Small airway fibroblasts from patients with chronic obstructive pulmonary disease exhibit cellular senescence.

Small airway disease (SAD) is a key early-stage pathology of chronic obstructive pulmonary disease (COPD). COPD is associated with cellular senescence whereby cells undergo growth arrest and express the senescence-associated secretory phenotype (SASP) leading to chronic inflammation and tissue remodeling. Parenchymal-derived fibroblasts have been shown to display senescent properties in COPD, however small airway fibroblasts (SAFs) have not been investigated. Therefore, this study investigated the role of these cells in COPD and their potential contribution to SAD. To investigate the senescent and fibrotic phenotype of SAF in COPD, SAFs were isolated from nonsmoker, smoker, and COPD lung resection tissue (n = 9-17 donors). Senescence and fibrotic marker expressions were determined using iCELLigence (proliferation), qPCR, Seahorse assay, and ELISAs. COPD SAFs were further enriched for senescent cells using FACSAria Fusion based on cell size and autofluorescence (10% largest/autofluorescent vs. 10% smallest/nonautofluorescent). The phenotype of the senescence-enriched population was investigated using RNA sequencing and pathway analysis. Markers of senescence were observed in COPD SAFs, including senescence-associated ß-galactosidase, SASP release, and reduced proliferation. Because the pathways driving this phenotype were unclear, we used cell sorting to enrich senescent COPD SAFs. This population displayed increased p21CIP1 and p16INK4a expression and mitochondrial dysfunction. RNA sequencing suggested these senescent cells express genes involved in oxidative stress response, fibrosis, and mitochondrial dysfunction pathways. These data suggest COPD SAFs are senescent and may be associated with fibrotic properties and mitochondrial dysfunction. Further understanding of cellular senescence in SAFs may lead to potential therapies to limit SAD progression.NEW & NOTEWORTHY Fibroblasts and senescence are thought to play key roles in the pathogenesis of small airway disease and COPD; however, the characteristics of small airway-derived fibroblasts are not well explored. In this study we isolate and enrich the senescent small airway-derived fibroblast (SAF) population from COPD lungs and explore the pathways driving this phenotype using bulk RNA-seq.

Hepcidin Is Essential for Alveolar Macrophage Function and Is Disrupted by Smoke in a Murine Chronic Obstructive Pulmonary Disease Model.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow-derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.

MicroRNA-570 is a novel regulator of cellular senescence and inflammaging.

Diseases of accelerated aging often occur together (multimorbidity), and their prevalence is increasing, with high societal and health care costs. Chronic obstructive pulmonary disease (COPD) is one such condition, in which one half of patients exhibit ≥4 age-related diseases. Diseases of accelerated aging share common molecular pathways, which lead to the detrimental accumulation of senescent cells. These senescent cells no longer divide but release multiple inflammatory proteins, known as the senescence-associated secretory phenotype, which may perpetuate and speed disease. Here, we show that inhibiting miR-570-3p, which is increased in COPD cells, reverses cellular senescence by restoring the antiaging molecule sirtuin-1. MiR-570-3p is induced by oxidative stress in airway epithelial cells through p38 MAP kinase-c-Jun signaling and drives senescence by inhibiting sirtuin-1. Inhibition of elevated miR-570-3p in COPD small airway epithelial cells, using an antagomir, restores sirtuin-1 and suppresses markers of cellular senescence (p16INK4a, p21Waf1, and p27Kip1), thereby restoring cellular growth by allowing progression through the cell cycle. MiR-570-3p inhibition also suppresses the senescence-associated secretory phenotype (matrix metalloproteinases-2/9, C-X-C motif chemokine ligand 8, IL-1ß, and IL-6). Collectively, these data suggest that inhibiting miR-570-3p rejuvenates cells via restoration of sirtuin-1, reducing many of the abnormalities associated with cellular senescence.-Baker, J. R., Vuppusetty, C., Colley, T., Hassibi, S., Fenwick, P. S., Donnelly, L. E., Ito, K., Barnes, P. J. MicroRNA-570 is a novel regulator of cellular senescence and inflammaging.

A Pilot Study of the Immunologic, Virologic, and Pathologic Consequences of Intra-anal 5% Imiquimod in HIV-1-Infected Men With High-Grade Squamous Intraepithelial Lesions.

