Defining the mechanism of galectin-3-mediated TGF-ß1 activation and its role in lung fibrosis.

Integrin-mediated activation of the profibrotic mediator transforming growth factor-ß1 (TGF-ß1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesized that galectin-3 potentiates TGF-ß1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-ß1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt-induced integrin-mediated TGF-ß1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvß1, αvß5, and αvß6, and to the TGFßRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3, which target the carbohydrate recognition domain. Galectin-3 binding to ß1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and ß1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-ß1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-ß1 stimulation, colocalization was detectable only in HLFs from IPF patients, suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients' reduced Col1a1, TIMP1, and hyaluronan secretion to a similar degree as TGF-ß type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-ß1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-ß1 signaling cascade.

Conformational pathology of the serpins: themes, variations, and therapeutic strategies.

Point mutations cause members of the serine protease inhibitor (serpin) superfamily to undergo a novel conformational transition, forming ordered polymers. These polymers characterize a group of diseases termed the serpinopathies. The formation of polymers underlies the retention of alpha(1)-antitrypsin within hepatocytes and of neuroserpin within neurons to cause cirrhosis and dementia, respectively. Point mutations of antithrombin, C1 inhibitor, alpha(1)-antichymotrypsin, and heparin cofactor II cause a similar conformational transition, resulting in a plasma deficiency that is associated with thrombosis, angioedema, and emphysema. Polymers of serpins can also form in extracellular tissues where they activate inflammatory cascades. This is best described for the Z variant of alpha(1)-antitrypsin in which the proinflammatory properties of polymers provide an explanation for both progressive emphysema and the selective advantage of this mutant allele. Therapeutic strategies are now being developed to block the aberrant conformational transitions and so treat the serpinopathies.

Residual Lung Abnormalities after COVID-19 Hospitalization: Interim Analysis of the UKILD Post-COVID-19 Study.

Rationale: Shared symptoms and genetic architecture between coronavirus disease (COVID-19) and lung fibrosis suggest severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may lead to progressive lung damage. Objectives: The UK Interstitial Lung Disease Consortium (UKILD) post-COVID-19 study interim analysis was planned to estimate the prevalence of residual lung abnormalities in people hospitalized with COVID-19 on the basis of risk strata. Methods: The PHOSP-COVID-19 (Post-Hospitalization COVID-19) study was used to capture routine and research follow-up within 240 days from discharge. Thoracic computed tomography linked by PHOSP-COVID-19 identifiers was scored for the percentage of residual lung abnormalities (ground-glass opacities and reticulations). Risk factors in linked computed tomography were estimated with Bayesian binomial regression, and risk strata were generated. Numbers within strata were used to estimate posthospitalization prevalence using Bayesian binomial distributions. Sensitivity analysis was restricted to participants with protocol-driven research follow-up. Measurements and Main Results: The interim cohort comprised 3,700 people. Of 209 subjects with linked computed tomography (median, 119 d; interquartile range, 83-155), 166 people (79.4%) had more than 10% involvement of residual lung abnormalities. Risk factors included abnormal chest X-ray (risk ratio [RR], 1.21; 95% credible interval [CrI], 1.05-1.40), percent predicted DlCO less than 80% (RR, 1.25; 95% CrI, 1.00-1.56), and severe admission requiring ventilation support (RR, 1.27; 95% CrI, 1.07-1.55). In the remaining 3,491 people, moderate to very high risk of residual lung abnormalities was classified at 7.8%, and posthospitalization prevalence was estimated at 8.5% (95% CrI, 7.6-9.5), rising to 11.7% (95% CrI, 10.3-13.1) in the sensitivity analysis. Conclusions: Residual lung abnormalities were estimated in up to 11% of people discharged after COVID-19-related hospitalization. Health services should monitor at-risk individuals to elucidate long-term functional implications.

Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.

