Lung ILC2s Research Articles

IQ motif-containing GTPase-activating protein 1 is essential for the optimal maintenance of lung ILC2s

Abstract Group 2 innate lymphoid cells (ILC2s) play critical roles in type 2 immunity and are crucial for pathogenesis of various types of inflammatory disease. IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein that is involved in multiple cellular functions such as cell survival and trafficking. While the roles for IQGAP1 in T and B lymphocytes have been uncovered, the physiological significance of IQGAP1 in innate lymphocytes remains to be elucidated. In the current study, we demonstrate that using bone marrow chimeras, the deficiency of IQGAP1 caused an impaired survival of lung ILC2s in a cell-intrinsic manner and that Iqgap1−/− mice displayed decreased accumulation of ILC2s after administration of papain and thereby reduced pathology of the disease. Moreover, Iqgap1−/− ILC2s showed a significantly enhanced apoptosis as compared to wild-type ILC2s under both steady-state and inflammatory conditions. Together these results identify for the first time that IQGAP1 is essential for homeostasis of ILC2s in the lung.

IL-33 and allergen exposure promote development of “memory-like” ILC2s in the Airways

Abstract The alarmin, IL-33 plays a critical role in the inception of lung inflammation and the exacerbation of allergic airway disease. Inhalation of environmental allergens and particulates is known to promote IL-33 release, and the cells responsive to this cytokine include group 2 innate lymphoid cells (ILC2s), dendritic cells and natural killer (NK) cells. In this study, we evaluated the effect of IL-33 and house dust mite allergen inhalation on the properties of lung ILC2s. Instillation of IL-33 into the airways of mice resulted in a marked elevation in number of ILC2s and eosinophils. Importantly, a notable feature of the inflammatory response was an increase in CD103 expression by ILC2 (from 3% to 45%). These CD103+ ILC2s appeared to represent memory-like ILC2s since they were highly responsive to IL-25 and produced significant levels of IL-9 and IL-13. Critically, inhalation of house dust mite allergen also increased CD103 expression by ILC2s. Given that CD103 expression can be induced by TGF-β, we next determined whether active TGF-β1 is released following IL-33-driven lung inflammation. Remarkably, the levels of active TGF-β1 present in the airways were markedly raised following IL-33 instillation, and this was responsible for a reduction in IFN-γ-producing NK cells and raised CD226 (DNAM-1) expression. Collectively, these findings reveal that IL-33 and allergen promote the accumulation of CD103+ memory-like ILC2s in the airways. A role for TGF-β in promoting CD103 expression while suppressing NK cell-mediated regulation of the inflammatory process is proposed.

COX inhibition enhances allergen-induced IL-33 release and ILC2 responses in mouse lungs

Abstract The cyclooxygenase (COX) metabolic pathway has regulatory functions in immune responses and inflammation. However, the effect of the COX pathway on innate airway inflammation induced by protease-containing allergens such as Alternaria alternata is not fully defined. Here we show that COX inhibition augmented innate lung type 2 responses after repeated airway exposures to Alternaria extract in mice. We treated wild type BALB/c and IL-33 KO mice with either the COX inhibitor indomethacin or vehicle in drinking water and challenged mice intranasally with Alternaria extract for 4 consecutive days to induce innate lung inflammation. We found that indomethacin significantly increased the numbers of group 2 innate lymphoid cells (ILC2) and IL-5+IL-13+ ILC2 in the lung. Indomethacin also increased type 2 cytokine (IL-5 and IL-13) responses, the percentages of eosinophils, and mucus production in the lung. IL-33 is required for Alt-induced lung ILC2 responses, and indomethacin did not change IL-5 and IL-13 expression in IL-33 KO mice. Consistently, indomethacin increased the release of IL-33 in bronchoalveolar lavage fluid after Alternaria challenge, suggesting that more IL-33 was available for ILC2 activation. Indomethacin also increased reactive oxygen species (ROS) production in the lung, providing a possible mechanism for the increased IL-33 release. Although contrasting effects of PGD2 and PGE2/PGI2 on ILC2 responses have been previously reported, the overall effect of COX pathway on ILC2 function is inhibitory in Alternaria extract-induced airway innate type 2 responses.

