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Surfactant protein D contributes to ocular defense against Pseudomonas aeruginosa in a murine model of dry eye disease.

Heimer SR, Evans DJ, Mun JJ, Stern ME, Fleiszig SM - PLoS ONE (2013)

Bottom Line: Anesthesia was maintained for 3 h post-inoculation.Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation.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.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, University of California, Berkeley, California, United States of America.

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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SP-D expression in EDE before and after P.aeruginosa challenge.Western immunoblot blot analysis of SP-D expression in pooled ocular surface washes from EDE and control mice (10 mice per group) after 5 days EDE induction, and before and 6 h after inoculation with P. aeruginosa strain PAO1 (109 cfu). To normalize for differences in tear volume, equivalent amounts of protein (2 µg) were used in the analysis (BCA protein assay). Purified recombinant SP-D (rSP-D, ∼43 kDa monomer), and a relevant number of bacteria suspended in PBS (5×103 cfu, see Fig. 2B), were included as positive and negative controls, respectively. SP-D expression in ocular surface washes was increased under EDE conditions before bacterial inoculation. The experiment was repeated once.
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pone-0065797-g003: SP-D expression in EDE before and after P.aeruginosa challenge.Western immunoblot blot analysis of SP-D expression in pooled ocular surface washes from EDE and control mice (10 mice per group) after 5 days EDE induction, and before and 6 h after inoculation with P. aeruginosa strain PAO1 (109 cfu). To normalize for differences in tear volume, equivalent amounts of protein (2 µg) were used in the analysis (BCA protein assay). Purified recombinant SP-D (rSP-D, ∼43 kDa monomer), and a relevant number of bacteria suspended in PBS (5×103 cfu, see Fig. 2B), were included as positive and negative controls, respectively. SP-D expression in ocular surface washes was increased under EDE conditions before bacterial inoculation. The experiment was repeated once.

Mentions: 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. To account for differences in tear volume, equivalent amounts of total protein from each sample were used for analyses. EDE mice showed increased expression of SP-D in ocular surface washes compared to normal controls prior to bacterial inoculation (Fig. 3). This difference was not seen the post-inoculation ocular surface washes, although the latter samples did contain an additional form of SP-D which we have also observed in a previous study [34]. The antibody against SP-D did not react with bacteria alone. These data show that EDE also increases expression of SP-D at the murine ocular surface.


Surfactant protein D contributes to ocular defense against Pseudomonas aeruginosa in a murine model of dry eye disease.

Heimer SR, Evans DJ, Mun JJ, Stern ME, Fleiszig SM - PLoS ONE (2013)

SP-D expression in EDE before and after P.aeruginosa challenge.Western immunoblot blot analysis of SP-D expression in pooled ocular surface washes from EDE and control mice (10 mice per group) after 5 days EDE induction, and before and 6 h after inoculation with P. aeruginosa strain PAO1 (109 cfu). To normalize for differences in tear volume, equivalent amounts of protein (2 µg) were used in the analysis (BCA protein assay). Purified recombinant SP-D (rSP-D, ∼43 kDa monomer), and a relevant number of bacteria suspended in PBS (5×103 cfu, see Fig. 2B), were included as positive and negative controls, respectively. SP-D expression in ocular surface washes was increased under EDE conditions before bacterial inoculation. The experiment was repeated once.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675081&req=5

pone-0065797-g003: SP-D expression in EDE before and after P.aeruginosa challenge.Western immunoblot blot analysis of SP-D expression in pooled ocular surface washes from EDE and control mice (10 mice per group) after 5 days EDE induction, and before and 6 h after inoculation with P. aeruginosa strain PAO1 (109 cfu). To normalize for differences in tear volume, equivalent amounts of protein (2 µg) were used in the analysis (BCA protein assay). Purified recombinant SP-D (rSP-D, ∼43 kDa monomer), and a relevant number of bacteria suspended in PBS (5×103 cfu, see Fig. 2B), were included as positive and negative controls, respectively. SP-D expression in ocular surface washes was increased under EDE conditions before bacterial inoculation. The experiment was repeated once.
Mentions: 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. To account for differences in tear volume, equivalent amounts of total protein from each sample were used for analyses. EDE mice showed increased expression of SP-D in ocular surface washes compared to normal controls prior to bacterial inoculation (Fig. 3). This difference was not seen the post-inoculation ocular surface washes, although the latter samples did contain an additional form of SP-D which we have also observed in a previous study [34]. The antibody against SP-D did not react with bacteria alone. These data show that EDE also increases expression of SP-D at the murine ocular surface.

Bottom Line: Anesthesia was maintained for 3 h post-inoculation.Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation.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.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, University of California, Berkeley, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus