ILC1s express the transcription factor T-bet and produce IFN-γ, a type 1 cytokine. Through their production of IFN-γ, ILC1s show antimicrobial activity and are involved in the clearance of intracellular infections. Compared to ILC2s and Ilc3s, ILC1s have not been well defined. They currently include IL-7Rα^- NK cells, because NK cells also express T-bet and produce IFN-γ; however, it is not clear whether a non-NK, IL-7Rα⁺ ILC1 lineage exists. A recent study identified a distinct ILC1 lineage in the intestinal lamina propria that expresses NKp46, NK1.1, and T-bet but lacks Eomesodermin, RORγt, and fate map. Interestingly, this newly described subset of ILC1s is the main producer of IFN-γ and TNF-α in response to IL-12 and intestinal infection by Toxoplasma gondii (32). Therefore, these results suggest that there might be an additional ILC1 subset distinct from NK cells.

Although the ILC1 lineages have not been well characterized, NK cells belong to the ILC1 family and have been studied thoroughly in the past. Therefore, we will focus on NK cells in the following discussion as analogous to the role of ILC1s in the development of asthma. In the context of asthma, NK cells have not been studied extensively and their roles are still not clear. This might be due to limitations of the methods used for specific depletion of NK cells. However, recent studies have shown that NK cells play an important role in the development of several forms of asthma. NK cells are responsible for lung inflammation during viral infections (33,34) and may regulate the pathogenesis of virus-induced asthma. For example, when humans and mice are infected with rhinoviruses, which cause a range of respiratory diseases, significantly higher amounts of IL-15 and IL-15Rα are detected in fluid from the nasal mucosa and in bronchial tissues (35). Since IL-15 is essential for the activation of NK cells as well as the development of ILC1s, it is possible that ILC1s are involved in the development of virus-associated asthma.

The role of NK cells in allergic asthma has been better studied. Korsgren et al. showed that depletion of NK cells using anti-NK1.1 antibody reduced asthma symptoms. In mice depleted of NK cells, eosinophilic infiltration and allergen-specific IgE production in the lung were greatly reduced (36). However, depletion of NK cells using anti-NK1.1 antibody can also deplete natural killer T cells, which are essential for the development of AHR. Therefore, it was not clear whether NK cells themselves could regulate the development of asthma. However, more recent studies of ovalbumin (OVA)-induced AHR have shown that NK cells are more numerous in OVA-challenged airways. Moreover, perforin-deficient mice failed to develop OVA-induced AHR, suggesting that NK cells are essential for priming OVA-induced AHR (37). Another study was done with a house dust mite (HDM)-induced asthma model. Allergic airway inflammation was induced by intranasal administration of HDM extract in wild-type mice and in NKG2D-deficient mice, which lack the receptor that activates NK cells. NKG2D-deficient mice were resistant to the development of HDM-induced AHR and showed reduced levels of eosinophils, serum IgE, and Th2 cells. Furthermore, transfer of NK cells into NKG2D-deficient mice restored HDM-induced airway inflammation, suggesting that NK cells might be critical for the development of allergen-induced AHR (38). Taken together, these studies suggest potential roles of ILC1s in the pathogenesis of asthma.

ILC2s were first reported among ILC lineages as non-T, non-B cells that proliferated upon administration of IL-25 (39,40) and induced Th2-like immune responses such as increased serum IgE level and eosinophil infiltration in the digestive tract. Non-T, non-B lymphocytes producing IL-5 and IL-13 were also detected in the lungs of patients with asthma, but their specific functions were unclear at the time (41). Then, in 2010, three different groups characterized ILCs that produced large quantities of IL-5 and IL-13 in response to IL-25 and IL-33 (8,9,42). The initial report indicated that these cells were found in lymphoid compartments of adipose tissue and in gut-associated lymphoid tissue. Although these cells had different surface markers and functions, they also shared some common surface markers and cytokine secretions (43). Now, a consensus has been reached to refer to all of these cells as ILC2s (8,9,40,44).

The cytokines IL-7, IL-25, and IL-33 seem to be important for the development of ILC2s. Transcription factors such as Id2, RORα, and GATA3 as well as Notch signaling also contribute to the development of ILC2s (18,19,27). Although there were subtle differences between the cells described in the previous reports, it seems that mouse ILC2s generally express CD25, CD90, and variable amounts of CD117 and CD127 and play critical roles in type 2 immune responses. In addition, ILC2s express variable amounts of CD278, ST2 (IL-1RL1), and IL-17BR.

