Myc-induced nuclear antigen constrains a latent intestinal epithelial cell-intrinsic anthelmintic pathway.

Expulsion of parasitic gastrointestinal nematodes requires diverse effector mechanisms coordinated by a Th2-type response. The evolutionarily conserved JmjC protein; Myc Induced Nuclear Antigen (Mina) has been shown to repress IL4, a key Th2 cytokine, suggesting Mina may negatively regulate nematode expulsion. Here we report that expulsion of the parasitic nematode Trichuris muris was indeed accelerated in Mina deficient mice. Unexpectedly, this was associated not with an elevated Th2- but rather an impaired Th1-type response. Further reciprocal bone marrow chimera and conditional KO experiments demonstrated that retarded parasite expulsion and a normal Th1-type response both required Mina in intestinal epithelial cells (IECs). Transcriptional profiling experiments in IECs revealed anti-microbial α-defensin peptides to be the major target of Mina-dependent retention of worms in infected mice. In vitro exposure to recombinant α-defensin peptides caused cytotoxic damage to whipworms. These results identify a latent IEC-intrinsic anthelmintic pathway actively constrained by Mina and point to α-defensins as important effectors that together with Mina may be attractive therapeutic targets for the control of nematode infection.

A SNP uncoupling Mina expression from the TGFß signaling pathway.

INTRODUCTION: Mina is a JmjC family 2-oxoglutarate oxygenase with pleiotropic roles in cell proliferation, cancer, T cell differentiation, pulmonary inflammation, and intestinal parasite expulsion. Although Mina expression varies according to cell-type, developmental stage and activation state, its transcriptional regulation is poorly understood. Across inbred mouse strains, Mina protein level exhibits a bimodal distribution, correlating with inheritance of a biallelic haplotype block comprising 21 promoter/intron 1-region SNPs. We previously showed that heritable differences in Mina protein level are transcriptionally regulated. METHODS: Accordingly, we decided to test the hypothesis that at least one of the promoter/intron 1-region SNPs perturbs a Mina cis-regulatory element (CRE). Here, we have comprehensively scanned for CREs across a Mina locus-spanning 26-kilobase genomic interval. RESULTS: We discovered 8 potential CREs and functionally validated 4 of these, the strongest of which (E2), residing in intron 1, contained a SNP whose BALB/c-but not C57Bl/6 allele-abolished both Smad3 binding and transforming growth factor beta (TGFß) responsiveness. CONCLUSIONS: Our results demonstrate the TGFß signaling pathway plays a critical role in regulating Mina expression and SNP rs4191790 controls heritable variation in Mina expression level, raising important questions regarding the evolution of an allele that uncouples Mina expression from the TGFß signaling pathway.

An NLRP3 inflammasome-triggered Th2-biased adaptive immune response promotes leishmaniasis.

Leishmaniasis is a major tropical disease that can present with cutaneous, mucocutaneous, or visceral manifestation and affects millions of individuals, causing substantial morbidity and mortality in third-world countries. The development of a Th1-adaptive immune response is associated with resistance to developing Leishmania major (L. major) infection. Inflammasomes are key components of the innate immune system that contribute to host defense against bacterial and viral pathogens; however, their role in regulating adaptive immunity during infection with protozoan parasites is less studied. Here, we demonstrated that the NLRP3 inflammasome balances Th1/Th2 responses during leishmaniasis. Mice lacking the inflammasome components NLRP3, ASC, or caspase 1 on a Leishmania-susceptible BALB/c background exhibited defective IL-1ß and IL-18 production at the infection site and were resistant to cutaneous L. major infection. Moreover, we determined that production of IL-18 propagates disease in susceptible BALB/c mice by promoting the Th2 cytokine IL-4, and neutralization of IL-18 in these animals reduced L. major titers and footpad swelling. In conclusion, our results indicate that activation of the NLRP3 inflammasome is detrimental during leishmaniasis and suggest that IL-18 neutralization has potential as a therapeutic strategy to treat leishmaniasis patients.

