Original Contribution
Nrf2 is a critical modulator of the innate immune response in a model of uveitis

https://doi.org/10.1016/j.freeradbiomed.2009.04.033Get rights and content

Abstract

Uveitis is an inflammatory condition that can lead to blindness. It is therefore important to understand the pathophysiology against which to develop targeted therapy. Herein, we tested whether the oxidant-responsive transcription factor Nrf2 is involved in regulating the innate immune response and oxidative damage in the LPS uveitis model. As shown by dihydroethidium staining, intraperitoneally injected LPS increased reactive oxygen species in the retina and iris-ciliary body of Nrf2+/+ and Nrf2−/− mice. After LPS injection, ICAM-1, IL-6, TNF-α, COX-2, iNOS, and MCP-1 mRNAs were increased more in the retina and iris-ciliary body of Nrf2−/− than in those of Nrf2+/+ mice. NQO-1 and GCLM, two Nrf2-responsive antioxidant enzymes, had reduced expression in Nrf2+/+ retinas after LPS injection, but no change in expression in Nrf2−/− mice. The number of FITC–Con A-labeled leukocytes adherent to the retinal vascular endothelium increased after LPS treatment in both Nrf2+/+ and Nrf2−/− mice compared to control injections, with more adherent leukocytes in Nrf2−/− than in Nrf2+/+ mice. Pretreatment with the Nrf2 activator 1-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)imidazole increased antioxidant gene expression in the retina, reduced inflammatory mediator expression, and reduced leukocyte adherence to retinal vasculature after LPS treatment in Nrf2+/+ mice, but had no effect on Nrf2−/− mice. Treatment targeting the Nrf2 pathway may be a new therapy for uveitis.

Section snippets

Mice

Nrf2-deficient (Nrf2−/−) mice were generated as described [14] and backcrossed into the C57BL6 background. Wild-type (Nrf2+/+) and Nrf2−/− mice were fed an AIN-76A diet and water ad libitum and were housed under controlled conditions (25 °C, 12-h light–dark cycles). All experimental protocols were performed in accordance with National Institutes of Health (NIH) guidelines and were approved by the Johns Hopkins University Animal Care and Use Committee.

Treatment

Mice received a single intraperitoneal

Retinal and ciliary body ROS are increased in Nrf2-deficient mice given an LPS challenge

Using DHE histochemical staining for ROS, Fig. 1A shows that the neurosensory retinas of Nrf2+/+ and Nrf2−/− mice have a low level of detectable ROS at baseline. After LPS stimulation, the degree of detectable ROS increased dramatically in all layers of the neurosensory retina. Interestingly, the retinal pigmented epithelium, which has a robust antioxidant capability, shows little increase in ROS. The increase in ROS was significantly greater in Nrf2−/− than in Nrf2+/+ mice stimulated with LPS (

Discussion

In this study, we demonstrate that Nrf2 is a critical host factor in modulating the innate immune response to LPS stimulation in a murine model of uveitis. LPS increased ROS, disrupted the coordinated Nrf2-mediated antioxidant gene expression response, induced a cluster of inflammatory mediators in the neurosensory retina, and finally, enhanced leukocyte adhesion to the retinal vascular endothelium. Although we focused our studies on the retina, we found a similar response in the ciliary body.

Acknowledgments

This work is supported by Grant EY14005 (J.T.H.) and The Robert Bond Welch Professorship (J.T.H.); a Bausch and Laumb Fellowship Award (N.N.), the Keio University Medical Science Fund (N.N.), and The Uehara Memorial Foundation (N.N.); NIH HL081205 (S.B.), NIH/NHLBI SCCOR Grant P50HL084945 (S.B.), and a Clinical Innovator Award from FAMRI (S.B.); NIH Grant CA-78814 (M.S.) and a grant from Reata Pharmaceuticals (M.S.); and generous gifts from Ric and Sandy Forsythe, the Kwok family, the Merlau

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