COVID-19 pathogenesis is associated with an exaggerated immune response. However, the specific cellular mediators and inflammatory components driving diverse clinical disease outcomes remain poorly understood. We undertook longitudinal immune profiling on both whole blood and peripheral blood mononuclear cells (PBMCs) of hospitalized patients during the peak of the COVID-19 pandemic in the UK. Here, we report key immune signatures present shortly after hospital admission that were associated with the severity of COVID-19. Immune signatures were related to shifts in neutrophil to T cell ratio, elevated serum IL-6, MCP-1 and IP-10, and most strikingly, modulation of CD14+ monocyte phenotype and function. Modified features of CD14+ monocytes included poor induction of the prostaglandin-producing enzyme, COX-2, as well as enhanced expression of the cell cycle marker Ki-67. Longitudinal analysis revealed reversion of some immune features back to the healthy median level in patients with a good eventual outcome. These findings identify previously unappreciated alterations in the innate immune compartment of COVID-19 patients and lend support to the idea that therapeutic strategies targeting release of myeloid cells from bone marrow should be considered in this disease. Moreover, they demonstrate that features of an exaggerated immune response are present early after hospital admission suggesting immune-modulating therapies would be most beneficial at early timepoints.

These findings support the concept of localized nasal allergy in 'non-atopic' rhinitis subjects. We propose the term 'entopy' to define this phenomenon and believe that this concept has a wider implication for localized allergic responses in other mucosal sites.

Background Emerging studies indicate that some COVID-19 patients suffer from persistent symptoms including breathlessness and chronic fatigue; however the long-term immune response in these patients presently remains ill-defined. Methods Here we describe the phenotypic and functional characteristics of B and T cells in hospitalised COVID-19 patients during acute disease and at 3-6 months of convalescence. Findings We report that the alterations in B cell subsets observed in acute COVID-19 patients were largely recovered in convalescent patients. In contrast, T cells from convalescent patients displayed continued alterations with persistence of a cytotoxic programme evident in CD8 + T cells as well as elevated production of type-1 cytokines and IL-17. Interestingly, B cells from patients with acute COVID-19 displayed an IL-6/IL-10 cytokine imbalance in response to toll-like receptor activation, skewed towards a pro-inflammatory phenotype. Whereas the frequency of IL-6 + B cells was restored in convalescent patients irrespective of clinical outcome, recovery of IL-10 + B cells was associated with resolution of lung pathology. Conclusions Our data detail lymphocyte alterations in previously hospitalized COVID-19 patients up to 6 months following hospital discharge and identify 3 subgroups of convalescent patients based on distinct lymphocyte phenotypes, with one subgroup associated with poorer clinical outcome. We propose that alterations in B and T cell function following hospitalisation with COVID-19 could impact longer term immunity and contribute to some persistent symptoms observed in convalescent COVID-19 patients. Funding Provided by UKRI, Lister Institute of Preventative Medicine, The Wellcome Trust, The Kennedy Trust for Rheumatology Research and 3M Global Giving.

Epidemiological studies suggest that a hookworm infection producing 50 eggs/gram of feces may protect against asthma. We conducted a dose-ranging study to identify the dose of hookworm larvae necessary to achieve 50 eggs/gram of feces for therapeutic trials of asthma. Ten healthy subjects without asthma or airway hyperresponsiveness to inhaled methacholine received 10, 25, 50, or 100 Necator americanus larvae administered double blind to an area of skin on the arm. Subjects were seen weekly for 12 weeks and were then treated with mebendazole. Skin itching at the entry site and gastrointestinal symptoms were common at higher doses. Lung function did not change. Levels of blood eosinophils and IgE increased transiently, and levels of IgG increased progressively. All doses resulted in at least 50 eggs/gram of feces in the eight subjects who completed the study. Infection with 10 N. americanus larvae is well tolerated, elicits a modest host eosinophil response, and is potentially suitable for use in preliminary clinical therapeutic trials.

Little is known about the impact of viral infections on lung matrix despite its important contribution to mechanical stability and structural support. The composition of matrix also indirectly controls inflammation by influencing cell adhesion, migration, survival, proliferation and differentiation. Hyaluronan is a significant component of the lung extracellular matrix and production and degradation must be carefully balanced. We have discovered an imbalance in hyaluronan production following resolution of a severe lung influenza virus infection, driven by hyaluronan synthase 2 from epithelial cells, endothelial cells and fibroblasts. Furthermore hyaluronan is complexed with inter-α-inhibitor heavy chains due to elevated TNF-stimulated gene 6 expression and sequesters CD44-expressing macrophages. We show that intranasal administration of exogenous hyaluronidase is sufficient to release inter-α-inhibitor heavy chains, reduce lung hyaluronan content and restore lung function. Hyaluronidase is already used to facilitate dispersion of co-injected materials in the clinic. It is therefore feasible that fibrotic changes following severe lung infection and inflammation could be overcome by targeting abnormal matrix production.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Neutrophilic airway inflammation is a feature of chronic obstructive pulmonary disease (COPD). Inhaled lipopolysaccharide (LPS) challenge in healthy non‐smokers has been used to assess the anti‐inflammatory effects of novel drugs on neutrophilic airway inflammation.WHAT THIS STUDY ADDS• We performed inhaled LPS challenge in smokers to study an inflammatory response in subjects who more closely resemble COPD patients. Inhaled LPS caused reproducible pulmonary inflammation in smokers, associated with changes in systemic biomarkers. Inhaled LPS appears to be a suitable model for studying exacerbations of COPD.AIMS Lipopolysaccharide (LPS) is a TLR4 agonist which activates NFκB dependent cytokine production. We investigated LPS inhalation in healthy smokers as a model of COPD bacterial exacerbations. We studied safety, reproducibility, the translocation of the NFκB subunit p65 in sputum cells and changes in systemic biomarkers of inflammation.METHODS Twelve smokers inhaled 5 and 30 µg LPS and safety was monitored over 24 h. IL‐6, CRP, CCl‐18, SP‐D, CC‐16 and β‐defensin 2 were measured in serum samples collected at baseline, 4, 8 and 24 h. Sputum was induced at baseline, 6 and 24 h for cell counts and p65 expression. Repeated challenges were performed after a 2 week interval in 10 smokers.RESULTS LPS inhalation was well tolerated. Significant increases occurred in sputum neutrophil counts with both doses, with a maximum increase of 21.5% at 6 h after 30 µg which was reproducible, ri (intraclass correlation coefficient) = 0.88. LPS increased sputum cell nuclear p65 translocation and phospho‐p65 expression. All of the serum biomarkers increased following challenge but with different temporal patterns.DISCUSSION Inhaled LPS challenge in smokers causes pulmonary and systemic inflammation that involves NFκB activation. This appears to be a suitable model for studying bacterial exacerbations of COPD.

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalised COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g. IL6, CCL20) or clinical hallmarks of COVID-19 (e.g. neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Analysis of an independent cohort of 108 patients from a different centre (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover hospital blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19.

Genotoxicity can be assessed by monitoring expression of a GADD45a-GFP reporter in the human lymphoblastoid cell line TK6. A flow cytometric method has been developed to effectively distinguish GFP fluorescence from coloured and fluorescent test samples as well from the S9 liver extracts used to generate metabolites from pro-genotoxins. The method includes the use of propidium iodide exclusion for the determination of cellular viability. This paper describes the method development, the derivation of decision thresholds for the identification of genotoxins using the method, and presents data from a 56-compound validation study of the method. The results illustrate that the method permitted the detection of the majority of pro-genotoxins tested and, importantly, the high specificity of the GADD45a-GFP assay was maintained.