A novel synthetic triterpenoid derived from oleanolic acid, 2‐cyano‐3,12‐dioxoolean‐1,9‐dien‐28‐oic acid (CDDO), has been shown to be a more potent antitumor and antiinflammatory agent than its natural plant‐derived analogs ( 5 , 6 ). The C‐28 methyl ester of CDDO, CDDO‐Me, also known as bardoxolone methyl, has been shown to inactivate STAT‐3 signaling ( 7 , 8 ), inhibit mitochondrial electron transport via perturbations in inner mitochondrial membrane integrity ( 9 ), block the NF‐κB pathway ( 10 ), induce apoptosis by disrupting intracellular redox balance ( 11 ), induce the proapoptotic Bax protein ( 12 ), inhibit the activation of ERK‐1/2 ( 13 ), and block Bcl‐2 phosphorylation ( 12 ). Additionally, CDDO‐Me protects against lipopolysaccharide (LPS)–induced inflammatory responses via activation of the NF‐E2–related factor 2 (Nrf2)–dependent antioxidative pathway ( 14 ). Very recently, CDDO‐Me has been shown to effectively sustain increases in the estimated glomerular filtration rate in patients with advanced chronic kidney disease and type 2 diabetes mellitus in a phase II clinical trial ( 15 ). Given the fact that CDDO‐Me can target multiple signaling pathways in multiple cell types, we examined whether it had the potential to suppress lymphoproliferation, autoantibody production, and renal inflammation in murine lupus.

Systemic lupus erythematosus (SLE) is a highly complex autoimmune disease characterized by hyperproliferation and hyperactivation of lymphocytes, autoantibody production, and eventually end‐organ damage. Several lines of evidence have indicated that specific signaling pathways are involved in the pathogenesis of lupus. For example, overexpression of both phosphatidylinositol 3‐kinase (PI3K) ( 1 ) and the antiapoptotic molecule Bcl‐2 ( 2 ), as well as haplo‐insufficiency of the tumor suppressor PTEN ( Pten +/− ) ( 3 ) have been shown to cause lymphoproliferative lupus. Our previous studies demonstrated that multiple signaling pathways are up‐regulated in lupus B cells, including the AKT pathway, MAPK pathway, JAK/STAT pathway, cyclin‐dependent kinase pathway, NF‐κB pathway, and pathways downstream of some antiapoptotic Bcl‐2 family members ( 4 ). Given that lupus is associated with the activation of multiple signaling axes, therapies targeting multiple activated signaling cascades may prove to be more effective in curtailing this disease.

Twenty‐four–hour urine samples were collected using metabolic cages. The total amount of urinary protein was assayed using a Coomassie‐based assay (Pierce). When the mice were killed, the kidneys were fixed, sectioned, and stained with hematoxylin and eosin and periodic acid–Schiff. At least 100 glomeruli per section were examined by light microscopy for evidence of inflammation and/or tissue damage, and graded as previously described ( 18 ), in a blinded manner. The occurrence of any mesangiopathic, capillary hyaline, proliferative, membranous, or crescentic glomerular changes was also noted.

Cellular ROS detection was performed using a flow cytometry–based method with a DCFDA‐Cellular Reactive Oxygen Species Detection Assay Kit (catalog no. ab113851; Abcam). For the in vivo experiments, splenocytes were harvested from MRL/ lpr mice after treatment with CDDO‐Me, placebos, or dexamethasone. An aliquot of 1.5 × 10 5 cells was stained with 20 μ M 2′,7′‐dichlorofluorescein diacetate (DCF‐DA) or antibodies to cell surface markers (B220, CD4, and CD11b). DCF was excited with a 488‐nm laser and detected at 535 nm. For the in vitro experiments, splenocytes were isolated from 4‐month‐old MRL/ lpr mice and treated with a STAT‐3 inhibitor (1 μ M cucurbitacin I; Santa Cruz Biotechnology) or MEK‐1 inhibitor (50 μ M PD98059; Cell Signaling Technology) for 4 hours. These cells were then stained with 20 μ M DCF‐DA or antibodies to cell surface markers (B220, CD4, and CD11b) for flow cytometric analysis.

