the COPDGene (Genetic Epidemiology of COPD) study participants. The association of metformin use with the percentage of emphysema and adjusted lung density was estimated by using a linear mixed model. Measurements and Main Results: Metformin protected against CS-induced pulmonary inflammation and airspace enlargement; small airway remodeling, glomerular shrinkage, oxidative stress, apoptosis, telomere damage, aging, dysmetabolism in vivo and in vitro; and ER stress. The AMPK (AMP-activated protein kinase) pathway was central to metformin's protective action. Within COPDGene, participants receiving metformin compared with those not receiving it had a slower progression of emphysema (-0.92%; 95% confidence interval [CI], -1.7% to -0.14%; P = 0.02) and a slower adjusted lung density decrease (2.2 g/L; 95% CI, 0.43 to 4.0 g/L; P = 0.01). Conclusions: Metformin protected against CS-induced lung, renal, and muscle injury; mitochondrial dysfunction; and unfolded protein responses and ER stress in mice. In humans, metformin use was associated with lesser emphysema progression over time. Our results provide a rationale for clinical trials testing the efficacy of metformin in limiting emphysema progression and its systemic consequences.

ADAM9 enhances Th17 cell differentiation and autoimmunity by activating TGF-ß1.

The a disintegrin and metalloproteinase (ADAM) family of proteinases alter the extracellular environment and are involved in the development of T cells and autoimmunity. The role of ADAM family members in Th17 cell differentiation is unknown. We identified ADAM9 to be specifically expressed and to promote Th17 differentiation. Mechanistically, we found that ADAM9 cleaved the latency-associated peptide to produce bioactive transforming growth factor ß1, which promoted SMAD2/3 phosphorylation and activation. A transcription factor inducible cAMP early repressor was found to bind directly to the ADAM9 promoter and to promote its transcription. Adam9-deficient mice displayed mitigated experimental autoimmune encephalomyelitis, and transfer of Adam9-deficient myelin oligodendrocyte globulin-specific T cells into Rag1-/- mice failed to induce disease. At the translational level, an increased abundance of ADAM9 levels was observed in CD4+ T cells from patients with systemic lupus erythematosus, and ADAM9 gene deletion in lupus primary CD4+ T cells clearly attenuated their ability to differentiate into Th17 cells. These findings revealed that ADAM9 as a proteinase provides Th17 cells with an ability to activate transforming growth factor ß1 and accelerates its differentiation, resulting in aberrant autoimmunity.

Hedgehog interacting protein (HHIP) represses airway remodeling and metabolic reprogramming in COPD-derived airway smooth muscle cells.

Although HHIP locus has been consistently associated with the susceptibility to COPD including airway remodeling and emphysema in genome-wide association studies, the molecular mechanism underlying this genetic association remains incompletely understood. By utilizing Hhip+/- mice and primary human airway smooth muscle cells (ASMCs), here we aim to determine whether HHIP haploinsufficiency increases airway smooth muscle mass by reprogramming glucose metabolism, thus contributing to airway remodeling in COPD pathogenesis. The mRNA levels of HHIP were compared in normal and COPD-derived ASMCs. Mitochondrial oxygen consumption rate and lactate levels in the medium were measured in COPD-derived ASMCs with or without HHIP overexpression as readouts of glucose oxidative phosphorylation and aerobic glycolysis rates. The proliferation rate was measured in healthy and COPD-derived ASMCs treated with or without 2-DG. Smooth muscle mass around airways was measured by immunofluorescence staining for α-smooth muscle actin (α-SMA) in lung sections from Hhip+/- mice and their wild type littermates, Hhip+/+ mice. Airway remodeling was assessed in Hhip+/- and Hhip+/- mice exposed to 6 months of cigarette smoke. Our results show HHIP inhibited aerobic glycolysis and represses cell proliferation in COPD-derived ASMCs. Notably, knockdown of HHIP in normal ASMCs increased PKM2 activity. Importantly, Hhip+/- mice demonstrated increased airway remodeling and increased intensity of α-SMA staining around airways compared to Hhip+/+ mice. In conclusion, our findings suggest that HHIP represses aerobic glycolysis and ASMCs hyperplasia, which may contribute to the increased airway remodeling in Hhip+/- mice.

