An Ocular Commensal Protects against Corneal Infection by Driving an Interleukin-17 Response from Mucosal γδ T Cells

Abstract

Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but proof of a living, resident ocular microbiome remains elusive. Here, we used a mouse model of ocular surface disease to reveal that commensals were present in the ocular mucosa and had functional immunological consequences. We isolated one such candidate commensal, Corynebacterium mastitidis, and showed that this organism elicited a commensal-specific interleukin-17 response from γδ T cells in the ocular mucosa that was central to local immunity. The commensal-specific response drove neutrophil recruitment and the release of antimicrobials into the tears and protected the eye from pathogenic Candida albicans or Pseudomonas aeruginosa infection. Our findings provide direct evidence that a resident commensal microbiome exists on the ocular surface and identify the cellular mechanisms underlying its effects on ocular immune homeostasis and host defense.

Keywords: IL-17; host defense; microbiome; mucosal immunity; ocular commensal bacteria; ocular surface disease; γδ T cells.

Figure 1. Locally produced IL-17 recruits neutrophils to the conjunctiva

(A) Neutrophils were quantified by flow cytometry from the conjunctiva of WT, Il17a^-/-, Il17f^-/-, or Il17a^-/-Il17f^-/- mice under steady state conditions. (B) Flow plots represent IL-17A production in cells from the conjunctiva after 4 hour PMA and ionomycin stimulation. TCR staining in the right plot represents the gated IL17A⁺ population in the left plot. Pie chart shows the contribution of IL-17A from each cell subset. (C) WT mice were given single subconjunctival injection of anti-IL-17A & F in one eye and PBS in the contralateral eye. After 48 hours, conjunctiva was harvested and assessed for neutrophils by flow cytometry. Data are representative of ≥5 (A) or 2 experiments (B & C). Symbols represent individual mice. Statistical significance was determined using an (A) unpaired two-way students t test and (C) paired one-way students t test.

Figure 2. Microbial stimulation of conjunctival T cells results in production of IL-17A from γδ T cells

(A-C) Single-cell suspensions from the conjunctiva of mice from NIH, Jackson Laboratories (“JAX”), Taconic Biosciences (“TAC”), or Charles River (“CR”) were either assessed for (A) neutrophil numbers by flow cytometry or (B & C) IL-17A production after a 4 hours stimulation with PMA and ionomycin in the presence of brefeldin A for 4 hours. (D) C. mastitidis cultured on TSA plates. The streaks are ophthalmic gentamicin gel (Gentak™) applied to the agar showing bacterial sensitivity. (E) Frozen sections of whole eyes (with eyelids) were stained with fluorescent probes against Corynebacterium spp. Pictures are representative of 3 mice. (F & G) Lysates from C. mastitidis or S. aureus were incubated with FACS isolated CD11b⁺CD11c⁺and Thy1⁺ cells from conjunctival tissue. After 72 hours, (F) supernatants were collected and IL-17A was measured by ELISA. (G) brefeldin A was added the last 6 hours of culture, and γδ TCR and IL-17A expression was assessed by flow cytometry. (H) 1 × 10⁴ αβ, Vγ4⁺ γδ , and Vγ4^- γδ Nr4a1^GFP reporter mice were incubated with 1 × 10⁵ splenic DCs pulsed with 3 μg for 48 hours. Numbers represent GFP expression ± SEM in cell populations as assessed by flow cytometry. Data are from triplicate wells in an individual experiment. (I) 1×10⁴ FACS isolated γδ T cells from the cervical LN and 1×10⁵ MACS isolated splenic CD11c⁺ cells were incubated with 3 μg of C. mast lysate and either αIL-1R or αCD1d for 72 hours. Brefeldin A was added the last 6 hours of culture and IL-17A was assessed by flow cytometry. (J) Lysates from C. mastitidis or LPS were incubated with PBMCs from healthy donors for 48 hours at 37°C. Supernatants were collected and assessed for IL-17A by ELISA. Data is pooled from 7 healthy donors from 3 independent experiments (Statistical significance was determined by ANOVA). Bars in A, B, D and F represent the mean ± SEM. (A & C) Symbols represent individual mice from two experiments. (H & I) Data are representative of 2 or 3 independent experiments, respectively. See also Figures S1 & S2.

Figure 3. Disruption of ocular bacteria reduces the immune signature in conjunctival tissue

(A-C) WT mice were treated topically with PBS or gentamicin ophthalmic gel daily for 6 days. (A) RNA from conjunctival tissue control WT, antibiotic WT, Germ Free (GF) mice was assessed using Nanostring Technologies' Immunology Gene panel. Bars represent the mean fold change ± SEM in gene expression compared to WT controls. (B) Single-cell suspensions from conjunctival tissue were stimulated with PMA/Ionomycin in the presence of brefeldin A for 4 hours. After stimulation intracellular IL-17A and the γδ TCR was assessed by flow cytometry. (C) Conjunctival neutrophils were quantified by flow cytometry. Bars represent the mean number ± SEM of neutrophils from each eye (n≥10 from 4 independent experiments, statistical significance was determined using Students T test). (D) The ocular surface was washed with 10μl of PBS and diluted in 190 μl of PBS. Dilutions were then assessed for S100A8 concentration using ELISA. Symbols represent the concentration from an individual mouse. Statistical significance was determined using ANOVA. See also Figure S3.