OBJECTIVE: Alpha-1 antitrypsin deficiency (AATD) is a common, potentially lethal inborn disorder caused by mutations in alpha-1 antitrypsin (AAT). Homozygosity for the 'Pi*Z' variant of AAT (Pi*ZZ genotype) causes lung and liver disease, whereas heterozygous 'Pi*Z' carriage (Pi*MZ genotype) predisposes to gallstones and liver fibrosis. The clinical significance of the more common 'Pi*S' variant remains largely undefined and no robust data exist on the prevalence of liver tumours in AATD. DESIGN: Baseline phenotypes of AATD individuals and non-carriers were analysed in 482 380 participants in the UK Biobank. 1104 participants of a multinational cohort (586 Pi*ZZ, 239 Pi*SZ, 279 non-carriers) underwent a comprehensive clinical assessment. Associations were adjusted for age, sex, body mass index, diabetes and alcohol consumption. RESULTS: Among UK Biobank participants, Pi*ZZ individuals displayed the highest liver enzyme values, the highest occurrence of liver fibrosis/cirrhosis (adjusted OR (aOR)=21.7 (8.8-53.7)) and primary liver cancer (aOR=44.5 (10.8-183.6)). Subjects with Pi*MZ genotype had slightly elevated liver enzymes and moderately increased odds for liver fibrosis/cirrhosis (aOR=1.7 (1.2-2.2)) and cholelithiasis (aOR=1.3 (1.2-1.4)). Individuals with homozygous Pi*S mutation (Pi*SS genotype) harboured minimally elevated alanine aminotransferase values, but no other hepatobiliary abnormalities. Pi*SZ participants displayed higher liver enzymes, more frequent liver fibrosis/cirrhosis (aOR=3.1 (1.1-8.2)) and primary liver cancer (aOR=6.6 (1.6-26.9)). The higher fibrosis burden was confirmed in a multinational cohort. Male sex, age ≥50 years, obesity and the presence of diabetes were associated with significant liver fibrosis. CONCLUSION: Our study defines the hepatobiliary phenotype of individuals with the most relevant AATD genotypes including their predisposition to liver tumours, thereby allowing evidence-based advice and individualised hepatological surveillance.

Real-world clinical applicability of pathogenicity predictors assessed on SERPINA1 mutations in alpha-1-antitrypsin deficiency.

The growth of publicly available data informing upon genetic variations, mechanisms of disease, and disease subphenotypes offers great potential for personalized medicine. Computational approaches are likely required to assess a large number of novel genetic variants. However, the integration of genetic, structural, and pathophysiological data still represents a challenge for computational predictions and their clinical use. We addressed these issues for alpha-1-antitrypsin deficiency, a disease mediated by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin. We compiled a comprehensive database of SERPINA1 coding mutations and assigned them apparent pathological relevance based upon available data. "Benign" and "pathogenic" variations were used to assess performance of 31 pathogenicity predictors. Well-performing algorithms clustered the subset of variants known to be severely pathogenic with high scores. Eight new mutations identified in the ExAC database and achieving high scores were selected for characterization in cell models and showed secretory deficiency and polymer formation, supporting the predictive power of our computational approach. The behavior of the pathogenic new variants and consistent outliers were rationalized by considering the protein structural context and residue conservation. These findings highlight the potential of computational methods to provide meaningful predictions of the pathogenic significance of novel mutations and identify areas for further investigation.

Aberrant disulphide bonding contributes to the ER retention of alpha1-antitrypsin deficiency variants.

Mutations in alpha1-antitrypsin (AAT) can cause the protein to polymerise and be retained in the endoplasmic reticulum (ER) of hepatocytes. The ensuing systemic AAT deficiency leads to pulmonary emphysema, while intracellular polymers are toxic and cause chronic liver disease. The severity of this process varies considerably between individuals, suggesting the involvement of mechanistic co-factors and potential for therapeutically beneficial interventions. We show in Hepa1.6 cells that the mildly polymerogenic I (Arg39Cys) AAT mutant forms aberrant inter- and intra-molecular disulphide bonds involving the acquired Cys39 and the only cysteine residue in the wild-type (M) sequence (Cys232). Substitution of Cys39 to serine partially restores secretion, showing that disulphide bonding contributes to the intracellular retention of I AAT. Covalent homodimers mediated by inter-Cys232 bonding alone are also observed in cells expressing the common Z and other polymerising AAT variants where conformational behaviour is abnormal, but not in those expressing M AAT. Prevention of such disulphide linkage through the introduction of the Cys232Ser mutation or by treatment of cells with reducing agents increases Z AAT secretion. Our results reveal that disulphide interactions enhance intracellular accumulation of AAT mutants and implicate the oxidative ER state as a pathogenic co-factor. Redox modulation, e.g. by anti-oxidant strategies, may therefore be beneficial in AAT deficiency-associated liver disease.

hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency.