BACKGROUND: Imiquimod can be used to treat internal anal high-grade squamous intraepithelial lesions. In HIV-1-infected individuals there is a theoretical concern for increased HIV replication in anorectal tissue secondary to imiquimod-induced mucosal inflammation. OBJECTIVE: The purpose of this study was to assess local virologic, immunologic, and pathologic effects of imiquimod treatment in HIV-infected individuals. DESIGN: This was a pilot study at a single academic center. SETTINGS: The study was conducted at the University of Pittsburgh Anal Dysplasia Clinic. PATIENTS: HIV-1-infected individuals with biopsy-confirmed internal anal high-grade squamous intraepithelial lesions were included. INTERVENTION: Imiquimod cream was prescribed for intra-anal use 3 times per week for 9 weeks. MAIN OUTCOME MEASURES: Anal human papillomavirus typing, anal and rectal tissue HIV-1 RNA and DNA quantification, cytokine gene expression, and anal histology were measured. RESULTS: Nine evaluable participants (1 participant was lost to follow-up) were all white men with a median age of 46 years (interquartile range = 12 y) and a median CD4 T-cell count of 480 cells per cubic millimeter (interquartile range = 835). All were taking antiretroviral therapy, and 7 of 9 had HIV-1 RNA <50 copies per milliliter. The median dose of imiquimod used was 27.0 (interquartile range = 3.5), and there was a median of 11 days (interquartile range = 10 d) from last dose to assessment. There was no progression to cancer, no significant change in the number of human papillomavirus types detected, and no significant change in quantifiable cytokines/HIV-1 RNA or DNA levels in anal or rectal tissue. Seven (35%) of 20 high-grade lesions resolved to low-grade squamous intraepithelial lesions. LIMITATIONS: The study was limited by the small number of participants and variable time to final assessment. CONCLUSIONS: Intra-anal imiquimod showed no evidence of immune activation or increase in HIV-1 viral replication in anal and rectal tissue and confirmed efficacy for intra-anal high-grade squamous intraepithelial lesion treatment morbidity. See Video Abstract at http://links.lww.com/DCR/A498.

Decreased phosphatase PTEN amplifies PI3K signaling and enhances proinflammatory cytokine release in COPD.

The phosphatidylinositol 3-kinase (PI3K) pathway is activated in chronic obstructive pulmonary disease (COPD), but the regulatory mechanisms for this pathway are yet to be elucidated. The aim of this study was to determine the expression and role of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the PI3K pathway, in COPD. PTEN protein expression was measured in the peripheral lung of COPD patients compared with smoking and nonsmoking controls. The direct influence of cigarette smoke extract (CSE) on PTEN expression was assessed using primary lung epithelial cells and a cell line (BEAS-2B) in the presence or absence of l-buthionine-sulfoximine (BSO) to deplete intracellular glutathione. The impact of PTEN knockdown by RNA interference on cytokine production was also examined. In peripheral lung, PTEN protein was significantly decreased in patients with COPD compared with the subjects without COPD (P < 0.001) and positively correlated with the severity of airflow obstruction (forced expiratory volume in 1-s percent predicted; r = 0.50; P = 0.0012). Conversely, phosphorylated Akt, as a marker of PI3K activation, showed a negative correlation with PTEN protein levels (r = -0.41; P = 0.0042). In both primary bronchial epithelial cells and BEAS-2B cells, CSE decreased PTEN protein, which was reversed by N-acetyl cysteine treatment. PTEN knockdown potentiated Akt phosphorylation and enhanced production of proinflammatory cytokines, such as IL-6, CXCL8, CCL2, and CCL5. In conclusion, oxidative stress reduces PTEN protein levels, which may result in increased PI3K signaling and amplification of inflammation in COPD.

A Pilot Study of the Prevalence of Anal Human Papillomavirus and Dysplasia in a Cohort of Patients With IBD.

