ne w s a n d v ie w s
Inflammatory ILC2 cells: disguising themselves as progenitors? Shigeo Koyasu
npg
© 2015 Nature America, Inc. All rights reserved.
Type 2 cytokine–producing innate lymphoid cells (ILC2 cells) can respond to interleukins IL-25 and IL-33. Distinct subsets are now recognized as inflammatory ILC2 cells and natural ILC2 cells that differ in their responses to these cytokines.
G
roup 2 innate lymphoid cells (ILC2 cells) have an important role in early innate responses to helminth infection and are involved in the pathophysiology of various allergic inflammatory responses, including asthma. There has been speculation as to whether the ILC2 group is composed of a single cell type or multiple cell types with distinct responsiveness to cytokines. Huang et al. in this issue of Nature Immunology now demonstrate that interleukin 25 (IL-25) induces a novel type of ILC2 cells that they call ‘inflammatory ILC2 cells’ (iILC2 cells)1. The characterization of iILC2 cells may resolve the enigma of variation among ILC2 cells. Multicellular parasites such as helminths are known to be too large for phagocytes to deal with, but the mechanisms of innate immune responses to helminths have been obscure. Accumulating evidence has indicated the importance of epithelial cell–derived cytokines such as IL-25, IL-33 and thymic stromal lymphopoietin, as well as type 2 cytokines such as IL-4, IL-5 and IL-13. Lineage marker–negative (Lin–) c-Kit+FcεR1– non-B, non-T cells of the innate immune system, which are present in Rag2–/– mice that lack B cells and T cells, are reported to appear during the initial stages of helminth infection and to produce type 2 cytokines in an IL-25-dependent manner2. In 2010, the identity of this long-sought cell type was revealed3–5. Natural helper cells (NH cells) were identified in adipose tissues of naive mice as Lin–c-Kit+Sca-1+ cells that respond to IL-33 by producing large amounts of type 2 cytokines, including IL-5, IL-6, and IL-13, but Shigeo Koyasu is with the RIKEN Center for Integrative Medical Sciences, Yokohama, Japan. e-mail: [email protected]
not IL-4 (ref. 3). NH cells do not respond to IL-25 alone, although a combination of IL-2 and IL-25 strongly activates these cells3,6. In contrast, IL-25 potently induces similar cells called ‘nuocytes’4 and ‘innate helper type 2 cells’ (Ih2 cells)5, identified in cytokinereporter mice in which activation-induced cytokine expression leads to the identification of cells expressing fluorescent proteins such as
CLP
CILP
green fluorescent protein. Thus, nuocytes and Ih2 cells are probably in an activated stage, while NH cells are in a resting stage. In addition, there are some differences between these cells in their tissue localization and surfacemarker-expression patterns. They are now collectively called ‘ILC2 cells’7. Huang et al. reexamine the effects of IL-25 and IL-33 on the induction and activation of ILC2
cNK ILC1
ILC3 CHILP
ILCP KLRG1Int GATA-3? RORα?
LTi
GATA-3? RORα?
IL-25
KLRG1Int +
IL-25R IL-33R+
lo Activated IL-25R+ IL-33R nILC2
nILC2 IL-33
? hi
KLRG1 TGF-β
ILC3 -like
IL-2 IL-7 IL-25R+ IL-33R –
KLRG1
KLRG1
iILC2 IL-6
?
Int
nILC2 -like IL-25R+ IL-33R+
IL-33
Int
Activated nILC2 -like IL-25R – IL-33R+
Figure 1 Characteristics and function of iILC2 cells. iILC2 cells are induced by the administration IL-25 in vivo, but their origin and requisite transcription factors are unknown (dashed arrow). iILC2 cells change their phenotype to become nILC2-like cells in vitro and in vivo, but it is unknown whether iILC2 cells are progenitors of nILC2 cells. nILC2-like cells derived from iILC2 cells are quite similar to nILC2 cells (double dashed lines), but iILC2 cells are not observed in naive mice. iILC2 cells become ILC3-like cells under ‘TH17’ conditions in vitro and in vivo, while nILC2 cells show little plasticity. CLP, common lymphoid progenitor; CILP, common ILC progenitor; CHiLP, common ‘helper-like’ ILC progenitor; ILCP, ILC progenitor; cNK, conventional natural killer cell; LTi, lymphoid tissue–inducer cell.
nature immunology volume 16 number 2 february 2015
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© 2015 Nature America, Inc. All rights reserved.
ne w s and v ie w s cells and find that in the lungs, IL-25 potently induces Lin–IL-33R–KLRG1hi iILC2 cells, which are not induced by IL-33 (ref. 1). IL-33, but not IL-25, increases the number of Lin–IL33R+KLRG1int cells present in naive mice, which are likely to be NH cells. The authors call these IL-33-responsive cells ‘nILC2 cells’ (for ‘natural ILC2 cells’). They confirm the dependence of iILC2 cells on IL-25 through results showing that iILC2 cells are not induced in mice that lack the receptor IL-25R (Il17rb–/– mice) but are induced in mice deficient in the receptor IL-33R (Il1rl1–/– mice). iILC2 cells express the receptor subunit IL-7Rα, and a combination of the cytokines IL-3 and SCF is unable to maintain this cell population, which indicates that iILC2 cells are distinct from MPPtype2 cells, another type 2 innate cell of the myeloid lineage6,8. iILC2 cells are also induced in other tissues and organs, such as the spleen, lymph nodes and liver1. As has been observed for nILC2 cells3,5,9, the induction of iILC2 cells is severely impaired in both mice deficient in the common γ-chain (Il2rg–/– mice) and IL-7Rα-deficient mice. iILC2 cells are functionally similar to nILC2 cells in that they can be maintained in culture medium containing IL-2 and IL-7, constitutively produce small amounts of IL-5 and IL-13, and produce large amounts of IL-5 and IL-13 in response to stimulation with IL-25 or IL-33. As observed for nILC2 cells3, a combination of the phorbol ester PMA and ionomycin induces IL-4 at a protein level, which is not observed after stimulation with IL-25 or IL-33. There are some important characteristics of iILC2 cells that are not observed for nILC2 cells. IL-25-induced iILC2 cells are IL-17RB+ (IL-25R+) but IL-33R– and unresponsive to IL-33. After cultivation with IL-2 and IL-7 in vitro, however, iILC2 cells acquire expression of IL-33R, downregulate expression of the activation marker KLRG1, and develop into cells with characteristics similar to those of nILC2 cells. Cultivation with IL-33 in the presence of IL-2 and IL-7 potently downregulates the expression of IL-25R, while cultivation with IL-2, IL-7 and/or IL-25 maintains IL-25R expression (Fig. 1). Similar conversion of iILC2 into nILC2-like cells is also observed in vivo. When adoptively transferred into Il2rg–/–Rag2–/– mice, IL-33R–KLRG1hi iILC2 cells change their phenotype to IL-33R+KLRG1int. On the basis of these observations, Huang et al. propose that IL-25induced iILC2 cells are transient progenitors of nILC2 cells or of cells similar to nILC2 cells1. They further examine that possibility using a helminth infection model. Infection with
134
Nippostrongylus brasiliensis transiently induces iILC2 cells, followed by an increase in nILC2 cells in the lungs. Induced iILC2 cells as well as nILC2 cells produce IL-5 and IL-13 to protect mice from infection with N. brasiliensis. Adoptive transfer of either iILC2 cells or nILC2 cells suppresses worm burden and egg production in Il2rg–/–Rag2–/– mice, which succumb to N. brasiliensis infection without transfer of these cells. Transferred iILC2 cells are also converted into nILC2-like cells in Il2rg–/–Rag2–/– mice during infection with N. brasiliensis, which supports the idea that iILC2 cells are transient progenitors of nILC2 cells. It is of interest to compare iILC2 cells to nuocytes4 and Ih2 cells5, both of which are observed as type 2 cytokine–producing cells during helminth infection or after the administration of IL-25 or IL-33 to cytokine-reporter mice. IL-25-induced nuocytes and Ih2 cells are probably identical to iILC2 cells, which are distinct from nILC2 cells that are more closely related to NH cells that do not respond to IL-25 alone3,6. However, helminth infection probably collectively induces nuocytes and Ih2 cells and activates NH cells. It is notable that the localization of IL-25-induced cells and that of IL-33-induced cells appears to be different in the small intestine5. It is therefore likely that helminth-induced nuocyte populations include both iILC2 cells and nILC2 cells, whereas IL-25-induced nuocytes and IL-33-induced nuocytes correspond to iILC2 cells and activated NH cells, respectively. NH cells are probably nILC2 cells in naive mice. Another interesting characteristic of iILC2 cells is that they have low expression of RORγt, which is a pivotal transcription factor for the expression of IL-17 by ILC3 cells9, whereas nILC2 cells do not express RORγt. Indeed, stimulation of iILC2 cells with PMA and ionomycin results in the expression of IL-17 at the protein level, and many cells coexpress IL-13 and IL-17. Cultivation of iILC2 cells under conditions like those that induce polarization into the TH17 subset of helper T cells further enhances their ability to produce IL-17. The authors confirm IL-17 production in vivo, as iILC2 cells transferred into Il2rg–/–Rag2–/– mice produce IL-17 upon infection with Candida albicans. iILC2-derived IL-17 protects Il2rg–/–Rag2–/– mice from the weight loss that results from infection with C. albicans. These results indicate that iILC2 cells have plasticity in their cytokine-production patterns depending upon the environmental cytokine milieu (Fig. 1). In contrast, nILC2 cells show much less plasticity than do iILC2 cells.
Outstanding questions remain. What is the origin of iILC2 cells? Published studies have identified common progenitors of ILCs and common progenitors of helper-like ILCs (CHILPs)8. It is unknown whether CHILPs or CHILP-like progenitors are activated by IL-25 to become iILC2 cells. Huang et al. show that iILC2 cells are not induced in the bone marrow, where CHILPs are present1. nILC2 cells differentiate from CHILPs with the aid of specific transcription factors such as GATA-3 and RORα8. Are these transcription factors involved in the induction of iILC2 cells? It is intriguing that intravenous administration of IL-25 induces iILC2 cells in many tissues and organs, but intranasal administration of IL-25 fails to induce iILC2 cells in the lungs1, which indicates that progenitors of iILC2 cells are not present in the lungs. The plasticity of iILC2 cells and their ability to convert into IL-17-producing cells could be important in vivo, but the mechanisms by which such conversion is triggered are totally unknown. Does coinfection with helminths and fungi induce the in vivo conversion of iILC2 cells into IL-17-producing cells? Does the transcription factor Gfi1 have a role in these conversion processes, since a lack of Gfi1 leads to the expression of IL-17 by nILC2 cells10? iILC2 cells have high expression of KLRG1, which contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. Is KLRG1 involved in a negative feedback mechanism in iILC2 function? It has been suggested that E-cadherin is a ligand for KLRG1 and that the interaction of these molecules sends bidirectional signals11. Do epithelial cells suppress iILC2 function to avoid excess activation of type 2 innate immune responses? Future studies should resolve those issues. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper. 1. Huang, Y. et al. Nat. Immunol. 16, 161–169 (2015). 2. Hurst, S.D. et al. J. Immunol. 169, 443–453 (2002). 3. Moro, K. et al. Nature 463, 540–544 (2010). 4. Neill, D.R. et al. Nature 464, 1367–1370 (2010). 5. Price, A.E. et al. Proc. Natl. Acad. Sci. USA 107, 11489–11494 (2010). 6. Saenz, S.A. et al. Nature 464, 1362–1366 (2010). 7. Spits, H. et al. Nat. Rev. Immunol. 13, 145–149 (2013). 8. Saenz, S.A. et al. J. Exp. Med. 210, 1823–1837 (2013). 9. Diefenbach, A., Colonna, M. & Koyasu, S. Immunity 41, 354–365 (2014). 10. Spooner, C.J. et al. Nat. Immunol. 14, 1229–1236 (2013). 11. Banh, C., Fugère, C. & Brossay, L. Blood 114, 5299–5306 (2009).
volume 16 number 2 february 2015 nature immunology
Inflammatory ILC2 cells: disguising themselves as progenitors? Shigeo Koyasu
npg
© 2015 Nature America, Inc. All rights reserved.
Type 2 cytokine–producing innate lymphoid cells (ILC2 cells) can respond to interleukins IL-25 and IL-33. Distinct subsets are now recognized as inflammatory ILC2 cells and natural ILC2 cells that differ in their responses to these cytokines.
G
roup 2 innate lymphoid cells (ILC2 cells) have an important role in early innate responses to helminth infection and are involved in the pathophysiology of various allergic inflammatory responses, including asthma. There has been speculation as to whether the ILC2 group is composed of a single cell type or multiple cell types with distinct responsiveness to cytokines. Huang et al. in this issue of Nature Immunology now demonstrate that interleukin 25 (IL-25) induces a novel type of ILC2 cells that they call ‘inflammatory ILC2 cells’ (iILC2 cells)1. The characterization of iILC2 cells may resolve the enigma of variation among ILC2 cells. Multicellular parasites such as helminths are known to be too large for phagocytes to deal with, but the mechanisms of innate immune responses to helminths have been obscure. Accumulating evidence has indicated the importance of epithelial cell–derived cytokines such as IL-25, IL-33 and thymic stromal lymphopoietin, as well as type 2 cytokines such as IL-4, IL-5 and IL-13. Lineage marker–negative (Lin–) c-Kit+FcεR1– non-B, non-T cells of the innate immune system, which are present in Rag2–/– mice that lack B cells and T cells, are reported to appear during the initial stages of helminth infection and to produce type 2 cytokines in an IL-25-dependent manner2. In 2010, the identity of this long-sought cell type was revealed3–5. Natural helper cells (NH cells) were identified in adipose tissues of naive mice as Lin–c-Kit+Sca-1+ cells that respond to IL-33 by producing large amounts of type 2 cytokines, including IL-5, IL-6, and IL-13, but Shigeo Koyasu is with the RIKEN Center for Integrative Medical Sciences, Yokohama, Japan. e-mail: [email protected]
not IL-4 (ref. 3). NH cells do not respond to IL-25 alone, although a combination of IL-2 and IL-25 strongly activates these cells3,6. In contrast, IL-25 potently induces similar cells called ‘nuocytes’4 and ‘innate helper type 2 cells’ (Ih2 cells)5, identified in cytokinereporter mice in which activation-induced cytokine expression leads to the identification of cells expressing fluorescent proteins such as
CLP
CILP
green fluorescent protein. Thus, nuocytes and Ih2 cells are probably in an activated stage, while NH cells are in a resting stage. In addition, there are some differences between these cells in their tissue localization and surfacemarker-expression patterns. They are now collectively called ‘ILC2 cells’7. Huang et al. reexamine the effects of IL-25 and IL-33 on the induction and activation of ILC2
cNK ILC1
ILC3 CHILP
ILCP KLRG1Int GATA-3? RORα?
LTi
GATA-3? RORα?
IL-25
KLRG1Int +
IL-25R IL-33R+
lo Activated IL-25R+ IL-33R nILC2
nILC2 IL-33
? hi
KLRG1 TGF-β
ILC3 -like
IL-2 IL-7 IL-25R+ IL-33R –
KLRG1
KLRG1
iILC2 IL-6
?
Int
nILC2 -like IL-25R+ IL-33R+
IL-33
Int
Activated nILC2 -like IL-25R – IL-33R+
Figure 1 Characteristics and function of iILC2 cells. iILC2 cells are induced by the administration IL-25 in vivo, but their origin and requisite transcription factors are unknown (dashed arrow). iILC2 cells change their phenotype to become nILC2-like cells in vitro and in vivo, but it is unknown whether iILC2 cells are progenitors of nILC2 cells. nILC2-like cells derived from iILC2 cells are quite similar to nILC2 cells (double dashed lines), but iILC2 cells are not observed in naive mice. iILC2 cells become ILC3-like cells under ‘TH17’ conditions in vitro and in vivo, while nILC2 cells show little plasticity. CLP, common lymphoid progenitor; CILP, common ILC progenitor; CHiLP, common ‘helper-like’ ILC progenitor; ILCP, ILC progenitor; cNK, conventional natural killer cell; LTi, lymphoid tissue–inducer cell.
nature immunology volume 16 number 2 february 2015
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npg
© 2015 Nature America, Inc. All rights reserved.
ne w s and v ie w s cells and find that in the lungs, IL-25 potently induces Lin–IL-33R–KLRG1hi iILC2 cells, which are not induced by IL-33 (ref. 1). IL-33, but not IL-25, increases the number of Lin–IL33R+KLRG1int cells present in naive mice, which are likely to be NH cells. The authors call these IL-33-responsive cells ‘nILC2 cells’ (for ‘natural ILC2 cells’). They confirm the dependence of iILC2 cells on IL-25 through results showing that iILC2 cells are not induced in mice that lack the receptor IL-25R (Il17rb–/– mice) but are induced in mice deficient in the receptor IL-33R (Il1rl1–/– mice). iILC2 cells express the receptor subunit IL-7Rα, and a combination of the cytokines IL-3 and SCF is unable to maintain this cell population, which indicates that iILC2 cells are distinct from MPPtype2 cells, another type 2 innate cell of the myeloid lineage6,8. iILC2 cells are also induced in other tissues and organs, such as the spleen, lymph nodes and liver1. As has been observed for nILC2 cells3,5,9, the induction of iILC2 cells is severely impaired in both mice deficient in the common γ-chain (Il2rg–/– mice) and IL-7Rα-deficient mice. iILC2 cells are functionally similar to nILC2 cells in that they can be maintained in culture medium containing IL-2 and IL-7, constitutively produce small amounts of IL-5 and IL-13, and produce large amounts of IL-5 and IL-13 in response to stimulation with IL-25 or IL-33. As observed for nILC2 cells3, a combination of the phorbol ester PMA and ionomycin induces IL-4 at a protein level, which is not observed after stimulation with IL-25 or IL-33. There are some important characteristics of iILC2 cells that are not observed for nILC2 cells. IL-25-induced iILC2 cells are IL-17RB+ (IL-25R+) but IL-33R– and unresponsive to IL-33. After cultivation with IL-2 and IL-7 in vitro, however, iILC2 cells acquire expression of IL-33R, downregulate expression of the activation marker KLRG1, and develop into cells with characteristics similar to those of nILC2 cells. Cultivation with IL-33 in the presence of IL-2 and IL-7 potently downregulates the expression of IL-25R, while cultivation with IL-2, IL-7 and/or IL-25 maintains IL-25R expression (Fig. 1). Similar conversion of iILC2 into nILC2-like cells is also observed in vivo. When adoptively transferred into Il2rg–/–Rag2–/– mice, IL-33R–KLRG1hi iILC2 cells change their phenotype to IL-33R+KLRG1int. On the basis of these observations, Huang et al. propose that IL-25induced iILC2 cells are transient progenitors of nILC2 cells or of cells similar to nILC2 cells1. They further examine that possibility using a helminth infection model. Infection with
134
Nippostrongylus brasiliensis transiently induces iILC2 cells, followed by an increase in nILC2 cells in the lungs. Induced iILC2 cells as well as nILC2 cells produce IL-5 and IL-13 to protect mice from infection with N. brasiliensis. Adoptive transfer of either iILC2 cells or nILC2 cells suppresses worm burden and egg production in Il2rg–/–Rag2–/– mice, which succumb to N. brasiliensis infection without transfer of these cells. Transferred iILC2 cells are also converted into nILC2-like cells in Il2rg–/–Rag2–/– mice during infection with N. brasiliensis, which supports the idea that iILC2 cells are transient progenitors of nILC2 cells. It is of interest to compare iILC2 cells to nuocytes4 and Ih2 cells5, both of which are observed as type 2 cytokine–producing cells during helminth infection or after the administration of IL-25 or IL-33 to cytokine-reporter mice. IL-25-induced nuocytes and Ih2 cells are probably identical to iILC2 cells, which are distinct from nILC2 cells that are more closely related to NH cells that do not respond to IL-25 alone3,6. However, helminth infection probably collectively induces nuocytes and Ih2 cells and activates NH cells. It is notable that the localization of IL-25-induced cells and that of IL-33-induced cells appears to be different in the small intestine5. It is therefore likely that helminth-induced nuocyte populations include both iILC2 cells and nILC2 cells, whereas IL-25-induced nuocytes and IL-33-induced nuocytes correspond to iILC2 cells and activated NH cells, respectively. NH cells are probably nILC2 cells in naive mice. Another interesting characteristic of iILC2 cells is that they have low expression of RORγt, which is a pivotal transcription factor for the expression of IL-17 by ILC3 cells9, whereas nILC2 cells do not express RORγt. Indeed, stimulation of iILC2 cells with PMA and ionomycin results in the expression of IL-17 at the protein level, and many cells coexpress IL-13 and IL-17. Cultivation of iILC2 cells under conditions like those that induce polarization into the TH17 subset of helper T cells further enhances their ability to produce IL-17. The authors confirm IL-17 production in vivo, as iILC2 cells transferred into Il2rg–/–Rag2–/– mice produce IL-17 upon infection with Candida albicans. iILC2-derived IL-17 protects Il2rg–/–Rag2–/– mice from the weight loss that results from infection with C. albicans. These results indicate that iILC2 cells have plasticity in their cytokine-production patterns depending upon the environmental cytokine milieu (Fig. 1). In contrast, nILC2 cells show much less plasticity than do iILC2 cells.
Outstanding questions remain. What is the origin of iILC2 cells? Published studies have identified common progenitors of ILCs and common progenitors of helper-like ILCs (CHILPs)8. It is unknown whether CHILPs or CHILP-like progenitors are activated by IL-25 to become iILC2 cells. Huang et al. show that iILC2 cells are not induced in the bone marrow, where CHILPs are present1. nILC2 cells differentiate from CHILPs with the aid of specific transcription factors such as GATA-3 and RORα8. Are these transcription factors involved in the induction of iILC2 cells? It is intriguing that intravenous administration of IL-25 induces iILC2 cells in many tissues and organs, but intranasal administration of IL-25 fails to induce iILC2 cells in the lungs1, which indicates that progenitors of iILC2 cells are not present in the lungs. The plasticity of iILC2 cells and their ability to convert into IL-17-producing cells could be important in vivo, but the mechanisms by which such conversion is triggered are totally unknown. Does coinfection with helminths and fungi induce the in vivo conversion of iILC2 cells into IL-17-producing cells? Does the transcription factor Gfi1 have a role in these conversion processes, since a lack of Gfi1 leads to the expression of IL-17 by nILC2 cells10? iILC2 cells have high expression of KLRG1, which contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. Is KLRG1 involved in a negative feedback mechanism in iILC2 function? It has been suggested that E-cadherin is a ligand for KLRG1 and that the interaction of these molecules sends bidirectional signals11. Do epithelial cells suppress iILC2 function to avoid excess activation of type 2 innate immune responses? Future studies should resolve those issues. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper. 1. Huang, Y. et al. Nat. Immunol. 16, 161–169 (2015). 2. Hurst, S.D. et al. J. Immunol. 169, 443–453 (2002). 3. Moro, K. et al. Nature 463, 540–544 (2010). 4. Neill, D.R. et al. Nature 464, 1367–1370 (2010). 5. Price, A.E. et al. Proc. Natl. Acad. Sci. USA 107, 11489–11494 (2010). 6. Saenz, S.A. et al. Nature 464, 1362–1366 (2010). 7. Spits, H. et al. Nat. Rev. Immunol. 13, 145–149 (2013). 8. Saenz, S.A. et al. J. Exp. Med. 210, 1823–1837 (2013). 9. Diefenbach, A., Colonna, M. & Koyasu, S. Immunity 41, 354–365 (2014). 10. Spooner, C.J. et al. Nat. Immunol. 14, 1229–1236 (2013). 11. Banh, C., Fugère, C. & Brossay, L. Blood 114, 5299–5306 (2009).
volume 16 number 2 february 2015 nature immunology
