MAP Kinase Phosphatase-2 Plays a Critical Role in Response to Infection by Leishmania mexicana

In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2+/+ but not from MKP-2−/− mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2−/− macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE2 production. However surprisingly, in MKP-2−/− macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2−/− mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2−/− T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2−/− bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage

Taz^Neo germ cell differentiation fails before reaching the Pachytene stage of meiosis I.

<p>Sycp-3 immunostaining of nuclear spread from control (left) and Taz deficient P16 spermatocytes assessing the different stages of the Meiosis I prophase I during spermatocytes differentiation (leptotene, zygotene, pachytene). Scale bars: 50<i>μ</i>m. Proportion of cells in various prophase I stages in control or Taz deficient spermatocytes (lower right panels).</p

Leydig and Sertoli cells are not affected by Taz deficiency.

<p>Immunohistology sections of SV129P2 wild type (A,B, G,I) and Taz^<b>Neo</b> testis tubules (B,D, H and J) showing the staining of Leydig cell marker 3<i>β</i>-HSD (Hsd3b6) (A,D), sertoli cell nucleus Wt1 (B,D). Recipient Sertoli cell non-contribution in Taz^<b>Neo</b> testis tubules (E, F): low chimera section showing wild type and defective tubules as stained with Hprt (E) and adjacent section stained for Tubb3 showing sertoli cell cytoplasm (F). Taz^<b>Neo</b> semiferous tubules showed increased cell death. Caspase-3 staining of sections of SV129P2 wild type (G) and Taz^<b>Neo</b> (H) showing the occurrence of cell death (brown nuclear staining) in Taz^<b>Neo</b> seminiferous tubules. Ki67 immunostaining of the control tubule is found restricted to dividing spermatogonia in the basal region of the tubules (I) as Taz^<b>Neo</b> tubules display positive staining through the tubule (J). Scale bar: 250<i>μ</i>m.</p

Taz deficient seminiferous tubules showed increased DNA damage due to higher L1 retrotransposon activity.

<p>A, B: immunostaining of P16 nuclear spread from control (A) or Taz^<b>Neo</b> testis (B) using anti-<i>γ</i>H2ax (red) antibody. Dapi nuclear stain (blue). Scale bar 50<i>μ</i>m. C-J: Immunostaining of adult testis sections of control (C,E,G,I) and Taz^<b>Neo</b> testis (D,F,H,J) with anti-<i>γ</i>H2ax (C-F) or anti-Orf1-Line1 (G-J) antibody. White arrows: sex bodies seen on germ cells reaching Pachytene stage (E). Black arrow heads indicating typical punctuating staining of DNA damage (F). Black arrows: Orf1 Line1 nuclear staining (J). Scale bars: 250<i>μ</i>m.</p

Taz deficient chimeras fail to produce mature sperm.

<p>Left panels: Histology sections of control (A,C,E,G) and Taz^<b>Neo</b> tissue (B,D,F,H) showing the epididymis (A,B), P8 (C,D), P16 (E,F) and adult testis tubule (G,H). Sections through the epididymis show a complete lack of differentiated sperm in the lumen of the Taz^<b>Neo</b> epididymis (B). White arrowhead: multinuclear cells. Black arrows: vacuoles. Scale bars: 250<i>μ</i>m. Right panels (I, J, K) show the high contribution to chimerism of Taz deficient cells in chimera testis: HPRT staining of wild type SV129P2 adult testis (I), of a low level of chimerism testis (J) (defective tubule harbouring numerous vacuoles is negative for HPRT staining*) and of a high level of chimerism testis (K). Scale bar: 250<i>μ</i>m</p

Taz^Neo spermatocytes do not differentiate to the round spermatid stage.

<p>Immunohistology sections of SV129P2 wild type (A,B,E,F,G) and TAZ^<b>Neo</b> testis (C,D,H, I and J) showing the staining of sperm germ cells marker, Dazl (A,C), Primordial germ cell marker, Vasa (B,D), mature germ cell markers Actl7B (E,H), Hook1 (F,I) and Pgk2 (G,J). Double arrows on each of the wild-type sections (A,B,E,F,G) indicate the zone of differentiating spermatozoa absent in the Taz^<b>Neo</b> tubules. Scale bar: 250<i>μ</i>m</p

Taz^Neo ES cells don’t express spermiogenesis markers when differentiated in vitro.

<p>A: Top Panel: Scheme of the introduction of the flox stop Dazl construct at the HPRT locus before and after Cre deletion and recombination at the lox P sites (black arrow head). B: RT-PCR of Taz and various sperm differentiation markers (Prm1, Tnp2, Dmc-1, Sycp-3, Sycp-1) with cDNA extracted from HM1 parental ES clones or Taz^<b>Neo</b> ES cells before or after 19 days differentiation. C: Western blotting of wild type or Taz deficient ES cell clone protein extract with or without 19 days differentiation for Dazl, Sycp-3 or Vasa. <i>β</i>-actin is used to show equal loading.</p