ILC2 Cells Research Articles

THE ROLE OF GROUP 3 INNATE LYMPHOID CELLS (ILC3) IN GM-CSF/CSF2RB-DEPENDENT INTESTINAL HOMEOSTASIS IN CROHN’S DISEASE

Abstract Inflammatory Bowel Disease (IBD) is comprised of two subtypes, Crohn’s disease (CD) and ulcerative colitis (UC). Genome-wide association studies (GWAS) of IBD identified 240 loci significantly associated with IBD risk. CSF2RB, one of the identified IBD-associated genes and the receptor for GM-CSF signaling, is responsible for maintaining intestinal homeostasis through retinoic acid (RA) production by macrophages and dendritic cells. GM-CSF (Sargramostim) treatment showed a promising result for preventing intestinal injury and inflammation in both a murine and our novel zebrafish model. However, due to GM-CSF-neutralizing antibodies present in patients, Sargramostim failed in a large cohort Phase II clinical trial. This GM-CSF-dependent homeostasis relies on the balance of innate lymphoid cells (ILCs). In human patients, we report that during inflammation in CD, there is a decrease of NCR+ ILC3s while proinflammatory ILC1s rapidly increase. Neutralizing GM-CSF auto-antibodies were detected in the pre-disease stage (ILC1 low & ILC3 low) in CD patients, a different result from CSF2RB frameshift allele carriers (ILC1 low & ILC3 high) (Figure 1). We developed a novel zebrafish model to mimic the acute and chronic nature of IBD disease pathogenicity in vivo. In our model, human GM-CSF treatment significantly alleviated intestinal injury but showed no effect during mucosal healing. With a CRISPR knockout csf2rb zebrafish line, we showed this protective effect is CSF2RB-dependent. Using single-cell technologies, DSS-induced injury increases rorc+ ILC3s. These ILC3 cell proportion changes were diminished in csf2rb-/- zebrafish (Figure 2). Figure 1. Distinct ILC subpopulations between anti-GM-CSF antibodies and CSFRB mutation carriers. Scatter plot of the PC1 values of the ILC3 gene list (ILC3-PC) (y axis) against the PC1 values of the ILC1 gene list (ILC1-PC) (x axis). Both ILC1-PC and ILC3-PC were derived from PCA analysis of signature gene lists of ILC1 and ILC3 cell types against a sub-dataset of the bulk mRNAseq samples from the MSCCR. Red dot indicate patient with anti-GM-CSF antibody. The logarithm antibody titer was shown with the intensity of red color. The green dots indicate the CSF2RB heterozygote carriers. Figure 2. GM-CSF protects against DSS-induced intestinal injury and regulates ILC3. (A-B) Treatment of human GM-CSF significantly reduces intestinal injury in dose-dependent manner in WT (A) but not CSF2RB-/- (B). ***, P< 0.001 (C) Analyzing scRNAseq data of zebrafish intestine cells, RORC+ (ILC3) cell proportion over total immune cells increases with DSS injury and decreases with human GM-CSF co-treatment. There is no RORC+ cell proportion change in CSF2RB-/-. (D) Modeling of GM-CSF in preventing ILC3 expansion and attenuating ILC3-related proinflammatory cytokines during intestinal injury. Our studies illuminate mechanisms of GM-CSF in perpetuating gut homeostasis through maintaining and regulating ILC3s. This novel aspect of regulating IBD pathogenesis through ILCs may lead to novel therapeutic targets.

Berberine ameliorates DSS-induced intestinal mucosal barrier dysfunction through microbiota-dependence and Wnt/β-catenin pathway.

Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder of the colon, and it has become one of the world-recognized medical problems as it is recurrent and refractory. Berberine (BBR) is an effective drug for UC treatment. However, the underlying mechanism and targets remain obscure. In this study, we systematically investigated the therapeutic effect and its mechanism of BBR in ameliorating DSS-induced mouse colitis. Expectedly, the colon inflammation was significantly relieved by BBR, and microbiota depletion by antibiotic cocktail significantly reversed the therapeutic effect. Further studies showed that BBR can regulate the abundance and component of bacteria, reestablish the broken chemical and epithelial barriers. Meanwhile, BBR administration dramatically decreased ILC1 and Th17 cells, and increased Tregs as well as ILC3 in colonic tissue of DSS-induced mice, and it was able to regulate the expression of various immune factors at the mRNA level. Moreover, a proteomic study revealed that Wnt/β-catenin pathway was remarkably enhanced in colonic tissue of BBR-treated mice, and the therapeutic effect of BBR was disappeared after the intervention of Wnt pathway inhibitor FH535. These results substantially revealed that BBR restores DSS-induced colon inflammation in a microbiota-dependent manner, and BBR performs its protective roles in colon by maintaining the structure and function of the intestinal mucosal barrier, regulating the intestinal mucosal immune homeostasis and it works through the Wnt/β-catenin pathway. Importantly, these findings also provided the proof that BBR serves as a potential gut microbiota modulator and mucosal barrier protector for UC prevention and therapy.

Development of Humanized Mouse Models for Studying Human NK Cells in Health and Disease.

Humanized mice, which we define as immunodeficient mice that have been reconstituted with a human immune system, represent promising preclinical models for translational research and precision medicine as they allow modeling and therapy of human diseases in vivo. The first generation of humanized mice showed insufficient development, diversity and function of human immune cells, in particular human natural killer (NK) cells and type 1 innate lymphoid cells (ILC1). This limited the applicability of humanized mice for studying ILC1 and NK cells in the context of human cancers and immunotherapeutic manipulation. However, since 2014, several next-generation humanized mouse models have been developed that express human IL-15 either as a transgene or knock-in (NOG-IL15, NSG-IL15, NSG-IL7-IL15, SRG-15) or show improved development of human myeloid cells, which express human IL-15 and thereby promote human NK cell development (NSG-SGM3, MISTRG, BRGSF). Here we compare the various next-generation humanized mouse models and describe the methodological procedures for creating mice with a functioning human immune system and how they can be used to study and manipulate human NK cells in health and disease.

Role of interleukin 5-induced eosinophils in interleukin 33-triggered airway inflammation in mice.

Interleukin (IL)-5 is essential for allergen induced eosinophilic airway inflammation, but not activation of T helper type 2 (Th2) cells in the lung. Although an excessive Th2 reaction is observed without IL-5 signaling, the mechanisms have remained unknown. To evaluate the negative-feedback mechanism in eosinophilic airway inflammation, we examined IL-33 triggered eosinophilic airway inflammation in IL-5Rα-/- mice. Mice were administered intranasal IL-33 for 3 days. Airway hyperresponsiveness (AHR) was evaluated and bronchoalveolar lavage (BAL) was performed 24 h after the last IL-33 treatment. The number of inflammatory cells and cytokine levels in the BAL fluid (BALF) were analyzed, and histologic examination was performed. Compared with IL-33 treated wild-type (WT) mice, intranasal administration of IL-33 in IL-5Rα-/- mice reduced eosinophilic airway inflammation, AHR, and basement membrane thickening, while we found excessive IL-33 induced IL-5 and IL-13 production in the airway without IL-5 signaling. The numbers of eosinophils with a ringshaped nucleus (resident) and segmented nucleus (inflammatory) were increased in WT mice, but not in IL-5Rα-/- mice following intranasal administration of IL-33, and the numbers of SiglecF-positive eosinophils with (resident) or without (inflammatory) expression of CD62L were also significantly increased by IL-33 treatment in WT mice, but not in IL5Rα-/- mice. The number of ILC2 cells in the BALF was significantly higher in IL-33 treated IL-5Rα-/- mice than in IL-33 treated WT mice. These findings suggest the possibility that IL-5 induced eosinophils contribute to the negative-feedback mechanisms in IL-33 induced ILC2 mediated airway inflammation.

