Hypoxia induces adrenomedullin from lung epithelia, stimulating ILC2 inflammation and immunity.

Hypoxia contributes to airway inflammation and remodeling in several lung diseases; however, exactly how hypoxic pulmonary epithelium regulates allergic inflammation remains to be fully characterized. Here, we report that conditional deletion of the E3 ubiquitin ligase VHL in lung epithelial cells resulted in exacerbated type 2 responses accompanied by selective increase of group 2 innate lymphoid cells (ILC2s) at steady state and following inflammation or helminth infection. Ablation of expression of the hypoxia-inducible factor 2α (HIF2α) significantly reversed VHL-mediated ILC2 activation. VHL deficiency in lung epithelial cells caused increased expression of the peptide hormone adrenomedullin (ADM), and our data suggest that HIF2α controls Adm expression. ADM directly promoted ILC2 activation both in vitro and in vivo. Our findings indicate that the hypoxic response mediated by the VHL-HIF2α axis is critical for control of pulmonary type 2 responses by increasing ADM expression in lung epithelia, causing ILC2 activation.

The deubiquitinase CYLD controls protective immunity against helminth infection by regulation of Treg cell plasticity.

BACKGROUND: Type 2 immunity can be modulated by regulatory T (Treg) cell activity. It has been suggested that the deubiquitinase cylindromatosis (CYLD) plays a role in the development or function of Treg cells, implying that it could be important for normal protective immunity, where type 2 responses are prevalent. OBJECTIVE: We sought to investigate the role of CYLD in Treg cell function and TH2 cell immune responses under steady-state conditions and during helminth infection. METHODS: Foxp3-restricted CYLD conditional knockout (KO) mice were examined in mouse models of allergen-induced airway inflammation and Nippostrongylus brasiliensis infection. We performed multiplex magnetic bead assays, flow cytometry, and quantitative PCR to understand how a lack of CYLD affected cytokine production, homing, and suppression in Treg cells. Target genes regulated by CYLD were identified and validated by microarray analysis, coimmunoprecipitation, short hairpin RNA knockdown, and transfection assays. RESULTS: Treg cell-specific CYLD KO mice showed severe spontaneous pulmonary inflammation with increased migration of Treg cells into the lung. CYLD-deficient Treg cells furthermore produced high levels of IL-4 and failed to suppress allergen-induced lung inflammation. Supporting this, the conditional KO mice displayed enhanced protection against N brasiliensis infection by contributing to type 2 immunity. Treg cell conversion into IL-4-producing cells was due to augmented mitogen-activated protein kinase and nuclear factor κB signaling. Moreover, Scinderin, a member of the actin-binding gelsolin family, was highly upregulated in CYLD-deficient Treg cells, and controlled IL-4 production through forming complexes with mitogen-activated protein kinase kinase/extracellular receptor kinase. Correspondingly, both excessive IL-4 production in vivo and the protective role of CYLD-deficient Treg cells against N brasiliensis were reversed by Scinderin ablation. CONCLUSIONS: Our findings indicate that CYLD controls type 2 immune responses by regulating Treg cell conversion into TH2 cell-like effector cells, which potentiates parasite resistance.

The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR.

The mechanisms by which the sensitivity of naive CD4+ T cells to stimulation by the cognate antigen via the T cell antigen receptor (TCR) determines their differentiation into distinct helper T cell subsets remain elusive. Here we demonstrate functional collaboration of the ubiquitin E3 ligases Itch and WWP2 in regulating the strength of the TCR signal. Mice lacking both Itch and WWP2 in T cells showed spontaneous autoimmunity and lung inflammation. CD4+ T cells deficient in Itch and WWP2 exhibited hypo-responsiveness to TCR stimulation and a bias toward differentiation into the TH2 subset of helper T cells. Itch and WWP2 formed a complex and cooperated to enhance TCR-proximal signaling by catalyzing the conjugation of atypical ubiquitin chains to the phosphatase SHP-1 and reducing the association of SHP-1 with the tyrosine kinase Lck. These findings indicate that targeted ubiquitination regulates the strength of the TCR signal and differentiation toward the TH2 lineage.