BACKGROUND & AIMS: α1-Antitrypsin deficiency (A1ATD) is an autosomal recessive disorder caused by mutations in the SERPINA1 gene. Individuals with the Z variant (Gly342Lys) retain polymerised protein in the endoplasmic reticulum (ER) of their hepatocytes, predisposing them to liver disease. The concomitant lack of circulating A1AT also causes lung emphysema. Greater insight into the mechanisms that link protein misfolding to liver injury will facilitate the design of novel therapies. METHODS: Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes provide a novel approach to interrogate the molecular mechanisms of A1ATD because of their patient-specific genetic architecture and reflection of human physiology. To that end, we utilised patient-specific hiPSC hepatocyte-like cells (ZZ-HLCs) derived from an A1ATD (ZZ) patient, which faithfully recapitulated key aspects of the disease at the molecular and cellular level. Subsequent functional and "omics" comparisons of these cells with their genetically corrected isogenic-line (RR-HLCs) and primary hepatocytes/human tissue enabled identification of new molecular markers and disease signatures. RESULTS: Our studies showed that abnormal A1AT polymer processing (immobilised ER components, reduced luminal protein mobility and disrupted ER cisternae) occurred heterogeneously within hepatocyte populations and was associated with disrupted mitochondrial structure, presence of the oncogenic protein AKR1B10 and two upregulated molecular clusters centred on members of inflammatory (IL-18 and Caspase-4) and unfolded protein response (Calnexin and Calreticulin) pathways. These results were validated in a second patient-specific hiPSC line. CONCLUSIONS: Our data identified novel pathways that potentially link the expression of Z A1AT polymers to liver disease. These findings could help pave the way towards identification of new therapeutic targets for the treatment of A1ATD. LAY SUMMARY: This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease α1-antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human-induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance.

Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization.

Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate-and therefore polymerize-more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the "breach" region and "shutter" region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.

Update on alpha-1 antitrypsin deficiency: New therapies.

α1-Antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin within the endoplasmic reticulum of hepatocytes. The retention of mutant protein causes hepatic damage and cirrhosis whilst the lack of an important circulating protease inhibitor predisposes the individuals with severe α1-antitrypsin deficiency to early onset emphysema. Our work over the past 25years has led to new paradigms for the liver and lung disease associated with α1-antitrypsin deficiency. We review here the molecular pathology of the cirrhosis and emphysema associated with α1-antitrypsin deficiency and show how an understanding of this condition provided the paradigm for a wider group of disorders that we have termed the serpinopathies. The detailed understanding of the pathobiology of α1-antitrypsin deficiency has identified important disease mechanisms to target. As a result, several novel parallel and complementary therapeutic approaches are in development with some now in clinical trials. We provide an overview of these new therapies for the liver and lung disease associated with α1-antitrypsin deficiency.

Defining the mechanism of polymerization in the serpinopathies.

The serpinopathies result from the ordered polymerization of mutants of members of the serine proteinase inhibitor (serpin) superfamily. These polymers are retained within the cell of synthesis where they cause a toxic gain of function. The serpinopathies are exemplified by inclusions that form with the common severe Z mutant of α(1)-antitrypsin that are associated with liver cirrhosis. There is considerable controversy as to the pathway of serpin polymerization and the structure of pathogenic polymers that cause disease. We have used synthetic peptides, limited proteolysis, monoclonal antibodies, and ion mobility-mass spectrometry to characterize the polymerogenic intermediate and pathological polymers formed by Z α(1)-antitrypsin. Our data are best explained by a model in which polymers form through a single intermediate and with a reactive center loop-ß-sheet A linkage. Our data are not compatible with the recent model in which polymers are linked by a ß-hairpin of the reactive center loop and strand 5A. Understanding the structure of the serpin polymer is essential for rational drug design strategies that aim to block polymerization and so treat α(1)-antitrypsin deficiency and the serpinopathies.