In the first preventive experiment, CDDO‐Me was diluted in sesame oil. Two‐month‐old B6. Sle1.Sle3 mice (n = 20) received CDDO‐Me at a final dose of 3 mg/kg or vehicle alone (i.e., placebo), by oral gavage 3 times per week for a period of 60 days. In a confirmatory preventive study, 2‐month‐old MRL/ lpr mice (n = 8 per group) were orally administered CDDO‐Me (3 mg/kg), sesame oil, water, or dexamethasone (1 mg/kg) 3 times a week for 60 days. Serum and 24‐hour urine samples were obtained on days 0, 14, and 60. All serum samples were subjected to ELISA for autoantibodies, and urine samples were assayed for total protein as previously described ( 17 , 18 ). On day 60, when the mice were killed, the cellular composition of the spleen and lymph nodes was determined by flow cytometry, and the kidneys were examined for pathology, as described below. In addition, the expression of various signaling molecules in the spleens and kidneys of the treated mice was assayed by Western blotting, as described above. In the treatment experiment, 7‐month‐old NZM2410 mice with proteinuria (n = 4–7 per group) were treated with CDDO‐Me (3 mg/kg) or placebo by oral gavage 5 times per week for a period of 60 days. Proteinuria, autoantibody production, spleen weight, and survival rates were assessed.

Purified B cells or T cells were lysed using 20 m M Tris HCl (pH 7.5), 150 m M NaCl, 1 m M Na 2 EDTA, 1 μg/ml leupeptin, 1% Triton X‐100, 1 m M phenylmethylsulfonyl fluoride, and 1 m M Na 3 VO 4 . Total protein was quantified by the Bradford method, and 10 μg was loaded per lane onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. The following primary antibodies were used: p‐STAT3 S727 (catalog no. 9134), p‐ERK‐1/2 T202/Y204 (catalog no. 4376), ERK‐1/2 (catalog no. 4695), p‐NF‐κB S536 (catalog no. 3031), p‐MEK‐1/2 S217/221 (catalog no. 9121), MEK‐1/2 (catalog no. 9122), AKT (catalog no. 9272), p‐AKT T308 (catalog no. 9275), α‐tubulin (catalog no. 2144) (all from Cell Signaling Technology), β‐actin (catalog no. RGM2; Advanced ImmunoChemical), and Bcl‐2 (catalog no. sc‐23960; Santa Cruz Biotechnology). Antibodies to Nrf2 (catalog no. ab71890), NAD(P)H quinone oxidoreductase 1 (NQO‐1; catalog no. ab2346), glutamate cysteine ligase catalytic subunit (catalog no. ab53179), and glutamate cysteine ligase modifier subunit (catalog no. ab81445) were purchased from Abcam. Horseradish peroxidase–conjugated secondary antibodies and an ECL Plus detection kit (Amersham) were used to develop the blot. For Western blot analysis of purified B cells and T cells, cells were pooled from 3–4 mice for each lane. The respective band intensities were measured using ImageJ software version 1.37 (National Institutes of Health; online at http://rsb.info.nih.gov/ij ), and normalized against the corresponding β‐actin or α‐tubulin levels. Where samples from different strains were compared, normalized band intensities were expressed as ratios, relative to the corresponding levels in B6 mice.

The anti–double‐stranded DNA (anti‐dsDNA), antihistone, and antihistone/DNA ELISAs were carried out as previously described ( 17 ). Raw optical density was converted to units/milliliter, using a positive control serum derived from a B6. Sle1.lpr mouse, and arbitrarily setting the reactivity of a 1:100 dilution of this serum to 100 units/ml. Test sera with reactivity stronger than the standard were diluted further and reassayed. The glomerular‐binding ELISA was performed as described previously ( 17 ), using sonicated rat glomeruli as the substrate.

Spleens were harvested from mice postmortem, and single‐cell suspensions were prepared by crushing spleens between frosted glass slides. Red blood cells were lysed using ACK lysis buffer (Invitrogen) followed by 2 washes in phosphate buffered saline plus 0.5% bovine serum albumin. B220+ B cells were purified by positive selection from this total splenocyte suspension with B220 microbeads (Miltenyi Biotec). CD4+ T cells were purified by positive selection from total splenocytes with CD4 microbeads according to the recommendations of the manufacturer (Miltenyi Biotec).

C57BL/6 (B6), MRL/ lpr, and NZM2410 mice were obtained from The Jackson Laboratory and bred in our animal facility. The derivation of B6‐congenic mice bearing different NZM2410‐derived lupus susceptibility intervals has been described previously ( 16 ). B6. Sle1.Sle3 mice, which are bicongenic for the 2 lupus susceptibility intervals Sle1 and Sle3 , were previously derived by intercrossing the respective monocongenic strains ( 4 ). All mice used for this study were bred and housed in a specific pathogen–free colony at the University of Texas Southwestern Medical Center at Dallas Department of Animal Resources. Both male and female mice were used, and any observed sex differences are noted. Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas.

