Functional evaluation of gene silencing on macrophages derived from U937 cells using interference RNA (shRNA) in a model of macrophages infected withLeishmania(Viannia)braziliensis

Abstract

Leishmaniasis development is multifactorial; nonetheless, the establishment of the infection, which occurs by the survival and replication of the parasite inside its main host cell, the macrophage, is mandatory. Thus, the importance of studying the molecular mechanisms involved in the Leishmania-macrophage interaction is highlighted. The aim of this study was to characterize a cellular model of macrophages derived from U937 cells that would allow for the identification of infection phenotypes induced by genetic silencing with interference RNA in the context of macrophages infected with Leishmania (Viannia) braziliensis. The model was standardized by silencing an exogenous gene (gfp), an endogenous gene (lmna) and a differentially expressed gene between infected and non-infected macrophages (gro-β). The silencing process was successful for the three genes studied, obtaining reductions of 88·9% in the GFP levels, 87·5% in LMNA levels and 74·4% for Gro-β with respect to the corresponding control cell lines. The cell model revealed changes in the infection phenotype of the macrophages in terms of number of amastigotes per infected macrophage, number of amastigotes per sampled macrophage and percentage of infected macrophages as a result of gene silencing. Thus, this cell model constitutes a research platform for the study of parasite-host interactions and for the identification of potentially therapeutic targets.

Lamin A/C and the Immune System: One Intermediate Filament, Many Faces

“…Macrophages thus have a dual effect on Leishmania infection, since they contribute to the eradication of internalized parasites but also provide a safe place for Leishmania replication [ 129 , 130 ]. The proportion of human macrophage U937 cells infected with L. braziliensis is reduced by shRNA silencing of lamin A/C, and the lamin A/C silencing also reduces the number of parasites per sampled macrophage, suggesting that lamin A/C plays a role in the prevalence of Leishmania parasites in macrophages [ 131 ]. Further experiments are needed to corroborate these results in primary macrophages and DCs and to discern how lamin A/C affects parasite eradication and division in macrophages ( Figure 3 d).…”

The Nuclear Envelope as a Regulator of Immune Cell Function

“…The c-Fos protein, which influences macrophage responses in cancer (45)(46)(47), was shown to interact with and be regulated by lamin A/C (25). Another study demonstrates that lamin A/C gene silencing reduces the number of Leishmania parasites per sampled macrophage and reduces the percentage of infected macrophages in Leishmaniasis (48). Generally, macrophages express higher levels of lamin A/C compared to some other immune cells, which may contribute to macrophages' proinflammatory actions and comparably longer life span than other innate immune cell types (49).…”

Defining host–pathogen interactions employing an artificial intelligence workflow

“…To date, these methods have not been adapted for the field of host–pathogen interaction. Typically, HCI based fluorescent imaging data from a host–pathogen interaction experiment is analyzed by classical image segmentation (Osaka et al, 2012; Schmutz et al, 2013; Kühbacher et al, 2015; Ovalle-Bracho et al, 2015). Occasionally segmentation combined with machine learning based on calculated features has been employed (Kreibich et al, 2015).…”