Androgen receptor activity underlies sex differences in lung-resident ILC2 functional responses during influenza virus infection

Abstract Lung-resident group 2 innate lymphoid cells (ILC2s) produce type 2 cytokines and facilitate tissue repair in response to alarmins such as IL-33 released during respiratory virus infection, but they also may be functionally suppressed by type 1 cytokines. Lung ILC2s express notably high levels of intracellular androgen receptor (AR) compared to ILC1s, T and B cells. To test the hypothesis that females and males show differential ILC2 responses in influenza virus (IAV) infection, we analyzed the numbers and functional responses of lung ILCs in IAV infected mice. On days 3–10 post-infection, lungs of female mice contained greater numbers of ILC2s and ILC1s compared to males. However, the female ILC2s were preferentially suppressed at the peak of infection, with a dampened type 2 program manifest as attenuated proliferation, decreased IL-5 and amphiregulin production, reduced expression of GATA-3 and IL-33R and increased surface IFNGR. IFNG levels in the lung were comparable between sexes at day 7 post-infection, suggesting intrinsic differences in ILC2 responses to interferons. Indeed, naïve female ILC2s showed higher expression of IFNGR and higher phospho-STAT1 levels following stimulation by IFNG. ILC2s in naive male mice with lymphocyte-restricted AR deficiency displayed levels of IFNGR comparable to female mice, suggesting AR activity underlies sex differences in intrinsic IFNGR expression. Early life orchiectomy revealed that endogenous androgens decreased ILC2 numbers but protected males from suppression of ILC2s in IAV infection. Taken together, our data show that AR activity preserves canonical ILC2 function in males during IAV infection and may help to explain the documented human gender differences in immunity to IAV.

TSLP and IL-33 reciprocally promote each other's lung protein expression and ILC2 receptor expression to enhance innate type-2 airway inflammation.

BackgroundThe epithelial cell‐derived danger signal mediators thymic stromal lymphopoietin (TSLP) and IL‐33 are consistently associated with adaptive Th2 immune responses in asthma. In addition, TSLP and IL‐33 synergistically promoted group 2 innate lymphoid cell (ILC2) activation to induce innate allergic inflammation. However, the mechanism of this synergistic ILC2 activation is unknown.MethodsBALB/c WT and TSLP receptor‐deficient (TSLPR−/−) mice were challenged intranasally with Alternaria extract (Alt‐Ext) or PBS for 4 consecutive days to evaluate innate airway allergic inflammation. WT mice pre‐administered with rTSLP or vehicle, TSLPR−/− mice, and IL‐33 receptor‐deficient (ST2−/−) mice were challenged intranasally with Alt‐Ext or vehicle once or twice to evaluate IL‐33 release and TSLP expression in the lung. TSLPR and ST2 expression on lung ILC2 were measured by flow cytometry after treatment of rTSLP, rIL‐33, rTSLP + rIL‐33, or vehicle.ResultsThymic stromal lymphopoietin receptor deficient mice had significantly decreased the number of lung ILC2 expressing IL‐5 and IL‐13 following Alt‐Ext‐challenge compared to WT mice. Further, eosinophilia, protein level of lung IL‐4, IL‐5, and IL‐13, and airway mucus score were also significantly decreased in TSLPR−/− mice compared to WT mice. Endogenous and exogenous TSLP increased Alt‐Ext‐induced IL‐33 release into BALF, and ST2 deficiency decreased Alt‐Ext‐induced TSLP expression in the lung. Further, rTSLP and rIL‐33 treatment reciprocally increased each other's receptor expression on lung ILC2 in vivo and in vitro.ConclusionThymic stromal lymphopoietin and IL‐33 signaling reciprocally enhanced each other's protein release and expression in the lung following Alt‐Ext‐challenge and each other's receptor expression on lung ILC2 to enhance ILC2 activation.

