Background: Eosinophils develop from CD34 + progenitor cells in the bone marrow under the influence of interleukin (IL)-5. Several cell types produce IL-5, including type 2 innate lymphoid cells (ILC2s). The alarmin cytokine IL-33 is known to activate ILC2s in mucosal tissues, but little is known about IL-33-responsive ILC2s in the bone marrow in allergen-induced airway inflammation. Methods: Wild type (WT) and Rag1 deficient (Rag1 −/−) mice, which lack mature T and B cells, received intranasal doses of papain to induce acute allergic inflammation. In some experiments, mice were pre-treated with anti-IL-5 prior to the papain challenge. Furthermore, recombinant IL-33 was administered to WT mice, Rag1 −/− mice, lymphocyte deficient mice (Rag2 −/− Il2rg −/−) and to ex vivo whole bone marrow cultures. Bone marrow eosinophils and ILC2s were analyzed by flow cytometry. Eosinophil count was assessed by differential cell count and secreted IL-5 from bone marrow cells by ELISA. Results: Intranasal administration of papain or IL-33 increased the number of mature eosinophils in the bone marrow despite the absence of adaptive immune cells in Rag1 −/− mice. In parallel, an increased number of eosinophils was observed in the airways together with elevated levels of Eotaxin-2/CCL24. Bone marrow ILC2s were increased after papain or IL-33 administration, whereas ILC2s was found to be increased at baseline in Rag1 −/− mice compared to WT mice. An upregulation of the IL-33 receptor (ST2) expression on bone marrow ILC2s was observed after papain challenge in both Rag1 −/− and WT mice which correlated to increased number of bone marrow eosinophilia. Furthermore, an increased number of ST2 + mature eosinophils in the bone marrow was observed after papain challenge, which was further dependent on IL-5. In addition, bone marrow-derived ILC2s from both mouse strains produced large amounts of IL-5 ex vivo after IL-33 stimulation of whole bone marrow cultures. In contrast, IL-33-induced bone marrow and airway eosinophilia were abolished in the absence of ILC2s in Rag2 −/− Il2rg −/− mice and no production of IL-5 was detected in IL-33-stimulated bone marrow cultures. Boberg et al. ILC2s and the IL-33/ST2 Axis Conclusion: These findings establish bone marrow ILC2s and the IL-33/ST2 axis as promising targets for modulation of uncontrolled IL-5-dependent eosinophilic diseases including asthma.

Type 2 innate lymphoid cells (ILC2s) and their adaptive counterpart type 2 T helper (TH2) cells respond to interleukin-33 (IL-33) by producing IL-5, which is a crucial cytokine for eosinophil development in the bone marrow. The aim of this study was to determine if bone marrow ILC2s, TH cells, and eosinophils are locally regulated by IL-33 in terms of number and activation upon exposure to the common aeroallergen house dust mite (HDM). Mice that were sensitized and challenged with HDM by intranasal exposures induced eosinophil development in the bone marrow with an initial increase of IL5Rα+ eosinophil progenitors, following elevated numbers of mature eosinophils and the induction of airway eosinophilia. Bone marrow ILC2s, TH2, and eosinophils all responded to HDM challenge by increased IL-33 receptor (ST2) expression. However, only ILC2s, but not TH cells, revealed increased ST2 expression at the onset of eosinophil development, which significantly correlated with the number of eosinophil progenitors. In summary, our findings suggest that airway allergen challenges with HDM activates IL-33-responsive ILC2s, TH cells, and eosinophils locally in the bone marrow. Targeting the IL-33/ST2 axis in allergic diseases including asthma may be beneficial by decreasing eosinophil production in the bone marrow.

The alarmin cytokine interleukin (IL)-33 plays an important proinflammatory role in type 2 immunity and can act on type 2 innate lymphoid cells (ILC2s) and type 2 T helper (TH2) cells in eosinophilic inflammation and asthma. The mechanistic target of rapamycin (mTOR) signaling pathway drives immune responses in several inflammatory diseases, but its role in regulating bone marrow responses to IL-33 is unclear. The aim of this study was to determine the role of the mTORC1 signaling pathway in IL-33-induced bone marrow ILC2 responses and its impact on IL-33-induced eosinophilia. Wild-type mice were intranasally exposed to IL-33 only or in combination with the mTORC1 inhibitor, rapamycin, intraperitoneally. Four groups were included in the study: saline-treated (PBS)+PBS, rapamycin+PBS, PBS+IL-33 and rapamycin+IL-33. Bronchoalveolar lavage fluid (BALF), serum and bone marrow cells were collected and analyzed by differential cell count, enzyme-linked immunosorbent assay and flow cytometry. IL-33 induced phosphorylation of the mTORC1 protein rpS6 in bone marrow ILC2s both ex vivo and in vivo. The observed mTOR signal was reduced by rapamycin treatment, indicating the sensitivity of bone marrow ILC2s to mTORC1 inhibition. IL-5 production by ILC2s was reduced in cultures treated with rapamycin before stimulation with IL-33 compared to IL-33 only. Bone marrow and airway eosinophils were reduced in mice given rapamycin before IL-33-exposure compared to mice given IL-33 only. Bone marrow ILC2s responded to IL-33 in vivo with increased mTORC1 activity and rapamycin treatment successfully decreased IL-33-induced eosinophilic inflammation, possibly by inhibition of IL-5-producing bone marrow ILC2s. These findings highlight the importance of investigating specific cells and proinflammatory pathways as potential drivers of inflammatory diseases, including asthma.

The polarization of CD4+ T cells into different T helper subsets is an important process in many diseases, including asthma. Part of the adaptive immune system, T cells are responsible for propagating signals to alert and prime the immune system. MicroRNAs (miRNAs) are small non-coding RNAs that act on numerous targets in the cell to regulate a variety of cellular processes, including roles in T cell polarization. In this study, we aimed to identify genes dysregulated in peripheral blood mononuclear cells from individuals with asthma. Moreover, we sought to examine miRNAs that may regulate the candidate genes and explore their functional relationship. Utilizing a focused gene array, we identified the serum/glucocorticoid-regulated kinase 1 (SGK1) gene to be upregulated in circulating peripheral blood mononuclear cells, which included T cells, from individuals with asthma. Several miRNAs were bioinformatically identified to target SGK1, but miR-19a was the only screened candidate that negatively correlated to SGK1 expression. Further analysis of the miR-19a-SGK1 relationship showed a negative correlation in CD4+ T cells in situ and direct binding in vitro during T cell activation. Moreover, we observed a negative correlation of miR-19a and SGK1 during early type 2 polarization of CD4+ naïve human T cells. Thus, we suggest that miR-19a has a role in binding and regulating SGK1 transcript levels during T cell development.

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