Ananda S. Mirchandani

IL-33 Amplifies the Polarization of Alternatively Activated Macrophages That Contribute to Airway Inflammation

Alternatively activated macrophages (AAM) play a crucial role in type 2 immunity. Mice deficient in ST2, a receptor for the latest member of the IL-1 family, IL-33, have impaired type 2 immune responses. We therefore reasoned that IL-33/ST2 signaling may be involved in the differentiation and activation of AAM during airway inflammation. We report here that IL-33 changed the quiescent phenotype of alveolar macrophages toward an AAM phenotype that expressed mannose receptor, IL-4Rα, and produced high levels of CCL24 and CCL17 in an IL-13-dependent manner during IL-33-induced airway inflammation. Neutralization of AAM-derived CCL24 led to an amelioration of IL-33-induced eosinophilia in the lungs. Moreover, depletion of alveolar macrophages reduced IL-33-induced airway inflammation. Additionally, the attenuated OVA-induced airway inflammation in ST2−/− mice was associated with a decrease in AAM differentiation. In vitro, IL-33 amplified IL-13-induced polarization of alveolar- and bone marrow-derived macrophage toward an AAM phenotype by increasing the expression of arginase I, Ym1, as well as the production of CCL24 and CCL17. IL-13/IL-4Rα signaling was crucial for IL-33-driven AAM amplification by inducing the expression of ST2L. Finally, we showed that IL-33 was more abundantly expressed in the lung epithelial cells of asthma patients than those from healthy controls, suggesting that IL-33 may be involved in lung macrophage activation in clinical asthma. Taken together, we demonstrate here that IL-33/ST2 plays a significant role in the amplification of AAM polarization and chemokine production which contribute to innate and Ag-induced airway inflammation.

Type 2 Innate Lymphoid Cells Drive CD4+ Th2 Cell Responses

CD4+ T cells have long been grouped into distinct helper subsets on the basis of their cytokine-secretion profile. In recent years, several subsets of innate lymphoid cell have been described as key producers of these same Th-associated cytokines. However, the functional relationship between Th cells and innate lymphoid cells (ILCs) remains unclear. We show in this study that lineage-negative ST2+ICOS+CD45+ type 2 ILCs and CD4+ T cells can potently stimulate each other’s function via distinct mechanisms. CD4+ T cell provision of IL-2 stimulates type 2 cytokine production by type 2 ILCs. By contrast, type 2 ILCs modulate naive T cell activation in a cell contact–dependent manner, favoring Th2 while suppressing Th1 differentiation. Furthermore, a proportion of type 2 ILCs express MHC class II and can present peptide Ag in vitro. Importantly, cotransfer experiments show that type 2 ILCs also can boost CD4+ T cell responses to Ag in vivo.

Interleukin-33 and the function of innate lymphoid cells

Interleukin (IL)-33 is a member of the IL-1 cytokine family that has been shown to play an important role in the induction and effector phases of type 2 immune responses. Both innate and adaptive immunity are regulated by IL-33, and many studies have shown disease-associated functions for this cytokine. Recently, IL-33 has been implicated in the function of novel innate lymphocyte populations that regulate both protective responses in parasitic infections and allergic airway inflammation. Here, we discuss recent data highlighting the dual roles of IL-33 in protective and deleterious immune responses.

IL-33 induces innate lymphoid cell–mediated airway inflammation by activating mammalian target of rapamycin

Background The IL-1 family cytokine IL-33 is involved in the induction of airway inflammation in allergic patients and after viral infection. Several cell types, including CD4+ TH2 cells and the recently described type 2 innate lymphoid cells (ILCs), are targets for IL-33, yet the mechanisms by which this cytokine modulates their activation are not clear. Objectives Our goal was to investigate a role for mammalian target of rapamycin (mTOR) signaling in the activation of TH2 and ILC responses and the induction of airway inflammation by IL-33. Methods We biochemically determined the effect of IL-33 on mTOR activation in TH2 cells and ILCs and examined the effect of this signaling pathway in vivo using a murine model of IL-33–induced lung inflammation. Results We found that IL-33 induces mTOR activation through p110δ phosphoinositide 3-kinase and that blockade of the mTOR pathway inhibited IL-33–induced IL-5 and IL-13 production by TH2 cells and ILCs. Furthermore, use of a ribosomal protein S6 kinase 1 inhibitor implicated a role for ribosomal protein S6 kinase 1 in IL-33–induced mTOR-dependent cytokine production. Intranasal administration of IL-33 to wild-type mice induced airway inflammation, whereas adoptive transfer of wild-type ILCs to IL-33 receptor–deficient (St2−/−) mice recapitulated this response. Importantly, coadministration of the mTOR inhibitor rapamycin reduced IL-33–dependent ILC, macrophage, and eosinophil accumulation; cytokine secretion; and mucus deposition in the airways. Conclusion These data reveal a hitherto unrecognized role of mTOR signaling in IL-33–driven, ILC-dependent inflammation in vivo and suggest that manipulation of this pathway might represent a target for therapeutic intervention for airway inflammation.

