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The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. GP63 has been found to be involved not only in the cleavage and degradation of various kinases and transcription factors, but also to be the major molecule modulating host negative regulatory mechanisms involving for instance protein tyrosine phosphatases (PTPs). Those latter being well recognized for their pivotal role in the regulation of a great number of signaling pathways. In this review article, we are providing a complete overview about the role of Leishmania GP63 in the mechanisms underlying the subversion of macrophage signaling and functions.

Leishmania parasites have evolved sophisticated mechanisms to subvert macrophage immune responses by altering the host cell signal transduction machinery, including inhibition of JAK/STAT signalling and other transcription factors such as AP-1, CREB and NF-κB. AP-1 regulates pro-inflammatory cytokines, chemokines and nitric oxide production. Herein we show that upon Leishmania infection, AP-1 activity within host cells is abolished and correlates with lower expression of 5 of the 7 AP-1 subunits. Of interest, c-Jun, the central component of AP-1, is cleaved by Leishmania. Furthermore, the cleavage of c-Jun is dependent on the expression and activity of the major Leishmania surface protease GP63. Immunoprecipitation of c-Jun from nuclear extracts showed that GP63 interacts, and cleaves c-Jun at the perinuclear area shortly after infection. Phagocytosis inhibition by cytochalasin D did not block c-Jun down-regulation, suggesting that internalization of the parasite might not be necessary to deliver GP63 molecules inside the host cell. This observation was corroborated by the maintenance of c-Jun cleavage upon incubation with L. mexicana culture supernatant, suggesting that secreted, soluble GP63 could use a phagocytosis-independent mechanism to enter the host cell. In support of this, disruption of macrophage lipid raft microdomains by Methyl β-Cyclodextrin (MβCD) partially inhibits the degradation of full length c-Jun. Together our results indicate a novel role of the surface protease GP63 in the Leishmania-mediated subversion of host AP-1 activity.

While natural CD4(+)Foxp3(+) regulatory T (nT(REG)) cells have long been viewed as a stable and distinct lineage that is committed to suppressive functions in vivo, recent evidence supporting this notion remains highly controversial. We sought to determine whether Foxp3 expression and the nT(REG) cell phenotype are stable in vivo and modulated by the inflammatory microenvironment. Here, we show that Foxp3(+) nT(REG) cells from thymic or peripheral lymphoid organs reveal extensive functional plasticity in vivo. We show that nT(REG) cells readily lose Foxp3 expression, destabilizing their phenotype, in turn, enabling them to reprogram into Th1 and Th17 effector cells. nT(REG) cell reprogramming is a characteristic of the entire Foxp3(+) nT(REG) population and the stable Foxp3(NEG) T(REG) cell phenotype is associated with a methylated foxp3 promoter. The extent of nT(REG) cell reprogramming is modulated by the presence of effector T cell-mediated signals, and occurs independently of variation in IL-2 production in vivo. Moreover, the gut microenvironment or parasitic infection favours the reprogramming of Foxp3(+) T(REG) cells into effector T cells and promotes host immunity. IL-17 is predominantly produced by reprogrammed Foxp3(+) nT(REG) cells, and precedes Foxp3 down-regulation, a process accentuated in mesenteric sites. Lastly, mTOR inhibition with the immunosuppressive drug, rapamycin, stabilizes Foxp3 expression in T(REG) cells and strongly inhibits IL-17 but not RORγt expression in reprogrammed Foxp3(-) T(REG) cells. Overall, inflammatory signals modulate mTOR signalling and influence the stability of the Foxp3(+) nT(REG) cell phenotype.

