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.

Data from clinical studies indicate that ocular use of topical antibiotics correlates positively with fungal infection, suggesting that disruption of the interactions between the immune system and ocular surface microbes renders the eye susceptible to disease (). In the present study we identified Corynebacterium mastitidis (C. mast) as a candidate resident commensal and dissected the immunological interactions that led to an enhancement of host defense at the ocular surface. Using a mouse model, we demonstrated that C. mast, a commensal organism found in both humans and mice (), was able to uniquely colonize the ocular surface and caused γδ T cells within the ocular mucosa to produce interleukin 17 (IL-17). This interaction resulted in the production and release of antimicrobial molecules into the tears and confered protection from invasive Candida albicans and Pseudomonas aeruginosa infection of the ocular surface. We used Koch’s postulates (originally coined to link a microbe to a disease phenotype) as criteria to support that the protective phenotype we observed is directly attributable to the presence of C. mast. Our findings indicate that true commensalism with benefit to the host can exist at the ocular surface and uncover the importance of the local γδ T cell response in this process. We suggest that tuning of the local immune response by commensals may be necessary to maintain immune homeostasis in the ocular mucosa and may play a broad role in diseases of the ocular surface.

Immune function within the conjunctiva is provided by the eye-associated lymphoid tissue, which is the mucosal tissue of the ocular surface, and includes the lacrimal gland, conjunctiva, and tear ducts. While the lacrimal glands and tear ducts are primarily responsible for the production and drainage of tears, the conjunctiva is thought to help mediate immune responses to promote the health and integrity of the cornea (). Within the conjunctiva are immune follicles (), similar to the isolated lymphoid follicles of the gut (ILFs) (). These develop after birth and contain all the necessary cells for the generation of immune responses, including antigen-presenting cells (APCs) of myeloid origin, B cells, and T cells, up to 50% of which are γδ T cells (). In experimental mouse models, cells comprising the conjunctival follicles can respond to antigenic stimulation, innate receptor stimulation, and topical application of bacteria (). Thus, microbes can trigger local immune responses at the ocular surface.

The ocular surface is a mucosal tissue that lies at the interface between the environment and the host immune system and is continually exposed to microbes such as bacteria, fungi, and viruses. It has long been a question whether the ocular mucosa, similar to other mucosal sites, harbors a regular consortium of microbes representing a microbiome. This is contentious because proof of a living microbiome associated with ocular tissue is lacking. The ability of the ocular surface environment to support a resident microbiome is in question due to constant tear washing and the profoundly antimicrobial nature of ocular secretions that contain lysozyme, antimicrobial peptides, immunoglobulin A, complement, and other substances (). Genetic analyses as well as conjunctival swabs reveal limited amounts (in number and variety) of non-pathogenic and pathogenic bacteria at the ocular surface, but a regular microbial “signature” remains elusive (). Current experimental limitations are unable to resolve whether these bacteria are resident (long-term colonizers as opposed to transient, originating from the environment), metabolically active, or even alive. Although there is evidence that bacteria at the ocular surface may affect disease (), this does not speak to their status as ocular surface commensals, or how they interact with the immune system to be functionally relevant to local immunity. Rather, these studies conclude that gut microbes might play a major role in disease outcome.

As association of C. mast with the ocular surface of Jax mice appeared to induce an immune signature and expansion of γδ T cells in previously C. mast-negative Jax mice, we asked whether this gain of function would translate to enhanced host resistance to infection with pathogenic organisms. 3 weeks after C. mast-association of Jax mice, their tear fluid became more effective at killing C. albicans in vitro compared to the tears of non-associated Jax mice. This was dependent on γδ T cells, as their depletion reduced IL-17 within the eye-draining lymph nodes ( Figure S6 ) and abrogated the enhanced fungal killing ability ( Figure 7 A). Next, the ocular surface was inoculated with 5 × 10CFU of C. albicans and the ability of C. albicans to establish infection was examined. At 8 hr after inoculation, mice without C. mast already had fungal filaments embedded in ocular tissue, whereas mice with C. mast had pristine corneas ( Figure 7 B). In line with this, there was significantly more C. albicans DNA found in corneas of C. mast-negative mice ( Figure 7 C). To expand our observations from fungal infection to bacterial infection as well, we inoculated C. mast-negative and C. mast-positive Jax mice with Pseudomonas aeruginosa, a common bacterial pathogen of the human ocular surface, and measured bacterial burden and corneal pathology 48 hr after inoculation. As with C. albicans, mice colonized with C. mast had a significantly lower bacterial burden in their eyes and a lower pathology score ( Figures 7 D and 7E). These data demonstrate that C. mast as an ocular commensal modulated ocular immunity and maintained a γδ T cell-dependent IL-17 response that protected the ocular surface from infection by invasive C. albicans and P. aeruginosa.