Activation of TLR3 Inhibits Innate Type 2 Immune Responses Induced by Alternaria in the Airways

Group 2 innate lymphoid cells (ILC2s) likely play a major role in allergic immune responses in the airways. However, our knowledge is limited as to how the activities and functions of these ILC2s are regulated in vivo. TLR3 recognizes viral double-stranded RNA, and polycytidylic acid (Poly(I:C)) serves a specific and potent TLR3 agonist. The goal of this project was to investigate the effects of TLR3 activation on ILC2s. Naïve BALB/c mice and IL-5-reporter mice (IL-5-Venus mice) were pretreated with Poly(I:C) intranasally (i.n.) for 6 to 24 hours and then exposed i.n. to Alternaria extract. IL-33 release and innate type 2 immune responses were analyzed at 1 hour or 4.5 hours after the exposure to Alternaria. In addition, lung single cell suspensions were cultured in vitro to address the mechanisms. Intranasal exposure to Alternaria induced IL-5 and IL-13 production in the lungs. The experiments with Il5-reporter mice suggested that Lin-CD25+CD44hi lung ILC2s are the major source of IL-5. Pretreatment with Poly(I:C) significantly inhibited Alternaria-induced production of IL-5 and IL-13, and reduced the proportion of Il5-expressing ILC2s. Poly(I:C) did not inhibit Alternaria-induced IL-33 release into the BAL fluids, but promoted IFN-g and IFN-a production in the lungs. IFN-g and IFN-a suppressed type 2 cytokine productions by lung single cells stimulated with IL-33 in vitro. TLR3 activation inhibits ILC2-driven innate type 2 immune response to Alternaria likely through the production of IFNs. Activation of TLRs may antagonize the innate and type 2 immunostimulatory activities of airborne allergens.

A critical role for c-Myc in group 2 innate lymphoid cell activation.

Asthma is a complicated chronic inflammatory disorder characterized by airway inflammation and bronchial hyperresponsiveness. Group 2 innate lymphoid cells (ILC2) are tissue-resident innate effector cells that can mediate airway inflammation and hyperresponsiveness through production of IL-5, IL-13 and VEGFA. ILC2 in asthma patients exhibit an activated phenotype. However, molecular pathways that control ILC2 activation are not well understood. MYC expression was examined in ILC2 sorted from peripheral blood of healthy controls and asthma patients or cultured with or without activating cytokines. CRISPR knockout technique was used to delete c-Myc in primary murine lung ILC2 or an ILC2 cell line. Cell proliferation was examined, gene expression pattern was profiled by genome-wide microarray analysis, and direct gene targets were identified by Chromatin immunoprecipitation (ChIP). ILC2 responses, airway inflammation and airway hyperresponsiveness were examined in Balb/c mice challenged with Alternaria extracts, with or without treatment with JQ1. ILC2 from asthma patients expressed increased amounts of MYC. Deletion of c-Myc in ILC2 results in reduced proliferation, decreased cytokine production, and reduced expression of many lymphocyte activation genes. ChIP identified Stat6 as a direct gene target of c-Myc in ILC2. In vivo inhibition of c-Myc by JQ1 treatment repressed ILC2 activity and suppressed Alternaria-induced airway inflammation and AHR. c-Myc expression is upregulated during ILC2 activation. c-Myc is essential for ILC2 activation and their in vivo pathogenic effects. These findings suggest that targeting c-Myc may unlock novel strategies to combat asthma or asthma exacerbation.

IQ motif-containing GTPase-activating protein 1 is essential for the optimal maintenance of lung ILC2s.

Group 2 innate lymphoid cells (ILC2s) play critical roles in type 2 immunity and are crucial for pathogenesis of various types of inflammatory disease. IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein that is involved in multiple cellular functions such as cell survival and trafficking. While the roles for IQGAP1 in T and B lymphocytes have been uncovered, the physiological significance of IQGAP1 in innate lymphocytes remains to be elucidated. In the current study, we demonstrate that using bone marrow chimeras, the deficiency of IQGAP1 caused an impaired survival of lung ILC2s in a cell-intrinsic manner and that Iqgap1-/- mice displayed decreased accumulation of ILC2s after administration of papain and thereby reduced the pathology of the disease. Moreover, Iqgap1-/- ILC2s showed a significantly enhanced apoptosis as compared to wild-type ILC2s under both steady-state and inflammatory conditions. Together these results identify for the first time that IQGAP1 is essential for homeostasis of ILC2s in the lung.

Single-cell analysis of RORα tracer mouse lung reveals ILC progenitors and effector ILC2 subsets

Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor–related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2− population different from conventional IL-18Rα−ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets.

Single-cell analysis of RORα tracer mouse lung reveals ILC progenitors and effector ILC2 subsets.

Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor-related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2- population different from conventional IL-18Rα-ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets.

IL-33 Therapy Prevents Acute Lung Injury after Transplantation Via IL-9-Producing Type 2 Innate Lymphoid Cells Induction

Idiopathic pneumonia syndrome (IPS) is a noninfectious acute lung injury, often fatal, following allogeneic hematopoietic cell transplantation (HCT). Similar to graft-versus-host disease (GVHD), IPS is mediated by type 1 cytopathic T cells accompanied with high levels of proinflammatory cytokines. We previously showed that elevated plasma soluble Stimulation-2 (sST2), which acts as a decoy receptor for IL-33, is a risk factor of death by GVHD (N. Engl. J. Med, 2013) or by IPS (Biol Blood Marrow Transplant, 2018). ST2 blockade of the excess of sST2 with a neutralizing antibody or small molecules released plasmatic IL-33, increasing its availability to cytoprotective T cells expressing the transmembrane molecule form of ST2, such as regulatory T cells (Tregs) reducing the type 1 proinflammatory T cell-response (Sci Transl Med, 2015; JCI Insight, 2019). The membrane-ST2 is also expressed on type 2 innate lymphoid cells (ILC2s), mostly present in lungs. Herein, we first confirmed in a cohort of 673 HCT patients that plasma sST2 measured 14 days following HCT is increased 10 fold in IPS patients (n=22) as compared to controls with no IPS/GVHD (n=271), and is 6 fold higher as compared to GVHD patients (n=380) (Figure 1A). Patients with IPS and high sST2 levels above the median of 200 ng/ml, were significantly more likely to die than patients with lower sST2 levels (Figure 1B). In a therapeutic translational purpose, we then inquired if local administration of IL-33 via intranasal route at a dose of 500 ng/mouse daily (5 doses from day -1 to +3) will ameliorate the recipients' pulmonary function tests in a major-mismatched B6 → Balb/c HCT murine model. Allogeneic recipients that received IL-33 improved their lung compliance (C), lung resistance (R), and elastance (E) as compared to vehicle treated mice (Figure 2A). Based on our patients' data, we further explored the sST2/IL-33 ratio. Although the treatment was local, plasma IL-33 increased at day +7 post-HCT and therefore the sST2/IL-33 ratio was significantly decreased in IL-33 treated mice (Figure 2B). Parraleling this decrease, both systemic IFNγ and TNFα at day +7 post-HCT were significantly lower in mice treated with IL-33 compared to vehicle treated mice (Figure 2B). Findings in the plasma were also correlated with a local decrease of IFNγ secretion in the bronchoalveolar lavage of IL-33 treated mice (not shown). The frequencies and numbers of donor CD45.1+ IFNg+CD4+ and IFNg+CD8+ donor T cells in the lungs of IL-33 treated mice were also significantly decreased as compared to vehicle treated mice (Figure 2C). We next sought to determine if IL-33 had an impact on recipient ILC2s (CD45.2+Lin-CD90.2+GATA3+ST2+). As shown in Figure 2D, recipient mice treated with IL-33 had significant higher frequencies of lung ILC2s at day +7 post-HCT compared to mice treated with vehicle. RNA-seq analysis of sorted ILC2s from the lungs of naïve GATA3 reporter mice treated with IL-33 showed increased Il9 and PU.1 transcripts in lung ILC2s, validated at the protein level in allogeneic mice treated with IL-33 as compared to allogeneic vehicle treated mice in which ILC2s were undetectebale (Figure 2D). As antibody (Ab) injection is more clinically relevant than local cytokine instillation, and since we have shown that anti-ST2 Ab results in IL-33 increase, we tested this in a minor-mismatched B6 → C3H.SW HCT murine model, and respectively treated mice with anti-ST2 Ab 100μg/dose every other day (6 doses total) or anti-ST2 Ab 200μg/dose for 2 doses at days -1 and +1 or isotype 100μg/dose for 6 doses. Prophylactic administration of anti-ST2 Ab with 6 doses and 2 doses significantly decreases mortality as compared to isotype with six doses allowing a better survival than the peritransplant administration (Figure 3A). Plasma IL-33 increased in both anti-ST2 treated groups vs. isotype (Figure 3B). Consistently, both plasma IFNγ and TNFα were significantly decreased in anti-ST2 Ab treated groups (Figures 3C, 3D). Percentages of cytopathic lung donor CD4+IFNγ+ and CD8+IFNγ+ T cells were decreased (Figure 3E) while cytoprotective lung recipient total, IL-9+, and PU.1+ ILC2s were increased in anti-ST2 Ab treated groups vs. isotype (Figures 3F, 3G). Tregs in both anti-ST2 Ab treated groups were concomitantly increased (Figure 3H). We concluded that not only is sST2 a prognostic biomarker for IPS but it is also a promising therapeutic target that may prevent IPS via IL-33 induced IL-9 secreting ILC2s. Disclosures Paczesny: Viracor Eurofins Clinical Diagnostic: Patents & Royalties.

