Protein profiles of tears from HSV-1 infected individuals may show increased levels of proinflammatory cytokines, alterations in signaling molecules and hormone levels, presence of autoantibodies, factors involved in apoptosis, and many other changes [
32]. Therefore, alterations in tear protein profiles are indicative of the processes underlying HSV-1 epithelial keratitis, and identification of marker proteins can provide information on the disease severity, as well as on the underlying pathology.
In this study, we identified 1275 proteins in the tear from three HSV-1 epithelial keratitis patients and three healthy control subjects using nLC-MS/MS.
Zhou et al has reported a high-confidence human tear proteome reference set which contains 1543 proteins (from four healthy subiects) [
33]. By comparing our tear proteome data with their tear proteome data reference set, we found that 1026 proteins (80.5% of total 1275 proteins) were common, while 952 proteins were identified in healthy control subjects, and 596 proteins were identified in HSV-1 epithelial keratitis patients. By comparing the differences in the tear proteomes between HSV-1 epithelial keratitis patients and healthy control subjects, 326 proteins were found to be unique to HSV-1 epithelial keratitis patients. This result suggest that infection of the cornea with HSV-1 leads to significant change in the protein composition of tears, which may be due to the abnormal secretion of lacrimal glands and corneal cell damage [
9]. GO enrichment analysis suggest that these unique proteins do not only participate in the host immune response, but also in cell damage and stress response. Through cluster analysis, we obtained several proteins with highly abundance and repetition rate in the tear from three HSV-1 epithelial keratitis patients (Fig.
3b-c). Most of these proteins are related to inhibition of viral infection, inflammatory response and cell damage, and may serve as a target for studying the pathogenesis and treatment of HSV-1 epithelial keratitis. It has to be noted that although some proteins (IL-6, S100A8 and VEGFA) were present in low abundance or in one or two patients only, they also play an important role in the process of HSV-1 keratitis [
29‐
31].
HSV-1 epithelial keratitis is thought to be caused by the virus replicating in and destroying epithelial cells [
2]. The lesions start as punctuate vesicular eruptions in the corneal epithelium, progressing to a stellate erosion, but quickly coalesce into dendritic shaped lesions [
34]. In order to suppress the infection and replication of the virus, the expression of various inflammatory factors and antiviral proteins were increased in corneal epithelial cells [
35]. Our mass spectrometry data showed the spectrum of several inflammatory factors were increased in tears of HSV-1 epithelial keratitis patients, such as IL1A and IL12B (Fig.
3 and S
1 Table). ELISA also confirmed that IL1A and IL12B were significantly elevated in patients with HSV-1 epithelial keratitis (Fig.
4). The current research shows that IL1A and IL-12B are important proinflammatory cytokines in HSV-1 epithelial keratitis and are not only produced by activated macrophages and lymphocytes, but also by corneal epithelium and keratocytes [
36]. When corneal epithelial cells are damaged by virus invasion, IL1 and IL12B are released as early warning signal [
37]. Local release of IL1A and/or IL12B triggers a series of events including an increase in the activity of inflammatory mediators such as prostaglandins, leukotrienes, and nitric oxide [
38,
39]. At the same time, upregulated inflammatory mediators promote the accumulation of neutrophils, macrophages, Langerhans cells, and lymphocytes [
38,
39]. In addition to increased expression of inflammatory factors, related antiviral proteins were also increased in tears of HSV-1 epithelial keratitis patients. Human DEFB4A (also named β-defensin-2), an inducible, anti-microbial peptide with a molecular mass of 4–6 kD, acts as an endogenous antibiotic in the defense against microbial infections [
40]. DEFB4A is only found occasionally in healthy ocular surface tissue, but it is found more frequently in infected/inflamed ocular surface tissue [
41]. CAMP (also named LL-37), the sole know member of the cathelicidin family of peptide expressed in humans, is a multifunctional host defense molecule essential for normal immune responses to infection and tissue injury [
42]. Studies have shown that corneal and conjunctival epithelia express CAMP as part of mucosal innate immunity to protect against bacterial and viral ocular infections [
43]. Notably, in a rabbit model of corneal injury, epithelial expression of CAMP was upregulated and levels of CAMP in the tears correlated with wound closure [
44]. Such observations have led to the suggestion that DEFB4A and CAMP may be important for ocular surface wound healing.
This study is the first report of tear proteome data from HSV-1 epithelial keratitis, which may deepen our understanding of molecular mechanisms behind HSV-1 epithelial keratitis. However, there are some possible limitations in this study, including the small sample size, the lack of clinical data related to disease severity. In addition, due to the limited volume of tear fluid, the collection technique will affect the protein profile of the tear sample. Future, we will focus on the correlation between the unique proteins of HSV-1 epithelial keratitis and the severity and/or prognosis of the disease. This requires a larger sample size and follow-up of patients to assess the clinical significance of these markers.