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Praveen Yerramothu, Ajay Kumar Vijay, Mark Willcox; Macrophage polarization in bacterial keratitis: A link with NLRP3 inflammasome. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3319. doi: https://doi.org/.
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Macrophages play important role in inflammation and infection. Macrophages are known to produce the NLRP3 inflammasome, which produces IL-1β a key pro-inflammatory mediator. Macrophages can polarise into M1, which associated with production inflammation, and M2, associated with resolution of disease, types. The polarisation of macrophages during P. aeruginosa keratitis is not known, nor is the consequence of inhibiting the production of the NLRP3 inflammasome, and so these factors were studied in the current study.
Eyes of 8-10-week-old female BALB/c and C57BL/6 mice were infected with Pseudomonas aeruginosa (6294, PAo1, 6206 and ATCC 19660). The NLRP3 inflammasome inhibitor MCC950 (10µL) or sterile phosphate buffered saline (PBS; 10µL) was topically applied twice a day to the infected eye. Eyes were collected 24 and 72 hours post infection to perform immunostaining of macrophages and to determine the levels of IL-1β by immunosorbent assay. Macrophages were manually enumerated by a masked observer using Image J 1.50b software.
Both M1 and M2 polarized macrophages were identified in both mice strains infected with P. aeruginosa 6294, 6206 and ATCC 19660 after 72 hours. Mice infected with P. aeruginosa o1 only had M1 macrophages in their corneas after 72 hours. In mice infected with all the P. aeruginosa strains there was a significant reduction (p<0.05) in macrophage numbers (In BALB/c, PBS vs MCC950; PA 6294 1296±434 vs 69.1±9; ATCC 19660 1879±580 vs 182.6±38.5) after MCC950 treatment. This reduction in macrophage numbers was due to significant inhibition (p<0.05) in the numbers (PA 6294 512±98 vs 45±6; ATCC 19660 1171.25±396.33 vs 113±12) of M1 macrophages. In comparison, the numbers of M2 macrophages significantly increased (p<0.05) after MCC950 treatment. M1 and M2 polarization only occurred at the 72-hour time point, not after 24 hours of infection. The levels of IL-1β produced during infection were significantly lower (p<0.05) after MCC950 treatment with all the strains of P. aeruginosa at 24 (BALB/c, ATCC 19660, 266.07±7098.62 vs 3954.6±263.6) and 72 hours (15162.18±9450 vs 8812.2±1378.33).
Macrophages entre the cornea during P. aeruginosa keratitis in mice, and M1 and M2 polarization occurs 24 hours after infection. The NLRP3 inflammasome may play an important role in macrophage polarization.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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