IRF-7 Is a Critical Regulator of Type 2 Innate Lymphoid Cells in Allergic Airway Inflammation

Allergic asthma is a highly prevalent airway disease triggered by hyperresponsiveness to inhaled allergens. Interferon regulatory factor 7 (IRF7) has been shown to be highly expressed in nasal aspirates from children with asthma. Type 2 innate lymphoid cells (ILC2s) represent the major player in allergic airway inflammation. The role of IRF7 in ILC2-driven asthma remains to be explored. Here, we report that IRF7 expression in murine lung ILC2s is dramatically induced upon papain or interleukin-33 (IL-33) stimulation. ILC2s from asthma patients display a much higher level of IRF7 than those from healthy donors. Deficiency of IRF7 in mice significantly impairs the expansion and function of lung ILC2s in multiple models of allergic asthma. Furthermore, the regulation of ILC2s by IRF7 is cell intrinsic and mediated by the transcription factor Bcl11b. These observations identify IRF7 as a regulator of lung ILC2s, whichmay have immunotherapeutic value in allergic asthma.

Calcitonin Gene-Related Peptide Negatively Regulates Alarmin-Driven Type 2 Innate Lymphoid Cell Responses

Neuroimmune interactions have emerged as critical modulators of allergic inflammation, and type 2 innate lymphoid cells (ILC2s) are an important cell type for mediating these interactions. Here, we show that ILC2s expressed both the neuropeptide calcitonin gene-related peptide (CGRP) and its receptor. CGRP potently inhibited alarmin-driven type 2 cytokine production and proliferation by lung ILC2s both invitro and invivo. CGRP induced marked changes in ILC2 expression programs invivo and invitro, attenuating alarmin-driven proliferative and effector responses. A distinct subset of ILCs scored highly for a CGRP-specific gene signature after invivo alarmin stimulation, suggesting CGRP regulated this response. Finally, we observed increased ILC2 proliferation and type 2 cytokine production as well as exaggerated responses to alarmins in mice lacking the CGRP receptor. Together, these data indicate that endogenous CGRP is a critical negative regulator of ILC2 responses invivo.

Activation through toll-like receptor 2 on group 2 innate lymphoid cells can induce asthmatic characteristics.