The myeloid-related proteins (MRPs) 8/14 are small proteins mainly produced by neutrophils, which have been reported to induce NO production in macrophages. On the other hand, Leishmania survives and multiplies within phagocytes by inactivating several of their microbicidal functions. Whereas MRPs are rapidly released during the innate immune response, their role in the regulation of Leishmaniasis is still unknown. In vitro experiments revealed that Leishmania infection alters MRP-induced signaling, leading to inhibition of macrophage functions (NO, TNF-α). In contrast, MRP-primed cells showed normal signaling activation and NO production in response to Leishmania infection. Using a murine air-pouch model, we observed that infection with L. major induced leukocyte recruitment and MRP secretion comparable to LPS-treated mice. Depletion of MRPs significantly reduced these inflammatory events and augmented both parasite load and footpad swelling during the first 8 weeks post-infection, as also observed in MRP KO mice. On the contrary, mouse treatment with recombinant MRPs (rMRPs) had the opposite effect. Collectively, our results suggest that rapid secretion of MRPs by neutrophils at the site of infection may protect uninfected macrophages and favor a more efficient innate inflammatory response against Leishmania infection. In summary, our study reveals the critical role played by MRPs in the regulation of Leishmania infection and how this pathogen can subvert its action.

While natural CD4.sup.+ Foxp3.sup.+ regulatory T (nT.sub.REG) cells have long been viewed as a stable and distinct lineage that is committed to suppressive functions in vivo, recent evidence supporting this notion remains highly controversial. We sought to determine whether Foxp3 expression and the nT.sub.REG cell phenotype are stable in vivo and modulated by the inflammatory microenvironment. Here, we show that Foxp3.sup.+ nT.sub.REG cells from thymic or peripheral lymphoid organs reveal extensive functional plasticity in vivo. We show that nT.sub.REG cells readily lose Foxp3 expression, destabilizing their phenotype, in turn, enabling them to reprogram into Th1 and Th17 effector cells. nT.sub.REG cell reprogramming is a characteristic of the entire Foxp3.sup.+ nT.sub.REG population and the stable Foxp3.sup.NEG T.sub.REG cell phenotype is associated with a methylated foxp3 promoter. The extent of nT.sub.REG cell reprogramming is modulated by the presence of effector T cell-mediated signals, and occurs independently of variation in IL-2 production in vivo. Moreover, the gut microenvironment or parasitic infection favours the reprogramming of Foxp3.sup.+ T.sub.REG cells into effector T cells and promotes host immunity. IL-17 is predominantly produced by reprogrammed Foxp3.sup.+ nT.sub.REG cells, and precedes Foxp3 down-regulation, a process accentuated in mesenteric sites. Lastly, mTOR inhibition with the immunosuppressive drug, rapamycin, stabilizes Foxp3 expression in T.sub.REG cells and strongly inhibits IL-17 but not ROR[gamma]t expression in reprogrammed Foxp3.sup.- T.sub.REG cells. Overall, inflammatory signals modulate mTOR signalling and influence the stability of the Foxp3.sup.+ nT.sub.REG cell phenotype.

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of <49 and ≥20 KDa ( M. hominis ) and <102 and ≥58 KDa ( M. fermentans ) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.

The myeloid-related proteins (MRPs) 8/14 are small proteins mainly produced by neutrophils, which have been reported to induce NO production in macrophages. On the other hand, Leishmania survives and multiplies within phagocytes by inactivating several of their microbicidal functions. Whereas MRPs are rapidly released during the innate immune response, their role in the regulation of Leishmaniasis is still unknown. In vitro experiments revealed that Leishmania infection alters MRP-induced signaling, leading to inhibition of macrophage functions (NO, TNF-α). In contrast, MRP-primed cells showed normal signaling activation and NO production in response to Leishmania infection. Using a murine air-pouch model, we observed that infection with L. major induced leukocyte recruitment and MRP secretion comparable to LPS-treated mice. Depletion of MRPs significantly reduced these inflammatory events and augmented both parasite load and footpad swelling during the first 8 weeks post-infection, as also observed in MRP KO mice. On the contrary, mouse treatment with recombinant MRPs (rMRPs) had the opposite effect. Collectively, our results suggest that rapid secretion of MRPs by neutrophils at the site of infection may protect uninfected macrophages and favor a more efficient innate inflammatory response against Leishmania infection. In summary, our study reveals the critical role played by MRPs in the regulation of Leishmania infection and how this pathogen can subvert its action.