(A) Three weeks after the final inoculation, the ocular surface of both eyes from a single mouse was washed with 10 μL of PBS and fungicidal activity was tested in vitro. Bars represent the mean percent increase of fungal survival over control ± SEM.

The ability to stably colonize the ocular surface and induce a local immune signature was not a general property possessed by all bacteria and may not apply to other Corynebacterium spp. We inoculated the conjunctiva of Jax mice with equal numbers of C. mast, C. bovis, C. glutamicum, or Staphylococcus epidermidis, a skin commensal that is frequently identified on the eye. After 3 weeks, only C. mast was detectable on the ocular surface, and only C. mast-inoculated mice exhibited enhanced numbers of neutrophils, IL-17, and γδ T cell activation in the conjunctiva ( Figure 6 ). These findings appear to reveal a unique ability of C. mast to colonize and induce an immune response in ocular tissue.

Jax mice ocularly colonized with C. mast displayed no ocular surface pathology and acquired an enhanced immune response within the ocular tissue, which included increases in expression of multiple immune-related genes ( Figure 5 E), expansion of Vγ4IL-17-producing γδ T cells ( Figures 5 F and 5G), and enhanced neutrophil recruitment ( Figure 5 H). In addition, IL-17-producing γδ T cells were expanded within the eye-draining cervical lymph nodes and displayed an activated phenotype ( Figure 5 I). In contrast, there was little change in the IL-17 production and activation phenotype of γδ T cells in peripheral lymphoid compartments, supporting the local nature of the γδ T cell response to ocular C. mast colonization ( Figure S5 ).

The ability of bacteria at the ocular surface to affect local immunity did not prove that they are a true resident commensal, similar to the commensals populating other mucosal tissues. To demonstrate this, it was essential to establish stable commensalism in a mouse strain that was not colonized with C. mast. SPF mice at major vendors such as Jackson Laboratories appeared to lack detectable colonization with C. mast and the associated C. mast-dependent immune responses ( Figures 2 and 5 ). Using a modified colonization protocol (), we were able to establish commensalism in ocular tissue by lightly dabbing the conjunctiva with a cotton swab and applying 1 × 10CFU of C. mast once every 3 days for a total of 3 inoculations. The inoculation procedure induced a stable colonization that could be detected by FISH or swabbing as long as 5 weeks after the final inoculation. Of note, co-housing of non-colonized Jax mice with C. mastmice from our facility for up to 8 weeks did not result in ocular C. mast transmission in terms of culturable bacteria or associated immune response, supporting the notion that this was true commensalism rather than self-re-inoculation from skin or feces. However, all offspring of an inoculated dam had detectable C. mast at the ocular surface when tested 8 weeks after birth, confirming vertical transmission of C. mast to the ocular mucosa within the colony ( Figures 5 C and 5D).

To link the C. mast-dependent protective effects to Vγ4γδ T cells, we eliminated them from NIH-bred mice by antibody depletion. This resulted in a significant reduction in conjunctival IL-17 production as well as in fungal-killing ability of the tear fluid ( Figures 5 A and 5B ). These data suggested that Vγ4T cells, which recognized C. mast, produced sufficient levels of IL-17 to directly enhance the production of anti-microbial components of tears. Therefore, colonization with C. mast should confer a protective γδ T cell-IL-17 phenotype.

(I) Cervical lymph nodes were harvested and either assessed by flow cytometry for IL-17A production after 4 hr 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 hi CD62L lo cells ± SEM.

(A) NIH-bred mice were depleted of Vγ4T cells 4 days before sacrifice, as described in STAR 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-17cells 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.

Based on the data depicted in Figure 3 , combined with reports in the literature implicating antibiotic use in the rise of ocular fungal infection (), we wished to examine whether disruption of ocular bacteria would lead to a higher susceptibility to fungal infections of the ocular surface. First, we tested in vitro the ability of tear fluid from unmanipulated or gentamicin-treated WT mice to kill C. albicans, the most common human fungal pathogen, compared to tears of immunodeficient Il17aIl17fand TcrbTcrdmice, both of which were colonized with C. mast as determined by culture (data not shown) and FISH data ( Figure S4 ). Significantly more viable C. albicans remained alive and culturable after a 1 hr incubation with tear fluid from antibiotic-treated than from untreated WT mice, resembling the immunodeficient situation ( Figure 4 A). We next used a model of ocular Candida albicans infection to assess host defense in vivo. C. albicans was applied to the ocular surface of anesthetized mice after gently dabbing the conjunctiva with a cotton swab, and corneas were collected for analysis after 15 hr. Antibiotic-treated mice had 10-fold more C. albicans DNA in corneal homogenates as detected by qPCR ( Figure 4 B) as well as pronounced fungal penetration into the cornea and tissue destruction ( Figures 4 C and S4 ), resembling the situation in immunodeficient mice. In the aggregate, our results thus far suggested that bacteria at the ocular surface, which can be eliminated by antibiotic treatment, were necessary to tune local host defense for protection against fungal infection.

(B and C) Then the WT groups, Il17a −/− Il17f −/− , and Tcrb −/− Tcrd −/− mice were ocularly infected with 5 × 10 5 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 5 μL of PBS and remained on the surface for 30 min until mice awoke. 15 hr after infection, mice were sacrificed.

If bacterial presence is responsible for the immune signature in the conjunctiva, it should be reduced if ocular surface bacteria are eliminated. We examined conjunctival tissue of germ-free (GF) mice as well as of mice from our specific-pathogen-free (SPF) facility that were treated topically for 6 days with gentamicin ophthalmic gel (0.3%, Gentak), a topical antibiotic commonly prescribed for human ocular infection () to which C. mast is susceptible in vitro ( Figure 2 D). The antibiotic-treated as well as the GF mice had reductions in gene expression compared to WT controls including reductions in Il17a and Il17f as well as downstream effectors of IL-17: Cxcl1, Cxcl3, Cxcl10, S100a8, and S100a9 ( Figures 3 A and S3 A). Consistent with the gene expression data, we saw concurrent reductions in IL-17-producing γδ T cells ( Figure 3 B) and neutrophil recruitment to the ocular mucosa ( Figure 3 C) in gentamicin-treated mice. Additionally, S100A8, a major anti-microbial peptide of the ocular surface when dimerized with S100A9 (), was routinely found in the tears of WT control mice but was not detected in mice treated with gentamicin gel or in Il17aIl17for TcrbTcrdmice ( Figure 3 D). We concluded from these data that local bacteria at the ocular surface maintained ocular immunity, and transient disruption of bacteria via antibiotics resulted in a reduction in immune-related mechanisms. In addition, T-cell-generated IL-17 appears to be critical for the production and release of the anti-microbial S100A8 into the tears, suggesting that bacteria-T cell interaction was essential for antimicrobial immunity at the ocular surface. Similarly, in conjunctival tissue of germ-free (GF) mice, neutrophils and IL-17 were reduced ( Figures S3 B and S3C), as is soluble immunoglobulin (sIgA) in the tears and lacrimal gland ( Figure S3 D) and these mice had a poorly developed lacrimal gland cytokine response ( Figures S3 E and S3F).

