Topical Antibiotics Reduce CD11c+ Cell Numbers in the Healthy Murine Cornea and Modulate their Response to Contact Lens Wear

doi:10.21203/rs.3.rs-1304551/v1

Previously we reported contact lens-induced CD11c + cell responses in healthy mouse corneas, a phenomenon that also occurs in humans. To test involvement of ocular-associated bacteria, we examined the impact of topical antibiotics on corneal CD11c + cell populations during 24 h of lens wear. Corneas were treated with gentamicin and ofloxacin (0.3%) or gentamicin alone, some also treated prior to lens wear (24 h). Contralateral PBS-treated eyes served as controls. CD11c-YFP mice allowed CD11c + cell visualization. Viable ocular- or lens-associated bacteria were labeled using FISH (16S rRNA-targeted probe) or click-chemistry (alkDala). Antibiotic treatment reduced baseline CD11c + cell numbers without lens wear and suppressed CD11c + cell responses to lens wear if corneas were both pretreated and treated during wear. Few bacteria colonized corneas or lenses under any circumstances. Conjunctival commensals were significantly reduced by antibiotics with or without lens wear, but minimally impacted by lens wear alone. Deliberate inoculation with conjunctival commensals triggered CD11c + cell responses irrespective of antibiotic pretreatment. These results suggest that while lens wear does not necessarily increase quantifiable numbers of conjunctival commensals, those neutralized by antibiotics play a role in lens-associated CD11c + cell responses and maintaining baseline CD11c + cell populations.

The healthy cornea of the eye is remarkably resistant to bacterial infection but can be rendered susceptible by injury or contact lens wear. Research by us and others has shown that this resistance depends on a constitutive defensive combination of tear fluid, epithelial and basal lamina barrier function, and the ability of resident immune cells to detect and respond to injury, microbes or their ligands¹. In this regard, the cornea contains a small resident population of bone-marrow derived dendritic cells and macrophages in the stroma and epithelium^2,3, with centrally-located stromal dendritic cells being primarily immature precursors that can mature and acquire MHC Class II expression during inflammatory events ^2,3.

The mechanisms by which contact lens wear predisposes to corneal infection by P. aeruginosa and other bacteria are yet to be resolved. Recently, we reported the development of a murine model of contact lens-associated P. aeruginosa keratitis in which lens wear was associated with a corneal para-inflammatory response involving CD11c+ (dendritic cell) infiltration/activation by 24 h and Ly6G + cell (neutrophil) infiltration after 5–6 days ⁴, despite corneas remaining free of visible pathology. Importantly, contact lens-associated corneal para-inflammatory responses involving Langerhans cells have also been reported in humans ^5,6.

Previously, we showed that CD11c + cells in murine corneas respond to P. aeruginosa challenge in vivo, contributing to IL-1R-dependent epithelial barrier function against bacterial adhesion after superficial injury ⁷. Here, we sought to determine the role of bacteria in triggering corneal CD11c + responses to contact lens wear. While healthy corneas do not host resident viable bacteria ⁸, adjacent tissues are colonized with a microbiome. For example, the conjunctiva hosts numerous resident bacteria including Staphylococcus spp, and Corynebacterium spp. ^9,10, the latter shown to be important for corneal immune responses during active infectious disease ¹¹. Contact lens wear can potentially alter bacterial colonization in the conjunctiva ¹², and cornea ¹³, including via lens binding ^14,15. Indeed. in the murine lens-wear model, and in humans, worn lenses were found colonized by bacteria typical of those found on the skin and conjunctiva^4,14,16. To explore the involvement of bacteria in corneal CD11c + responses to lens wear, CD11c+-YFP mice were treated with topical antibiotics with and without contact lens wear, and imaging used to quantify CD11c + responses in addition to conjunctival and lens colonization by viable bacteria. Results showed that topical antibiotic treatment can reduce resident CD11c + cell numbers and suppress their responses to contact lens wear. They also showed that corneal CD11c + cell responses could be induced in healthy corneas using conjunctival commensals. Taken together, these results suggest the involvement of commensal bacteria in driving the CD11c + cell responses.

