Abstract
Purpose: :
The wet mucosal surface of the cornea is exposed to diverse and rapidly changing environmental factors. Under these conditions, corneal epithelial cells (CEC) provide a stable barrier against a wide range of commensal and environmental microorganisms, varying in pathogenicity. CECs also act as microorganism detectors, relaying information via paracrine signaling that is critical for ocular protection. We hypothesize that CECs can distinguish between organisms that are acutely pathogenic and non- to mildly pathogenic in order to engage a specific optimized response, geared toward limiting inflammatory damage. Staphylococcus aureus is a good example of an opportunistic pathogen capable of inducing ulcerative keratitis. S. aureus secretes a host of virulence factors which promote survival within the tear film and perturb normal cell structure/function. To test whether CECs can distinguish S. aureus from a non-pathogenic organism, the transcriptional programs elicited by S. aureus and Micrococcus luteus were compared. Additional analyses with isogenic wild type and non-toxigenic S. aureus strains were used to address the influence of toxins on the programmed response.
Methods: :
Monolayers of human CECs were co-cultured for 6 and 8h with S. aureus (RN6390, ALC135) and M. luteus (KC142). Treated cultures were evaluated for transcriptional changes relative to untreated controls using Affy GeneChip U133 Plus 2, Ingenuity Pathway Analysis software and RT-PCR.
Results: :
CECs produced significantly more STC1 and NDRG1 in response to S. aureus than the M. luteus (3-4x). Both gene products have been implicated in stabilizing endothelial barrier function. Increased expression was also noted in a GPC-regulator RGS2 (12x) and a transcriptional regulator NR4A2 (7x) involved in inflammation. S. aureus-treated CECs expressed lower levels of TNF (5x), HB-EGF (5x) and CXC1,2,3 (3-4x). cFOS expression was repressed 60x in M. luteus co-cultures relative to uninfected CECs but was unchanged by S. aureus. Diminished expression of several cFOS-regulated genes (EGR1, HMOX1, and NDRG1) was also noted. Toxins did not play a role in these programmed responses.
Conclusions: :
CECs have specific programmed responses to different microorganisms. S. aureus induces factors which likely stabilize barrier function and minimize strong proinflammatory signals like TNF.
Keywords: bacterial disease • Staphylococcus • keratitis