Transcriptomics analysis highlights potential ways in human pathogenesis in Leishmania braziliensis infected with the viral endosymbiont LRV1

Abstract

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression.

Fig 1. In vitro macrophage infection with L (V.) braziliensis with and without the viral endosymbiont (LRV).

(A) human monocyte-derived macrophages infected with LbLRV1+; (B) human monocyte-derived macrophages infected with LbLRV1-; (C) control and (D) phagocytic index. Values represent the mean and standard error of 3 donors. *P<0.05, compared to the presence of the LRV1 viral endosymbiont (data presented with T-test).

Fig 2. Venn diagram of the compared groups.

Similarities between groups are represented in the overlapping portions of the circles, while differences are represented in the non-overlapping portions of the circles from LbLRV1- vs. control, LbLRV1+ vs. control and LbLRV1+ vs. LbLRV1- groups showing inside the circles the upregulated genes in red and downregulated genes in blue.

Fig 3. Heatmap of the top 10 up and downregulated genes.

(A) Comparison between LbLRV1- vs control, (B) LbLRV1+ vs control, and (C) and LbLRV1+ vs. LbLRV1-. Genes with high expression are indicated in red and low expression are indicated in blue. Hierarchical clustering was performed using the Euclidean distance to obtain gene clusters.

Fig 4. Volcano plots of the top 10 up and downregulated genes from LbLRV1- vs.

LbLRV1- vs. control (A), LbLRV1+ vs. control (B), and LbLRV1+ vs. LbLRV1- (C). Upregulated genes are indicated in red and the downregulated ones in blue.

Fig 5. Heatmap of gene signature related to LRV1 presence.

The row z-score values of normalized expression were generated to emphasize the difference of each gene between the samples/groups. The groups and donors are represented on the top-horizontal side, while the pathways related to each gene are represented on the left-vertical side. Hierarchical clustering was performed using the Euclidean distance to obtain gene clusters.

Fig 6. Relative analysis of mRNA expression of the genes from the IFNI signaling pathway using RT-qPCR.

The ISG15, IFIT1, IFT2, IFT3, IFTM3, and IFI6 genes that were upregulated in the microarray were validated by RT-qPCR. The results were expressed as relative gene expression (Fold of GAPDH gene) and represent the mean ± SEM of three to five independent volunteers. *p<0.05 compared to control, and #p<0.05 compared to LbLRV1- (Data were presented with ANOVA followed by Tukey post-test).

Fig 7. Relative analysis of mRNA expression of the OAS/RNase L oligoadenylate synthase signaling pathway using RT-qPCR.

The OAS1, OAS2, OAS3, and OASL genes that were upregulated in the microarray were validated by RT-qPCR. The results were expressed as relative gene expression (Fold of GAPDH gene) and represent the mean ± SEM of three to five independent volunteers. *p<0.05 compared to control, and #p<0.05 compared to LbLRV1- (Data were presented with ANOVA followed by Tukey post-test).

Fig 8. CCL18 quantification by ELISA.

Supernatant from the incubation of human monocyte-derived macrophages infected with LbLRV1+, LbLRV1- or without Leishmania (control) was used to determine the cytokines levels. The results were expressed as pg/mL of CCL18 produced and represent the mean ± SEM of three to five independent volunteers. *p<0.05 compared to control, and #p<0.05 compared to LbLRV1- (Data were presented with ANOVA followed by Tukey post-test).

Fig 9. Th1, Th2, and Th17 cytokines quantification by CBA.

Supernatant from the incubation of human monocyte-derived macrophages infected with LbLRV1+, LbLRV1- or without Leishmania (control) was used to determine the cytokines levels. The results were expressed as mean fluorescence intensity (MFI) and represent the mean ± SEM of three to five independent volunteers. *p<0.05 compared to control, and #p<0.05 compared to LbLRV1- (Data were presented with ANOVA followed by Tukey post-test).

Fig 10. LDH quantification.

Supernatant from the incubation of human monocyte-derived macrophages infected with LbLRV1+, LbLRV1- or without Leishmania (control) was used to determine the LDH levels. The results were expressed as U/L of LDH produced and represent the mean ± SEM of three to five independent volunteers. *p<0.05 compared to control, and #p<0.05 compared to LbLRV1- (Data were presented with ANOVA followed by Tukey post-test).