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.

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Highlights► TLR-4 activation by Leishmania mexicana promastigotes and CPB-deficient amastigotes. ► Prolonged, TLR-4 dependent iNOS and COX-2 expression by L. mexicana promastigotes. ► Enhanced TLR-4 dependent-arginase-1 expression. ► Regulation of IL-12 induction by an arginase-1 dependent mechanism.

Immunologically intact BALB/c mice infected with Leishmania mexicana develop non-healing progressively growing lesions associated with a biased Th2 response while similarly infected IL-4Rα-deficient mice fail to develop lesions and develop a robust Th1 response. In order to determine the functional target(s) for IL-4/IL-13 inducing non-healing disease, the course of L. mexicana infection was monitored in mice lacking IL-4Rα expression in specific cellular compartments. A deficiency of IL-4Rα expression on macrophages/neutrophils (in LysMcreIL-4Rα−/lox animals) had minimal effect on the outcome of L. mexicana infection compared with control (IL-4Rα−/flox) mice. In contrast, CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice infected with L. mexicana developed small lesions, which subsequently healed in female mice, but persisted in adult male mice. While a strong Th1 response was manifest in both male and female CD4+ T cell specific IL-4Rα−/− mice infected with L. mexicana, induction of IL-4 was manifest in males but not females, independently of CD4+ T cell IL-4 responsiveness. Similar results were obtained using pan-T cell specific (iLckcreIL-4Rα−/lox) IL-4Rα−/− mice. Collectively these data demonstrate that upon infection with L. mexicana, initial lesion growth in BALB/c mice is dependent on non-T cell population(s) responsive to IL-4/IL-13 while progressive infection is dependent on CD4+ T cells responsive to IL-4.

Dysfunctional intracellular enzymatic activity is believed to be an underlying cause of a myriad of diseases. We present the first use of surface enhanced Raman scattering (SERS) as a detection technique capable of reporting intracellular activity of a specific enzyme. Careful choice of reagents allowed the preparation of high resolution cellular activity maps highlighting the specific conversion of the commonly used ELISA reagent 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal), by wild type β-galactosidase enzymes. Further, through co-addition of X-Gal substrate and inhibitors we were able to demonstrate that intracellular substrate conversion occurred predominantly through an enzymatically specific pathway. The data presented therefore supports the application of SERS probes as sensitive, specific sensors of biochemical activity and demonstrates the use of SERS probes for the first time as beacons capable of high resolution subcellular localisation of native enzymes.

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 Toxoplasma gondii 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 T. gondii 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.

Leishmaniasis encompasses a large spectrum of clinical diseases which, depending upon the parasite species and the host's immune response, can have various outcomes (16). Our understanding of the factors that lead to this diversity of clinical symptoms has, in large part, come from studies using murine models and particularly Leishmania major. While the majority of mouse strains can control infection with L. major, virtually all develop nonhealing lesions full of parasites when infected cutaneously with L. mexicana (4). Studies with the BALB/c mouse have indicated that the nonhealing response of this mouse strain to infection with L. major is LACK antigen dependent (13), while the nonhealing response to L. mexicana is independent of this antigen (27). Conversely, our studies have implicated cysteine peptidases (CPs) as major virulence factors for L. mexicana (reviewed in reference 17). Information about the apparent functions and importance of CPs in the host-parasite interaction was obtained via the generation of mutants deficient in the CPA and CPB genes (⌬cpa and ⌬cpb mutants). L. mexicana mutants deficient in the multicopy CPB gene array (⌬cpb mutants) have reduced virulence, with poor lesion growth, for BALB/c mice (2, 18). Only the reexpression of multiple CPB genes from a cosmid significantly restored virulence (8), suggesting that the multiple genes have complementary functions. CPA CPB double null mutant parasites (⌬cpa ⌬cpb mutants) were less infective for BALB/c mice than ⌬cpb mutants (2, 18), which not only implicates CPA as a virulence factor but also indicates that there is some redundancy in function between CPA and CPB. CPA, together with CPB1 and CPB2 (the CPB isoforms encoded by the first 2 genes of the 19-gene CPB tandem array), is expressed in metacyclic promastigotes, suggesting roles for these CPs in the virulence of this life cycle stage. Indeed, the L. mexicana ⌬cpa ⌬cpb mutants are defective in metacyclogenesis, due to an impairment of autophagy-dependent protein turnover required for differentiation (28).Small-molecule inhibitors of CPB have some efficacy against Leishmania, both in vitro and in vivo (24). These inhibitors are thought to be active against not only CPB but also CPA and CPC, and their toxicity to Leishmania may be a consequence of the inhibition of multiple enzymes. Nevertheless, these results confirm the importance of CPs in the host-parasite interaction. Treatment with a natural CP inhibitor, cystatin, promoted a protective response against Leishmania infection and a switch from a predominately Th2 to a Th1 response (7). Leishmania lacks cystatins, but the parasite possesses an unusual inhibitor of CPs (designated ICP), which does not occur in mammals (22). Leishmania ICP has been shown to be a potent inhibitor

We have recently demonstrated that MAP kinase phosphatase 2 (MKP-2) deficient C57BL/6 mice, unlike their wild-type counterparts, are unable to control infection with the protozoan parasite Leishmania mexicana. Increased susceptibility was associated with elevated Arginase-1 levels and reduced iNOS activity in macrophages as well as a diminished TH1 response. By contrast, in the present study footpad infection of MKP-2−/− mice with L. major resulted in a healing response as measured by lesion size and parasite numbers similar to infected MKP-2+/+ mice. Analysis of immune responses following infection demonstrated a reduced TH1 response in MKP-2−/− mice with lower parasite specific serum IgG2b levels, a lower frequency of IFN-γ and TNF-α producing CD4+ and CD8+ T cells and lower antigen stimulated spleen cell IFN-γ production than their wild-type counterparts. However, infected MKP-2−/− mice also had similarly reduced levels of antigen induced spleen and lymph node cell IL-4 production compared with MKP-2+/+ mice as well as reduced levels of parasite-specific IgG1 in the serum, indicating a general T cell hypo-responsiveness. Consequently the overall TH1/TH2 balance was unaltered in MKP-2−/− compared with wild-type mice. Although non-stimulated MKP-2−/− macrophages were more permissive to L. major growth than macrophages from MKP-2+/+ mice, reflecting their reduced iNOS and increased Arginase-1 expression, LPS/IFN-γ activation was equally effective at controlling parasite growth in MKP-2−/− and MKP-2+/+ macrophages. Consequently, in the absence of any switch in the TH1/TH2 balance in MKP-2−/− mice, no significant change in disease phenotype was observed.