E3 Ligase VHL Promotes Group 2 Innate Lymphoid Cell Maturation and Function via Glycolysis Inhibition and Induction of Interleukin-33 Receptor.

Group 2 innate lymphoid cells (ILC2s) are a specialized subset of lymphoid effector cells that are critically involved in allergic responses; however, the mechanisms of their regulation remain unclear. We report that conditional deletion of the E3 ubiquitin ligase VHL in innate lymphoid progenitors minimally affected early-stage bone marrow ILC2s but caused a selective and intrinsic decrease in mature ILC2 numbers in peripheral non-lymphoid tissues, resulting in reduced type 2 immune responses. VHL deficiency caused the accumulation of hypoxia-inducible factor 1α (HIF1α) and attenuated interleukin-33 (IL-33) receptor ST2 expression, which was rectified by HIF1α ablation or inhibition. HIF1α-driven expression of the glycolytic enzyme pyruvate kinase M2 downmodulated ST2 expression via epigenetic modification and inhibited IL-33-induced ILC2 development. Our study indicates that the VHL-HIF-glycolysis axis is essential for the late-stage maturation and function of ILC2s via targeting IL-33-ST2 pathway.

E3 Ubiquitin Ligase VHL Regulates Hypoxia-Inducible Factor-1α to Maintain Regulatory T Cell Stability and Suppressive Capacity.

Foxp3(+) regulatory T (Treg) cells play a critical role in immune homeostasis; however, the mechanisms to maintain their function remain unclear. Here, we report that the E3 ubiquitin ligase VHL is essential for Treg cell function. Mice with Foxp3-restricted VHL deletion displayed massive inflammation associated with excessive Treg cell interferon-γ (IFN-γ) production. VHL-deficient Treg cells failed to prevent colitis induction, but converted into Th1-like effector T cells. VHL intrinsically orchestrated such conversion under both steady and inflammatory conditions followed by Foxp3 downregulation, which was reversed by IFN-γ deficiency. Augmented hypoxia-inducible factor 1α (HIF-1α)-induced glycolytic reprogramming was required for IFN-γ production. Furthermore, HIF-1α bound directly to the Ifng promoter. HIF-1α knockdown or knockout could reverse the increased IFN-γ by VHL-deficient Treg cells and restore their suppressive function in vivo. These findings indicate that regulation of HIF-1α pathway by VHL is crucial to maintain the stability and suppressive function of Foxp3(+) T cells.

Cutting edge: progesterone directly upregulates vitamin d receptor gene expression for efficient regulation of T cells by calcitriol.

The two nuclear hormone receptor ligands progesterone and vitamin D (vit.D) play important roles in regulating T cells. The mechanism that connects these two hormones in regulating T cells has not been established. In this study, we report that progesterone is a novel inducer of vit.D receptor (VDR) in T cells and makes T cells highly sensitive to calcitriol. At the molecular level, the induction by progesterone is mediated by two progesterone receptor-binding elements in the intron region after the first noncoding exon of the human VDR gene. Increased expression of VDR by progesterone allows highly sensitive regulation of T cells by vit.D even when vit.D levels are suboptimal. This novel regulatory pathway allows enhanced induction of regulatory T cells but suppression of Th1 and Th17 cells by the two nuclear hormones. The results have significant ramifications in effective regulation of T cells to prevent adverse immune responses during pregnancy.

BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid.

CCR9 and α4ß7 are the major trafficking receptors for lymphocyte migration to the gut, and their expression is induced during lymphocyte activation under the influence of retinoic acid (RA). We report here that BATF (basic leucine zipper transcription factor, ATF-like), an AP-1 protein family factor, is required for optimal expression of CCR9 and α4ß7 by T helper cells. BATF-deficient (knockout [KO]) mice had reduced numbers of effector T and regulatory T cells in the intestine. The intestinal T cells in BATF KO mice expressed CCR9 and α4ß7 at abnormally low levels compared with their wild-type (WT) counterparts, and BATF KO CD4(+) T cells failed to up-regulate the expression of CCR9 and α4ß7 to WT levels in response to RA. Defective binding of RARα and histone acetylation at the regulatory regions of the CCR9 and Itg-α4 genes were observed in BATF KO T cells. As a result, BATF KO effector and FoxP3(+) T cells failed to populate the intestine, and neither population functioned normally in the induction and regulation of colitis. Our results establish BATF as a cellular factor required for normal expression of CCR9 and α4ß7 and for the homeostasis and effector functions of T cell populations in the intestine.

