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Leishmaniasis is a disease caused by the protozoan parasite Leishmania and is known to affect millions of individuals worldwide. In recent years, we have established the critical role played by Leishmania zinc-metalloprotease GP63 in the modulation of host macrophage signalling and functions, favouring its survival and progression within its host. Leishmania major lacking GP63 was reported to cause limited infection in mice, however, it is still unclear how GP63 may influence the innate inflammatory response and parasite survival in an in vivo context. Therefore, we were interested in analyzing the early innate inflammatory events upon Leishmania inoculation within mice and establish whether Leishmania GP63 influences this initial inflammatory response. Experimentally, L . major WT ( L . major WT ), L . major GP63 knockout ( L . major KO ), or L . major GP63 rescue ( L . major R ) were intraperitoneally inoculated in mice and the inflammatory cells recruited were characterized microscopically and by flow cytometry (number and cell type), and their infection determined. Pro-inflammatory markers such as cytokines, chemokines, and extracellular vesicles (EVs, e.g. exosomes) were monitored and proteomic analysis was performed on exosome contents. Data obtained from this study suggest that Leishmania GP63 does not significantly influence the pathogen-induced inflammatory cell recruitment, but rather their activation status and effector function. Concordantly, internalization of promastigotes during early infection could be influenced by GP63 as fewer L . major KO amastigotes were found within host cells and appear to maintain in host cells over time. Collectively this study provides a clear analysis of innate inflammatory events occurring during L . major infection and further establish the prominent role of the virulence factor GP63 to provide favourable conditions for host cell infection.

Leishmaniasis is a disease caused by the protozoan parasite Leishmania known to affect millions of individuals worldwide. In recent years, we have established the critical role played by Leishmania zinc-metalloprotease GP63 (GP63) in the modulation of host macrophage signalling and functions. In an immunological context, it favours survival and progression of the parasite within its host. Whereas Leishmania major knock out for GP63 caused limited infection in mice, it is still unclear how GP63 may influence the innate inflammatory response and parasite survival in an in vivo context. Therefore, we were interested in analyzing the early innate inflammatory events upon Leishmania inoculation within mice and establish whether Leishmania GP63 influences this initial inflammatory response. Experimentally, four groups of mice were injected intraperitoneally with PBS, L. major wild-type (L. majorWT), L. major GP63 knockout (L. majorKO) or L. major GP63 rescue (L. majorR). Six hours post-inoculation, intraperitoneal lavages were performed, and the cell suspension was collected for further analysis. We counted the total live recruited inflammatory cells, and cytospin slides were prepared to identify the cell types present in the lavage. Flow cytometry was also performed to verify the cell types and the populations of macrophages. In addition, the collected cells were plated and studied ex vivo to determine the percentage of infected macrophages and neutrophils within 48 hours after the infection. Centrifugation was used to isolate the supernatant and cytokine/chemokine contents were measured. Furthermore, we performed transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and proteomic analysis on the exosome content released in the supernatant by the cells recruited to the peritoneal cavity. Data collected suggest that all Leishmania cause similar inflammatory cell recruitment. However, cytokine/chemokine results show variabilities between groups and are not sufficient to explain why GP63 KO parasites cause a less aggressive infection in vivo. GP63 may be involved in the internalization of promastigotes during early infection because there is a trend observed where less GP63KO amastigotes were found within host cells upon initial hours of infection. L. major deficient in GP63 appear to be significantly less able to confer same level of infection as L. majorWT and L. majorR. Collectively this study provides a clear analysis of innate inflammatory events occurring during L. major infection and the role of the virulence factor GP63, as well as permit better understanding of the cellular and molecular mechanisms underlying the Leishmania infection process, which could lead to the development of new ways to protect humans against this pathogen.

Leishmaniasis is a disease caused by the protozoan parasite Leishmania and is known to affect millions of individuals worldwide. In recent years, we have established the critical role played by Leishmania zinc-metalloprotease GP63 in the modulation of host macrophage signalling and functions, favouring its survival and progression within its host. Leishmania major lacking GP63 was reported to cause limited infection in mice, however, it is still unclear how GP63 may influence the innate inflammatory response and parasite survival in an in vivo context. Therefore, we were interested in analyzing the early innate inflammatory events upon Leishmania inoculation within mice and establish whether Leishmania GP63 influences this initial inflammatory response. Experimentally, L. major WT (L. majorWT), L. major GP63 knockout (L. majorKO), or L. major GP63 rescue (L. majorR) were intraperitoneally inoculated in mice and the inflammatory cells recruited were characterized microscopically and by flow cytometry (number and cell type), and their infection determined. Pro-inflammatory markers such as cytokines, chemokines, and extracellular vesicles (EVs, e.g. exosomes) were monitored and proteomic analysis was performed on exosome contents. Data obtained from this study suggest that Leishmania GP63 does not significantly influence the pathogen-induced inflammatory cell recruitment, but rather their activation status and effector function. Concordantly, internalization of promastigotes during early infection could be influenced by GP63 as fewer L. majorKO amastigotes were found within host cells and appear to maintain in host cells over time. Collectively this study provides a clear analysis of innate inflammatory events occurring during L. major infection and further establish the prominent role of the virulence factor GP63 to provide favourable conditions for host cell infection.