IL-22 and its interaction with amino acid and glycolipid metabolite in polycystic ovary syndrome (PCOS) patients

IL-22 and its interaction with amino acid and glycolipid metabolite in polycystic ovary syndrome (PCOS) patients

ILC2s induce adaptive Th2-type immunity in different stages of tuberculosis through the Notch-GATA3 pathway

The study is to investigate the roles of group 2 innate lymphoid cells (ILC2s) in different courses of tuberculosis (TB). Serum and PBMCs were respectively isolated from the TST negative, LTBI (latent TB infection), ATB (active TB) and RTB (recurrent TB) patients. Flow cytometry was used to detect Th1, Th2 and ILC2s in the peripheral blood. The mRNA and protein levels of GATA3, RORα, CRTH2, Hes1, Notch1, NF-κB, and ID2 were detected by qRT-PCR and Western blotting. ILC2 cells from ATB and RTB patients were stimulated with rJagged2 or DAPT in vitro, and co-cultured with CD4+ T cells from TST negative group. ELISA was used to detect cytokine levels. The results showed that compared with the TST negative or LTBI group, Th2 cells and serum IL-4 in ATB group increased dramatically, accompanied by an increase of Th2/Th1 ratio in ATB patients, especially in RTB group. However, ILC2s in the ATB and RTB group increased significantly, along with increased GATA3, RORα, and CRTH2 levels. After rJagged2 stimulation in vitro, the levels of Hes1, Notch1, NF-κB, RORα, GATA3 and ID2 and those of IL-4, IL-5 and IL-13 were significantly increased. These effects were abrogated by DAPT treatment. Then, ILC2s, especially those from RTB patients, induced Th2-type immune response after co-culturing with CD4+ T cells. In conclusion, our results suggest that ILC2s may promote Th2-type immune response in different stages of TB via the Notch-GATA3 pathway.

Type 1 Innate Lymphoid Cells Limit the Antitumoral Immune Response

Natural killer (NK) cells are known to be able to kill established tumor cell lines, but important caveats remain regarding their roles in the detection and elimination of developing primary tumors. Using a genetic model of selective ILC1 and NK cell deficiency, we showed that these cells were dispensable for tumor immunosurveillance and immunoediting in the MCA-induced carcinogenesis model. However, we were able to generate primary cell lines derived from MCA-induced tumors with graded sensitivity to NK1.1+ cells (including NK cells and ILC1). This differential sensitivity was associated neither with a modulation of intratumoral NK cell frequency, nor the capacity of tumor cells to activate NK cells. Instead, ILC1 infiltration into the tumor was found to be a critical determinant of NK1.1+ cell-dependent tumor growth. Finally, bulk tumor RNAseq analysis identified a gene expression signature associated with tumor sensitivity to NK1.1+ cells. ILC1 therefore appear to play an active role in inhibiting the antitumoral immune response, prompting to evaluate the differential tumor infiltration of ILC1 and NK cells in patients to optimize the harnessing of immunity in cancer therapies.

Third-party type 2 innate lymphoid cells prevent and treat GI tract GvHD

AbstractAcute graft-versus-host disease (aGVHD), mediated by the recognition of host major histocompatibility complex/peptide polymorphisms by donor T cells, remains a significant complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). aGVHD most commonly involves the gastrointestinal tract, liver, and skin; symptomatic aGVHD is treated with corticosteroids. Steroid-nonresponsive aGVHD is a significant problem for patients undergoing allo-HSCT, with <15% of these patients alive 1 year after diagnosis. Previously, we found that the infusion of donor innate lymphoid type 2 (ILC2) cells could prevent and treat aGVHD of the lower gastrointestinal tract with no effect on the graft-versus-leukemia response. This approach for clinical translation is cumbersome, as it would require the generation of donor-derived ILC2 cells for each recipient. Thus, the ability to use third-party ILC2 cells would provide an “off-the-shelf” reagent that could be used to treat and/or prevent aGVHD. Here, we show that third-party ILC2 cells enhance the survival of allo-HSCT recipients. Treatment required at least 4 weekly infusions of ILC2 cells. Mechanistically, we show that ILC2 cell function was completely lost if the cells could not express both interleukin-13 (IL-13) and amphiregulin. Finally, we show that the activity of IL-13 has a greater dependence on the expression of the IL-13R on host rather than donor bone marrow cells. The ability to generate third-party ILC2 cells offers a new avenue for the prevention of aGVHD.

CD127+ CD94+ innate lymphoid cells expressing granulysin and perforin are expanded in patients with Crohn\u2019s disease

Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn’s disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-β strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn’s disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn’s disease patients and may play a role in the pathology of the disease.