The C. mast lysate was able to elicit a rapid (48 hr) IL-17 production from PBMCs of healthy human donors ( Figure 2 J), suggesting presence of immunity to this organism. Overall, these data illustrated that C. mast, which was isolated from murine conjunctivae, stimulated IL-17 production from mouse γδ T cells and human PBMCs.

We asked whether the interaction of C. mast with γδ T cells leading to IL-17 production was purely innate or involved an interaction with the γδ TCR. To examine this, we used Nr4a1reporter mice, in which intensity of GFP fluorescence reflects the Nur77 gene expression and hence the strength of antigen receptor signaling (). Three populations of T cells (αβ T cells, Vγ4γδ T cells, and Vγ4γδ T cells) were isolated by FACS from the cervical lymph nodes of Nr4a1reporter mice bred in our colony. These cells were stimulated with C. mast lysate-pulsed splenic CD11cDCs for 48 hr. Although C. mast can stimulate αβ T cells (), we noted that Vγ4γδ T cells also expressed GFP, suggesting that γδ T cells can also respond to C. mast and that the responding population is the Vγ4subset ( Figure 2 H). IL-17 production by the Vγ4 population was dependent on presence of the dendritic cells, and no IL-17 was induced by stimulation of γδ T cells alone (data not shown). Antibody blockade of IL-1 signaling or of the non-classical MHC molecule CD1d inhibited IL-17 production from Vγ4 T cells, supporting involvement of a TCR-dependent interaction ( Figure 2 I).

We hypothesized that IL-17-inducing ocular surface bacteria might be present in our mouse colony but absent in vendor colonies, similarly to the case of segmented filamentous bacteria (SFB), which are found in Taconic but not in Jackson mice (). We therefore spread homogenized conjunctivae of mice from our facility on various agar plates. Similar to previous reports (), various Staphylococci spp. could be cultured from murine conjunctival tissue (limited in numbers of CFU; data not shown). In addition, on trypticase soy agar (TSA) plates that had been incubated for 7 days, we observed numerous small, translucent colonies representing a Gram, gentamicin-sensitive bacterium ( Figure 2 D), which we subsequently identified by 16S sequencing as Corynebacterium mastitidis (C. mast; Figure S2 ). Corynebacterium spp. are frequently found in the ocular tissue in human individuals and C. mast is a known skin commensal (). This organism was consistently cultured from conjunctiva of C57BL/6 mice from our colony as well as from mice sent to us by a collaborator at Washington University, but could not be cultured from conjunctiva of vendor mice. The bacteria could also be detected by Corynebacterium-specific fluorescence in situ hybridization (FISH) () in NIH mice but not in vendor mice ( Figure 2 E). To assess whether the C. mast we identified was stimulatory to immune cells, we co-incubated C. mast lysate with fluorescence activated cell sorting (FACS)-isolated CD11bCD11cdendritic cells and Thy1cells from ocular tissue of C57BL/6 mice from our colony. C. mast lysate induced IL-17 production from γδ T cells in a dose-dependent manner, which was not the case when the γδ T cells were incubated with Staphylococcus aureus lysate ( Figures 2 F and 2G).

In the course of these studies, we noted that wild-type (WT) C57BL/6 mice bred in our facility consistently had a significantly higher neutrophil presence in the conjunctiva during steady-state conditions compared to mice from major commercial vendors ( Figure 2 A). This correlated with significantly more IL-17cells in the conjunctiva of our mice ( Figure 2 B). As shown in Figure 1 , most of the IL-17-producing cells in the conjunctiva in steady state were γδ T cells; of these about half were Vγ4subset (by positive staining with the anti-Vγ4 antibody UC3-10A6) ( Figure 2 C;). Of the non-Vγ4 IL-17 producers, less than 5% were γδ T cells while the remaining were αβ T cells and ILCs. In parallel, a higher percentage of γδ T cells in the cervical lymph nodes of mice from our facility demonstrated an activated phenotype (CD44CD62L) compared to parallel γδ T cells from vendor mice ( Figure S1 ).

