Severe asthma is a heterogeneous disease. The mechanisms driving airway pathology when type 2 (T2) cytokine activity is suppressed remain poorly understood. This study aimed to provide insight by identifying the airway molecular pathways of T2 biomarker-high and -low severe asthma. We analysed clinical and transcriptomic data from bronchial biopsies and brushes in the UK Refractory Asthma Stratification Programme multi-centre severe asthma cohort (18 corticosteroid-resistant T2 biomarker-high [T2-high], 23 T2 biomarker-intermediate [T2-intermediate], 11 T2 biomarker-low [T2-low]) plus 20 healthy controls pre- and post-treatment with high-dose inhaled corticosteroids (ICS). Many genes dysregulated in asthma vs. health were concordantly dysregulated in healthy subjects receiving ICS. Severe asthma as a whole, independent of confounding by ICS, was characterised by upregulation of mucins, CEACAM5, typical T2-genes (POSTN, CLCA1, CCL26), epithelial mast cell genes, and CPA4. T2-high severe asthma demonstrated upregulated T2-dependent genes, epithelial barrier and keratin genes, adaptive immune responses, and impaired ciliary function. T2-low asthma showed upregulated Th1- and IL-17-associated genes (IDO1, CXCL10, GBP1, LAG3), interferon-γ signalling, neuroimmune pathways, airway smooth muscle-related genes, and neutrophil enrichment. T2-intermediate asthma exhibited a mixed molecular profile sharing features of T2-high and T2-low endotypes, with selective expression of the pathogen defence and antiviral response genes. The results were validated using bronchoscopy data from the U-BIOPRED Consortium. This study defines airway molecular endotypes of severe asthma associated with T2 biomarker high and low phenotypes, independent of corticosteroid effects. These findings offer insights for severe asthma management and the development of targeted biologic therapies.

Background: Melioidosis is a serious infection caused by the bacterium Burkholderia pseudomallei with a case fatality rate of up to 40% in Northeast Thailand. Diabetes increases the risk of developing melioidosis by 12-fold. A similar, but less marked, relationship with diabetes is seen in tuberculosis (TB) patients, with a 3-fold increased risk of developing TB in people with diabetes. However, the mechanisms underlying increased susceptibility are not fully understood. Methods: 81 acute melioidosis patients from Northeast Thailand and 151 TB patients from South Africa, Indonesia, Romania and Peru alongside uninfected control cohorts were studied by whole blood RNA sequencing. Both supervised and unsupervised data analysis approaches were performed including differential gene expression (DGE) analysis, pathway analyses, and weighted gene co-expression network analysis (WGCNA). Results: Diabetes status was associated with a hyper-inflammatory response to both melioidosis and TB, with increased neutrophil and platelet degranulation, and exaggerated activation of coagulation and scavenger activation pathways, alongside decreased phosphoinositide-3-kinase protein kinase B (P13K-Akt) signalling. In melioidosis, changes with diabetes were subtle but also included increased tumour necrosis factor (TNF) signalling via nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and enhancement of endoplasmic reticulum stress and unfolded protein responses. Diabetes-related changes were more distinct in TB, with marked reduction of interferon signalling responses. Conclusion: Diabetes is associated with enhanced non-specific inflammatory responses in both melioidosis and TB and an impaired interferon-mediated response to TB, with implications for future host-directed therapies.

Chronic hepatitis C (CHC) can progress to cirrhosis and hepatocellular carcinoma (HCC). This study aimed to identify genetic determinants and host-viral interactions that drive this progression to inform risk stratification and personalised treatment strategies. We performed a genome-wide association study (GWAS) of cirrhosis (2,829 cases and 1,515 CHC controls), followed by a GWAS of HCC (706 cases and 2,152 cirrhosis controls). We performed cis-eQTL mapping and deconvolution in liver tissue of HCV-infected (136 CHC and 54 cirrhosis) patients to investigate gene expression regulation and cellular heterogeneity. Additionally, ten polygenic risk scores (PRS) for non-viral liver diseases were tested in 3,406 infected individuals. We identified the missense risk variant rs738409 in PNPLA3 and a protective variant (rs4386418) in XKR3 in genotype 1-infected patients that were significantly associated with cirrhosis but not HCC progression. HLA fine-mapping identified two amino acids in HLA-DQB1*03:01 and HLA-DRB1*13:01 associated with cirrhosis risk. No genome-wide significant association was observed for HCC, and loci previously linked to non-viral HCC did not replicate. The eQTL analysis revealed 2,060 genes under cis-regulatory control and 129 whose effects were modified by cirrhosis. An intronic eQTL lowered PNPLA3 expression, but was not linked to cirrhosis risk. Deconvolution revealed expansion of plasma cells and macrophages and depletion of hepatocytes in CHC, with further immune-stromal remodelling in cirrhosis. All PRS showed a significant association with cirrhosis risk but not HCC progression. Cirrhosis in CHC shares genetic architecture with non-viral liver diseases but also displays virus-specific risk variants. Cirrhosis risk involves genetic factors that differ from those underlying progression to HCC.