Mina: a Th2 response regulator meets TGFß.

The JmjC protein Mina is an important immune response regulator. Classical forward genetics first discovered its immune role in 2009 in connection with the development of T helper 2 (Th2) cells. This prompted investigation into Mina's role in the two best-studied contexts where Th2 responses are essential: atopic asthma and helminth expulsion. In work focused on a mouse model of atopic asthma, Mina deficiency was found to ameliorate airway hyper-resistance and pulmonary inflammation. And, in a case-control study genetic variation at the human MINA locus was found to be associated with the development of childhood atopic asthma. Although the underlying cellular and molecular mechanism of Mina's involvement in pulmonary inflammation remains unknown, our recent work on parasitic helminth expulsion suggests the possibility that, rather than T cells, epithelial cells responding to TGFß may play the dominant role. Here we review the growing body of literature on the emerging Mina pathway in T cells and epithelial cells and attempt to set these into a broader context.

Transcriptional activation of Mina by Sp1/3 factors.

Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5' region. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays--reporter, gel shift and chromatin immunoprecipitation--to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.

Dynamic regulatory network controlling TH17 cell differentiation.

Despite their importance, the molecular circuits that control the differentiation of naive T cells remain largely unknown. Recent studies that reconstructed regulatory networks in mammalian cells have focused on short-term responses and relied on perturbation-based approaches that cannot be readily applied to primary T cells. Here we combine transcriptional profiling at high temporal resolution, novel computational algorithms, and innovative nanowire-based perturbation tools to systematically derive and experimentally validate a model of the dynamic regulatory network that controls the differentiation of mouse TH17 cells, a proinflammatory T-cell subset that has been implicated in the pathogenesis of multiple autoimmune diseases. The TH17 transcriptional network consists of two self-reinforcing, but mutually antagonistic, modules, with 12 novel regulators, the coupled action of which may be essential for maintaining the balance between TH17 and other CD4(+) T-cell subsets. Our study identifies and validates 39 regulatory factors, embeds them within a comprehensive temporal network and reveals its organizational principles; it also highlights novel drug targets for controlling TH17 cell differentiation.

Enhanced susceptibility of Ago1/3 double-null mice to influenza A virus infection.

RNA interference (RNAi) is a critical component of many cellular antiviral responses in plants, invertebrates, and mammals. However, its in vivo role in host protection from the negative-sense RNA virus influenza virus type A (flu) is unclear. Here we have examined the role of RNAi in host defense to flu by analyzing Argonaute 1 and 3 double-knockout mice deficient in components of the RNA-induced silencing complex. Compared to littermate controls, flu-infected double-knockout mice exhibited increased mortality, consistent with more severe alveolitis and pneumonitis. These data indicate that optimal resistance to flu requires Argonaute 1 and/or 3. Enhanced mortality of double-knockout mice was not associated either with increased viral replication or with differential pulmonary recruitment or function of innate and adaptive immune cells. Given the absence of detectable immune defects, our results support the notion that the enhanced flu susceptibility of double-knockout mice arises from an intrinsic impairment in the ability of lung cells to tolerate flu-elicited inflammation.

Evolution of IL4 and pathogen antagonism.

Interleukin-4 (IL4) is a pleiotropic cytokine involved in host protection from gastrointestinal nematodes. Here, we review the structure, function, and evolutionary history of IL4. Cumulative evidence indicates that over 100 million years of eutherian mammalian evolution, IL4 has experienced multiple episodes of positive selection. We argue that IL4 may have evolved in conflict with pathogen-derived antagonists, and therefore diversified to escape antagonism while being constrained to maintain binding to its cellular receptors. Selective pressure driving IL4 diversification may have arisen from ancient episodes of conflict with parasitic worm-derived IL4 antagonists. Descendants of such antagonists may still equip the armamentarium of contemporary gastrointestinal nematodes.

