Mycobacterium tuberculosis dormancy in retinal pigment epithelium

Abstract

Purpose : In tuberculosis uveitis, previous studies have shown Mycobacterium tuberculosis (Mtb) localization in retinal pigment epithelium (RPE). This study aims to demonstrate that Mtb expresses a dormant phenotype within RPE cells.

Methods : Mtb H37Ra cells (ATCC) were cultured in Middlebrook 7H9 broth. ARPE-19 cells (ATCC) were cultured in Dulbecco's Modified Eagle Medium and Ham’s Nutrient Mixture F-12 supplemented with 10% fetal bovine serum (FBS). THP-1 cells (ATCC) were cultured in Roswell Park Memorial Institute 1640 Medium with 10% FBS.

Cell cultures were treated daily with DETA-NONOate, a nitric oxide (NO) donor. Denatured DETA was used as a control. Serial dilutions of the treated cells were grown on Middlebrook 7H10 agar to determine colony forming units (CFU). A quantitative glucose-6-phosphate dehydrogenase (G6PD) cytotoxicity assay was performed to analyze the toxic effects of NO on RPE cells.

RPE cells were infected with Mtb in a midlogarithmic growth phase for 8 hours with a multiplicity of infection (MOI) of 10. Mtb-infected RPE cultures were treated daily with 500 μM DETA-NONOate. Cells were fixed, lysed, and mRNA was extracted. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to monitor tgs1 and DosR expression and compared to a control group. Mtb bacilli within RPE cells were stained with Auramine O and Nile Red to visualize triacylglyceride (TAG) accumulation.

Results : CFU data for Mtb showed that concentrations of approximately 250 μM of DETA-NONOate resulted in stable Mtb viability with no significant cytotoxicity to THP-1 cells (p=1.00) or RPE cells (p=0.65). RT-qPCR revealed a significant increase in the expression of two dormancy markers of Mtb, tgs1 and DosR, within RPE cells with further increased levels in DETA-NONOate-treated RPE.

Conclusions : The upregulated expression of tgs1 and DosR by Mtb organisms within RPE cells suggests that the RPE may provide an environment favorable to Mtb dormancy, particularly under oxidative stress. Reactivation of the dormant organisms may lead to recurrent uveitis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Mtb bacilli significantly increased expression of tsg1 after phagocytosis by RPE cells, as determined by RT-qPCR. Exposure to NO further increased tgs1 mRNA levels.

 

Mtb bacilli significantly increased expression of DosR genes after phagocytosis by RPE cells. Exposure to NO further increased DosR mRNA levels. DosR expression fell slightly after 2 days but remained above baseline.