MKP-2^−/− and MKP-2^+/+ splenocyte IFN-γ production was similar through the acute and chronic stages of infection.

<p>Splenocytes from <i>T. gondii</i> infected mice were stimulated with TLA (5 µg/ml) and the supernatants assessed for IFN-γ by ELISA. Each value represents the mean of four animals per experimental group ± SEM. All experiments were carried out on at least two occasions.</p

MKP-2 deletion does not impair T cell responses during infection with <i>T. gondii</i>.

<p>T cell responses were determined by flow cytometry. Cells were surface-stained for CD3, CD4 and CD8 and intracellular for IFN-γ and TNF-α. Live cells were gated on forward (FSC) versus side scatter (SSC). T cells were first gated for CD3 and then sub-gated for either CD4 or CD8 and subsequently their respective antigen-specific intracellular cytokine production, following stimulation with TLA (10 µg/ml). The specificity of the intracellular staining was ensured by analysing the respective isotype controls and normalizing samples accordingly (A). Populations of CD3⁺ CD4⁺ T cells (B) and CD3⁺ CD8⁺ T cells (C) single or double positive for IFN-γ and TNF-α were determined using FlowJo software. Each value represents the mean of four animals per experimental group ± SEM. All experiments were carried out on at least two occasions.</p

Systemic serum nitrite levels are reduced and tissue arginase-1 expression is enhanced in MKP-2^−/− compared with MKP-2^+/+ mice infected with <i>T. gondii</i>.

<p>Serum from infected animals was assessed for its nitrite content by Griess assay. Each value represents the mean from ten animals per experimental group ± SEM (A). Splenocyte cell lysates were prepared and assayed for Arginase-1 by western blot. Cells were lysed in sample buffer and protein concentrations measured for each mouse. Equal amounts/animal were pooled and 20 µg loaded for each lane (B). **P<0.005. All experiments were carried out on at least two occasions.</p

NO inhibition by L-NAME enhances susceptibility of MKP-2^+/+ but not MKP-2^−/− mice to <i>T. gondii</i> infection.

<p>Mice were pre-treated with L-NAME and subsequently treated with L-NAME (100 mg per mouse) daily following infection with 20,000 Prugniaud tachyzoites expressing firefly luciferase. Mortality was measured over 12 days (A). Mice were imaged every second day. (B) Represents day 8 post-infection. The parasite burden was determined by measuring the total flux (photons/second) for each group (C). Each value represents the mean of five mice per group ± SEM. *P<0.05. All experiments were carried out on at least two occasions.</p

Arginase-1 inhibition by nor-NOHA enhances susceptibility of MKP-2^−/− but not MKP-2^+/+ mice to <i>T. gondii</i> infection.

<p>Mice were pre-treated with nor-NOHA (200 mg/kg) and treated daily following infection with 20,000 FLUC Prugniaud tachyzoites (A). Mice were imaged every second day post-infection. (B) Represents day 8 post-infection. Total flux (photons/second) was determined for each animal to determine parasite burden (C). Each value represents the mean of five mice per group ± SEM. *P<0.05. All experiments were carried out on at least two occasions.</p

MKP-2 deficient macrophages display deficient iNOS activity but do not display an increased susceptibility to infection.

<p>BMD macrophages were treated as appropriate with L-NAME, nor-NOHA, LPS and IFN-γ and subsequently infected with Prugniaud tachyzoites expressing YFP. After 24 and 72 h parasite burdens was determined by measuring YFP fluorescence (A). Supernatants from cultures were assayed for nitrite content by Griess assay (B). Each value represents four replicates ± SEM. ***P<0.005. All experiments were carried out on at least two occasions.</p

MKP-2 deficient macrophages display enhanced arginase-1 expression and activity.

<p>BMD macrophage cell lysates were examined for arginase-1 expression following infection (A). BMD macrophages were treated as appropriate with L-NAME, nor-NOHA, LPS and IFN-γ and subsequently infected with Prugniaud tachyzoites expressing YFP (B). At 24 hours post-infection supernatants were assayed for arginase-1 activity as described in Methods. Each value represents four replicates ± SEM. **P<0.01 ***P<0.005.</p

MAP Kinase Phosphatase-2 Plays a Key Role in the Control of Infection with <i>Toxoplasma gondii</i> by Modulating iNOS and Arginase-1 Activities in Mice

<div><p>The dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2) has recently been demonstrated to negatively regulate macrophage arginase-1 expression, while at the same time to positively regulate iNOS expression. Consequently, MKP-2 is likely to play a significant role in the host interplay with intracellular pathogens. Here we demonstrate that MKP-2^−/− mice on the C57BL/6 background have enhanced susceptibility compared with wild-type counterparts following infection with type-2 strains of <i>Toxoplasma gondii</i> as measured by increased parasite multiplication during acute infection, increased mortality from day 12 post-infection onwards and increased parasite burdens in the brain, day 30 post-infection. MKP-2^−/− mice did not, however, demonstrate defective type-1 responses compared with MKP-2^+/+ mice following infection although they did display significantly reduced serum nitrite levels and enhanced tissue arginase-1 expression. Early resistance to <i>T. gondii</i> in MKP-2^+/+, but not MKP-2^−/−, mice was nitric oxide (NO) dependent as infected MKP-2^+/+, but not MKP-2^−/− mice succumbed within 10 days post-infection with increased parasite burdens following treatment with the iNOS inhibitor L-NAME. Conversely, treatment of infected MKP-2^−/− but not MKP-2^+/+ mice with nor-NOHA increased parasite burdens indicating a protective role for arginase-1 in MKP-2^−/− mice. In vitro studies using tachyzoite-infected bone marrow derived macrophages and selective inhibition of arginase-1 and iNOS activities confirmed that both iNOS and arginase-1 contributed to inhibiting parasite replication. However, the effects of arginase-1 were transient and ultimately the role of iNOS was paramount in facilitating long-term inhibition of parasite multiplication within macrophages.</p></div