Immunity to many human and murine gastrointestinal helminth parasites requires interleukin-4 (IL-4)-directed type 2 helper (TH2) differentiation of CD4+ T cells to elicit type-2 immunity. Despite a good understanding of the inflammatory cascade elicited following helminth infection, the initial source of IL-4 is unclear. Previous studies using the rat helminth parasite Nippostronglyus brasiliensis, identified an important role for basophil-derived IL-4 for TH2 differentiation. However, basophils are redundant for TH2 differentiation following infection with the natural helminth parasite of mice Heligmosomoides polygyrus, indicating that other sources of IL-4 are required. In this study using H. polygyrus, which is controlled by IL-4-dependent immunity, we identified that group-2 innate lymphoid cells (ILC2s) produced significant amounts of IL-4 and IL-2 following H. polygyrus infection. Leukotriene D4 was sufficient to stimulate IL-4 secretion by ILC2s, and the supernatant from activated ILC2s could potently drive TH2 differentiation in vitro in an IL-4-dependent manner. Furthermore, specific deletion of IL-4 from ILC2s compromised TH2 differentiation in vivo. Overall, this study highlights a previously unrecognized and important role for ILC2-derived IL-4 for TH2 differentiation in a natural TH2-dependent model of human helminthiasis.

The transcription factor c-Maf induces the anti-inflammatory cytokine IL-10 in CD4+ T cells in vitro. However, the global effects of c-Maf on diverse immune responses in vivo are unknown. Here we show that c-Maf regulates IL-10 production in CD4+ T cells in TH1 (malaria), TH2 (allergy) and TH17 (autoimmunity) disease models in vivo. Although CD4-targeted Maf-deficient mice showed greater pathology in TH1 and TH2 responses, TH17-mediated pathology was reduced, with accompanying decreased TH17 and increased Foxp3+ regulatory T cells. Bivariate genomic footprinting elucidated the c-Maf transcription factor network, including enhanced NFAT activity, leading to the identification and validation of c-Maf as a negative regulator of IL-2. Decreased Rorc resulting from c-Maf deficiency was dependent on IL-2, explaining the in vivo observations. Thus, c-Maf is a positive and negative regulator of cytokine gene expression, with context-specific effects that allow each immune response to occur in a controlled yet effective manner.

Significance The rising prevalence of allergic diseases throughout the world demands new approaches to treat this inflammatory disorder. CD4 + Th2 cells orchestrate the allergic cascade, stimulating IgE production, activating innate cells, and stimulating local tissue. This study took a comprehensive approach to identify the unique transcriptional features of pathogenic Th2 cells with the aim of identifying novel molecular regulators. Highly purified Th1, Th2, Th9, Th17, and Treg cells isolated from mice with allergy, infection, and autoimmunity identified unique mRNA and microRNAs (miRNAs) expressed in Th2 cells. Functional and mechanistic studies using miRNA-deficient mice, luciferase assays, miRNA inhibitors, and siRNA in combination with state-of-the-art adoptive transfer systems, identified a critical role for miR-155–regulated S1pr1 in the pathogenesis of Th2-mediated allergy.

Interleukin-10 (IL-10) is an important regulatory cytokine required to control allergy and asthma. IL-10-mediated regulation of T cell-mediated responses was previously thought to occur indirectly via antigen-presenting cells. However, IL-10 can act directly on regulatory T cells and T helper type 17 (Th17) cells. In the context of allergy, it is therefore unclear whether IL-10 can directly regulate T helper type 2 (Th2) cells and whether this is an important regulatory axis during allergic responses. We sought to determine whether IL-10 signaling in CD4 Th2 cells was an important mechanism of immune regulation during airway allergy. We demonstrate that IL-10 directly limits Th2 cell differentiation and survival in vitro and in vivo. Ablation of IL-10 signaling in Th2 cells led to enhanced Th2 cell survival and exacerbated pulmonary inflammation in a murine model of house dust mite allergy. Mechanistically, IL-10R signaling regulated the expression of several genes in Th2 cells, including granzyme B. Indeed, IL-10 increased granzyme B expression in Th2 cells and led to increased Th2 cell death, identifying an IL-10-regulated granzyme B axis in Th2 cells controlling Th2 cell survival. This study provides clear evidence that IL-10 exerts direct effects on Th2 cells, regulating the survival of Th2 cells and severity of Th2-mediated allergic airway inflammation.