Since this was a relatively long‐term study, and since CDDO‐Me cripples multiple signaling axes, one concern may be potential side effects. After 60 days of drug administration (either in the preventive or treatment experiment), body weights of mice from both the CDDO‐Me group and the placebo group were similar (data from the preventive experiment are available from the corresponding author upon request). Likewise, all blood cell counts, including white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell distribution width index, mean platelet volume, neutrophils, lymphocytes, and eosinophils remained similar between the 2 groups (data are available from the corresponding author upon request). Platelet counts in the 2 groups were also comparable. These results indicate that the mice experienced no apparent hematologic side effects or weight loss as a result of CDDO‐Me administration. In addition, we monitored liver function by assaying aspartate aminotransferase (AST); however, there were no significant changes in the CDDO‐Me–treated group (AST activity 75.7 ± 6.5 units/liter) compared to the placebo group (74.9 ± 5.8 units/liter).

Finally, to test the therapeutic efficacy of CDDO‐Me after disease onset, we carried out an additional treatment experiment by administering CDDO‐Me to a third strain of mice with lupus (7‐month‐old NZM2410 mice; n = 4–7 mice per group) for a period of 60 days. These mice already had proteinuria at the beginning of the study. Once again, CDDO‐Me was effective in improving survival and reducing cellularity, circulating antibodies, and proteinuria (data are available from the corresponding author upon request). Thus, CDDO‐Me appears to be therapeutically effective even when administered after disease onset.

A–C, Significant reduction in cellular reactive oxygen species (ROS) in both splenic CD4+ T cells ( B ) and CD11b+ cells ( C ) but not B220+ B cells ( A ) isolated from 4‐month‐old MRL/ lpr mice treated with methyl‐2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐ 28‐oate (CDDO‐Me). D–F, Positive correlation of the reduction in ROS with the decreased phosphorylation of ERK‐1/2 ( D ) and MEK ( E ), and negative correlation of the reduction in ROS with the increase in NF‐E2–related factor 2 (Nrf2) ( F ). DCF‐DA = 2′,7′‐dichlorofluorescein diacetate; DEX = dexamethasone.

To investigate if CDDO‐Me has any modulatory effect on oxidative stress in murine lupus, we examined the level of ROS in the splenocytes using a DCF‐DA kit as described in Materials and Methods . We found that the ROS levels, as indicated by the percentage of DCF‐DA+ cells, were significantly reduced among both CD4+ T cells and CD11b+ cells in the CDDO‐Me–treated group, compared to the other groups, including the dexamethasone‐treated mice (Figures 5 A–C) (additional results are available from the corresponding author upon request). We also found that in CD4+ T cells, the ROS levels positively correlated with the phosphorylation of ERK‐1/2 and MEK, but negatively correlated with Nrf2 levels, suggesting that the pathways by which CDDO‐Me influences lymphocyte signaling may be mechanistically linked to the pathways that generate ROS (Figures 5 D–F). To determine if the up‐regulation or phosphorylation of the signaling molecules MEK‐1 and STAT‐3 may be mechanistically linked to altered ROS in murine lupus, we determined the percentage of DCF‐DA+ cells among splenocytes following in vitro inhibition of MEK‐1 or STAT‐3; however, we did not find any significant association between ROS levels and MEK‐1 or STAT‐3 activation (data are available from the corresponding author upon request).

The drug control group that received dexamethasone also demonstrated disease improvement after treatment, comparable to the efficacy noted with CDDO‐Me. The sesame oil vehicle by itself did not have any significant impact on the disease, since the mice treated with sesame oil exhibited phenotypes that were comparable to the mice that received water only. We further examined the signaling status in both B cells and T cells in the mouse spleen after CDDO‐Me treatment. The phosphorylation of MEK and ERK‐1/2 was down‐regulated, whereas the antioxidative molecule Nrf2 was up‐regulated in both lymphocyte compartments (Figure 4 D).

Amelioration of disease by methyl‐2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oate (CDDO‐Me) in the MRL/ lpr mouse model of lupus. A, Significant reduction in splenomegaly in mice treated with CDDO‐Me for 60 days compared to mice treated with water or sesame oil. Significantly reduced spleen weight was also noted in the mice treated with dexamethasone (DEX). B and C, Significant decrease in anti–double‐stranded DNA (anti‐dsDNA) antibody levels ( B ) and proteinuria and glomerulonephritis (GN) score ( C ) in mice treated with CDDO‐Me compared to mice treated with water or sesame oil. In A–C, data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines inside the boxes represent the median. Lines outside the boxes represent the 10th and 90th percentiles. D, Reduced phosphorylation of MEK and ERK‐1/2 and increased phosphorylation of NF‐E2–related factor 2 (Nrf2) in both splenic B cells and T cells from mice treated with CDDO‐Me. ∗ = P < 0.05.