Progesterone suppresses the mTOR pathway and promotes generation of induced regulatory T cells with increased stability.

While induced FoxP3(+) T cells (iTreg cells) are promising cellular therapeutics to treat inflammatory diseases, a limitation in utilizing iTreg cells prepared in vitro is their low stability in inflammatory conditions. Progesterone (P4) is an immune regulatory nuclear hormone with a potent Treg induction activity. We reasoned that this function of progesterone would be utilized to generate iTreg cells with highly suppressive activity and improved stability in vivo. Here we generated iTreg cells with progesterone in vitro and found that progesterone generates iTreg cells that are highly stable in inflammatory conditions. Moreover, P4-induced iTreg cells highly express latency-associated peptide TGF-ß1 and are efficient in regulating inflammation in multiple tissues, whereas control iTreg cells induced with TGF-ß1 alone are less stable and ineffective in suppressing inflammation. The function of progesterone in inducing iTreg cells with improved regulatory activity is associated with the function of P4 in suppressing the mTOR pathway. Moreover, the function of progesterone in inducing FoxP3(+) T cells is decreased but not completely abolished on nuclear progesterone receptor-deficient T cells, suggesting that both nuclear and nonnuclear progesterone receptors are involved in mediating the function. We conclude that P4 can be utilized to generate iTreg cells with a high therapeutic potential in treatment of tissue inflammation.

Optimal population of FoxP3+ T cells in tumors requires an antigen priming-dependent trafficking receptor switch.

FoxP3(+) T cells populate tumors and regulate anti-tumor immunity. The requirement for optimal population of FoxP3(+) regulatory T cells in tumors remains unclear. We investigated the migration requirement and stability of tumor-associated FoxP3(+) T cells. We found that only memory, but not naïve, FoxP3(+) T cells are highly enriched in tumors. Almost all of the tumor-infiltrating FoxP3(+) T cells express Helios, an antigen associated either with thymus-generated FoxP3(+) T cells or activated T cells in the periphery. The tumor-infiltrating FoxP3(+) T cells largely lack CD62L and CCR7, two trafficking receptors required for T cell migration into secondary lymphoid tissues. Instead, the tumor infiltrating FoxP3(+) T cells highly express memory/tumor-associated CCR8 and CXCR4. Antigen priming is required for induction of this trafficking receptor phenotype in FoxP3(+) T cells and only antigen primed, but not antigen-inexperienced naive, FoxP3(+) T cells can efficiently migrate into tumors. While the migration of FoxP3(+) T cells into tumors was a readily detectable event, generation of induced FoxP3(+) T cells within tumors was unexpectedly inefficient. Genetic marking of current and ex-FoxP3(+) T cells revealed that tumor-infiltrating FoxP3(+) T cells are highly stable and do not readily convert back to FoxP3(-) T cells. Taken together, our results indicate that population of tumors with thymus-generated FoxP3(+) T cells requires an antigen priming-dependent trafficking receptor switch in lymphoid tissues.

Progesterone promotes differentiation of human cord blood fetal T cells into T regulatory cells but suppresses their differentiation into Th17 cells.