Interferon-γ (IFN-γ) production by natural killer (NK) cells and cytotoxic lymphocytes is a key component of innate and adaptive immune responses. Because inhibitor of κB-ζ (IκBζ), a Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) inducible transcription factor, regulates IFN-γ production in KG-1 cells, we tested IκBζ's role in the classic lymphocyte pathway of IL-12/IL-18–induced IFN-γ. Upon stimulation with IL-12/IL-18, monocyte-depleted human peripheral blood lymphocytes expressed the 79-kDa form of IκBζ and released IFN-γ. CD56+ NK cells were shown to be the IκBζ-producing lymphocyte subpopulation, which also released abundant IFN-γ in response to IL-12/IL-18. Importantly, IκBζ was undetectable in CD56− lymphocytes where IFN-γ release was 10-fold lower. In addition, small interfering RNA knockdown of IκBζ suppressed IFN-γ expression in CD56+ cells. The association of IκBζ with the IFN-γ promoter was documented by chromatin immunoprecipitation. IFN-γ promoter activity from IκBζ overexpression was confirmed by luciferase reporter assay. Finally, IκBζ coprecipitated with p65 and p50 NF-κB in NK cells in response to IL-12/IL-18, suggesting that IκBζ's effects on IFN-γ promoter activity are coregulated by NF-κB. These results suggest that IκBζ functions as an important regulator of IFN-γ in human NK cells, further expanding the class of IκBζ-modulated genes.

IL-6 is a pleiotropic cytokine implicated in the pathogenesis of disorders such as sepsis and cancer. We noted that human monocytes are excellent producers of IL-6 as compared with monocyte-derived macrophages. Because macrophages from molecule containing ankyrin repeats induced by LPS (MAIL) knockout animals have suppressed IL-6 production, we hypothesized that regulation of MAIL is key to IL-6 production in humans and may explain the differences between human monocytes and macrophages. To test this hypothesis fresh human monocytes and monocyte-derived macrophages were compared for MAIL expression in response to LPS. LPS-induced monocyte MAIL expression was highly inducible and transient. Importantly for our hypothesis MAIL protein expression was suppressed during differentiation of monocytes to macrophages. Of note, the human MAIL protein detected was the 80 kDa MAIL-L form and human MAIL showed nuclear localization. Human MAIL-L bound to p50 subunit of the NF-κB and increased IL-6 luciferase promoter activity in a cEBPβ, NF-κB, and AP-1-dependent fashion. Like the differences in MAIL induction, monocytes produced 6-fold more IL-6 compared with macrophages (81.7 ± 29.7 vs 12.6 ± 6.8 ng/ml). Furthermore, suppression of MAIL by small interfering RNA decreased the production of IL-6 significantly in both THP-1 cells and in primary monocytes. Costimulation of monocytes with LPS and muramyl dipeptide induced an enhanced IL-6 response that was suppressed by siMAIL. Our data suggests that MAIL is a key regulator of IL-6 production in human monocytes and plays an important role in both TLR and NOD-like receptor ligand induced inflammation.

Pelly et al. use novel mouse reporter systems to show that a proportion of Th2 cells develop from Foxp3-expressing cells in an IL-4–dependent manner, highlighting the potential to subvert T reg cell–mediated suppression in favor of type 2 immunity.

Parasitic helminths establish chronic infections in mammalian hosts. Helminth/Plasmodium co-infections occur frequently in endemic areas. However, it is unclear whether Plasmodium infections compromise anti-helminth immunity, contributing to the chronicity of infection. Immunity to Plasmodium or helminths requires divergent CD4+ T cell-driven responses, dominated by IFNγ or IL-4, respectively. Recent literature has indicated that Th cells, including Th2 cells, have phenotypic plasticity with the ability to produce non-lineage associated cytokines. Whether such plasticity occurs during co-infection is unclear. In this study, we observed reduced anti-helminth Th2 cell responses and compromised anti-helminth immunity during Heligmosomoides polygyrus and Plasmodium chabaudi co-infection. Using newly established triple cytokine reporter mice (Il4gfpIfngyfpIl17aFP635), we demonstrated that Il4gfp+ Th2 cells purified from in vitro cultures or isolated ex vivo from helminth-infected mice up-regulated IFNγ following adoptive transfer into Rag1–/– mice infected with P. chabaudi. Functionally, Th2 cells that up-regulated IFNγ were transcriptionally re-wired and protected recipient mice from high parasitemia. Mechanistically, TCR stimulation and responsiveness to IL-12 and IFNγ, but not type I IFN, was required for optimal IFNγ production by Th2 cells. Finally, blockade of IL-12 and IFNγ during co-infection partially preserved anti-helminth Th2 responses. In summary, this study demonstrates that Th2 cells retain substantial plasticity with the ability to produce IFNγ during Plasmodium infection. Consequently, co-infection with Plasmodium spp. may contribute to the chronicity of helminth infection by reducing anti-helminth Th2 cells and converting them into IFNγ-secreting cells.