Dry eye disease (DED) is a complex and multifactorial disorder of the ocular surface with an estimated global prevalence of 20–50% worldwide [1,2]. Patients with dry eye have symptoms of eye irritation, redness and visual discomfort that can have negative impact on daily productivity and quality of life [2,3]. DED is primarily characterized by perturbation in the tear secreting lacrimal gland or lipid producing meibomian gland of the ocular functional unit [4]. Lacrimal secretory insufficiency leads to aqueous deficient dry eye, while meibomian impairment leads to evaporative dry eye [5]. Often, these DED subtypes are not mutually exclusive but coexist as a hybrid, presenting etiologies of reduction in tear secretions and excessive tear loss termed mixed dry eye [6]. It is reported that mixed dry eye is highly common with 30–70% of dry eye patients suffer from this form of disease [6].

The histopathologic features of the ocular surface in DED are defective epithelium, goblet cell (GC) density loss with low mucus production, accompanied by ocular tissue inflammation and epitheliopathy [1,4]. These morphologic changes described in humans and experimental animal models with dry eye are depicted by decreased corneal epithelial barrier function to fluorescein dye uptake, reduced conjunctival GC mucin staining, elevated conjunctival CD4+ T-lymphocyte cell infiltration and increased conjunctival epithelial cell apoptosis [[7], [8], [9], [10], [11], [12], [13], [14], [15]].

Currently available management options of DED are palliative measures including tear replenishment approaches that provide temporary relief of symptoms and topical anti-inflammatory medications that are restricted by limited efficacy and intolerable side effects with continued use [6]. As such, there exists a great need to develop new treatments that can be used as a standalone or adjunctive therapy, to resolve the burden of DED.

Histatin (Hst) antimicrobial peptides are major constituents of the primordial innate immunity [16]. They are endogenous, low weight, histidine-rich cationic proteins that consist of at least 12 structurally related known members [17]. Hsts are encoded by HTN1 and HTN3 genes, which initially produce two full length polypeptides, Hst1 and Hst3 [18]. Subsequently, through posttranslational proteolytic cleavage and processing of these parent Hsts, all other smaller Hsts are generated; Hst2 from Hst1 and Hst4 to 12 from Hst3 [18]. Hsts presence has only been found in humans and in higher primates, where they were originally discovered in salivary secretions and later identified to be also present in tears [19,20]. Interestingly, patients with severe DED demonstrated lower levels of Hst peptides in ocular surface washings [20]. Functionally, Hsts are primary homeostatic control agents of the host defense system [21]. While in addition to their well-established killing activities against a broad range of pathogenic microorganisms, Hsts have also been shown to have therapeutic efficacy in models of diseases associated with epithelial injury and inflammation like gingivitis and periodontitis [22,23]. Because these diseases are of oral mucosal surface that draw parallels in commonalities with ocular mucosa and share similarities with DED of epithelial damage and pro-inflammatory reaction that improved with Hsts application, we sought to translate the pro-epithelial and anti-inflammatory functions of Hsts to the ocular system [[24], [25], [26], [27]].

In our previously published studies utilizing ocular epithelial cell culture systems and murine models, we reported that Hst1 and Hst5 are able to enhance wound closure rate in human corneal epithelial (HCE) monolayer via stimulation of cell migration and spreading [28,29]. Hst5 can also promote corneal surface repair in a mouse model of corneal epithelial injury [29]. Moreover, we demonstrated Hst1 inhibitory effect on lipopolysaccharides (LPS)-induced inflammatory signaling and pro-inflammatory mediator production in macrophages known to play critical role in inflammation of the ocular surface [30].

Although Hsts exhibited significant epithelial wound healing and anti-inflammatory results in experimental ocular platforms, there are no well described studies evaluating the effect of Hsts in models of dry eye. Several animal models of dry eye exist, each with attendant attributes and limitations [[31], [32], [33]]. A mouse model of dry eye using scopolamine and desiccating stress (SDS) has been described and validated in the literature and has interesting real-world implications [[34], [35], [36]]. This is a well-accepted model of dry eye that incorporates low relative humidity and high air flow draft, in combination with administration of an anticholinergic agent [35,37,38]. This is a useful model to integrate multiple different features of the complex dry eye seen in many patients, which many of whom have both aqueous deficient and evaporative mixed mechanism DED [6], though the model does not incorporate meibomian gland disease directly.

The purpose of this current study was to test the hypothesis that Hst5 peptide is efficacious in mitigating dry eye phenotypic parameters; namely, corneal epithelial cell permeability, conjunctival mucin-producing GC population loss, conjunctival inflammatory CD45+ immune cell infiltration and conjunctival apoptotic epithelial cell death in the in-vivo SDS mouse model of DED.