Nuclear Factor Erythroid 2-related Factor 2 Deficiency Exacerbates Lupus Nephritis in B6/lpr mice by Regulating Th17 Cell Function

Abstract

Lupus nephritis (LN) is the major clinical manifestation of systemic lupus erythematosus. LN is promoted by T helper 17 (Th17) cells, which are the major pro-inflammatory T cell subset contributing to autoimmunity regulation. Nuclear factor erythroid 2-related factor 2 (NRF2) is critical for suppressing reactive oxygen species (ROS) and relieving oxidant stress by regulating antioxidant gene expression. Previous studies have demonstrated that Nrf2 deficiency promotes drug-induced or spontaneous LN. However, whether NRF2 regulates Th17 function during LN development is still unclear. In this study, we introduced Nrf2 deficiency into a well-known LN model, the B6/lpr mouse strain, and found that it promoted early-stage LN with altered Th17 activation. Th17 cells and their relevant cytokines were dramatically increased in these double-mutant mice. We also demonstrated that naïve T cells from the double-mutant mice showed significantly increased differentiation into Th17 cells in vitro, with decreased expression of the Th17 differentiation suppressor Socs3 and increased phosphorylation of STAT3. Our results demonstrated that Nrf2 deficiency promoted Th17 differentiation and function during LN development. Moreover, our results suggested that the regulation of Th17 differentiation via NRF2 could be a therapeutic target for the treatment of subclinical LN patients.

Figure 1. Survival rates, renal function, and anti-dsDNA autoantibody production.

During the course of 9 months, the survival of mice with different genotypes was monitored (a). Enzyme-linked immunosorbent assay analyses of the serum anti-dsDNA antibody level (n = 11) were carried at 3 months (b) and 6 months (c) of age. To analyse the development of renal insufficiency, we measured the serum levels of blood urea nitrogen (d and e) and total protein (f and g) at different times (n = 8). Data values represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. Histopathologic analysis of renal tissues.

Representative photomicrographs of kidneys from B6.Nrf2^−/−lpr/lpr, B6/lpr, B6.Nrf2^−/−, and WT B6 mice (n = 6) at 6 months old are shown. The sections were stained with H&E and PAS (a) All images were taken at 400× total magnification. Glomerular injury was scored from 0 to 4 and interstitial injury from 0 to 4. Shown are scores for the mice of each genotype at 6 months old (c). (b) Immunostaining of immunocomplex deposition (IgG and IgM) in kidney at 6 months old. Data values represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Composition of lymphocytes in spleen.

Spleens and splenic lymphocytes were isolated from B6.Nrf2^−/−lpr/lpr, B6/lpr, B6.Nrf2^−/−, and WT B6 mice at 6 months old (n = 9) and 3 months old (n = 7) and counted, then analysed by flow cytometry. (a) The spleens of the Nrf2^−/−lpr/lpr mice were the largest in comparison with those of other genotypes of mice at 6 months old. (b) The total number of lymphocytes in the B6.Nrf2^−/−lpr/lpr mice were increased at both 3 and 6 months. The proportion of CD3⁺CD4⁺ T cells was significantly increased in the B6.Nrf2^−/−lpr/lpr mice compared with that in the B6.lpr mice (c). (d) At 6 months of age, in the B6.Nrf2^−/−lpr/lpr mice, the proportions of CD3⁺CD4⁺ T cells and CD3⁺CD8⁺ T cells among splenic lymphocytes were higher than those in the other mouse genotypes. The Nrf2^−/−lpr/lpr mice had more B cells in spleen (e). Data values represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Cytokine levels in the peripheral blood, kidney, and macrophages.

(a) The levels of IL-17, G-CSF, IL-12p7, and IL-1β in serum as determined by a Bio-Plex Pro™ Mouse Cytokine 23-Plex Panel. (b) Cytokine expression in macrophages activated using lipopolysaccharides as measured by qRT-PCR. (c) The expression of Th17-related cytokines in kidneys as determined by qRT-PCR. Mice were aged 6 months. n = 6 for each group. Data values represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Splenic Th17 cell population and cytokine expression.

Lymphocytes were collected from B6.Nrf2^−/−lpr/lpr (n = 4), B6/lpr (n = 4), B6.Nrf2^−/− (n = 3), and WT B6 (n = 4) mice at 6 months of age and activated with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (1 μg/mL). (a and b) Representative flow cytometry plots and data showing a higher proportion of RORγt⁺CD3⁺CD4⁺Th17 cells among splenic CD4⁺ cells in the Nrf2^−/−lpr/lpr mice than in the mice of other genotypes. (c) Representative flow cytometry plots demonstrating interleukin-17A (IL-17A) production by CD3⁺CD4⁺ T cells from mice of four different genotypes. (d) The percentage of T cells producing IL-17A was markedly increased in the Nrf2^−/−lpr/lpr mice. (e and f) The mRNA levels of cytokines associated with Th17 in splenic cells. Data values represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6. In vitro Th17 cell differentiation.

Naïve CD4⁺ T cells were collected from mice of different genotypes and activated by plate-bound CD3 and CD28, then polarised into Th17 cells by treatment with IL-6 and TGF-β for 5 days. (a) Representative flow cytometry plots showing a higher proportion of RORγt⁺CD3⁺CD4⁺ Th17 cells in the Nrf2^−/−lpr/lpr mice than in mice of other genotypes. (b) qRT-PCR analysis of Th17 cell-associated cytokines and (d) Socs3 transcripts. (c) Western blot analysis of pY-STAT3 in naïve CD4⁺ T cells cultured in Th17 cell-conditioned media. Data values represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.