Th2 cells are believed to be at the center of allergic asthma pathology. Thus, previous studies of asthma paid little attention to other cell types. However, there is evidence that other cell types are important for inducing asthma. In human patients with asthma, viral infections can precipitate virtually all asthma symptoms regardless of the presence of allergy, suggesting that innate cells rather than adaptive cells (such as Th2 cells) are critical for this type of asthma. For example, Il1rl1^-/- mice, which lack the IL-33 receptor, developed near-normal AHR responses after sensitization and challenge with OVA. However, when Il1rl1^-/- mice were infected with influenza A virus (H1N1), AHR was greatly reduced (11). Even in the absence of adaptive immunity (e.g., in Rag2^-/- mice), AHR was induced by influenza A virus. This airway hyperreactivity depended on ILC2s expressing c-Kit, Sca-1, Thy1.2 (CD90), and ST2 (11). Moreover, depletion of ILC2s in Rag2^-/- mice using anti-Thy1.2 antibody abolished the influenza-induced AHR response. These responses were dependent on IL-13 production, since adoptive transfer of IL-13-secreting ILC2s restored influenza-induced AHR in Il13^-/- recipients. In another study, Hong et al. infected 6-day-old neonatal mice with rhinovirus and found that the virus promoted IL-25 production, which in turn induced activation of ILC2s. In addition, proliferation of IL-13-secreting ILC2s was observed in rhinovirus-infected neonatal mice but not in adult mice. Treatment with anti-IL-25 neutralizing antibody attenuated ILC2 proliferation, mucous hypersecretion, and airway responsiveness (45). It has also been suggested that infection with respiratory syncytial virus might result in the subsequent development of asthma, independent of allergen challenge (46).

The studies described above show that ILC2s play a pathological role during virus-induced asthma. However, another study has suggested that ILC2s may also contribute to lung homeostasis. In mice infected with influenza virus (PR8), ILCs accumulated in the lung, and depletion of ILCs resulted in loss of airway epithelial integrity, diminished lung function, and impaired airway remodeling. ILCs seem to contribute to lung homeostasis by producing amphiregulin (47). Taken together, these results indicate that ILC2s may have a dual function, either inducing inflammation or restoring airway cell integrity depending on the strength of the pathogen and the length of infection.

As mentioned previously, ILC2s secrete high levels of IL-5 and IL-13, which suggests they might play an important role in allergic asthma as well as virus-induced asthma, since type 2 cytokines are believed to play a critical role in the development of allergen-induced AHR. In contrast to the virus model, Il1rl1^-/- mice had normal AHR responses after sensitization and challenge with OVA, suggesting that ILC2s may not be required for allergen-induced AHR, although ILC2s clearly accumulated in the lungs during allergen challenge (11). Using an OVA sensitization and airway challenge method, Kearly et al. showed that persistent AHR was correlated with the continued presence of Th2 cells (48). They found that the ST2-IL-33 pathway was important, since antibody against ST2 reduced IL-4 and IL-13 production as well as allergic inflammation and AHR. They concluded that Th2 cells are the primary cell type that cause AHR. However, ILC2s also rely on the ST2-IL-33 pathway, therefore, they are capable of inducing type 2 allergic airway inflammation.

While studies with allergen-induced asthma models have focused on allergen-specific Th2 cells, ILC2s also contribute to type 2 allergic airway disease. Treatment with recombinant IL-25 or IL-33 induced the proliferation of IL-5- and IL-13-producing ILC2s in the lungs and mediastinal lymph nodes (49,50,51,52,53). Klein et al. showed that both ILC2s and Th2 cells are the major sources of IL-5 and IL-13 in HDM-induced and OVA-induced asthma (53). Halim et al. showed that intranasal administration of protease-containing allergens (papain) induced eosinophilia and mucus hyperproduction in Rag1^-/- mice (which have ILCs) but not Rag2^-/- Il2rg^-/- mice (which lack all types of ILCs). Adoptive transfer of ILC2s enabled Rag2^-/- Il2rg^-/- mice to respond to papain. Papain damages stromal cells, which can then release IL-33 and thymic stromal lymphopoietin (TSLP), which in turn can activate ILC2s (54). Wilhelm et al. administered papain into the lungs and found IL-9-producing ILC2s, a new subset of ILC2s (55). These IL-9-producing cells required IL-2 and IL-33 (but not IL-25) to produce IL-5 and IL-13, and blockade of IL-9 resulted in reduced expression of IL-5 and IL-13, suggesting that ILC2s may initially produce IL-9 and then mature into IL-5- and IL-13-producing cells. In a papain-induced asthma model, ILC2s were the main source of type 2 cytokine production.

Another ubiquitous fungal allergen, Alternaria alternata, also induced IL-33 production in the lung, which in turn induced the proliferation of Lin^-CD25⁺CD44^hi ILC2s. The production of IL-5 and IL-13 by ILC2s in response to A. alternata was nearly abolished in Il1rl1^-/- mice, suggesting that ILC2s mediate asthma in an IL-33-dependent manner (50). While all of these studies together suggest that ILC2s play essential roles in allergen-induced airway disease, at this point it is unclear whether ILC2s alone are sufficient for inducing allergic asthma. Further study of the precise requirement for ILC2s in allergic lung disease is needed.