Type 2 innate lymphoid cells (ILC2s) are one of the sources of IL-5 and IL-13 in allergic airway inflammation. Innate immune receptors such as Toll-like receptors (TLRs) expressed on epithelial cells could contribute to ILC2 activation through IL-33 production, but a direct effect of TLRs on ILC2s remains to be elucidated. We hypothesized that TLRs can directly activate lung ILC2s and participate in the pathogenesis of asthma. After intranasal administration of IL-33 to wild-type (WT), TLR2KO and TLR4KO female mice, ILC2s were isolated from harvested lungs. ILC2s were incubated with IL-2 and TLR stimulants (pam3csk4 (PAM), house dust mite extract (HDM)). In some experiments, TLR2 or dectin-1 signalling inhibitors were used. As an in vivo model, the mice were treated with IL-33 and rested until lung recruitment of eosinophils regressed. Then they were treated intranasally with PAM+HDM or vehicle and analysed. In vitro stimulation of isolated ILC2s showed that PAM could induce IL-13 and IL-5 production, and HDM had a synergistic effect on this stimulation. Both effects were dependent on TLR2 and NF-κB signalling. PAM+HDM stimulation of WT mice led to increased ILC2s, airway hyperresponsiveness and increased levels of both neutrophils and eosinophils in bronchoalveolar lavage fluid. These observations were dependent on TLR2. TLR2 can directly activate lung ILC2s, an effect that is augmented by HDM. Asthmatic characteristics mediated through the TLR2 pathway were evident inthe in vivo mice model. These data implicate a new pathway of ILC2 activation in the pathogenesis of asthma.

Dietary Fiber-Induced Microbial Short Chain Fatty Acids Suppress ILC2-Dependent Airway Inflammation.

Group 2 Innate lymphoid cells (ILC2) contribute significantly to allergic inflammation. However, the role of microbiota on ILC2s remains to be unraveled. Here we show that short chain fatty acids (SCFAs), such as butyrate, derived from fermentation of dietary fibers by the gut microbiota inhibit pulmonary ILC2 functions and subsequent development of airway hyperreactivity (AHR). We further show that SCFAs modulate GATA3, oxidative phosphorylation, and glycolytic metabolic pathways in pulmonary ILC2s. The observed phenotype is associated with increased IL-17a secretion by lung ILC2s and linked to enhanced neutrophil recruitment to the airways. Finally, we show that butyrate-producing gut bacteria in germ-free mice effectively suppress ILC2-driven AHR. Collectively, our results demonstrate a previously unrecognized role for microbial-derived SCFAs on pulmonary ILC2s in the context of AHR. The data suggest strategies aimed at modulating metabolomics and microbiota in the gut, not only to treat, but to prevent lung inflammation and asthma.

Essential role of CD4+T cells for the activation of group 2 innate lymphoid cells during respiratory syncytial virus infection in mice.

Aim: To investigate whether and how CD4+T cells contribute to ILC2 activation during respiratory syncytial virus (RSV) infection. Methods: The methods of flow cytometry, quantitative PCR and ELISA were used in the present study. Results: Depletion of CD4+T cells diminished the numbers of lung ILC2s as well as their ability to produce type 2 cytokines. CD4+T cell-mediated ILC2 activation is related to IL-2. The main cellular source of IL-2 was CD4+T cells. Depletion of CD4+T cells decreased IL-2 levels in the lungs of RSV-infected mice. IL-2 can directly stimulate ILC2 proliferation and promote ILC2s to produce cytokines. Treatment of mice with neutralizing anti-IL-2 monoclonal antibodies diminished ILC2 activation. Conclusion: These results suggest that CD4+T cells contribute to RSV-induced ILC2 activation partly via producing IL-2.

