Leishmania aethiopica cell‐to‐cell spreading involves caspase‐3, AkT, and NF‐κB but not PKC‐δ activation and involves uptake of LAMP‐1‐positive bodies containing parasites

Abstract

Development of human leishmaniasis is dependent on the ability of intracellular Leishmania parasites to spread and enter macrophages. The mechanism through which free promastigotes and amastigotes bind and enter host macrophages has been previously investigated; however, little is known about intracellular trafficking and cell-to-cell spreading. In this study, the mechanism involved in the spreading of Leishmania aethiopica and Leishmania mexicana was investigated. A significant increase in phosphatidylserine (PS) exhibition, cytochrome C release, and active caspase-3 expression was detected (P < 0.05) during L. aethiopica, but not L. mexicana spreading. A decrease (P < 0.05) of protein kinase B (Akt) protein and BCL2-associated agonist of cell death (BAD) phosphorylation was also observed. The nuclear factor kappa-lightchain enhancer of activated B cells (NF-kB) signaling pathway and pro-apoptotic protein protein kinase C delta (PKC-d) were downregulated while inhibition of caspase-3 activation prevented L. aethiopica spreading. Overall suggesting that L. aethiopica induces host cell's apoptosis during spreading in a caspase-3-dependent manner. The trafficking of amastigotes within macrophages following cell-to-cell spreading differed from that of axenic parasites and involved co-localization with lysosomal-associated membrane protein 1 (LAMP-1) within 10 min postinfection. Interestingly, following infection with axenic amastigotes and promastigotes, co-localization of parasites with LAMP-1-positive structures took place at 1 and 4 h, respectively, suggesting that the membrane coat and LAMP-1 protein were derived from the donor cell. Collectively, these findings indicate that host cell apoptosis, demonstrated by PS exhibition, caspase-3 activation, cytochrome C release, downregulation of Akt, BAD phosphorylation, NF-kB activation, and independent of PKC-d expression, is involved in L. aethiopica spreading. Moreover, L. aethiopica parasites associate with LAMP-rich structures when taken up by neighboring macrophages.Abbreviations 7AAD, 7-aminoactinomycin D; Akt, protein kinase B; BAD, BCL2-associated agonist of cell death; CF, PKC-d cleaved fragment; CL, cutaneous leishmaniasis; EEA 1, early endosome antigen 1; HI-FBS, heat-inactivated fetal bovine serum; IF1, intermediate form 1; LAMP-1, lysosomal-associated membrane protein 1; NF-kB, nuclear factor kappa-light-chain enhancer of activated B cells; PKC-d, protein kinase C delta; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine; RA, retinoic acid; SEM, scanning electron microscope; Ser, serine; Thr, threonine.

Discovering the Potent Inhibitors Against Babesia bovis in vitro and Babesia microti in vivo by Repurposing the Natural Product Compounds

“…Fascinatingly, EVs (Extracellular vesicles) from parasite or Trypanosoma cruzi -infected macrophages interacting with TLR2 were able to elicit translocation of NF-κB and, as a consequence, to alter the EVs, the gene expression of proinflammatory cytokines, and STAT-1 and STAT-3 signaling pathway ( 41 ). In leishmaniasis, the nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB) signaling pathway and pro-apoptotic protein kinase C delta (PKC-δ) were downregulated, while inhibition of caspase-3 activation prevented L. aethiopica spreading ( 42 ). On the other hand, STAT-5 is also related to promote T cell proliferation and differentiation with immune-related proteins, while spleen continued to initiate immune responses to combat the infection of B. microti ( 43 ).…”

The Role of Nuclear Factor Kappa B (NF-κB) in the Immune Response against Parasites

“…This is dependent on Leishmania protease gp63. p35 is involved in the expression of specific chemokines and the parasite has been proposed to induce those favorable to its survival [ 98 , 109 ]. Cleavage of p65 to p35 has also been reported following dendritic cell infection by L. infantum promastigotes [ 111 ].…”

“…Cleavage of p65 to p35 has also been reported following dendritic cell infection by L. infantum promastigotes [ 111 ]. L. aethiopica amastigotes have been shown to manipulate a variety of signaling pathways, including NF-κB, in human macrophages during spreading [ 109 ]. This results in reduced p65 expression and phosphorylation (Ser32/36) of IκB, indicating downregulation of NF-κB.…”

“…This results in reduced p65 expression and phosphorylation (Ser32/36) of IκB, indicating downregulation of NF-κB. This down-regulation of NF-κB leading to cell apoptosis is expected to facilitate the spread of the parasite [ 109 ]. Conversely, decreased expression of IκB was also reported which is expected to induce p65 translocation and NF-κB pathway activation and may indicate pleiotropic impacts of Leishmania on NF-κB pathways.…”

“…This is supported by the results of Nogueira et al who evaluated responses to various Leishmania species ( L. braziliensis , L. infantum , L. amazonensis ) and showed that only L. amazonensis was able to induce p65 translocation to the nucleus and a pro-inflammatory response in a particular laboratory model [ 108 ]. The variability among species to cleave p65 to p35 should also be noted [ 98 , 109 ]. Species-specific and stage-specific variations, as well as the development of effective immune responses complicate the situation; nevertheless, from the known data emerges a pattern which generally indicates that Leishmania modulates the NF-κB pathway towards a dampening of TNF-α expression and production of an environment that facilitates parasite survival.…”