DNA Methylation in macrophages infected with Leishmania spp. in different culture conditions

Abstract

Leishmania modulate the host cell epigenome, including DNA methylation. This work aimed to explore the DNA methylation pattern in infected macrophages with L. (Viannia) braziliensis, L. (Leishmania) amazonensis and L. (L.) infantum. We performed a genome-wide methylation analysis in macrophages, cultured in presence/absence of interleukin 6 (IL-6) and infected with the three species for a period of 72hs. Upon 72hs infection, sample groups of L. (V.) braziliensis - and L. (L.) infantum-infected macrophages were treated with Glucantime and Amphotericin B for 48hs, respectively. Uninfected macrophages and macrophages treated with heat-killed Leishmania were included as controls. Several CpG islands alterations were identified upon infection and among species. The methylome analysis showed that L. (L.) amazonensis and L. (L.) infantum clustered together separately from L. (V.) braziliensis. The identified alterations were mainly associated with cytoskeleton organization. We also detected that the DNA methylation pattern of ten, six and eight CGIs for each aforementioned species slightly changed in a culture environment with IL-6, whereas treatment led to distinct DNA methylation profiles respect to untreated samples. Interestingly, some altered CGIs showed a re-establishment towards the control methylation pattern in L. (L.) infantum (69%, 11 out of 16) and L. (V.) braziliensis (36%, 4 out of 11). The identified alterations suggest a species-specific parasite/host interaction probably leading to gene expression regulation. The discovery of these methylation alterations addresses further functional studies and suggests them as potential therapeutic targets.

Keywords: Amphotericin B; CpG islands; DNA methylation alterations; Glucantime; Leishmania infection; Leishmania-associated alterations; Leishmania-host cell interaction.

Graphical abstract

Figure 1.

Experimental plan, DNA methylation analysis workflow and infection efficacy. (A) The control groups included uninfected macrophages and macrophages treated with heat-killed Leishmania. Macrophages were cultured in the absence or presence of IL-6 and infected with L. (V.) braziliensis, L. (L.) amazonensis and L. (L.) infantum. At 72hs post-infection cells were harvested for DNA extraction. Simultaneously, macrophages infected with L. (V.) braziliensis and L. (L.) infantum were treated during 48hs with Glucantime and Amphotericin B, respectively. Figure created using Servier Medical Art images. (B) Scheme of differential methylation analyses among conditions. The arrows indicate the paired comparisons. Abbreviations: control A, uninfected macrophages (CtA) and control B, macrophages with heat-killed Leishmania (CtB); all infected samples (ALL-INF); L. (L.) amazonensis-, L. (L.) infantum – and L. (V.) braziliensis – infected samples in absence and presence of IL-6 (AM and AIL; I and IIL; BR and BIL); treated L. (L.) infantum and L. (V.) braziliensis samples (IA and BG). (C) Macrophages infected with L. (L.) amazonensis, L. (L.) infantum and L. (V.) braziliensis amastigotes. Arrows indicate the amastigotes.

Figure 2.

Leishmania-associated CGI alterations. (A) Unsupervised hierarchical clustering analysis based on the β value of each Leishmania species-infected sample and controls in triplicate. Abbreviations: L. (L.) amazonensis (AM1, AM2 and AM3); L. (L.) infantum (I1, I2 and I3); L. (V.) braziliensis (BR1, BR2 and BR3); uninfected control sample A (CtA1, CtA2 and CtA3) and B, macrophages with heat-killed Leishmania (CtB1, CtB2). (B) Volcano plots showing differentially methylated CGIs in L. (L.) amazonensis-, L. (L.) infantum – and L. (V.) braziliensis-infected samples compared to CtA. Significant hypermethylated and hypomethylated CGIs are indicated in red and blue. (C) VennDiagram of the altered CGIs in Leishmania species, showing overlapping and species-specific CGIs.

Figure 3.

Reactome pathway analysis. (A) Chord diagram showing the significantly altered pathways associated with each Leishmania species, including the number of the identified altered genes within the outer ring. The pathways are located on the right outer ring, whereas L. (L.) amazonensis (red), L. (L.) infantum (green) and L. (V.) braziliensis (blue) are on the left outer ring and on the right inner ring overlapping with the aberrant pathways. Colour beams link the pathway with the Leishmania species. Enrichment dot plots showing the significant altered pathways with the number of the identified genes in L. (L.) amazonensis (B), L. (L.) infantum (C) and L. (V.) braziliensis (D). The name of the Leishmania infection pathways, altered in L. (L.) infantum infection, is highlighted in green.

Figure 4.

DNA methylation of altered CGIs in treated and untreated macrophages. Heatmaps showing beta methylation values of CGIs in control, treated and untreated samples for L. (L.) infantum (A) and L. (V.) braziliensis (C) infection in triplicate. Abbreviations: untreated and treated L. (L.) infantum (I1, I2 and I3; IA1, IA2 and IA3); untreated and treated L. (V.) braziliensis (BR1, BR2 and BR3; BG1, BG2 and BG3); control A, uninfected macrophages (CtA1, CtA2 and CtA3) and B, macrophages with heat-killed Leishmania (CtB1, CtB2). Dot plots of mean beta values, resulting from the average of the triplicates for each altered CGI in L. (L.) infantum (B) and L. (V.) braziliensis (D). Asterisks indicate significant differences between infected and CtA samples (nominal p value < 0.05 and |Δβ| > 0.05).

Figure 5.

Leishmania-associated alterations at promoters. Volcano plots showing differentially methylated promoters in three comparisons: L. (L.) amazonensis-infected macrophages vs uninfected macrophages (A), L. (L.) infantum-infected macrophages vs uninfected macrophages (B) and L. (V.) braziliensis-infected macrophages vs uninfected macrophages (C). Significant hypermethylated and hypomethylated CGIs are indicated in red and blue.