Topical antibiotic application decreases CD11c + cell numbers in the healthy murine cornea. Prior to exploring topical antibiotic effects on contact lens-associated CD11c + responses, a control experiment was performed to determine if antibiotics affected baseline numbers of resident corneal CD11c + cells. CD11c-YFP mice were treated topically with a cocktail of gentamicin and ofloxacin (0.3% of each in solution), or gentamicin alone, from 0–6 h and corneal CD11c + cell numbers imaged and quantified at 24 h (Methods Procedure 1). Figure 1 shows that both antibiotic treatments caused a reduction of corneal CD11c + cell numbers versus PBS-treated contralateral controls. PBS (44 ± 4 cells) versus antibiotic cocktail (28 ± 3 cells, 6 % decrease) or gentamicin (33 ± 2 cells,25 % decrease) (P < 0.0001 One-Way ANOVA Tukey's multiple comparisons test, Fig. 1a & b). There was no significant difference between antibiotic cocktail and gentamicin alone. FISH labeling confirmed an absence of viable bacteria on antibiotic-treated corneas as for healthy murine corneas (Supplemental Fig. S1).

Topical gentamicin application modulates contact lens-associated CD11c + cell responses. Having shown that gentamicin alone was not significantly different from its combination with ofloxacin, here we used gentamicin to explore the role of bacteria in contact lens-associated CD11c + cell responses (increased number and changes in morphology) in the murine cornea after 24 h of lens wear ⁴. We tested if topical gentamicin treatment could abrogate that response. Mice were divided into PBS or gentamicin treated groups and fitted with a contact lens on one eye for 24 h (Methods Procedure 2). Contralateral eyes served as no lens wear controls. Figure 2 shows that 24 h of contact lens wear was associated with a significant increase in CD11c + cell numbers in both the PBS control group (~ 44% increase) and the gentamicin-treated group (~ 36% increase) (Fig. 2a & b). However, no significant change in CD11c + cell morphology (circularity) was observed under any condition (Fig. 2c). Thus, despite a reduction in baseline CD11c + cells with gentamicin treatment, contact lens-induced CD11c + cell responses after 24 h remained intact.

Since potential antibiotic effects may require extended exposure time, the experiment shown in Fig. 2 was repeated but eyes were first pretreated with either PBS or gentamicin (Methods Procedure 3). Under these conditions, topical gentamicin blocked the 24 h contact lens-associated corneal CD11c + cell response (Fig. 3a & b). If only gentamicin pretreatment was applied, the 24 h contact lens-associated CD11c + cell response remained intact. This experiment also showed that the reduction in corneal CD11c + cells induced by topical gentamicin was maintained for 24 h after antibiotic removal. As above, no significant differences in CD11c + cell morphology were detected between groups (Fig. 3c).

Antibiotic treatment reduced the number of viable conjunctival bacteria detected by alkDala labeling with and without contact lens wear. In both of the above experiments (Figs. 2 & 3), FISH or alkDala labeling of bacteria on the cornea revealed very few bacteria under all conditions with no significant differences between PBS or gentamicin treated groups and with or without contact lens wear for 24 h (Supplemental Fig. S2), a finding consistent with our previous work ^4,8,17. Also consistent with our previous work, FISH labeling of worn lenses revealed bacterial binding to worn lenses but without a difference between groups (Supplemental Fig. S2). Thus, increases in corneal CD11c + cell numbers associated with 24 h of contact lens wear (Figs. 2 & 3) and their suppression by antibiotic treatment (Fig. 3) were not associated with any changes in the number of viable bacteria detected by FISH on the cornea or worn lenses.

Thus, the adjacent conjunctiva was examined using the same experimental conditions. AlkDala labeling alone was used given its distinctive (bacteria-specific) labeling of the filamentous conjunctival bacteria in mouse eyes observed in our previous studies^8,17, and because we previously found FISH to be problematic for the conjunctiva. The filamentous nature of the conjunctival bacterial population required quantifying area of alkDala florescence per field of view rather than individual bacterial cells. As shown in Fig. 4, there was a significant decrease in bacterial fluorescence in gentamicin treated eyes versus PBS controls with and without contact lens wear, showing a reduction in the population detectable. These results suggested a potential relationship between the conjunctival population of commensals and the corneal CD11c + cell responses during gentamicin treatment with and without lens wear (comparing Fig. 4a & b with Figs. 2b & 3b, respectively).

Addition of commensal conjunctival bacteria restores CD11c + cell numbers in antibiotic treated murine corneas. Having found that changes in corneal CD11c + cells associated with gentamicin treatment and contact lens wear correlated with the detection of viable conjunctival bacteria using alkDala labeling, we next explored if we could complement the CD11c + response using conjunctival commensal inoculation. Thus, post-gentamicin treated eyes (healthy corneas) were inoculated in vivo with Gram-positive commensals (mouse-derived C. mastitidis, and human-derived coagulase-negative staphylococci). We also used the opportunistic pathogen P. aeruginosa that we previously showed can induce CD11c + cell responses in mouse corneas⁷. After 4 h exposure to ∼10¹¹ CFU/mL bacteria, corneal CD11c + cell numbers were quantified ex vivo. Fig. 5 shows that the exposure of previously gentamicin-treated eyes to Gram-positive commensal bacteria restored corneal CD11c + numbers to the same level as control non-antibiotic treated eyes after bacterial challenge, i.e. showing that the reduction in CD11c + cells induced by prior antibiotic treatment could be complemented by the addition of conjunctival commensals (Fig. 5). While P. aeruginosa also increased corneal CD11c + cell numbers, levels in gentamicin treated eyes, remained below those of control untreated eyes suggesting a commensal is needed to complement the CD11c + cell response after antibiotic treatment.

Previously, we reported development of a murine model of contact lens wear in which we demonstrated lens-associated corneal para-inflammation after 5–6 days of wear, that was preceded by infiltration and activation of corneal CD11c+ (dendritic) cells after 24 h ⁴. The latter response mimicked those observed for Langerhans cells during human lens wear ^18,19. Here, we investigated the potential role of microbial triggers in contact lens-induced CD11c + responses in the murine model. Using topical application of broad-spectrum antibiotics to target ocular-associated bacteria with and without lens wear, results showed that antibiotic exposure reduced baseline CD11c + cell levels in healthy corneas after 24 h and suppressed corneal CD11c + cell responses to 24 h of contact lens wear but only if corneas were pretreated with antibiotic and treatment continued during lens wear. In both cases, this correlated with reduced numbers of viable conjunctival-associated bacteria. Adding commensal bacteria to antibiotic-treated eyes restored corneal CD11c + cell numbers: not the case with addition of the potential pathogen P. aeruginosa. Together these findings suggest a role for ocular-associated commensal flora in maintaining CD11c + cell homeostasis and in enabling contact lens-associated para-inflammation. They also appear to be important for the induction of the CD11c + response to P. aeruginosa challenge which we previously showed contributes to resisting P. aeruginosa colonization if the cornea is superficially-injured⁷.

How might topical antibiotics mediate the CD11c + phenotypes observed in this study? While a combination of ofloxacin and gentamicin was used initially, gentamicin alone was found effective. This is an aminoglycoside antibiotic, with bactericidal activity against many Gram-negative bacteria and some medically-important Gram-positive bacteria²⁰. Since there are very few viable bacteria on the murine cornea⁸ (confirmed in the present study), or on contact lenses, the most likely mechanism is that the antibiotic interfered with conjunctival commensals, either directly reducing their viability and/or interfering with their growth or metabolism. Indeed, there was a significant decrease in conjunctival alkDala (peptidoglycan cell wall) labeling after gentamicin treatment closely correlating with changes in baseline levels of CD11c + cells and their responses to contact lens wear.

Involvement of conjunctival-associated commensals in the observed CD11c + phenotypes would not be surprising as others have shown conjunctival-associated bacteria (C. mastitidis) to be important for corneal immune responses against P. aeruginosa infection via effects on gamma-delta T cells ²¹. However, the CD11c + cell response as a result of lens wear, a completely different phenotype occurring in healthy rather than diseased corneas, could involve different mechanisms. Indeed, other possibilities include that topical gentamicin affects commensal bacteria beyond the eye. The nasolacrimal drainage system exits the eye and drains tear fluid into the back of the nose and throat so topically added gentamicin solution at this very high concentration (3 mg/ml) could influence microbiota of the nasopharynx or intestinal tract, the latter well known to influence immune responses at distant mucosal sites including the conjunctiva^22–24. Another potential explanation is that topical gentamicin influenced corneal CD11c + cell numbers and responses to lens wear independently of effects on bacteria. Indeed, aminoglycosides and other antibiotics are known to have a multitude of direct impacts on immune cell function^25–28. Further studies will be needed to delineate the respective roles of bacteria, conjunctival or other, versus other potential mechanisms, in mediating the CD11c + phenotypes observed.

Even if topical ocular gentamicin does mediate the CD11c + phenotypes observed in our study via an impact on microbes, it cannot be assumed that bactericidal effects are exclusively involved. In this study, the presence of numerous filamentous forms of murine conjunctival bacteria⁸ necessitated the quantification of total alkDala (peptidoglycan cell wall) fluorescence rather than distinguishing individual bacterial cells. Thus, it remains possible that alkDala fluorescence with gentamicin exposure resulted from reduced cell wall synthesis while bacteria remained viable. Another possibility is that gentamicin inhibited bacterial protein synthesis (part of its established mechanism of action²⁰) interfering with the expression of bacterial ligands, metabolites, or other secreted factors that mediate the CD11c + responses. In this regard, the rapid return of CD11c + responses to contact lens wear after gentamicin removal (Fig. 3b) is interesting and may reflect a rapid resumption of bacterial ligand/metabolite/secreted factor expression by conjunctival commensals. It is also interesting that baseline levels of CD11c + cell numbers did not recover in the same time frame after gentamicin removal (Fig. 3b) suggesting the possibility that distinct mechanisms mediate these CD11c + cell phenotypes, or that responsible bacterial factors were trapped under or bound to the contact lens that enabled a response. Related to that possibility, contact lens-associated CD11c + responses (Figs. 2b & 3b) occurred with little or no observable increase in alkDala fluorescence of conjunctival commensals (Fig. 4a & b) suggesting that commensal bacteria involvement might not be via direct triggering of contact lens-associated CD11c + cell responses. In other words, some other trigger associated with contact lens wear might instead stimulate already existing conjunctival commensals to drive lens-associated CD11c + responses independently of peptidoglycan cell wall synthesis, e.g. expression of ligands, metabolites etc. Given how dramatically a contact lens can alter the ocular surface environment, conjunctival (or other) commensals may play an even more complex role being only one part of a system regulating responsiveness and homeostasis of corneal CD11c + cells. Our results showing that gentamicin exposure reduced corneal CD11c + responses to P. aeruginosa but not to known commensals (Fig. 5) would be consistent with a more complex mechanism.

In summary, the results of this study show that topical antibiotic application modulates corneal CD11c + cells influencing their constitutive (baseline) levels and ability to respond to contact lens wear or P. aeruginosa challenge. While the results implicate commensal conjunctival bacteria in these CD11c + phenotypes, further studies will be needed to determine the nature of their contribution which the data suggest might be complex.

Mice. Transgenic wild-type C57BL/6 female mice (6–8 weeks old) were used that express both a CD11c-YFP reporter (CD11c-positive cells, e.g. dendritic cells, yellow) and mT/mG knock in (cell membranes, red) ^4,7. At the end of each experiment, or if mice presented with excessive weight loss, distress, signs of keratitis, euthanasia was performed using isoflurane inhalation (5%) for 10 min, or combined administration (intra-peritoneal injection) of ketamine (80–100 mg/Kg) and xylazine (5-10mg/Kg), either method followed by cervical dislocation. Eyes were enucleated for fixation and imaging. All procedures involving animals were carried out in accordance with the standards established by the Association for the Research in Vision and Ophthalmology, under a protocol AUP-2019-06-12322 approved by the Animal Care and Use Committee, University of California Berkeley. This protocol adheres to PHS policy on the humane care and use of laboratory animals, and the guide for the care and use of laboratory animals. This study is reported in accordance with the ARRIVE guidelines (Animals in Research: Reporting In-Vivo Experiments).

