**3. The role of ocular surface inflammation**

Understanding the role of inflammation on the cellular and molecular level may lead to improve treatment options for DED patients. Ocular inflammation may be regarded as both a cause and effect of DED. The current understanding of the mechanism responsible is, that the repeating desiccating stress accompanied by hyperosmolarity induces the immune system reaction leading to the chronic inflammation and apoptosis of ocular surface cells. It is important to acknowledge, that different form of DED, such as SS-DED (SS—Sjogren syndrome) and non-SS DED are related to various inflammatory microenvironment. Also, there are significant differences between an acute and chronic state of DED in term of the inflammatory response.

#### *Dry Eye Disease: Chronic Ocular Surface Inflammation DOI: http://dx.doi.org/10.5772/intechopen.114118*

It is also worth emphasizing that regardless of the underlining cause, DED and ocular surface allergy (OA) share common pathognomonic pathways [10]. The chronic and acute reaction of the immune system in atopy, OA, and DED is related to ocular inflammation on the cellular level and its impact on the molecular homeostasis. Inflammatory biomarkers, which are significantly elevated in both conditions include matrix metalloproteinase-9 (MMP-9), Interferon-gamma (INFγ), IL-1α, IL-2, IL-6, IL-8, IL-17, and IL-22. Moreover, cytokines previously regarded as specific to OA are also elevated in DED, and those include IL-4, IL-5, and IL-13. MMP-9 is a proteolytic enzyme, expressed by eosinophils, correlated to the epithelial, and conjunctival cells interruption. MMP-9 level may be assessed in DED and OA patients using commercially available tests. IFN-γ is an inflammatory cytokine secreted by numerous cells such as epithelial cells, CD+T cells, and NK cells. It is one of the major indicators of the ocular surface inflammation. IL-17, IL-22, and IL-6 are the known effector cytokines of Th17 lymphocytes, which are characteristic for both DED and OA [11].

In vivo confocal microscopy (IVCM) allowed us to broaden our knowledge regarding the cellular changes in DED. Specific changes are as follows: the increased stromal nerve thickness and tortuosity, the decreased density of basal epithelial cells, stromal keratocytes, and subbasal nerves, and the presence of dendritic cells, leukocytes, activated keratocytes, and increased level of epithelial and stromal reflectivity [12, 13]. All those features revealed by the IVCM exam, which are characteristic for DED provides a direct, clinical proof, the inflammation plays a crucial role in DED pathogenesis.

On a molecular level, it has been established that proinflammatory cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-22, TNFα, tumor necrosis factor α, and INFγ) are over-expressed in the tear film and ocular surface of patients DED [14].

#### **3.1 Cytokines**

Inflammation in DED may begin as an acute immune reaction in response to desiccating stress. Mitogen-activated protein (MAP) kinases and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) play a crucial role in initiating and maintaining the immune reaction, leading to the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 on the ocular surface. Activation of toll-like receptor 4 (TLR4) also causes the activation and secretion of pro-inflammatory cytokines, such as IL-1β. At the same time, regardless of the TLR4-mediated pathway, the release of reactive oxygen species (ROS) induces activation of caspase-8 and NLRP3 inflammasome, also promoting the IL-1β release. The process leads to increased expression of MMP-9, a proteolytic enzyme known to break the epithelial corneal barrier and cause punctate keratitis.

### **3.2 CD+ T cells**

There is growing evidence from experimental, animal, and human studies that CD+ T cells play a crucial role in DED pathogenesis. The initiating factor results in the loss of the ocular surface immune homeostasis, and the activation of CD4+ T cells are the leading factors of the tear film instability, hyperosmolarity, ocular surface damage, and neurosensory abnormalities. It is well proved that CD+ T cells differentiate in response to the local microenvironment of cytokines and are defined by their transcription factor expression. With an excess concentration of INFγ and IL-12, CD+ T cells differentiate into Th1 lymphocytes, while in the concentration of Il-6 and TGF-β

(transforming growth factor beta), they may differentiate into Th17 lymphocytes. Further, ocular surface damage is caused by proinflammatory cytokines released by Th1 and Th17 lymphocytes, which stimulate the production of MMP-3, and MMP-9. Th17 lymphocytes produce Il-17, which damages the epithelial barrier function and causes apoptosis. Moreover, Th17 lymphocytes are characterized by phenotypic and functional plasticity, which lineage throughout the disease initiation, perpetuation, and sustention. Th17 cells are plastic and can differentiate into Th1 or Th2 subsets depending on environmental stimuli. Recently a new, autoimmune model of DED pathogenesis was proposed based on the concept of Th17 cells mediated disruption of ocular surface immune homeostasis that leads to DED [14]. This model is presented in **Table 2**.

### **3.3 Differences in inflammatory response in relation DED form and chronicity**

There is a difference between an acute and chronic DED in terms of the inflammation activation. Chronic DED is principally mediated by effector memory of Th17 cells because Th17 cells persist in chronic phase of DED. After the resolution of acute inflammation on the ocular surface, a part of effector Th17 cells pool (both eTh17 and eTh17/1 cells) converts into long-lived memory Th17 cells (mTh17). This population of cells is responsible for chronic inflammation. Based on animal studies it was proved, that aged mice (12–14 months) develop a more severe DED than in young mice (6–8 weeks). Aged mice had increased frequencies of conjunctival and draining lymph nodes Th17 cells compared to young mice [15]. Therefore, anti-IL-15 was proposed to reduce the memory of Th17 cells and further the severity of DED.

There is also a difference between immune response in Sjogren (SS-DED) and non-Sjogren DED (non-SS DED). The main feature of SS-DED is the lymphocytic infiltration of the lacrimal glands. The subpopulation of cells consists of primarily CD4+ T cells with minor number of B cells. Several immune mechanisms are common for both DED forms, including Th17 cells activation and overexpression of cytokines such as IL-6, IL-17, and IL-22. However, there is a significant difference in the levels of


5. Ocular surface damage, punctate epitheliopathy caused by Th17 cells through humoral response: IL-17, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF)

#### **Table 2.**

*The autoimmune model of DED pathogenesis based on [14].*

## *Dry Eye Disease: Chronic Ocular Surface Inflammation DOI: http://dx.doi.org/10.5772/intechopen.114118*

CXCL chemokine (chemokine C-X-C motif) ligand family and macrophage inflammatory proteins chemokine family. Above are highly expressed in SS-DED compared to non-SS DED. Paired box protein Pax-6 (PAX6) is one of the conjunctival protein biomarkers associated with an increased ocular surface damage. Downregulation of PAX6 in SS-DED was significantly related with epithelial damage [11].