Background: Mepolizumab is an anti-IL-5 mAb treatment for severe eosinophilic asthma that reduces asthma exacerbations. Residual airway inflammation with mepolizumab therapy may lead to persistent exacerbations. Oral corticosteroids remain the main treatment for these residual exacerbations. Objective: Our study aimed to explore the corticosteroid responsiveness of airway inflammation after mepolizumab treatment to find potentially treatable inflammatory mechanisms beyond the IL-5 pathway. Methods: The MAPLE trial was a multicenter, randomized, double-blind, placebo-controlled, crossover study of 2 weeks of high-dose oral prednisolone treatment at stable state in 27 patients treated with mepolizumab for severe eosinophilic asthma. We analyzed paired sputum (n = 16) and plasma (n = 25) samples from the MAPLE trial using high-throughput Olink proteomics. We analyzed additional sputum proteins using ELISA. Results: In patients receiving mepolizumab, prednisolone significantly downregulated sputum proteins related to type 2 inflammation and chemotaxis including IL-4, IL-5, IL-13, CCL24, CCL26, EDN, CCL17, CCL22, OX40 receptor, FCER2, and the ST2 receptor. Prednisolone also downregulated cell adhesion molecules, prostaglandin synthases, mast cell tryptases, MMP1, MMP12, and neuroimmune mediators. Neutrophilic pathways were upregulated. Type 2 proteins were also downregulated in plasma, combined with IL-12, IFN-g, and IP-10. IL-10 and amphiregulin were upregulated. Conclusions: At stable state, prednisolone has broad antiinflammatory effects on top of mepolizumab. These effects are heterogeneous and may be clinically relevant in residual exacerbations. (J Allergy Clin Immunol 2024;154:1146-58.)

Background Mepolizumab is an anti-interleukin-5 monoclonal antibody for severe eosinophilic asthma (SEA). The additional effects of prednisolone to mepolizumab, on molecular mechanisms in the airways and blood are poorly understood. Aim Determine the transcriptomic and proteomic effects of prednisolone versus placebo on the airways and blood in patients with SEA treated with mepolizumab. Methods MAPLE was a randomized, double-blind, placebo-controlled crossover trial of prednisolone at stable state in adults with SEA after mepolizumab (Yang F, JACI Pract 2022;10:2925–34.e12). Prednisolone had a minor effect on FEV1 but not on symptoms. Sputum and blood samples were taken before and after high dose prednisolone and placebo in patients treated with mepolizumab. These underwent O-link expression analysis of 1536 proteins. A paired comparison of normalised protein expression for 1536 proteins in sputum and serum were compared in a linear mixed effects model, with Benjamini-Hochberg correction for multiple testing. Nasal scrape samples were taken for transcriptomic analysis after prednisolone and placebo in patients treated with mepolizumab. RNA was extracted (Qiagen) and good quality samples sequenced (Illumina Novaseq). We identified differentially expressed genes with paired t-tests with Benjamini-Hochberg correction for multiple testing. Results 21 participants had paired serum, and 14 had paired sputum, before and after both prednisolone and placebo. Prednisolone significantly downregulated 173 and 229 proteins and upregulated 63 and 140 proteins in sputum and serum respectively. Downregulated proteins in sputum included IL-4, IL-5, IL-13, chemokines, and signatures of mast cells, prostaglandin synthesis, and alternatively activated macrophages. Up-regulated proteins included FKBP5, typical of steroid treatment. 6 people had paired nasal epithelial samples comparing prednisolone to placebo. 28 genes were down-regulated by prednisolone included leukocyte chemotaxis, mast cell tryptase and the 15-lipoxygenase pathway. Conclusions Prednisolone in addition to mepolizumab suppresses type-2 pathways unaffected by IL-5 inhibition in the sputum and blood proteome, and nasal transcriptome. These findings support the notion that the type-2 airway epithelium remains active in mepolizumab-treated patients. The relationship of these additional effects to longer term clinical outcome is unknown. Please refer to page A286 for declarations of interest related to this abstract.