Diversifying selection and functional analysis of interleukin-4 suggests antagonism-driven evolution at receptor-binding interfaces.

BACKGROUND: Interleukin-4 (IL4) is a secreted immunoregulatory cytokine critically involved in host protection from parasitic helminths 1. Reasoning that helminths may have evolved mechanisms to antagonize IL4 to maximize their dispersal, we explored mammalian IL4 evolution. RESULTS: This analysis revealed evidence of diversifying selection at 15 residues, clustered in epitopes responsible for IL4 binding to its Type I and Type II receptors. Such a striking signature of selective pressure suggested either recurrent episodes of pathogen antagonism or ligand/receptor co-evolution. To test the latter possibility, we performed detailed functional analysis of IL4 allotypes expressed by Mus musculus musculus and Mus musculus castaneus, which happen to differ at 5 residues (including three at positively selected sites) in and adjacent to the site 1 epitope that binds the IL4Ralpha subunit shared by the Type I and Type II IL4 receptors. We show that this intra-species variation affects the ability of IL4 neither to bind IL4 receptor alpha (IL4Ralpha) nor to signal biological responses through its Type I receptor. CONCLUSIONS: Our results - reminiscent of clustered positively selected sites revealing functionally important residues at host-virus interaction interfaces - are consistent with IL4 having evolved to avoid recurrent pathogen antagonism, while maintaining the capacity to bind and signal through its cognate receptor. This work exposes what may be a general feature of evolutionary conflicts fought by pathogen antagonists at host protein-protein interaction interfaces involved in immune signaling: the emergence of receptor-binding ligand epitopes capable of buffering amino acid variation.

Cutting edge: Dab2 is a FOXP3 target gene required for regulatory T cell function.

FOXP3-expressing regulatory T (Treg) cells are vital for maintaining peripheral T cell tolerance and homeostasis. The mechanisms by which FOXP3 target genes orchestrate context-dependent Treg cell function are largely unknown. In this study we show that in mouse peripheral lymphocytes the Drosophila Disabled-2 (Dab2) homolog, a gene that is involved in enhancing TGFbeta responses, is exclusively expressed in FOXP3+ regulatory T cells. Dab2 is a direct target of FOXP3, and regulatory T cells lacking DAB2 are functionally impaired in vitro and in vivo. However, not all aspects of Treg cell function are perturbed, and DAB2 appears to be dispensable for Treg cell function in maintaining naive T cell homeostasis.

Mina, an Il4 repressor, controls T helper type 2 bias.

T helper type 2 (T(H)2) bias, which is the propensity of naive CD4(+) T cells to differentiate into interleukin 4 (IL-4)-secreting T(H)2 cells, is a genetic trait that affects susceptibility to infectious, autoimmune and allergic diseases. T(H)2 bias correlates with the amount of IL-4 initially secreted by newly activated helper T cells that feeds back positively through the pathway of the IL-4 receptor and the transcription factors STAT6 and GATA-3 to drive T(H)2 development. Here we identify Mina, a member of the jumonji C (JmjC) protein family, as a genetic determinant of T(H)2 bias. Mina specifically bound to and repressed the Il4 promoter. Mina overexpression in transgenic mice impaired Il4 expression, whereas its knockdown in primary CD4(+) T cells led to Il4 derepression. Our findings collectively provide mechanistic insight into an Il4-regulatory pathway that controls helper T cell differentiation and genetic variation in T(H)2 bias.

Intergenic transcription is not required in Th2 cells to maintain histone acetylation and transcriptional permissiveness at the Il4-Il13 locus.

Noncoding RNA transcripts mapping to intergenic regions of the Il4-Il13 locus have been detected in Th2 cells harboring transcriptionally permissive Il4 and Il13 genes but not in Th1 cells where these genes are repressed. This correlation has given rise to the idea that intergenic transcription may be involved in maintaining the "open" chromatin structure of the Il4-Il13 locus in Th2 cells. We present evidence from real-time RT-PCR, nuclear run on, chromatin immunoprecipitation and 5,6-dichlorobenzimidazole 1-beta-D-ribofuranoside-mediated transcriptional inhibition analyses that argue against this hypothesis. Instead, our results are consistent with an alternative role for intergenic transcription in the maintenance of transcriptional silence in Th1-primed cells.

