Clearance of Pseudomonas aeruginosa from a Healthy Ocular Surface Involves Surfactant Protein D and Is Compromised by Bacterial Elastase in a Murine Null-Infection Model

School of Optometry, University of California, Berkeley, CA 94720-2020, USA.
Infection and immunity (Impact Factor: 3.73). 04/2009; 77(6):2392-8. DOI: 10.1128/IAI.00173-09
Source: PubMed


Our previous studies showed that surfactant protein D (SP-D) is present in human tear fluid and that it can protect corneal
epithelial cells against bacterial invasion. Here we developed a novel null-infection model to test the hypothesis that SP-D
contributes to the clearance of viable Pseudomonas aeruginosa from the healthy ocular surface in vivo. Healthy corneas of Black Swiss mice were inoculated with 107 or 109 CFU of invasive (PAO1) or cytotoxic (6206) P. aeruginosa. Viable counts were performed on tear fluid collected at time points ranging from 3 to 14 h postinoculation. Healthy ocular
surfaces cleared both P. aeruginosa strains efficiently, even when 109 CFU was used: e.g., <0.01% of the original inoculum was recoverable after 3 h. Preexposure of eyes to bacteria did not enhance
clearance. Clearance of strain 6206 (low protease producer), but not strain PAO1 (high protease producer), was delayed in
SP-D gene-targeted (SP-D−/−) knockout mice. A protease mutant of PAO1 (PAO1 lasA lasB aprA) was cleared more efficiently than wild-type PAO1, but this difference was negligible in SP-D−/− mice, which were less able to clear the protease mutant. Experiments to study mechanisms for these differences revealed that
purified elastase could degrade tear fluid SP-D in vivo. Together, these data show that SP-D can contribute to the clearance
of P. aeruginosa from the healthy ocular surface and that proteases can compromise that clearance. The data also suggest that SP-D degradation
in vivo is a mechanism by which P. aeruginosa proteases could contribute to virulence.

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    • "We have previously shown that SP-D, a member of the collectin family of innate defense molecules, is present in tear fluid and the corneal epithelium and plays a role in ocular defense against P. aeruginosa [34], [36]. Thus, SP-D levels were assessed in EDE mice and controls after 5 days of EDE induction, and before and after (6 h) inoculation with 109 cfu PAO1. "
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    ABSTRACT: Dry eye disease can cause ocular surface inflammation that disrupts the corneal epithelial barrier. While dry eye patients are known to have an increased risk of corneal infection, it is not known whether there is a direct causal relationship between these two conditions. Here, we tested the hypothesis that experimentally-induced dry eye (EDE) increases susceptibility to corneal infection using a mouse model. In doing so, we also examined the role of surfactant protein D (SP-D), which we have previously shown is involved in corneal defense against infection. Scopolamine injections and fan-driven air were used to cause EDE in C57BL/6 or Black Swiss mice (wild-type and SP-D gene-knockout). Controls received PBS injections and were housed normally. After 5 or 10 days, otherwise uninjured corneas were inoculated with 10(9) cfu of Pseudomonas aeruginosa strain PAO1. Anesthesia was maintained for 3 h post-inoculation. Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation. SP-D was measured by Western immunoblot, and corneal pathology assessed from 6 h to 4 days. EDE mice showed reduced tear volumes after 5 and 10 days (each by ∼75%, p<0.001) and showed fluorescein staining (i.e. epithelial disruption). Surprisingly, there was no significant difference in corneal pathology between EDE mice and controls (∼10-14% incidence). Before bacterial inoculation, EDE mice showed elevated SP-D in ocular washes. After inoculation, fewer bacteria were recovered from ocular washes of EDE mice (<2% of controls, p = 0.0004). Furthermore, SP-D knockout mice showed a significant increase in P. aeruginosa corneal colonization under EDE conditions. Taken together, these data suggest that SP-D contributes to corneal defense against P. aeruginosa colonization and infection in EDE despite the loss of barrier function to fluorescein.
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    • "Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that does not normally infect healthy epithelial-lined tissue surfaces in vivo1. However, epithelial cells derived from these tissues become exquisitely sensitive to a variety of virulence mechanisms of P. aeruginosa and other opportunistic pathogens if grown in cell culture media in vitro23. "
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    ABSTRACT: Mucosal epithelial cells, including those at the ocular surface, resist infection by most microbes in vivo but can be susceptible to microbial virulence in vitro. While fluids bathing mucosal surfaces (e.g. tears) contain antimicrobials, potentially pathogenic microbes often thrive in these fluids, suggesting that additional mechanisms mediate epithelial resistance in vivo. Here, tear fluid acted directly upon epithelial cells to enhance their resistance to bacterial invasion and cytotoxicity. Resistance correlated with tear fluid-magnified activation of NFκB and AP-1 transcription factors in epithelial cells in response to bacterial antigens, suggesting priming of innate defense pathways. Further analysis revealed differential regulation of potential epithelial cell defense genes by tears. siRNA knockdown confirmed involvement of at least two factors, RNase7 and ST-2, for which tears increased mRNA levels, in protection against bacterial invasion. Thus, the role of mucosal fluids in defense can include modulation of epithelial immunity, in addition to direct effects on microbes.
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