How Does the Cornea Guard Against Infection and Regulate Inflammation?

The human cornea is a remarkable structure, and not just for its refractive capabilities. Though constantly exposed to opportunistic and potentially pathogenic microbes in the environment, it rarely becomes inflamed or infected, indicating the presence of a formidable, highly effective protection system.

In 2012, University of California, Berkeley, ophthalmic researcher Connie Tam, PhD, led a team that discovered a previously unknown component of that complex protection system — a structural protein called cytokeratin 6a (K6a), expressed by corneal epithelium, that also possesses novel antimicrobial properties.

Short phosphorylated fragments of K6a are potent, broad-spectrum bactericides, capable of binding to and killing a variety of ocular pathogens, including Pseudomonas aeruginosa, Staphylococcus aureas and Streptococcus pyogenes.

Since joining Cleveland Clinic’s Cole Eye Institute in 2014, Dr. Tam’s ongoing research is focused on better understanding the surprising corneal protective role of K6a and its peptide fragments. For instance, Dr. Tam is exploring how and where the protein fragments are generated, how they rapidly mobilize to a potential infection site, whether fragments can differentially target microbes, and whether K6a’s capabilities might be harnessed in new human antimicrobial agents.

Her latest findings, presented at the 2019 Association for Research in Vision and Ophthalmology (ARVO) annual meeting, reveal K6a’s ability to control local inflammation in the cornea, whether from infectious or non-infectious causes.

“We would never have thought that a keratin protein, which is shifting back and forth between its polymerized and subunit forms in the epithelial cell, could be an active player in the host defense and immunoregulatory mechanisms,” Dr. Tam says. “Perhaps understanding more about how it works can help us develop new therapies for infectious and inflammatory diseases.”

A Formidable Barrier

The healthy cornea’s ability to resist epithelial penetration by virtually all microbes and to constantly clear pathogenic bacteria from its surface points to the existence of a unique constitutive function distinct from the adaptive immune system. Ocular immune homeostasis is crucial, since corneal inflammation, if not prevented or constrained, could reduce optical clarity, alter refractive shape and ultimately result in permanent vision impairment or blindness.

Some elements of the cornea’s constitutive patho-protective capacity previously have been identified, including antimicrobial proteins in tears, and antimicrobial peptides (AMPs) of the β-defensin and cathelicidin classes produced by ocular surface epithelial cells. AMPs are the eukaryotic equivalents of antibiotics and work by binding to and lethally disrupting the permeability of microbial membranes. They also have a cell-signaling and mobilization capacity, serving to link the innate and adaptive immune systems. Comprehension of AMPs’ functionality and interactions is still evolving.

Dr. Tam suspected that additional ocular antimicrobial elements existed, helping to maintain immune homeostasis. She investigated, mining an immortalized cell line derived from human corneal epithelium for evidence of constitutively expressed AMPs.

Fractionation and mass spectrometry revealed AMPs derived from K6a, a sub-type of one of the more than 50 known human keratin proteins.

Keratins are key structural elements in multiple cell and tissue types (hair, teeth, nails, skin and mucosal epithelia) that maintain cell integrity and resilience. Fibrous bundles of keratin provide mechanical support and protection to the cell. They also are involved in wound healing and tissue regeneration. Keratins are known to regulate diverse cell functions, including gene and protein expression and cell growth, survival and differentiation.

Structurally, keratin sub-type K6a forms the cytoskeleton of epithelial cells, including those in the cornea.

Synthetic analogues of the natural K6a-dervied AMPs (KAMPs) that Dr. Tam tested showed bactericidal activity against several potential ocular pathogens, including Pseudomonas aeruginosa, Staphylococcus aureas and Streptococcus pyogenes. Dr. Tam confirmed KAMPs’ in vitro antimicrobial capabilities in vivo; mouse corneas with deficient K6a production or topically treated with a proteasome inhibitor showed impaired bacterial clearance efficiency in vivo and increased bacterial adherence on the surface ex vivo.

Tagged KAMPs tracked with fluorescence microscopy and high-resolution transmission electron microscopy revealed that when the peptide bonded with P. aeruginosa bacterial cells, it caused them to become permeable, lose motility and die within hours, although the precise mechanism is unknown.

In subsequent research, Dr. Tam showed that the human cornea’s exposure to bacterial ligands prompts remodeling of the cornea’s K6a filamentous network, via depolymerization of the keratin cytoskeleton and production of KAMPs in the cytosol. That remodeling is a host defense response involving direct up-regulation of KAMP production by the ubiquitin-proteosome system, without an appreciable increase in K6a gene expression or proteasome activity — a new role for proteasome-mediated proteolysis in innate immunity defense.

A Role in Inflammation Control

Dr. Tam’s newest findings, presented at ARVO 2019, shed more light on K6a’s immunoregulatory capabilities. Using human cells and mouse models, she found another important role of cytosolic K6a — controlling local inflammation driven by both infectious and non-infectious causes.

Specifically, when Dr. Tam used CRISPR-Cas9 genome-editing technology to delete K6a proteins in human corneal epithelial cells, and then stimulated the epithelial cells with bacterial outer-membrane molecules called lipopolysaccharides (LPS), the result was increased production of proinflammatory and chemotactic cytokines.

In mouse models of P. aeruginosa- or LPS-induced keratitis and sterile corneal epithelial wound healing, increased cytokine and chemokine levels in the K6a-deficient corneas worsened clinical outcomes. The negative manifestations included extensive neutrophil infiltration, irreversible tissue damage, impaired wound healing and, in the case of infectious keratitis, increased bacterial load.

Although inflammation is the body’s natural immune reaction to injury and microbial insults, unconstrained inflammatory responses can irreversibly damage tissue structure and, in the case of the cornea, pose a great risk of vision loss and blindness.

Common risk factors for corneal inflammation (keratitis) include contact lens use, corneal trauma, tear deficiency and diabetes. The Centers for Disease Control and Prevention estimates that at least one million clinical visits for keratitis occur annually in the United States, causing significant medical and socioeconomic burdens.

In clinical practice, topical corticosteroids are the current standard of care for corneal inflammation. Although powerful, corticosteroids function non-specifically to inhibit the host immune response and are associated with serious side effects, such as elevated intraocular pressure, corneal thinning, cataracts and secondary infection. Therefore, new and more targeted therapeutic options for controlling inflammation are necessary to improve patient care and clinical outcomes.

Building upon her newest findings, Dr. Tam will investigate the immunoregulatory mechanisms of cytosolic K6a, as she believes increased understanding of K6a’s molecular role will aid development of tissue-specific anti-inflammatory drugs with fewer side effects than corticosteroids.