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Eosinophils, best known for their role in anti-parasitic responses, have recently been shown to actively participate in tissue homeostasis and repair. Their regulation must be tightly controlled, as their absence or hyperplasia is associated with chronic disease (e.g. asthma or inflammatory bowel disease). In the context of skeletal muscle, eosinophils play a supportive role after acute damage. Indeed, their depletion leads to strong defects in skeletal muscle regeneration and, in the absence of eosinophil-secreted interleukin (IL) 4 and IL13, fibro-adipogenic progenitors fail to support muscle stem cell proliferation. However, the role of eosinophils in muscular dystrophy remains elusive. Although it has been shown that eosinophils are present in higher numbers in muscles from mdx mice (a mouse model for Duchenne muscular dystrophy), their depletion does not affect muscle histopathology at an early age. Here, we evaluated the impact of hyper-eosinophilia on the development of fibrofatty infiltration in aged mdx mice and found that muscle eosinophilia leads to defects in muscle homeostasis, regeneration and repair, and eventually hastens death.

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

Allergic disease is on the rise and yet the underlying cause and risk factors are not fully understood. While lifesaving in many circumstances, the use of antibiotics and the subsequent disruption of the microbiome are positively correlated with the development of allergies. Here, we describe the use of the antibiotic vancomycin in combination with the papain-induced mouse model of allergic disease that allows for the assessment of microbiome perturbations and the impact on allergy development.

Allergic disease is on the rise and yet the underlying cause and risk factors are not fully understood. While lifesaving in many circumstances, the use of antibiotics and the subsequent disruption of the microbiome are positively correlated with the development of allergies. Here, we describe the use of the antibiotic vancomycin in combination with the papain-induced mouse model of allergic disease that allows for the assessment of microbiome perturbations and the impact on allergy development.

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.

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.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer, chronic infections and inflammatory conditions. In an individual with a tumor burden, MDSCs accumulate in peripheral lymphoid organs and within the tumor microenvironment. In several human cancers, and in mouse tumor models, the presence of high numbers of MDSCs correlates with tumor growth, metastasis and an overall poor prognosis. Approaches that impair the activity or deplete MDSCs have shown promise in animal models. Thus, MDSCs are an attractive target for therapeutic intervention. We have developed an immunomagnetic method for the isolation of untouched mouse MDSCs. Using CyTOF analysis of cell surface antigens on spleen cells from tumor bearing mice, we developed a cocktail of antibodies that targets non-MDSCs for removal using a column-free, negative selection protocol. Our method isolates CD45+CD11b+Gr1+ cells with the following purity and recovery from spleen, BM and blood: MDSCs (CD11b+Gr1+) from tumor-bearing miceTissuePurity (±SD)Recovery (±SD)Spleen94 ± 2.1%50 ± 12%Blood99 ± 0.6%55 ± 7.0%Bone marrow96 ± 1.0%68 ± 5.6% (naïve) As a functional test, naïve mouse splenocytes were labeled with a proliferation dye and the T cells were activated with anti-CD3 and anti-CD28 antibodies. MDSCs isolated from tumor-bearing mice were added at different ratios and after 3 days of culture proliferation of CD4+ and CD8+ T cells was assessed by flow cytometry. This assay showed a dose-dependent suppression of both CD4+ and CD8+ T cells by the isolated MDSCs. Our new EasySep™ Mouse MDSC (CD11b+Gr1+) Isolation Kit offers a fast and easy method that could facilitate MDSC discoveries that impact a wide range of diseases.

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair. The transcription factor RORα is essential for ILC2 development but it is also highly expressed by ILC3s where its function remains poorly defined. Previously, we found that Rorasg/sg bone marrow transplant (BMT) mice were protected from intestinal fibrosis in a Salmonella--induced model of Crohn’s disease and that this is due to defective cytokine production by ILC3s. In the current study, whole transcriptome sequencing analysis reveals a striking downregulation of ILC3 signature genes in RORα-deficient ILC3s isolated from Salmonella infected mice. In particular, the expression of genes involved in sensing an inflammatory milieu is attenuated. Moreover, we find that Rorasg/sg ILC3s fail to express IL-17A following ex vivo stimulation with IL-23 and IL-1β. Consistent with these observations, we also find that Rorasg/sg BMT mice are more susceptible to Citrobacter rodentium infection due to attenuated expression of IL-22 and impaired induction of antimicrobial peptides. Collectively, our data suggests that RORα plays a key role in preserving functional ILC3s by modulating their ability to sense environmental cues that stimulate the efficient production of cytokines. These observations also suggest that targeting ILC3 cells could be of therapeutic benefit in fibrosis associated with Crohn’s disease.