Surface-bound matrix metalloproteinase-8 on macrophages: Contributions to macrophage pericellular proteolysis and migration through tissue barriers.

OBJECTIVE: MMP-8 binds to surface-bound tissue inhibitor of metalloproteinase-1 (TIMP-1) on PMNs to promote pericellular proteolysis during the development of inflammatory diseases associated with tissue destruction. Little is known about the biology of MMP-8 in macrophages. We tested the hypotheses that: (1) MMP-8 and TIMP-1 are also expressed on the surface of activated macrophages, (2) surface-bound MMP-8 on macrophages promotes TIMP-resistant pericellular proteolysis and macrophage migration through tissue barriers, and (3) MMP-8 binds to surface-bound TIMP-1 on macrophages. METHODS: Surface MMP-8 and TIMP-1 levels were measured on human monocyte-derived macrophages (MDM) and/or murine macrophages using immunostaining, biotin-labeling, and substrate cleavage methods. The susceptibility of membrane-bound Mmp-8 on activated macrophages from wild-type (WT) mice to TIMPs was measured. Migration of WT and Mmp-8-/- macrophages through models of tissue barriers in vitro and the accumulation of peritoneal macrophages in WT versus Mmp-8-/- mice with sterile peritonitis was compared. Surface levels of Mmp-8 were compared on activated macrophages from WT and Timp-1-/- mice. RESULTS: Lipopolysaccharides and a cluster of differentiation 40 ligand increased surface MMP-8 and/or TIMP-1 staining and surface type I collagenase activity on MDM and/or murine macrophages. Activated Mmp-8-/- macrophages degraded less type I collagen than activated WT macrophages. The surface type-I collagenase activity on WT macrophages was resistant to inhibition by Timp-1. Peritoneal macrophage accumulation was similar in WT and Mmp-8-/- mice with sterile acute peritonitis. However, Mmp-8-/- macrophages migrated less efficiently through models of tissue barriers (especially those containing type I collagen) than WT cells. Activated WT and Timp-1-/- macrophages had similar surface-bound Mmp-8 levels. CONCLUSIONS: MMP-8 and TIMP-1 are expressed on the surface of activated human MDM and murine macrophages, but Mmp-8 is unlikely to bind to surface-bound Timp-1 on these cells. Surface-bound MMP-8 contributes to TIMP-resistant monocyte/macrophage pericellular proteolysis and macrophage migration through collagen-containing tissue barriers.

Long-term safety and efficacy of tezacaftor-ivacaftor in individuals with cystic fibrosis aged 12 years or older who are homozygous or heterozygous for Phe508del CFTR (EXTEND): an open-label extension study.

