Comparative transcriptomic analysis of long noncoding RNAs in Leishmania-infected human macrophages

Abstract

It is well established that infection with Leishmania alters the host cell's transcriptome. Since mammalian cells have multiple mechanisms to control gene expression, different molecules, such as noncoding RNAs, can be involved in this process. MicroRNAs have been extensively studied upon Leishmania infection, but whether long noncoding RNAs (lncRNAs) are also altered in macrophages is still unexplored. We performed RNA-seq from THP-1-derived macrophages infected with Leishmania amazonensis (La), L. braziliensis (Lb), and L. infantum (Li), investigating a previously unappreciated fraction of macrophage transcriptome. We found that more than 24% of the total annotated transcripts and 30% of differentially expressed (DE) RNAs in Leishmania-infected macrophage correspond to lncRNAs. LncRNAs and protein coding RNAs with altered expression are similar among macrophages infected with the Leishmania species. Still, some species-specific alterations could occur due to distinct pathophysiology in which Li infection led to a more significant number of exclusively DE RNAs. The most represented classes among DE lncRNAs were intergenic and antisense lncRNAs. We also found enrichment for immune response-related pathways in the DE protein coding RNAs, as well as putative targets of the lncRNAs. We performed a coexpression analysis to explore potential cis regulation of coding and antisense noncoding transcripts. We identified that antisense lncRNAs are similarly regulated as its neighbor protein coding genes, such as the BAALC/BAALC-AS1, BAALC/BAALC-AS2, HIF1A/HIF1A-AS1, HIF1A/HIF1A-AS3 and IRF1/IRF1-AS1 pairs, which can occur as a species-specific modulation. These findings are a novelty in the field because, to date, no study has focused on analyzing lncRNAs in Leishmania-infected macrophage. Our results suggest that lncRNAs may account for a novel mechanism by which Leishmania can control macrophage function. Further research must validate putative lncRNA targets and provide additional prospects in lncRNA function during Leishmania infection.

Keywords: Leishmania; RNA-seq; THP-1; gene expression; host-parasite interaction; lncRNA; macrophage; transcriptomics.

FIGURE 1

Number of differentially expressed protein coding and long noncoding RNAs subtypes in Leishmania-infected THP-1 macrophages. (A) Intersection plot representing the number of significantly upregulated lncRNAs and protein coding genes from La-, Lb- and Li-infected THP-1 macrophages (N = 5); (B) Intersection plot representing the number of significantly downregulated lncRNAs and protein coding genes from La-, Lb- and Li-infected THP-1 macrophages (N = 5); (C) Number of transcripts of each lncRNA type classified using the GRCh38.p13 database. The transcripts from the GRCh38.p13 database that lack validation studies are referred to as novel transcripts. IG: intergenic, AS: antisense; OT: overlapping transcript; IT: intronic transcript; NC: non-classified.

FIGURE 2

Enriched Reactome pathways for La, Lb, and Li infection in THP-1 macrophages. The plot represents the normalized enrichment score (NES) by both color and bubble size, as indicated in the figure legend, of significantly altered level 3 Reactome pathways based in the protein coding genes of THP-1 macrophages infected with La, Lb or Li.

FIGURE 3

Alluvial plots representing enriched pathways and their related protein coding genes modulated in Leishmania-infected THP-1 macrophages. The alluvial plots show the significantly differentially expressed mRNAs in La (blue lines), Lb (yellow lines) and Li (red lines)-infected macrophages for 5 enriched pathways (A) Reactive oxygen species (ROS) and reactive nitrogen species (RNS) production in phagocytes (B) Detoxification of ROS (C) Cellular response to hypoxia (D) Tumor necrosis factor (TNF) signaling and (E) TNF receptor 2 (TNFR2) non-canonical nuclear factor kappa B (NF-κB).

FIGURE 4

Coexpression of LncRNAs and mRNAs modulated upon Leishmania infection in THP-1 macrophages. Scatter plots represent the log2 fold-change values determined by RNA-seq of protein coding (X axis) and lncRNAs (Y axis) of (A) L. amazonensis-infected (B) L. braziliensis-infected (C) L. infantum-infected macrophages; (D) illustrations representing the genomic position of 10 coregulated lncRNA-mRNA pairs, in which grey represents protein-coding gene, purple represent AS-lncRNA and the numbers depict genomic position in the sense and antisense complementary DNA strand. AS: antisense; DT: divergent transcript; IT: intronic transcript; Chr: chromosome.