Topical antibiotic treatment. One or both eyes of each mouse were treated topically with a 5 µl drop of antibiotic solution: a combination (cocktail) of gentamicin and ofloxacin (Falcon Pharmaceuticals. Fort Worth, TX USA) in solution (0. % of each), or gentamicin alone in solution (0. %) for 3 applications during time 0–6 h. Control eyes were treated with PBS. Eyes were examined for various phenotypes at 24 h. In some experiments, eyes were pretreated with antibiotic solution or PBS using the same timing prior to the above treatment.

Contact lens fitting. In some experiments, mice were fitted with custom-manufactured silicone hydrogel contact lens as previously described ⁴. Contact lenses were fitted only onto the right eye and the contralateral eye used as a non-lens wearing control. Before fitting, contact lenses were removed from their packaging solution and soaked for 1 h in sterile phosphate buffered saline (PBS). After lens insertion under isoflurane anesthesia, mice were single-housed without enrichment to prevent lens removal. Pure-o’Cel paper bedding (The Andersons Inc., Maumee OH) was used to reduce dust level. For overnight lens wear mice were returned to the animal care facility. Mice were monitored daily for lens retention, and evidence of pathology, e.g. discharge or corneal opacity. Any mouse that lost its contact lens was excluded from further experimentation.

Fluorescein in-situ hybridization (FISH). FISH-labeling was performed as previously described using universal 16S rRNA-targeted gene [Alexa488]-GCTGCCTCCCGTAGGAGT-[Alexa488] (Eurofins Genomics) ⁸. Contact lenses or enucleated eyes were fixed in paraformaldehyde (2%) for 24 h then stored in PBS at 4ºC until processed for FISH. Samples were washed in 80% EtOH, 95% EtOH then PBS for 10 min each with rotation, placed in hybridization buffer [NaCl (0.9 M), Tris-HCl (20 mM, pH 7.2) and SDS (0.01%)] for incubation (55 ºC, 30 min), then incubated overnight at 55^oC with the 16S rRNA probe (100 nM). Samples were transferred to wash buffer solution [NaCl (0.9 M) and Tris-HCl (20 mM, PH 7.2)], then washed three times for 10 min each with rotation. For imaging, contact lenses were flat-mounted with Prolong Gold™ and eyes were whole-mounted in a 47 mm Petri dish filled with PBS to cover the eyeball completely. Bacteria were imaged and quantified using ImageJ with maximum intensity projections.

AlkDala (Alkyne-Functionalized D-Alanine) Labeling. Viable bacteria on the murine ocular surface were also labeled using an alkyne functionalized D-alanine (alkDala) biorthogonal probe as previously described ^8,29,30. Freshly enucleated eyes were incubated in a solution of alkDala (10 mM) in Dulbecco’s Modified Eagle Medium (DMEM) at 37◦C for 2 h then transferred to pre-chilled 70% EtOH then fixed for 20 min at − 20◦C. After a thorough rinse in PBS, eyes were permeabilized in PBS with Triton-X100 (0.5%) for 10 min with shaking at room temperature and washed 3 times for 5 min each in PBS with Triton-X100 (0.1%) and BSA (3%) with shaking. Eyes were transferred to click-labeling cocktail [in PBS, TBTA (100 µM), CuSO₄ (1 mM), sodium ascorbate (2 mM), a 488 nm azide-fluorophore (10 µM), and BSA (0.1 mg/mL)] for 1 h with shaking at room temperature. Eyes were imaged as described below.

