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Background Early life represents a major risk window for asthma development. However, the mechanisms controlling the threshold for establishment of allergic airway inflammation in early life are incompletely understood. Airway macrophages (AMs) regulate pulmonary allergic responses and undergo TGF-β–dependent postnatal development, but the role of AM maturation factors such as TGF-β in controlling the threshold for pathogenic immune responses to inhaled allergens remains unclear. Objective Our aim was to test the hypothesis that AM-derived TGF-β1 regulates pathogenic immunity to inhaled allergen in early life. Methods Conditional knockout (Tgfb1ΔCD11c) mice, with TGF-β1 deficiency in AMs and other CD11c+ cells, were analyzed throughout early life and following neonatal house dust mite (HDM) inhalation. The roles of specific chemokine receptors were determined by using in vivo blocking antibodies. Results AM-intrinsic TGF-β1 was redundant for initial population of the neonatal lung with AMs, but AMs from Tgfb1ΔCD11c mice failed to adopt a mature homeostatic AM phenotype in the first weeks of life. Evidence of constitutive TGF-β1 signaling was also observed in pediatric human AMs. TGF-β1–deficient AMs expressed enhanced levels of monocyte-attractant chemokines, and accordingly, Tgfb1ΔCD11c mice exposed to HDM throughout early life accumulated CCR2-dependent inflammatory CD11c+ mononuclear phagocytes into the airway niche that expressed the proallergic chemokine CCL8. Tgfb1ΔCD11c mice displayed augmented TH2, group 2 innate lymphoid cell, and airway remodeling responses to HDM, which were ameliorated by blockade of the CCL8 receptor CCR8. Conclusion Our results highlight a causal relationship between AM maturity, chemokines, and pathogenic immunity to environmental stimuli in early life and identify TGF-β1 as a key regulator of this., Graphical abstract

We previously generated a transgenic mouse line expressing skin-specific IL-33 (IL33tg mice) and showed that IL-33 elicits group 2 innate lymphoid cell (ILC2)–dependent atopic dermatitis–like skin inflammation. ILC2s are believed to be tissue-resident cells under steady-state conditions, but the dynamics of ILC2 migration are not fully understood. We sorted ILC2s from the skin and draining lymph nodes of IL33tg mice and analyzed their transcriptomes using the single-cell RNA sequencing technique, which revealed that the skin ILC2s had split into two clusters: circulating ILC2 and skin-resident ILC2. The circulating ILC2s expressed H2-related major histocompatibility complex class II genes. Conversely, the skin-resident ILC2s demonstrated increased mRNA expression of the ICOS, IL-5, and IL-13. Next, we tracked ILC2 migration using IL33tg–Kikume Green-Red mice. Exposing the IL33tg–Kikume Green-Red mice’s inflamed skin to violet light allowed us to label the circulating ILC2s in their skin and track the ILC2 migration from the skin to the draining lymph nodes. Cutaneous local innate responses could transition to systemic type 2 responses by migrating the activated ILC2s from the skin into the draining lymph node. Conversely, the skin-resident ILC2s produced a large number of cytokines. Thus, the skin ILC2s turned out to be a heterogeneous cell population.

Background Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2), is expressed in human ILC2. However, the function of CRTH2 in these cells is unclear. Objectives We sought to determine the role of PGD2 and CRTH2 in human ILC2 and compare it with that of established ILC2 activators, IL-25 and IL-33. Methods The effects of PGD2, IL-25, IL-33 on the cell migration, cytokine production, gene regulation and receptor expression of ILC2 were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR and QuantiGene assays. The effects of PGD2 under physiological conditions were evaluated by using the supernatant from activated mast cells. Results PGD2 binding to CRTH2 induced ILC2 migration, production of type 2 cytokines and many other cytokines. ILC2 activation via CRTH2 also up-regulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD2 on ILC2 could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. Conclusions PGD2 is an important and potent activator of ILC2 via CRTH2 mediating strong pro-allergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges innate and adaptive pathways in human ILC2.