BACKGROUND: Tezacaftor-ivacaftor is an approved cystic fibrosis transmembrane conductance regulator (CFTR) modulator shown to be efficacious and generally safe and well tolerated over 8-24 weeks in phase 3 clinical studies in participants aged 12 years or older with cystic fibrosis homozygous for the Phe508del CFTR mutation (F/F; study 661-106 [EVOLVE]) or heterozygous for the Phe508del CFTR mutation and a residual function mutation (F/RF; study 661-108 [EXPAND]). Longer-term (>24 weeks) safety and efficacy of tezacaftor-ivacaftor has not been assessed in clinical studies. Here, we present results of study 661-110 (EXTEND), a 96-week open-label extension study that assessed long-term safety, tolerability, and efficacy of tezacaftor-ivacaftor in participants aged 12 years or older with cystic fibrosis who were homozygous or heterozygous for the Phe508del CFTR mutation. METHODS: Study 661-110 was a 96-week, phase 3, multicentre, open-label study at 170 clinical research sites in Australia, Europe, Israel, and North America. Participants were aged 12 years or older, had cystic fibrosis, were homozygous or heterozygous for Phe508del CFTR, and completed one of six parent studies of tezacaftor-ivacaftor: studies 661-103, 661-106, 661-107, 661-108, 661-109, and 661-111. Participants received oral tezacaftor 100 mg once daily and oral ivacaftor 150 mg once every 12 h for up to 96 weeks. The primary endpoint was safety and tolerability. Secondary endpoints were changes in lung function, nutritional parameters, and respiratory symptom scores; pulmonary exacerbations; and pharmacokinetic parameters. A post-hoc analysis assessed the rate of lung function decline in F/F participants who received up to 120 weeks of tezacaftor-ivacaftor in studies 661-106 (F/F) and/or 661-110 compared with a matched cohort of CFTR modulator-untreated historical F/F controls from the Cystic Fibrosis Foundation Patient Registry. Primary safety analyses were done in all participants from all six parent studies who received at least one dose of study drug during this study. This study was registered at ClinicalTrials.gov (NCT02565914). FINDINGS: Between Aug 31, 2015, to May 31, 2019, 1044 participants were enrolled in study 661-110 from the six parent studies of whom 1042 participants received at least one dose of study drug and were included in the safety set. 995 (95%) participants had at least one TEAE; 22 (2%) had TEAEs leading to discontinuation; and 351 (34%) had serious TEAEs. No deaths occurred during the treatment-emergent period; after the treatment-emergent period, two deaths occurred, which were both deemed unrelated to study drug. F/F (106/110; n=459) and F/RF (108/110; n=226) participants beginning tezacaftor-ivacaftor in study 661-110 had improvements in efficacy endpoints consistent with parent studies; improvements in lung function and nutritional parameters and reductions in pulmonary exacerbations observed in the tezacaftor-ivacaftor groups in the parent studies were generally maintained in study 661-110 for an additional 96 weeks. Pharmacokinetic parameters were also similar to those in the parent studies. The annualised rate of lung function decline was 61·5% (95% CI 35·8 to 86·1) lower in tezacaftor-ivacaftor-treated F/F participants versus untreated matched historical controls. INTERPRETATION: Tezacaftor-ivacaftor was generally safe, well tolerated, and efficacious for up to 120 weeks, and the safety profile of tezacaftor-ivacaftor in study 661-110 was consistent with cystic fibrosis manifestations and with the safety profiles of the parent studies. The rate of lung function decline was significantly reduced in F/F participants, consistent with cystic fibrosis disease modification. Our results support the clinical benefit of long-term tezacaftor-ivacaftor treatment for people aged 12 years or older with cystic fibrosis with F/F or F/RF genotypes. FUNDING: Vertex Pharmaceuticals Incorporated.

Long-term safety and efficacy of lumacaftor-ivacaftor therapy in children aged 6-11 years with cystic fibrosis homozygous for the F508del-CFTR mutation: a phase 3, open-label, extension study.

