Chagas Disease Megaesophagus Patients Carrying Variant MRPS18B P260A Display Nitro-Oxidative Stress and Mitochondrial Dysfunction in Response to IFN-γ Stimulus

Figures

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  Figure 1. Schematic drawing describing the workflow of analysis.

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  Figure 2. Sanger chromatograms of patients with or without the damaging variant MRPS18B. The confirmatory sequencing was performed by Sanger sequencing. The DNA of each patient was extracted using QIAamp DNA Blood Maxi Kit (Qiagen, Germantown, WI, USA) and 25 ng of DNA was used for the amplification of MRPS18B gene. PCR quality was assessed by gel electrophoresis and all samples generated a single band corresponding to MRPS18B. Then, samples were sequenced by Sanger sequencing by Genewiz. Representative images of Sanger chromatograms of the 5 patients reveal the change in the nucleotide. The red arrow indicates the base change from cytosine to guanine.

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  Figure 3. ATP production and nitro-oxidative stress after IFN-γ treatment in carriers or non-carriers of MRPS18B c.688 C > G MRPS18B p. P230A mitochondrial gene mutation. EBV-LCL were obtained from the PBMCs of the patients and infected with EBV for transformation. The EBV-LCL from the patients were stimulated in vitro with IFN-γ. We measured the production of nitrite (A), carried out relative quantification of nitrated proteins (B), ATP (C) and mtROS (D), after the stimulation with IFN-γ 25 ng/mL with MitoSOX™ Red, Griess Reagent Kit, Dot Blot, ATP Determination Kit and the anti-3-nitrotyrosine antibody. We here depict the average ratio between IFN-γ-treated vs. unstimulated cells. Each dot is a patient with ≥6 independent measurements. Experiments were repeated several times independently. As we had only one CME patient G/G, statistics were carried out only between C/C patients and C/G patients. Two-way ANOVA statistics were performed between C/C and C/G. ** = p < 0.01, **** = p < 0.0001.

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  Figure 4. (A). Mitochondrial ROS increase in IFN-γ-treated EBV-LCL from CME07 (homozygous MRPS18B G/G). A total of 15 × 104 EBV-LCL cells were stimulated with IFN-γ 25 ng/mL for 24 h and cell nuclei were stained with Hoechst 33342 (blue), the nuclei of dead cells were stained with ReadyProbes™ NucGreen (green), also used to measure cell viability, and superoxide was measured with MitoSOX™ (red) (B). Positive control: 560 µM of antimycin A. Magnification 200×. NS: not stimulated. Statistical analysis performed using the Mann–Whitney test. ** = p < 0.01.

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  Figure 5. Viability of EBV-LCL cells after treatment with IFN-γ for 24 h. The cell viability of patient-specific EBV-LCL was quantified using the ReadyProbes™ NucGreen dye after 24 h of treatment with IFN-γ (25 ng/mL). Cell viability of EBV-LCL from patients with EMC with the mutation in HOM (G/G), HET (C/G) and non-carriers (C/C). Each dot is an independent measurement. Statistical analysis performed using the Mann–Whitney test.