BACKGROUND: Defective cell-mediated immunity increases the risk of human papillomavirus-associated anal dysplasia and cancer. There is limited information on anal canal disease in patients with IBD. OBJECTIVE: The purpose of this study was to assess anal/vaginal human papillomavirus and anal dysplasia prevalence in patients with IBD. DESIGN: Patients had an anal examination before routine colonoscopy. SETTINGS: The study was conducted at a tertiary IBD referral center. PATIENTS: We studied a convenience sample of sexually active male and female patients with IBD who were not on biological therapy. INTERVENTION: Anal examination, anal and vaginal human papillomavirus testing, anal cytology, and high-resolution anoscopy/biopsy were carried out. MAIN OUTCOME MEASURES: Anal and vaginal human papillomavirus types, anal cytology, and biopsy grade were measured. RESULTS: Twenty-five male and 21 female evaluable participants, 31 with Crohn's disease, 14 with ulcerative colitis, and 1 with indeterminate colitis, were predominantly white (91.3%), treatment experienced (76.1%), an average age of 38.1 years (range, 22.0-66.0 y), and had an average length of IBD diagnosis of 9.3 years (range, 1.0-33.0 y). Eighteen (39.1%) had an abnormal perianal examination and 3 (6.5%) had an abnormal digital examination. Forty-one (89.1%) had anal human papillomavirus, 16 with a single type and 25 with multiple types (range, 2-5 types). Human papillomavirus type 16 was most common (65.2%), followed by human papillomavirus types 11 and 45 (37.0% each). Nineteen of 21 (90.5%) women had vaginal human papillomavirus. Overall, 21 (45.7%) had abnormal anal cytology. Thirty three (71.7%) had ≥1 anal biopsy (9 had multiple), with dysplasia diagnosed in 28 (60.9%) and high-grade and low-grade squamous intraepithelial lesions diagnosed in 4 (8.7%) and 24 (43.5%). LIMITATIONS: No control group was included, and no detailed sexual history was taken. CONCLUSIONS: A high prevalence of anal and vaginal human papillomavirus and anal dysplasia was demonstrated in the study population outcomes. See Video Abstract at http://links.lww.com/DCR/A379.

Topical application of trichloroacetic acid is efficacious for the treatment of internal anal high-grade squamous intraepithelial lesions in HIV-positive men.

OBJECTIVES: To assess the efficacy of topical 80% trichloroacetic acid (TCA) to treat internal anal high-grade squamous intraepithelial lesions (HSILs) in HIV-positive individuals. METHODS: All patients who attended the University of Pittsburgh Anal Dysplasia Clinic for treatment of biopsy-proven internal anal HSIL with topical TCA between July 1, 2009, and June 30, 2012, and who had 1 or more follow-up visits to assess treatment efficacy were included in the analysis. Recurrence of HSIL was assessed in July 1, 2013. RESULTS: A total of 98 HSILs from 72 patients were treated, and 77 (78.6%) resolved to normal epithelium or low-grade SIL during follow-up. Forty-eight (49.0%) and 27 (27.6%) of lesions resolved with 1 and 2 TCA treatments, respectively, whereas 1 lesion (1%) each resolved with 3 and 4 TCA treatments. Twenty-one (21.4%) lesions in 20 patients remained without resolution after TCA treatments. These patients were offered an alternative treatment. During follow-up, 8 (15.1%) of 53 patients had a lesion that recurred at the index site (11/53 [20.8%], inclusive of adjacent lesions) and 17 patients had new lesions diagnosed. CONCLUSIONS: Topical TCA is an efficacious treatment of internal anal HSIL in an anal dysplasia clinic setting with high-resolution anoscopy capacity. Advantages of TCA for this recurrent disease process include the following: low cost, no requirement for special equipment beyond that for high-resolution anoscopy, and painless application procedure. A larger prospective comparative study would better define efficacy and patient acceptability between treatment methods.

Coordinated nasal mucosa-mediated immunity accelerates recovery from COVID-19.