Imaging. Confocal imaging of the mouse cornea (whole eyes ex vivo), or worn contact lenses, was performed using a 20x/1.00 NA water-dipping objective with an upright Olympus Fluoview FV1000 microscope. Whole mounted eyes were imaged using a 512 nm laser for detection of CD11c+-YFP cells (Yellow Fluorescence Protein) and a 559 nm laser for detecting red fluorescent cell membranes. A 488 nm laser was used to detect bacteria labeled with FISH or alkDala. For Z stacks at 1.00 µm steps, images were collected from 4 or more random fields per sample. 3-D image reconstruction (reducing a 3-D image into 2-D) was achieved by projecting the maximum intensity of each pixel in a specific channel to the z plane. Image-J (MorpholibJ tools collection) and Imaris (Bitplane) were used for image processing and analysis. For circularity measurements, morphological image analysis was performed using 3D segmentation in ImageJ and parameters related to z-projections used (circularity) to exclude artifacts due to lower z resolution.

Bacteria. Three bacterial species were used in this study. Corynebacterium mastiditis ¹¹ (kindly provided by Dr. Anthony St. Leger, University of Pittsburgh, PA), a clinical isolate of coagulase-negative Staphylococcus spp. and Pseudomonas aeruginosa strain PAO1F. Bacteria were grown on tryptic soy agar plates at 37°C for ∼16 h. A bacterial inoculum was prepared by suspension in PBS to a concentration of ∼10¹¹ CFU/ml that was confirmed by viable counts. To inoculate mouse eyes, mice were anesthetized using ketamine (80–100 mg/Kg) and dexmedetomidine (0.25–0.5 mg/Kg) and the cornea of one eye was inoculated with 5 µl of bacterial suspension. An additional 5 µl of bacterial suspension was re-inoculated onto the corneas each hour up to 4 h (4 inoculations per eye in total). Mice remained anesthetized and covered under a heat lamp for the entire 4 h period. Subsequently, mice were subject to euthanasia and eyes examined by quantitative imaging.

Experimental procedures.

Procedure 1. Mice were divided into experimental groups containing 3–4 mice. One group was treated with antibiotic solution (cocktail), another with gentamicin alone, in one eye as described above. Contralateral eyes were treated with PBS as a control. After 24 h, eyes were imaged ex vivo to measure corneal CD11c + cell numbers or subject to FISH labeling to determine if viable bacteria were present.

Procedure 2. Two groups of 3–4 mice were used. One group was treated with gentamicin solution (timing as above) in both eyes, the other group similarly treated in both eyes with PBS as a control. For the antibiotic treated group, after the first application of antibiotic, contact lens fitting was performed on one eye followed by remaining antibiotic applications. Contralateral eyes served as non-lens wearing antibiotic-treated controls. The PBS control group was treated similarly. One eye fitted with a contact lens followed by remaining PBS applications, contralateral eyes served as non-lens wearing PBS-treated controls.

Procedure 3. Similar to Procedure 2 with two groups of 3–4 mice: antibiotic treated group versus PBS control group. However, prior to each experiment, each group was pretreated with either antibiotic or PBS with the same timing as used for the primary experiment. In some experiments, however, antibiotic pretreatment was followed by contact lens application with PBS or PBS alone.

Statistical analysis. Data analysis was performed using Prism 9.0 for Mac, Microsoft Excel 2010, and the Statistical Package for Social Science for Mac version 27.0 (SPSS, Inc, Chicago, IL). The distribution of data was assessed by the normality test (Shapiro-Wilk test and Kolmogorov-Smirnov test), and since most data was normally distributed, it was expressed as the mean with standard deviation. One-way or Two-way ANOVA with Tukey’s multiple comparisons test for post-hoc analysis. P values less than 0.05 were considered significant.

Mice. Transgenic wild-type C57BL/6 female mice (6–8 weeks old) were used that express both a CD11c-YFP reporter (CD11c-positive cells, e.g. dendritic cells, yellow) and mT/mG knock in (cell membranes, red) ^4,7. At the end of each experiment, or if mice presented with excessive weight loss, distress, signs of keratitis, euthanasia was performed using isoflurane inhalation (5%) for 10 min, or combined administration (intra-peritoneal injection) of ketamine (80–100 mg/Kg) and xylazine (5-10mg/Kg), either method followed by cervical dislocation. Eyes were enucleated for fixation and imaging. All procedures involving animals were carried out in accordance with the standards established by the Association for the Research in Vision and Ophthalmology, under a protocol AUP-2019-06-12322 approved by the Animal Care and Use Committee, University of California Berkeley. This protocol adheres to PHS policy on the humane care and use of laboratory animals, and the guide for the care and use of laboratory animals. This study is reported in accordance with the ARRIVE guidelines (Animals in Research: Reporting In-Vivo Experiments).