Mepolizumab is an anti-interleukin-5 monoclonal antibody treatment for severe eosinophilic asthma (SEA) that reduces asthma exacerbations. Residual airway inflammation on mepolizumab may lead to persistent exacerbations. Oral corticosteroids have broad anti-inflammatory effects and remain the main treatment for these residual exacerbations. Our study aimed to explore the nature and corticosteroid-responsiveness of airway inflammation after mepolizumab treatment to find potentially treatable inflammatory mechanisms. The MAPLE trial was a multi-centre, randomized, double-blind, placebo-controlled, crossover study of 2 weeks of high-dose oral prednisolone treatment at stable state in patients treated with mepolizumab for SEA. We analysed sputum and plasma samples from the MAPLE trial using high-throughput Olink proteomics. We also analysed plasma microRNA, sputum proteins using ELISA, and nasal mucosal bulk RNA sequencing. In patients receiving mepolizumab, prednisolone significantly downregulated sputum proteins related to type-2 inflammation and chemotaxis including IL-4, IL-5, IL-13, CCL24, CCL26, EDN, CCL17, CCL22, OX40 receptor, FCER2, and the ST2 receptor. Prednisolone also downregulated cell adhesion molecules, prostaglandin synthases, mast cell tryptases, MMP1, MMP12, and neuroimmune mediators. Tissue repair and neutrophilic pathways were upregulated. Type-2 proteins were also downregulated in plasma, combined with IL-12, IFN-γ, and IP-10. IL-10 and amphiregulin were upregulated. In the nasal transcriptome, prednisolone suppressed genes involved in leucocyte chemotaxis, mast cell tryptase, 15-lipoxygenase and MMP12. By contrast, mepolizumab differentially regulated only Galectin-10 in plasma and no sputum proteins, and in nasal tissue affected genes related to cilia, keratinisation, extracellular matrix formation, and IL-4/13 signalling. At stable state, prednisolone has broad anti-inflammatory effects on top of mepolizumab.### Competing Interest StatementIH has received a conference travel grant from GSK. FY and SED have received speaker fees from AstraZeneca. JC, VB, and EM report no declarations of interest. AA is currently an employee of AZ. PJM has received support to attend educational meetings from Chiesi. JB has received personal fees from NuvoAir, and a research grant to his Institute from AstraZeneca, outside the submitted work. CB has received speakers fees from AZ and GSK and has received advisory board fees from AZ. LH has received grants from GSK, Astra Zeneca, Roche/Genentech, has given lectures supported by Astra Zeneca, Sanofi, Circassia, GlaxoSmithKline, has received travel grants from AstraZeneca and GSK, and has honoraria for Advisory Board Meetings from Novartis, Roche/Genentech, GSK, Teva and Celltrion. IDP has received speakers honoraria for speaking at sponsored meetings from Astra Zeneca, Boehringer Inglehiem, Aerocrine, Almirall, Novartis, Teva, Chiesi, Sanofi/Regeneron, Menarini and GSK and payments for organising educational events from AZ, GSK, Sanofi/Regeneron and Teva. He has received honoraria for attending advisory panels with Genentech, Sanofi/Regeneron, Astra Zeneca, Boehringer Ingelheim, GSK, Novartis, Teva, Merck, Circassia, Chiesi and Knopp and payments to support FDA approval meetings from GSK. He has received sponsorship to attend international scientific meetings from Boehringer Ingelheim, GSK, Astra Zeneca, Teva and Chiesi. He has received a grant from Chiesi to support a phase 2 clinical trial in Oxford. He is co-patent holder of the rights to the Leicester Cough Questionnaire and has received payments for its use in clinical trials from Merck, Bayer and Insmed. In 2014-5 and 2019-20 he was an expert witness for a patent dispute involving Astra Zeneca and Teva. CEB has received grants and consultancy fees from 4D Pharma, Areteia, AstraZeneca, Chiesi, Genentech, GlaxoSmithKline, Mologic, Novartis, Regeneron Pharmaceuticals, Roche and Sanofi. RC has received lecture fees from GSK, AZ, Teva, Chiesi, Sanofi and Novartis; honoraria for Advisory Board Meetings from GSK, AZ and Celltrion; sponsorship to attend international scientific meetings from Chiesi, Sanofi and GSK and a research grant to her Institute from AZ for a UK multi-centre study. TSCH has received grants from the Wellcome Trust, grants from The Guardians of the Beit Fellowship, and grants from the NIHR Oxford Biomedical Research Centre during the conduct of the study; and grants from Pfizer Inc., grants from University of Oxford, personal fees from Astra Zeneca, personal fees from TEVA, personal fees from Peer Voice outside the submitted work. ### Clinical TrialThis study is registered on Clinicaltrials.gov ([NCT03610685][1]).### Funding StatementThis study was funded jointly by the Medical Research Council (MRC) UK (MR/M016579/1) and industrial partners within the MRC Refractory Asthma Stratification Programme consortium and by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). Proteomics and transcriptomics analysis was funded by GSK (ID: 215294). TSCH is supported by a Wellcome Trust Fellowship (211050/Z/18/z). All authors had full access to the full data in the study and accept responsibility to submit for publication. ### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The study was approved by the Medical Ethics Committee (West of Scotland Research Ethics Service 3, Reference number 18/WS/0060). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAnonymised patient level data analysed and presented in this study are available from the corresponding author on reasonable request, providing the request meets local ethical and research governance criteria after publication. Data will be available immediately after publication for 10 years. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03610685&atom=%2Fmedrxiv%2Fearly%2F2024%2F02%2F15%2F2024.02.14.24302812.atom

