The inflammasome NLRP3 plays a protective role against a viral immunopathological lesion

Abstract

Herpes simplex 1 infection of the eye can cause blindness with lesions in the corneal stroma largely attributable to inflammatory events that include components of both adaptive and innate immunity. Several innate immune responses are triggered by herpes simplex 1, but it is unclear how such innate events relate to the subsequent development of stromal keratitis. In this study, we compared the outcome of herpes simplex 1 ocular infection in mice unable to express NLRP3 because of gene knockout (NLRP3(-/-)) to that of wild-type mice. The NLRP3(-/-) mice developed more-severe and earlier stromal keratitis lesions and had higher angiogenesis scores than did infected wild-type animals. In addition, NLRP3(-/-) mice generated an increased early immune response with heightened chemokines and cytokines, including interleukin-1β and interleukin-18, and elevated recruitment of neutrophils. Increased numbers of CD4(+) T cells were seen at later stages of the disease in NLRP3(-/-) animals. Reduction in neutrophils prevented early onset of the disease in NLRP3(-/-) animals and lowered levels of bioactive interleukin-1β but did not lower bioactive interleukin-18. In conclusion, our results indicate that NLRP3 has a regulatory and beneficial role in herpetic stromal keratitis pathogenesis.

Keywords: HSV; inflammation; stromal keratitis.

Figure 1.. NLRP3^−/− animals have an early onset and more-severe disease.

C57BL/6 (WT) and NLRP3^−/− animals were scarified and infected with HSV (HSV infected) and scarified but uninfected (scratched control). The disease progression was analyzed throughout time in a blinded manner using a scale described in the “Materials and Methods.” (A) The progression of SK and angiogenesis lesion severity was significantly increased in the NLRP3^−/− compared with the WT mice. (B) Representative eye photos show increased SK lesions and angiogenesis development in NLRP3^−/− mice compared with WT mice on d 7 and 15 PI. Those eyes were processed for cryosections, and H&E staining was carried out on 6-μm sections. Histopathology pictures were taken at original magnification ×40 microscope augmentation. Data are representative of 3 independent experiments and show means ± sem (n = 8 mice/group). Inf., infected. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.

Figure 2.. Mice lacking NLRP3 present bioactive levels of IL-1β and IL-18 following ocular challenge with HSV.

C57BL/6 (WT) and NLRP3^−/− mice were infected with HSV, and corneal samples were processed to measure IL-1β and IL-18 by ELISA and activated caspase 1 by FLICA assay. (A) Quantification of mature IL-1β was performed at different time points. On d 7 PI, NLRP3^−/− mice had significantly increased levels of matured IL-1β. (B) Quantification of matured IL-18 protein was performed at different time points. Matured IL-18 was similar between NLRP3^−/− and WT animals. (C) Representative histogram of FLICA⁺ cells gated on total CD45⁺ PI cells infiltrated in the corneas of WT and NLRP3^−/− animals. Spleen of naïve mice was used as an isotype control. Data are representative of 2 independent experiments and show means ± sem (n = 3; each sample is representative of 5 corneas). *P ≤ 0.05.

Figure 3.. NLRP3 deficiency increases proinflammatory cytokines and chemokines C57BL/6 (WT) and NLRP3^−/− mice corneas were scarified and infected with HSV.

On d 2 and 15 PI, corneas were collected. (A) Relative fold change in mRNA expression of IL-6, IL-12, TNF-α, MIP2, KC, IL-17, and IL-10 was examined by RT-qPCR and compared between both groups. (B) Quantification of VEGF was measured by ELISA on d 2 and 15 PI. NLRP3^−/− mice had significantly increased levels of VEGF at both time points. Data represent means ± sem from 3 different independent experiments (n = 3; each sample is representative of 5 corneas). Inf., infected ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.

Figure 4.. Corneal viral titers and neutrophil infiltration in HSV infected eyes of NLRP3^−/− and WT animals.

C57BL/6 (WT) and NLRP3^−/− mice were scarified and infected with HSV. (A) Corneal tissue was collected on d 0, 2, 7 and 10 PI, and titration was performed by standard plaque assay as described in the “Materials and Methods” section. Titers were calculated as log¹⁰ PFU/ml. Data are representative of 3 independent experiments and show means ± sem (n = 8 mice/group). (B and C) Corneas were collected at different time points to analyze the neutrophil infiltration throughout the disease. Numbers of total neutrophil infiltration (B) and representative FACS plots and percentages (C) are shown for d 2, 7, and 15 PI. At all time points, neutrophil infiltration was significantly increased in NLRP3^−/− compared with WT mice. Data are representative of 3 independent experiments and show means ± sem (n = 6; each sample is representative of 2 corneas). KO, knock out. **P ≤ 0.01, *P ≤ 0.05.