BACKGROUND: The safety and efficacy of 24 weeks of lumacaftor-ivacaftor combination therapy in children aged 6-11 years with cystic fibrosis homozygous for the F508del-CFTR mutation was previously shown in two phase 3 studies. Here, we report long-term safety and efficacy data. METHODS: In this phase 3, open-label, multicentre, extension study (study 110), we examined the long-term safety, tolerability, and efficacy of lumacaftor-ivacaftor in children pooled from two phase 3 parent studies (open-label study 011B and randomised, placebo-controlled study 109). The study was conducted at 61 clinics in the USA, Australia, Belgium, Canada, Denmark, France, Germany, Sweden, and the UK. Children with cystic fibrosis homozygous for the F508del-CFTR mutation who had received lumacaftor-ivacaftor or placebo in the parent studies were treated with lumacaftor-ivacaftor for up to 96 weeks; those who had received the combination therapy in the parent studies (the treatment-to-treatment group) received up to 120 weeks of treatment in total. Participants aged 6-11 years at the start of the parent study received lumacaftor 200 mg-ivacaftor 250 mg orally once every 12 h; those aged 12 years or older received lumacaftor 400 mg-ivacaftor 250 mg orally once every 12 h. The primary endpoint was safety and tolerability in all children who had received at least one dose of the study drug. Secondary endpoints included change from baseline in lung clearance index 2·5% (LCI2·5), sweat chloride concentration, body-mass index, and Cystic Fibrosis Questionnaire-Revised respiratory domain score. This extension study is registered with ClinicalTrials.gov, NCT02544451, and has been completed. FINDINGS: The extension study ran from Aug 13, 2015, to Aug 17, 2018. Of 239 children who enrolled in the study and received at least one dose of lumacaftor-ivacaftor, 215 (90%) completed 96 weeks of treatment. Most children (236 [99%] of 239 children) had adverse events that were mild (49 [21%] of 239) or moderate (148 [62%] of 239) in severity, and there was a low rate of adverse events leading to treatment discontinuation. The most frequently reported adverse events were common manifestations or complications of cystic fibrosis, such as cough and pulmonary exacerbation, or were consistent with the known safety profile of lumacaftor-ivacaftor in older children and adults. No new safety concerns were identified with extended lumacaftor-ivacaftor treatment. Children in the placebo-to-treatment group had improvements in efficacy endpoints consistent with those observed in the parent studies. Improvements observed in children treated with lumacaftor-ivacaftor in the parent study were generally maintained in the extension study. INTERPRETATION: Lumacaftor-ivacaftor therapy in children homozygous for F508del-CFTR who initiated treatment at age 6-11 years was generally safe and well tolerated, and efficacy was sustained for up to 120 weeks. These data support the long-term use of lumacaftor-ivacaftor to treat children aged 6 years and older who are homozygous for the F508del-CFTR mutation. FUNDING: Vertex Pharmaceuticals Incorporated.

A disintegrin and metalloproteinase domain-15 deficiency leads to exaggerated cigarette smoke-induced chronic obstructive pulmonary disease (COPD)-like disease in mice.

A disintegrin and metalloproteinase domain-15 (ADAM15) is expressed by cells implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), but its contributions to COPD are unknown. To address this gap, ADAM15 levels were measured in samples from cigarette smoke (CS)-versus air-exposed wild-type (WT) mice. CS-induced COPD-like disease was compared in CS-exposed WT, Adam15-/-, and Adam15 bone marrow chimeric mice. CS exposure increased Adam15 expression in lung macrophages and CD8+ T cells and to a lesser extent in airway epithelial cells in WT mice. CS-exposed Adam15-/- mice had greater emphysema, small airway fibrosis, and lung inflammation (macrophages and CD8+ T cells) than WT mice. Adam15 bone marrow chimera studies revealed that Adam15 deficiency in leukocytes led to exaggerated pulmonary inflammation and COPD-like disease in mice. Adam15 deficiency in CD8+ T cells was required for the exaggerated pulmonary inflammation and COPD-like disease in CS-exposed Adam15-/- mice (as assessed by genetically deleting CD8+ T cells in Adam15-/- mice). Adam15 deficiency increased pulmonary inflammation by rendering CD8+ T cells and macrophages resistant to CS-induced activation of the mitochondrial apoptosis pathway by preserving mTOR signaling and intracellular Mcl-1 levels in these cells. These results strongly link ADAM15 deficiency to the pathogenesis of COPD.

A phase 3, randomized, double-blind, parallel-group study to evaluate tezacaftor/ivacaftor in people with cystic fibrosis heterozygous for F508del-CFTR and a gating mutation.