Regulation of type 17 helper T-cell function by nitric oxide during inflammation

Type 17 helper T (Th17) cells are implicated in the pathogenesis many of human autoimmune diseases. Development of Th17 can be enhanced by the activation of aryl hydrocarbon receptor (AHR) whose ligands include the environmental pollutant dioxin, potentially linking environmental factors to the increased prevalence of autoimmune disease. We report here that nitric oxide (NO) can suppress the proliferation and function of polarized murine and human Th17 cells. NO also inhibits AHR expression in Th17 cells and the downstream events of AHR activation, including IL-22, IL-23 receptor, and Cyp1a1. Conversely, NO did not affect the polarization of Th17 cells from mice deficient in AHR. Furthermore, mice lacking inducible nitric oxide synthase (Nos2−/−) developed more severe experimental autoimmune encephalomyelitis than WT mice, with elevated AHR expression, increased IL-17A, and IL-22 synthesis. NO may therefore represent an important endogenous regulator to prevent overexpansion of Th17 cells and control of autoimmune diseases caused by environmental pollutants.

Novel allergic asthma model demonstrates ST2-dependent dendritic cell targeting by cypress pollen

BACKGROUND Cypress pollen causes respiratory syndromes with different grades of severity, including asthma. IL-33, its receptor ST2, and dendritic cells (DCs) have been implicated in human respiratory allergy. OBJECTIVE We sought to define a new mouse model of allergy to cypress pollen that recapitulates clinical parameters in allergic patients and to evaluate the implications of DCs and the IL-33/ST2 pathway in this pathology. METHODS BALB/c mice, either wild-type or ST2 deficient (ST2(-/-)), were sensitized and challenged with the Cupressus arizonica major allergen nCup a 1. Local and systemic allergic responses were evaluated. Pulmonary cells were characterized by means of flow cytometry. DCs were stimulated with nCup a 1 and tested for their biological response to IL-33 in coculture assays. RESULTS nCup a 1 causes a respiratory syndrome closely resembling human pollinosis in BALB/c mice. nCup a 1-treated mice exhibit the hallmarks of allergic pathology associated with pulmonary infiltration of eosinophils, T cells, and DCs and a dominant TH2-type immune response. IL-33 levels were increased in lungs and sera of nCup a 1-treated mice and in subjects with cypress allergy. The allergen-specific reaction was markedly reduced in ST2(-/-) mice, which showed fewer infiltrating eosinophils, T cells, and DCs in the lungs. Finally, stimulation of DCs with nCup a 1 resulted in ST2 upregulation that endowed DCs with increased ability to respond to IL-33-mediated differentiation of IL-5- and IL-13-producing CD4 T cells. CONCLUSIONS Our findings define a novel preclinical model of allergy to cypress pollen and provide the first evidence of a functionally relevant linkage between pollen allergens and TH2-polarizing activity by DCs through IL-33/ST2.

Innate Lymphoid Cells in Type 2 Immune Responses

In recent years, several distinct innate lymphoid cell populations (ILC) have been characterized in mice and humans. Group 2 ILC function as a rapid responder population in type 2 immune responses. Thus, a wealth of data has implicated an important role for ILC2 in immunity to parasitic infection and in immune pathology in inflammatory and allergic responses. In this review, we describe recent progress in our understanding of the development and ontogeny of ILC2 populations and the mechanisms by which these cells function in a variety of infection and disease settings. Finally, we emphasize recent findings indicating functional interactions between these innate cells and their adaptive CD4+ Th2 cell counterparts.

