Leishmania kinetoplast DNA contributes to parasite burden in infected macrophages: Critical role of the cGAS-STING-TBK1 signaling pathway in macrophage parasitemia

Abstract

Leishmania parasites harbor a unique network of circular DNA known as kinetoplast DNA (kDNA). The role of kDNA in leishmania infections is poorly understood. Herein, we show that kDNA delivery to the cytosol of Leishmania major infected THP-1 macrophages provoked increased parasite loads when compared to untreated cells, hinting at the involvement of cytosolic DNA sensors in facilitating parasite evasion from the immune system. Parasite proliferation was significantly hindered in cGAS- STING- and TBK-1 knockout THP-1 macrophages when compared to wild type cells. Nanostring nCounter gene expression analysis on L. major infected wild type versus knockout cells revealed that some of the most upregulated genes including, Granulysin (GNLY), Chitotriosidase-1 (CHIT1), Sialomucin core protein 24 (CD164), SLAM Family Member 7 (SLAMF7), insulin-like growth factor receptor 2 (IGF2R) and apolipoprotein E (APOE) were identical in infected cGAS and TBK1 knockout cells, implying their involvement in parasite control. Amlexanox treatment (a TBK1 inhibitor) of L. major infected wild type cells inhibited both the percentage and the parasite load of infected THP-1 cells and delayed footpad swelling in parasite infected mice. Collectively, these results suggest that leishmania parasites might hijack the cGAS-STING-TBK1 signaling pathway to their own advantage and the TBK1 inhibitor amlexanox could be of interest as a candidate drug in treatment of cutaneous leishmaniasis.

Keywords: 2'3'-cGAMP; H151; STING; TBK1; amlexanox; cGAS; kinetoplast DNA (kDNA); leishmania.

Figure 1

Effect of Cytosolic kDNA delivery on in vitro Leishmania infection. (A) AFM images of the purified kDNA networks with scanning areas of 5x5 μm (left) and 2.5x2.5 μm (right). PMA-differentiated THP-1 cells (5x10⁵ cells/ml) were incubated with CFSE labeled parasites (5x10⁶ parasites/ml) for 24 hours in the presence or absence of kDNA. Percentage of infected cells (B) and total parasite loads (C) were evaluated by flow cytometry. Numbers on x-axis labels indicate concentration of kDNA (μg/ml). Data represent the average of two independent experiments ± SD. All groups were compared statistically by one-way ANOVA followed by Dunnet`s multiple comparison test. (D) Fluorescence microscopy images of infected, SYTO Green stained THP-1 cells (from left to right, 10X 20X and 40X) untreated (upper panels) or treated with 1 μg/ml kDNA. Images are representatives of two independent experiments. ns=not significant, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001

Figure 2

Kinetics of Leishmania infection in WT versus cGAS-, STING- or TBK1-knockout THP-1 cells. Low dose [(A) and (B) or high dose (C) and (D)] PMA differentiated THP-1 cells were infected with eGFP expressing L. major parasites at a MOI of 1:10 (macrophage:parasite). At indicated time points (6,24,48 and 72 h), infection percentages and parasite loads were quantified using a flow cytometer. Data presented in graphs represent the average of two independent experiments with technical replicates ± SD. Statistical comparisons to WT cells were based on two-way ANOVA followed by Tukey’s multiple comparison test. ns, not significant, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 3

Effect of the STING agonist 2’3’-cGAMP, the STING antagonist H151 and recombinant IFN-β on in vitro Leishmania infection. Differentiated WT [(A), (B) and (C)], cGAS- [(D)] or STING- (E) knockout THP-1 cells were infected with eGFP expressing L. major parasites at a MOI of 1:10 (macrophage:parasite). Infection percentages and parasite loads were quantified using a flow cytometer 24 h later. H151 [2 or 4 µg/ml; (A) and (B)], cGAMP [10, 20 or 40 µg/ml; (B), (C), (D) and (E)] or recIFN-β [50 ng/ml; (C), (D) and (E)] were added 8, 2 and 8 hours prior to infection, respectively. Data represent average ± SD. All groups were compared statistically by one-way ANOVA followed by Dunnet`s multiple comparison test. ns, not significant, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 4

Differentially expressed genes in infected WT versus cGAS and TBK1 knockout cells. (A) Volcano plots of differentially expressed genes between L. major infected WT and knockout cell lines (n=3). Upregulated (fold change ≥ 2) and downregulated (fold change ≤ 2) genes are shown in red and blue, respectively. Dashed lines represent p=0.01 and dotted lines show p=0.05. The log2FC is plotted on the x-axis, and the negative log10FDR (adjusted p-value) is plotted on the y-axis. (B) Heatmaps showing the top 10 most up- or down-regulated genes in the infected knockout cells with respect to infected WT controls. Six of the shared upregulated genes and 4 of the shared downregulated genes are enclosed in boxes (red boxes, upregulated; blue boxes, downregulated). Statistical analysis for differentially expressed genes were performed using nSolver Analysis software (v4, NanoString Technologies, Inc., U.S.A.) and nCounter^® Advanced Analysis Software (v2, NanoString Technologies, Inc., U.S.A.).

Figure 5

Amlexanox-induced inhibition of L. major infection in WT THP1 cells in a 3-day infection model. Differentiated THP-1 cells were infected with eGFP expressing L. major parasites at a MOI of 1:10 (macrophage:parasite). Infection percentages and parasite loads were quantified using a flow cytometer at the indicated time points. Amlexanox was added either 1 hour prior to infection or 24 h post infection (late amlexanox). (A) and (B) show representative flow cytometry density and histogram plots, corresponding to infected cell percent and parasite load, respectively. (C) Data presented in graphs are representative of two independent experiments with similar results Amlexanox was added at different time points in treated groups (purple, 1 h pre-infection; red, 24 h post-infection). 72 h data were statistically compared to untreated group using one-way ANOVA followed by Bonferroni’s multiple comparison test. (D) Footpad swelling in L. major infected mice treated or not with amlexanox starting on day 41 post-infection for or a duration of 12 days. Results were based on digital caliper measurement of footpad depth and width of DMSO injected controls (n=3) and amlexanox injected (n=4) treatment group. Data were statistically compared using multiple unpaired t tests. ns, not significant, *p≤0.05, ***p≤0.001, ****p≤0.0001.