KEY CONCEPTS
- •
Inflammatory mediators are among the key contributing and modifiable factors in keratoconus (KC).
- •
A significant increase in the inflammatory factors and immune components in the corneal tissues, tear fluid, and/or blood of KC patients is observed.
- •
Endogenous inflammation dampeners such as vitamin D and vitamin D receptors are reduced in KC.
- •
KC is strongly associated with immune disorders such as atopy and allergy that are known to increase itch factors and subsequent eye rubbing, a key risk factor in KC pathogenesis.
- •
Topical anti-inflammatory treatments and management of atopy or allergic conditions have been shown to reduce the levels of inflammatory factors, along with stabilizing the disease in KC patients.
Introduction
The long adhered to definition of keratoconus (KC) has described it to be a noninflammatory corneal ectatic disease. KC is considered a noninflammatory condition, and the definition has remained so despite the growing evidence of altered inflammatory factors in the KC cornea. This is due to the lack of the “cardinal signs” of inflammation (redness, heat, pain swelling) expect for the loss of function in the majority of KC patients. Studies over the last two decades have demonstrated the relationship between KC and a variety of dysregulated inflammatory factors, both local (ocular surface, cornea) and systemic, along with the increase in prevalence of KC in subjects with immunological conditions, such as atopy and allergy. Management of inflammation and/or associated immune conditions has shown cessation or stabilization of disease progression, suggesting the pivotal role of inflammatory factors in the pathogenesis of KC. Therefore, it is apparent that the focal ectatic condition of the human cornea in KC is mediated by inflammatory factors. This chapter discusses the altered inflammatory factors in KC and their relevance in pathogenesis, and offers insights into dampening of the local inflammatory milieu in the management of KC.
Status of Inflammatory Factors in Keratoconus
Altered levels and expression of various inflammatory factors in the tear fluid, corneal tissues, cultured primary corneal cells, aqueous humor, serum, and/or saliva of KC patients have been reported. Fig. 11.1 summarizes the factors altered in the various types of sample in KC.
TEAR FLUID
The levels of classical proinflammatory factors including interleukin (IL)-1α/β, IL-6, , IL-8, , , tumor necrosis factor alpha (TNF-α), IL-17A, , C-C motif chemokine ligand 5 (CCL5), and nerve growth factor (NGF) , have been found to be increased in the tear fluid of KC patients. Tear fluid immunoglobulin E (IgE) and allergy-associated cytokines such as IL-4, IL-5, , and IL-13 were also elevated in KC patients. A group of other inflammatory factors that were reported to be elevated in tear fluid of KC patient in at least one study include epidermal growth factor (EGF), IL-2, IL-3, IL-21, IL-23, interferon alpha (IFN-α), interferon gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein-1 alpha/beta (MIP1-α/β), E-selectin, soluble intercellular adhesion molecule 1 (sICAM1), S100A6, and plasminogen activator inhibitor type 1 (PAI-1). An antiinflammatory factor, IL-10, was reported to be either increased or decreased , , in the tear fluid of KC patients. IL-11, interferon γ-induced protein (IP-10), , secretory immunoglobulin A (sIgA), and serum albumin were lower in the tear fluid of KC patients compared with controls. The key matrix metalloproteinase, MMP-9, , , , , , , was higher in the tear fluid of KC patients. The other MMPs that were also higher in tear fluid of KC patients include MMP-1, , MMP-2, MMP-3, MMP-7, and MMP-13. , Importantly, the endogenous regulator of MMPs, tissue inhibitor of metalloproteinases 1 (TIMP1), was lower in the tear fluid of KC patients compared with controls. , More recently, altered immune cell proportions on the ocular surface of KC patients compared with normal controls were also reported. These observations clearly indicate an increased inflammatory factor milieu on the ocular surface of KC patients.
