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

<div><p>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 <i>Trichuris muris</i> 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.</p></div

Drak2 Does Not Regulate TGF-β Signaling in T Cells.

Drak2 is a serine/threonine kinase expressed highest in T cells and B cells. Drak2-/- mice are resistant to autoimmunity in mouse models of type 1 diabetes and multiple sclerosis. Resistance to these diseases occurs, in part, because Drak2 is required for the survival of autoreactive T cells that induce disease. However, the molecular mechanisms by which Drak2 affects T cell survival and autoimmunity are not known. A recent report demonstrated that Drak2 negatively regulated transforming growth factor-β (TGF-β) signaling in tumor cell lines. Thus, increased TGF-β signaling in the absence of Drak2 may contribute to the resistance to autoimmunity in Drak2-/- mice. Therefore, we examined if Drak2 functioned as a negative regulator of TGF-β signaling in T cells, and whether the enhanced susceptibility to death of Drak2-/- T cells was due to augmented TGF-β signaling. Using several in vitro assays to test TGF-β signaling and T cell function, we found that activation of Smad2 and Smad3, which are downstream of the TGF-β receptor, was similar between wildtype and Drak2-/- T cells. Furthermore, TGF-β-mediated effects on naïve T cell proliferation, activated CD8+ T cell survival, and regulatory T cell induction was similar between wildtype and Drak2-/- T cells. Finally, the increased susceptibility to death in the absence of Drak2 was not due to enhanced TGF-β signaling. Together, these data suggest that Drak2 does not function as a negative regulator of TGF-β signaling in primary T cells stimulated in vitro. It is important to investigate and discern potential molecular mechanisms by which Drak2 functions in order to better understand the etiology of autoimmune diseases, as well as to validate the use of Drak2 as a target for therapeutic treatment of these diseases

Smad2 translocation is not enhanced in <i>Drak2-/-</i> T cells compared to wildtype T cells.

<p>Wildtype and <i>Drak2-/-</i> CD4⁺ cells were negatively selected with Miltenyi magnetic beads and stimulated on anti-CD3-coated coverglass slides along with soluble anti-CD28 for 24 hours. Half of the cells were treated with TGF-β for the final 20 minutes of culture. Cells were fixed, permeabilized, and stained with DAPI, Phalloidin, and anti-Smad2. Images were collected via confocal microscopy. n = 2 mice per group. Data are representative of two independent experiments.</p

Enhanced susceptibility to death of <i>Drak2-/-</i> T cells compared to wildtype T cells is independent of TGF-β signaling <i>in vitro</i>.

<p>A) CD4⁺CD25^-CD44^lo or B) CD8⁺CD25^-CD44^lo naïve cells were purified from wildtype, <i>Drak2-/-</i>, <i>DNRII</i>, and <i>DNRII</i>. <i>Drak2-/-</i> mice and stimulated with anti-CD3 and anti-CD28 for 2–3 days. The percent of nonviable CD4⁺ or CD8⁺ T cells is shown. Cells were obtained from one mouse per group and tested in quadruplicate. Data are representative of four separate experiments. <i>**P < 0</i>.<i>01</i>, <i>***P < 0</i>.<i>001</i> (Mann-Whitney <i>U</i>-test).</p

Smad2 and Smad2/3 complex phosphorylation is not enhanced in <i>Drak2-/-</i> T cells compared to wildtype T cells.

<p>A) Wildtype and <i>Drak2-/-</i> splenocytes, and FACS sorted naïve CD4⁺ and CD8⁺ T cells were stimulated for 2 hours with anti-CD3 and anti-CD28, with or without 2 ng/ml TGF-β for one additional hour. Cells were lysed and analyzed by western blot with antibodies specific for Smad2, phosphorylated Smad2, and HSP90 as a loading control. Cells were pooled from 9 wildtype and 8 <i>Drak2-/-</i> mice. Data are representative of two independent experiments. B) Wildtype and <i>Drak2-/-</i> splenocytes were stimulated for 2 hours with anti-CD3 and anti-CD28 with or without increasing concentrations of TGF-β for one additional hour. The cells were harvested, stained with antibodies specific for CD4, CD8, and pSmad2/3, and analyzed by flow cytometry. The average mean fluorescence intensity (MFI) of pSmad2/3 expression is shown for 3 mice per group. There was no significant difference in the response of the wildtype and <i>Drak2-/-</i> cells according to the Mann-Whitney <i>U</i>-test. Data are representative of 3 independent experiments.</p

TGF-β-mediated regulatory T cell induction is not altered in the absence of <i>Drak2</i>.

<p>A) CD4⁺CD25^-CD44^lo naïve cells were purified from wildtype and <i>Drak2-/-</i> mice and stimulated with 1μg/ml anti-CD3 and 1μg/ml anti-CD28 with 20ng/ml IL-2 alone or plus 10-fold TGF-β titrations for 3 days. The A) percent and B) number of Foxp3⁺ cells of electronically gated CD4⁺ cells is shown. There was no significant difference in the response of the wildtype and <i>Drak2-/-</i> cells according to the Mann-Whitney <i>U</i>-test.</p

TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and <i>Drak2-/-</i> T cells.

<p>A) CD4⁺CD25^-CD44^lo naïve cells were purified from <i>OT-II</i> and <i>OT-II</i>.<i>Drak2-/-</i> mice and stimulated with irradiated splenocytes loaded with 10μM OVA₃₂₃ peptide in the presence or absence of 10-fold TGF-β titrations for three days. The number of live, divided Foxp3^-CD4⁺ cells are shown for each titration. Cells were obtained from one <i>OT-II</i> or <i>OT-II</i>.<i>Drak2-/-</i> mouse and tested in quadruplicate. Data are representative of five separate experiments. B) CD8⁺CD25^-CD44^loCD62L^hi naïve cells were purified from <i>OT-I</i> and <i>OT-I</i>.<i>Drak2-/-</i> mice and stimulated with splenocytes loaded with 100pM OVA₂₅₇ peptide in the presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow cytometry. The number of live, divided CD8⁺ cells are shown for each titration. Cells were obtained from one <i>OT-I</i> or <i>OT-I</i>.<i>Drak2-/-</i> mouse and tested in quadruplicate. Data are representative of three separate experiments. There was no significant difference in the response of the wildtype and <i>Drak2-/-</i> cells according to the Mann-Whitney <i>U</i>-test.</p

TGF-β-mediated responses to opposing cytokines are comparable between wildtype and <i>Drak2-/-</i> T cells.

<p>CD8⁺CD25^-CD44^loCD62L^hi naïve cells were purified from <i>OT-I</i> and <i>OT-I</i>.<i>Drak2-/-</i> mice and stimulated with 100nM OVA₂₅₇–pulsed splenocytes for 2 days. Cells were harvested and replated at equal numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells were harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8⁺ cells and B) percent Annexin V⁺ of CD8⁺ cells are shown for each cytokine condition. Cells were obtained from one <i>OT-I</i> or <i>OT-I</i>.<i>Drak2-/-</i> mouse and tested in quadruplicate. Data are representative of two independent experiments. <i>*P < 0</i>.<i>05</i> (Mann-Whitney <i>U-</i>test).</p