Progesterone, a key female sex hormone with pleiotropic functions in maintenance of pregnancy, has profound effects on regulation of immune responses. We report in this work a novel function of progesterone in regulation of naive cord blood (CB) fetal T cell differentiation into key T regulatory cell (Treg) subsets. Progesterone drives allogeneic activation-induced differentiation of CB naive, but not adult peripheral blood, T cells into immune-suppressive Tregs, many of which express FoxP3. Compared with those induced in the absence of progesterone, the FoxP3(+) T cells induced in the presence of progesterone highly expressed memory T cell markers. In this regard, the Treg compartment in progesterone-rich CB is enriched with memory-type FoxP3(+) T cells. Moreover, CB APCs were more efficient than their peripheral blood counterparts in inducing FoxP3(+) T cells. Another related function of progesterone that we discovered was to suppress the differentiation of CB CD4(+) T cells into inflammation-associated Th17 cells. Progesterone enhanced activation of STAT5 in response to IL-2, whereas it decreased STAT3 activation in response to IL-6, which is in line with the selective activity of progesterone in generation of Tregs versus Th17 cells. Additionally, progesterone has a suppressive function on the expression of the IL-6 receptor by T cells. The results identified a novel role of progesterone in regulation of fetal T cell differentiation for promotion of immune tolerance.

FoxP3+ regulatory T cells restrain splenic extramedullary myelopoiesis via suppression of hemopoietic cytokine-producing T cells.

Extramedullary myelopoiesis occurs in peripheral organs such as spleen and produces many types of myeloid cells with diverse functions in response to inflammation and infection. It is increased during immune responses and chronic inflammation and is a significant factor in regulating inflammatory diseases and immunity. Increased myeloid cells are found in FoxP3-deficient mice but the mechanism has been unclear. We investigated the mechanism by which FoxP3(+) regulatory T cells regulate the extramedullary myelopoiesis. We found that Ab or genetic depletion of FoxP3(+) regulatory T cells greatly increased the number of the myeloid progenitors in spleen during immune responses. Consistently, the splenic myelopoiesis was effectively suppressed by increased numbers of natural or induced FoxP3(+) regulatory T cells. We demonstrated that myelopoiesis is positively regulated by splenic CD4(+) T cells that produce myelopoietic cytokines (GM-CSF and IL-3), and these effector CD4(+) T cells are induced from naive CD4(+) T cells in response to antigenic stimulation. FoxP3(+) regulatory T cells were able to effectively suppress the differentiation of naive T cells into myelopoietic cytokine-producing T cells. This suppression was found to be dependent on cell contact but independent of TGFbeta. Unlike splenic myelopoiesis, marrow myelopoiesis is not significantly affected by FoxP3(+) regulatory T cells. We conclude that FoxP3(+) T cells can negatively regulate splenic extramedullary myelopoiesis by suppressing the naive T cell differentiation into myelopoietic cytokine-producing CD4(+) T cells. Our results provide new insights into regulation of extramedullary myelopoiesis.

FoxP3+ T cells undergo conventional first switch to lymphoid tissue homing receptors in thymus but accelerated second switch to nonlymphoid tissue homing receptors in secondary lymphoid tissues.

Forkhead box P3 (FoxP3)-positive T cells are a specialized T cell subset for immune regulation and tolerance. We investigated the trafficking receptor switches of FoxP3(+) T cells in thymus and secondary lymphoid tissues and the functional consequences of these switches in migration. We found that FoxP3(+) T cells undergo two discrete developmental switches in trafficking receptors to migrate from primary to secondary and then to nonlymphoid tissues in a manner similar to conventional CD4(+) T cells as well as unique to the FoxP3(+) cell lineage. In the thymus, precursors of FoxP3(+) cells undergo the first trafficking receptor switch (CCR8/CCR9-->CXCR4-->CCR7), generating mostly homogeneous CD62L(+)CCR7(+)CXCR4(low)FoxP3(+) T cells. CXCR4 expression is regained in FoxP3(+) thymic emigrants in the periphery. Consistent with this switch, recent FoxP3(+) thymic emigrants migrate exclusively to secondary lymphoid tissues but poorly to nonlymphoid tissues. The FoxP3(+) thymic emigrants undergo the second switch in trafficking receptors for migration to nonlymphoid tissues upon Ag priming. This second switch involves down-regulation of CCR7 and CXCR4 but up-regulation of a number of memory/effector type homing receptors, resulting in generation of heterogeneous FoxP3(+) T cell subsets expressing various combinations of trafficking receptors including CCR2, CCR4, CCR6, CCR8, and CCR9. A notable difference between the FoxP3(+) and FoxP3(-) T cell populations is that FoxP3(+) T cells undergo the second homing receptor switch at a highly accelerated rate compared with FoxP3(-) T cells, generating FoxP3(+) T cells with unconventionally efficient migratory capacity to major nonlymphoid tissues.