CORNEAL TISSUES AND CULTURED CORNEAL FIBROBLASTS
Inflammatory cellular infiltrates were observed in the corneal tissue sections from a subset of KC patients. , In addition, elevated expressions of inflammatory factors such as IL-1α/β, , IL-6, , IL-17A, TNF-α, , S100A8, MMP-1, MMP-2, MMP-8, and MMP-9, , , with potential to modulate extracellular matrix (ECM) remodeling, were higher and TIMP1 was lower in the corneal epithelium and/or stroma of KC patients. The expressions of TNF-α, IL-6, and MMP-9 were higher in the epithelium/stroma from the ectatic region (cone) compared with the nonectatic region (periphery) of the KC cornea. Further, the levels of IL-1α/β, IL-6, MMP-1, , MMP-2, , MMP-3, and MMP-9 , were also higher in cultured primary corneal fibroblasts/keratocytes from KC patients compared with non-KC controls. These findings corroborate the inflammatory milieu findings in the tear fluid of KC patients mentioned earlier.
Cell surface receptor expressions that are known to mediate inflammatory signaling such as toll-like receptors (TLR2, TLR4), TNF receptor 2 (TNFR2), EGF receptor, and hepatocyte growth factor (HGF) receptor-/c-Met have been found to be higher in KC corneas. Transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), transcription factor p65 (RelA), and RAR-related orphan receptor gamma (RORγ) that regulate the production of proinflammatory cytokines such as IL-6, TNF-α, and IL-17A were also higher in KC epithelium. In addition, the endogenous negative modulator of inflammatory response, the vitamin D receptor, was decreased in KC epithelium. A higher expression of Fas ligand (FasL)—apoptosis inducer or regulator of cell death—was also observed in KC cornea, which may be related to decrease in keratocyte density in KC. These findings indicate an aberrant cellular inflammatory factor profile in the KC cornea.
BLOOD, SALIVA, AND AQUEOUS HUMOR
Systemic alteration in some of the inflammatory factors has also been reported in KC. IL-1β, IL-6, TNF-α, MMP-9, and IgE were found to be higher in the serum of KC patients. Altered immune cell proportions in the blood such as neutrophil to lymphocyte ratio (NLR), , platelets to lymphocyte ratio (PLR), and monocyte to high-density lipoprotein cholesterol ratio (MHR), indicative of oxidative stress and systemic inflammation, were higher in the KC patients compared with controls. The expression of inflammatory signaling mediators such as TLR2, TLR4, and NFκB was raised in the peripheral blood leukocytes of KC patients. An increase in the systemic inflammatory status in KC patients has been observed with a decrease in serum vitamin D, a major endogenous inflammatory modulator. IL-8, IL-16, and stem cell factor (SCF) were increased in the saliva and transforming growth factor beta 2 (TGFβ2) was higher in the aqueous humor of KC patients compared with controls.
GENETIC VARIATIONS
Genetic alterations such as single nucleotide polymorphisms (SNPs) in collagens and lysyl oxidase, and endogenous collagen cross-linking enzymes, have been implicated as predisposing factors in KC. , Genetic variants in inflammatory genes have also been associated with increased predisposition to KC. SNPs in IL-1α (rs2071376) and IL-1β (rs1143627, rs16944, rs16944, s1143627) genes were associated with an increased risk of development and severity of KC. TNF-α promoter polymorphism rs1800629 was significantly associated with the development of KC with increased levels of TNF-α in the serum and expressions of TNF-α, TNFR2, RelA, and IL-6 in corneal tissues. Polymorphism (rs763110) in FASL gene was associated with increased occurrence of KC. A single nucleotide variation was also reported in IL-17B gene in the KC patients, thus indicating a genetic basis for altered inflammatory factors in some KC patients.