Figure 5.. NLRP3^−/−mice exhibit increased corneal and lymph node cellular infiltrates at d 7 PI.

C57BL/6 (WT) and NLRP3^−/− animals were infected with HSV. On d 7 PI, corneas and DLN were collected and stimulated with PMA/ionomycin for 4 h. (A) Representative FACS plots and percentages (left) and numbers of CD4⁺ T cells and CD4⁺ IFN-γ secreting cells from pooled corneas. (B) Representative FACS plots and percentages (left) and numbers of total CD4⁺T cells (right), CD4⁺ IFN-γ and IL-17 from lymph nodes. Data are representative of 3 independent experiments and show means ± sem (n = 8). In the case of corneas each sample is representative of 3 corneas. SSC, side scatter. **P ≤ 0.01, *P ≤ 0.05.

Figure 6.. NLRP3^−/− mice exhibit increased corneal and lymph node cellular infiltrates on d 15 PI.

C57BL/6 (WT) and NLRP3^−/− animals were infected with HSV. On d 15 PI, corneas and lymph nodes were collected and processed for stimulation with PMA/ionomycin for 4 h. (A) Representative FACS plots and percentages and numbers of CD4⁺ T cells and CD4⁺ IFN-γ secreting cells from corneas taken on d 15 PI. (B) Representative FACS plots and percentages and numbers of total CD4⁺ T cells. CD4⁺ IFN-γ and IL-17 from lymph nodes were determined on 15 d PI. Cell ratios are for total numbers of T_regs per Th1 in lymph nodes (C) and corneas (D) on d 15 PI. Data are representative of 3 independent experiments and show means ± sem (n = 8). In the case of corneas, each sample is representative of 3 corneas. SSC, side scatter. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.

Figure 7.. NLRP3^−/− animals have high levels of neutrophil-derived proteases C57BL/6 (WT) and NLRP3^−/− mice corneas were scarified and infected with HSV.

(A–C) On d 2 PI, corneas were collected and relative fold change in mRNA expression of neutrophil elastase (A), proteinase-3 (B), and cathepsin (C) was examined by qRT-PCR and compared between both groups. Data represent means ± sem from 3 different independent experiments (n = 3; each sample is representative of 5 corneas). Inf., infected., **P ≤ 0.01, *P ≤ 0.05.

Figure 8.. Reduction of the early neutrophilic infiltrate prevents the early onset of the disease in NLRP3^−/− animals.

C57BL/6 (WT) and 2 groups of NLRP3^−/− animals were scarified in the eye and infected with HSV. (A) Using mAbs against Ly6G from d −1 to d 6 PI, neutrophils were reduced in 1 group of NLRP3^−/− animals (NLRP3^−/− TRT). The other 2 groups, including NLRP3^−/− and WT mice, were used as controls (NLRP3^−/− control and WT control) and treated with isotype control (IgG2b) Abs from d −1 to d 6 PI. (B) Representative FACS plots and percentages of corneas collected on d 7 PI show that mouse anti-Ly6G treatment effectively reduced the infiltration of neutrophils in corneas. (C) Representative histopathologic pictures taken at original magnification ×40 microscope augmentation show that NLRP3^−/− TRT animals had less cellular infiltration and fewer neutrophils than NLRP3^−/− control mice. (D) The progression of SK and angiogenesis was evaluated throughout the disease, and both were significantly increased in NLRP3^−/− control compared with NLRP3^−/− TRT and WT control animals. (E and F) Quantification of bioactive IL-18 and IL-1β protein on d 7 PI by ELISA. NLRP3^−/− mice treated with anti-Ly6G had similar levels of bioactive IL-18 to NLRP3^−/− and WT control. However, bioactive concentrations of IL-1β were reduced in NLRP3^−/− TRT compared with NLRP3^−/− control WT and NLRP3^−/− animals. Data are representative of 2 independent experiments and show means ± sem (n = 8 mice/group). TRT, treated. **P ≤ 0.01, *P ≤ 0.05.