Trypanosoma cruzi Dysregulates piRNAs Computationally Predicted to Target IL-6 Signaling Molecules During Early Infection of Primary Human Cardiac Fibroblasts

Figures

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  Figure 1. Mapping differentially expressed piRNAs to specific genetic regions of IL6 and SOCS3. The targets of differentially expressed piRNAs in PHCF challenged with T. cruzi were computationally predicted using the miRanda algorithm. In the genetic cartoons, exons represented by blocks (blue) are connected by horizontal lines representing introns, while the 5′ and 3′ UTRs are represented as thinner blocks (orange) at the extremities of each gene, respectively. (A) IL6 gene cartoon (5′ to 3′ orientation) showing the position where the differentially expressed piR_004506 is predicted to map. (B) Cartoon of SOCS3 gene (3′ to 5′ orientation) showing positions where known piRNAs piR_001356 and piR_017716 map to the 3′ UTR region of SOCS3.

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  Figure 2. Validation of piRNAs and target transcript expression during early T. cruzi infection in PHCF. Small and total RNAs purified from serum-starved PHCF monolayers challenged with invasive T. cruzi trypomastigotes for different time points were used to validate (A) IL6, (B) SOCS3 transcripts, (C) piR_004506, (D) piR_001356, and (E) piR_017716. Relative expression of each transcript was normalized against ribosomal protein lateral stalk subunit P0 and beta-2-microglobulin as housekeeping genes. Relative expression of each piRNA was normalized against U6. Each value is the mean of biological triplicates performed in technical duplicates ± SEM. Visualization was created, and statistical significance was calculated using Student’s t-test in GraphPad Prism. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

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  Figure 3. Cytokine array analysis evaluating IL-6 signaling proteins in condition media of T. cruzi challenged PHCF. Condition media of PHCF challenged with T. cruzi at multiple time points were incubated with cytokine array membranes C6 and C7. (A) Membranes showing the location of controls (green borders) cytokines and inflammatory molecules spotted onto a human cytokine array membranes. Signals of IL-6, IL-6R, and gp-130 at 0, 1, 3, and 6 h are indicated by numbers 1-3, respectively. Spots were quantified using ImageJ and normalized to positive control spots (green borders). Bar graphs depict fold change of (B) IL-6, (C) IL-6R, and (D) gp-130 mean spot densities relative to positive controls in condition media of PHCF T. cruzi challenged cells. Mean values of biological replicates ± SEM are shown. Visualization was created, and statistical significance was calculated using Student’s t-test in GraphPad Prism. *p<0.05, **p<0.01.

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  Figure 4. Expression of downstream IL-6 signaling proteins during early T. cruzi infection of PHCF. Whole cell lysates from PHCF challenged with T. cruzi at various time points were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with Abs against (A, upper panel) p-STAT3, and STAT3, (B, upper panel) SOSC3, and GADPDH and developed with the corresponding IRDye conjugated secondary Ab. The developed blots were scanned using the infrared fluorescence detection Odyssey Imaging System. The normalized fold changes were determined and plotted in the bar graph (A, lower panel) p-STAT3 Y705 and (B, lower panel) SOCS3. Graph bars represent mean values ± SEM from three independent biological replicates. Visualization was created, and statistical significance was calculated using Student’s t-test in GraphPad Prism. *p<0.05, ***p<0.001.

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  Figure 5. Biological interaction network of IL-6 signaling proteins. Network constructed based on genes from GeneMANIA using IL6, IL6R, gp130/IL6ST, STAT3, and SOCS3 as the query input. The gray connections represent protein-protein and pathway-level interactions between the query proteins and the expanded gene set. Predicted piRNA interactions are highlighted in orange. Molecular entities were categorized into type (node color), degree of connection to other nodes (node size), and molecular function (node shape).

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  Figure 6. Model of suggested IL-6 signaling during early T. cruzi infection of PHCF. IL-6 binds to sIL-6R to enact receptor activation. The complex binds to mbgp-130 receptor, which becomes activated and leads to JAK phosphorylation. Activated p-JAK phosphorylates STAT3 at tyrosine Y705 to facilitate homodimerization of p-STAT3 transcription factors. The p-STAT3 homodimer is translocate to the nucleus to initiate the transcription of target genes such as SOCS3. SOCS3 traditionally inhibits IL-6 signaling. However, increased piRNA expression potentially targets and decrease SOCS3 transcript and protein expression during early infection. Accordingly, increased expression of piRNA targeting IL-6 will attenuate IL-6 transcript, protein expression and secretion during early infection of PHCF. This image was created with the assistance of BioRender.