Melioidosis, a neglected tropical infection caused by Burkholderia pseudomallei, , commonly presents as pneumonia or sepsis with mortality rates up to 50% despite appropriate treatment. A better understanding of the early host immune response to melioidosis may lead to new therapeutic interventions and prognostication strategies to reduce disease burden. Whole blood transcriptomic signatures in 164 patients with melioidosis and in 70 patients with other infections hospitalized in northeastern Thailand enrolled within 24 hours following hospital admission were studied. Key findings were validated in an independent melioidosis cohort. Melioidosis was characterized by upregulation of interferon (IFN) signaling responses compared with other infections. Mortality in melioidosis was associated with excessive inflammation, enrichment of type 2 immune responses, and a dramatic decrease in T cell-mediated immunity compared with survivors. We identified and independently confirmed a 5-gene predictive set classifying fatal melioidosis (validation cohort area under the receiver operating characteristic curve 0.83; 95% CI, 0.67-0.99). This study highlights the intricate balance between innate and adaptive immunity during fatal melioidosis and can inform future precision medicine strategies for targeted therapies and prognostication in this severe infection.

BACKGROUND:The airway microbiome in severe asthma has not been characterised at species-level by metagenomic sequencing, nor have the relationships between specific species and mucosal immune responses in 'type-2 low', neutrophilic asthma been defined. We performed an integrated species-level metagenomic data with inflammatory mediators to characterise prevalence of dominant potentially pathogenic organisms and host immune responses. METHODS:Sputum and nasal lavage samples were analysed using long-read metagenomic sequencing with Nanopore and qPCR in two cross-sectional adult severe asthma cohorts, Wessex (n = 66) and Oxford (n = 30). We integrated species-level data with clinical parameters and 39 selected airway proteins measured by immunoassay and O-link. RESULTS:The sputum microbiome in health and mild asthma displayed comparable microbial diversity. By contrast, 23% (19/81) of severe asthma microbiomes were dominated by a single respiratory pathogen, namely H. influenzae (n = 10), M. catarrhalis (n = 4), S. pneumoniae (n = 4) and P. aeruginosa (n = 1). Neutrophilic asthma was associated with H. influenzae, M. catarrhalis, S. pneumoniae and T. whipplei with elevated type-1 cytokines and proteases; eosinophilic asthma with higher M. catarrhalis, but lower H. influenzae, and S. pneumoniae abundance. H. influenzae load correlated with Eosinophil Cationic Protein, elastase and IL-10. R. mucilaginosa associated positively with IL-6 and negatively with FGF. Bayesian network analysis also revealed close and distinct relationships of H. influenzae and M. catarrhalis with type-1 airway inflammation. The microbiomes and cytokine milieu were distinct between upper and lower airways. CONCLUSIONS:This species-level integrated analysis reveals central, but distinct associations between potentially pathogenic bacteria and airways inflammation in severe asthma.

