Cellular Sources and Immune Functions of Interleukin-9

PROGRESS

TH2 cells or whether TH2‑cell derived

IL‑4 supports the development of TH9 cells Cellular sources and immune from either naive CD4+ T cells or T 2 H cells. Currently, only one group has reported functions of interleukin‑9 low but detectable co-expression of IL‑4 and IL‑9 during in vitro differentiation of T 2 cells6. Surprisingly, to date there have Randolph J. Noelle and Elizabeth C. Nowak H been no reports addressing whether these Abstract | Interleukin‑9 (IL‑9) has attracted renewed interest owing to the cytokines are co-expressed by effector CD4+ T cell populations that differentiate in vivo identification of its expression by multiple T helper (TH) cell subsets, including TH2 during a TH2‑type immune response. cells, TH9 cells, TH17 cells and regulatory T (TReg) cells. Here, we provide a broad overview of the conditions that are required for cells to produce IL‑9 and describe TH9 cells. TH9 cells are a recently described the cellular targets and nature of the immune responses that are induced by IL‑9. subset of TH cells that express IL‑9 but not

other TH cell lineage-specific cytokines or transcription factors7,8 (FIG. 2). The most Interleukin‑9 (IL‑9) was originally identi‑ same study showed that T cells were the definitive work that supports the existence fied in mice as a T cell growth factor and is main producers of IL‑9 in vivo and that and functional relevance of TH9 cells in vivo a member of the common γ‑chain-receptor the levels of IL‑9 expression correlated has come from the study of mice with a cytokine family, with other members includ‑ with the expansion of TH2 cell populations. T cell-specific deletion of the transcription ing IL‑2, IL‑4, IL‑7, IL‑15 and IL‑21. The IL9 Futhermore, blockade of IL‑4, which is a factor PU.1. These mice do not develop gene loci of both humans and mice have a crucial mediator of TH2 cell differentiation, IL‑9‑dependent allergic inflammation in similar organization, consisting of five exons, could suppress IL‑9 production5. Owing the lungs and have only low levels of IL‑9 in and share a 55% amino acid homology at the to the recent characterization of TH9 cells bronchoalveolar lavage fluid, but still develop protein level. The IL‑9 receptor consists of (which express IL‑9 but not the key signa‑ normal numbers of TH2 cells. This finding the cytokine-specific IL‑9 receptor α-chain ture cytokines of other TH cell subsets, such indicates that, at least in this setting, TH9 cells 1 (IL‑9Rα) and the γ-chain . IL‑9‑induced as IL‑4 (TH2 cells), interferon‑γ (IFNγ) are the main T cell subset expressing IL‑9 and 6 receptor activation promotes the cross- (TH1 cells) and IL‑17 (TH17 cells)) it is now that PU.1 is crucial for their development . phosphorylation of Janus kinase 1 (JAK1) unclear whether the correlation between Another transcription factor that is associ‑ and JAK3. This cross-phosphorylation leads IL‑9 levels and TH2 cell numbers is due to ated with TH9 cell development both in vitro to the downstream activation of signal trans‑ the direct production of this cytokine by and in vivo is interferon-regulatory factor 4 9 ducer and activator of transcription (STAT) (IRF4) , which also regulates TH2 and TH17 complexes, specifically STAT1 homodimers, cell differentiation10–12. Characterization IL-9 STAT5 homodimers and STAT1–STAT3 IL-9Rα γ-chain of TH9 cells in vitro has shown that their heterodimers2–4 (FIG. 1). Although the con‑ generation is dependent on transforming tribution of IL‑9 to the activation of these growth factor-β (TGFβ) and IL‑4 (REF. 13). pathways in primary cells has not been fully JAK1 JAK3 The addition of IL‑25 along with these two elucidated, it may be of relevance in vivo. cytokines can further enhance IL‑9 pro‑