Topical antibiotic treatment. One or both eyes of each mouse were treated topically with a 5 µl drop of antibiotic solution: a combination (cocktail) of gentamicin and ofloxacin (Falcon Pharmaceuticals. Fort Worth, TX USA) in solution (0. % of each), or gentamicin alone in solution (0. %) for 3 applications during time 0–6 h. Control eyes were treated with PBS. Eyes were examined for various phenotypes at 24 h. In some experiments, eyes were pretreated with antibiotic solution or PBS using the same timing prior to the above treatment.

Contact lens fitting. In some experiments, mice were fitted with custom-manufactured silicone hydrogel contact lens as previously described ⁴. Contact lenses were fitted only onto the right eye and the contralateral eye used as a non-lens wearing control. Before fitting, contact lenses were removed from their packaging solution and soaked for 1 h in sterile phosphate buffered saline (PBS). After lens insertion under isoflurane anesthesia, mice were single-housed without enrichment to prevent lens removal. Pure-o’Cel paper bedding (The Andersons Inc., Maumee OH) was used to reduce dust level. For overnight lens wear mice were returned to the animal care facility. Mice were monitored daily for lens retention, and evidence of pathology, e.g. discharge or corneal opacity. Any mouse that lost its contact lens was excluded from further experimentation.

Fluorescein in-situ hybridization (FISH). FISH-labeling was performed as previously described using universal 16S rRNA-targeted gene [Alexa488]-GCTGCCTCCCGTAGGAGT-[Alexa488] (Eurofins Genomics) ⁸. Contact lenses or enucleated eyes were fixed in paraformaldehyde (2%) for 24 h then stored in PBS at 4ºC until processed for FISH. Samples were washed in 80% EtOH, 95% EtOH then PBS for 10 min each with rotation, placed in hybridization buffer [NaCl (0.9 M), Tris-HCl (20 mM, pH 7.2) and SDS (0.01%)] for incubation (55 ºC, 30 min), then incubated overnight at 55^oC with the 16S rRNA probe (100 nM). Samples were transferred to wash buffer solution [NaCl (0.9 M) and Tris-HCl (20 mM, PH 7.2)], then washed three times for 10 min each with rotation. For imaging, contact lenses were flat-mounted with Prolong Gold™ and eyes were whole-mounted in a 47 mm Petri dish filled with PBS to cover the eyeball completely. Bacteria were imaged and quantified using ImageJ with maximum intensity projections.

AlkDala (Alkyne-Functionalized D-Alanine) Labeling. Viable bacteria on the murine ocular surface were also labeled using an alkyne functionalized D-alanine (alkDala) biorthogonal probe as previously described ^8,29,30. Freshly enucleated eyes were incubated in a solution of alkDala (10 mM) in Dulbecco’s Modified Eagle Medium (DMEM) at 37◦C for 2 h then transferred to pre-chilled 70% EtOH then fixed for 20 min at − 20◦C. After a thorough rinse in PBS, eyes were permeabilized in PBS with Triton-X100 (0.5%) for 10 min with shaking at room temperature and washed 3 times for 5 min each in PBS with Triton-X100 (0.1%) and BSA (3%) with shaking. Eyes were transferred to click-labeling cocktail [in PBS, TBTA (100 µM), CuSO₄ (1 mM), sodium ascorbate (2 mM), a 488 nm azide-fluorophore (10 µM), and BSA (0.1 mg/mL)] for 1 h with shaking at room temperature. Eyes were imaged as described below.

