ILC2 Expansion Research Articles

IFN-γ Blocks Development of an Asthma Phenotype in Rhinovirus-Infected Baby Mice by Inhibiting Type 2 Innate Lymphoid Cells.

Early-life wheezing-associated infections with rhinovirus (RV) have been associated with asthma development in children. We have shown that RV infection of 6-day-old mice induces mucous metaplasia and airways hyperresponsiveness, which is dependent on IL-13, IL-25, and type 2 innate lymphoid cells (ILC2s). Infection of immature mice fails to induce lung IFN-γ expression, in contrast to mature 8-week-old mice with a robust IFN-γ response, consistent with the notion that deficient IFN-γ production in immature mice permits RV-induced type 2 immune responses. We therefore examined the effects of intranasal IFN-γ administration on RV-induced ILC2 expansion and IL-13 expression in 6-day-old BALB/c and IL-13 reporter mice. Airway responses were assessed by histology, immunofluorescence microscopy, quantitative polymerase chain reaction, ELISA, and flow cytometry. Lung ILC2s were also treated with IFN-γ ex vivo. We found that, compared with untreated RV-infected immature mice, IFN-γ treatment attenuated RV-induced IL-13 and Muc5ac mRNA expression and mucous metaplasia. IFN-γ also reduced ILC2 expansion and the percentage of IL-13-secreting ILC2s. IFN-γ had no effect on the mRNA or protein expression of IL-25, IL-33, or thymic stromal lymphoprotein. Finally, IFN-γ treatment of sorted ILC2s reduced IL-5, IL-13, IL-17RB, ST2, and GATA-3 mRNA expression. We conclude that, in immature mice, IFN-γ inhibits ILC2 expansion and IL-13 expression in vivo and ex vivo, thereby attenuating RV-induced mucous metaplasia. These findings demonstrate the antagonistic function of IFN-γ on ILC2 expansion and gene expression, the absence of which may contribute to the development of an asthma-like phenotype after early-life RV infection.

MicroRNA-155 is a critical regulator of type 2 innate lymphoid cells and IL-33 signaling in experimental models of allergic airway inflammation

Allergic airway inflammation is triggered by allergen exposure through several steps including release of IL-33, which promotes cytokine (IL-5, IL-13) production by type 2 innate lymphoid cells (ILC2s). MicroRNA (miR)-155 has recently been described to regulate adaptive responses in allergic inflammation. However, the role of miR-155 in the regulation of ILC2s remains unexplored. We sought to elucidate the contribution of miR-155 in ILC2 expansion using experimental murine models of allergic airway inflammation. To determine the role of miR-155 in the regulation of ILC2s in allergic airway inflammation, miR-155 deficient (miR-155-/-) and wild-type (WT) mice were subjected to acute or chronic allergen-induced inflammation or treated with recombinant IL-33. miR-155 was 10-fold upregulated in WT-derived ILC2s in response to IL-33. Furthermore, miR-155-/- mice demonstrated impaired lung IL-33 levels in response to allergen challenge and the number of ILC2s was significantly reduced in allergen-challenged miR-155-/- mice compared with WT mice. Exogenous IL-33 treatment revealed that miR-155 is needed for IL-33-induced ILC2 expansion and eosinophilic airway inflammation. Indeed, ILC2s from IL-33-challenged miR-155-/- lungs exhibited impaired proliferation, GATA-3 expression, and IL-13 production as compared with IL-33-challenged WT ILC2s. Our findings for the first time demonstrate that ILC2s and IL-33 signaling are regulated by miR-155 in allergicairway inflammation.

The ETS1 transcription factor is required for the development and cytokine-induced expansion of ILC2.

Group 2 innate lymphoid cells (ILC2s) are a subset of ILCs that play a protective role in the response to helminth infection, but they also contribute to allergic lung inflammation. Here, we report that the deletion of the ETS1 transcription factor in lymphoid cells resulted in a loss of ILC2s in the bone marrow and lymph nodes and that ETS1 promotes the fitness of the common progenitor of all ILCs. ETS1-deficient ILC2 progenitors failed to up-regulate messenger RNA for the E protein transcription factor inhibitor ID2, a critical factor for ILCs, and these cells were unable to expand in cytokine-driven in vitro cultures. In vivo, ETS1 was required for the IL-33-induced accumulation of lung ILC2s and for the production of the T helper type 2 cytokines IL-5 and IL-13. IL-25 also failed to elicit an expansion of inflammatory ILC2s when these cells lacked ETS1. Our data reveal ETS1 as a critical regulator of ILC2 expansion and cytokine production and implicate ETS1 in the regulation of Id2 at the inception of ILC2 development.