Background Group 2 innate lymphoid cells (ILC2s) are a potential innate source of type 2 cytokines in the pathogenesis of allergic conditions. Epithelial cytokines (IL-33, IL-25, and thymic stromal lymphopoietin [TSLP]) and mast cell mediators (prostaglandin D2 [PGD2]) are critical activators of ILC2s. Cysteinyl leukotrienes (cysLTs), including leukotriene (LT) C4, LTD4, and LTE4, are metabolites of arachidonic acid and mediate inflammatory responses. Their role in human ILC2s is still poorly understood. Objectives We sought to determine the role of cysLTs and their relationship with other ILC2 stimulators in the activation of human ILC2s. Methods For ex vivo studies, fresh blood from patients with atopic dermatitis and healthy control subjects was analyzed with flow cytometry. For in vitro studies, ILC2s were isolated and cultured. The effects of cysLTs, PGD2, IL-33, IL-25, TSLP, and IL-2 alone or in combination on ILC2s were defined by using chemotaxis, apoptosis, ELISA, Luminex, quantitative RT-PCR, and flow cytometric assays. The effect of endogenous cysLTs was assessed by using human mast cell supernatants. Results Human ILC2s expressed the LT receptor CysLT1, levels of which were increased in atopic subjects. CysLTs, particularly LTE4, induced migration, reduced apoptosis, and promoted cytokine production in human ILC2s in vitro. LTE4 enhanced the effect of PGD2, IL-25, IL-33, and TSLP, resulting in increased production of type 2 and other proinflammatory cytokines. The effect of LTE4 was inhibited by montelukast, a CysLT1 antagonist. Interestingly, addition of IL-2 to LTE4 and epithelial cytokines significantly amplified ILC2 activation and upregulated expression of the receptors for IL-33 and IL-25. Conclusion CysLTs, particularly LTE4, are important contributors to the triggering of human ILC2s in inflammatory responses, particularly when combined with other ILC2 activators.

Background Group 2 innate lymphoid cells (ILC2s) are involved in the initial phase of type 2 inflammation and can amplify allergic immune responses by orchestrating other type 2 immune cells. Prostaglandin (PG) E2 is a bioactive lipid that plays protective roles in the lung, particularly during allergic inflammation. Objective We set out to investigate how PGE2 regulates human ILC2 function. Methods The effects of PGE2 on human ILC2 proliferation and intracellular cytokine and transcription factor expression were assessed by means of flow cytometry. Cytokine production was measured by using ELISA, and real-time quantitative PCR was performed to detect PGE2 receptor expression. Results PGE2 inhibited GATA-3 expression, as well as production of the type 2 cytokines IL-5 and IL-13, from human tonsillar and blood ILC2s in response to stimulation with a combination of IL-25, IL-33, thymic stromal lymphopoietin, and IL-2. Furthermore, PGE2 downregulated the expression of IL-2 receptor α (CD25). In line with this observation, PGE2 decreased ILC2 proliferation. These effects were mediated by the combined action of E-type prostanoid receptor (EP) 2 and EP4 receptors, which were specifically expressed on ILC2s. Conclusion Our findings reveal that PGE2 limits ILC2 activation and propose that selective EP2 and EP4 receptor agonists might serve as a promising therapeutic approach in treating allergic diseases by suppressing ILC2 function., Graphical abstract

Background Respiratory syncytial virus (RSV) is a major health care burden with a particularly high worldwide morbidity and mortality rate among infants. Data suggest that severe RSV-associated illness is in part caused by immunopathology associated with a robust type 2 response. Objective We sought to determine the capacity of RSV infection to stimulate group 2 innate lymphoid cells (ILC2s) and the associated mechanism in a murine model. Methods Wild-type (WT) BALB/c, thymic stromal lymphopoietin receptor (TSLPR) knockout (KO), or WT mice receiving an anti-TSLP neutralizing antibody were infected with the RSV strain 01/2-20. During the first 4 to 6 days of infection, lungs were collected for evaluation of viral load, protein concentration, airway mucus, airway reactivity, or ILC2 numbers. Results were confirmed with 2 additional RSV clinical isolates, 12/11-19 and 12/12-6, with known human pathogenic potential. Results RSV induced a 3-fold increase in the number of IL-13–producing ILC2s at day 4 after infection, with a concurrent increase in total lung IL-13 levels. Both thymic stromal lymphopoietin (TSLP) and IL-33 levels were increased 12 hours after infection. TSLPR KO mice did not mount an IL-13–producing ILC2 response to RSV infection. Additionally, neutralization of TSLP significantly attenuated the RSV-induced IL-13–producing ILC2 response. TSLPR KO mice displayed reduced lung IL-13 protein levels, decreased airway mucus and reactivity, attenuated weight loss, and similar viral loads as WT mice. Both 12/11-19 and 12/12-6 similarly induced IL-13–producing ILC2s through a TSLP-dependent mechanism. Conclusion These data demonstrate that multiple pathogenic strains of RSV induce IL-13–producing ILC2 proliferation and activation through a TSLP-dependent mechanism in a murine model and suggest the potential therapeutic targeting of TSLP during severe RSV infection., Graphical abstract