Innate recognition network driving herpes simplex virus-induced corneal immunopathology: role of the toll pathway in early inflammatory events in stromal keratitis

Abstract

Ocular infection with herpes simplex virus (HSV) sets off an array of events that succeed in clearing virus from the cornea but leaves the tissue with a CD4(+) T-cell-orchestrated chronic inflammatory lesion that impairs vision. We demonstrate that Toll-like receptor (TLR) signaling forms a part of the recognition system that induces the syndrome that eventually culminates in immunopathology. Accordingly, in a comparison of the outcomes of infection in wild-type (WT) mice and those lacking TLR function, it was apparent that the absence of TLR2 and, to a lesser extent, TLR9 resulted in significantly diminished lesions. Similarly, mice lacking the adapter molecule MyD88 were resistant to lesion development, but such animals were also unable to control infection, with most succumbing to lethal encephalitis. The susceptibility of TLR4(-/-) animals was also evaluated. These animals developed lesions, which were more severe, more rapidly than did WT animals. We discuss the possible mechanisms by which early recognition of HSV constituents impacts the subsequent development of immunopathological lesions.

FIG. 1.

Induced TLR ligands in HSV-infected corneas. (A) Corneal buttons from uninfected mice were harvested, and isolated total mRNAs were tested by reverse transcription-PCR for TLR2, TLR4, and TLR9. All three could be demonstrated in the naïve cornea. The DC2.4 cell line was used as a positive control. (B) Single-cell suspensions of corneal cells were prepared from four corneas at 24 h p.i. Intracellular staining was done with FITC-labeled anti-Hsp70 antibody, as mentioned in Materials and Methods. The number of Hsp70-expressing cells was determined by fluorescence-activated cell sorting. Rat IgG was used as the isotype control. The number in each upper right corner represents the percentage of Hsp70 cells/1 × 10⁵ corneal cells at 24 h p.i. (C) C57BL/6 mice were infected with 2 × 10⁴ PFU of HSV-1 RE. At the indicated time points, four corneas from infected mice were collected and total mRNAs were extracted. Real-time PCR was performed to compare the expression of mouse β-defensin 2 (mBD2) and mouse β-defensin 3 (mBD-3) mRNAs in the infected corneas at different times p.i. as described in Materials and Methods. The bar diagram demonstrates the relative increases (n-fold) in the expression of mouse β-defensin 2 and mouse β-defensin 3 compared to those in naïve corneas.

FIG. 2.

Reduced SK lesions and neovascularization in TLR2^−/−, TLR2/4d^−/−, and MyD88^−/− mice compared to those in TLR9^−/−, TLR4^−/−, and WT mice. (A) Bar diagram demonstrating the percentage severity of SK in each group of mice (n = 16) infected with 5 × 10³ PFU HSV-1 RE at day 15 p.i. TLR2^−/−, TLR2/4d^−/−, and MyD88^−/− mice showed a reduced incidence (herpetic stromal keratitis score, ≥3) of disease compared to TLR9^−/−, TLR4^−/−, and WT animals. (B) Mean herpetic stromal keratitis lesion scores at day 8 and day 15 p.i. of WT, TLR4^−/−, TLR9^−/−, TLR2/4d^−/−, TLR2^−/−, and MyD88^−/− mice infected with a 5 × 10³-PFU infection dose. **, P ≤ 0.001; *, P ≤ 0.05 (statistically significant differences compared to WT controls). (C) Mean angiogenesis lesion scores at day 8 and day 15 p.i. of WT, TLR4^−/−, TLR9^−/−, TLR2/4d^−/−, TLR2^−/−, and MyD88^−/− mice infected with a 5 × 10³-PFU infection dose. **, P ≤ 0.001; *, P ≤ 0.05 (statistically significant differences compared to WT control animals).

FIG. 3.

Compromised inflammatory cell influx into the corneas of TLR2^−/−, TLR9^−/−, TLR2/4d^−/−, and MyD88^−/− mice compared to TLR4^−/− and WT mice at 48 h p.i. Mice were infected with 5 × 10³ PFU HSV-1 RE. Mice were terminated at 48 h p.i., and eyes were processed for cryo-sections. Hematoxylin and eosin staining was carried out on 6-μm sections. Dot plots associated with the sections represent the Gr1⁺ CD11b⁺ cells in the WT and TLR4^−/− corneas (Gr1⁺ on x axis and CD11b⁺ on y axis). The numbers in the dot plots denote the percentages of inflammatory cells expressing both the Gr1⁺ and CD11b⁺ markers.

FIG. 4.

Compromised cell infiltration into the TG of TLR2^−/− and MyD88^−/− mice compared to that into the TG of TLR4^−/− and WT mice. Single-cell suspensions of TG cells were prepared from four TG at 48 h and 96 h p.i. for each group of mice. The cells were stained with FITC-labeled anti-Gr-1, -CD11c, and -F4/80 and with phycoerythrin-labeled CD11b antibody. The number of positive cells was determined by fluorescence-activated cell sorting. The bars represent the numbers of cells/4 × 10⁵ cells from the TG at 48 h and 96 h p.i. *, P ≤ 0.05; **, P ≤ 0.01 (statistically significant difference compared to WT animals).

FIG. 5.

Reduced levels of cytokines and angiogenic factors in the corneas of TLR2^−/− and MyD88^−/− mice compared to those in the corneas of TLR4^−/− and WT mice. At the indicated time points, six corneas/group were processed to measure the IL-6, IL-1β, MIP-2, and VEGF levels. Levels of IL-6, IL-1β, MIP-2, and VEGF were estimated from the supernatants of corneal lysates of WT, TLR2^−/−, TLR2/4d^−/−, TLR4^−/−, TLR9^−/−, and MyD88^−/− animals infected with 5 × 10³ PFU HSV-1 RE by an antibody capture ELISA as outlined in Materials and Methods. *, P ≤ 0.05; ** P ≤ 0.01 (statistically significant difference compared to WT animals).

FIG. 6.

Increased expression of proinflammatory cytokines and reduced levels of IL-10 in TLR4^−/− animals compared to those in WT animals. At the indicated time points, six corneas/group and cervical DLNs from individual mice (n = 4) were processed to measure the IL-10, IL-6, and IFN-γ levels of WT and TLR4^−/− animals infected with 5 × 10³ PFU HSV-1 RE. Cytokines were measured by antibody capture ELISA as outlined in Materials and Methods. *, P ≤ 0.05 (statistically significant differences compared to WT animals).

FIG. 7.

Reduced expression of COX-2 in TLR2^−/− animals compared to that in WT animals. Single-cell suspensions of corneal cells were prepared from two corneas (n = 8) at day 3 and day 6 p.i. Intracellular COX-2 staining was carried out as described in Materials and Methods. In panels A and B, bar diagrams demonstrate the total number of COX-2⁺ cells and the total number of COX-2⁺ Gr1⁺ cells/1 × 10⁵ corneal cells, respectively. *, P ≤ 0.05 (statistically significant differences compared to WT animals).