ICOS regulates the pool of group 2 innate lymphoid cells under homeostatic and inflammatory conditions in mice.

Group 2 innate lymphoid cells (ILC2s) are innate effectors playing an important role in the defense against helminthic infections and in the pathogenesis of allergic inflammation. Cytokines have been identified as the major stimuli driving ILC2 activation and expansion. Conversely, it is unclear whether costimulatory molecules contribute to regulation of ILC2 functions. ILC2s display high expression of inducible T-cell costimulator (ICOS), which belongs to the CD28 superfamily, and which has been shown to control late effector T-cell functions, and is of utmost importance for the humoral immune response. However, the biological function of ICOS expression on ILC2s is unknown. Here, we show that ICOS signaling in mice regulates ILC2 homeostasis independently of T cells and B cells, by promoting proliferation and accumulation of mature ILC2s in lung and intestine. In a model of IL-33-induced airway inflammation, ICOS controls ILC2 activation and eosinophil infiltration in the lung. Our data identify a role of ICOS in innate immunity and indicate that not only cytokines, but also costimulatory pathways such as those involving ICOS, can contribute to regulate the ILC2 pool. Thus, ICOS costimulation blockade, which is currently under clinical evaluation for inhibiting the humoral immune response, could also target innate inflammatory circuits.

ICOS promotes group 2 innate lymphoid cell activation in lungs

Group 2 innate lymphoid cells (ILC2s) are newly identified, potent producers of type 2 cytokines, such as IL-5 and IL-13, and contribute to the development of allergic lung inflammation induced by cysteine proteases. Although it has been shown that inducible costimulator (ICOS), a costimulatory molecule, is expressed on ILC2s, the role of ICOS in ILC2 responses is largely unknown. In the present study, we investigated whether the interaction of ICOS with its ligand B7-related protein-1 (B7RP-1) can promote ILC2 activation. Cytokine production in ILC2s purified from mouse lungs was significantly increased by coculture with B7RP-1-transfected cells, and increased cytokine production was inhibited by monoclonal antibody-mediated blocking of the ICOS/B7RP-1 interaction. ILC2 expansion and eosinophil influx induced by papain, a cysteine protease antigen, in mouse lungs were significantly abrogated by blocking the ICOS/B7RP-1 interaction. Dendritic cells (DCs) in the lungs expressed B7RP-1 and the number of DCs markedly increased with papain administration. B7RP-1 expression on lung DCs was reduced after papain administration. This downregulation of B7RP-1 expression may be an indication of ICOS/B7RP-1 binding. These results indicate that ILC2s might interact with B7RP-1-expressing DCs in allergic inflammatory lung, and ICOS signaling can positively regulate the protease allergen-induced ILC2 activation followed by eosinophil infiltration into the lungs.

ICOS deficiency results in defective ILC2 expansion, impaired Th2 responses, and increased mortality after bleomycin-induced lung injury (HUM1P.264)

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease causing irreversible lung scarring and loss of pulmonary function. RNA analysis has shown that patients with IPF have reduced ICOS expression in peripheral blood mononuclear cells. We now show that ICOS surface expression on CD4 T cells positively correlates with lung function in patients with IPF. However, whether decreased ICOS is a result of disease progression or playing a role in the pathogenesis is unclear. Therefore, using bleomycin to induce pulmonary fibrosis in mice, we found that ICOS was decreased in peripheral CD4 T cells similar to our findings in patients. In contrast, ICOS was increased on lung CD4 T cells by 3d post-bleomycin and remained elevated through 21d at the height of collagen deposition. Compared to wild-type B6 mice, ICOS-deficient mice had increased weight loss and mortality after bleomycin challenge, suggesting a protective role for ICOS. Strikingly, ILC2s expanded in the lung at 3d post-bleomycin in B6 mice, but not in ICOS-deficient mice. In B6 mice, ILC2 expansion was associated with CD4 T cell infiltration at 5d and Th2 cytokine production in the draining lymph nodes at 21d. However, ICOS-deficient mice that survived the initial bleomycin insult failed to recruit T cells to the lungs and had less IL-13 in the draining lymph nodes. Overall, these results indicate that ICOS expression promotes lung tissue repair by promoting both innate and adaptive type-2 immune responses.