Critical role of OX40/OX40L in ILC2-mediated activation of CD4+T cells during respiratory syncytial virus infection in mice

CD4+T cells are crucial cellular source of type 2 cytokines and responsible for RSV-induced asthma-like symptoms and asthma exacerbations. However, the mechanism for regulating the activation of CD4+T cells during RSV infection is not clear completely. We show in this study that infection with RSV may induce an expansion and activation of CD4+T cells in the lungs of BALB/c mice. RSV-induced CD4+T cell expansion and activation seems to depend upon the pulmonary group 2 innate lymphoid cells (ILC2s), since adoptive transfer of lung ILC2s can enhance not only the numbers of CD4+T cells but also the cytokine production by CD4+T cells. Interestingly, blockade of the contact between ILC2s and CD4+T cells, may significantly diminish the CD4+T cell expansion and cytokine production, suggesting that membrane molecules may be involved in ILC2-regulated CD4+T cell activation. In fact, infection with RSV resulted in an increase in the numbers of OX40+CD4+T cells as well as OX40L+ILC2s in the lungs of mice. Moreover, the mRNA expressions of OX40 and OX40L as well as the levels of OX40 and OX40L proteins in the lung CD4+T cells and ILC2s were elevated respectively. When co-culture of CD4+T cells with ILC2s in the presence of anti-OX40L antibody, the cytokine productions by CD4+T cells were reduced markedly, suggesting that lung ILC2s may regulate RSV-induced CD4+T cell expansion and activation perhaps via OX40/OX40L interaction.

MicroRNA-146a negatively regulates IL-33 in activated group 2 innate lymphoid cells by inhibiting IRAK1 and TRAF6.

Type II innate lymphoid cells (ILC2) play a very important role in the pathogenesis of allergic asthma. This study aims to investigate whether miR-146a inhibition of asthma is related with interleukin (IL)-33 signaling path way in ILC2 and the underlying mechanisms. Asthma mice model was induced by ovalbumin. miRNA146a mimics was administrated to asthma mice or transfected to activated ILC2 purified from asthma mice lung. RT-PCR was used to detect miRNA146a level in lung tissue and ILC2. IL-5 and IL-13 levels in culture supernatant were detected by flow cytometry. Interleukin-1 receptor-associated kinase 1 (IRAK1), TNF receptor-associated factor 6 (TRAF6), signal transducer and activator of transcription 1 (STAT1) protein expression levels were detected by western blot. miR-146a directly inhibited ILC2 function and suppressed ILC2 proliferation both in vivo and in vitro. During stimulation of ILC2, miR-146a expression gradually increased with a decrease of cell proliferation. Modulation of ILC2 function by miR-146a may depend on IL-33/interleukin 1 receptor-like 1 (IL1RL1 or ST2) signaling through inhibiting IRAK1 and TRAF6.miR-146a can inhibit IRAK1 and TRAF6, downstream molecules of ST2 signal pathway, thereby negatively regulate IL-33/ST2-activated ILC2 to inhibit asthma. Targeting miR-146 maybe a novel strategy for the treatment of allergic asthma.

Compartmentalized effects of aging on group 2 innate lymphoid cell development and function.

The effects of aging on innate immunity and the resulting impacts on immunosenescence remain poorly understood. Here, we report that aging induces compartmentalized changes to the development and function of group 2 innate lymphoid cells (ILC2), an ILC subset implicated in pulmonary homeostasis and tissue repair. Aging enhances bone marrow early ILC2 development through Notch signaling, but the newly generated circulating ILC2 are unable to settle in the lungs to replenish the concomitantly declining mature lung ILC2 pool in aged mice. Aged lung ILC2 are transcriptomically heterogeneous and functionally compromised, failing to produce cytokines at homeostasis and during influenza infection. They have reduced expression of Cyp2e1, a cytochrome P450 oxidase required for optimal ILC2 function. Transfer of lung ILC2 from young mice enhances resistance to influenza infection in old mice. These data highlight compartmentalized effects of aging on ILC and indicate that targeting tissue‐resident ILCs might unlock therapies to enhance resistance to infections and diseases in the elderly.

Unconventional ST2- and CD127-negative lung ILC2 populations are induced by the fungal allergen Alternaria alternata

We demonstrate that unconventional ST2- and CD127-negative ILC2 populations are present in mouse lung and are induced by Alternaria, suggesting commonly used ILC2 identification practices do not accurately enumerate the total burden of type 2 cytokine producing ILC2s.