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Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica.

Macrophages (Mφ) and dendritic cells are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a strategy employed by multiple pathogens to ensure their survival in the infected cell. Leishmania promastigotes have been shown to protect Mφ, neutrophils, and dendritic cells from both natural and induced apoptosis. Nevertheless, the effect of the infection with Leishmania amastigotes in the apoptosis of these cell populations has not been established, which results are very important since amastigotes persist in cells for many days and are responsible for sustaining infection in the host. As shown in this study, apoptosis of monocyte-derived dendritic cells (moDC) induced by treatment with camptothecin was downregulated by infection with L. mexicana amastigotes from 42.48 to 36.92% as detected by Annexin-V binding to phosphatidylserine. Also, the infection of moDC with L. mexicana amastigotes diminished the fragmentation of DNA as detected by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, and changes in cell morphology were analyzed by electron microscopy. The observed antiapoptotic effect was found to be associated with an 80% reduction in the presence of active caspase-3 in infected moDC. The capacity of L. mexicana amastigotes to delay apoptosis induction in the infected moDC may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.

The regulatory effect of mast cells on the pathogenesis of leishmaniasis is unclear. We report a comparative analysis of TLR2 membrane expression, TNF-α, IL-10 and MIP-1α production, and granule release of bone marrow-derived mast cells (BMMCs) from susceptible BALB/c and resistant C57BL/6 mice, stimulated in vitro with Leishmania mexicana lipophosphoglycan (LPG). We studied the kinetics of mast cell degranulation and parasite numbers in lesions of both mouse strains infected with L. mexicana. We found that BMMCs of C57BL/6 mice expressed more TLR2 and produced higher levels of both cytokines and MIP-1α, whereas BALB/c BMMCs significantly augmented their granule release. Lesions of BALB/c mice showed higher levels of degranulated mast cells at 3 h of infection, whereas after 3 days of infection, the number of degranulated mast cells in C57BL/6 was higher than in BALB/c lesions. Throughout infection, BALB/c mice harboured more parasites. The regulatory effect of mast cells seems to depend on the genetic background of the host: mast cells of BALB/c mice facilitate disease progression due to an augmented inflammatory response early in the infection, whereas mast cells of C57BL/6 mice produce cytokines that regulate inflammation and maintain an elevated number of immune cells in the lesions, promoting disease control.

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