Therapeutic Potential of Innate Lymphoid Cells for Multiple Myeloma Therapy.

Simple SummaryMultiple myeloma (MM) is the second most common haematological malignancy. Despite huge progress associated with the introduction of new antimyeloma drugs, MM remains an incurable disease. In this review, we discuss the role of the innate lymphoid system, its role in the pathogenesis of the disease, and the mechanisms by which innate lymphoid cells (ILC) can theoretically achieve therapeutic benefit in MM treatment.Innate lymphoid cells (ILCs) are a recently identified family of lymphocyte-like cells lacking a specific antigen receptor. They are part of the innate immune system. They play a key role in tissue homeostasis and also control inflammatory and neoplastic processes. In response to environmental stimuli, ILCs change their phenotype and functions, and influence the activity of other cells in the microenvironment. ILC dysfunction can lead to a wide variety of diseases, including cancer. ILC can be divided into three subgroups: ILC Group 1, comprising NK cells and ILC1; Group 2, including ILC2 alone; and Group 3, containing Lymphoid Tissue inducers (LTi) and ILC3 cells. While Group 1 ILCs mainly exert antitumour activity, Group 2 and Group 3 ILCs are protumorigenic in nature. A growing body of preclinical and clinical data support the role of ILCs in the pathogenesis of multiple myeloma (MM). Therefore, targeting ILCs may be of clinical benefit. In this manuscript, we review the available data on the role of ILCs in MM immunology and therapy.

Effect of parasympathetic inhibition on expression of ILC2 cells in a mouse model of allergic rhinitis

BackgroundWe wanted to investigate whether parasympathetic inhibition affected the expression of type 2 innate lymphoid cells (ILC2s) in the nasal mucosa of a mouse model of allergic rhinitis (AR). MethodsThirty male C57BL/6 mice were randomly divided into 3 groups: control group, AR group, AR-treated group. AR nasal symptoms were assessed on a semi-quantitative scale according to the frequencies of nose rubbing and sneezing and the degree of rhinorrhea. The expression of cytokines protein in serum was detected by enzyme linked immunosorbent assay (ELISA). The number of ILC2s in nasal mucosa was detected by immunofluorescence double staining assay. Quantitative real-time Polymerase Chain Reaction (qPCR) was used to detect the expression of ILC2-associated factor in nasal mucosa. ResultsThe symptom scores of the AR group were significantly higher than those of the control group and AR-treated group. The expression levels of mouse ovalbumin (OVA) specific IgE, IL4, IL5, and IL13 in the serum of AR group were significantly higher than those in the control group and AR-treated group. The number of ILC2s and the gene expression of ILC2s related factors GATA3, CD25 and CD90 (Thy1) in the nasal mucosa of the AR group were significantly higher than those of the control group and AR-treated group. ConclusionsWe found that parasympathetic inhibition relieved AR symptoms and inhibited immune response of AR mice. ILC2s play an important role in the occurrence and development of AR, and parasympathetic nerve inhibition reduced the number of ILC2s and inhibited the cytokines expression by ILC2s. Our data provide information on the mechanism of action of parasympathetic inhibition in AR.

A Gata3 enhancer necessary for ILC2 development and function

The type 2 helper effector program is driven by the master transcription factor GATA3 and can be expressed by subsets of both innate lymphoid cells (ILCs) and adaptive CD4+ T helper (Th) cells. While ILC2s and Th2 cells acquire their type 2 differentiation program under very different contexts, the distinct regulatory mechanisms governing this common program are only partially understood. Here we show that the differentiation of ILC2s, and their concomitant high level of GATA3 expression, are controlled by a Gata3 enhancer, Gata3 +674/762, that plays only a minimal role in Th2 cell differentiation. Mice lacking this enhancer exhibited defects in several but not all type 2 inflammatory responses, depending on the respective degree of ILC2 and Th2 cell involvement. Our study provides molecular insights into the different gene regulatory pathways leading to the acquisition of the GATA3-driven type 2 helper effector program in innate and adaptive lymphocytes.