Data are pooled from 7 healthy donors from 3 independent experiments (statistical significance was determined by ANOVA). Bars in (A), (B), (H), and (J) represent the mean ± SEM. (A and B) Symbols represent individual mice from two experiments. (H and I) Data are representative of 2 or 3 independent experiments, respectively. See also Figures S1 and S2

(I) 1 × 10 4 FACS-isolated γδ T cells from the cervical LN and 1 × 10 5 MACS-isolated splenic CD11c + cells were incubated with 3 μg of C. mast lysate and either αIL-1R or αCD1d for 72 hr. Brefeldin A was added the last 6 hr of culture and IL-17A was assessed by flow cytometry.

(F and G) Lysates from C. mastitidis or S. aureus were incubated with FACS isolated CD11b + CD11c + and Thy1 + cells from conjunctival tissue. After 72 hr, (F) supernatants were collected and IL-17A was measured by ELISA. (G) brefeldin A was added the last 6 hr of culture, and γδ TCR and IL-17A expression was assessed by flow cytometry.

(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 and C) IL-17A production after a 4 hr stimulation with PMA and ionomycin in the presence of brefeldin A for 4 hr.

Experiments aimed at identifying the IL-17-producing cell population in the conjunctiva revealed that IL-17 was produced by Thy1cells ( Figure 1 B). Consistently, γδ T cells accounted for more than 50% of the IL-17-producing cells in the steady state, with lower amounts being produced by αβ T cells and with a modest contribution by TCR-negative cells, consistent with the recently described group 3 innate lymphoid cells (ILC3) ( Figure 1 B). Neutralization of IL-17 by a subconjunctival injection of anti-IL-17 antibodies was followed after only 48 hr by a significant reduction in neutrophils in the injected eye, compared to the contralateral eye that received an injection of PBS ( Figure 1 C). These data suggested that IL-17 is continually produced by γδ T cell locally and that this production was required to maintain neutrophil presence in the conjunctiva during steady state.

Interleukin (IL)-17 plays a pathogenic role in ocular surface diseases such as dry eye disease (DED) and various forms of infectious keratitis (). However, in our facility, mice deficient in IL-17A alone or in both IL-17A and IL-17F harbored significantly fewer neutrophils in the conjunctiva at steady state compared to WT controls and single Il17fmice ( Figure 1 A). About 50% of Il17aIl17f(but not Il17aor Il17f) mice developed ocular surface inflammation, which was prevented by including antibiotics in their drinking water. Il17aIl17fmice were previously reported to be prone to spontaneous Staphylococcus aureus infection (). This suggested that, while IL-17A is dominant among the two cytokines in recruiting neutrophils, both contribute to control the outgrowth of inflammation-causing bacteria.

Discussion

In the current study, we present evidence that may help resolve the long-standing controversy of whether a resident microbiome exists at the ocular surface. While many published reports now freely use the terminology “resident microbiota” in relation to bacteria detected on the ocular surface, evidence that these organisms have not arrived minutes ago from the surroundings and will not be promptly eliminated by the ocular antimicrobial environment has not been provided previously. As proof of concept, we demonstrated that a specific commensal, Corynebacterium mastitidis, stably colonized the ocular surface and enhanced the host’s ability to resist pathogenic fungal and bacterial infections. Lack of horizontal transmission upheld the notion that C. mast was a bona fide ocular commensal and that its persistence was not a result of continuous self-re-inoculation from skin or feces. We further dissected its interaction with the local conjunctival immune system and demonstrated that its presence had functional consequences for host defense at the ocular surface. As an ocular commensal conferring a beneficial (rather than a disease) phenotype, C. mast satisfied all four of Koch’s postulates for a causative agent, namely: (1) C. mast was present in mice displaying a distinct immune signature in the conjunctiva but was absent in mice without this immune phenotype; (2) C. mast could be isolated from ocular tissue of mice displaying the phenotype and grown in pure culture; (3) inoculation of C. mast into mice free of C. mast colonization conferred the protective immune phenotype; and (4) C. mast could be re-isolated from the inoculated hosts which display the protective immune phenotype.