Imaging. Confocal imaging of the mouse cornea (whole eyes ex vivo), or worn contact lenses, was performed using a 20x/1.00 NA water-dipping objective with an upright Olympus Fluoview FV1000 microscope. Whole mounted eyes were imaged using a 512 nm laser for detection of CD11c+-YFP cells (Yellow Fluorescence Protein) and a 559 nm laser for detecting red fluorescent cell membranes. A 488 nm laser was used to detect bacteria labeled with FISH or alkDala. For Z stacks at 1.00 µm steps, images were collected from 4 or more random fields per sample. 3-D image reconstruction (reducing a 3-D image into 2-D) was achieved by projecting the maximum intensity of each pixel in a specific channel to the z plane. Image-J (MorpholibJ tools collection) and Imaris (Bitplane) were used for image processing and analysis. For circularity measurements, morphological image analysis was performed using 3D segmentation in ImageJ and parameters related to z-projections used (circularity) to exclude artifacts due to lower z resolution.

Bacteria. Three bacterial species were used in this study. Corynebacterium mastiditis ¹¹ (kindly provided by Dr. Anthony St. Leger, University of Pittsburgh, PA), a clinical isolate of coagulase-negative Staphylococcus spp. and Pseudomonas aeruginosa strain PAO1F. Bacteria were grown on tryptic soy agar plates at 37°C for ∼16 h. A bacterial inoculum was prepared by suspension in PBS to a concentration of ∼10¹¹ CFU/ml that was confirmed by viable counts. To inoculate mouse eyes, mice were anesthetized using ketamine (80–100 mg/Kg) and dexmedetomidine (0.25–0.5 mg/Kg) and the cornea of one eye was inoculated with 5 µl of bacterial suspension. An additional 5 µl of bacterial suspension was re-inoculated onto the corneas each hour up to 4 h (4 inoculations per eye in total). Mice remained anesthetized and covered under a heat lamp for the entire 4 h period. Subsequently, mice were subject to euthanasia and eyes examined by quantitative imaging.

Experimental procedures.

Procedure 1. Mice were divided into experimental groups containing 3–4 mice. One group was treated with antibiotic solution (cocktail), another with gentamicin alone, in one eye as described above. Contralateral eyes were treated with PBS as a control. After 24 h, eyes were imaged ex vivo to measure corneal CD11c + cell numbers or subject to FISH labeling to determine if viable bacteria were present.

Procedure 2. Two groups of 3–4 mice were used. One group was treated with gentamicin solution (timing as above) in both eyes, the other group similarly treated in both eyes with PBS as a control. For the antibiotic treated group, after the first application of antibiotic, contact lens fitting was performed on one eye followed by remaining antibiotic applications. Contralateral eyes served as non-lens wearing antibiotic-treated controls. The PBS control group was treated similarly. One eye fitted with a contact lens followed by remaining PBS applications, contralateral eyes served as non-lens wearing PBS-treated controls.

Procedure 3. Similar to Procedure 2 with two groups of 3–4 mice: antibiotic treated group versus PBS control group. However, prior to each experiment, each group was pretreated with either antibiotic or PBS with the same timing as used for the primary experiment. In some experiments, however, antibiotic pretreatment was followed by contact lens application with PBS or PBS alone.

Statistical analysis. Data analysis was performed using Prism 9.0 for Mac, Microsoft Excel 2010, and the Statistical Package for Social Science for Mac version 27.0 (SPSS, Inc, Chicago, IL). The distribution of data was assessed by the normality test (Shapiro-Wilk test and Kolmogorov-Smirnov test), and since most data was normally distributed, it was expressed as the mean with standard deviation. One-way or Two-way ANOVA with Tukey’s multiple comparisons test for post-hoc analysis. P values less than 0.05 were considered significant.

Data availability

All data generated or analyzed in this study are included in this published article and its Supplementary Information files.

Acknowledgements

This work was supported by the National Institutes of Health EY030350 (SMJF).

Author contributions

AD, JL, and TT performed the experiments.AD, DE and SF analyzed the data. AD, DE and SF wrote the manuscript. DE and SF designed the study and supervised the project.

Competing financial interests

The authors have no conflicts of interest to disclose in relation to the work presented.

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No competing interests reported.

Topical Antibiotics Reduce CD11c+ Cell Numbers in the Healthy Murine Cornea and Modulate their Response to Contact Lens Wear

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