Tumor necrosis factor (TNF) is pleiotropic cytokine involved in cellular transport and activation, pathogen resistance, and regulation of immune/inflammatory responses. Given its potent effects activating itself and other proteins (such as caspases and cathepsins) and inducing cellular death, the levels of TNF are very low in healthy subjects [1]. Several cell types, including epithelial cells, can produce TNF, while macrophages are the greatest producers of TNF [1].

Aging is a complex biological process that affects multiple organs, including the eye and lacrimal gland. Inflammaging is a chronic low-grade inflammation that happens with aging, where circulating high levels of certain cytokines and proteases negatively correlate with mortality. Both cathepsin S and TNF are related aging biomarkers. TNF is a pleiotropic cytokine involved in inflammation, activation, and formation of ectopic lymphoid organs. TNF also activates cathepsin S, a protease involved in extracellular matrix remodeling and MHC II presentation [2,3]. Elevated levels of cathepsin S activity are present in tears of dry eye subjects and elderly mice [4,5].

Inflammaging is a low-grade, sterile inflammation that accompanies aging [6]. Cytokines involved in inflammaging are IFN-γ, IL-1β, TNF, and cathepsins [7]. The increase of proinflammatory cytokines has been linked to morbidity and mortality in elderly people. In centenarians, increased TNF and cathepsin S serum levels were correlated with frailty and increased mortality [[8], [9], [10], [11]].

One of the most prevalent diseases in the aged population is dry eye disease (DED), characterized by a multifactorial and self-perpetuating inflammation of the lacrimal gland function unit [12]. Sjögren Syndrome (SS) is the prototype autoimmune DED in which the exocrine glands are affected by immune cell infiltrates and the production of cytokines, leading to dry eye and dry mouth symptoms. The immune cell infiltrates in lacrimal and salivary glands consist of B and T cells that are organized in tertiary lymphoid organs (also named ectopic lymphoid structures) that lead destroy lacrimal glands structure and lead to sustained inflammation and production of autoantibodies [13,14].

Polymorphisms for TNF and TNFAIP3 alleles have been described in SS [[15], [16], [17], [18]]. Furthermore, in animal models of SS, increased levels of TNF have been observed [19]. Since the aging population of people grows every year a better understanding of age-related DED and its treatment strategies are necessary.

We and others have shown that aged C57BL/6J mice develop spontaneous DED. During aging, dry eye disease has chronic inflammation characterized by increased corneal and conjunctival barrier disruption and goblet cell loss, and lacrimal glands inflammation. Like animal models of SS, aged lacrimal glands also have increased levels of TNF and ectopic lymphoid structures [[20], [21], [22], [23]]. It has been shown that aged Tnf−/− mice have decreased age-related inflammatory changes [[24], [25], [26], [27]]. However, the effects of Tnf genetic deletion in the aged lacrimal glands have not been investigated.

Recently, anti-TNF therapies have emerged (e.g., TNF antagonists: infliximab, adalimumab, etanercept) as successful in treating certain autoimmune diseases, such as rheumatoid arthritis and ankylosing spondylitis [28]. However, there is a paucity of anti-TNF agents designed specifically for topical application in DED. Infliximab eye drops improved clinical signs and goblet cell density in mice subjected to desiccating stress [29]. Recent positive effects of topical tanfanercept (previously known as HL036), a TNF blocker, have been reported [30,31]. In mice subjected to desiccating stress, topical tanfanercept improved goblet cell density and ameliorated corneal staining [30,31]. Tanfanercept is currently in Phase III Clinical Trials for dry eye disease, but it has yet to be tested in aged mice.

We hypothesize that TNF is a critical mediator not only in SS-associated DED but also in age-related DED. First, we determined the effects of genetic deletion of TNF on the formation of ectopic lymphoid structures during age-related DED and cytokine production. Second, we examined the effects of topical administration of tanfanercept in age-related DED. Herein we demonstrated that TNF participates in age-related DED by stimulating the production of cytokines involved in the germinal center formation and cathepsin S expression promoting ectopic lymphoid structures formation. We also showed that topical administration of tanfanercept can improve age-related goblet cell loss, decrease cytokine production in tears, while decreasing ectopic lymphoid structures and infiltration of certain B cell subtypes.