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Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. To investigate the early developmental pathways that leads to the generation of lung ILC2, I divided adult bone marrow lymphoid primed multipotent progenitor (LMPPs) into CD127⁻ (LMPP-s) and CD127⁺ (LMPP+s) subsets and compared them with Ly6D⁻ and Ly6D⁺ common lymphoid progenitors (CLPs). Although, all lymphocytes are thought to develop from CLPs, LMPP+s differentiated into T cells and ILCs more rapidly and efficiently than other progenitors in transplantation assays. These results suggested that some ILCs and T cells may develop from LMPP+s via CLP-independent pathways. To investigate whether distinct ILC2 subsets mediate the distinct ILC2 functions and elucidate their developmental relationship, we generated RORα lineage tracer mice and analyzed them by single cell RNA sequencing, flow cytometry and functional assays. Adult lung ILC2s were divided into IL-18Rα⁺ST2⁻ and IL-18Rα⁻ST2⁺ subsets. The former had an immature ILC phenotype, produced little cytokines and contained ILC progenitor like cells expressing Tcf7, whereas the latter was conventional ILC2s. Neonatal lung conventional ILC2s (IL-18Rα⁻ST2⁺) were divided into two distinct effector subsets and an IL-18Rα⁺ST2⁻Tcf7⁺ progenitor-like subset. The two effector subsets were defined by the expression of ICOS and KLRG1, and they differentially produced the growth-factor amphiregulin and type 2 cytokines. Therefore, effector ILC2s diverge into tissue-repairing and pro-inflammatory subsets, which differ in transcriptional and phenotypic properties. The IL-18Rα⁺ST2⁻Tcf7⁺ cells are likely IL-18 responsive lung ILC progenitors, which may contribute to ILC-poiesis in neonatal and inflamed lungs.

Group 2 innate lymphoid cells (ILC2s) primarily reside on mucosal surfaces and produce copious amounts of IL-5 and IL-13 upon activation by epithelium-derived cytokines, such as IL-33, leading to inflammation characterized by eosinophilia. Elevated numbers of ILC2s are found in the peripheral blood of patients with allergic diseases including asthma, suggesting their involvement in the disease. Epidemiological studies have shown higher prevalence of asthma in women than men during reproductive age, but the mechanism is largely unknown. By using flow cytometric analyses, I found that post-pubertal female lung ILC2s are more responsive to IL-33 stimulation than male ILC2s. Gene expression analyses of purified ILC2s and measurement of epithelium-derived cytokines in the lung demonstrated ILC2 intrinsic and lung environmental differences between naïve female and male lungs, suggesting a more activated state of female ILC2s compared to male ILC2s at steady state conditions.ILC2s have previously been shown to be tissue resident at steady state as well as during inflammation. However, recent reports have demonstrated migratory potential of ILC2s upon activation. Intranasal IL-33 administration into mice caused expansion of ILC2s not only in the lung but also in the blood and liver. Parabiosis experiments showed that ILC2s migrate out of the lung to the liver through circulation. Lung-derived ILC2s potently produced IL-5, IL-13 and IL-6, inducing eosinophilia and mild fibrosis. In contrast, intranasal IL-33 pre-treatment attenuated concanavalin A-induced acute hepatitis and cirrhosis.Overall, these results highlight the complexity of ILC2 regulation and ILC2-mediated local and systemic immunity. These considerations need to be taken into account when investigating ILC2s in human diseases.

Airway allergic diseases predominantly originate from early life exposure to allergens, but the mechanism behind this is unclear. Group 2 innate lymphoid cells (ILC2s) are critical for innate and adaptive immune responses to airway allergens in adult mice. Therefore, I hypothesized that ILC2s play an essential role in neonatal lung immunity. ILC2s quickly seed the mouse lung after birth and begin proliferating and producing IL-13 and IL-5 around postnatal day 10-14, inducing eosinophilia. Neonatal pups deficient for IL-33, an epithelial-derived alarmin that activates ILC2s, have fewer lung ILC2s and eosinophils at day 10 than wildtype (WT) pups. The amount of IL-33 in WT pups is high at day 10 and decreases after day 15, correlating with a contraction of ILC2s into adulthood. Thus, spontaneous release of IL-33 into neonatal lungs provokes activation of ILC2s and eosinophilia. Neonatal ILC2s are more responsive to intranasal (I.N) protease allergen papain than adult ILC2s and respond more intensely upon a secondary challenge in adulthood. Furthermore, ILC2s drive neonatal Th2-biased cell differentiation. I.N OVA antigen treatments into day 10 ILC2-deficient pups transplanted with OT-II adult CD4⁺ T cells results in fewer IL-4⁺IL-13⁺ CD4⁺ OT-II T cells in the lung-draining lymph node than in WT pups. Therefore, ILC2s play a role in neonatal sensitization to allergens leading to persistent allergic disease. The majority of neonatal ILC2s incorporate BrdU, and the BrdU-labeled neonatal ILC2s persist into adulthood, comprising almost 30% of adult lung ILC2s. To determine the effect of neonatal IL-33 exposure on ILC2 function, I.N IL-33 was given to IL-33-deficient (KO) and wildtype (WT) adult mice. While there was no difference in the number of lung ILC2s, there were fewer IL-5⁺IL-13⁺ ILC2s in IL-33KO than IL-33WT mice. I.N IL-33 into IL-33KO pups rescued the impaired response in adulthood. Microarray analysis showed cell-intrinsic differences between IL-33KO and IL-33WT ILC2s. Overall, weak activation by endogenous IL-33 of neonatal lung ILC2s may “train” ILC2s to respond strongly to allergen exposure later in life. These results place ILC2s as crucial components of neonatal lung immunity and should be taken into consideration when developing therapeutics for the prevention of allergic disease development.

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