Inhibitory effect of human meibomian gland epithelial cells on the growth rate of Pseudomonas aeruginosa.

Abstract

Purpose : We recently discovered that human meibomian gland epithelial cells (HMGECs) are enriched with genes encoding antimicrobial peptides, including leukocyte-associated immunoglobulin-like receptor-1, S100 calcium binding proteins A8 and 9 (i.e. calprotectin), elafin, and secretory leukocyte peptidase inhibitor. We hypothesize that peptides such as these, if translated, would account for the apparent HMG resistance to bacterial infection in obstructive MG dysfunction, the leading cause of dry eye disease. To begin to test this hypothesis, we examined whether HMGECs have the ability to suppress the growth rate of the gram-negative bacteria, Pseudomonas (P.) aeruginosa, in vitro.

Methods : Immortalized (I) HMGECs were cultured in serum-containing or serum-free media for 7 days. Harvested cells were centrifuged, suspended in 1 ml of low osmolarity solution, incubated at 20^oC, then lyophilized. Reconstituted lysate samples (30 ul of 1 mg/ml protein) in sextuplicate were added to 96-well plates, followed by addition of 120 ul of P. aeruginosa 6294 (generous gift of Dr. Mihaela Gadjeva, Brigham and Women's Hospital, Boston, MA), which had been grown in LB (Luria-Bertani) media overnight at 37^oC, and diluted to an optical density at 600 nm (OD600) of 0.2. Plates were incubated at 37^oC, and bacterial growth was monitored by a turbidity assay with periodic OD600 readings in a spectrophotometer. Experiments were performed three times and included positive (growth inhibition with gentamycin) and negative (vehicle and non-specific protein) controls.

Results : Our results demonstrate that IHMGEC lysates inhibit the growth rate of P. aeruginosa in vitro. After 4.5 hours of incubation at 37 ^oC, the IHMGEC lysates had induced a significant (p<0.05), 17% decrease in the rate of bacterial growth as compared to that in control cultures. Following 15.6 hours of bacterial exposure, the IHMGEC lysate had significantly (p<.0001) reduced the P. aeruginosa growth rate by 21%. This ability of IHMGECs to suppress bacterial growth was expressed by cells that were cultured in serum-containing (i.e. differentiating), but not serum-free (i.e. proliferating), media.

Conclusions : Our findings support our hypothesis that IHMGECs contain antimicrobial activity. In future studies, we will aim to identify the intracellular factors of HMGECs capable of inhibiting bacterial growth.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.