This article stems from a literature review project given to the final year optometry students at the University of Johannesburg, class of 2017. What follows is the paper which received the best mark including some editing and additions.– Dirk Booysen , September 2017-

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Abstract

Keratoconus is traditionally defined as a non-inflammatory, bilateral, progressive ectasia of the cornea. Although inflammation is typically characterized by pain, redness, swelling and heat, these signs are not present in the avascular cornea. However, loss of function (as well as pain in the later stages), one of the other cardinal signs of inflammation is present. Recent research has shown increased pro- inflammatory and decreased anti-inflammatory mediators in the tears of keratoconic patients,which indicate that inflammation may play a role in the pathogenesis of keratoconus.

This article will summarise some of the more recent research findings.

Introduction

Keratoconus is a clinical term used to describe a progressive, non-inflammatory, bilateral, predominantly degenerative disease, with mechanically induced trauma accelerating its course.Progression results in focal thinning and steepening of the cornea. Changes in refractive error follow and eventually lead to an abnormal conical corneal shape ^1-4, with impaired visual acuity.

Traditionally, keratoconus has been classified as a non-inflammatory disease because of the lack of classical signs of inflammation, which include, heat, redness, swelling and pain. However, loss of function, another cardinal sign attributed to Virchow, is present⁵.  Histologically, the corneal tissues of keratoconic patients lack marked cellular infiltration and neovascularization seen in chronic inflammation ⁶. Inflammation can be seen as a process characterized by the release and activation of toxic cellular mediators. These mediators promote tissue injury and result in some, but not all of the clinical signs of inflammation. Given the wide range of inflammatory mediators or cytokines associated with keratoconus, it may be more appropriate, to at least classify keratoconus as a quasi-inflammatory or inflammatory-related disease⁵. It is difficult to distinguish primary disease mechanisms from secondary inflammatory or degenerative effects. It might also be possible that the clinical appearance of keratoconus is related to a number of unrelated environmental or genetic effects ⁵. For example, eye rubbing related to keratoconus could increase the corneal temperature, over express the levels of pro-inflammatory cytokines, as well as proteinases in the tear film. Therefore, it can cause epithelial thinning with repercussions in every layer in the cornea ².

Evidence of inflammation

Nishtala et al., (2016) conducted an enzyme linked immunosorbent analysis of capillary collected tears in keratoconus patients. The tears showed elevated levels of inflammatory markers such as: IL-6, TNF-α and MMP-9 as well as higher gelatinolytic and collagenolytic activity. They concluded that a variety of matrix metalloproteinases (MMP) -1, -3, -7, -13, interleukins (IL) -4, -5, -6, -8 and tumour necrosis factor (TNF) –α and –ß are elevated in the tears of keratoconic patients. These inflammatory markers are generally released at the site of an injury by the innate immune system to activate an inflammatory response⁷.

Various other reports have associated keratoconus with alterations of cytokines in the tear film ¹. Cytokines are produced by many cell populations, but the predominant producers are helper T cells (Th) and macrophages. Cytokines can be pro-inflammatory or anti-inflammatory. Pro-inflammatory cytokines such as interleukin IL-1 (IL-1α and IL- 1β) show increased levels in patients with keratoconus,while anti-inflammatory cytokines such as lipophilin levels are decreased ^{1, 8}.

IL-1 is secreted by the corneal epithelium after tissue damage or apoptosis and the subtype IL- 1α is present after corneal trauma and inflammation. In addition to these cytokines , Lema and Duran(2005) found, that there is an overexpression of IL-6, tumour necrosis factor (TNF)-α and matrix metalloproteinase (MMP-9) (respectively 3,2 and 10 times higher) in the tears of keratoconicpatients,compared to the tears of normal individuals⁹. Matrix metalloproteinase are enzymes responsible for the degradation of extracellular matrix proteins and are secreted in response to cytokines and growth factors¹. Galvis et al., (2015) stated that thinning and ectasia of the cornea are related to a degraded extracellular matrix, involving inflammatory events such as increased levels of MMP-9, IL-6 and TNF-α, as well as an increase in oxidative stress ⁶. Immuno-inflammatory cells such as macrophages, leucocytes and antigen presenting cells, have been observed in the epithelium and stroma of a keratoconic cornea¹. The neutrophil to lymphocyte ratio (NLR) has also been found to be significantly higher in the serum of patients with progressive keratoconus.

