SUBCLINICAL INFLAMMATION

MORE THAN MEETS THE EYE

A look at subclinical inflammation, including whether and how it is affected by contact lens wear.

CAMERON POSTNIKOFF, MASC, & JASON J. NICHOLS, OD, MPH, PHD

Inflammation is classically defined by four characteristics: redness, heat, swelling, and pain. In terms of the ocular surface, we usually associate inflammation most with conjunctival redness or with the presence of observable infiltrates. However, how often do you see patients who complain of ocular discomfort, either with or without contact lens wear, who seem to have no clinical cues to explain their symptoms? These symptoms may possibly be driven by inflammation, but at a scale that is not currently observable with our clinical capabilities—this defines subclinical inflammation.

Subclinical inflammation describes pathologies that seem to escape our current clinical metrics, yet may be playing a major role in ocular surface pathology. It is also important to note that there are many subclinical inflammatory processes that are related to homeostasis and everyday life. Without these mechanisms, our eyes would be continuously infected and attacked by the many pathogens that we encounter in our environment. This article will provide a glimpse into the inflammatory state that exists on our ocular surface and how these factors may be responsible for some of the symptoms that our patients encounter.

WHY IS THE OCULAR SURFACE INFLAMMATORY?

The ocular surface, comprised of the lids, tears, and corneal and conjunctival epithelium, is in a state of constant change, with cell growth and cell death, constant tear replenishment, and commensal (or non-pathogenic) bacteria that live in our tears and eyelids. Our eyes are also constantly attacked by allergens, dust, pathogenic bacteria, ultraviolet (UV) rays, and an assortment of other objects, both large and small.

As a result, our eyes need to be capable of dealing with all of these insults, with the number one goal of preserving vision. Innate factors—such as blinking, facial geometry, and tear replenishment—all serve as a first-line defense against foreign invaders.

At a subclinical level, the tears are full of proteins and lipid mediators that help resolve more harmful invaders. The tears contain more than 1,500 different proteins, ranging from a few such as lysozyme and lactoferrin that are highly abundant, to many obscure and scare proteins whose purpose at the ocular surface has yet to be determined (Zhou et al, 2012).

This means that there are a seemingly infinite number of processes that are ongoing at any period in time. However, there are probably only a few that significantly affect a contact lens wearer. Importantly, contact lenses also have an impact on the entire tear film and ocular surface environment by affecting blinking, not to mention tear film properties such as stability, evaporation, temperature, thickness, volume, pH, and osmolarity. Each of these individual factors may also trigger inflammatory changes in and of themselves.

Here is a look at two different mechanisms that underlie subclinical inflammation at the ocular surface: leukocyte dynamics from conjunctival-associated lymphoid tissue; and cellular turnover, with combined extracellular matrix turnover.

Leukocyte Dynamics The immune system is predominantly thought of as two systems that work together to maintain health and combat disease—namely, the innate and adaptive immune systems. The innate system deals with non-specific defense and is mediated by the aforementioned innate factors such as blinking and orbital geometry. At a cellular level, this system is thought to be mediated by natural killer cells, mast cells, monocytes, macrophages, and neutrophils. These kinds of cells are primarily involved in foreign body recognition and pathogen resolution.

The adaptive immune system is specific and is also referred to as the acquired immune system, given that it has a long-lasting memory to allow for protective immunity. This system is mediated primarily by lymphocytes, both those that mature in the thymus (T cells) as well as those that mature in the bone marrow (B cells).

Both of these systems should therefore work in harmony to provide protection from invaders, provide immunity to non-harmful substances, and overall form our complete immune system. But, what happens in an environment that doesn’t elicit classical inflammatory immune responses, such as the eye?

The eye is considered to be an immune-privileged organ, which is a term that has taken on many meanings and connotations over time. Overall, immune privilege means that there is a tolerance of antigens without an inflammatory immune response. Immune privilege was a concept that was first proven at the ocular surface where it was found that a corneal transplant could be well-tolerated between two individuals of the same species, allowing for the first successful organ transplant (Niederkorn and Stein-Streilein, 2010).

Over time, the meaning of immune privilege became confused with the idea that those sites were capable of resolving inflammation without leukocyte-mediated immune responses. It is important to note that the eye is not devoid of immune cells. Rather, the ocular surface is served by conjunctival-associated lymphoid tissue and is surveilled by neutrophils in the closed eye (See the sidebar on page 31 for more information about the conditions in the closed eye).

Conjunctival associated lymphoid tissue resides primarily in the upper eyelid (Figure 1), which allows for plasma cells, dendritic cells, and T cells to continuously sample the tears for antigens (Knop and Knop, 2010). This immune tissue is part of the ocular surface adaptive immune system.