Figure 4. Local antibiotic treatment compromises host resistance to Candida albicans at the ocular surface

WT mice were topically treated with PBS or gentamicin ophthalmic gel daily for 6 days. After 6 days, (A) the ocular surface of both eyes from a single mouse was washed with 10ul of PBS and fungicidal activity was tested in vitro. Bars represent the mean percentage ± SEM increase in fungal survival after 60 minutes compared to WT controls. (B & C) Then the WT groups, Il17a^-/-Il17f^-/-, and Tcrb Tcrd^-/- mice were ocularly infected with 5 × 10⁵ CFU of Candida albicans. Briefly, mice were anesthetized and the ocular surface was gently dabbed with gauze. C. albicans (strain SC5314) was then applied in 5μl of PBS and remained on the surface for 30 minutes until mice awoke. Fifteen hours after infection, mice were sacrificed. (B) qPCR was used to measure the concentration of fungal DNA in corneal homogenates. Symbols represent individual mice pooled from 2 independent experiments. Statistical significance was determined using Kruskal-Wallis Test. (C) Representative PAS stains of corneas at the end point. See also Figure S4.

Figure 5. Ocular colonization with C. mastitidis induces immunity at the ocular surface and the eye draining lymph nodes

(A) NIH bred mice were depleted of Vy4⁺ T cells 4 days before sacrifice, as described in Methods. Conjunctival single-cell suspensions were stimulated with PMA and ionomycin + brefeldin A and IL-17 production was assessed by flow. Bars represent the mean ± SEM of IL-17⁺ cells after stimulation. Symbols represent individual mice from a single experiment that is representative of two experiments. Pie Charts represent the percent contribution ± SEM of IL-17 production by the various cell populations. (B) Tear wash from these mice was tested for fungicidal activity in vitro, as in Fig 4A. (C-I) Mice from JAX Laboratories were given PBS or were inoculated with 1 × 10⁸ CFU of C. mast. once every three days, totaling three inoculations. (C) After three weeks, frozen sections of whole eyes (with eyelids) were stained with fluorescent probes against Corynebacterium spp. Pictures are representative of 3 mice. (D) After 5 weeks, swabs of conjunctiva were assessed for C. mast. CFU. Swabs were also taken from co-housed mice or 8 wk-old mice born from ocular inoculated dams. (E-I) Three weeks after the final inoculation, mice were sacrificed and the conjunctiva was assessed for (E) immunology-related gene expression, (F) T cells, (G) IL-17A⁺ cells after 4 hour PMA and ionomycin stimulation, and (H) neutrophil numbers. (C) Bars represent the mean fold change in gene expression ± SEM over uninoculated mice (5 mice from 2 independent experiments) as determined by the Nanostring Mouse Immunology Panel. (F) Representative flow plots displaying the frequency of Vγ4⁺ γδ T cells of Thy1⁺ cells in the conjunctiva ± SEM (6 mice from 2 independent experiments. (G & H) Bars represent the mean (G) frequency of IL-17⁺ cells or (H) number of neutrophils ± SEM in the conjunctiva after inoculation with C. mast (n≥6 from at least 2 experiments). (I) Cervical lymph nodes were harvested and either assessed by flow cytometry for IL-17A production after 4 hour PMA and ionomycin stimulation or for the expression of T cell activation markers, CD44 and CD62L. Symbols represent individual mice and bars represent the mean frequency of IL-17A⁺ cells or CD44^HICD62L^LOW cells ± SEM. Statistical significance was determined using students T test (G & H) or ANOVA (I). See also Figure S5.

Figure 6. C. mastitidis, but not other bacteria, persists at the ocular surface and induces immunity

Mice from JAX Laboratories were given PBS or were inoculated with 1 × 10⁸ CFU of the noted bacteria once every three days totaling three inoculations. After 3 weeks, mice were sacrificed and the conjunctiva was assessed for (A) live bacteria (CFU), (B) neutrophils, and (C) IL-17A producing cells. (D) Cervical lymph nodes were harvested and either assessed by flow cytometry for the expression of T cell activation markers, CD44 and CD62L. Symbols represent individual mice from 2 independent experiments. Bars represent the mean (A) CFU of bacteria (B) number of neutrophils or (C) frequency of IL-17A⁺ cells or (D) frequency of CD44^HICD62L^LOW cells ± SEM. Statistical significance was determined using One-Way ANOVA.

Figure 7. Immunity induced by local C. mast colonization protects the ocular surface from pathogenic fungal or bacterial infection

Mice from JAX Laboratories were given PBS or were inoculated as described previously. Three weeks after the final inoculation, (A) the ocular surface of both eyes from a single mouse was washed with 10ul of PBS and fungicidal activity was tested in vitro. (B & C) The ocular surface was then ocularly infected with 5 × 10⁵ CFU of C. albicans (strain SC5314) and corneas were harvested after 8 hours. (B) Representative PAS stains of cornel sections. (C) qPCR was used to measure the concentration of fungal DNA in corneal homogenates. (D & E) After inoculation with C. mast, mice were ocularly infected with 1×10⁶ CFU of Pseudomonas aeruginosa (strain 6294) After 48 hours, (D) bacterial burden and (E) pathology scores were assessed. Symbols represent samples from individual mice from 2 independent experiments. Statistical significance was determined using Mann-Whitney Test (A & E) or students T test (C & D). See also Figure S6.