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.

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.

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.

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.

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.

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.

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.

Non-invasive testing for liver pathology in alpha-1 antitrypsin deficiency.

BACKGROUND: Many patients with alpha-1 antitrypsin deficiency (A1ATD) receive care in respiratory clinics without access to specialist hepatology expertise. Liver disease can develop asymptomatically, and non-invasive markers of fibrosis may help identify patients who require definitive assessment with liver biopsy. We evaluated the utility of non-invasive markers of liver fibrosis in A1ATD to guide testing in settings without ready access to hepatology expertise. METHODS: Patients attending the London A1ATD service undergo assessment using blood tests to calculate the 'APRI' and 'FIB-4' score, liver ultrasound and Fibroscan. Liver biopsy is offered to patients who have abnormal liver function tests with abnormal liver ultrasound and/or liver stiffness >6 kPa on Fibroscan. Liver biopsies were assessed for the presence of A1AT, steatosis, fibrosis and inflammation. RESULTS: 75 patients with A1ATD had results for analysis, 56% were female, age 16-82 years. 75% of patients had Fibroscan <6 kPa, 19% had Fibroscan 6-7.9 kPa and 6%>8 kPa. There was a significant correlation between FIB-4 and Fibroscan (r=0.244, p=0.035). Fibroscan >6 kPa corresponded to a FIB-4 score of >1.26. However, FIB-4 >1.26 had poor sensitivity (47%), specificity (32%) and positive-predictive value (PPV; 36%) to identify Fibroscan >6 kPa. The negative-predictive value (NPV) was stronger at 81%. APRI data were similar. Twelve patients underwent liver biopsy, with 11 reports available for analysis. Six had FIB-4 scores<1.26 and five had Fibroscan of <6 kPa. A1AT was present in 64% of biopsies, steatosis in 82%, mild fibrosis in 36%, moderate fibrosis in 9% and severe fibrosis in 9%. CONCLUSION: A combination of liver ultrasound and non-invasive fibrosis tests can help identify patients with A1ATD liver injury. However, APRI and FIB-4 scores alone had poor sensitivity and specificity to justify use as an independent tool for liver pathology in A1ATD.

The structural basis for Z α1-antitrypsin polymerization in the liver.

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α1-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α1-antitrypsin as "polymer" chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α1-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α1-antitrypsin.

Relationship of CT densitometry to lung physiological parameters and health status in alpha-1 antitrypsin deficiency: initial report of a centralised database of the NIHR rare diseases translational research collaborative.

OBJECTIVES: To establish a database network for the study of alpha-1 antitrypsin deficiency (AATD) and compare the results to CT lung density as the most direct measure of emphysema. DESIGN: A central electronic database was established to permit the upload of anonymised patient data from remote sites. Prospectively collected CT data were recorded onto disc, anonymised, analysed at the coordinating centre and compared with the clinical features of the disease. SETTING: Tertiary referral centres with expertise in the management of AATD focused on academic Biomedical Research Units and Wellcome Clinical Research Facilities. PARTICIPANTS: Data were collected from 187 patients over 1 year from eight UK academic sites. This included patient demographics, postbronchodilator physiology, health status and CT. Analysis was undertaken at the coordinating centre in Birmingham. RESULTS: Patient recruitment in the 12 months reached 94% of target (set at 200) covering the whole spectrum of the disease from those with normal lung function to very severe chronic obstructive lung disease. CT scan suitable for analysis was available from 147 (79%) of the patients. CT density, analysed as the threshold for the lowest 15% of lung voxels, showed statistically significant relationships with the objective physiological parameters of lung function as determined by spirometric Global Initiative for Chronic Obstructive Lung Disease (GOLD) severity staging (p<0.001) and carbon monoxide gas transfer (p<0.01). Density also correlated with subjective measures of quality of life (p=0.02). CONCLUSIONS: Establishment of the network for data collection and its transfer was highly successful facilitating future collaboration for the study of this rare disease and its management. CT densitometry correlated well with the objective clinical features of the disease supporting its role as the specific marker of the associated emphysema and its severity. Correlations with subjective measures of health, however, were generally weak indicating other factors play a role.