Association Between Inflammatory Factors and Disease Characteristics
The association between the levels of certain altered inflammatory factors and KC-related corneal clinical indices suggests the plausibility of their role in KC pathogenesis. IL-1β has exhibited a positive correlation with disease severity and a negative correlation with central corneal thickness (CCT), corneal hysteresis (CH), and corneal resistance factor (CR). A positive relationship between IL-6 levels and disease stage, , maximum curvature power on front of cornea (K max ), and steep keratometric reading (K2), along with a negative relationship with CCT, CH, and Klyce/Maeda KC index was observed. Similarly, a positive association between TNF-α levels and the Belin/Ambrósio display enhanced ectasia total derivation value (BAD-D), average curvature power on front of cornea (K mean ), K2, and deformation amplitude, as well as a negative association with thinnest corneal thickness, CH , and CR was observed. IL-4 levels positively correlated with disease severity and negatively with CCT, CH, and CR. IL-8 level positively correlated with BAD-D and negatively with corneal thickness at the thinnest point of the cornea. IFN-γ levels associated with progression exhibited a negative correlation with CCT, CH, and CR. NGF correlated positively with K2 index. CCL5 showed a positive association with disease stage and a negative association with CH and CCT. The expression of TLR2 and TLR4 had a positive association with K2 and coma along with a negative association with minimum thickness point of cornea. MMP-9 levels showed a positive relationship with severity of KC. , , , , MMP-9 levels positively correlated with K2, , BAD-D, and negatively with thinnest corneal thickness in KC. In addition, the disease progression in KC patients was related to increased tear MMP-9 levels. Similarly, MMP-13 was also associated with KC severity and progression. , Lysyl oxidase, a key enzyme that facilitates endogenous cross-linking between collagen and elastin fibrils, was reduced , in KC and demonstrated a decreasing trend with increasing grades of KC. The increased expression of TNF-α, IL-6, and MMP-9 in the ectatic region also exhibited a decrease in the levels of collagen and lysyl oxidase compared with the nonectatic region KC cornea, thus emphasizing the inverse relationship between inflammatory factors and ECM components in the KC cornea.
Inflammatory Factors and Keratoconus Pathogenesis
The KC cornea is characterized by biomechanical weakening and structural change in the form of steepening with consequent decrease in visual acuity. The key changes in the KC cornea are observed in the corneal epithelium, epithelial basement membrane, Bowman’s layer, and stroma. Ultrastructural changes in the KC cornea include reduced expression of ECM components such as collagens, altered lamellar organization, fibril features, proteoglycan profile, and fewer stromal keratocytes. , Although KC is an ectatic condition, fibrotic changes in the cornea have also been observed, , particularly in later stages of disease, owing to a compensatory response that contributes to KC pathology by causing scarring. This was supported by studies reporting higher levels of TGF-β—an antiinflammatory and profibrotic factor in KC. , , Ectasia and fibrosis are conditions at the opposite ends of the spectrum of ECM-associated diseases affecting different organs. The dynamic and reparative nature of the ECM remodeling process and the factors influencing them need to be determined to understand disease pathogenesis.
Inflammatory mediators and immune cells are known as one of the major factors that contribute to dysregulation in ECM modeling by modulating cellular signaling pathways that regulate the key ECM component production such as collagens, proteoglycans, cross-linking enzymes, and proteolysis enzymes. Environmental insults including oxidative stress, atopic conditions, and eye rubbing can induce the production of inflammatory factors on the ocular surface that can compromise barrier function and alter cellular metabolism and signaling, which can further amplify the level of inflammatory mediators. Eye rubbing is considered as an important risk factor associated with KC. Itch sensations are also mediated by inflammatory or immune factors that stimulate release of itch factors. Studies have demonstrated an increase in IL-6, TNF-α, and MMP-13 levels in tear fluid following eye rubbing even in normal eyes, , suggesting an inflammatory factor–associated mechanism in KC. Inflammatory factors such as IL-1β and FasL that are increased in the KC cornea mediate corneal tissue organization by regulating keratocyte apoptosis. , , Hence, increased activity of these factors may induce keratocyte apoptosis resulting in reduced keratocyte density and impacting ECM remodeling. TNF-α and/or IL-1β that are increased in KC are known to reduce collagen synthesis and lysyl oxidase expression. Altered collagen and abnormal arrangement are also due to increase in proteolytic enzymes and decreased endogenous levels of protease inhibitors. Inflammatory factors trigger tissue protease activity and progression in KC, which is supported by disease grade–dependent increase in the levels of MMP-9 in KC. , Interestingly, IL-17, a known inducer of MMP-9 and barrier function breakdown, , is also elevated in KC. These factors can collectively act on the stromal cells (keratocytes or fibroblasts) resulting in their altered function and density. The altered stromal cells affect the ECM remodeling processes with an increase in MMPs and decrease in collagens and endogenous cross-linking enzyme, thus causing structural weakening of the corneal stroma, KC initiation, and progression.
