Masaki Kashiwada

TH17 Cell Differentiation Is Regulated by the Circadian Clock

Lighting Up Immunity TH17 cells are CD4+ T helper cells that produce the proinflammatory cytokine interleukin-17. In the intestines, TH17 cells protect the host from fungal and bacterial infections, and their proinflammatory function is linked with autoimmune diseases including inflammatory bowel disease. Yu et al. (p. 727) show that the molecular circadian clock directly regulates the differentiation of TH17 cells in the intestine, which suggest that both nutrition and light are important environmental factors that directly regulate the immune response. Diurnal regulation of an immune cell lineage in the intestine protects against inflammatory disease in mice. Circadian clocks regulate numerous physiological processes that vary across the day-night (diurnal) cycle, but if and how the circadian clock regulates the adaptive immune system is mostly unclear. Interleukin-17–producing CD4+ T helper (TH17) cells are proinflammatory immune cells that protect against bacterial and fungal infections at mucosal surfaces. Their lineage specification is regulated by the orphan nuclear receptor RORγt. We show that the transcription factor NFIL3 suppresses TH17 cell development by directly binding and repressing the Rorγt promoter. NFIL3 links TH17 cell development to the circadian clock network through the transcription factor REV-ERBα. Accordingly, TH17 lineage specification varies diurnally and is altered in Rev-erbα−/− mice. Light-cycle disruption elevated intestinal TH17 cell frequencies and increased susceptibility to inflammatory disease. Thus, lineage specification of a key immune cell is under direct circadian control.

IL-4-induced transcription factor NFIL3/E4BP4 controls IgE class switching

IL-4 signaling promotes IgE class switching through STAT6 activation and the induction of Ig germ-line ε (GLε) transcription. Previously, we and others identified a transcription factor, Nfil3, as a gene induced by IL-4 stimulation in B cells. However, the precise roles of nuclear factor, IL-3-regulated (NFIL3) in IL-4 signaling are unknown. Here, we report that NFIL3 is important for IgE class switching. NFIL3-deficient mice show impaired IgE class switching, and this defect is B-cell intrinsic. The induction of GLε transcripts after LPS and IL-4 stimulation is significantly reduced in NFIL3-deficient B cells. Expression of NFIL3 in NFIL3-deficient B cells restores the impairment of IgE production, and overexpression of NFIL3 in the presence of cycloheximide induces GLε transcripts. Moreover, NFIL3 binds to Iε promoter in vivo. Together, these results identify NFIL3 as a key regulator of IL-4-induced GLε transcription in response to IL-4 and subsequent IgE class switching.

NFIL3/E4BP4 is a key transcription factor for CD8α+ dendritic cell development

Antigen presentation by mature dendritic cells (DCs) is the first step for initiating adaptive immune responses. DCs are composed of heterogeneous functional subsets; however, the molecular mechanisms that regulate differentiation of specific DC subsets are not understood. Here, we report that the basic leucine zipper transcription factor NFIL3/E4BP4 is essential for the development of CD8α(+) conventional DCs (cDCs). Nfil3(-/-) mice specifically lack CD8α(+) cDCs but not CD8α(-) cDCs or plasmacytoid DCs in lymphoid tissues. Flt3 ligand-dependent generation of CD8α(+) cDCs in lymphoid tissues and CD8α(+)-equivalent cDCs from Nfil3(-/-) bone marrow cells was also impaired. NFIL3 regulates CD8α(+) cDC development in part through Batf3 expression. Importantly, Nfil3(-/-) mice exhibited impaired cross-priming of CD8(+) T cells against cell-associated antigen, a process normally performed by CD8α(+) cDCs, and failed to produce IL-12 after TLR3 stimulation. Thus, NFIL3 plays an essential role in the development of CD8α(+) cDCs.

Deletion of SOCS7 leads to enhanced insulin action and enlarged islets of Langerhans

NIDDM is characterized by progressive insulin resistance and the failure of insulin-producing pancreatic beta cells to compensate for this resistance. Hyperinsulinemia, inflammation, and prolonged activation of the insulin receptor (INSR) have been shown to induce insulin resistance by decreasing INSR substrate (IRS) protein levels. Here we describe a role for SOCS7 in regulating insulin signaling. Socs7-deficient mice exhibited lower glucose levels and prolonged hypoglycemia during an insulin tolerance test and increased glucose clearance in a glucose tolerance test. Six-month-old Socs7-deficient mice exhibited increased growth of pancreatic islets with mildly increased fasting insulin levels and hypoglycemia. These defects correlated with increased IRS protein levels and enhanced insulin action in cells lacking SOCS7. Additionally, SOCS7 associated with the INSR and IRS1--molecules that are essential for normal regulation of insulin action. These data suggest that SOCS7 is a potent regulator of glucose homeostasis and insulin signaling.

NFIL3 Is a Regulator of IL-12 p40 in Macrophages and Mucosal Immunity

Regulation of innate inflammatory responses against the enteric microbiota is essential for the maintenance of intestinal homeostasis. Key participants in innate defenses are macrophages. In these studies, the basic leucine zipper protein, NFIL3, is identified as a regulatory transcription factor in macrophages, controlling IL-12 p40 production induced by bacterial products and the enteric microbiota. Exposure to commensal bacteria and bacterial products induced NFIL3 in cultured macrophages and in vivo. The Il12b promoter has a putative DNA-binding element for NFIL3. Basal and LPS-activated NFIL3 binding to this site was confirmed by chromatin immunoprecipitation. LPS-induced Il12b promoter activity was inhibited by NFIL3 expression and augmented by NFIL3-short hairpin RNA in an Il12b-bacterial artificial chromosome-GFP reporter macrophage line. Il12b inhibition by NFIL3 does not require IL-10 expression, but a C-terminal minimal repression domain is necessary. Furthermore, colonic CD11b+ lamina propria mononuclear cells from Nfil3−/− mice spontaneously expressed Il12b mRNA. Importantly, lower expression of NFIL3 was observed in CD14+ lamina propria mononuclear cells from Crohn’s disease and ulcerative colitis patients compared with control subjects. Likewise, no induction of Nfil3 was observed in colons of colitis-prone Il10−/− mice transitioned from germ-free to a conventional microbiota. In conclusion, these experiments characterize NFIL3 as an Il12b transcriptional inhibitor. Interactions of macrophages with the enteric microbiota induce NFIL3 to limit their inflammatory capacity. Furthermore, altered intestinal NFIL3 expression may have implications for the pathogenesis of experimental and human inflammatory bowel diseases.

Immunoreceptor Tyrosine-Based Inhibitory Motif of the IL-4 Receptor Associates with SH2-Containing Phosphatases and Regulates IL-4-Induced Proliferation

Immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been implicated in the negative modulation of immunoreceptor signaling pathways. The IL-4R α-chain (IL-4Rα) contains a putative ITIM in the carboxyl terminal. To determine the role of ITIM in the IL-4 signaling pathway, we ablated the ITIM of IL-4Rα by deletion and site-directed mutagenesis and stably expressed the wild-type (WT) and mutant hIL-4Rα in 32D/insulin receptor substrate-2 (IRS-2) cells. Strikingly, 32D/IRS-2 cells expressing mutant human (h)IL-4Rα were hyperproliferative in response to IL-4 compared with cells expressing WT hIL-4Rα. Enhanced tyrosine phosphorylation of Stat6, but not IRS-2, induced by hIL-4 was observed in cells expressing mutant Y713F. Using peptides corresponding to the ITIM of hIL-4Rα, we demonstrate that tyrosine-phosphorylated peptides, but not their nonphosphorylated counterparts, coprecipitate SH2-containing tyrosine phosphatase-1, SH2-containing tyrosine phosphatase-2, and SH2-containing inositol 5′-phosphatase. The in vivo association of SH2-containing inositol 5′-phosphatase with IL-4Rα was verified by coimmunoprecipitation with anti-IL-4Rα Abs. These results demonstrate a functional role for ITIM in the regulation of IL-4-induced proliferation.

NFIL3/E4BP4 controls type 2 T helper cell cytokine expression

Type 2 T helper (TH2) cells are critical for the development of allergic immune responses; however, the molecular mechanism controlling their effector function is still largely unclear. Here, we report that the transcription factor NFIL3/E4BP4 regulates cytokine production and effector function by TH2 cells. NFIL3 is highly expressed in TH2 cells but much less in TH1 cells. Production of interleukin (IL)‐13 and IL‐5 is significantly increased in Nfil3−/− TH2 cells and is decreased by expression of NFIL3 in wild‐type TH2 cells. NFIL3 directly binds to and negatively regulates the Il13 gene. In contrast, IL‐4 production is decreased in Nfil3−/− TH2 cells. Increased IL‐13 and IL‐5 together with decreased IL‐4 production by antigen‐stimulated splenocytes from the immunized Nfil3−/− mice was also observed. The ability of NFIL3 to alter TH2 cytokine production is a T‐cell intrinsic effect. Taken together, these data indicate that NFIL3 is a key regulator of TH2 responses.

Downstream of Tyrosine Kinases-1 and Src Homology 2-Containing Inositol 5′-Phosphatase Are Required for Regulation of CD4 + CD25 + T Cell Development

The adaptor protein, downstream of tyrosine kinases-1 (Dok-1), and the phosphatase SHIP are both tyrosine phosphorylated in response to T cell stimulation. However, a function for these molecules in T cell development has not been defined. To clarify the role of Dok-1 and SHIP in T cell development in vivo, we compared the T cell phenotype of wild-type, Dok-1 knockout (KO), SHIP KO, and Dok-1/SHIP double-knockout (DKO) mice. Dok-1/SHIP DKO mice were runted and had a shorter life span compared with either Dok-1 KO or SHIP KO mice. Thymocyte numbers from Dok-1/SHIP DKO mice were reduced by 90%. Surface expression of both CD25 and CD69 was elevated on freshly isolated splenic CD4+ T cells from SHIP KO and Dok-1/SHIP DKO, suggesting these cells were constitutively activated. However, these T cells did not proliferate or produce IL-2 after stimulation. Interestingly, the CD4+ T cells from SHIP KO and Dok-1/SHIP DKO mice produced higher levels of TGF-β, expressed Foxp3, and inhibited IL-2 production by CD3-stimulated CD4+CD25− T cells in vitro. These findings suggest Dok-1 and SHIP function in pathways that influence regulatory T cell development.

Positive Regulation of Interleukin-4-mediated Proliferation by the SH2-containing Inositol-5′-phosphatase

The SH2-containing inositol 5′-phosphatase (SHIP) is tyrosine-phosphorylated in response to cytokines such as interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor. SHIP has been shown to modulate negatively these cytokine signalings; however, a potential role in IL-4 signaling remains uncharacterized. It has been recently shown that IL-4 induces tyrosine phosphorylation of SHIP, implicating the phosphatase in IL-4 processes. Tyrosine kinases, Jak1 and Jak3, involved in IL-4 signaling can associate with SHIP, yet only Jak1 can tyrosine-phosphorylate SHIP when co-expressed. In functional studies, cells overexpressing wild type SHIP are found to be hyperproliferative in response to IL-4 in comparison to parental cells. In contrast, cells expressing catalytically inactive form, SHIP(D672A), show reduced proliferation in response to IL-4. These changes in IL-4-induced proliferation correlate with alterations in phosphatidylinositol 3,4,5-triphosphate levels. However, no differential activation of STAT6, Akt, IRS-2, or p70S6k, in response to IL-4, was observed in these cells. These data suggest that the catalytic activity of SHIP acts in a novel manner to influence IL-4 signaling. In addition, these data support recent findings that suggest there are uncharacterized signaling pathways downstream of phosphatidylinositol 3,4,5-triphosphate.

Fes mediates the IL-4 activation of insulin receptor substrate-2 and cellular proliferation

Although Jak kinases are essential for initiating cytokine signaling, the role of other nonreceptor tyrosine kinases in this process remains unclear. We have examined the role of Fes in IL-4 signaling. Examination of Jak1-deficient cell lines demonstrates that Jak1 is required for the activation of Fes by IL-4. Experiments studying signaling molecules activated by IL-4 receptor suggest that IL-4 signaling can be subdivided into Fes-dependent and Fes-independent pathways. Overexpression of kinase-inactive Fes blocks the IL-4 activation of insulin receptor substrate-2, but not STAT6. Fes appears to be a downstream kinase from Jak1/Jak3 in this process. Further examination of downstream signaling demonstrates that kinase-inactive Fes inhibits the recruitment of phosphoinositide 3-kinase to the activated IL-4 receptor complex and decreases the activation of p70S6k kinase in response to IL-4. This inhibition correlates with a decrease in IL-4-induced proliferation. In contrast, mutant Fes does not inhibit the activation of Akt by IL-4. These data demonstrate that signaling pathways activated by IL-4 require different tyrosine kinases. This differential requirement predicts that specific kinase inhibitors may permit the disruption of specific IL-4-induced functions.