Multi-center evaluation of baseline neutrophil-to-lymphocyte (NLR) ratio as an independent predictor of mortality and clinical risk stratifier in idiopathic pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal disorder with a variable disease trajectory. The aim of this study was to assess the potential of neutrophil-to-lymphocyte ratio (NLR) to predict outcomes in IPF. Methods: We adopted a two-stage discovery (n = 71) and validation (n = 134) design using patients from the UCL partners (UCLp) cohort. We then combined discovery and validation cohorts and included an additional 794 people with IPF, using real-life data from 5 other UK centers, to give a combined cohort of 999 patients. Data were collected from patients presenting over a 13-year period (2006-2019) with mean follow up of 3.7 years (censoring: 2018-2020). Findings: In the discovery analysis, we showed that high values of NLR (>/ = 2.9 vs < 2.9) were associated with increased risk of mortality in IPF (HR 2.04, 95% CI 1.09-3.81, n = 71, p = 0.025). This was confirmed in the validation (HR 1.91, 95% CI 1.15-3.18, n = 134, p = 0.0114) and combined cohorts (HR 1.65, n = 999, 95% CI 1.39-1.95; p < 0·0001). NLR correlated with GAP stage and GAP index (p < 0.0001). Stratifying patients by NLR category (low/high) showed significant differences in survival for GAP stage 2 (p < 0.0001), however not for GAP stage 1 or 3. In a multivariate analysis, a high NLR was an independent predictor of mortality/progression after adjustment for individual GAP components and steroid/anti-fibrotic use (p < 0·03). Furthermore, incorporation of baseline NLR in a modified GAP-stage/index, GAP-index/stage-plus, refined prognostic ability as measured by concordance (C)-index. Interpretation: We have identified NLR as a widely available test that significantly correlates with lung function, can predict outcomes in IPF and refines cohort staging with GAP. NLR may allow timely prioritisation of at-risk patients, even in the absence of lung function. Funding: Breathing Matters, GSK, CF Trust, BLF-Asthma, MRC, NIHR Alpha-1 Foundation.

Safety, tolerability, and efficacy of pirfenidone in patients with rheumatoid arthritis-associated interstitial lung disease: a randomised, double-blind, placebo-controlled, phase 2 study.

BACKGROUND: Interstitial lung disease is a known complication of rheumatoid arthritis, with a lifetime risk of developing the disease in any individual of 7·7%. We aimed to assess the safety, tolerability, and efficacy of pirfenidone for the treatment of patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD). METHODS: TRAIL1 was a randomised, double-blind, placebo-controlled, phase 2 trial done in 34 academic centres specialising in interstitial lung disease in four countries (the UK, the USA, Australia, and Canada). Adults aged 18-85 years were eligible for inclusion if they met the 2010 American College of Rheumatology and European Alliance of Associations for Rheumatology criteria for rheumatoid arthritis and had interstitial lung disease on a high-resolution CT scan imaging and, when available, lung biopsy. Exclusion criteria include smoking, clinical history of other known causes of interstitial lung disease, and coexistant clinically significant COPD or asthma. Patients were randomly assigned (1:1) to receive 2403 mg oral pirfenidone (pirfenidone group) or placebo (placebo group) daily. The primary endpoint was the incidence of the composite endpoint of a decline from baseline in percent predicted forced vital capacity (FVC%) of 10% or more or death during the 52-week treatment period assessed in the intention-to-treat population. Key secondary endpoints included change in absolute and FVC% over 52 weeks, the proportion of patients with a decline in FVC% of 10% or more, and the frequency of progression as defined by Outcome Measures in Rheumatoid Arthritis Clinical Trials (OMERACT) in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02808871. FINDINGS: From May 15, 2017, to March 31, 2020, 231 patients were assessed for inclusion, of whom 123 patients were randomly assigned (63 [51%] to the pirfenidone group and 60 [49%] to the placebo group). The trial was stopped early (March 31, 2020) due to slow recruitment and the COVID-19 pandemic. The difference in the proportion of patients who met the composite primary endpoint (decline in FVC% from baseline of 10% or more or death) between the two groups was not significant (seven [11%] of 63 patients in the pirfenidone group vs nine [15%] of 60 patients in the placebo group; OR 0·67 [95% CI 0·22 to 2·03]; p=0·48). Compared with the placebo group, patients in the pirfenidone group had a slower rate of decline in lung function, measured by estimated annual change in absolute FVC (-66 vs -146; p=0·0082) and FVC% (-1·02 vs -3·21; p=0·0028). The groups were similar with regards to the decline in FVC% by 10% or more (five [8%] participants in the pirfenidone group vs seven [12%] in the placebo group; OR 0·52 [95% CI 0·14-1·90]; p=0·32) and the frequency of progression as defined by OMERACT (16 [25%] in the pirfenidone group vs 19 [32%] in the placebo group; OR 0·68 [0·30-1·54]; p=0·35). There was no significant difference in the rate of treatment-emergent serious adverse events between the two groups, and there were no treatment-related deaths. INTERPRETATION: Due to early termination of the study and underpowering, the results should be interpreted with caution. Despite not meeting the composite primary endpoint, pirfenidone slowed the rate of decline of FVC over time in patients with RA-ILD. Safety in patients with RA-ILD was similar to that seen in other pirfenidone trials. FUNDING: Genentech.