Figure 1. Immune environment of the tears in contact lens wear.

An antigen is considered a toxin or foreign substance, which may induce an immune reaction through the production of antibodies. Antibody production is primarily conferred by B cells (of which plasma cells are a differentiated form), and the importance of antibodies relates to immobilization or neutralization of pathogens.

While B cells are in charge of antibodies, T cells may be either cytotoxic (CD8+) or helper (CD4+), which essentially means that T cells have an ability to coordinate and resolve immune reactions. Both B and T cells may also have memory functions to remember, or be prepared for, antigens that they have encountered previously, which could either allow for tolerance or for an expedited immune response. T cells primarily communicate through the use of cytokines, which are a group of small proteins vital to immune cell signalling.

While this explanation is an oversimplification, it is important to recognize that all of these actions are performed on a daily basis. Further, plasma cells and T cells of the ocular surface are constantly releasing antibodies and cytokines into the tear film, all of which could interact with and be affected by lens wear.

Cell and Extracellular Matrix Turnover While the adaptive immune system primarily contributes to the subclinical inflammatory environment of the tears from the eyelids, cellular turnover is primarily a factor for the production of inflammatory mediators from the cornea. Complete turnover of the corneal epithelium occurs in about seven to 10 days, with basal cells constantly replenishing to form new superficial cells that eventually are shed from the ocular surface into the tear film (Hanna et al, 1961). The cells that enter the tear film thus introduce membrane phospholipids and extracellular matrix components in the process. Generically, these factors may be referred to as metabolic by-products.

Phospholipids themselves are not inflammatory, but phospholipids can be easily broken down into arachidonic acid via phospholipases, which are common in the tear film (Figure 2). Arachidonic acid can then be further broken down to lipid mediators, specifically known as eicosanoids, thromboxanes, or prostaglandins. All of these lipid mediators may be involved in cell-signaling and inflammation. Collagen breakdown, facilitated by enzymes called matrix metalloproteinases (MMPs), may also result in the production of inflammatory tripeptides, such as proline-glycine-proline (PGP), which are known to recruit neutrophils.

Figure 2. Cell and extracellular matrix turnover of the corneal epithelium may lead to the production of inflammatory lipid and protein mediators.

While tear turnover is often fast enough to clear away these immune factors, the introduction of a contact lens splits the tear film into the post-lens tear film and the pre-lens tear film (Nichols and King-Smith, 2003). Those tears that remain trapped under the lens have demonstrated reduced ability to exchange with the pre-lens tear film (Muntz et al, 2015) and may facilitate an accumulation or concentration of inflammatory factors under the lid. Further, a contact lens may alter the environment of the tear film by absorbing components of the tear film or by acting as a delivery device for lens solution components (Gorbet and Postnikoff, 2013).

Any of the above mechanisms may be affected by a contact lens. Additionally, a disturbance to ocular surface homeostasis could affect leukocyte activation or cytokine and antibody production. Further, contact lenses could affect cell turnover, matrix turnover, or the removal of these mediators from the ocular surface. These are only two mechanisms of many both homeostatic and immune mechanisms that occur at the ocular surface. As a result, it is extremely difficult to find the source of lens intolerance or discomfort. And, it is imperative that more research is done to better elucidate these factors.

WHEN WILL SUBCLINICAL BECOME CLINICAL?

The term “subclinical” implies that the phenomena are currently out of the scope of being evaluated or managed in current eyecare practice. However, there are new innovations that are being considered and investigated to better diagnose and treat subclinical inflammation related to contact lens wear.

Over time, our imaging techniques have greatly improved to allow us to see changes in blood flow and leukocyte populations, allowing us to better diagnose or observe inflammatory processes. One specific idea is to examine changes in blood supply to the ocular surface using high-resolution cameras.

NEUTROPHILS AND THE CLOSED EYE

Eyelid closure during sleep is a very significant event in the diurnal regulation of the eye. At the retina, it is important that the eyelids are closed to allow for the production and regulation of melatonin. At the ocular surface, the entire immune environment changes, and the tears become markedly more inflammatory. Specifically, there is an altered tear film proteome, with increases in matrix metalloproteinases, complement activation products, pro-inflammatory cytokines, and an influx of almost 1 million neutrophils (Gorbet et al, 2015; Sack et al, 2000).