To determine if CDDO‐Me attenuates disease in other murine lupus strains that are genetically different from B6. Sle1.Sle3 mice, we administered CDDO‐Me to 2‐month‐old MRL/ lpr mice, another strain that develops spontaneous lupus. In addition, to clarify if the vehicle sesame oil by itself has any modulatory effects on murine lupus, we included another control group that received only water. In this validation study, we also included a “positive control” group for drug treatment, in which the lupus‐prone mice were treated with dexamethasone. Dexamethasone is a glucocorticoid, and some glucocorticoids are standard therapy for human lupus ( 21 ). Once again, we found that CDDO‐Me significantly reduced splenomegaly, compared to placebo (sesame oil) (Figure 4 A). As expected, we observed a significant reduction in anti‐dsDNA antibody levels (Figure 4 B), proteinuria, and GN score (Figure 4 C) in the CDDO‐Me–treated group compared to the control groups.

Up‐regulation of the NF‐E2–related factor 2 (Nrf2) signaling pathway in the kidneys of B6. Sle1.Sle3 mice after methyl‐2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oate (CDDO‐Me) treatment. Kidneys were collected from both CDDO‐Me–treated and placebo‐treated female B6. Sle1.Sle3 mice, and kidney lysates were used for Western blot analysis. Western blot results were quantified using ImageJ software and plotted as bar graphs. Bars show the mean ± SEM band intensity (n = 4–5 mice per group). ∗∗∗ = P < 0.001 versus placebo‐treated mice, by Student's t ‐test. NQO‐1 = NAD(P)H quinone oxidoreductase 1; GCLC = glutamate cysteine ligase catalytic subunit; GCLM = glutamate cystein ligase modifier subunit.

Kidney damage can be triggered via oxidative or inflammatory signals. In this context, CDDO‐Me has previously been shown to protect against LPS‐induced inflammatory responses via activation of the Nrf2‐dependent antioxidative pathway ( 14 ). Hence, we examined flash‐frozen renal tissue from the CDDO‐Me–treated mice for evidence of enhanced Nrf2 activation. As shown in Figure 3 , the antioxidative modulator, Nrf2, and its target, NQO‐1, were both significantly increased in the mouse kidneys after CDDO‐Me treatment. The Nrf2 regulator glutamate cysteine ligase catalytic subunit also showed a trend toward enhanced expression after CDDO‐Me treatment. These results suggest that CDDO‐Me may also protect against renal damage in murine lupus nephritis by altering the intrarenal redox balance.

To determine if CDDO‐Me treatment similarly impacted signaling cascades in splenic T cells, splenic CD4+ T cells were isolated, and their lysates were analyzed as described above. The activation/phosphorylation of ERK‐1/2, MEK‐1/2, and STAT‐3 were significantly ameliorated in CD4+ T cells by CDDO‐Me treatment (Figure 2 B). Both AKT and NF‐κB showed a trend toward reduced phosphorylation, but these differences did not reach statistical significance. Taken together, the data shown in Figures 2 A and B provide strong evidence that multiple signaling pathways were inhibited in both splenic B cells and CD4+ T cells following CDDO‐Me administration.

Inhibition of selected signaling axes in splenic B cells ( A ) and T cells ( B ) from female B6. Sle1.Sle3 mice treated with methyl‐2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oate (CDDO‐Me). At the end of the 60‐day CDDO‐Me treatment period (i.e., when all mice were 4 months of age), splenic B cells or T cells were purified using magnetic beads, and cell lysates were examined for signaling status by Western blot analysis. Western blot results were quantified using ImageJ software and plotted as bar graphs. Bars show the mean ± SEM band intensity (n = 10 mice per group). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by Student's t ‐test.

Currently, the major therapeutic use of CDDO‐Me is to inactivate signaling pathways underlying cell growth and cell proliferation in cancer. To investigate the effects of CDDO‐Me administration on lymphocyte signaling, splenic B220+ B cells were purified with magnetic beads, and B cell lysates were analyzed by Western blot analysis for several signaling axes. Our results demonstrate that MEK‐1/2 activation was significantly dampened in splenic B cells from the CDDO‐Me–treated mice (Figure 2 A). In addition, CDDO‐Me treatment appeared to diminish the activation of NF‐κB, STAT‐3, and to a lesser extent Akt, although these differences were not statistically significant.