MTOR signaling mediates ILC3-driven immunopathology

Innate lymphoid cells (ILCs) have a protective immune function at mucosal tissues but can also contribute to immunopathology. Previous work has shown that the serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1) is involved in generating protective ILC3 cytokine responses during bacterial infection. However, whether mTORC1 also regulates IFN-γ-mediated immunopathology has not been investigated. In addition, the role of mTORC2 in ILC3s is unknown. Using mice specifically defective for either mTORC1 or mTORC2 in ILC3s, we show that both mTOR complexes regulate the maintenance of ILC3s at steady state and pathological immune response during colitis. mTORC1 and to a lesser extend mTORC2 promote the proliferation of ILC3s in the small intestine. Upon activation, intestinal ILC3s produce less IFN-γ in the absence of mTOR signaling. During colitis, loss of both mTOR complexes in colonic ILC3s results in the reduced production of inflammatory mediators, recruitment of neutrophils and immunopathology. Similarly, treatment with rapamycin after colitis induction ameliorates the disease. Collectively, our data show a critical role for both mTOR complexes in controlling ILC3 cell numbers and ILC3-driven inflammation in the intestine.

Topical application of celastrol alleviates atopic dermatitis symptoms mediated through the regulation of thymic stromal lymphopoietin and group 2 innate lymphoid cells

ABSTRACT Atopic dermatitis is a chronic inflammatory skin disease, of which incidence is closely related to exposure to environmental pollutants and allergens. Thymic stromal lymphopoietin (TSLP) plays an important role in the early stages of atopic dermatitis development by inducing Th2 immune responses. In addition, TSLP regulates activation of group 2 innate lymphoid cells (ILC2), promoting the pathogenesis of atopic dermatitis. The aim of this study was to investigate whether celastrol alleviated atopic dermatitis symptoms by regulating TSLP expression and ILC2 stimulation. Celastrol suppressed TSLP production in mouse keratinocyte cells by inhibiting NF-ĸB activation. Topical application of celastrol significantly improved atopic dermatitis symptoms induced by house dust mite (HDM) in NC/Nga mice as determined by dermatitis score and histological assessment. Celastrol decreased the levels of TSLP in atopic dermatitis skin lesions of HDM-stimulated NC/Nga mice. Celastrol reduced levels of Th2 cytokines including IL-4, IL-5, and IL-13 in atopic dermatitis skin lesions of NC/Nga mice. Further, celastrol significantly reduced ILC2 population in atopic dermatitis skin lesions of NC/Nga mice. These results indicate that topical application of celastrol improved atopic dermatitis symptoms by lowering TSLP levels and concomitant immune responses. Data demonstrated that reduced TSLP levels and associated lower number of ILC2 cells alleviate atopic dermatitis symptoms induced by house dust mite.

A Novel Process for the Expansion of Functional Human ILC2 Cells for GCHD Prevention

