Dysbiosis Modulates Ocular Surface Inflammatory Response to Liposaccharide

Abstract

Purpose: The purpose of this study was to investigate the inflammatory response of cornea and conjunctiva to topically applied lipopolysaccharide (LPS) in mice with and without antibiotic (antibiotic cocktail, ABX) induced dysbiosis.

Methods: Dysbiosis was induced by oral antibiotics for 14 days in a group of conventional female C57BL/6J (B6) mice. 16S rRNA sequencing investigated microbiome composition. Intestinal microbiome differences were assessed using 16S rRNA sequencing of fecal pellet DNA. Blood was collected after euthanasia. CD86 expression in draining nodes was examined by flow cytometry. At day 15, a single dose of LPS or vehicle was topically applied to ABX and naïve mice. Corneal epithelium and conjunctiva were obtained after 4 hours and processed for gene expression analysis. A separate group of germ-free (GF) B6 mice was also topically challenged with LPS.

Results: Antibiotic treatment significantly decreased intestinal diversity and increased serum levels of LPS. This was accompanied by a significant increase in CD86+MHC II+CD11c+CD11b+ cells in draining nodes. Compared to vehicle, topically applied LPS increased IL-1β, TNF-α, and CXCL10 mRNA transcripts in cornea and IL-1β, TNF-α, and CXCL10 in the conjunctiva in conventional and antibiotic-treated groups. However, there was higher TNF-α, CXCL10, and IL-12 expression in the cornea of LPS-treated ABX mice compared to LPS-treated mice with intact microbiota. LPS stimulation on GF conjunctiva mirrored the results in ABX mice, although greater IL-12 and IFN-γ expression was observed in GF conjunctiva compared to conventional LPS-treated mice.

Conclusions: Acute depletion of commensals through antibiotics or germ-free environment worsens the inflammatory response to LPS.

Figure 1

Antibiotic treatment induces a profound change in microbiome. C57BL/6J mice were treated with a cocktail of broad-spectrum antibiotics (ABX: ampicillin, gentamicin, metronidazole, neomycin, and vancomycin) dissolved in drinking water with artificial sweetener (Splenda) ad libitum. Stools were collected before treatment (naïve) and after 14 days (14D) of continuous oral cocktail and processed as described in Materials and Methods for 16S analysis. (A, B) Number of observed OTUs and Shannon Diversity Index scores (B) showing α diversity before (naïve) and after oral antibiotics (ABX) for 14 days (14D; n = 8). Mann-Whitney U test. (C) Principal coordinate analysis (PCoA) plot using the Bray-Curtis similarity measure. Each symbol represents an individual sample from naive and ABX-treated mice. (D) The relative abundance of major bacterial taxa before (naïve) and after oral antibiotics (ABX) for 14 days (14D). (E) Comparison of the significant relative abundance of different genera among groups before (naïve) and after oral antibiotics (ABX) for 14 days (14D). The dotted line divides significant genera that decreased (left) or increased (right) after treatment. For OTUs that do not have a genus classification, the family classification is given. Mann-Whitney U tests with false discovery rate correction. The P values shown in the graph were rounded to the largest value, as shown.

Figure 2

Antibiotic cocktail increases the frequency of antigen-presenting cells and their maturation status. (A) Single-cell preparations from lymph nodes were prepared and analyzed by flow cytometry. Lymphocytes were identified by forward and side scatter properties (SSC), single-cell gates were drawn, dead cells were excluded. CD11c⁺ cells were then plotted versus MHC II and further gated into MHC II and CD11b⁺ cells. (B) Representative histograms of CD11c⁺MHC II⁺CD11b⁺ showing CD86 fluorescence intensity. FMO, fluorescence minus one. (C) Accumulative data of flow cytometry analysis showing the frequency of CD11c⁺MHC II⁺ and median fluorescence intensity (MFI) of CD86 in cervical lymph nodes. Mean ± SEM, n = 4 mice/group, biological replicates from two independent experiments were averaged. Mann-Whitney U test.

Figure 3

Oral antibiotic treatment worsens the inflammatory response to LPS in the cornea. (A) Conventionally housed (CON) C57BL/6 mice were subjected to a cocktail of oral antibiotics (ABX) in drinking water for 14 days or drank normal water. On the 15th day, mouse eyes were treated topically with LPS or endotoxin-free water (vehicle) and mice were euthanized after 4 hours to analyze cytokine/chemokine expression. A separate group of germ-free (GF) mice also received LPS or vehicle. (B) Relative fold expression changes of IL-1β, tumor necrosis factor-α (TNF-α), CXCL10, and IL-12 mRNA in the cornea. Bar graphs show mean ± SEM of a representative experiment with four to six samples/group (the experiment was repeated once with similar results). *P < 0.05, **P < 0.01, ***P < 0.001, P < 0.0001 intragroup comparison vehicle versus LPS. Kruskal-Wallis followed by Tukey's comparison test.

Figure 4

Dysbiosis worsens the inflammatory response to LPS in the conjunctiva. Mice were treated as described in Figure 3. Relative fold expression changes of IL-1β, IL-6, TNF-α, IL-12, IFN-γ, and CXCL10 mRNA in the conjunctiva. Bar graphs show mean ± SEM of a representative experiment with four to six samples/group (the experiment was repeated once with similar results). *P < 0.05, **P < 0.01, ***P < 0.001, P < 0.0001 intragroup comparison vehicle versus LPS. Kruskal-Wallis followed by Tukey's comparison test.