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. 2014 Oct 16;55(11):7457-66.
doi: 10.1167/iovs.14-15411.

Correlation between human tear cytokine levels and cellular corneal changes in patients with bacterial keratitis by in vivo confocal microscopy

Affiliations

Correlation between human tear cytokine levels and cellular corneal changes in patients with bacterial keratitis by in vivo confocal microscopy

Takefumi Yamaguchi et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: We investigated bilateral tear cytokine levels in patients with unilateral bacterial keratitis (BK) as associated with in vivo confocal microscopic (IVCM) alterations in corneal nerves and dendritiform immune cells (DCs).

Methods: A total of 54 (13 BK, 13 contralateral, 28 healthy controls) tear samples was collected prospectively and analyzed by multiplex microbeads assay. The IVCM of the central cornea was performed on the same day, and assessed for corneal nerve and DC alterations.

Results: Interleukin-1β, IL-6, and IL-8 were significantly elevated only in affected eyes (66.6 ± 26.8, 7174 ± 2430, and 810 ± 315 ρg/mL, respectively; P = 0.04, P < 0.001, and P < 0.001, respectively), compared to healthy controls (13.0 ± 4.0, 171.8 ± 32.1, and 56.5 ± 33.8 ρg/mL). Levels of chemokine ligand 2 (CCL-2), IL-10, and IL-17a were elevated only in contralateral eyes (813 ± 478, 86.7 ± 38.3, and 3350 ± 881 ρg/mL, respectively; P = 0.02, P = 0.01, and P = 0.04, respectively), compared to controls (73.7 ± 25.3, 17.5 ± 4.9, and 1350 ± 337 ρg/mL). Triggering receptor expressed on myeloid cells (TREM)-1 was significantly elevated in affected (551 ± 231 ρg/mL, P = 0.02) and contralateral unaffected (545 ± 298 ρg/mL, P = 0.03) eyes compared to controls (31.3 ± 12.4 ρg/mL). The density of DCs was significantly increased in affected (226.9 ± 37.3 cells/mm(2), P < 0.001) and unaffected (122.3 ± 23.7 cells/mm(2), P < 0.001) eyes compared to controls (22.7 ± 5.9 cells/mm(2)). Sub-basal nerve density significantly decreased in affected (3337 ± 1615 μm/mm(2), P < 0.001) and contralateral (13,230 ± 1635 μm/mm(2), P < 0.001) eyes compared to controls (21,200 ± 545 μm/mm(2)). Levels of IL-1β, IL-6, and IL-8 were significantly correlated with DC density (R = 0.40, R = 0.55, and R = 0.31, all P < 0.02) and nerve density (R = -0.30, R = -0.53, and R = -0.39, all P < 0.01).

Conclusions: Proinflammatory tear cytokines are elevated bilaterally in patients with unilateral BK, and are correlated strongly with alterations in DCs and nerve density as detected by IVCM.

Keywords: corneal infection; cytokines; dendritic cells; infectious keratitis; tears.

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Figures

Figure 1
Figure 1
Slit-lamp photographs and in vivo confocal microscopy images. The representative slit-lamp photographs of a normal control eye (A), a clinically affected eye with unilateral BK (B, C), and its contralateral clinically unaffected eye (D). The IVCM images of a healthy control eye (E), a clinically affected eye with unilateral BK (F), and its contralateral clinically unaffected eye (G). Scale bars: 100 μm.
Figure 2
Figure 2
Corneal sub-basal nerves and dendritiform cells. The nerve density (A) and number of nerves (B) in eyes with unilateral BK and contralateral eyes were significantly decreased compared to normal eyes (P < 0.001, 1-way ANOVA). The DC density (C) and mean DC area (D) were significantly higher in eyes with unilateral BK and in contralateral unaffected eyes than in eyes of healthy controls (P < 0.001, 1-way ANOVA). A P value of less than 0.05 was considered statistically significant. Graph bars: represent average SE.
Figure 3
Figure 3
Tear cytokine levels. The IL-1β, IL-6, and IL-8 levels in affected BK eyes (P = 0.04, P < 0.001, and P < 0.001), but not contralateral eyes (P > 0.05), were significantly increased compared to controls. In contrast, IL-10, IL-17a, and CCL-2 were only significantly increased in contralateral unaffected eyes (P = 0.01, P = 0.04, and P = 0.02), but not in affected eyes (P > 0.05), compared to controls. The level of TREM-1 was significantly increased in affected eyes with BK (P = 0.02), as well as in contralateral unaffected eyes (P = 0.03), compared to normal eyes. There were no significant differences in levels of IL-1Ra, IL-2, IL-7, GM-CSF, and FGF-2 among the groups (P > 0.05). Error bars: represent the mean value for each group. A P value of less than 0.05 was considered statistically significant.
Figure 4
Figure 4
Correlation between tear cytokine levels and DC density. Levels of IL-1β (R = 0.40, P = 0.001 [A]), IL-6 (R = 0.55, P < 0.001 [B]), IL-8 (R = 0.31, P = 0.01 [C]), and IL-17a (R = 0.34, P = 0.01 [D] significantly correlated with DC density. A P value of less than 0.05 was considered statistically significant.
Figure 5
Figure 5
Correlation between cytokine levels and DC morphology. The number of dendrites was significantly correlated with IL-1β (R = −0.51, P = 0.004 [A]). The DC field was significantly correlated with IL-7 (R = −0.31, P = 0.01 [B]). A P value of less than 0.05 was considered statistically significant.
Figure 6
Figure 6
Correlation between nerve density and cytokine levels. Nerve density was significantly inversely correlated with IL-1β (R = −0.30, P = 0.01 [A]), IL-6 (R = −0.53, P < 0.001 [B]), IL-8 (R = −0.39, P = 0.001 [C]), and TREM-1 (R = −0.35, P = 0.005 [D]). A P value of less than 0.05 was considered statistically significant.
Figure 7
Figure 7
Alterations of DC density and tear cytokines over time. In 3 cases (case 8–10 in Table 2), tear samples and IVCM were obtained on the same day at visits 1 and 2. In all of these 3 patients, the BK improved clinically after the antibiotic treatment started. The representative IVCM images at visits 1 and 2 (A). Size bars: represent 100 μm. The DC density, IL-1β, IL-6, and IL-17a in the affected eyes decreased from visit 1 to visit 2. The DC density, IL-1β, and IL-6 in the contralateral eyes gradually increased from visit 1 to visit 2, whereas IL-17a in the contralateral eyes increased at visit 1 and decreased at visit 2 (B).

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