Living with your biome: how the bacterial microbiome impacts ocular surface health and disease.

Introduction: Microbiome research has grown exponentially but the ocular surface microbiome (OSM) remains an area in need of further study. This review aims to explore its complexity, disease-related microbial changes, and immune interactions, and highlights the potential for its manipulation as a therapeutic for ocular surface diseases. Areas Covered: We introduce the OSM by location and describe what constitutes a normal OSM. Second, we highlight aspects of the ocular immune system and discuss potential immune microbiome interactions in health and disease. Finally, we highlight how microbiome manipulation may have therapeutic potential for ocular surface diseases. Expert Opinion: The ocular surface microbiome varies across its different regions, with a core phyla identified, but with genus variability. A few studies have linked microbiome composition to diseases like dry eye but more research is needed, including examining microbiome interactions with the host. Studies have noted that manipulating the microbiome may impact disease presentation. As such, microbiome manipulation via diet, oral and topical pre and probiotics, and hygienic measures may provide new therapeutic algorithms in ocular surface diseases.

The Secreted Ly6/uPAR-Related Protein 1 (Slurp1) Modulates Corneal Angiogenic Inflammation Via NF-κB Signaling.

Purpose: Previously we demonstrated that the secreted Ly-6/uPAR related protein 1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an antiangiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal response to silver nitrate (AgNO3) cautery. Methods: Five days after AgNO3 cautery, we compared the wild-type (WT) and Slurp1X-/- mouse (1) corneal neovascularization (CNV) and immune cell influx by whole-mount immunofluorescent staining for CD31 and CD45, (2) macrophage and neutrophil infiltration by flow cytometry, and (3) gene expression by quantitative RT-PCR. Quantitative RT-PCR, immunofluorescent staining, and immunoblots were employed to evaluate the expression, phosphorylation status, and subcellular localization of NF-κB pathway components. Results: Unlike the WT, the Slurp1X-/- corneas displayed denser CNV in response to AgNO3 cautery, with more infiltrating macrophages and neutrophils and greater upregulation of the transcripts encoding VEGFA, MMP2, IL-1b, and vimentin. At 2, 7, and 10 days after AgNO3 cautery, Slurp1 expression was significantly downregulated in the WT corneas. Compared with the WT, naive Slurp1X-/- CE displayed increased phosphorylation of IKK(a/b), elevated phosphorylation of IκB with decreased amounts of total IκB, and higher phosphorylation of NF-κB, suggesting that NF-κB signaling is constitutively active in naive Slurp1X-/- corneas. Conclusions: Enhanced angiogenic inflammation in AgNO3 cauterized Slurp1X-/- corneas and constitutively active status of NF-κB signaling in the absence of Slurp1 suggest that Slurp1 modulates corneal angiogenic inflammation via NF-κB signaling.

Genetic Manipulation of Corynebacterium mastitidis to Better Understand the Ocular Microbiome.

Purpose: Corynebacterium spp. are Gram-positive bacteria commonly associated with the ocular surface. Corynebacterium mastitidis was isolated from mouse eyes and was demonstrated to induce a beneficial immune response that can protect the eye from pathogenic infection. Because eye-relevant Corynebacterium spp. are not well described, we generated a C. mast transposon (Tn) mutant library to gain a better understanding of the nature of eye-colonizing bacteria. Methods: Tn mutagenesis was performed with a custom Tn5-based transposon that incorporated a promoterless gene for the fluorescent protein mCherry. We screened our library using flow cytometry and enzymatic assays to identify useful mutants that demonstrate the utility of our approach. Results: Fluorescence-activated cell sorting (FACS) of mCherry+ bacteria allowed us to identify a highly fluorescent mutant that was detectable on the murine ocular surface using microscopy. We also identified a functional knockout that was unable to hydrolyze urea, UreaseKO. Although uric acid is an antimicrobial factor produced in tears, UreaseKO bacterium maintained an ability to colonize the eye, suggesting that urea hydrolysis is not required for colonization. In vitro and in vivo, both mutants maintained the potential to stimulate protective immunity as compared to wild-type C. mast. Conclusions: In sum, we describe a method to genetically modify an eye-colonizing microbe, C. mast. Furthermore, the procedures outlined here will allow for the continued development of genetic tools for modifying ocular Corynebacterium spp., which will lead to a more complete understanding of the interactions between the microbiome and host immunity at the ocular surface.

Mucosal immunology of the ocular surface.

The eye is a sensory organ exposed to the environment and protected by a mucosal tissue barrier. While it shares a number of features with other mucosal tissues, the ocular mucosal system, composed of the conjunctiva, Meibomian glands, and lacrimal glands, is specialized to address the unique needs of (a) lubrication and (b) host defense of the ocular surface. Not surprisingly, most challenges, physical and immunological, to the homeostasis of the eye fall into those two categories. Dry eye, a dysfunction of the lacrimal glands and/or Meibomian glands, which can both cause, or arise from, sensory defects, including those caused by corneal herpes virus infection, serve as examples of these perturbations and will be discussed ahead. To preserve vision, dense neuronal and immune networks sense various stimuli and orchestrate responses, which must be tightly controlled to provide protection, while simultaneously minimizing collateral damage. All this happens against the backdrop of, and can be modified by, the microorganisms that colonize the ocular mucosa long term, or that are simply transient passengers introduced from the environment. This review will attempt to synthesize the existing knowledge and develop trends in the study of the unique mucosal and immune elements of the ocular surface.