Goodman and Lefrancois, 1989 Goodman T.

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et al. NK cells promote Th-17 mediated corneal barrier disruption in dry eye. +CD11b+ DCs on the non-classical MHC class 1 molecule CD1d. Our data also supported a role for innate stimulation, as evidenced by the requirement for IL-1 in order to elicit IL-17 production in the γδ T-DC cocultures. An interesting next step would involve identifying the putative antigenic epitope(s) and second signals involved in regulating the γδ T cell response to C. mast in the conjunctiva. Nevertheless, the current data demonstrated that in response to these stimuli, Vγ4 T cells accumulated in the conjunctiva and eye-draining LN, where they expressed surface activation markers (CD44hi, CD62Llo) and produced IL-17, conferring resistance to colonization with pathogenic organisms. A similar protective relationship is found in the gut between SFB and CD4+ T cells, which prevent C. rodentium infection, and between S. epidermidis and CD8+ T cells in the skin, which prevent C. albicans infection ( Ivanov et al., 2009 Ivanov I.I.

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et al. Commensal-dendritic-cell interaction specifies a unique protective skin immune signature. Our data also uncovered a nonredundant function of γδ T cells, particularly the Vγ4 subset, in IL-17 production in response to C. mast and in host defense at the ocular surface. High numbers of γδ T cells are typically found at barrier surfaces (gut, lung, skin, reproductive tract) and they appear to be somewhat specialized in terms of their TCR usage and diversity. For example, Vγ5 is found in mouse skin, Vγ7 in gut, Vγ6 in the uterus, and Vγ4 in human gut (). As innate T cells, γδ T cells can produce cytokines in response to innate receptor stimulation and previous work has also shown potential recognition of antigens through the γδ TCR (), but little is known about the antigens or MHC restrictions involved in these interactions. High numbers of γδ T cells are also observed in the conjunctiva (), but no study has thus far investigated what selects and activates them. Our data suggested that C. mast potentially possessed antigen(s) that triggered Vγ4 T cells at least in part through a TCR-driven interaction. The C. mast-derived antigen appears to be presented to the Vγ4 TCR by CD11cCD11bDCs on the non-classical MHC class 1 molecule CD1d. Our data also supported a role for innate stimulation, as evidenced by the requirement for IL-1 in order to elicit IL-17 production in the γδ T-DC cocultures. An interesting next step would involve identifying the putative antigenic epitope(s) and second signals involved in regulating the γδ T cell response to C. mast in the conjunctiva. Nevertheless, the current data demonstrated that in response to these stimuli, Vγ4 T cells accumulated in the conjunctiva and eye-draining LN, where they expressed surface activation markers (CD44, CD62L) and produced IL-17, conferring resistance to colonization with pathogenic organisms. A similar protective relationship is found in the gut between SFB and CD4T cells, which prevent C. rodentium infection, and between S. epidermidis and CD8T cells in the skin, which prevent C. albicans infection (). Thus, in this regard as well, C. mast appeared to behave similarly to other commensals.

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Koh A.Y. Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization. Widespread use of antibiotics is a known risk factor associated with vaginal candidiasis (); however, a causal connection to elimination of bacterial flora has not yet been demonstrated. Antibiotic-induced dysbiosis of the intestinal microbes, specifically in Bacteroides spp., is linked to a reduction in the cathelicidin antimicrobial peptide, CRAMP (LL-37 in humans), which results in the outgrowth of intestinal Candida spp. leading to other systemic pathologies (). In this study, we showed that C. mast acted in a similar fashion by eliciting the production of IL-17 from γδ T cells and its downstream effectors like the antimicrobial S100A8-S100A9 dimer, which was released into the tears. This process enhanced the killing ability of tear fluid, which directly contributed to the defense of the ocular surface from not only C. albicans but also of P. aeruginosa, a leading cause of bacteria-induced corneal infection and potential blindness.