IL-25 inhibits atherosclerosis development in apolipoprotein E deficient mice.

ObjectiveIL-25 has been implicated in the initiation of type 2 immunity and in the protection against autoimmune inflammatory diseases. Recent studies have identified the novel innate lymphoid type 2 cells (ILC2s) as an IL-25 target cell population. The purpose of this study was to evaluate if IL-25 has any influence on atherosclerosis development in mice.Methods and ResultsAdministration of 1 μg IL-25 per day for one week to atherosclerosis-prone apolipoprotein (apo)E deficient mice, had limited effect on the frequency of T cell populations, but resulted in a large expansion of ILC2s in the spleen. The expansion was accompanied by increased levels of anti-phosphorylcholine (PC) natural IgM antibodies in plasma and elevated levels of IL-5 in plasma and spleen. Transfer of ILC2s to apoE deficient mice elevated the natural antibody-producing B1a cell population in the spleen. Treatment of apoE/Rag-1 deficient mice with IL-25 was also associated with extensive expansion of splenic ILC2s and increased plasma IL-5, suggesting ILC2s to be the source of IL-5. Administration of IL-25 in IL-5 deficient mice resulted in an expanded ILC2 population, but did not stimulate generation of anti-PC IgM, indicating that IL-5 is not required for ILC2 expansion but for the downstream production of natural antibodies. Additionally, administration of 1 μg IL-25 per day for 4 weeks in apoE deficient mice reduced atherosclerosis in the aorta both during initiation and progression of the disease.ConclusionsThe present findings demonstrate that IL-25 has a protective role in atherosclerosis mediated by innate responses, including ILC2 expansion, increased IL-5 secretion, B1a expansion and natural anti-PC IgM generation, rather than adaptive Th2 responses.

Neonatal rhinovirus induces mucous metaplasia and airways hyperresponsiveness through IL-25 and type 2 innate lymphoid cells

Early-life human rhinovirus infection has been linked to asthma development in high-risk infants and children. Nevertheless, the role of rhinovirus infection in the initiation of asthma remains unclear. We hypothesized that, in contrast to infection of mature BALB/c mice, neonatal infection with rhinovirus promotes an IL-25-driven type 2 response, which causes persistent mucous metaplasia and airways hyperresponsiveness. Six-day-old and 8-week-old BALB/c mice were inoculated with sham HeLa cell lysate or rhinovirus. Airway responses from 1 to 28 days after infection were assessed by using quantitative PCR, ELISA, histology, immunofluorescence microscopy, flow cytometry, and methacholine responsiveness. Selected mice were treated with a neutralizing antibody to IL-25. Compared with mature mice, rhinovirus infection in neonatal mice increased lung IL-13 and IL-25 production, whereas IFN-γ, IL-12p40, and TNF-α expression was suppressed. In addition, the population of IL-13-secreting type 2 innate lymphoid cells (ILC2s) was expanded with rhinovirus infection in neonatal but not mature mice. ILC2s were the major cell type secreting IL-13 in neonates. Finally, anti-IL-25 neutralizing antibody attenuated ILC2 expansion, mucous hypersecretion, and airways responsiveness. These findings suggest that early-life viral infection could contribute to asthma development by provoking age-dependent, IL-25-driven type 2 immune responses.

The TNF-family cytokine TL1A promotes allergic immunopathology through group 2 innate lymphoid cells.

The TNF-family cytokine TL1A (TNFSF15) costimulates T cells and promotes diverse T-cell dependent models of autoimmune disease through its receptor DR3. TL1A polymorphisms also confer susceptibility to inflammatory bowel disease. Here we find that allergic pathology driven by constitutive TL1A expression depends on IL-13, but not T, NKT, mast cells or commensal intestinal flora. Group 2 innate lymphoid cells (ILC2) express surface DR3 and produce IL-13 and other type 2 cytokines in response to TL1A. DR3 is required for ILC2 expansion and function in the setting of T cell dependent and independent models of allergic disease. By contrast, DR3 deficient ILC2 can still differentiate, expand and produce IL-13 when stimulated by IL-25 or IL-33, and mediate expulsion of intestinal helminths. These data identify costimulation of ILC2 as a novel function of TL1A important for allergic lung disease, and suggest that TL1A may be a therapeutic target in these settings.