RORγt is a ligand-dependent nuclear hormone receptor expressed in several cell types such as Th17 cells and the type 3 ILCs (ILC3s) (29,56,57). RORγt-dependent ILCs can be divided into at least three different subpopulations based on their functional characteristics. LTi cells, the first subpopulation to be identified among the type 3 ILCs, represent the prototypic cell type of the RORγt⁺ family of ILCs (58). These cells are RORγt⁺ and IL-7Rα⁺ and are mainly involved in secondary lymphoid organ formation (59). Approximately half of fetal LTi cells express CD4, while half of them do not, suggesting that there might be a precursor-progeny relationship between CD4^- and CD4⁺ LTi cells (60). The second group of ILC3s is the IL-17-producing ILC3s. These cells were found in the intestine of fetal mouse (61) and in human fetal lymph nodes (62). IL-17-producing ILC3s are also present in the intestine of adult mice (10) and humans (63) under inflammatory conditions. IL-17-producing ILC3s do not express NKp46, suggesting that these cells are distinct from IL-22-producing NKp46⁺ cells. The third group of ILC3s is the IL-22-producing ILC3s. IL-22-producing ILC3s express the natural cytotoxicity receptor (NCR), which is NKp44 in humans (64) and NKp46 in mice (65,66). Owing to their NK-associated receptor expression, the ILC3 and ILC1 (NK cells) lineages are considered closer to each other than to the ILC2 lineage; however, expression of RORγt and production of type 3 cytokines such as IL-17 and/or IL-22 should be considered standard markers of type 3 ILCs.

Asthma is one of the representative immune diseases induced by type 2 cytokines produced by Th2 cells and ILC2s. The role of IL-17A or IL-22 in the development of asthma is controversial, since IL-17A may either inhibit or exacerbate allergic asthma (5,67). However, recent studies have indicated that IL-17A can directly cause AHR (68,69). These studies have shown that cytokine production by Th17 cells or direct administration of recombinant IL-17 can induce airway inflammation and AHR by inducing contraction of smooth muscle cells. Therefore, IL-17 might have a pathogenic role in airway disease, particularly non-allergic asthma.

Obesity is usually associated with several diseases, such as type 2 diabetes mellitus, cardiovascular disease, liver disease, and cancer (70). Interestingly, obesity is also a major risk factor for the development of asthma, particularly a severe, steroid-resistant form of asthma (71,72). Thus, these findings point to unknown mechanisms of non-allergic asthma that differ from those of allergic asthma.

Interestingly, some obese people show asthma symptoms with increasing levels of IL-17A. Recently, a role for IL-17A-producing ILC3s in AHR associated with obesity was proposed (23). Kim et al. demonstrated that obese mice fed a high-fat diet spontaneously developed AHR and had significant numbers of IL-17-producing cells in their lungs. The majority of IL-17A-producing cells were Lin^-Thy1.2⁺Sca-1⁺CCR6⁺ILC3s. In this study, it was first shown that ILC3s were present in the lung and mediated the development of AHR. These IL-17-producing ILC3s were distinct from ILC1s and ILC2s, since they did not express T-bet or produce IFN-γ and did not produce IL-5 or IL-13. Obesity-induced AHR is independent of adaptive immune cells, since Rag1^-/- mice fed a high-fat diet became obese, had more IL-17-producing ILC3s in the lungs, and developed AHR. Just as IL-33 and IL-25 are key cytokines that can induce proliferation of ILC2s, IL-1β could promote proliferation of IL-17-producing ILC3s in the lung (but not IL-22-producing ILC3s). Furthermore, adoptive transfer of IL-17-producing ILC3s into Rag2^-/- Il2rg^-/- mice (which lack all types of ILCs) restored IL-1β-induced AHR, indicating that IL-17-producing ILC3s by themselves can induce AHR. The production of IL-1β required activation of the NLRP3 inflammasome, since Nlrp3^-/- mice fed the high-fat diet became obese but did not develop AHR. Secretion of the active form of IL-1β, which is mediated by the NLRP3 inflammasome, was increased in M1 macrophages and the lungs of obese mice and induced the proliferation of IL-17-producing ILC3s. Moreover, blocking IL-1β signaling with a short treatment with anakinra (an IL-1R antagonist) abrogated development of AHR in the obese mice and greatly reduced the number of IL-17-producing ILC3s in the lung.

Although there is only one study so far showing that ILC3s are related to the development of asthma, it is possible that ILC3s may also be involved in the development of other forms of asthma, such as those induced by viruses or allergens, since Th17 and Th22 cells are considered pathogenic in asthma. However, the precise roles of each ILC lineage (ILC1s, ILC2s, and ILC3s) remain to be determined.