Toll-like receptor 2 regulates CXC chemokine production and neutrophil recruitment to the cornea in Onchocerca volvulus/Wolbachia-induced keratitis

Abstract

The filarial nematode Onchocerca volvulus is the causative organism of river blindness. Our previous studies demonstrated an essential role for endosymbiotic Wolbachia bacteria in corneal disease, which is characterized by neutrophil infiltration into the corneal stroma and the development of corneal haze. To determine the role of Toll-like receptors (TLRs) in neutrophil recruitment and activation, we injected a soluble extract of O. volvulus containing Wolbachia bacteria into the corneal stromata of C57BL/6, TLR2-/-, TLR4-/-, TLR2/4-/-, and TLR9-/- mice. We found an essential role for TLR2, but not TLR4 or TLR9, in neutrophil recruitment to the cornea and development of corneal haze. Furthermore, chimeric mouse bone marrow studies showed that resident bone marrow-derived cells in the cornea can initiate this response. TLR2 expression was also essential for CXC chemokine production by resident cells in the cornea, including corneal fibroblasts, and for neutrophil activation. Taken together, these findings indicate that Wolbachia activates TLR2 on resident bone marrow-derived cells in the corneal stroma to produce CXC chemokines, leading to neutrophil recruitment to the corneal stroma, and that TLR2 mediates O. volvulus/Wolbachia-induced neutrophil activation and development of corneal haze.

FIG. 1.

O. volvulus-induced keratitis in TLR2^−/−, TLR4^−/−, and TLR2/4^−/− mice. OvAg was injected into the corneal stromata of C57BL/6, TLR2^−/−, TLR4^−/−, and TLR2/4^−/− mice. After 18 h, mice were sacrificed and corneas were examined as described in the text. (A) Single-cell suspensions were prepared from the corneas, and total neutrophils were detected by flow cytometry using the monoclonal antibody NIMP-R14. (B) Five-micrometer-thick corneal sections were immunostained using the same antibody, and cells were counted directly. (C) Corneas were examined by in vivo confocal microscopy. Values were exported to Excel and graphed, and the area under the curve was used to represent corneal haze. Statistically significant differences were seen between OvAg-injected C57BL/6, TLR2^−/−, and TLR2/4^−/− mice (P < 0.0001). Data points represent individual corneas, and the experiment is representative of four repeat experiments.

FIG. 2.

Wolbachia-induced keratitis in TLR2^−/− and TLR9^−/− mice. (A) Corneas of C57BL/6 and TLR2^−/− mice were injected with 10,000 Wolbachia bacteria. After 18 h, the numbers of neutrophils in corneal sections were determined by immunohistochemistry, and corneal haze was examined by in vivo confocal microscopy. Data points represent individual corneas, and comparison between C57BL/6 and TLR2^−/− mice was statistically significant for neutrophils and corneal haze (P < 0.01). (B) C57BL/6 and TLR9^−/− corneas were injected with 4 μl HBSS, 10 μg/ml OvAg, or 10,000 Wolbachia bacteria. After 18 h, corneal haze was measured and the number of neutrophils per 5-μm section was determined by immunohistochemistry. Data points represent individual corneas. No statistically significant differences were noted between C57BL/6 and TLR9^−/− mice for any parameter (P > 0.05).

FIG. 3.

CXC chemokine production by corneal fibroblasts and whole corneas. (A and B) Corneas from C57BL/6 and TLR2^−/− mice were injected with 4 μg OvAg or HBSS (4 μl). After 6 h, corneas were dissected and sonicated, and CXCL1/KC and CXCL2/MIP-2 were measured by ELISA. (C) Corneal fibroblasts (MK-T cells) were incubated with 2, 10, or 50 μg/ml OvAg, with Wolbachia bacteria, or with 200 ng/ml Pam3Cys or LPS, and CXCL1/KC in supernatants was measured after 4 h of incubation. Results are shown as means plus standard errors of the means. The experiment was repeated twice with similar results.

FIG. 4.

O. volvulus-induced keratitis in TLR2^−/− bone marrow chimeric mice. Bone marrow cells from donor C57BL/6 EGFP⁺ and TLR2^−/− mice were used to reconstitute sublethally irradiated recipient C57BL/6 or TLR2^−/− mice. After 2 weeks, chimeric mice were injected intrastromally with OvAg or saline (HBSS) and, 18 h later, were examined by fluorescence microscopy. Corneal sections were examined for neutrophils by immunohistochemistry. (A) Representative corneas from irradiated C57BL/6 mice reconstituted with C57BL/6 EGFP⁺ bone marrow cells. After 2 weeks, mice were either left untreated (naïve) or examined 24 h after injection with either saline (HBSS) or OvAg. (B) Total neutrophils per corneal section. Data points represent individual corneas; these data points were combined from three repeat experiments.

FIG. 5.

CXC chemokine production and L-selectin expression on peritoneal neutrophils. Neutrophils were purified to 98% purity from peritoneal lavage cells and stimulated for 6 h with 10 μg/ml OvAg or culture medium alone. (A) CXCL1/KC and CXCL2/MIP-2 in the culture supernatants were measured by ELISA (P < 0.001 between OvAg-stimulated neutrophils from C57BL/6 versus TLR2^−/− mice). (B) Surface L-selectin expression was measured by flow cytometry of gated populations of NIMP-R14⁺ cells to determine the percentage of positive neutrophils and the mean fluorescence intensity (MFI). Data are means plus standard errors of the means for triplicate wells and are representative of three repeat experiments (P < 0.01 between medium control and Wolbachia- or Pam3Cys-treated cells.).