Fig 1.
Neutrophils purified from whole blood of healthy male donors were infected with L.
infantum. Representative image of (A) field of cytocentrifuge slide (100x magnification) comprised predominantly of neutrophils and (B) FACS of purified cells from sample 2, 59.2% were CD45+ leukocytes, thus analyzed for CD45+CD16+ resulting in 91,6% of neutrophils. Representative fields of cytocentrifuge slides (100x magnification) following a 3-hour culture period from (C) control samples comprising predominantly uninfected neutrophils and (D) neutrophils infected with the promastigote form of L. infantum. Black arrows indicate internalized Leishmania parasites, within parasitophorous vacuoles in neutrophils.
Fig 2.
Transcripts modulated by L. infantum in human neutrophils.
In vitro infection by L. infantum (INF) decreased expression of 202 transcripts and increased expression of 10 transcripts in human neutrophils when compared to control (CT). (A) Volcano plot shows 212 DEGs at log2(FC)±0.58 and FDR≤0.05 between human neutrophils infected and non-infected with L. infantum. Significantly upregulated transcripts (red dots) are in the upper right square of the graph (positive log2(FC) value) and significantly downregulated (green dots) are in the upper left square of the graph (negative log2(FC) value). (B) Heatmap shows normalized values for 212 differentially expressed transcripts (log2(FC)±0.58; FDR≤0.05). Transcripts were grouped by hierarchical clustering. This analysis was performed using heatmap3 package in RStudio and color scale represents z-score (green indicates lower expression, whereas red indicates higher expression).
Table 1.
Pathway enrichment analysis for differentially expressed genes following in vitro infection of human neutrophils by L. infantum.
Fig 3.
Heatmap of miRNAs modulated by L. infantum in human neutrophils.
MiRNAs were grouped by hierarchical clustering. A total of 197 miRNAs were increased and 92 were decreased following in vitro infection of neutrophils with L. infantum (INF) when compared to control (CT). Heatmap shows corrected signal for 289 differentially expressed miRNAs (FC±2; FDR≤0.01). Analysis was performed using heatmap3 package in RStudio and color scale represents z-score (green indicates lower expression, whereas red indicates higher expression).
Fig 4.
Target prediction of upregulated and downregulated miRNAs following in vitro infection of human neutrophils by L. infantum.
(A) Differentially expressed miRNAs were divided according to expression patterns (up- or downregulated). DIANA-miRPath v3.0 (https://dianalab.e-ce.uth.gr/html/mirpathv3/index.php?r=mirpath) web platform was used to access three target prediction databases (TarBase, microT-CDS and TargetScan) for both, and then TarBase plus microT-CDS∩TargetScan predicted targets were compared to transcripts in our RNA-Seq analysis (p-value≤0.05). (B) 556 downregulated transcripts (p-value≤0.05) were found as potential targets of upregulated miRNAs. (C) 193 upregulated transcripts (p-value≤0.05) were found as potential targets of downregulated miRNAs.
Table 2.
Pathway enrichment analysis for downregulated transcripts, targets of upregulated miRNAs, following in vitro infection of human neutrophils by L. infantum.
Fig 5.
Heatmap of lncRNAs modulated by L. infantum following in vitro infection of human neutrophils.
LncRNAs were grouped by hierarchical clustering. One lncRNA (HIF1A-AS3) was up regulated after in vitro infection with L. infantum (INF), while five (PELATON, AC090559.1, SERPINB9P1, MIR3945HG and LINC01093) were downregulated when compared to control (CT). Heatmap shows normalized values for six differentially expressed transcripts (log2(FC)±0.58; FDR≤0.05), classified as lncRNAs, using BioMart documentation (release 103). Analysis was performed using heatmap3 package in RStudio and color scale represents z-score (green indicates lower expression, whereas red indicates higher expression).
Fig 6.
Network of co-expressed mRNAs/lncRNA and their enriched pathways, following in vitro infection of human neutrophils by L. infantum.
LncRNA SERPINB9P1 and partner mRNAs, all positively correlated (|r|≥0.8; p-value≤0.05 and ndG≤-0.20) and downregulated after in vitro infection with L. infantum (green color), are shown. Pathways obtained after enrichment analysis of co-expressed transcripts are also represented. Diamond shape corresponds to lncRNA, ellipses correspond to mRNAs and rectangles to pathways. Analysis was performed using Cytoscape software (version 3.9.0).
Fig 7.
Network of co-expressed mRNAs/lncRNA present in Leishmaniasis Pathway.
mRNA/lncRNA pairs, positively correlated (|r|≥0.8; p-value≤0.05 and ndG≤-0.10) enriching the Leishmaniasis pathway are presented, color of the lines varies according to correlation value. The higher |r|, darker the line. The color of the shapes varies according to log2fold change (FC) value. Darker green color represents lower expression. Diamond shape corresponds to lncRNA and ellipses correspond to mRNAs. Analysis was performed using Cytoscape software (version 3.9.0).
Fig 8.
Regulation of the Leishmaniasis pathway following in vitro infection of human neutrophils by L.
infantum. L. infantum infection impairs multiple immune signaling pathways, negatively affecting processes such as phagocytosis, apoptosis, and NO production, and can also impair pro-inflammatory signaling in human neutrophils. This suggests that L. infantum can regulate host response through increased expression of miRNAs. Green color represents downregulation of mRNA and correlated lncRNAs, red color indicates upregulation of miRNAs, molecule shape was based on the online tool https://targetexplorer.ingenuity.com/, and track functionality was based on KEGG database.
Table 3.
Leishmaniasis pathway.