Recently, we have shown that type 2 mouse innate lymphoid cells (ILC2) ameliorate acute GVHD in a mouse model (Bruce et al, 2017). Moreover, it has been shown by us and others that not only is the recovery of ILC2 cells slow in both mouse and humans allogeneic recipients but also the presence of active human ILC2 cells in correlates with lower incidence of acute GVHD. Thus, we sought out to determine if human ILC2 cells could be enriched and expanded in vitro for potential use as a therapy to prevent or treat acute GVHD in humans. Toward this goal, we utilized human peripheral blood cells from apheresis units. We compared three methods to enrich ILC2 cells: RosetteSep kit, EasySep kit and EasySep NK56 kit. In the apheresis unit, ILC2 cells ranged from 0.023-0.062% of the total cells. In comparing the three enrichment methods, the percentage of enriched ILC2 cells varied from 1.1-6.9% with the EasySep ILC2 kit giving the best yields (6.9% and 4.7e4 total ILC2 cells). Interestingly, although the RosetteSep had the lowest percentage enriched, it had a higher yield than the EasySep NK Kit due to the higher yield of total cells (3.4 e6 compared to 4.3 e5 with 3.7 e4 and 2.2e4 ILC2 cells respectively). We next investigated optimal culture conditions. Our initial culture conditions were with α-MEM media with a cytokine cocktail of IL-2, IL-33 and IL-25. Using these conditions, the range of expansion was 500-1500 fold from the initial number of ILC2 at the end of the culture on Day 10. We tested a number of different interventions for optimization: addition of OP9-DL1 as a stromal cell component, addition of IL-7, and addition of TSLP. These results showed that OP9DL1, IL-7 or TSLP did not improve expansion or change the phenotype of the ILC2 cells at the end of the 10 day culture. We also tested different medias (α-MEM and IMDM) with different concentrations of the cytokines (based on the results by Camelo et al, 2017). We found that cells that were enriched using the RosetteSep and then cultured in α-MEM media with 50 ng/ml of IL-33, IL-25 and 20 IU/Ml IL-2 resulted in the best ILC2 expansion (>1000-fold) within 10 days. The final product was >90% pure ILC2 cells (lineage negative, CD161 and Gata-3 expression). These cells produced IL-13 and IL-5 in culture upon stimulation with PMA/ionomycin but did not produce IFN-gamma. Notably, between 50-80% of the ILC2 cells lost expression of CRTH2 (CD294) while in culture, similar to results found by Camelo et al. (2017). A representative experiment is shown in Figure 1.We tested whether injection of human ILC2 cells ameliorated acute GVHD in a humanized mouse model. In this model, PBMCs are used as a positive control for GVHD. Two different approaches were taken. In the first approach we used third party hILC2 cells. On Day 0, irradiated female NSG mice received 15e6 PBMCs +/- 15e6 ILC2 cells. Mice were monitored for GVHD scores and survival. Recipients that received human ILC2 cells had significantly higher percentage mean initial body weight ratios at each time point beginning at Day 8 (P<0.05). At one month, 32% of controls (n=19) had succumbed to GVHD versus 12.5% (n=8) who had received third party human ILC2 cells. In the second approach we used donor-specific hILC2 cells, Irradiated (Day -1) male NSG mice were given 8e6 PBMC with or without donor matched ILC2 cells in 1:1 ratio on day 0. We found a single injection of matched ILC2 cells increased median survival by 10 days increasing survival from 19 days to 29 days for ILC2 treated animals (p<0.01). Taken together, we have discovered a novel method to rapidly expand human ILC2 cells in vitro. We have found that a single dose of hILC2 cells is active to decrease the severity of xenogeneic acute GVHD. These findings provide a rationale basis for the development of hILC2 cells to treat/prevent GI tract GvHD.Figure 1. Apheresis cells were enriched using the ILC2 RosetteSep kit and were cultured in α-MEM media with 50 ng/ml of IL-33, IL-25 and 20 IU/Ml IL-2 for 10 days. The predominant population was ILC2 cells (A) that expressed GATA3 (B; ILC2 shown in red, negative control in blue) and upon stimulation with PMA and ionomycin expressed IL-13 but not IFN-gamma (C). The cells expanded >1000 fold in 10 days (D).Figure 2. Survival of NSG mice following xeno-transplantation with PBMCs and donor matched expanded ILC2. Kaplan-Meier plot of survival following allo-SCT (n=4 per group) Log-rank (Mantel-cox) test used. [Display omitted] DisclosuresSerody:Merck: Research Funding.

ILC2 Cells Promote Th2 Cell Differentiation in AECOPD Through Activated Notch-GATA3 Signaling Pathway.

This study is to investigate the capacity of type 2 innate lymphoid cells (ILC2s) in regulating the Th2 type adaptive immune response of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The study enrolled healthy people, stable chronic obstructive pulmonary disease (COPD) patients, and AECOPD patients. Flow cytometry was used to detect Th2 and ILC2 cells in the peripheral blood. In addition, ILC2s from the peripheral blood of AECOPD patients were stimulated with PBS, IL-33, Jagged1, DAPT, IL-33+Jagged1, IL-33+DAPT, and IL-33+Jagged-1+DAP in vitro. The levels of cytokines in the culture supernatant were detected by ELISA and the culture supernatant was used to culture CD4 + T cells. The mRNA and protein levels of Notch1, hes1, GATA3, RORα, and NF-κB of ILC2s were detected by real-time PCR and Western blot. The proportion of Th2 and ILC2s was significantly increased in the peripheral blood of AECOPD patients, alone with the increased Notch1, hes1, and GATA3 mRNA levels. In vitro results showed that the mRNA and protein levels of Notch1, hes1, GATA3 and NF-κB were significantly increased after stimulation with Notch agonist, meanwhile, the level of type 2 cytokines were increased in the supernatant of cells stimulated with Notch agonist, and significantly promoted differentiation of Th2 cells in vitro. Disruption of Notch pathway weakened GATA3 expression and cytokine production, and ultimately affected the differentiation of Th2 cells. In conclusion, our results suggest that ILC2s can promote Th2 cell differentiation in AECOPD via activated Notch-GATA3 signal pathway.