BACKGROUND: Tezacaftor (TEZ)/ivacaftor (IVA) is an approved CFTR modulator shown to be efficacious and generally safe and well tolerated in people ≥12 years of age with cystic fibrosis (CF) homozygous for the F508del-CFTR mutation or heterozygous for the F508del-CFTR mutation and a residual function mutation. Although previous studies with IVA alone showed clinical benefits in people with CFTR gating mutations, TEZ/IVA has not yet been evaluated in a Phase 3 study of participants heterozygous for F508del-CFTR and a gating mutation (F/gating genotypes). Here, we present results from a randomized, double-blind, IVA-controlled, parallel-group, Phase 3 study assessing the efficacy, safety, and pharmacokinetics (PK) of TEZ/IVA in participants ≥12 years of age with F/gating genotypes. METHODS: Enrolled participants entered a 4-week IVA run-in period to create a stable IVA baseline. Participants were then randomized to receive IVA or TEZ/IVA for 8 weeks in an active comparator treatment period (ACTP). The primary endpoint was absolute change in percent predicted forced expiratory volume in 1 second (ppFEV1). Key secondary endpoints were relative change in ppFEV1 and absolute change in CF Questionnaire-Revised respiratory domain score. Secondary endpoints included absolute change in sweat chloride (SwCl) concentration, PK parameters, and safety. All endpoints except PK parameters and safety were assessed from baseline through Week 8. RESULTS: Sixty-nine participants (92.0%) in the IVA group and 75 participants (98.7%) in the TEZ/IVA group completed treatment. No improvements were seen in efficacy endpoints from baseline at the end of the IVA run-in period through the end of the ACTP in the IVA group. No significant differences in ppFEV1 or any key secondary endpoint were observed between the IVA and TEZ/IVA groups. SwCl concentrations decreased more in the TEZ/IVA versus IVA group during the ACTP. The safety profile and PK parameters of TEZ/IVA were consistent with those of previous studies in participants ≥12 years of age with CF. CONCLUSIONS: This Phase 3 study showed that the dual-combination regimen of TEZ/IVA demonstrated clinical efficacy but did not have significantly greater clinical efficacy than IVA alone in participants ≥12 years of age with F/gating genotypes. However, as reported in other studies, TEZ/IVA was generally safe and well tolerated (NCT02412111).

A phase 3, double-blind, parallel-group study to evaluate the efficacy and safety of tezacaftor in combination with ivacaftor in participants 6 through 11 years of age with cystic fibrosis homozygous for F508del or heterozygous for the F508del-CFTR mutation and a residual function mutation.

BACKGROUND: The CFTR modulator tezacaftor/ivacaftor was efficacious and generally safe and well tolerated in Phase 3 studies in participants ≥12 years of age with cystic fibrosis (CF) homozygous for the F508del-CFTR mutation or heterozygous with a residual function-CFTR mutation (F/F or F/RF respectively). We evaluated tezacaftor/ivacaftor's efficacy and safety over 8 weeks in participants 6 through 11 years of age with these mutations. METHODS: Participants were randomized 4:1 to tezacaftor/ivacaftor or a blinding group (placebo for F/F, ivacaftor for F/RF). The primary endpoint was within-group change from baseline in the lung clearance index 2·5 (LCI2·5) through Week 8. Secondary endpoints were change from baseline in sweat chloride (SwCl), cystic fibrosis questionnaire-revised (CFQ-R) respiratory domain score, and safety. RESULTS: Sixty-seven participants received at least one study drug dose. Of those, 54 received tezacaftor/ivacaftor (F/F, 42; F/RF, 12), 10 placebo, and 3 ivacaftor; 66 completed the study. The within-group change in LCI2·5 was significantly reduced (improved) by -0·51 (95% CI: -0·74, -0·29). SwCl concentration decreased (improved) by -12·3 mmol/L and CFQ-R respiratory domain score increased (improved, nonsignificantly) by 2·3 points. There were no serious adverse events (AEs) or AEs leading to tezacaftor/ivacaftor discontinuation or interruption. The most common AEs (≥10%) in participants receiving tezacaftor/ivacaftor were cough, headache, and productive cough. CONCLUSIONS: Tezacaftor/ivacaftor improved lung function (assessed using LCI) and CFTR function (measured by SwCl concentration) in participants 6 through 11 years of age with F/F or F/RF genotypes. Tezacaftor/ivacaftor was safe and well tolerated; no new safety concerns were identified.

