Transcriptional analysis of THP-1 cells infected with Leishmania infantum indicates no activation of the inflammasome platform

Abstract

Leishmaniasis is caused by intracellular parasites transmitted to vertebrates by sandfly bites. Clinical manifestations include cutaneous, mucosal or visceral involvement depending upon the host immune response and the parasite species. To assure their survival inside macrophages, these parasites developed a plethora of highly successful strategies to manipulate various immune system pathways. Considering that inflammasome activation is critical for the establishment of a protective immune response in many parasite infections, in this study we determined the transcriptome of THP-1 cells after infection with L. infantum, with a particular focus on the inflammasome components. To this end, the human cell line THP-1, previously differentiated into macrophages by PMA treatment, was infected with L. infantum promastigotes. Differentiated THP-1 cells were also stimulated with LPS to be used as a comparative parameter. The gene expression signature was determined 8 hours after by RNA-seq technique. Infected or uninfected THP-1 cells were stimulated with nigericin (NIG) to measure active caspase-1 and TNF-α, IL-6 and IL-1β levels in culture supernatants after 8, 24 and 48 hours. L. infantum triggered a gene expression pattern more similar to non-infected THP-1 cells and very distinct from LPS-stimulated cells. Some of the most up-regulated genes in L. infantum-infected cells were CDC20, CSF1, RPS6KA1, CD36, DUSP2, DUSP5, DUSP7 and TNFAIP3. Some up-regulated GO terms in infected cells included cell coagulation, regulation of MAPK cascade, response to peptide hormone stimulus, negative regulation of transcription from RNA polymerase II promoter and nerve growth factor receptor signaling pathway. Infection was not able to induce the expression of genes associated with the inflammasome signaling pathway. This finding was confirmed by the absence of caspase-1 activation and IL-1β production after 8, 24 and 48 hours of infection. Our results indicate that L. infantum was unable to activate the inflammasomes during the initial interaction with THP-1 cells.

Fig 1. Global transcriptional expression patterns from L. infantum-infected cells or LPS-stimulated cells.

A) The heat map was performed by using the Euclidean distance method with complete linkage for all samples (M1, M2, M3 are triplicates of non-stimulated cells; M4, M5, M6 are triplicates of LPS-stimulated cells; M7, M8, M9 are triplicates of L. infantum-infected cells). B) The principal component analysis (PCA) was performed with normalized data: PC1 (axis x) shows the variance (%) among the groups (non-stimulated, LPS-stimulated and infected cells) and PC2 (axis y) shows the variance (%) among the samples. C) The MA-plot shows the distribution of the gene expression between L. infantum-infected cells or LPS-stimulated cells and non-stimulated cells. The axis X shows the mean of normalized counts and axis Y shows the comparison by Log2fold change (Log2FC). Red dots correspond to genes up-regulated (above 0) and down-regulated genes (bellow 0) based on the False Discovery Rate (FDR<0.01).

Fig 2. The top 30 genes most significantly up and down expressed by THP-1 cells infected with L. infantum.

The heatmap shows the 30 most significantly up-regulated genes in L. infantum-infected cells (A) and the 30 most significantly down-regulated genes in L. infantum-infected cells (B) in comparison to non-stimulated cells after 8 hours incubation. The matrix was made by DESeq2 based on the Log2 fold-change (FC>0).

Fig 3. Functional enrichment analysis of L. infantum-infected cells in relation to unstimulated cells.

Gene Ontology (GO) enrichment analysis summarized by REVIGO showing 9 biological process enriched (parents GO terms) in up-regulated genes (FDR<0.01) in L. infantum-infected cells after 8 hours. Highly similar GO terms are linked by edges, where the line width indicates the degree of similarity.

Fig 4. Active caspase-1 in THP-1 cells infected with L. infantum.

THP-1 cells previously differentiated into macrophages were infected with L. infantum (1:10) and the percentage of cells expressing active caspase-1 (A), the mean fluorescence intensity (MFI) (B) and representative histograms plots (C) were evaluated 8, 24 and 48 hours after infection. In addition to L. infantum, were also tested other stimuli as LPS + nigericin (LPS + NIG) and L. infantum + nigericin (L.inf. + NIG). Cultures were stimulated with nigericin during 1 hour before the endpoint of the tests. The results are representative of three independent experiments performed in triplicate. Results are expressed as mean ± SD: a vs. LPS+NIG, b vs. L. inf + NIG, p<0.05.

Fig 5. Production of cytokines by THP-1 cells infected with L. infantum.

THP-1 cells previously differentiated into macrophages were infected with L. infantum (1:10) and levels of IL-1β (A), TNF-α (B) and IL-6 (C) were evaluated in the supernatants of the cultures after 8, 24 and 48 hours of incubation. In addition to L. infantum, the following stimuli were tested: LPS, nigericin (NIG), LPS + nigericin (LPS + NIG) and L. infantum + nigericin (L. inf + NIG). Cultures were stimulated with nigericin during 1 hour before the endpoint of the tests. The results are representative of three independent experiments performed in triplicate. Data are expressed as mean ± SD: p <0.05 a vs. LPS + NIG, b vs. LPS.