Tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6), originally identified among gene products induced in fibroblasts by TNF, is an ∼35 kDa protein secreted by a wide range of cell types [1]. TSG-6 has been shown to be constitutively expressed by epidermal cells of the skin, by α and β cells of islets of Langerhans, by astrocytes in the central nervous system (CNS), by fibroblasts in the amniotic membrane and by umbilical cord mesenchymal stem cells [[2], [3], [4], [5], [6]]. Additionally, TSG-6 is constitutively produced and stored in neutrophils and mast cells and released upon proinflammatory signals [7,8]. TSG-6 is readily upregulated by many cell types in response to inflammatory cytokines, for example, stromal cells, such as, fibroblasts and smooth muscle cells, and inflammatory cells, such as, monocytes, macrophages, and myeloid dendritic cells [8,9]. TSG-6 has also been shown to be upregulated in multiple pathological conditions, such as, in arthritis, asthma, spinal cord and brain injury and in serum during bacterial sepsis [[10], [11], [12], [13], [14]]. A growing body of evidence suggests TSG-6 is an anti-inflammatory molecule that plays a protective role during pathological inflammatory events [9,[15], [16], [17]]. Specifically, TSG-6 has been shown to inhibit neutrophil migration [18] and suppress resident immune cell signaling [15,16]. Studies using TSG-6 null mice correlated the loss of TSG-6 expression with an increased inflammatory response in an arthritis model, with the subsequent rescue of TSG-6 expression decreasing the inflammatory response and joint damage associated with arthritis [19]. Further, TSG-6 overexpression reduces the severity of collagen induced arthritis [20], and localized TSG-6 overexpression inhibits local inflammation and joint destruction associated with arthritis [21]. Additionally, TSG-6 null mice have been shown to present increased inflammation and an increased area of glial scarring following brain stab wounds [22], while, the administration of TSG-6 has been shown to improve memory following traumatic brain injury in mice [23]. Seminal studies by the Prockop group established that many of the therapeutic benefits of mesenchymal stem cell (MSC) administration comes from the secretion of TSG-6 [24]. Using a myocardial infarction model, they were able to show that intravenously administered MSCs get trapped in the lungs and locally express TSG-6, which, in turn, is systemically distributed, leading to reduced inflammation and myocardial damage [24]. Thus, MSCs located in the lungs were able to treat a myocardial infarction model by increasing the systemic distribution of TSG-6 [24]. However, under certain physiological conditions, TSG-6 has also been shown to potentiate inflammatory responses. For example, TSG-6 is essential for the propagation of acute eosinophilic pulmonary inflammation and airway hyperresponsiveness, and TSG-6 expression contributes to the pathogenesis of asthma [10]. TSG-6 has been shown to modulate pro-inflammatory signaling pathways by binding to bone morphogenetic proteins (BMPs) [[25], [26], [27]], C-X-C Motif Chemokine Ligand 8 (CXCL8) [28] and other chemokines [29].

Given the well-established anti-inflammatory properties of TSG-6, over the years various studies have explored the administration of exogenous TSG-6 to limit tissue damage in various pathological conditions (reviewed in Ref. [15]). For example, Watanabe and colleagues administered mesenchymal stem/stromal cells (MSCs) overexpressing TSG-6 via intravenous administration as a means of continuously delivering systemic TSG-6 after traumatic brain injuries (TBI), with a reduction in the size of the lesion after cortical impact injuries [23]. Moreover, intraperitoneal administration of TSG-6 was shown to ameliorate acute liver damage caused by CCL4 administration [30]. Additionally, intratracheal instillation of TSG-6 in mice has been shown to prevent LPS-induced lung injury and neutrophil sequestration, and increase survival [31]. Besides that, exogenous administration of TSG-6 ameliorates liver fibrosis [30], provides neuroprotection against subarachnoid hemorrhage induced early brain injury [32] and reduces the severity of colitis [33]. The therapeutic potential of TSG-6 has also been explored in the context of the eye. For example, Oh et al. demonstrated that injecting human recombinant TSG-6 into the anterior chamber of the eye reduces inflammation and damage after mechanical injury through debridement and chemical injury with 100 % ethanol to the cornea and limbus using the rat model [16]. Lee et al. has also shown that topical administration of TSG-6 protects the ocular surface exposed to dry eye disease [34]. Recently, Day and colleagues demonstrated that topical application of the Link module from human TSG-6 (Link_TSG6) dose-dependently reduced corneal epithelial defects in a dry eye disease mouse model, while increasing tear production and conjunctival goblet cell density [35]. This group went on to establish the pharmaceutical company Link Biologics Limited to produce and commercialize the medical use of a TSG-6 based drug for treating inflammatory and tissue-degenerative disorders, including dry eye disease and osteoarthritis [36].

During normal physiology and in pathological conditions, TSG-6 interacts and binds to hyaluronan (HA), a major component of the ECM, via a Link module domain [37]. HA has a central role in many pathophysiological processes, such as, in inflammation, wound healing, regeneration, tissue remodeling, tumor progression and metastasis [38]. The mechanisms by which HA mediates biological processes is complex, and the size of the HA chains directly dictates its physiological functions [[39], [40], [41]]. Additionally, the association of HA with a variety of proteins and proteoglycans can further affect its physiological functions. For example, TSG-6 can directly crosslink HA [42], which changes the structural organization, biophysical properties and physiological functions of HA, for example, enhancing HA-CD44 interactions on leukocytes [43]. In addition to directly interacting with HA, TSG-6 also catalyzes the covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) and pre-α-inhibitor (PαI) onto HA chains forming HA-HC complexes [44,45], and we have recently shown that these HA-HC complexes exist in the cornea [46]. During inflammatory processes and/or tissue injury, increased vascular permeability leads to an increase in IαI/PαI leaking into tissues from the circulation, and as mentioned above, local TSG-6 expression is usually increased during inflammation and following injury. Studies have shown that TSG-6 transfers HCs onto HA whenever HA is in the presence of IαI/PαI and TSG-6, and the formation of HA-HC/TSG-6 complexes has been associated with ECM stabilization during pathological conditions, with a proposed anti-inflammatory and protective role [6]. HA-HC/TSG-6 complexes have also been shown to suppress inflammation and prevent rejection, for example, preventing xenograft rejection of human umbilical cord mesenchymal stem cells when transplanted into the mouse cornea [23]. In stark contrast, the formation of HA-HC/TSG-6 complexes have been shown to contribute to pathology in the respiratory system, such as in asthma and lung disease [10,15].

Thus, TSG-6 has been shown to play an important role in inflammatory responses, wound healing, and tissue regeneration in many biology systems, with mostly an anti-inflammatory role, however under certain physiological conditions, TSG-6 can potentiate an immune response. Importantly, various studies have explored the therapeutic applications of TSG-6 and TSG-6 based therapies for treating various ocular disorders, including various types of corneal injuries. However, to the best of our knowledge, the function of endogenous TSG-6 has yet to be explored in the cornea, during homeostasis and after injury. We hereby investigated whether TSG-6 is expressed during homeostasis and the expression profile and function of TSG-6 following different types of corneal injuries.