Here, we summarize the cellular sources duction by T cells in vitro. TH9 cells from of IL‑9, focusing principally on T cells. IL‑25‑deficient mice have decreased IL‑9 We also describe the main targets of IL‑9 expression and reduced airway inflamma‑ STAT1 STAT3 STAT5 in vivo and discuss the downstream effects tion in a model of asthma14. There have of IL‑9‑induced signalling on leukocyte Activation of STAT1 and STAT5 homodimers also been conflicting reports that various function and overall immunity. and STAT1–STAT3 heterodimers cytokines, such as IL‑1β, IL‑6, IL‑10, IL‑12, IL‑21, IFNα and IFNβ, may have an additive Cellular sources of IL‑9 Figure 1 | The IL‑9 receptor signalling com‑ effect in promoting T 9 cell generation in plex. Interleukin‑9 (IL‑9)Natur activatese Reviews a heterodimeric| Immunology H T 2 cells. IL‑9 was first linked to T helper 2 the presence of TGFβ and IL‑4 in vitro15,16. H receptor that consists of the IL‑9 receptor α-chain (TH2) cells, which express IL‑4, IL‑5 and In addition, culturing human memory (IL‑9Rα) and the γ- chain and promotes the cross- IL‑13, following the report that levels of phosphorylation of Janus kinase 1 (JAK1) and JAK3. T cells in the presence of TGFβ (alone or in IL‑9 expression were high in the TH2‑prone This leads to the activation of signal transducer combination with IL‑1β, IL‑4, IL‑6, IL‑12, BALB/c mouse strain but low in the and activator of transcription 1 (STAT1), STAT3 and IL‑21 or IL‑23) was shown to promote IL‑9

TH1‑prone C57BL/6 mouse strain during STAT5 and the upregulation of IL‑9‑inducible production with or without co-expression infection with Leishmania major. The gene transcription. of IFNγ and IL‑17 (REFS 16,17).

TH17 cells. There have also been reports as well as in purified TReg cell populations release of numerous pre-formed media‑ 18,21 that TH17 cells can produce IL‑9. In vivo dif‑ in vitro . However, other groups did not tors. Two of these products, histamine and ferentiated TH17 cells, which were identified report co-expression of FOXP3 and IL‑9 in IL‑1β, can promote further IL‑9 produc‑ by their expression of either IL‑17F or the T cell cultures that were generated in vitro8,19. tion23,24. As IL‑9 is a growth factor for mast

TH17‑associated transcription factor retinoic Similarly, there have been conflicting data cells, it is thought that these pathways pro‑ acid receptor-related orphan receptor-γt from studies of TReg cells from healthy mote the survival and expansion of mast (RORγt), could produce IL‑9 after re-stimu‑ human donors15–17. As one group showed cells during an active immune response. lation with phorbol 12-myristate 13-acetate that retinoic acid can downregulate IL‑9 18 22 and ionomycin . In addition, some groups expression by TReg cells , a possible explana‑ Natural killer T cells. Studies using natural also reported co-expression of IL‑17 and tion for the discrepancy between the data is killer T (NKT) cells from naive mice have 18,19 IL‑9 during TH17 cell generation in vitro . that there may be differences in the levels of shown that following stimulation with However, IL‑23, which is important for the retinoic acid present in cultures in vitro and IL‑2, but not IL‑15, these cells can produce expansion of TH17 cells after their initial dif‑ in the local microenvironments in vivo. IL‑9. IL‑2 stimulation also led to some co- ferentiation, has been shown to inhibit the For all of the above CD4+ T cell subsets, expression of IL‑4, IL‑5 and IL‑13, but not expression of IL‑9 by T cells in mice19; there‑ TGFβ seems to be the unifying factor for IFNγ, by IL‑9‑producing NKT cells25. In a fore, this finding suggests that the expression promoting IL‑9 production; an inability mouse model of allergic airway inflammation, of IL‑9 by TH17 cells could be transient and to respond to TGFβ in vivo prevents IL‑9 deficiency of CD1d‑restricted NKT cells may decrease over time. expression by T cells8. The main exception correlated with decreased IL‑9 expression

Human TH17 cells have also been shown to this observation is that TGFβ does not and reduced mast cell recruitment to the to produce IL‑9 in vitro. After multiple seem to be necessary for IL‑9 production by lungs. This finding suggested that NKT cells 26 rounds of differentiation in vitro, some TH17 TH2 cells; however, one possible explanation can promote IL‑9 responses in vivo . cells acquire expression of IL‑9. In memory for this finding could be the presence of In addition, NKT cells that have under‑

T cells, TH17‑promoting cytokines, such endogenous TGFβ in the culture media gone transformation to nasal NKT cell as IL‑1β and IL‑21, can also enhance the that is used for TH2 cell differentiation. lymphoma cell lines also produce IL‑9; in co-production of IL‑9 and IL‑17. However, this setting, IL‑9 functions as an autocrine these cultures also contain IL‑9‑producing Mast cells as a source of IL‑9. Although growth factor. Histological sections from T cells that do not co-express IL‑17 and are mainly studied as a target of IL‑9, mast patients with nasal NKT cell lymphomas 16 presumably bona fide TH9 cells . cells have also been reported to pro‑ contained large numbers of infiltrating duce this cytokine. Production occurs IL‑9‑producing NKT cells, which suggested 27 TReg cells. There have been contradictory in an autocrine manner in response to that IL‑9 may promote disease progression . reports regarding the expression of IL‑9 by IL‑9‑induced signals and as a consequence It is currently unclear whether IL‑9 can also

TReg cells. Data from mice have shown of the crosslinking of IgE molecules on have effects on other cell types in the nasal that co-expression of forkhead box P3 the surface of mast cells. Crosslinking of mucosa of these patients. For example, the

(FOXP3) and IL‑9 can occur in TReg cells surface IgE molecules results in mast cell effects of IL‑9 on mast cells might contribute that are found in tolerant allografts in vivo20, degranulation and leads to the immediate to cancer progression by promoting the pro‑ duction of angiogenic factors that increase the vasculature of the tumour. Naive + CD4 T cell Immune cell targets of IL‑9 The downstream effects of IL‑9 have been Cytokines IL-4 TGFβ TGFβ TGFβ IL-12 IL-6 primarily associated with mast cells; however, required IL-4 IL-6 IL-2 TNF this does not preclude IL‑9 from exerting for TH cell IL-21 effects on other cell types. Here, we summa‑ differentiation IL-23 rize the observed effects of IL‑9 on mast cells TGFβ (FIG. 3) IL-4 and other cell types . However, as there TH2 TH9 TH17 TReg TH1 TH22 is currently no way to definitively confirm the expression of IL‑9Rα on specific, rather Transcription GATA3 PU.I RORγt FOXP3 T-bet AHR than heterogenous, cell populations, it can be factors associated IRF4 IRF4 RORα difficult to determine the relative importance with T cell subset H IRF4 of each individual cell type. Cytokines IL-4 IL-9 IL-9 IL-9 IFNγ IL-22 expressed by IL-5 IL-10 IL-17 IL-10 TNF TNF Mast cells. One of the main targets of IL‑9 is T cell subset IL-9 IL-21 IL-35 H Not known to the mast cell and, as mentioned earlier, initial IL-10 TNF TGFβ produce IL-9 IL-13 studies described a role for IL‑9 in promot‑ ing the expansion of mast cell populations28. Figure 2 | Expression of IL‑9 by distinct T cell subsets. Different differentiation pathways of effector CD4+ T cell subsets are shown, as well as the transcription factors that are necessary for their differentia‑ Subsequent work in mice that were deficient tion and some of the cytokines that they produce. Interleukin‑9 (IL‑9) has beenNatur reportede Reviews to |be Immunolog expressedy for both IL‑9 and IL‑9Rα showed that IL‑9 is not required for the generation of mast by T helper 2 (TH2), TH9, TH17 and regulatory T (TReg) cell subsets, and all of these subsets, except TH2 cells, require transforming growth factor-β (TGFβ) for IL-9 production. AHR, aryl hydrocarbon receptor; cell precursors, as the basal numbers of mast FOXP3, forkhead box P3; GATA3, GATA-binding protein 3; IFNγ, interferon-γ; IRF4, interferon-regulatory cells in these mice were normal. However, factor 4; ROR, retinoic acid receptor-related orphan receptor; TNF, tumour necrosis factor. mice that were deficient for IL‑9 or IL‑9Rα

IL-9 decreased production of IL‑12 and IFNγ in response to treatment with extracts from Mycobacterium tuberculosis. Under these conditions, it was also shown that the addi‑ tion of IL‑9‑specific blocking antibodies to Mast cell TReg cell TH17 cell APC the cultures upregulates PBMC production of IL‑12 (REF. 39). Overall, these data suggest that IL‑9 might limit the capacity of APCs