A novel monoclonal antibody to characterize pathogenic polymers in liver disease associated with alpha1-antitrypsin deficiency.

UNLABELLED: Alpha(1)-antitrypsin is the most abundant circulating protease inhibitor. The severe Z deficiency allele (Glu342Lys) causes the protein to undergo a conformational transition and form ordered polymers that are retained within hepatocytes. This causes neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. We have developed a conformation-specific monoclonal antibody (2C1) that recognizes the pathological polymers formed by alpha(1)-antitrypsin. This antibody was used to characterize the Z variant and a novel shutter domain mutant (His334Asp; alpha(1)-antitrypsin King's) identified in a 6-week-old boy who presented with prolonged jaundice. His334Asp alpha(1)-antitrypsin rapidly forms polymers that accumulate within the endoplasmic reticulum and show delayed secretion when compared to the wild-type M alpha(1)-antitrypsin. The 2C1 antibody recognizes polymers formed by Z and His334Asp alpha(1)-antitrypsin despite the mutations directing their effects on different parts of the protein. This antibody also recognized polymers formed by the Siiyama (Ser53Phe) and Brescia (Gly225Arg) mutants, which also mediate their effects on the shutter region of alpha(1)-antitrypsin. CONCLUSION: Z and shutter domain mutants of alpha(1)-antitrypsin form polymers with a shared epitope and so are likely to have a similar structure.

Therapeutic target-site variability in α1-antitrypsin characterized at high resolution.

The intrinsic propensity of α(1)-antitrypsin to undergo conformational transitions from its metastable native state to hyperstable forms provides a motive force for its antiprotease function. However, aberrant conformational change can also occur via an intermolecular linkage that results in polymerization. This has both loss-of-function and gain-of-function effects that lead to deficiency of the protein in human circulation, emphysema and hepatic cirrhosis. One of the most promising therapeutic strategies being developed to treat this disease targets small molecules to an allosteric site in the α(1)-antitrypsin molecule. Partial filling of this site impedes polymerization without abolishing function. Drug development can be improved by optimizing data on the structure and dynamics of this site. A new 1.8 Å resolution structure of α(1)-antitrypsin demonstrates structural variability within this site, with associated fluctuations in its upper and lower entrance grooves and ligand-binding characteristics around the innermost stable enclosed hydrophobic recess. These data will allow a broader selection of chemotypes and derivatives to be tested in silico and in vitro when screening and developing compounds to modulate conformational change to block the pathological mechanism while preserving function.

The Induction of Alpha-1 Antitrypsin by Vitamin D in Human T Cells Is TGF-ß Dependent: A Proposed Anti-inflammatory Role in Airway Disease.

Background: Vitamin D upregulates anti-inflammatory and antimicrobial pathways that promote respiratory health. Vitamin D synthesis is initiated following skin exposure to sunlight, however nutritional supplementation can be required to address deficiency, for example during the winter months or due to cultural constraints. We recently reported that 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment induced alpha-1 antitrypsin (AAT) expression in CD4+, but not CD8+ T cells, with evidence supporting an immunoregulatory role. Research Question: To understand the relationship between vitamin D, lung AAT levels and T lymphocytes further we investigated whether TGF-ß is required as a co-factor for 1,25(OH)2D3-induced upregulation of AAT by vitamin D in CD8+ T cells in vitro and correlated circulating vitamin D levels with lung AAT levels in vivo. Results: 1,25(OH)2D3 in combination with TGF-ß1 increased AAT expression by CD8+ T cells, as well as VDR and RXRα gene expression, which may partly explain the requirement for TGF-ß. CD4+ T cells may also require autocrine stimulation with TGF-ß as a co-factor since 1,25(OH)2D3 was associated with increased TGF-ß bioactivity and neutralisation of TGF-ß partially abrogated 1,25(OH)2D3-induced SERPINA1 gene expression. Neither CD4+ nor CD8+ T cells responded to the circulating vitamin D precursor, 25-hydroxyvitamin D3 for induction of SERPINA1, suggesting that local generation of 1,25(OH)2D3 is required. Transcriptional gene profiling studies previously demonstrated that human bronchial epithelial cells rapidly increased TGF-ß2 gene expression in response to 1,25(OH)2D3. Here, human epithelial cells responded to precursor 25(OH)D3 to increase bioactive TGF-ß synthesis. CD8+ T cells responded comparably to TGF-ß1 and TGF-ß2 to increase 1,25(OH)2D3-induced AAT. However, CD8+ T cells from adults with AAT-deficiency, homozygous for the Z allele of SERPINA1, were unable to mount this response. AAT levels in the airways of children with asthma and controls correlated with circulating 25(OH)D3. Conclusions: Vitamin D increases AAT expression in human T cells and this response is impaired in T cells from individuals homozygous for the Z allele of SERPINA1 in a clinic population. Furthermore, a correlation between circulating vitamin D and airway AAT is reported. We propose that vitamin D-induced AAT contributes to local immunomodulation and airway health effects previously attributed to vitamin D.

