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The COVID-19 pandemic continues to challenge the capacities of hospital ICUs which currently lack the ability to identify prospectively those patients who may require extended management. In this study of 90 ICU COVID-19 patients, we evaluated serum levels of four cytokines (IL-1β, IL-6, IL-10 and TNFα) as well as standard clinical and laboratory measurements. On 42 of these patients (binned into Initial and Replication Cohorts), we further performed CyTOF-based deep immunophenotyping of peripheral blood mononuclear cells with a panel of 38 antibodies. All measurements and patient samples were taken at time of ICU admission and retrospectively linked to patient clinical outcomes through statistical approaches. These analyses resulted in the definition of a new measure of patient clinical outcome: patients who will recover after short ICU stays (< 6 days) and those who will subsequently die or recover after long ICU stays (> 6 days). Based on these clinical outcome categories, we identified blood prognostic biomarkers that, at time of ICU admission, prospectively distinguish, with 91% sensitivity and 91% specificity (positive likelihood ratio 10.1), patients in the two clinical outcome groups. This is achieved through a tiered evaluation of serum IL-10 and targeted immunophenotyping of monocyte subsets, specifically, CD11clow classical monocytes. Immunophenotyping revealed clear predictors of clinical outcome in COVID-19 providing a highly sensitive and specific prognostic test that could prove useful in guiding clinical resource allocation.Graphical Abstract

Innate lymphoid cells (ILCs) are a recently identified subset of leukocytes that play a central role in pathogen surveillance and resistance, modulation of immune response, and tissue repair. They are remarkably similar to CD4+ T-helper subsets in terms of function and transcription factors required for their development but are distinguished by their lack of antigen-specific receptors. Despite their similarities, the absence of a surface T-cell receptor (TCR) and presence of ILCs and precursors in adult bone marrow has led to speculation that ILCs and T cells develop separately from lineages that branch at the point of precursors within the bone marrow. Considering the common lineage markers and effector cytokine profiles shared between ILCs and T cells, it is surprising that the status of the TCR loci in ILCs was not fully explored at the time of their discovery. Here, we demonstrate that a high proportion of peripheral tissue ILC2s have TCRγ chain gene rearrangements and TCRδ locus deletions. Detailed analyses of these loci show abundant frameshifts and premature stop codons that would encode nonfunctional TCR proteins. Collectively, these data argue that ILC2 can develop from T cells that fail to appropriately rearrange TCR genes, potentially within the thymus.

The cytokine IL-22 is rapidly induced at barrier surfaces where it regulates host-protective antimicrobial immunity and tissue repair but can also enhance disease severity in some chronic inflammatory settings. Using the chronic Salmonella gastroenteritis model, Ab-mediated neutralization of IL-22 impaired intestinal epithelial barrier integrity and, consequently, exaggerated expression of proinflammatory cytokines. As disease normally resolved, neutralization of IL-22 caused luminal narrowing of the cecum—a feature reminiscent of fibrotic strictures seen in Crohn disease patients. Corresponding to the exaggerated immunopathology caused by IL-22 suppression, Salmonella burdens in the gut were reduced. This enhanced inflammation and pathogen clearance was associated with alterations in gut microbiome composition, including the overgrowth of Bacteroides acidifaciens. Our findings thus indicate that IL-22 plays a protective role by limiting infection-induced gut immunopathology but can also lead to persistent pathogen colonization.

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium ΔAroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium ΔAroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair but can also contribute to chronic inflammatory diseases. The transcription factor RORα is required for ILC2 development but is also highly expressed by other ILC subsets where its function remains poorly defined. We previously reported that Rorasg/sg bone marrow chimeric mice (C57BL/6J) were protected from Salmonella-induced intestinal fibrosis due to defective ILC3 responses. In this study, single-cell RNA analysis of ILCs isolated from inflamed tissues indicates that RORα perturbation led to a reduction in ILC3 lineages. Furthermore, residual Rorasg/sg ILC3s have decreased expression of key signature genes, including Rorc and activating cytokine receptors. Collectively, our data suggest that RORα plays a key role in preserving functional ILC3s by modulating their ability to integrate environmental cues to efficiently produce cytokines.

The cytokine IL-22 is rapidly induced at barrier surfaces where it regulates host-protective antimicrobial immunity and tissue repair but can also enhance disease severity in some chronic inflammatory settings. Using the chronic