MKP-2 deficient mice infected with <i>L. major</i> have a reduced T_H2 response.

<p>Serum of <i>L. major</i> infected MKP-2^−/− (open column) and MKP-2^+/+ (closed column) mice was analysed for parasite-specific antibody isotype IgG1 (A). The titres were calculated as reciprocal dilution of the half-maximal absorption at 450 nm. In order to measure T_H2 responses, 3 MKP-2^−/−, 3 MKP-2^+/+ and 3 BALB/c mice were given 2×10⁶<i>L. major</i> promastigotes subcutanteously into the hind foot pad. At peak of infection, mice were sacrificed and spleen and popliteal lymph node removed. Spleens were analysed individually and lymph nodes pooled within each group. CD4⁺ T cells were magnet-purified from spleens using a negative isolation kit. Splenocytes (B, C), lymph node cells (D) and purified CD4⁺ T cells (E) were re-stimulated either with antigen pulsed DC, PMA/Ionomycin, or with unpulsed DC (Medium control) in the presence of IL-4 receptor blocking antibody for 72 h and supernatants were analysed immediately for IL-4. Error bars show standard error of the mean (SEM). *<i>P</i><0.05 one-tailed unpaired t test.</p

A sustained T_H1/T_H2 balance in MKP-2 deficient mice is due to general T cell hypo-responsiveness against <i>L. major</i>.

<p>Antibody titres for IgG2b were divided by respective titres for IgG1 for each individual mouse and for both time points. Higher values are indicative for higher IgG2b levels and lower values reflect a higher IgG1 response. The bar indicates the median of each group (A). The percent reduction of the mean for each measured parameter (IFN-γ, IL-4 and IFN-γ/TNF-α⁺ CD4⁺ T cells) of MKP-2^−/− mice was calculated against the mean values of MKP-2^+/+ mice ( = 100%) (B). If both immune responses (T_H1, (IFN-γ/TNF-α⁺ CD4⁺ T cells and IFN-γ) versus T_H2 (IL-4) are reduced to a similar extend (B), the balance between disease promoting T_H2 and the protective T_H1 response is not disturbed. Since the mice are on a C57BL/6 background the T_H1 response will outweigh the T_H2 response and consequently result in a healing phenotype (C).</p

MKP-2 deficient mice infected with <i>L. major</i> have a reduced T_H1 response.

<p>Splenocytes from <i>L. major</i> infected MKP-2^−/− (open columns) and MKP-2^+/+ (closed columns) mice were re-stimulated either with antigen pulsed DC, PMA/Ionomycin, ConA or with unpulsed DC (Medium control) for 6 h (FACS) or 48 h (ELISA). Cells were stained for T cell surface markers CD3, CD4 and CD8 as well as intracellular cytokines IFN-γ and TNF-α or their respective isotypes (A). Percentages of IFN-γ single and IFN-γ/TNF-α double producing parasite-specific CD4⁺ (B) and CD8⁺ (C) T cells are shown after normalization to medium and isotype controls. Supernatants of 48 h T cell re-stimulations were analyzed for IFN-γ in a sandwich ELISA and values were normalized to medium controls (D). Serum of infected mice was collected and analysed for parasite-specific antibody isotype IgG2b. The titres were calculated as reciprocal dilution of the half-maximal absorption at 450 nm (E). Error bars are shown as standard error of the mean (SEM). *<i>P</i><0.05, **<i>P</i><0.01; one-tailed Mann Whitney U test. The data shown is representative of two independent experiments.</p

MKP-2 deficiency does not alter the course of infection with <i>L. major</i>.

<p>Ten MKP-2^−/− (open symbols) and ten MKP-2^+/+ mice (closed symbols) were given with 2×10⁶<i>L. major</i> promastigotes subcutaneously into the left hind foot pad. The right uninfected foot pad was used for reference. Lesion development was monitored by measuring the footpad thickness (A) over time using a dial gauge calliper. At peak of infection (day 42, B) and after the onset of healing (day 88, C) five mice were sacrificed at random and parasite burdens in foot pad, popliteal lymph nodes and spleens were determined using a limiting dilution assay. Detection limit was 65 for spleens and 6500 for lymph node and foot pads. Error bars are shown as standard error of the mean (SEM). The data shown is representative of at least three independent experiments.</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

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

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