S21 Type-2 Innate lymphoid cells induce CD4 T helper cell type-2 immune functions

Introduction Type-2 innate lymphoid cells (ILC2) are a novel subset of immune cells characterised by their responsiveness to interleukin (IL)-33 and their production of type-2 cytokines (including IL-5, IL-9, IL-13). ILC2 have a variety of roles in lung inflammation and repair. Their interaction with innate immune cells has been shown, however their influence on the adaptive immune system remains unknown. Given the important roles of CD4 T helper (Th) cells in the lung and in conditions such as asthma, it is vital to determine whether ILC2 can influence their functions. Aim To determine the interactions of lung-derived ILC2 cells on naïve Th functions. Method BALB/c mice were treated with IL-33 intranasally for 5 days and ILC2 cells were sorted using fluorescence-activated cell sorting (FACS). Naïve Th cells were sorted by FACS from ST2 knockout mice (lacking the IL-33 receptor). Cells were co-cultured in the presence of anti-CD3 and anti-CD28 antibody for 72 hours. Intracellular cytokines were determined by FACS following phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Th cells were differentiated from ILC2 by their expression of CD4 and lack of ST2. Results Naïve Th cells expression of type-2 cytokines increases significantly when cultured with ILC2 cells (Fig.1). Th cell IL-4 expression increases 3-fold in co-culture, whilst IL-5 and IL-13 expression are enhanced 30- and nearly 20-fold, respectively. This effect is completely abrogated when cells are separated with a semi-porous membrane. Furthermore, ILC2 are able to enhance T cell responses in vivo in the lungs of BALB/c mice. Abstract S21 Figure 1. Th cells express type-2 cytokines when co-cultured with ILC2. Naïve Th cells (CD4+CD44lo) were sorted by FACS from ST2 knockout mice and were cultured alone (A) or with ILC2 (Lin*CD45+ICOS+ST2+) sorted from the lungs of IL-33-treated mice (B) for 72 hours in the presence of anti-CD3/CD28. Th cells intracellular cytokines were determined by FACS following 4 hours stimulation with PMA/ionomycin. CD4 cells were identified by presence of CD4 and lack of ST2. Discussion These data demonstrate that ILC2 are able to drive a Th2-phenotype in naïve Th cells directly. Furthermore, this effect is contact-dependent. These data demonstrate for the first time that ILC2 are capable of driving type-2 immune responses by influencing Th cell responses and hence provide an important link between lung innate and adaptive immune responses and a possible novel mechanism for their role in asthma.

S35 Rapamycin inhibits IL-33-induced, nuocyte-driven airway inflammation

Introduction IL-33 is an innate cytokine that promotes Th2 responses in both the innate and the adaptive immune systems, with an established role in allergic airway inflammation.1 The signalling pathway of IL-33/ ST2 is incompletely understood and the cells driving IL-33-mediated inflammation have remained elusive. Nuocytes, also known as natural helper cells, are a novel subpopulation of lineage negative innate cells that respond to IL-33 and IL-2.2 Rapamycin is a macrolide antibiotic that allosterically blocks mTOR, a serine-threonine kinase involved in numerous cellular signalling pathways. Aim To determine the role of mTOR in IL-33-induced airway inflammation and the effect of rapamycin on IL-33-induced nuocytes in the lung. Method BALB/c mice were treated with 1 μg of IL-33 intranasally for 5 consecutive days in the presence or absence of rapamycin. Bronchoalveolar lavage (BAL) for cellular and cytokine analysis was performed. Fluorescence-activated cell sorting (FACS) of lung digests were analysed for intracellular IL-5 and cell surface markers. Results IL-33 induced profound airway cellular infiltration noted in the BAL that was significantly inhibited by rapamycin. Cytokine levels from BAL fluid were also significantly reduced in mice treated with IL-33+ rapamycin. FACS analysis of lung digests demonstrated that IL-33 induced the expansion of lineage negative cells, in keeping with a population of nuocytes, which were the main source of IL-5 in the lung. Furthermore, this population of cells was suppressed by rapamycin. Discussion Intranasal IL-33 drives mTOR-dependant airway inflammation. Nuocytes are the main source of IL-5 in IL-33-driven airway inflammation. Rapamycin inhibits the production of IL-5 and IL-13 in vivo as well as the expansion of nuocytes in the lung.Abstract S35 Figure 1 BALB/c mice were treated intranasally with 1 μg IL-33 in the presence or absence of 1 mg/kg rapamycin for 5 consecutive days. The mice were sacrificed on day 6 and BAL total cell counts were performed. ***=p<0.001.