Introduction: Understanding the interplay of immune mediators in relation to clinical outcomes during acute infection has the potential to highlight immune networks critical to symptom recovery. The objective of the present study was to elucidate the immune networks critical to early symptom resolution following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Methods: In a community-based randomised clinical trial comparing inhaled budesonide against usual care in 139 participants with early onset SARS-CoV-2 (the STOIC study; clinicaltrials.gov identifier NCT04416399), significant clinical deterioration (reported need for urgent care, emergency department visit, hospitalisation: the primary outcome), self-reported symptom severity (Influenza Patient-Reported Outcome questionnaire) and immune mediator networks were assessed. Immune mediator networks were determined using pre-defined mathematical modelling of immune mediators, determined by the Meso Scale Discovery U-Plex platform, within the first 7 days of SARS-CoV-2 infection compared to 22 healthy controls. Results: Interferon- and chemokine-dominant networks were associated with high viral burden. Elevated levels of the mucosal network (chemokine (C-C motif) ligand (CCL)13, CCL17, interleukin (IL)-33, IL-5, IL-4, CCL26, IL-2, IL-12 and granulocyte-macrophage colony-stimulating factor) was associated with a mean 3.7-day quicker recovery time, with no primary outcome events, irrespective of treatment arm. This mucosal network was associated with initial nasal and throat symptoms at day 0. Conclusion: A nasal immune network is critical to accelerated recovery and improved patient outcomes in community-acquired viral infections. Overall, early prognostication and treatments aimed at inducing epithelial responses may prove clinically beneficial in enhancing early host response to virus.

Single cell quantification of microRNA from small numbers of non-invasively sampled primary human cells.

Expression levels of microRNAs (miRNAs) in single cells are low and conventional miRNA detection methods require amplification that can be complex, time-consuming, costly and may bias results. Single cell microfluidic platforms have been developed; however, current approaches are unable to absolutely quantify single miRNA molecules expressed in single cells. Herein, we present an amplification-free sandwich hybridisation assay to detect single miRNA molecules in single cells using a microfluidic platform that optically traps and lyses individual cells. Absolute quantification of miR-21 and miR-34a molecules was achieved at a single cell level in human cell lines and validated using real-time qPCR. The sensitivity of the assay was demonstrated by quantifying single miRNA molecules in nasal epithelial cells and CD3+ T-cells, as well as nasal fluid collected non-invasively from healthy individuals. This platform requires ~50 cells or ~30 µL biofluid and can be extended for other miRNA targets therefore it could monitor miRNA levels in disease progression or clinical studies.

IL-36 receptor agonist and antagonist imbalance drives neutrophilic inflammation in COPD.

Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating alternative therapies. Here, we show that COPD subjects have increased IL-36γ and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared with control subjects. IL-36γ is derived from small airway epithelial cells (SAEC) and is further induced by a viral mimetic, whereas IL-36Ra is derived from macrophages. IL-36γ stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36γ, thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, which was inhibited by exogenous IL-36Ra. The use of a therapeutic antibody that inhibits binding to the IL-36R attenuated IL-36γ-driven inflammation and cellular crosstalk. We have demonstrated a mechanism for the amplification and propagation of neutrophilic inflammation in COPD and have shown that blocking this cytokine family via a IL-36R neutralizing antibody could be a promising therapeutic strategy in the treatment of COPD.

Early Th2 inflammation in the upper respiratory mucosa as a predictor of severe COVID-19 and modulation by early treatment with inhaled corticosteroids: a mechanistic analysis.