Draft Reference Genome Sequence of Corynebacterium mastitidis RC, an Ocular Commensal, Isolated from Mouse Conjunctiva.

Here, we report the genome sequence of a protective commensal, Corynebacterium mastitidis RC, isolated from mouse conjunctiva. The C. mastitidis RC genome sequence is 2,153,054 bp in size and 96.95% complete, and we believe that it can contribute to the understanding of the functional immune attributes of the ocular commensal microbiome.

Nonretinoid chaperones improve rhodopsin homeostasis in a mouse model of retinitis pigmentosa.

Rhodopsin-associated (RHO-associated) retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified nonretinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules toward the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Furthermore, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL), indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that nonretinoid chaperones are promising drug candidates in treating RHO-associated RP.

Sensory Nerve Retraction and Sympathetic Nerve Innervation Contribute to Immunopathology of Murine Recurrent Herpes Stromal Keratitis.

Purpose: Herpes stromal keratitis (HSK) represents a spectrum of pathologies which is caused by herpes simplex virus type 1 (HSV-1) infection and is considered a leading cause of infectious blindness. HSV-1 infects corneal sensory nerves and establishes latency in the trigeminal ganglion (TG). Recently, retraction of sensory nerves and replacement with "unsensing" sympathetic nerves was identified as a critical contributor of HSK in a mouse model where corneal pathology is caused by primary infection. This resulted in the loss of blink reflex, corneal desiccation, and exacerbation of inflammation leading to corneal opacity. Despite this, it was unclear whether inflammation associated with viral reactivation was sufficient to initiate this cascade of events. Methods: We examined viral reactivation and corneal pathology in a mouse model with recurrent HSK by infecting the cornea with HSV-1 (McKrae) and transferring (intravenous [IV]) human sera to establish primary infection without discernible disease and then exposed the cornea to UV-B light to induce viral reactivation. Results: UV-B light induced viral reactivation from latency in 100% of mice as measured by HSV-1 antigen deposition in the cornea. Further, unlike conventional HSK models, viral reactivation resulted in focal retraction of sensory nerves and corneal opacity. Dependent on CD4+ T cells, inflammation foci were innervated by sympathetic nerves. Conclusions: Collectively, our data reveal that sectoral corneal sensory nerve retraction and replacement of sympathetic nerves were involved in the progressive pathology that is dependent on CD4+ T cells after viral reactivation from HSV-1 latency in the UV-B induced recurrent HSK mouse model.

Local Immune Control of Latent Herpes Simplex Virus Type 1 in Ganglia of Mice and Man.

Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen. HSV-1 genomes persist in trigeminal ganglia neuronal nuclei as chromatinized episomes, while epithelial cells are typically killed by lytic infection. Fluctuations in anti-viral responses, broadly defined, may underlay periodic reactivations. The ganglionic immune response to HSV-1 infection includes cell-intrinsic responses in neurons, innate sensing by several cell types, and the infiltration and persistence of antigen-specific T-cells. The mechanisms specifying the contrasting fates of HSV-1 in neurons and epithelial cells may include differential genome silencing and chromatinization, dictated by variation in access of immune modulating viral tegument proteins to the cell body, and protection of neurons by autophagy. Innate responses have the capacity of recruiting additional immune cells and paracrine activity on parenchymal cells, for example via chemokines and type I interferons. In both mice and humans, HSV-1-specific CD8 and CD4 T-cells are recruited to ganglia, with mechanistic studies suggesting active roles in immune surveillance and control of reactivation. In this review we focus mainly on HSV-1 and the TG, comparing and contrasting where possible observational, interventional, and in vitro studies between humans and animal hosts.

A whole-mount immunohistochemistry protocol for detection of mouse corneal nerves.

A cornea is innervated by sensory nerves, which branch into thick trunks, subbasal plexuses, and sensory endings. Appropriate assessment of nerve structure in a tissue provides a more complete understanding of the role of nerves in health and disease. Here, we present a whole-mount immunohistochemistry protocol that facilitates evaluation of nerve architecture throughout the mouse cornea. The fixation step in this protocol allows for reliable detection of nerve structures within the cornea and likely other tissues. For complete details on the use and execution of this protocol, please refer to Yun et al, (2020).

The Rcs Stress Response System Regulator GumB Modulates Serratia marcescens-Induced Inflammation and Bacterial Proliferation in a Rabbit Keratitis Model and Cytotoxicity In Vitro.

In this study, we tested the hypothesis that the conserved bacterial IgaA-family protein, GumB, mediates microbial pathogenesis associated with Serratia marcescens ocular infections through regulation of the Rcs stress response system. The role of the Rcs system and bacterial stress response systems for microbial keratitis is not known, and the role of IgaA proteins in mammalian pathogenesis models has only been tested with partial-function allele variants of Salmonella. Here, we observed that an Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h and demonstrated a notable reduction in inflammation based on inflammatory signs, including the absence of hypopyons, and proinflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid. We observed that bacteria with an inactivated Rcs stress response system induced high levels of ocular inflammation and restored corneal virulence to the gumB mutant. The high virulence of the ΔrcsB mutant was dependent upon the ShlA cytolysin transporter ShlB. Similar results were found for testing the cytotoxic effects of wild-type and mutant bacteria on a human corneal epithelial cell line in vitro. Together, these data indicate that GumB regulates virulence factor production through the Rcs system, and this overall stress response system is a key mediator of a bacterium's ability to induce vision-threatening keratitis.