Activated inflammatory infiltrate in HSV-1-infected corneas without herpes stromal keratitis

Abstract

Purpose: To investigate herpes stromal keratitis (HSK) immunopathology by studying HSV-1-infected corneas that fail to develop HSK.

Methods: Plaque assay quantified HSV-1 in the tear film of infected mice. FACS analysis enumerated corneal leukocytic infiltrate and characterized infiltrate phenotypically after staining for activation and regulatory T cell (Treg) markers and for markers of antigen-presenting cell (APC) maturation. Treg cells were depleted in vivo using anti-CD25 mAb. Luminex analysis quantified the amount of cytokines and chemokines expressed in corneal tissue homogenate.

Results: Infected corneas without HSK exhibited a pronounced leukocytic infiltrate containing a significantly higher proportion and nearly identical absolute number of activated CD4+ T cells 15 days after infection when compared with those with HSK. Moreover, the frequency and absolute number of regulatory CD4+ T cells (Tregs) was lower in nondiseased corneas, and Treg depletion did not influence HSK incidence. The frequency of mature, immunogenic DCs and the ratio of mature DCs to CD4+ T cells were nearly identical in corneas with and without HSK. The authors observed a reduced population of neutrophils and reduced expression of neutrophil chemoattractants MIP-1beta and keratinocyte chemoattractant and the neutrophil-attracting cytokine IL-6 in corneas without HSK.

Conclusions: These findings demonstrate that HSV-1-infected corneas can retain clarity in the presence of a substantial secondary leukocytic infiltrate, that activated CD4+ T cells, while necessary, are not sufficient for HSK development, that susceptibility to HSK is not determined by Tregs, and that clinical disease correlates with the accumulation of a critical mass of neutrophils through chemoattraction.

Figure 1

Viral burden and clearance in corneal tear films were equivalent between HSV-infected corneas with and without HSK. Viral load and clearance from corneal tear films were assessed by standard plaque assay of eye swabs taken 2, 4, 6, 8, and 10 dpi. Data represent two independent experiments with at least 14 corneal samples per group.

Figure 2

Inflammatory cells infiltrate HSV-infected corneas without HSK but at reduced frequency compared to corneas with HSK. HSV-infected corneas with and without HSK at 15 dpi were disaggregated into single-cell suspensions and stained with anti-CD45, CD4, CD8α, CD11c, F4/80, and Gr-1 mAb. Cells were analyzed by flow cytometry. (A) Distinct live cell populations were identified based on FSC versus SSC, from which Gr-1^bright (PMNs), CD11c⁺ DCs, F4/80⁺ macrophages, and CD4⁺ and CD8α ⁺ T cells could be gated. Isotype controls were used to aid gating (data not shown). (B) The total number of infiltrating cells per cornea is shown. Data represent the average of at least eight corneas per group from two or more independent experiments. **P < 0.01; ***P < 0.001.

Figure 3

T cells in HSV-infected corneas without HSK are activated. Single-cell suspensions of HSV-infected corneas with and without HSK at 15 dpi were stained with anti-CD45, CD4, CD8α, and CD69 mAb. Flow cytometry was used to analyze the percentage (A) and number (B) of the total CD4⁺ and CD8α ⁺ T-cell populations that were CD69⁺. Data are representative of two independent experiments with at least seven mice per group. **P < 0.01; ***P < 0.001.

Figure 4

Few CD4⁺ T cells in HSV-infected nondiseased corneas are Foxp3⁺ Tregs. Single-cell suspensions of HSV-infected corneas with and without HSK at 15 dpi were stained with anti-CD45, CD4, and Foxp3 mAb. Flow cytometry was used to determine the total number of CD4⁺ T cells per cornea and to analyze the percentage of the total CD4⁺ T-cell population that was Foxp3⁺. Data are presented as the absolute number of CD4⁺Foxp3⁺ T cells per cornea and are representative of two independent experiments with at least seven mice per group. ***P < 0.001.

Figure 5

Resistance to HSK is not associated with elevated levels of the inhibitory cytokine IL-10. HSV-infected corneas with and without HSK at 15 dpi were dissected and sonicated in PBS + protease inhibitor, yielding a final volume of one diseased cornea or two nondiseased corneas per 600 μL. Multiplex bead array for IL-10 expression was performed on this tissue extract. Data are representative of two independent experiments, with n values of six corneas with HSK and six samples (two corneas pooled per sample) of corneas without HSK. **P < 0.01.

Figure 6

Depletion of CD4⁺CD25⁺ Tregs in HSV-infected corneas affects T-cell numbers in corneas but not HSK development. Mice were administered 100 μg anti-CD25 mAb (clone PC61) or anti-HLA-DR5 (control mAb) in 500 μL PBS by intraperitoneal injection 3 days before infection. (A) FACS dot plots comparing depleted corneal Treg infiltrate with a nondepleted control cornea. (B) At 15 dpi, single-cell suspensions of corneas that had developed HSK from both CD25-depleted and control mice were stained with anti-CD45, CD4, CD8α, Gr-1, and F4/80 mAb and were analyzed by flow cytometry to enumerate the total number of cells per cornea. Data are representative of two independent experiments, with n of 14 from depleted mice and 15 from control mice. *P < 0.05; ***P < 0.001.

Figure 7

HSV-infected nondiseased corneas have reduced expression of the APC and lymphocyte chemoattractant MCP-1. HSV-infected corneas with and without HSK at 15 dpi were dissected and sonicated in preparation for multiplex bead array analysis for expression of the chemokine MCP-1 (CCL2) in corneal tissue extract. Data are representative of 2 independent experiments, with n values of six corneas with HSK and six samples (two pooled corneas per sample) of corneas without HSK.

Figure 8

CD11c⁺ dendritic cells and F4/80⁺ macrophages infiltrating HSV-infected nondiseased corneas are mature rather than tolerogenic. APC infiltrate was assessed at 15 dpi in HSV-infected corneas with and without HSK by flow cytometry after staining with anti-CD45, CD11c, F4/80, CD8α, MHC class II (IA/IE), CD80, and CD40 mAb for (A, C) the frequency of APCs per cornea expressing MHC class II and costimulatory molecules and (B, D) the amount of surface expression of MHC class II, CD80, and CD40, presented as the ratio of mean fluorescence intensity of corneas without HSK to corneas with HSK. (A, B) CD11c⁺ cells. (C, D) F4/80⁺ cells. Data represent the average of values from two independent experiments, with n of at least seven corneas per group. *P < 0.05.

Figure 9

HSV-infected corneas without HSK have significantly reduced neutrophil chemoattractant expression. HSV-infected corneas with and without HSK at 15 dpi were dissected and sonicated for multiplex bead array analysis of corneal tissue extract. Results for corneas without HSK were halved to estimate the amount of protein per individual cornea. Representative data from one of two independent experiments are presented, with n values of six corneas with and six samples (two pooled corneas per sample) of corneas without HSK. *P < 0.05; **P < 0.01.