Leishmania parasites have the ability to modify macrophage signaling pathways in order to survive and multiply within its mammalian host. They are also known to invade other cells including neutrophils, fibroblasts and dendritic cells (DCs). DCs have an important role in immunity as the link between innate and adaptive immunity, necessary for the development of an effective response; however, the impact of Leishmania mexicana infection on DCs has been poorly studied. Herein, we report that Leishmania infection rapidly induced DC protein tyrosine phosphatases activity, leading to MAP kinases inactivation. In line with this, L. mexicana was found to decrease the nuclear translocation of transcription factors such as AP-1 and NF-κB. Concomitantly, L. mexicana-infected DCs showed reduced expression of several surface antigen-presenting and co-stimulatory molecules upon LPS stimulation. Leishmania-induced interference on DC maturation was further reflected by their reduced capacity to present OVA antigen to OVA-specific T cells, as shown by abrogation of IL-2 production by the T cells. Collectively, our data revealed that DC infection by L. mexicana appears to affect the cellular and immunological mechanisms necessary for the development of an effective and protective immune response, therefore favouring the survival and propagation of the parasite within its host.

Leishmania parasites have evolved sophisticated mechanisms to subvert macrophage immune responses by altering the host cell signal transduction machinery, including inhibition of JAK/STAT signalling and other transcription factors such as AP-1, CREB and NF-κB. AP-1 regulates pro-inflammatory cytokines, chemokines and nitric oxide production. Herein we show that upon Leishmania infection, AP-1 activity within host cells is abolished and correlates with lower expression of 5 of the 7 AP-1 subunits. Of interest, c-Jun, the central component of AP-1, is cleaved by Leishmania. Furthermore, the cleavage of c-Jun is dependent on the expression and activity of the major Leishmania surface protease GP63. Immunoprecipitation of c-Jun from nuclear extracts showed that GP63 interacts, and cleaves c-Jun at the perinuclear area shortly after infection. Phagocytosis inhibition by cytochalasin D did not block c-Jun down-regulation, suggesting that internalization of the parasite might not be necessary to deliver GP63 molecules inside the host cell. This observation was corroborated by the maintenance of c-Jun cleavage upon incubation with L. mexicana culture supernatant, suggesting that secreted, soluble GP63 could use a phagocytosis-independent mechanism to enter the host cell. In support of this, disruption of macrophage lipid raft microdomains by Methyl β-Cyclodextrin (MβCD) partially inhibits the degradation of full length c-Jun. Together our results indicate a novel role of the surface protease GP63 in the Leishmania-mediated subversion of host AP-1 activity.

Protozoan parasites of Leishmania genus are able to successfully infect their host macrophage due to multiple virulence strategies that result in its deactivation. Recent studies suggest Leishmania GP63 to be a critical virulence factor in modulation of many macrophage molecules, including protein tyrosine phosphatases (PTPs) and transcription factors (TFs). Additionally, we and others recently reported that Leishmania-released exosomes can participate in pathogenesis. Exosomes are 40-100 nm vesicles that are freed by many eukaryotic cells. To better understand the GP63-dependent immune modulation of the macrophage by Leishmania parasites and their exosomes, we compared the immunomodulatory properties of Leishmania major (WT) and L. major gp63-/- (KO) as well as their exosomes in vitro and in vivo. Importantly, we observed that Leishmania exosomes can modulate macrophage PTPs and TFs in a GP63-dependent manner. In addition, our qRT-PCR analyses showed that WT parasites were able to downregulate multiple genes involved in the immune response, especially cytokines and pattern recognition receptors. KO parasites showed a strongly reduced modulatory capacity compared to WT parasites. Furthermore, comparison of WT versus KO exosomes also showed divergences in alteration of gene expression, especially of chemokine receptors. In parallel, studying the in vivo inflammatory recruitment using a murine air pouch model, we found that exosomes have stronger proinflammatory properties than parasites and preferentially induce the recruitment of neutrophils. Finally, comparative proteomics of WT and KO exosomes surprisingly revealed major differences in their protein content, suggesting a role for GP63 in Leishmania exosomal protein sorting. Collectively our data clearly establish the crucial role of GP63 in dampening the innate inflammatory response during early Leishmania infection, and also provides new insights in regard to the role and biology of exosomes in Leishmania host-parasite interactions.