to induce TH1‑type immune responses. Promotes mast cell Promotes suppressive Induces TH17 cell Promotes TGFβ production growth and production functions proliferation and downregulates IL-12 IL‑9 and immunity of IL-1β, IL-5, IL-6, production IL-13 and TGFβ IL‑9 can function as both a positive and Figure 3 | Targets of IL‑9 function. Interleukin‑9 (IL‑9) has been shown to have various effects on negative regulator of immune responses different cell types. These effects include activating mast cells to secrete several products, including (TABLE 1). In general, it seems that IL‑9 IL‑13, which exerts its effects on the epithelial cells of the lung and gut. In addition, IL‑9 seems to have has detrimental roles during allergy and Nature Reviews | Immunology autoimmunity. However, during parasitic a direct effect on regulatory T (TReg) cells, T helper 17 (TH17) cells and antigen-presenting cells (APCs). TGFβ, transforming growth factor-β. infections, IL‑9 can help to clear the pathogen, and during skin transplantation, IL‑9 can promote the maintenance of a showed defective expansion and recruit‑ Antigen-presenting cells. Although a tolerant environment. ment of mast cell populations in response to careful analysis of the specific antigen- intestinal nematode infection or following presenting cell (APC) subsets that express Allergy. During some allergic responses in the induction of experimental autoimmune the IL‑9 receptor has not yet been carried the lung, which are traditionally thought of 18,29 encephalomyelitis (EAE) . IL‑9 can induce out, there are indications that professional as being TH2 cell-mediated, IL‑9 contributes mast cell production of TGFβ, which can APCs are also targets of IL‑9. During to disease by promoting mast cell expansion have pro-inflammatory downstream effects lipopolysaccharide-induced activation and production of IL‑13, which in turn pro‑ on neurons in a murine stroke model and on of a heterogenous population of macro‑ motes the release of mucus that contributes epithelial cells during intestinal inflamma‑ phages and monocytes, IL‑9 can promote to airway hyperresponsiveness33,35,40. The tion30,31. Following antigen-specific cross the expression of TGFβ; this results in a initial influx of mast cells in this model is linking of surface IgE molecules on mast decrease in the oxidative burst of these likely to be driven by IL‑9 that is derived 26 cells, IL‑9 can enhance mast cell expression cells, as well as in decreased expression of from NKT cells . In addition, TH9 cells also of several cytokines, including IL‑1β, IL‑5, tumour necrosis factor (TNF)37,38. In addi‑ seem to contribute to disease in this model, IL‑6, IL‑9, IL‑10 and IL‑13 (REF. 24). Of these tion, the culture of peripheral blood mono‑ as mice with PU.1‑deficient T cells and a cytokines, IL‑5 and IL‑13 are of particular nuclear cells (PBMCs), which contain both global IL‑25 deficiency are protected from interest, as the previously described direct T cells and monocytes, with IL‑9 leads to allergic airway inflammation6,14. Similarly, effects of IL‑9 in promoting eosinophilia and mucus production in the lung and Table 1 | Sources and targets of IL‑9 during immune responses the gut instead seem to be indirect effects mediated through the induction of these Immune model Sources of IL‑9 Targets of IL‑9 Effects of IL‑9 Refs cytokines by IL‑9 (REFS 32–36). Allergy Allergic airway NKT cells and Mast cells Promotes allergic 6,9,26, T cell subsets. There are some indications inflammation TH9 cells inflammation 33,36,40 that IL‑9 can target certain T cell subsets, Oral antigen-induced TH2 cells and Mast cells Promotes allergic 41 specifically, TH17 cells and TReg cells. In the anaphylaxis T 9 cells inflammation case of T 17 cells, IL‑9 seems to function as H H Autoimmunity an autocrine growth factor that facilitates the 19 expansion of T 17 cell populations in vitro . EAE TH17 cells and Mast cells and Promotes EAE 18, H T 9 cells T 17 cells 42–44 This is also supported by the decreased H H T 17 cells and T cells Inhibits EAE 19 accumulation of TH17 cells that is seen in H Reg 18 T 9 cells IL‑9Rα-deficient mice during EAE ; how‑ H ever, because this is a global defect in vivo, Type 1 diabetes TH17 cells Unknown Unknown 16 the possibility that this is an indirect effect of Parasitic infection IL‑9 deficiency cannot be entirely dismissed. Lung infection with TH2 cells and Mast cells No effect on 29 By contrast, IL‑9 does not seem to facili‑ Schistosoma mansoni T 9 cells granuloma formation tate the survival or expansion of T cells. H Reg Intestinal infection with T 2 cells and Mast cells Promotes parasite 45–47 Instead, standard suppression assays show H Trichuris muris TH9 cells expulsion that TReg cells from IL‑9Rα-deficient mice are less able to inhibit the proliferation of effec‑ Transplantation + 19 tor CD4 T cells in vitro . This suggests that Skin allograft TReg cells Mast cells and Promotes allograft 20,48 transplantation T cells tolerance IL‑9 can enhance the regulatory function of Reg EAE, experimental autoimmune encephalomyelitis; NKT, natural killer T; T , T helper; T , regulatory T. TReg cells through an unknown mechanism. H Reg

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