EZH2 and histone 3 trimethyl lysine 27 associated with Il4 and Il13 gene silencing in Th1 cells.

Differentiation of naïve CD4 T cells toward the T helper 1 (T(H)1) and T helper 2 (T(H)2) fates involves the transcriptional repression and enhancement, respectively, of Il4 and Il13, adjacent chromosome 11 genes encoding the canonical T(H)2 cytokines interleukin-4 and interleukin-13. Proper execution of this developmental fate choice during immune responses is critical to host defense and, when misregulated, leads to susceptibility to infectious microbes and to allergic and autoimmune diseases. Here, using chromatin immunoprecipitation and real time reverse transcription PCR we identify the Polycomb family histone methyltransferase EZH2 as the enzyme responsible for methylating lysine 27 of histone H3 at the Il4-Il13 locus of T(H)1 but not T(H)2 cells, implicating EZH2 in the mechanism of Il4 and Il13 transcriptional silencing.

A Leishmania major response locus identified by interval-specific congenic mapping of a T helper type 2 cell bias-controlling quantitative trait locus.

The propensity of naive CD4 T cells to become T helper (Th) type 2 cells correlates with susceptibility to infection by the protozoal parasite Leishmania major. Using genetic linkage analysis, we earlier identified Dice1 as a Th2 cell bias-controlling quantitative trait locus on chromosome 16. Using interval-specific congenic mapping, we now resolve Dice1 into two independent genetic loci, Dice1.1 and Dice1.2, which control Il4 expression from naive Th cells and thereby indirectly control Th2 cell bias. Interestingly, only one of the two congenic intervals containing Dice1.1 and Dice1.2, respectively, also contained an L. major response locus, indicating that L. major responsiveness can be insensitive to determinants that influence Th2 cell bias by controlling naive T cell Il4 expression. These results lay the groundwork for identifying the Dice1.1 and Dice1.2 genes controlling naive T cell Il4 expression and L. major responses, and for testing whether these control other Th2 cell-dependent processes such as worm expulsion, allergic asthma, and dermatitis.

Chromatin landscape dynamics of the Il4-Il13 locus during T helper 1 and 2 development.

Il4 and Il13 encode the canonical T helper 2 (TH2) cytokines responsible both for promoting immune responses against extracellular pathogens and, when misregulated, causing allergic and autoimmune disease. The expression potential of these genes undergoes developmentally programmed repression and enhancement during commitment of naïve CD4+ T cells to the mature T helper 1 (TH1) and TH2 fates, respectively. Thus, like the globin locus, the TH2 cytokine locus provides a highly tractable system to study a developmental fate choice leading to alternative transcriptional states of either silence or permissivity. We used quantitative chromatin immunoprecipitation and RT-PCR to correlate changes in the transcriptional states of Il4 and Il13 with markers of permissive chromatin across the Il4-Il13 locus in naïve CD4+ T cells undergoing TH1 and TH2 differentiation. We provide evidence that DNaseI hypersensitive site V in the Il4 3' enhancer is the likely target for signals maintaining Il4 and Il13 transcriptional permissivity in naïve cells. We also demonstrate rapid acquisition of differences in H3 acetylation between TH1- and TH2-primed cells, indicating a developmentally early role for cytokine signaling in the process of TH cell fate determination. Finally, we show that transcriptional repression correlates with the disappearance of permissive H3 modifications from everywhere in the Il4-Il13 locus except hypersensitive site IV, suggesting a critical role for this element in the maintenance of transcriptional repression. Our findings are consistent with a progressive regulatory element activation/deactivation model of TH1/TH2 development.