ADAM15 expression is increased in lung CD8+ T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction.

BACKGROUND: A disintegrin and metalloproteinase domain-15 (ADAM15) is expressed by activated leukocytes, and fibroblasts in vitro. Whether ADAM15 expression is increased in the lungs of COPD patients is not known. METHODS: ADAM15 gene expression and/or protein levels were measured in whole lung and bronchoalveolar lavage (BAL) macrophage samples obtained from COPD patients, smokers, and non-smokers. Soluble ADAM15 protein levels were measured in BAL fluid (BALF) and plasma samples from COPD patients and controls. Cells expressing ADAM15 in the lungs were identified using immunostaining. Staining for ADAM15 in different cells in the lungs was related to forced expiratory volume in 1 s (FEV1), ratio of FEV1 to forced vital capacity (FEV1/FVC), and pack-years of smoking history. RESULTS: ADAM15 gene expression and/or protein levels were increased in alveolar macrophages and whole lung samples from COPD patients versus smokers and non-smokers. Soluble ADAM15 protein levels were similar in BALF and plasma samples from COPD patients and controls. ADAM15 immunostaining was increased in macrophages, CD8+ T cells, epithelial cells, and airway α-smooth muscle (α-SMA)-positive cells in the lungs of COPD patients. ADAM15 immunostaining in macrophages, CD8+ T cells and bronchial (but not alveolar) epithelial cells was related inversely to FEV1 and FEV1/FVC, but not to pack-years of smoking history. ADAM15 staining levels in airway α-SMA-positive cells was directly related to FEV1/FVC. Over-expressing ADAM15 in THP-1 cells reduced their release of matrix metalloproteinases and CCL2. CONCLUSIONS: These results link increased ADAM15 expression especially in lung leukocytes and bronchial epithelial cells to the pathogenesis of COPD.

Tezacaftor/ivacaftor in people with cystic fibrosis heterozygous for minimal function CFTR mutations.

BACKGROUND: Tezacaftor/ivacaftor is a CFTR modulator approved to treat people with cystic fibrosis (pwCF) who are homozygous (F/F) or heterozygous for the F508del-CFTR mutation and a residual function mutation (F/RF). This randomized, double-blind, placebo-controlled Phase 3 study evaluated the efficacy, safety, tolerability, and pharmacokinetics (PK) of tezacaftor/ivacaftor in participants ≥12 years of age heterozygous for the F508del-CFTR mutation and a minimal function mutation (F/MF), which produces no CFTR protein or a protein unresponsive to tezacaftor/ivacaftor in vitro. METHODS: Participants were randomized 1:1 to receive tezacaftor/ivacaftor or placebo for 12 weeks. The primary endpoint was the absolute change from baseline in percent predicted forced expiratory volume in 1 second (ppFEV1) between the tezacaftor/ivacaftor and placebo groups through week 12. Key secondary endpoints included absolute change from baseline in CF Questionnaire-Revised respiratory domain scores and the number of pulmonary exacerbations through week 12 and the absolute change from baseline in body mass index at week 12. A prespecified interim analysis (IA) for futility was conducted when approximately 50% of a planned enrollment of 300 participants reached week 12 of the study. RESULTS: At the time of the IA, 83 participants were randomized to tezacaftor/ivacaftor and 85 to placebo; 165 participants completed treatment. The study failed to demonstrate that tezacaftor/ivacaftor significantly improved ppFEV1 or any of the key secondary endpoints and was terminated for futility. The safety profile and PK parameters of tezacaftor/ivacaftor were similar to those reported in prior studies in participants ≥12 years of age with CF. CONCLUSIONS: Tezacaftor/ivacaftor did not show a clinically meaningful benefit in participants with F/MF genotypes but was generally safe and well tolerated, consistent with the safety profile reported in other Phase 3 studies (NCT02516410).

ADAM (a Disintegrin and Metalloproteinase) 15 Deficiency Exacerbates Ang II (Angiotensin II)-Induced Aortic Remodeling Leading to Abdominal Aortic Aneurysm.