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et al. Corynebacterium lowii sp. nov. and Corynebacterium oculi sp. nov., derived from human clinical disease and an emended description of Corynebacterium mastitidis. C. mast is a known skin commensal; however, its presence in the conjunctiva has also been described (). Our findings showed that C. mast can actively colonize ocular tissue and rather than maintaining its better known rod-like morphology, it appeared to take on a filamentous morphology similar to Corynebacterium spp. reported in the oral cavity. Since some strains of bacteria filament due to stress, the filamentous nature of C. mast supported the notion that despite the ability to persist on the ocular surface, C. mast may perceive this environment as hostile. Indeed, other related strains of Corynebacteria, C. bovis and C. glutamicum, did not appear to be able to colonize the ocular surface. It will be of interest in future studies to dissect what properties make a microorganism into a successful ocular commensal and to examine the presence of C. mast in the human conjunctiva in health versus disease.

Kobayashi et al., 2015 Kobayashi T.

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Nagao K. Dysbiosis and Staphylococcus aureus colonization drives inflammation in atopic dermatitis. −/−Tcrd−/− and Il17a−/−Il17f−/− mice, which developed spontaneous ocular surface inflammation. This suggested, but did not prove, that under conditions of immune deficiency C. mast might act as a pathobiont. Although the load of Corynebacterium in eyes of immunodeficient mice by fluorescence quantitation was lower than in WT, this by itself neither supported nor excluded C. mast as a potential pathobiont. These mice also harbor other bacteria, including S. aureus, and the immune status of the host can disrupt the community compostion of resident bacteria (dysbiosis). We propose that deficient production of IL-17 and its downstream antibacterial mediators would diminish the ability of the host to control C. mast itself as well as other microbes, permitting development of pathology. An in-depth analysis of the complex host-bacterial interrelationships on the ocular surface of an immunodeficient host may provide insights into the factors that affect the balance between a mutualistic and a pathobiont situation. It is conceivable that C. mast might, under some conditions, act also as a pathobiont in the eye. Under conditions of dysbiosis in the skin, this and other Corynebacterium spp. are linked to increases in IL-17 and atopic dermatitis (). While immunosufficient WT mice harboring or colonized de novo with C. mast demonstrated no ocular surface pathology as a result, we did detect Corynebacterium by FISH in the conjunctiva of immunodeficient TcrbTcrdand Il17aIl17fmice, which developed spontaneous ocular surface inflammation. This suggested, but did not prove, that under conditions of immune deficiency C. mast might act as a pathobiont. Although the load of Corynebacterium in eyes of immunodeficient mice by fluorescence quantitation was lower than in WT, this by itself neither supported nor excluded C. mast as a potential pathobiont. These mice also harbor other bacteria, including S. aureus, and the immune status of the host can disrupt the community compostion of resident bacteria (dysbiosis). We propose that deficient production of IL-17 and its downstream antibacterial mediators would diminish the ability of the host to control C. mast itself as well as other microbes, permitting development of pathology. An in-depth analysis of the complex host-bacterial interrelationships on the ocular surface of an immunodeficient host may provide insights into the factors that affect the balance between a mutualistic and a pathobiont situation.

Many other questions arise that should be addressed in future investigations. It is currently unknown what makes C. mast able to persist and successfully colonize the ocular surface, whereas a number of related bacteria fail to do so. In addition, why this bacterium is not eliminated from the ocular surface by the IL-17 response that it itself induces bears further investigation. Further biological and structural investigation into this issue might help to better understand how microbes can evade the anti-microbial mechanisms to colonize ocular mucosa. Finally, it will be of interest to address why horizontal transmission is ineffective and how C. mast is transmitted from mother to offspring.

The ocular surface is an easily accessible site and the possibility of altering microbial communities at the ocular surface has exciting implications on ocular disease. Our findings provided proof of concept that resident ocular commensals exist and have consequences for the establishment and maintenance of ocular immune homeostasis; they furthermore identified a major mechanism whereby this is regulated through interaction of the commensal with local γδ T cells, which afforded protection from pathogenic microorganisms. These findings may have clinical implications not only on the use of antibiotics for ocular surface disease but also for the development of potential probiotic and prebiotic therapies for ocular disease.