Tensin1 expression and function in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) constitutes a major cause of morbidity and mortality. Genome wide association studies have shown significant associations between airflow obstruction or COPD with a non-synonymous SNP in the TNS1 gene, which encodes tensin1. However, the expression, cellular distribution and function of tensin1 in human airway tissue and cells are unknown. We therefore examined these characteristics in tissue and cells from controls and people with COPD or asthma. Airway tissue was immunostained for tensin1. Tensin1 expression in cultured human airway smooth muscle cells (HASMCs) was evaluated using qRT-PCR, western blotting and immunofluorescent staining. siRNAs were used to downregulate tensin1 expression. Tensin1 expression was increased in the airway smooth muscle and lamina propria in COPD tissue, but not asthma, when compared to controls. Tensin1 was expressed in HASMCs and upregulated by TGFß1. TGFß1 and fibronectin increased the localisation of tensin1 to fibrillar adhesions. Tensin1 and α-smooth muscle actin (αSMA) were strongly co-localised, and tensin1 depletion in HASMCs attenuated both αSMA expression and contraction of collagen gels. In summary, tensin1 expression is increased in COPD airways, and may promote airway obstruction by enhancing the expression of contractile proteins and their localisation to stress fibres in HASMCs.

An in vitro investigation of the inflammatory response to the strain amplitudes which occur during high frequency oscillation ventilation and conventional mechanical ventilation.

Children randomised in the neonatal period to high frequency oscillatory ventilation (HFOV) or conventional mechanical ventilation (CMV) in the United Kingdom Oscillation study (UKOS) had superior lung function at 11 to 14 years of age. During HFOV, much smaller tidal volumes, but a higher mean airway distending pressure is delivered, hence, a possible explanation for a volume dependent effect on long term lung function could be an increase in inflammation in response to higher tidal volumes and strains. We tested that hypothesis by assessing interleukin-6 (IL-6) and -8 (IL-8) release from A549 alveolar analogue cells following biaxial mechanical strain applied at 0.5 Hz occurring during conditions mimicking strain during CMV (5-20% strain) and conditions mimicking strain during HFOV (17.5% ±â€¯2.5% strain) for up to 4 h. Cyclic strain of 5-20%, occurring during CMV, increased levels of both IL-6 and IL-8 compared to unstrained controls, while 17.5% ±â€¯2.5% strain, occurring during HFOV, was associated with significantly lower levels of IL-6 (46.31 ±â€¯2.66 versus 56.79 ±â€¯3.73 pg/mL) and IL-8 (1340.2 ±â€¯74.9 versus 2522 ±â€¯248 pg/mL) secretion compared to conditions occurring during CMV at four hours. These results may provide a possible explanation for the superior lung function in 11-14-year-old children who had been supported in the neonatal period by HFOV.

Vitamin D (1,25(OH)2D3) induces α-1-antitrypsin synthesis by CD4+ T cells, which is required for 1,25(OH)2D3-driven IL-10.

Studies to identify novel immune-regulatory functions of active vitamin D (1,25(OH)2D3) in human CD4+ T cells revealed that 1,25(OH)2D3 potently induced expression of the gene SERPINA1, encoding the anti-protease α-1-antitrypsin. We confirmed α-1-antitrypsin protein expression by 1,25(OH)2D3-treated CD4+ T cells, but not in CD8+ T cells or monocytes. α-1-Antitrypsin promotes anti-inflammatory IL-10 synthesis in other immune cell populations. We therefore investigated its immune-regulatory effects in CD4+ T cells. Plasma-derived α-1-antitrypsin drove IL-10 synthesis by CD4+ T cells, which was not dependent on anti-protease activity, but appeared to require a serum-binding factor, since this could not be achieved with recombinant protein. α-1-Antitrypsin is reported to bind complement components, which regulate T cell function. A role for this interaction was therefore probed. Plasma-derived, but not recombinant α-1-antitrypsin contained C3a. Surface Plasmon Resonance and Microscale Thermophoresis demonstrated α-1-antitrypsin binding to C3a. Addition of C3a to CD4+ T cells cultured with recombinant α-1-antitrypsin restored induction of IL-10, whereas neutralisation of C3a abrogated IL-10 induced by plasma-derived α-1-antitrypsin. To interrogate an endogenous role for the α-1-antitrypsin-C3a axis in 1,25(OH)2D3-driven CD4+ T cell IL-10 synthesis, we treated cells from healthy or α-1-antitrypsin-deficient individuals (which transcribe SERPINA1 but do not secrete protein) with 1,25(OH)2D3. A significant correlation was identified between SERPINA1 and IL10 gene expression in healthy donor CD4+ T cells, which was absent in cells from α-1-antitrypsin-deficient individuals. Therefore, α-1-antitrypsin is required for 1,25(OH)2D3-induced IL-10 expression in CD4+ T cells, interacting with C3a to drive IL-10 expression.

