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The detection of Leishmania tarentolae in sympatric areas where Leishmania infantum is endemic raised questions regarding the protective effect exerted in dogs by L. tarentolae when in coinfection. This study aimed monitoring the in vitro gene expression of pro- (IFN- γ; TNF-α; IL-12) and anti-inflammatory (IL-4; IL-6; IL-10) cytokines in primary canine macrophages infected by L. tarentolae and L. infantum in single and in co-infections. Macrophages differentiated from dog blood mononuclear cells were infected with the L. tarentolae field-isolated (RI-325) and laboratory (LEM-124) strains, with L. infantum laboratory strain (IPT1), or both. Infection and the number of amastigotes per infected cell were evaluated microscopically by counting a total of 200 cells between 4 and 96 h. Cytokine gene expression was analyzed by real-time PCR from infected macrophages mRNA. Single infections presented higher expression of the cytokines IL-4 and IL-6, and lower of IL-12. Co-infections induced a lower gene expression of IL-4 and IL-6, and a higher gene expression of IL-12, correlating with the low amastigote burden despite the slight increase of infected cells. Data highlight the potential protective effect of L. tarentolae against L. infantum in co-infection by the reduced anti-inflammatory and increased pro-inflammatory cytokines gene expression, opening new perspectives for a canine vaccine development exploiting the non-pathogenic L. tarentolae . Supplementary Information The online version contains supplementary material available at 10.1038/s41598-024-78451-x.
The detection of Leishmania tarentolae in sympatric areas where Leishmania infantum is endemic raised questions regarding the protective effect exerted in dogs by L. tarentolae when in coinfection. This study aimed monitoring the in vitro gene expression of pro- (IFN- γ; TNF-α; IL-12) and anti-inflammatory (IL-4; IL-6; IL-10) cytokines in primary canine macrophages infected by L. tarentolae and L. infantum in single and in co-infections. Macrophages differentiated from dog blood mononuclear cells were infected with the L. tarentolae field-isolated (RI-325) and laboratory (LEM-124) strains, with L. infantum laboratory strain (IPT1), or both. Infection and the number of amastigotes per infected cell were evaluated microscopically by counting a total of 200 cells between 4 and 96 h. Cytokine gene expression was analyzed by real-time PCR from infected macrophages mRNA. Single infections presented higher expression of the cytokines IL-4 and IL-6, and lower of IL-12. Co-infections induced a lower gene expression of IL-4 and IL-6, and a higher gene expression of IL-12, correlating with the low amastigote burden despite the slight increase of infected cells. Data highlight the potential protective effect of L. tarentolae against L. infantum in co-infection by the reduced anti-inflammatory and increased pro-inflammatory cytokines gene expression, opening new perspectives for a canine vaccine development exploiting the non-pathogenic L. tarentolae . Supplementary Information The online version contains supplementary material available at 10.1038/s41598-024-78451-x.
In canine leishmaniosis endemic areas, Leishmania infantum may occur in sympatry with the non-pathogenic Leishmania tarentolae, which is associated to reptiles. The potential infectivity of L. tarentolae for mammals raises questions about the interactions between the two Leishmania species, and the potential cross-immune protection in dogs. This study aimed to assess the outcome of experimental L. tarentolae infection in dogs, determining: i) the anti-L. tarentolae antibody production, ii) the duration of the immunity and cytokine expression, and iii) the possible pathogenic effect in the canine host. Twelve purpose-bred beagle dogs were randomly allocated to three groups (intravenous inoculation, G1; intradermal inoculation, G2; negative control, G3). G1 and G2 dogs were inoculated twice (day 0, day 28) with 108 promastigotes of L. tarentolae strain (RTAR/IT/21/RI-325) isolated from a Tarentola mauritanica gecko. The animals were followed until day 206. Blood, serum, conjunctival swabs and lymph node aspirate samples were collected monthly and bone marrow, liver and spleen biopsies on day 91. Hematological and biochemical parameters were assessed monthly, as well as serology (IFAT and ELISA) and molecular identification of L. tarentolae. Mononuclear cells (PBMC) were obtained to assess the cytokine expression through in vitro stimulation or (re-) infection. Data from this study demonstrated that DNA from L. tarentolae is detectable up to 3 months post-infection, with seroconversion after day 28. Moreover, the non-pathogenic nature of L. tarentolae was confirmed, with a neutral Th1/Th2 polarization, and a possible shift to Th1 phenotype after derived macrophages (re-) infection, as demonstrated by the expression of IFN-gamma. Therefore, L. tarentolae demonstrated a great potential as a surrogate pathogen and/or immune-prophylaxis/immune-therapy against Leishmania infections in dogs and humans.
BackgroundInnate immune responses against infectious agents can act as triggers of inflammatory diseases. On the other hand, various pathogens have developed mechanisms for the evasion of the immune response, based on an inhibition of innate immunity and inflammatory responses. Inflammatory diseases could thus be controlled through the administration of pathogens or pathogen-derived molecules, capable of interfering with the mechanisms at the basis of inflammation. In this framework, the NLRP3 inflammasome is an important component in innate antimicrobial responses and a major player in the inflammatory disease. Parasites of the genus Leishmania are master manipulators of innate immune mechanisms, and different species have been shown to inhibit inflammasome formation. However, the exploitation of pathogenic Leishmania species as blockers of NLRP3-based inflammatory diseases poses safety concerns.MethodsTo circumvent safety issues associated with pathogenic parasites, we focused on Leishmania tarentolae, a species of Leishmania that is not infectious to humans. Because NLRP3 typically develops in macrophages, in response to the detection and engulfment microorganisms, we performed our experiments on a monocyte-macrophage cell line (THP-1), either wild type or knockout for ASC, a key component of NLRP3 formation, with determination of cytokines and other markers of inflammation.ResultsL. tarentolae was shown to possess the capability of dampening the formation of NLRP3 inflammasome and the consequent expression of pro-inflammatory molecules, with minor differences compared to effects of pathogenic Leishmania species.ConclusionThe non-pathogenic L. tarentolae appears a promising pro-biotic microbe with anti-inflammatory properties or a source of immune modulating cellular fractions or molecules, capable of interfering with the formation of the NLRP3 inflammasome.
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