OBJECTIVE: ADAM (a disintegrin and metalloproteinase) 15-a membrane-bound metalloprotease from the ADAM (disintegrin and metalloproteinase) family-has been linked to endothelial permeability, inflammation, and metastasis. However, its function in aortic aneurysm has not been explored. We aimed to determine the function of ADAM15 in the pathogenesis of aortic remodeling and aneurysm formation. Approach and Results: Male Adam15-deficient and WT (wild type) mice (10 weeks old), on standard laboratory diet, received Ang II (angiotensin II; 1.5 mg/kg per day) or saline (Alzet pump) for 2 or 4 weeks. Ang II increased ADAM15 in WT aorta, while Adam15-deficiency resulted in abdominal aortic aneurysm characterized by loss of medial smooth muscle cells (SMCs), elastin fragmentation, inflammation, but unaltered Ang II-mediated hypertension. In the abdominal aortic tissue and primary aortic SMCs culture, Adam15 deficiency decreased SMC proliferation, increased apoptosis, and reduced contractile properties along with F-actin depolymerization to G-actin. Ang II triggered a markedly greater increase in THBS (thrombospondin) 1 in Adam15-deficient aorta, primarily the medial layer in vivo, and in aortic SMC in vitro; increased SSH1 (slingshot homolog 1) phosphatase activity and cofilin dephosphorylation that promoted F-actin depolymerization and G-actin accumulation. rhTHBS1 (recombinant THBS1) alone was sufficient to activate the cofilin pathway, increase G-actin, and induce apoptosis of aortic SMCs, confirming the key role of THBS1 in this process. Further, in human abdominal aortic aneurysm specimens, decreased ADAM15 was associated with increased THBS1 levels and loss of medial SMCs. CONCLUSIONS: This study is the first to demonstrate a key role for ADAM15 in abdominal aortic aneurysm through regulating the SMC function, thereby placing ADAM15 in a critical position as a potential therapeutic target for abdominal aortic aneurysm.

CFTR regulates B cell activation and lymphoid follicle development.

BACKGROUND: Cystic fibrosis (CF) is an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that promotes persistent lung infection and inflammation and progressive loss of lung function. Patients with CF have increased lung lymphoid follicles (LFs) and B cell-activating factor of tumor necrosis factor family (BAFF) that regulates B cell survival and maturation. A direct role for CFTR in B cell activation and disease pathogenesis in CF remains unclear. METHODS: The number of LFs, BAFF+, TLR4+ and proliferation marker Ki67+ B cells in lung explants or resections from subjects with CF and normal controls was quantified by immunostaining. The role of CFTR in B cell activation and LF development was then examined in two independent cohorts of uninfected CFTR-deficient mice (Cftr -/-) and wild type controls. The number of lung LFs, B cells and BAFF+, CXCR4+, immunoglobulin G+ B cells was examined by immunostaining. Lung and splenocyte B cell activation marker and major histocompatibility complex class II (MHC class II) expression was quantified by flow cytometry. Inflammatory cytokine levels were measured in supernatants from isolated B cells from Cftr -/- and wild type mice stimulated in vitro with Pseudomonas aeruginosa lipopolysaccharide (LPS). RESULTS: There was a significant increase in well-formed LFs in subjects with CF compared to normal controls. Increased B cell activation and proliferation was observed in lung LFs from CF subjects as was quantified by a significant increase in B cell BAFF, TLR4 and Ki67 expression. Uninfected Cftr -/- mice had increased lung LFs and BAFF+ and CXCR4+ B cells compared to wild type controls. Lung B cells isolated from uninfected Cftr -/- mice demonstrated increased MHC class II expression. In vitro, isolated B cells from Cftr -/- mice produced increased IL-6 when stimulated with LPS compared to wild type controls. CONCLUSIONS: These data support a direct role for CFTR in B cell activation, proliferation and inflammatory cytokine production that promotes lung LF follicle development in cystic fibrosis.