TNF superfamily member TL1A elicits type 2 innate lymphoid cells at mucosal barriers.

Immune responses at mucosal barriers are regulated by innate type 2 lymphoid cells (ILC2s) that elaborate effector cytokines interleukins 5 and 13 (IL5 and IL13). IL25 and IL33 are key cytokines that support ILC2s; however, mice deficient in these pathways retain some functional ILC2s. Analysis of human and murine cells revealed that ILC2s highly express tumor necrosis factor (TNF)-receptor superfamily member DR3 (TNFRSF25). Engagement of DR3 with cognate ligand TL1A promoted ILC2 expansion, survival, and function. Exogenous protein or genetic overexpression of TL1A activated ILC2s independent of IL25 or IL33. Dr3−/− mice failed to control gut helminthic infections, and failed to mount ILC2 responses in the lung after nasal challenge with papain. Our data demonstrate a key role for TL1A in promoting ILC2s at mucosal barriers.

269 : TSLP driven expansion and activation of type 2 innate lymphoid cells is augmented by IL-25 and IL-33

TSLP, IL-25 and IL-33 are primarily epithelial derived cytokines which act on common immune cell populations and individually promote pro-inflammatory responses generally with a T H 2 bias. Type 2 innate lymphocytes (ILC2s) are a newly identified population of lineage negative innate immune cells which are present in a number of inflammatory disease settings capable of producing high levels of type 2 cytokines in response to stimulation with the IL-25, IL-33 and most recently TSLP. Previously we observed that in combination TSLP, IL-25 and IL-33 induced multiple pro-inflammatory factors in human and mouse CD4 + T-cell cultures dominated by high levels of IL-5 and IL-13 production provoking the hypothesis that these responses may be due to the expansion of ILC2 cells in these cultures. In this study we demonstrate TSLP in combination with IL-25 and IL-33 is capable of driving the expansion of ILC2 cells from normal donor peripheral blood cells including PBMC, negatively selected untouched CD4 + T-cell cultures, and sorted ILC2s. Expansion of ILC2 cells occurred in response to TSLP in combination with either IL-25 or IL-33 but was most abundant when all three cytokines were used in combination. ILC2 expansion was dependent upon the presence of TSLP as neither IL-25 nor IL-33 alone or in combination was capable of inducing a similar response. ILC2s expanded by TSLP, IL-25 and IL-33 produced high levels of IL-5 and IL-13 and expressed high surface levels of the TSLPR. The specificity of the responses were demonstrated utilizing CD4 + T-cell cultures from receptor deficient mice and cytokine specific inhibitors. Additionally sorting CD4 + T-cells to >99% purity abolished the observed ILC2 induction. ILC2s represent an additional population of innate immune cells which may have a profound influence on both developing and established immune responses in inflammatory settings where IL-25, IL-33 and TSLP are released.

Polycystic Kidney Disease

Group 2 innate lymphoid cells (ILC2s) are a subset of ILCs that play a protective role in the response to helminth infection, but they also contribute to allergic lung inflammation. Here, we report that the deletion of the ETS1 transcription factor in lymphoid cells resulted in a loss of ILC2s in the bone marrow and lymph nodes and that ETS1 promotes the fitness of the common progenitor of all ILCs. ETS1-deficient ILC2 progenitors failed to up-regulate messenger RNA for the E protein transcription factor inhibitor ID2, a critical factor for ILCs, and these cells were unable to expand in cytokine-driven in vitro cultures. In vivo, ETS1 was required for the IL-33–induced accumulation of lung ILC2s and for the production of the T helper type 2 cytokines IL-5 and IL-13. IL-25 also failed to elicit an expansion of inflammatory ILC2s when these cells lacked ETS1. Our data reveal ETS1 as a critical regulator of ILC2 expansion and cytokine production and implicate ETS1 in the regulation of Id2 at the inception of ILC2 development.