Development of lacrimal gland inflammation in the mouse model of herpes stromal keratitis

Abstract

Herpes stromal keratitis (HSK) is a chronic immunoinflammatory condition which develops in response to recurrent herpes simplex virus-1 (HSV-1) infection of the cornea. Patients with HSK often demonstrate the concurrence of corneal desiccation and the loss of blink reflex. However, the relationship between severity of HSK, level of basal tears and inflammation of the lacrimal gland is mostly unexplored. In this study, we compared these variables in extraorbital lacrimal gland (EoLG) after corneal HSV-1 infection in the C57BL/6J mouse model. Our results showed a significant reduction in the volume of tears in infected eyes during the development of HSK. Extensive architectural damage to EoLG, presumably caused by a massive influx of interferon-gamma secreting T cells, was observed during clinical disease period of HSK. A positive correlation between the decrease in tear volume, severity of HSK and the damage to EoLG were evident in infected mice. The presence of infectious virus measured in EoLG during pre-clinical, but not clinical disease period of HSK, suggested that viral cytopathic effects are not the major contributors of extensive damage seen in EoLG. Furthermore, topical administration of lacritin peptide delayed but did not prevent the decrease in tears in HSV-1 infected mice, and had no significant effect in either reducing the severity of HSK or T cell infiltration in EoLG of infected mice. Together, our results showed an interplay between the severity of HSK, inflammation of EoLG, and the reduced level of tears after corneal HSV-1 infection.

Keywords: HSV-1; Lacrimal gland; Lacritin and T cells; Tears.

Figure 1:. Inflammatory cell infiltrate in EoLG and the decrease in tear fluid volume during clinical phase of HSK.

A. Phenol-red thread images demonstrate the measurement of the volume of tear fluid in uninfected (naive) eyes and HSV-1 infected eyes at 10-day post-infection. The color change in thread from yellow to red depicts the tear volume. Data shown is the representation of three independent experiments. Scatter plot with colored dots represents the level of basal tears in an individual eye prior to and after the corneal HSV-1 infection. Each colored dot represents the tear volume in an individual eye prior to infection (Naive), and on 7-day and 10-day post-ocular infection (POI). Data shown is the sum of two independent experiments. (n= 4 eyes per group). The p values were calculated using one-way ANOVA followed by the Bonferroni’s multiple comparison test. (****p<0.0001 and *p=0.0347). Bar diagram depicts the level of tears in eyes with severe and mild HSK (n= 5 eyes per group). The p value was calculated using unpaired nonparametric Mann-Whitney test (**p=0.0079). B. Representative FACS plots denote the frequency of CD45+ cells (top panel) and CD4/CD8 T cells (bottom panel) in EoLG of HSV-1 infected mice on 4 and 10-day post-infection. Scatter plots demonstrate the frequency and absolute number of total leukocytes (CD45) and CD4/CD8 T cells in individual EoLG of infected mice on 4-day and 10-day POI. Data were analyzed using unpaired two-tailed student’s t-test with Welch’s correction (*** p=0.0008, ****p<0.0001). C. Representative hematoxylin and eosin (H&E) staining in the paraffin sections of EoLG excised at 4-day and 10-day post-infection. At 10-day post-infection, the inflammatory cell influx was compared between EoLG excreting tears to eyes with mild or severe HSK. Eye images depict the severity of corneal opacity. Labeled areas in EoLG excreting tears to eyes with severe HSK demonstrate (i) fatty infiltration, (ii) acinar atrophy and fibrosis, (iii) ductal dilation, (iv) inflammatory infiltrate in duct lumen, (v) stasis of tear fluid. (D) Representative FACS plots denoting the frequency of IFN-gamma secreting CD4 and CD8 T cells in EoLG providing tears to HSK developing eyes at 10-day post-infection. (E) Scatter plot with bar diagram shows the number of plaque forming units (PFU) of HSV-1 in EoLG at 5- and 10-day post-infection. *p=0.0357 was calculated by carrying out unpaired non-parametric Mann-Whitney test.

Figure 2.. Topical application of lacritin protein transiently increases the tears in HSV-1 infected eyes, but did not reduce HSK severity and the inflammatory influx in EoLG.

A. Schematic representing the treatment plan with topical application of lacritin and C-25 control peptide. B. Scatter plot represents the volume of tears measured between C-25 treated (open circles, n=11 eyes) and lacritin treated groups of infected mice (closed circles, n=12 eyes) at 6-, 8-, and 10-day post-ocular infection (POI). A significant increase in the tear volume is shown in lacritin treated mice at 8-day POI (**p=0.0084 calculated using unpaired t-test with Welch’s correction). Data shown are derived from two similar experiments. C. Scatter plot shows the corneal opacity score of individual eye from lacritin and C-25 treated groups of mice at 10-day POI. Data shown are pooled from two similar experiments. (open circles, n= 11 eyes and closed circles, n= 12 eyes) D. Representative FACS plots show the frequencies of CD45 and CD4/CD8 T cells in EoLG derived from C-25 and lacritin treated groups of infected mice at 10-day POI. Scatter plots demonstrate frequency and absolute number of CD45 and CD4/CD8 T cells in the lacrimal gland from both groups of infected mice at 10-day POI. Data shown in C and D were analyzed using unpaired two-tailed student’s t-test with Welch’s correction (ns represent p>0.05).