Regulatory T cell Itch reins in Th2 inflammation

Type 2 T helper (Th2) inflammation underlies common clinical disorders such as asthma, eczema and other atopic diseases. CD4+ 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 the transcription factors T-bet, Irf4 and STAT3 regulate Treg suppressive activity in Th1, Th2 and Th17 inflammation, respectively (Table 1).3,4,5 Indeed, Treg-specific Irf4 knockout mice have dysregulated Th2 responses. However, we have limited data on the specific molecular pathways that Tregs employ to control Th2 inflammation. Table 1 Role of selected proteins and the effect of their Treg-specific loss 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 (Itch−/−) mice develop skin-scratching, enlarged lymph nodes and spleen and inflammation of the lungs and gut.7 CD4+ T cells in these mice produce Th2 cytokines,8 and this Th2 skewing has been linked to the inability of naive CD4+ 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 wild-type 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) CD4+ T cell population. The authors used multiparameter flow cytometry to determine that splenic non-Treg CD4+ T cells from Itchfl/flFoxp3Cre mice demonstrated selectively dysregulated Th2 responses. CD4+ non-Tregs displayed an increased activation state characterized by a CD44hiCD62LloCD69+ phenotype and increased stimulation-induced production of Th2 cytokines (IL-4, IL-5 and IL-10). Production of IFN-γ (a Th1 cytokine) and IL-17 (a Th17 cytokine) was similar to Itch+/+Foxp3Cre 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 CD4+ T cells. Because Tregs are a relatively minor subpopulation of CD4+ 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 CD4+ T cells with culture supernatants from Itch-deficient Tregs; the naive CD4+ 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 CD4+ T cells with anti-IL-4 antibody-treated supernatant. These results suggest that IL-4 derived from Itch-deficient Tregs is the signal driving Th2 conversion in non-Treg CD4+ 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 ex-Foxp3 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 Itch-deficient 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 Itch+/+Foxp3Cre 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 CD4+CD45RBhi 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 CD4+ 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.