Use of peripheral neutrophil to lymphocyte ratio and peripheral monocyte levels to predict survival in fibrotic hypersensitivity pneumonitis (fHP): a multicentre retrospective cohort study.

The factors determining disease course and survival in fibrotic hypersensitivity pneumonitis (fHP) have not been fully elucidated.The aim of this study was to describe the characteristics of patients with fHP in a real-world cohort and investigate factors associated with worse outcomes. We aimed to explore the use of neutrophil to lymphocyte ratio (NLR) and peripheral blood monocyte levels in predicting mortality. METHODS: A retrospective, multicentre, observational UK cohort study. RESULTS: Patients with fHP were significantly younger than those with idiopathic pulmonary fibrosis (IPF) (median age fHP 73 vs IPF 75 years) and were much more likely to be woman (fHP 61% vs IPF 26%). In almost half of all fHP cases (49%, n=104/211), no causative antigen was identified from either the history or specific antigen testing. Overall, fHP was associated with a better survival than IPF, although median survival of both groups was poor (fHP 62 months vs IPF 52 months).IPF survival in patients with a high NLR was significantly lower than those with a low NLR (44 vs 83 months). A monocyte count ≥0.95 K/uL also predicted significantly poorer outcomes for patients with IPF compared with <0.95 K/uL (33 vs 57 months). In contrast, NLR and monocyte count did not predict survival in the fHP cohort. CONCLUSIONS: Although fHP has a statistically lower mortality than IPF, absolute survival time of both conditions is poor. High baseline NLR and absolute monocyte counts predict worse survival in IPF but not in fHP, highlighting the potential for divergence in their pathogenic mechanisms.

Understanding the burden of interstitial lung disease post-COVID-19: the UK Interstitial Lung Disease-Long COVID Study (UKILD-Long COVID).

INTRODUCTION: The COVID-19 pandemic has led to over 100 million cases worldwide. The UK has had over 4 million cases, 400 000 hospital admissions and 100 000 deaths. Many patients with COVID-19 suffer long-term symptoms, predominantly breathlessness and fatigue whether hospitalised or not. Early data suggest potentially severe long-term consequence of COVID-19 is development of long COVID-19-related interstitial lung disease (LC-ILD). METHODS AND ANALYSIS: The UK Interstitial Lung Disease Consortium (UKILD) will undertake longitudinal observational studies of patients with suspected ILD following COVID-19. The primary objective is to determine ILD prevalence at 12 months following infection and whether clinically severe infection correlates with severity of ILD. Secondary objectives will determine the clinical, genetic, epigenetic and biochemical factors that determine the trajectory of recovery or progression of ILD. Data will be obtained through linkage to the Post-Hospitalisation COVID platform study and community studies. Additional substudies will conduct deep phenotyping. The Xenon MRI investigation of Alveolar dysfunction Substudy will conduct longitudinal xenon alveolar gas transfer and proton perfusion MRI. The POST COVID-19 interstitial lung DiseasE substudy will conduct clinically indicated bronchoalveolar lavage with matched whole blood sampling. Assessments include exploratory single cell RNA and lung microbiomics analysis, gene expression and epigenetic assessment. ETHICS AND DISSEMINATION: All contributing studies have been granted appropriate ethical approvals. Results from this study will be disseminated through peer-reviewed journals. CONCLUSION: This study will ensure the extent and consequences of LC-ILD are established and enable strategies to mitigate progression of LC-ILD.