Gastrointestinal dysfunction is a common symptom of acute mountain sickness (AMS). The gut microbiota and γδ T cells play critical roles in intestinal disease. However, the mechanistic link between the microbiota and γδ T cells in hypoxia-induced intestinal injury remains unclear. Here, we show that hypoxia-induced intestinal damage was significantly alleviated after microbiota depletion with antibiotics. Hypoxia modulated gut microbiota composition by promoting antimicrobial peptides angiogenin-4 secretions. The abundance of Clostridium in the gut of mice after hypoxia significantly decreased, while the abundance of Desulfovibrio significantly increased. Furthermore, Desulfovibrio-derived phosphatidylethanolamine and phosphatidylcholine promoted γδ T cell activation. In CD1d-deficient mice, the levels of intraepithelial IL-17A and γδ T cells and intestinal damage were significantly decreased compared with those in wild-type mice under hypoxia. Mechanistically, phospholipid metabolites from Desulfovibrio are presented by intestinal epithelial CD1d to induce the proliferation of IL-17A-producing γδ T cells, which aggravates intestinal injury. Gut microbiota-derived metabolites promote hypoxia-induced intestinal injury via CD1d-dependent γδ T cells, suggesting that phospholipid metabolites and γδ T cells can be targets for AMS therapy.

Microbial challenges, such as widespread bacterial infection in sepsis, induce endotoxin tolerance, a state of hyporesponsiveness to subsequent infections. The participation of DNA methylation in this process is poorly known. In this study, we perform integrated analysis of DNA methylation and transcriptional changes following in vitro exposure to gram-negative bacterial lipopolysaccharide, together with analysis of ex vivo monocytes from septic patients. We identify TET2-mediated demethylation and transcriptional activation of inflammation-related genes that is specific to toll-like receptor stimulation. Changes also involve phosphorylation of STAT1, STAT3 and STAT5, elements of the JAK2 pathway. JAK2 pathway inhibition impairs the activation of tolerized genes on the first encounter with lipopolysaccharide. We then confirm the implication of the JAK2-STAT pathway in the aberrant DNA methylome of patients with sepsis caused by gram-negative bacteria. Finally, JAK2 inhibition in monocytes partially recapitulates the expression changes produced in the immunosuppressive cellular state acquired by monocytes from gram-negative sepsis, as described by single cell-RNA-sequencing. Our study evidences both the crucial role the JAK2-STAT pathway in epigenetic regulation and initial response of the tolerized genes to gram-negative bacterial endotoxins and provides a pharmacological target to prevent exacerbated responses.
Copyright © 2021 Morante-Palacios, Lorente-Sorolla, Ciudad, Calafell-Segura, Garcia-Gomez, Català-Moll, Ruiz-Sanmartín, Martínez-Gallo, Ferrer, Ruiz-Rodriguez, Álvarez-Errico and Ballestar.

While pro-inflammatory immune responses are a requirement to combat microbes, uncontrolled self-directed inflammatory immune responses are the hallmark of autoimmune diseases. Restoration of immunological tolerance involves both suppression of ongoing tissue-destructive immune responses and re-education of the host immune system. Both functionally immunosuppressive macrophages (M2) and regulatory T cells (Tregs) are implicated in these processes. Their mutual interaction is synergistic in this context and adoptive transfer of each cell type has been functioning as immunotherapy in experimental models, being particularly effective when using M2 macrophages generated with an optimized interleukin-4 (IL-4)/interleukin-10 (IL-10)/transforming growth factor-β (TGF-β) combination. As a prerequisite for eventual translation of M2 therapy into clinical settings we herein studied the induction, stability and mechanism of generation of human induced Tregs (iTregs) by M2 macrophages generated with IL-4/IL-10/TGF-β. The supernatants of monocyte-derived human M2 macrophages robustly induced FOXP3 and other Treg signature molecules such as CTLA-4 and IKZF4 in human naïve CD4 T cells. M2-induced iTregs displayed enhanced FOXP3 stability and low expression of pro-inflammatory cytokines interferon-γ and IL-17, as well as functional immunosuppressive activity compared with control T cells. The FOXP3-inducing activity was dependent on TGF-β, which was both expressed and captured with re-release by M2 macrophages into the soluble supernatant fraction, in which the TGF-β was not confined to extracellular vesicles such as exosomes. We propose that adoptive transfer of human M2 macrophages may be exploited in the future to induce Tregs in situ by delivering TGF-β, which could be developed as a therapeutic strategy to target autoimmune and other inflammatory diseases.

Mesenchymal stem cells (MSC) are a unique population of adult stem cells that have the capacity to differentiate into numerous cell types as well as the ability to modulate the immune system. As such, MSC represent a promising stem cell population for use in the clinical treatment of a range of disorders involving tissue regeneration as well as the immune system. The lack of accessibility to MSC is currently limiting the use of MSC in mainstream clinical treatment strategies. It is therefore imperative for the future success of stem cell-based treatment approaches that are more reliable, and accessible sources of MSC are identified. The present chapter describes a method for generating MSC-like cells from induced pluripotent stem cells (iPSC), with equivalent growth and functional properties to parental MSC populations.