MicroRNA expression profile in human macrophages in response to Leishmania major infection

Abstract

Background: Leishmania (L.) are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts.

Methodology/principal findings: We report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h). We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation.

Conclusions/significance: Our data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major infection. These results could contribute to better understanding of the dynamics of gene expression in host cells during leishmaniasis.

Figure 1. Hierarchical cluster analysis of deregulated miRNA expression in L. major-infected human primary macrophages.

The miRNA expression values are presented using a red-white-blue color scheme, with red data points indicating higher expression than median values, white indicating expression equal to the median, and blue indicating lower expression than the median. MiRNAs were analyzed based on their expression before and at different time points, post-infection (3, 6, 12 and 24 h) of primary human macrophages from three healthy donors (D1, D2 and D3).

Figure 2. Biological processes deduced from analysis of deregulated miRNA-targets in L. major-infected human macrophages at 3 h post-infection.

Yellow color gradient intensity correlates with up- or down-regulation levels. White nodes are not significantly overrepresented. The area of each node is proportional to the number of genes in the set annotated to the corresponding GO category. Interactions were visualized as a network using Cytoscape and BINGO plugin.

Figure 3. Negative correlation between expressions of an up-regulated set of miRNAs and their targeted chemokine transcripts.

Expression means of let-7a, miR-25, miR-26a, miR-140, miR-146a and miR-155 at 3 h and miR-23b and miR-132 at 6 h post-infection of three healthy donors (D1, D2 and D3; panel A) is negatively correlated with CCL2, CCL5, CXCL10, CXCL11 and CXCL12 mRNA mean levels at 12 and 24 h post-infection (panel B) in L. major-infected human macrophages. Results were expressed using the 2^−ΔΔCt method.

Figure 4. Time course of miRNA-210 and procaspase-3 expression levels in L. major infected human primary macrophages.

MiR-210 expression at 3, 6, 12, and 24 h in infected cells relatively to non-infected cells (panel A), after siRNA-control or HIF-1α-silencing transfections in non-infected and infected cells (panel B). Results were expressed using the 2^−ΔΔCt method. Stars (*) are indicated when results are statistically significant from control. One star indicates a p value <0.05; two stars indicate a p value <0.01 and three stars indicate a p value <0.001. Panel C represents abundance of pro-caspase-3 protein levels in time course parasite-infected macrophages of healthy donors revealed by western blot analysis. HSP27 was used as loading control. Ten µg of L. major lysate (latest lane) was used a negative control to ensure that anti-procaspase-3 antibody does not cross-react with parasite proteins. Data are representative of three independent experiments conducted on MDM derived from two to three different healthy donors. NI indicates non-infected cells and IF indicates infected cells.