Inflammatory Factors Modulation and Keratoconus Management
The management of early stage KC includes glasses and rigid contact lenses for visual rehabilitation. Strategies such as intracorneal ring segments or topography-guided treatments are also available to prevent disease progression. More recently, collagen cross-linking has evolved as one of the effective ways to stabilize the progression of the disease. However, corneal transplants are required to manage advanced stage KC to restore vision. Therefore it would be clinically prudent to detect KC at its earliest form and manage it by treating the associated risk factors in the patient. It is becoming apparent that allergy, atopy, eye rubbing, and aberrant inflammatory factors are key modifiable risk factors that impact the prognosis and treatment outcomes of KC.
Ocular allergy and eye rubbing are major risk factors that contribute to initiation and progression of KC owing to the increase in associated inflammatory factors. , Hence, controlling local and systemic allergy would aid in the reduction of eye rubbing and the related surge in inflammatory factors at the ocular surface. Modulation of inflammatory and immune-mediated mechanisms underpins the management of atopy and allergy. It includes the topical and systemic use of steroids, antihistamines, and leukotriene receptor antagonists. Targeting IgE, IL-4, and IL-13 by using monoclonal antibodies against these factors and mast cell stabilizing agents has been successful in the management of systemic allergy and atopic conditions. Since high IgE is associated with increasing severity of KC and progression, controlling it is important in improving ocular allergy status and associated KC. Identifying the causal allergen, adhering to allergen avoidance measures, and using allergen-specific immunotherapy such as sublingual immunotherapy (SLIT) reduce the specific IgE load, eye rubbing, and associated ocular surface inflammatory factors. SLIT shifts the T cell responses, that is, from Th2 to Th1, to facilitate the development of anergy or tolerance to the allergen. ,
Cyclosporine is an immunomodulatory agent that acts by inhibiting the intracellular calcineurin-mediating signaling that is required for the production of inflammatory factors. Use of cyclosporine in the management of autoimmune diseases is well known. Topical cyclosporine is also used to manage ocular surface inflammation in dry eye disease. Modulating the ocular surface inflammatory factor milieu using topical cyclosporine in KC patients has shown promise with stabilization of the disease. The use of cyclosporine also resulted in a significant reduction in the tear fluid MMP-9 levels in these patients. Collagen cross-linking procedures that resulted in stabilization of KC progression also resulted in a decrease in tear fluid inflammatory factors, including MMPs. , ,
It is important to note that higher expression of lysyl oxidase, an endogenous collagen cross-linking agent in the ectatic zone, has shown a favorable relationship with collagen cross-linking outcome. Hence, reducing the inflammatory factors that can reduce the levels of lysyl oxidase would be useful to improve the stabilization of disease. Targeting inflammatory or molecular factors such as MMPs and their precursors (e.g., IL-17) that are significantly dysregulated in the KC cornea using previously approved drugs can be beneficial. For example, doxycycline, a derivative of tetracycline known to reduce expression and activity of MMP and other inflammatory factors, is a potential agent. Similarly, batimastat, a hydroxamic acid–based MMP inhibitor, is the other probable agent that might be explored. IL-17 blockade using RORγ inhibitors can also be used. IL-17, reported to be higher in KC, is a known inducer of MMP-9 and corneal barrier function loss. , Finally, augmenting endogenous antiinflammatory mechanisms by vitamin D supplementation in KC patients deficient in vitamin D would be beneficial in prevention of disease progression.
Conclusion
Based on the mounting evidence regarding the multifaceted associations between inflammatory factors and KC ( Fig. 11.2 ), it is apparent that inflammatory mediators are among the key contributing and modifiable factors in KC. Hence, monitoring and managing inflammatory milieu changes, including subclinical inflammation, would be clinically beneficial in stabilizing the disease and improving clinical outcomes.