Overexpression of the coactivator bridge-1 results in insulin deficiency and diabetes.

Multiple forms of heritable diabetes are associated with mutations in transcription factors that regulate insulin gene transcription and the development and maintenance of pancreatic beta-cell mass. The coactivator Bridge-1 (PSMD9) regulates the transcriptional activation of glucose-responsive enhancers in the insulin gene in a dose-dependent manner via PDZ domain-mediated interactions with E2A transcription factors. Here we report that the pancreatic overexpression of Bridge-1 in transgenic mice reduces insulin gene expression and results in insulin deficiency and severe diabetes. Dysregulation of Bridge-1 signaling increases pancreatic apoptosis with a reduction in the number of insulin-expressing pancreatic beta-cells and an expansion of the complement of glucagon-expressing pancreatic alpha-cells in pancreatic islets. Increased expression of Bridge-1 alters pancreatic islet, acinar, and ductal architecture and disrupts the boundaries between endocrine and exocrine cellular compartments in young adult but not neonatal mice, suggesting that signals transduced through this coactivator may influence postnatal pancreatic islet morphogenesis. Signals mediated through the coactivator Bridge-1 may regulate both glucose homeostasis and pancreatic beta-cell survival. We propose that coactivator dysfunction in pancreatic beta-cells can limit insulin production and contribute to the pathogenesis of diabetes.

Interactions with p300 enhance transcriptional activation by the PDZ-domain coactivator Bridge-1.

Transcriptional coactivators are essential mediators of signal amplification in the regulation of gene expression in response to hormones and extracellular signals. We previously identified Bridge-1 (PSMD9) as a PDZ-domain coregulator that augments insulin gene transcription via interactions with the basic helix-loop-helix transcription factors E12 and E47, and that increases transcriptional activation by the homeodomain transcription factor PDX-1. In these studies, we find that transcriptional activation by Bridge-1 can be regulated via interactions with the histone acetyltransferase and nuclear receptor coactivator p300. In transfection assays, transcriptional activation by Bridge-1 is increased by the inhibition of endogenous histone deacetylase activity with trichostatin A, indicating that the transcriptional activation function of Bridge-1 can be regulated by histone modifications. The exogenous expression of p300 enhances the transcriptional activation by Bridge-1 in a dose-dependent manner. In contrast, the sequestration of p300 by the overexpression of the adenoviral protein E1A, but not by an E1A mutant protein that is unable to interact with p300, suppresses the transcriptional activation by Bridge-1. We demonstrate that p300 and Bridge-1 proteins interact in immunopre-cipitation and glutathione-S-transferase (GST) pull-down assays. Bridge-1 interacts directly with multiple regions within p300 that encompass C/H1 or C/H2 cysteine- and histidine-rich protein interaction domains and the histone acetyltransferase domain. Deletion or point mutagenesis of the Bridge-1 PDZ domain substantially reduces transcriptional activation by Bridge-1 and interrupts interactions with p300. We propose that p300 interactions with Bridge-1 can augment the transcriptional activation of regulatory target genes by Bridge-1.

H3N2 influenza virus transmission from swine to turkeys, United States.

In 1998, a novel H3N2 reassortant virus emerged in the United States swine population. We report the interspecies transmission of this virus to turkeys in two geographically distant farms in the United States in 2003. This event is of concern, considering the reassortment capacity of this virus and the susceptibility of turkey to infection by avian influenza viruses. Two H3N2 isolates, A/turkey/NC/16108/03 and A/turkey/MN/764/03, had 98.0% to 99.9% nucleotide sequence identity to each other in all eight gene segments. All protein components of the turkey isolates had 97% to 98% sequence identity to swine H3N2 viruses, thus demonstrating interspecies transmission from pigs to turkeys. The turkey isolates were better adapted to avian hosts than were their closest swine counterparts, which suggests that the viruses had already begun to evolve in the new host. The isolation of swine-like H3N2 influenza viruses from turkeys raises new concerns for the generation of novel viruses that could affect humans.