BACKGROUND: Community-based clinical trials of the inhaled corticosteroid budesonide in early COVID-19 have shown improved patient outcomes. We aimed to understand the inflammatory mechanism of budesonide in the treatment of early COVID-19. METHODS: The STOIC trial was a randomised, open label, parallel group, phase 2 clinical intervention trial where patients were randomly assigned (1:1) to receive usual care (as needed antipyretics were only available treatment) or inhaled budesonide at a dose of 800 µg twice a day plus usual care. For this experimental analysis, we investigated the nasal mucosal inflammatory response in patients recruited to the STOIC trial and in a cohort of SARS-CoV-2-negative healthy controls, recruited from a long-term observational data collection study at the University of Oxford. In patients with SARS-CoV-2 who entered the STOIC study, nasal epithelial lining fluid was sampled at day of randomisation (day 0) and at day 14 following randomisation, blood samples were also collected at day 28 after randomisation. Nasal epithelial lining fluid and blood samples were collected from the SARS-CoV-2 negative control cohort. Inflammatory mediators in the nasal epithelial lining fluid and blood were assessed for a range of viral response proteins, and innate and adaptive response markers using Meso Scale Discovery enzyme linked immunoassay panels. These samples were used to investigate the evolution of inflammation in the early COVID-19 disease course and assess the effect of budesonide on inflammation. FINDINGS: 146 participants were recruited in the STOIC trial (n=73 in the usual care group; n=73 in the budesonide group). 140 nasal mucosal samples were available at day 0 (randomisation) and 122 samples at day 14. At day 28, whole blood was collected from 123 participants (62 in the budesonide group and 61 in the usual care group). 20 blood or nasal samples were collected from healthy controls. In early COVID-19 disease, there was an enhanced inflammatory airway response with the induction of an anti-viral and T-helper 1 and 2 (Th1/2) inflammatory response compared with healthy individuals. Individuals with COVID-19 who clinically deteriorated (ie, who met the primary outcome) showed an early blunted respiratory interferon response and pronounced and persistent Th2 inflammation, mediated by CC chemokine ligand (CCL)-24, compared with those with COVID-19 who did not clinically deteriorate. Over time, the natural course of COVID-19 showed persistently high respiratory interferon concentrations and elevated concentrations of the eosinophil chemokine, CCL-11, despite clinical symptom improvement. There was persistent systemic inflammation after 28 days following COVID-19, including elevated concentrations of interleukin (IL)-6, tumour necrosis factor-α, and CCL-11. Budesonide treatment modulated inflammation in the nose and blood and was shown to decrease IL-33 and increase CCL17. The STOIC trial was registered with ClinicalTrials.gov, NCT04416399. INTERPRETATION: An initial blunted interferon response and heightened T-helper 2 inflammatory response in the respiratory tract following SARS-CoV-2 infection could be a biomarker for predicting the development of severe COVID-19 disease. The clinical benefit of inhaled budesonide in early COVID-19 is likely to be as a consequence of its inflammatory modulatory effect, suggesting efficacy by reducing epithelial damage and an improved T-cell response. FUNDING: Oxford National Institute of Health Research Biomedical Research Centre and AstraZeneca.

Leukocyte Function in COPD: Clinical Relevance and Potential for Drug Therapy.

Chronic obstructive pulmonary disease (COPD) is a progressive lung condition affecting 10% of the global population over 45 years. Currently, there are no disease-modifying treatments, with current therapies treating only the symptoms of the disease. COPD is an inflammatory disease, with a high infiltration of leukocytes being found within the lung of COPD patients. These leukocytes, if not kept in check, damage the lung, leading to the pathophysiology associated with the disease. In this review, we focus on the main leukocytes found within the COPD lung, describing how the release of chemokines from the damaged epithelial lining recruits these cells into the lung. Once present, these cells become active and may be driven towards a more pro-inflammatory phenotype. These cells release their own subtypes of inflammatory mediators, growth factors and proteases which can all lead to airway remodeling, mucus hypersecretion and emphysema. Finally, we describe some of the current therapies and potential new targets that could be utilized to target aberrant leukocyte function in the COPD lung. Here, we focus on old therapies such as statins and corticosteroids, but also look at the emerging field of biologics describing those which have been tested in COPD already and potential new monoclonal antibodies which are under review.

Cigarette smoke-induced impairment of autophagy in macrophages increases galectin-8 and inflammation.

Cigarette smoke impairs autophagy, an intracellular protein degradation system, but the consequences of this defect have not been fully elucidated, especially in macrophages. Dysfunctional alveolar macrophages play an important role in chronic obstructive pulmonary disease (COPD). Here we show that galectin-8, a danger receptor that identifies damaged intracellular host vesicles and initiates autophagosome engulfment, is elevated due to activation of autophagy by cigarette smoke extract (CSE) in macrophages. CSE impaired autophagic flux in PMA-differentiated U937 macrophage-like cells, resulting in intracellular accumulation of galectin-8 and the autophagic adaptor protein NDP52. COPD patients showed elevated levels of galectin-8 and NDP52 in the lung homogenates with significant increase in the serum galectin-8 levels in patients with frequent acute exacerbations. Soluble galectin-8 induced interleukin (IL)-6 release in bronchial epithelial cells via PI3Kα signalling. Thus, increased galectin-8 due to CSE-induced impaired autophagy may be involved in the pathogenesis of COPD and may be a biomarker of this disease.

IL36 is a critical upstream amplifier of neutrophilic lung inflammation in mice.

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases.