Cellular & Molecular Immunology (2014) 11, 126–128 ß 2014 CSI and USTC. All rights reserved 1672-7681/14 $32.00 www.nature.com/cmi

RESEARCH HIGHLIGHT

Regulatory T cell Itch reins in Th2 inflammation Benjamin D Singer and Franco R D’Alessio Cellular & Molecular Immunology (2014) 11, 126–128; doi:10.1038/cmi.2013.63; published online 17 February 2014

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ype 2 T helper (Th2) inflammation underlies common clinical disorders such as asthma, eczema and other atopic diseases. CD41 regulatory T cells (Tregs) suppress over-exuberant immune responses in a variety of inflammatory microenvironments, including Th2 conditions. The transcription factor forkhead box protein 3 (Foxp3) controls Treg development, function and maintenance; however, mechanisms through which Tregs mitigate immune activation during different inflammatory situations remain largely unknown. In their recent article, Jin et al. report that Itch, an E3 ubiquitin ligase, is required to prevent Tregs from acquiring Th2-like phenotype and function.1 The authors created a novel Treg-specific Itch-deficient mouse (Itchfl/flFoxp3Cre) that enabled careful examination of Treg Itch in controlling Th2 inflammatory responses. General Treg suppressive mechanisms involve contact-dependent inhibitory cell surface receptors (CTLA-4), competition for growth factors (CD25), hydrolysis of extracellular nucleotides (CD39, CD73) and induction of cell death (granzyme secretion).2 Additionally, Tregs suppress T cell skewing by acquiring specific T effector transcriptional programs that permit homing to a particular inflammatory microenvironment. Expression of Johns Hopkins University Division of Pulmonary and Critical Care Medicine, Baltimore, MD, USA Correspondence: Dr FR D’Alessio, Johns Hopkins University Division of Pulmonary and Critical Care Medicine, Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Fourth Floor, Baltimore, MD 21224, USA E-mail: [email protected] Received: 29 November 2013; Accepted: 2 December 2013

the transcription factors T-bet, Irf4 and STAT3 regulate Treg suppressive activity in Th1, Th2 and Th17 inflammation, respectively (Table 1).3–5 Indeed, Tregspecific Irf4 knockout mice have dysregulated Th2 responses. However, we have limited data on the specific molecular pathways that Tregs employ to control Th2 inflammation. Ubiquitin conjugation is a key immune regulatory mechanism. Post-translational ubiquitination leads to differential consequences for the substrate protein and results in diverse cellular responses.6 Itch is a HECT (homologous to E6-associated protein C terminus)-type E3 ubiquitin ligase involved in immune regulation. Itch knockout (Itch2/2) mice develop skin-scratching, enlarged lymph nodes and spleen and inflammation of the lungs and gut.7 CD41 T cells in these mice produce Th2 cytokines,8 and this Th2 skewing has been linked to the inability of naı¨ve CD41 T cells to convert into inducible Tregs. The Itchfl/flFoxp3Cre mouse allowed Jin et al. to study Itch in thymus-derived natural Tregs. The authors provide convincing evidence that Treg-specific Itch critically limits Th2 inflammation. Itchfl/flFoxp3Cre mice were normal at birth, but spontaneously developed systemic Th2 inflammation characterized by early mortality, as well as skin-scratching, splenomegaly, lymphadenopathy, and splenic, pulmonary, dermal, gastric and hepatic inflammation by 6 weeks of age. They also demonstrated hyper-responsiveness to an antigen-induced airway inflammation model of asthma (ovalbumin immunization and challenge). Notably, while wildtype Treg adoptive transfer ameliorated

pathology in wild-type mice that underwent ovalbumin immunization and challenge, adoptive transfer of Itch-deficient Tregs paradoxically exacerbated airway inflammation. These results confirm a Treg-specific role for Itch in restraining Th2 pathology. Itchfl/flFoxp3Cre mice showed Th2 skewing of their (Itch-sufficient) CD41 T cell population. The authors used multiparameter flow cytometry to determine that splenic non-Treg CD41 T cells from Itchfl/flFoxp3Cre mice demonstrated selectively dysregulated Th2 responses. CD41 non-Tregs displayed an increased activation state characterized by a CD44hiCD62LloCD691 phenotype and increased stimulation-induced production of Th2 cytokines (IL-4, IL-5 and IL-10). Production of IFN-c (a Th1 cytokine) and IL-17 (a Th17 cytokine) was similar to Itch1/1Foxp3Cre controls. Itchfl/flFoxp3Cre mice boosted antibody isotype class switching to IgG1 and IgE, which is a Th2-requiring phenomenon. Thus, Treg Itch controls Th2 responses in non-Treg CD41 T cells. Because Tregs are a relatively minor subpopulation of CD41 T cells, the authors reasoned that non-Treg cell types may also be responsible for the Th2 response to Treg-specific Itch loss. They treated naive CD41 T cells with culture supernatants from Itch-deficient Tregs; the naive CD41 T cells differentiated into IL-4-producing effector T cells with a Th2 mRNA expression profile. The authors then blocked induction of the Th2 phenotype by culturing naive CD41 T cells with anti-IL-4 antibodytreated supernatant. These results suggest that IL-4 derived from Itch-deficient

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Table 1 Role of selected proteins and the effect of their Treg-specific loss Protein

Role

Effect of deficiency in Tregs

T-bet Irf4 GATA3

Required for the development of Th1 cells Required for the development of Th2 cells Canonical Th2 transcription factor; required for Treg function

STAT3 Itch

Required for the development of Th17 cells E3 ubiquitin ligase involved in the regulation of Th2 responses

Unable to control Th1 immune responses3 Unable to control Th2 immune responses4 Decreased Foxp3, CTLA-4, GITR and CD25 expression; unable to prevent induction of systemic lymphoproliferative disease10 Unable to control Th17 immune responses5 Unrestrained development of Th2 pathology1

Abbreviations: Foxp3, forkhead box protein 3; Th2, Type 2 T helper; Treg, regulatory T cell.

Tregs is the signal driving Th2 conversion in non-Treg CD41 T cells. Thus, Treg-specific Itch loss leads Tregs to directly instruct Th2 cell differentiation via IL-4, at least in an in vitro system. Itch-deficient Tregs themselves also acquired Th2 properties. A Bio-Plex multicytokine assay revealed that anti-CD3/ CD28-stimulated Itch-deficient Tregs produced high levels of Th2 cytokines (IL-4, IL-5, IL-10 and IL-13). Flow cytometry of Itch-deficient Tregs demonstrated an activated phenotype with increased surface expression of GITR and ICOS but not CD25 or CTLA-4. Additionally, Itch-deficient Tregs were more resistant to activation-induced cell death and showed enhanced proliferation in culture. It remains controversial whether Tregs can be converted into ‘ex-Foxp3’ effector T helper cells or if pro-inflammatory ex-Foxp3 cells arise from promiscuous Foxp3 expression in non-Tregs.9 Jin et al. used a fate-mapping approach to determine that Itch-deficient Tregs did not convert to an ex-Foxp3 state with any more frequency than Itch-sufficient controls. However, Itch-deficient exFoxp3 cells exhibited an exuberant Th2 phenotype. Thus, although Treg instability did not account for the Th2 inflammation observed in Itchfl/flFoxp3Cre mice, Foxp3 loss exaggerated Th2 cytokine production by Itch-deficient cells. These experiments suggest that Foxp3 loss compounds the pro-Th2 functional modification caused by Itch deficiency. Pathogenic Th2 responses in Itch-deficient Tregs required GATA3 and STAT6, key signaling molecules involved in Th2 inflammation. The authors used short hairpin RNA to knock down Gata3 or Stat6 in Itchfl/flFoxp3Cre bone marrow cells and then transplanted the transfected cells