This supports a relationship between progressive keratoconus and an increased systemic inflammatory response¹⁰.  Additionally, TGF-ß2 has been found to be elevated in both the aqueous humour and corneal epithelial cells in keratoconic patients¹. Basal epithelial cells in keratoconic corneas show moderate-to-strong immunoreactivity for hepatocyte growth factor and it’s receptor ¹¹.The serum albumin levels in the tears of individuals with keratoconus were more than three times higherthan in individuals without keratoconus ⁸. The presence of serum albumin is indicative of blood-ocular barrier failure in conjunctival vessels and suggests the presence of conjunctival inflammation.

They also found a significant decrease in the levels of lipophilin A and C in the tears of keratoconic patients. Lipophilins are proteins with anti-inflammatory properties and suggest that inflammatory processes are more active on the ocular surface of keratoconic patients⁸. Balasubramanian (2012) found that there is a twofold decrease in the total protein levels in the tears of non-contact lens wearers with keratoconus, compared to patients without keratoconus. They also found that there is a significant reduction in immunoglobulin A and lactoferrin ¹².

Biomarkers	Function	Variation in Keratoconus
Lactoferrin – protease inhibitor	Anti-microbial and anti-inflammatory protein	Decreased in keratoconus patients. Lactoferrin down regulates the expression of cytokines (IL-1, IL-2, IL-6 and TNF-α) and proteinases. Suggests a disruption of the protective barrier function
Interleukin-1 (IL-1α, IL-1β) – inflammatory mediator	Promotion of pro-inflammatory cytokines, regulation of cell growth	Increased IL-1α and IL-1β expression. IL-1 is secreted by the corneal epithelium during corneal trauma inflammation and subsequent apoptosis. IL-1 stimulation stimulates production of prostaglandins (10x higher in keratoconus) and lowers collagen production. MMP activity in the cornea is partly regulated by IL-1
Interleukin-6 – inflammatory mediator	Pivotal role in stimulating several immune responses, such as eradication of infection and wound repair	Secreted by many cells including dendritic cells, endothelial cells, T-cells, and macrophages. IL-6 is increased in the tear fluid of keratoconics due to rubbing and contact lens wear. Directly related to corneal thinning.
Tumour necrosis factor-α – inflammatory mediator	TNF-α contributes to the production of IL-6 by the keratocytes, and induces the expression of MMP-9 in the cornea.	Increased in the tear film and cornea and fibroblasts of keratoconic patients. TNF-α interrupts the barrier function of the corneal epithelial cells. TNF-α can be produced by the corneal epithelium, keratocytes, and endothelial cells in response to corneal damage due to environmental factors
Interleukin-17 – inflammatory mediator	Pro-inflammatory cytokine	Elevated IL-17 stimulate fibroblasts to secrete various pro-inflammatory cytokines including IL-6, IL-8 as well as MMPs. IL-17 is produced by T helper 17 cells and upregulated by TGF-βand IL-6. IL-17 is not produced in normal corneas.
Transforming growth factor-β2– inflammatory mediator	Controls cell proliferation and differentiation in epithelial and endothelial cells	Interact with several collagen types and stimulate the secretion of MMPs which can influence the structure and collagen distribution in the cornea. In response to tissue injury TGF-β activation dives microfibroblasts to restore the integrity of the cornea by secreting extracellular matrix
Vascular endothelial growth factor and nerve growth factor (VEGF and NGF)	VEGF is associated with neovascularization and is highly expressed in corneas of healthy individuals. NGF plays a role in wound healing and affects epithelial cell proliferation	Lower levels of VEGF in tears and corneal samples of keratoconics. NGF is a normal constituent of the tear film and is increased in keratoconic corneas.
Metalloproteinases (MMPs)- proteolytic enzyme. Modulated by the cytokines  IL-1 and TGF-β	MMP-9 is responsible for degrading denatured collagen fibrils.	Increased in every structure of the cornea in keratoconus.MMPs influence the structure and collagen distribution in the cornea.
Serum albumin	Tear albumin can be considered a non-specific marker of ocular surface integrity. There are no reports to date that demonstrate any physiological role for albumin in tears. However, possible effects exerted by albumin may include anti-inflammatory properties or antioxidant effects.	Increased three fold. When tear albumin levels increase, it is synonymous with conjunctival inflammation as it signifies the disruption of the blood-ocular barrier.
Lipophilin A and C	Lipophilins are proteins with anti-inflammatory properties	Decrease in the levels of lipophilin A and C in the tears of keratoconus patients