Intranasal delivery of allergen in a nanoemulsion adjuvant inhibits allergen-specific reactions in mouse models of allergic airway disease.

Atopic diseases are an increasing problem that involve both immediate hypersensitivity reactions mediated by IgE and unique cellular inflammation. Many forms of specific immunotherapy involve the administration of allergen to suppress allergic immune responses but are focused on IgE-mediated reactions. In contrast, the effect of allergen-specific immunotherapy on allergic inflammation is complex, not entirely consistent and not well understood. We have previously demonstrated the ability of allergen administered in a nanoemulsion (NE) mucosal adjuvant to suppress IgE-mediated allergic responses and protect from allergen challenge in murine food allergy models. This activity was associated with decreases in allergen-specific IL-10 and reductions in allergic cytokines and increases in regulatory T cells. Here, we extend these studies to using 2 distinct models, the ovalbumin (OVA) and cockroach (CRA) models of allergic airway disease, which are based predominantly on allergic inflammation. Acute or chronic allergic airway disease was induced in mice using ovalbumin and cockroach allergen models. Mice received three therapeutic immunizations with allergen in NE, and reactivity to airway challenge was determined. Therapeutic immunization with cockroach or OVA allergen in NE markedly reduced pathology after airway challenge. The 2 models demonstrated protection from allergen challenge-induced pathology that was associated with suppression of Th2-polarized immune responses in the lung. In addition, the reduction in ILC2 numbers in the lungs of allergic mice along with reduction in epithelial cell alarmins, IL-25 and IL-33, suggests an overall change in the lung immune environment induced by the NE immunization protocol. These results demonstrate that suppression of allergic airway inflammation and bronchial hyper-reactivity can be achieved using allergen-specific immunotherapy without significant reductions in allergen-specific IgE and suggest that ILC2 cells may be critical targets for this activity.

Mapping Rora expression in resting and activated CD4+ T cells.

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.

Temporal overexpression of IL-22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis.

IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-γ+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.

Development of group-3 innate lymphoid cells via Tox2-dependent pathway in gut.

Abstract Innate lymphoid cells (ILCs) are immune cells that lack specific antigen receptors but possess similar effector functions as T cells. Interestingly, ILCs and T cells express many of the same transcription factors. One such factor is TOX, a sequence non-specific nuclear transcriptional regulator that is crucial for development of both CD4 T cells and ILCs. TOX-deficient mice have significantly reduced number of early innate lymphoid progenitors (EILP) and ILC progenitors (ILCP). Consequently, the mature NK cells, ILC1, ILC2 and ILC3 subsets are greatly reduced in Tox−/− mice. Interestingly ILC3 numbers in gut are not reduced by the absence of TOX. We have discovered that TOX2, another member of the TOX family of HMG-box proteins, is highly expressed in ILC precursors. We generated Tox2−/− mice and found they have reduced ILC3 in gut; however, ILC3 cells in spleen or lymph node are intact. We also observed ILCP in fetal gut and adult gut were significantly reduced in Tox2−/−mice suggesting TOX2 is required for gut ILC3 development. However, ILCP numbers in adult gut of Tox−/− mice were unaffected. We are currently performing single-cell RNA-sequencing on gut ILC3 from Tox2−/− and Tox−/− mice to transcriptionally characterize these cells and to identify the heterogeneity within the ILC3 population. Taken together, our results indicate the existence of at least two pathways of ILC development: a central pathway in bone marrow and fetal liver that is TOX dependent; and a peripheral pathway of development of ILC3 in gut that requires TOX2.