into lethally irradiated wild-type mice. Eight weeks later, splenic and lymph node Tregs were isolated and stimulated with anti-CD3/CD28 antibodies in vitro. These Tregs displayed increased Th2 cytokine production when harvested from mice transplanted with control short hairpin RNA-treated cells, whereas Gata3 or Stat6 knockdown completely abolished the Th2 response. These data point to GATA3 and STAT6 as essential molecules promoting the Th2 phenotype in Itchdeficient Tregs. The mechanisms through which Itch interacts with downstream molecules remain unknown. The authors’ experiments do not completely exclude a role for Itch in suppressing Th17 inflammation. They found no difference between Itchfl/flFoxp3Cre and Itch1/1Foxp3Cre mice in their susceptibility to experimental autoimmune encephalomyelitis induction, a model of Th17 inflammation. However, Itchfl/flFoxp3Cre mice exhibited an increased proportion of Th17 cells in their lungs. Further exploration of the role Itch plays in Th17 responses is warranted. Interestingly, Itchfl/flFoxp3Cre Tregs displayed normal Foxp3 expression and retained classical Treg suppressive activity. Itch-deficient Tregs suppressed in vitro proliferation of conventional T cells and abrogated an in vivo CD41CD45RBhi T cell transfer-mediated colitis model with equal potency to Itch-sufficient control Tregs. These results underscore the limitation of classical Treg functional assays in assessing Treg function, as Itchfl/flFoxp3Cre Tregs appeared normal in traditional assays yet clearly harbored significant pro-Th2 inflammatory properties. These findings raise several questions. Does Itch drive differential Treg phenotypes via its ubiquitin ligase properties and how might differential ubiquitination

lead to divergent cell responses? Specifically, how does Itch regulate GATA3 and STAT6? Given the essential role GATA3 plays in Treg function,10 does Itch’s regulation of GATA3 have more global effects on Treg activity outside of Th2 responses? Does Itch modulate Irf4 to promote Th2 skewing? Does cell-specific Itch loss in CD41 non-Tregs lead to immunopathology? What is the relevance of Itch or Treg-specific Itch in human disease states? Future studies will need to address these questions and work to translate basic discoveries into the clinical arena. In conclusion, Jin et al. used an elegant murine system to define Itch as a central molecular mechanism by which Tregs rein in Th2 responses. Translational studies are needed to confirm if Itch plays a role in human pathology and if Itch could be targeted for therapeutic benefit. If so, the implications of Jin et al.’s discoveries have importance for many Th2 conditions including asthma and allergic-spectrum disorders.

1 Jin HS, Park Y, Elly C, Liu YC. Itch expression by Treg cells controls Th2 inflammatory responses. J Clin Invest 2013; 123: 4923– 4934. 2 Shevach EM. Mechanisms of foxp3 1 T regulatory cell-mediated suppression. Immunity 2009; 30: 636–645. 3 Koch MA, Tucker-Heard G, Perdue NR, Killebrew JR, Urdahl KB, Campbell DJ. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat Immunol 2009; 10: 595–602. 4 Zheng Y, Chaudhry A, Kas A, deRoos P, Kim JM, Chu TT et al. Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T H 2 responses. Nature 2009; 458: 351–356. 5 Chaudhry A, Rudra D, Treuting P, Samstein RM, Liang Y, Kas A et al. CD41 regulatory T cells Cellular & Molecular Immunology

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128 control TH17 responses in a Stat3-dependent manner. Science 2009; 326: 986–991. 6 Bhoj VG, Chen ZJ. Ubiquitylation in innate and adaptive immunity. Nature 2009; 458: 430–437. 7 Liu YC. The E3 ubiquitin ligase Itch in T cell activation, differentiation, and tolerance. Semin Immunol 2007; 19: 197–205.

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8 Fang D, Elly C, Gao B, Fang N, Altman Y, Joazeiro C et al. Dysregulation of T lymphocyte function in itchy mice: a role for Itch in TH2 differentiation. Nat Immunol 2002; 3: 281– 287. 9 Miyao T, Floess S, Setoguchi R, Luche H, Fehling HJ, Waldmann H et al. Plasticity of Foxp31 T cells reflects promiscuous

Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells. Immunity 2012; 36: 262– 275. 10 Wang Y, Su MA, Wan YY. An essential role of the transcription factor GATA-3 for the function of regulatory T cells. Immunity 2011; 35: 337–348.

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