What about rubbing, dry eye, atopy and contact lens wear in keratoconus?

Rigid gas permeable contact lenses worn by keratoconic patients caused an increase in pro-inflammatory cytokines (IL-8, epithelial growth factor, IL-6 and TNF-α) inthe tears ¹¹.Eye rubbing also elevates the temperature of the cornea,which leads to anincreasein pro-inflammatory cytokine levels².Meibomian gland dysfunction (MGD) and aqueous deficiency can cause an abnormally high tear osmolarity. This leads to the production of inflammatory mediators in the tear layer and cornea tissue, which may aggravate inflammatory processes in keratoconus ⁵. In atopic conditions, the cytokine IL-10 (anti-inflammatory) is decreased,while IL-13 (pro-inflammatory) and TNF-α are increased⁵. Pro-inflammatory cytokines are released by epithelial cells in reaction to ultraviolet radiation. This may add to other inflammatory processes in the keratoconic cornea ⁵.

Conclusion

It is evident from the literature, that instead of a simple increase in pro-inflammatory cytokines in the tears and tissues of keratoconic patients, there may be a complex imbalance between pro-inflammatory and anti-inflammatory cytokines. Their regulatory functions can lead to an alteration in epithelial and stromal functions of the cornea. However, it is not clear to what extent the increase in cytokines can be attributed to keratoconus or to secondary factors. What must also be considered is the wound healing response caused by rubbing, contact lens wear, and tear film disturbance ⁵. At the time of writing,the pathophysiology of keratoconus can probablybest be classified as alterations in the stromal composition, imbalance of pro-inflammatory and anti-inflammatory molecules, imbalance of the enzymes that causes extracellular matrix degeneration and their inhibitors; oxidative stress and cellular hypersensitivity. These events can occur simultaneously and may present a positive feedback between one another ⁶.

Finally, several discrepancies impede our understanding of the inflammatory model of keratoconus. Despite the evidence collected form several studies, keratoconic corneas strikingly lack the histological and clinical features of inflammation. Furthermore, coexisting disease or mechanical trauma; such as allergies, MGD, dry eye, rubbing and contact lens wear potentially mask the true underlying inflammation in keratoconus. Ongoing research may provide tools for further investigation of keratoconus related inflammation. Elucidating the etiopathological mysteries of this common corneal malady, may enhance the possibility of beneficial anti-inflammatory interventions.

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*Dirk J Booysen. Dip Optom FOA(SA), TMOD (USA), MC Optom (UK), CAS (NECO), D Optom (Aston), FSLS. Private practice Krugersdorp South Africa, Senior Lecturer University of Johannesburg Department of Optometry

**Mirinka Celliers. Final year Optometry student (B Optom), University of Johannesburg Department of Optometry

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