A novel p40-independent function of IL-12p35 is required for progression and maintenance of herpes stromal keratitis

Abstract

PURPOSE. Interleukin (IL)-12p40 can couple with IL-12p35 or p19 chains to form the molecules IL-12p70 and IL-23, respectively, which promote T(H)1 cytokine responses. IL-12p35 can bind to EBI3 to form the anti-inflammatory molecule IL-35, but a proinflammatory function of IL-12p35 independent of IL-12p40 has not been described. Here such a function in a mouse model of herpes stromal keratitis (HSK), a CD4(+) T(H)1 cell-dependent corneal inflammation, is demonstrated. METHODS. Corneas of wild-type (WT), IL-12p40(-/-), IL-12p35(-/-), and IL-12p35(-/-)p40(-/-) (double knockout) mice were infected with the RE strain of HSV-1, and HSK was monitored based on corneal opacity, neovascularization, leukocytic infiltrate, and cytokine/chemokine levels. RESULTS. All mouse strains developed moderate HSK by 11 days after infection (dpi). However, from 11 to 21 dpi, HSK progressed in WT and IL-12p40(-/-) mice but regressed in IL-12p35(-/-) and IL-12p35(-/-)p40(-/-) mice. HSK regression was characterized by reductions in neutrophils and CD4(+) T cells and attenuation of blood vessels, which was associated with reduced levels of the chemokines KC (CXCL3), Mip-2 (CXCL2), and MCP-1 (CCL2) and the angiogenic factor vascular endothelial growth factor. CONCLUSIONS. HSK development does not require IL-12p40 and is thus independent of IL-12p70 and IL-23. However, late HSK progression does require a previously unrecognized IL-12p40-independent, proinflammatory function of IL-12p35.

Figure 1.

Mice lacking IL-12 develop HSK. IL-12p35^−/−, IL-12p40^−/−, IL-12p35^−/−p40^−/− (double knockout), and WT mice infected with HSV-1 RE were scored for HSK by slit-lamp examination from 7 to 21 dpi. Data shown reflect n values of at least five mice per group and are representative of two or more independent experiments.

Figure 2.

Mice lacking IL-12p35 have a reduced corneal leukocytic infiltrate. At 13, 17, and 21 dpi, corneas were dispersed into single-cell suspensions and stained with anti–CD45, CD4, and Gr-1 mAb. Cell suspensions were analyzed by flow cytometry. Data are represented as mean ± SEM number of CD4⁺ T cells (left axis) and GR-1^bright neutrophils (right axis). Data reflect the average of two independent experiments with n values of at least four corneas per group. *P < 0.05; **P < 0.01.

Figure 3.

IL-12p35^−/− corneas contain an increased Treg population during disease regression. Corneas were dispersed into single-cell suspensions at 13 and 17 dpi and were stained with anti–CD4, CD25, and Foxp3 mAb. Corneal suspensions were analyzed by flow cytometry. Data are represented as mean ± SEM percentages. CD25⁺FoxP3⁺ cells in the CD4⁺ T-cell population. Groups consisted of five or more corneas, and results reflect the average of two independent experiments. *P < 0.05; ***P < 0.001.

Figure 4.

Regulatory T cells do not cause disease attenuation in IL-12p35^−/− mice. IL-12p35^−/− and WT mice were approximately 80% depleted of Treg cells by treatment with anti–CD25 mAb (PC61) 3 days before infection with HSV-1 RE (A, comparing depletion in WT mice). WT and IL-12p35^−/− mice were followed up for HSK (data not shown and B, respectively). At 21 dpi, dispersed corneas were stained with anti–CD4, CD8, CD45, and GR-1 and were analyzed by flow cytometry (WT, C; IL-12p35^−/−, D). Data are represented as mean ± SEM number of cells per cornea. Results represent the average of two independent experiments with an n value of at least six mice per group. *P < 0.05.

Figure 5.

Absence of IL-12 alters expression of cytokines and chemoattractants in corneas. WT, IL-12p35^−/−, IL-12p40^−/−, and IL-12p35^−/−p40^−/− corneas were harvested at 17 dpi. Corneas were homogenized by sonic dismembranation in PBS + protease inhibitor and analyzed by multiplex bead array for cytokine and chemokine expression. Data are represented as mean ± SEM picogram per milliliter of analyte. Groups consisted of five or more corneas, and results were averaged between two independent experiments. *P < 0.05; **P < 0.01.