ABSTRACTRationaleThe effects of inhaled corticosteroids (ICS) on healthy airways are poorly defined.ObjectivesTo delineate the effects of ICS on gene expression in healthy airways, without confounding caused by changes in disease-related genes and disease-related alterations in ICS-responsiveness.MethodsRandomised open-label bronchoscopy study of high dose ICS therapy in 30 healthy adult volunteers randomised 2:1 to i) fluticasone propionate 500 mcg bd or ii) no treatment, for 4 weeks. Laboratory staff were blinded to allocation. Biopsies and brushings were analysed by immunohistochemistry, bulk RNA sequencing, DNA methylation array and metagenomics.Measurements and main resultsICS induced small between-group differences in blood and lamina propria eosinophil numbers, but not in other immunopathological features, blood neutrophils, FeNO, FEV1, microbiome or DNA methylation. ICS treatment upregulated 72 genes in brushings and 53 genes in biopsies, and downregulated 82 genes in brushings and 416 genes in biopsies. The most downregulated genes in both tissues were canonical markers of type-2 inflammation (FCER1A, CPA3, IL33, CLEC10A, SERPINB10 and CCR5), T cell-mediated adaptive immunity (TARP, TRBC1, TRBC2, PTPN22, TRAC, CD2, CD8A, HLA-DQB2, CD96, PTPN7), B cell immunity (CD20, immunoglobulin heavy and light chains), and innate immunity, including CD48, Hobit, RANTES, Langerin and GFI1. An IL-17-dependent gene signature was not upregulated by ICS.ConclusionsIn healthy airways, 4-week ICS exposure reduces gene expression related to both innate and adaptive immunity, and reduces markers of type-2 inflammation. This implies that homeostasis in health involves tonic type-2 signalling in the airway mucosa, which is exquisitely sensitive to ICS.

Supplementary figure 5. Expression of T cell markers and cytotoxic effectors by EC molecular subtype

Supplementary table 2. Antigen presentation pathway mutations in TCGA POLE proofreading-mutant ECs

Supplementary figure 1. Tumor infiltrating lymphocytes and Crohn's like reaction in POLE- mutant endometrioid endometrial cancers

Supplementary figure 6. T cell exhaustion markers according to tumor CD8A expression and EC molecular subtype

Supplementary figure 3. Number of TIA-1+ cytolytic T cells according to molecular subtype

Introduction Azithromycin reduces exacerbations in severe asthma,1 particularly if Haemophilus influenzae is present in the airways. Though this suggests antibacterial effects predominate, other antibiotics are not effective, implying other mechanisms including azithromycin's additional antiviral and immunomodulatory functions may be important, whilst antimicrobial resistance is a significant concern. Our metagenomic studies showed airway dominance by specific potentially pathogenic organisms as a common 'treatable trait' in severe asthma.2 We sought to determine the azithromycin mechanisms of action to enable better targeting azithromycin therapy, or non-antibacterial macrolide compounds in airways disease. Objective Characterise changes in airway mucosal immunology and microbiology using bronchoscopy before and after therapeutic intervention with azithromycin. Methods Patients with severe asthma despite optimised inhaled therapy underwent detailed clinical phenotyping and bronchoscopy before and 3 months post-initiation of clinically-indicated azithromycin (250 mg, three times weekly). Endobronchial samples and nasal brushings underwent single cell immune-profiling (10X genomics). Bronchoalveolar lavage (BAL) and nasal samples were stored for metagenomic sequencing with Nanopore and immunoassays. Results Eleven patients (mean age±SD, 51.8±16.8 years; 63% male) with severe asthma and high symptom burden (mean ACQ-5±SD, 2.9±1.1) underwent pre-/post-azithromycin bronchoscopy. H. influenzae was isolated in 36% on BAL culture pre-azithromycin. No pathogenic organisms were isolated post-azithromycin therapy. Analyses performed on 55,962 airways cells (n=6) revealed significant downregulation of gene ontology categories related to innate immune defence responses and cytokine mediated signalling pathways, with upregulation of genes encoding extracellular matrix components (p adj<1x10-7) post-azithromycin therapy (table 1). This corresponded to reduced expression of CXCL10 and S100A8, with upregulation of CST1 and mast cell CPA3. CD8 T cells underwent the greatest transcriptional reprogramming, downregulating heat shock proteins and pathways implicated in T cell activation and cytokine responses. Conclusions In addition to known antimicrobial effects, single cell transcriptomics reveals azithromycin suppresses Th1 and neutrophilic airways inflammation, modulates T cell functions and upregulates corticosteroid responsiveness markers (CST1, CPA3). In ongoing work, transcriptomic data from the full study population (n=11) will be analysed and integrated with single cell T cell repertoires, comparing upper and lower airway responses, and integrated with airway metagenomics. References Gibson, et al. Lancet. 2017 Aug 12;390(10095):659–668. http://dx.doi.org/10.2139/ssrn.4473145

Supplementary figure 4. CD20+ and FOXP3+ tumor infiltrate according to EC molecular subtype