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.

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.

Multicentre evaluation of multidisciplinary team meeting agreement on diagnosis in diffuse parenchymal lung disease: a case-cohort study.

BACKGROUND: Diffuse parenchymal lung disease represents a diverse and challenging group of pulmonary disorders. A consistent diagnostic approach to diffuse parenchymal lung disease is crucial if clinical trial data are to be applied to individual patients. We aimed to evaluate inter-multidisciplinary team agreement for the diagnosis of diffuse parenchymal lung disease. METHODS: We did a multicentre evaluation of clinical data of patients who presented to the interstitial lung disease unit of the Royal Brompton and Harefield NHS Foundation Trust (London, UK; host institution) and required multidisciplinary team meeting (MDTM) characterisation between March 1, 2010, and Aug 31, 2010. Only patients whose baseline clinical, radiological, and, if biopsy was taken, pathological data were undertaken at the host institution were included. Seven MDTMs, consisting of at least one clinician, radiologist, and pathologist, from seven countries (Denmark, France, Italy, Japan, Netherlands, Portugal, and the UK) evaluated cases of diffuse parenchymal lung disease in a two-stage process between Jan 1, and Oct 15, 2015. First, the clinician, radiologist, and pathologist (if lung biopsy was completed) independently evaluated each case, selected up to five differential diagnoses from a choice of diffuse lung diseases, and chose likelihoods (censored at 5% and summing to 100% in each case) for each of their differential diagnoses, without inter-disciplinary consultation. Second, these specialists convened at an MDTM and reviewed all data, selected up to five differential diagnoses, and chose diagnosis likelihoods. We compared inter-observer and inter-MDTM agreements on patient first-choice diagnoses using Cohen's kappa coefficient (κ). We then estimated inter-observer and inter-MDTM agreement on the probability of diagnosis using weighted kappa coefficient (κw). We compared inter-observer and inter-MDTM confidence of patient first-choice diagnosis. Finally, we evaluated the prognostic significance of a first-choice diagnosis of idiopathic pulmonary fibrosis (IPF) versus not IPF for MDTMs, clinicians, and radiologists, using univariate Cox regression analysis. FINDINGS: 70 patients were included in the final study cohort. Clinicians, radiologists, pathologists, and the MDTMs assigned their patient diagnoses between Jan 1, and Oct 15, 2015. IPF made up 88 (18%) of all 490 MDTM first-choice diagnoses. Inter-MDTM agreement for first-choice diagnoses overall was moderate (κ=0·50). Inter-MDTM agreement on diagnostic likelihoods was good for IPF (κw=0·71 [IQR 0·64-0·77]) and connective tissue disease-related interstitial lung disease (κw=0·73 [0·68-0·78]); moderate for non-specific interstitial pneumonia (NSIP; κw=0·42 [0·37-0·49]); and fair for hypersensitivity pneumonitis (κw=0·29 [0·24-0·40]). High-confidence diagnoses (>65% likelihood) of IPF were given in 68 (77%) of 88 cases by MDTMs, 62 (65%) of 96 cases by clinicians, and in 57 (66%) of 86 cases by radiologists. Greater prognostic separation was shown for an MDTM diagnosis of IPF than compared with individual clinician's diagnosis of this disease in five of seven MDTMs, and radiologist's diagnosis of IPF in four of seven MDTMs. INTERPRETATION: Agreement between MDTMs for diagnosis in diffuse lung disease is acceptable and good for a diagnosis of IPF, as validated by the non-significant greater prognostic separation of an IPF diagnosis made by MDTMs than the separation of a diagnosis made by individual clinicians or radiologists. Furthermore, MDTMs made the diagnosis of IPF with higher confidence and more frequently than did clinicians or radiologists. This difference is of particular importance, because accurate and consistent diagnoses of IPF are needed if clinical outcomes are to be optimised. Inter-multidisciplinary team agreement for a diagnosis of hypersensitivity pneumonitis is low, highlighting an urgent need for standardised diagnostic guidelines for this disease. FUNDING: National Institute of Health Research, Imperial College London.