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Original Investigation |

Replication of Mycobacterium Tuberculosis in Retinal Pigment Epithelium

Hossein Nazari, MD1; Petros C. Karakousis, MD2,3; Narsing A. Rao, MD1
[+] Author Affiliations
1University of Southern California Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles
2Department of Medicine, The Johns Hopkins University, Baltimore, Maryland
3Department of International Health, The Johns Hopkins University, Baltimore, Maryland
JAMA Ophthalmol. 2014;132(6):724-729. doi:10.1001/jamaophthalmol.2014.270.
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Importance  Mycobacterium tuberculosis is an important cause of posterior uveitis in tuberculosis-endemic regions. Clinical and histopathologic evidence suggests that retinal pigment epithelium (RPE) can harbor M tuberculosis. However, the mechanism of M tuberculosis phagocytosis and its growth in RPE is not clear.

Objective  To investigate M tuberculosis phagocytosis, replication, and cytopathic effects in RPE cells compared with macrophages.

Design, Setting, and Participants  Human fetal RPE and monocytic leukemia macrophage (THP-1) cell lines were cultured, and RPE and THP-1 cells were exposed to avirulent M tuberculosis H37Ra. Mycobacteria were added to RPE and THP-1 cells with a 5:1 multiplicity of infection. Nonphagocytized M tuberculosis was removed after 12 hours of exposure (day 0). Cells were harvested at days 0, 1, and 5 to count live and dead cells and intracellular mycobacteria. Toll-like receptor 2 (TLR2) and TLR4 expression was determined by immunohistochemistry; intracellular bacillary load, following TLR2 and TLR4 blockade.

Main Outcomes and Measures  Number of intracellular M tuberculosis, cell survival, and TLR2 and TLR4 expression in RPE and THP-1 cells following exposure to M tuberculosis.

Results  At day 0, an equal number of intracellular M tuberculosis was observed per THP-1 and RPE cells (0.45 and 0.35 M tuberculosis per RPE and THP-1 cells, respectively). Mean (SD) number of intracellular M tuberculosis at day 5 was 1.9 (0.03) and 3.3 (0.01) per RPE and THP-1 cells, respectively (P < .001). Viability of infected RPE was significantly greater than that of THP-1 cells at day 5 (viable cells: 17 [8%] THP-1 vs 73% [4%] RPE; P < .05). Expression of TLR2 and TLR4 was detected in both cell types after 12 hours of exposure. Inhibition of TLR2 and TLR4 reduced intracellular M tuberculosis counts in RPE but not in THP-1 cells.

Conclusions and Relevance  Mycobacterium tuberculosis is phagocytized by RPE to a similar extent as in macrophages. However, RPE cells are better able to control bacillary growth and RPE cell survival is greater than that of THP-1 cells following mycobacterial infection, suggesting that RPE can serve as a reservoir for intraocular M tuberculosis infection.

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Figures

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Figure 1.
Intra–Retinal Pigment Epithelium (RPE) Mycobacteria

Mycobacteria were stained with auramine-rhodamine and viewed under a fluorescent microscope. The RPE nuclei were stained with 4′,6-diamidino-2-phenylindole, and RPE cells were infected using a multiplicity of infection of 5:1. Images were acquired 36 hours (A) and 132 hours (B) after exposure to Mycobacterium tuberculosis. Original magnification ×40.

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Figure 2.
Number of Intracellular Viable Mycobacterium in Monocytic Leukemia Macrophage Cell Line (THP-1) and Retinal Pigment Epithelium (RPE) Cells

The initial Mycobacterium tuberculosis to live RPE and M tuberculosis to live THP-1 ratio was equivalent, but the ratio of M tuberculosis per live cell increased more robustly in THP-1 macrophages compared with RPE at day 5 (132 hours) (P < .001). Colony-forming unit determination and cellular viability measurements were performed in triplicate cultures. Limit lines indicate SD.

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Figure 3.
The Percentage of Live Retinal Pigment Epithelium (RPE) and Monocytic Leukemia Macrophage Cell Line (THP-1) in Cultures Infected for 12 Hours With Mycobacterium tuberculosis H37Ra

Macrophage (THP-1) cultures showed a higher proportion of nonviable cells at all time points. Limit lines indicate SD.

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Figure 4.
Toll-like receptor 2 (TLR2) and TLR4 Expression in Retinal Pigment Epithelium (RPE) and Monocytic Leukemia Macrophage Cell Line (THP-1) Cultures

The RPE and THP-1 cultures did not express TLR2 and TLR4 when examined 6 hours after Mycobacterium tuberculosis exposure. The M tuberculosis–infected THP-1 and RPE cultures were immunostained with anti-TLR2 and anti-TLR4 antibodies and viewed under a confocal fluorescent microscope. Both RPE and THP-1 stained for TLR2 and TLR4 at the 12-hour time point. Original magnification ×40.

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Figure 5.
Intracellular Mycobacteria Count in Monocytic Leukemia Macrophage Cell Line (THP-1) and Retinal Pigment Epithelium (RPE) Cells With and Without Toll-Like Receptor 2 (TLR2) and TLR4 Blockade

Colony-forming units (CFUs) with and without TLR2 and TLR4 blockade 36 hours after exposure to Mycobacterium tuberculosis H37Ra. The TLR2 and TLR4 were blocked by pretreating cells with oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine for 6 hours before exposure to M tuberculosis H37Ra for 12 hours. Limit lines indicate SD.aP = .13.bP = .003.

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