Neutrophils were classically considered to be simple effector cells of the innate immune system, mostly involved with phagocytosis and immobilization of microbes and other pathogens. This paradigm has recently shifted, however, with neutrophils demonstrating impressive heterogeneity and adaptive immune functions. Interestingly, the neutrophils isolated from the closed eye are an example of this heterogeneity, as they are markedly different from blood-isolated neutrophils and seem non-responsive to inflammatory stimuli (Gorbet et al, 2015). However, these cells appear more activated, which means they are a source of some of the inflammatory mediators in the tear film (Figure 3).

Figure 3. Neutrophils are highly granular cells and have the capability to release many potent inflammatory mediators, such as reactive oxygen species, matrix metalloproteinase-9 (MMP-9), neutrophil elastase, and phospholipase. Several of these components have been identified in the tears of contact lens wearers who are experiencing discomfort.

The link between these closed-eye neutrophils and the adaptive immune system has yet to be demonstrated, but it is hypothesized that these neutrophils perform sophisticated functions to maintain homeostasis of the tear film. They may also play a role in overnight contact lens wear, in which the risk of microbial keratitis following overnight lens wear is significantly increased (Stapleton et al, 2008).

Future research will focus on better understanding how closed-eye neutrophils contribute to ocular surface homeostasis and dysregulation. Additionally, researchers will need to determine whether these cells can be a target of diagnosis or therapy for ocular surface complications.

In inflammation, the commonly observed redness and swelling is due to blood vessel dilation, but that dilation can greatly vary in scale, and minute changes may not be readily observable. Figure 4 shows microvasculature in the upper tarsal conjunctiva, the lid margin, and the bulbar conjunctiva and follows how they change in contact lens wear (Deng et al, 2016). Notably, there is an observed increased in microvasculature density following six hours of lens wear. These changes have also demonstrated a correlation with contact lens discomfort—also primarily at the lid margin (Deng et al, 2016).

Figure 4. Changes in microvasculature following six hours of contact lens wear. Images represent the upper tarsal conjunctiva (top row), lid margin (middle row), and bulbar conjunctiva (bottom row). Note the increased microvascular density in the lid margin following lens wear. This image is reproduced with permission of the American Journal of Ophthalmology/Elsevier. The image was originally published in Deng et al, 2016.

The lid margin has been of recent interest in the contact lens field—with lid wiper epitheliopathy—as demonstrated by lid margin staining with lissamine green. This is experiencing mixed success as a clinical sign of contact lens discomfort (Efron et al, 2016).

Imaging of the lid margin with in vivo confocal microscopy has also demonstrated that this area may have a resident immune component (Knop et al, 2011). Further, in vivo confocal microscopy has also demonstrated an upregulation of Langerhans cells (dendritic cells) in the lid margin in subjects who have contact lens discomfort (Alzahrani et al, 2016). Interferometric methods may also have the potential ability to sense changes in tear film physiology associated with inflammation. Altogether, imaging modalities are incredibly important and advantageous for ocular surface investigation.

With recent developments in miniaturization, microfluidics, and nanotechnology, technology has given way to the creation of lab-on-a-chip devices to detect specific proteins, mediators, or changes in osmolarity. Such devices could become point-of-care diagnostics capable of sensing inflammatory mediators to determine whether subjects are at risk for lens discomfort or whether they may be responsive to specific therapies or interventions.

While this article does not discuss important considerations such as differences in contact lens materials or material design, one potential opportunity for treatment of subclinical inflammation is to use the lens itself. Contact lenses are highly adaptable and can be loaded with active molecules or coated with an active coating. Strategies such as these could be used to combat or prevent inflammation. Lastly, there is increasing interest in using contact lenses or contact lens cases as biosensors to detect any imbalance in tear film parameters; these imbalances could be related to either local or distal disease or dysregulation (Phan et al, 2016).

It’s important to note that this article presents only a broad overview regarding the immune environment of the ocular surface. It is imperative that researchers continue to address the basic science and the clinical investigation of subclinical inflammation to improve patient care and tolerance of contact lens materials.

However, it is clear that there is a lot of opportunity in the contact lens field with regard to inflammation of the ocular surface. And, it is bound to be an exciting few years as new biomarkers are identified, subclinical signs become clinical, and new technologies are developed to allow for improved prevention, detection, and treatment of contact lens-related inflammatory complications. CLS

For references, please visit www.clspectrum.com/references and click on document #252.

Cameron Postnikoff is a trained engineer and current vision science PhD student at the University of Alabama at Birmingham (UAB).
Dr. Nichols is an assistant vice president for industry research development and professor at the University of Alabama-Birmingham as well as editor-in-chief of Contact Lens Spectrum and editor of the weekly email newsletter Contact Lenses Today. He has received research funding from Johnson & Johnson Vision Care and honoraria from Shire.