After 60 days of treatment, the serum levels of IgG anti‐dsDNA, anti–single‐stranded DNA (anti‐ssDNA), antihistone, and antiglomerular antibodies were all significantly decreased in mice treated with CDDO‐Me compared to those treated with placebo. Prior to the initiation of CDDO‐Me treatment (day 0), basal levels of IgG anti‐dsDNA, anti‐ssDNA, antihistone, and antiglomerular antibodies were measured and found to be comparable in the 2 groups of mice (Figures 1 H–K). It is important to note that after CDDO‐Me treatment, all of the IgG antibody levels listed above were reversed to normal, similar to the phenotypes previously seen in healthy B6 mice ( 17 ).

Next, we examined whether the administration of CDDO‐Me reduces renal damage in murine lupus nephritis. On day 60 after placebo or CDDO‐Me treatment, urine was collected and tested for proteinuria. Compared to the placebo‐treated group, the CDDO‐Me–treated mice showed significantly reduced proteinuria (Figure 1 E). Examination of the mouse kidneys clearly demonstrated that CDDO‐Me treatment resulted in lower GN scores than placebo treatment. Microscopic analysis revealed increased cellularity in the glomeruli of mice treated with placebo compared to those treated with CDDO‐Me, indicating the presence of more inflammation and greater numbers of infiltrating cells in the placebo‐treated mice. CDDO‐Me treatment also led to reduced BUN levels, further indicating that renal function was improved in these mice (Figures 1 F and G). Most importantly, all parameters of renal disease were reversed to normal, similar to the phenotypes previously seen in healthy B6 mice ( 17 ).

Next, we investigated which cell populations were significantly suppressed by CDDO‐Me. As expected, among splenic T cells, the percentage of CD4+ T cells was decreased (mean ± SEM 12.1 ± 0.35% versus 15.1 ± 1.2%; P = 0.021), while the percentage of CD8+ T cells was increased (9.73 ± 0.4% versus 6.8 ± 1.1%; P = 0.023) in the CDDO‐Me–treated group (Figure 1 C). The absolute number of total splenic CD4+ T cells was also decreased (18.7 ± 3.8 million versus 39.0 ± 2.0 million; P < 0.0001) in the CDDO‐Me–treated group (Table 1 ). Within the CD4+ T cell compartment, the activated population (CD69+) was significantly deceased in the CDDO‐Me–treated group compared to the control group (Figure 1 C and Table 1 ). Of note, besides the dramatic reduction in and deactivation of CD4+ T cells, the absolute cell numbers (if not percentages) of splenic B220+ B cells (both mature and immature B cells, and B1a cells) were also decreased with CDDO‐Me treatment (Table 1 ). B cell activation, as gauged by surface CD86 expression, was also markedly reduced following CDDO‐Me treatment (6.48 ± 0.42 versus 8.72 ± 0.45 mean fluorescence intensity units; P < 0.002) (data not shown). Importantly, after CDDO‐Me treatment, the cell numbers and activation status of splenic B cells and T cells and their subsets were reversed to normal, similar to the phenotypes previously seen in healthy B6 mice ( 17 ).

Attenuation of disease in B6. Sle1.Sle3 mice with spontaneous lupus treated with methyl‐2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oate (CDDO‐Me). Two‐month‐old female B6. Sle1.Sle3 mice (n = 20 per group) were treated with CDDO‐Me or placebo (sesame oil) as indicated. A and B , Amelioration of splenomegaly, as indicated by spleen weight ( A ) and splenic cell number ( B ) in mice after 60 days of treatment with CDDO‐Me. In A, circles represent individual mice; horizontal lines show the mean. In B, bars show the mean ± SEM. C, Suppression of the expansion of activated CD4+ T cells in B6. Sle1.Sle3 mice examined after 60 days of treatment. Flow cytometry plots show results from a representative experiment. Values are the percent of cells. R2 = gate of CD8+ cells; R3 = gate of CD4+ cells. D, Hematoxylin and eosin staining of kidney sections from a mouse treated with CDDO‐Me and a mouse treated with placebo. Results are representative of several similar experiments (n = 10). Original magnification × 20. E–G, Reduction in proteinuria ( E ), glomerulonephritis (GN) score ( F ), and blood urea nitrogen (BUN) levels ( G ) in B6. Sle1.Sle3 mice after 60 days of treatment with CDDO‐Me. H–K, Attenuation of serum levels of IgG anti–double‐stranded DNA ( H ), anti–single‐stranded DNA (anti‐ssDNA) ( I ), antihistone ( J ), and antiglomerular antibodies ( K ) in B6. Sle1.Sle3 mice treated with CDDO‐Me. In E–K, data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines inside the boxes represent the median. Lines outside the boxes represent the 10th and 90th percentiles. Data are shown for mice at the ages of 2 months (day 0) and 4 months (day 60). Serially diluted sera from B6. Sle1.lpr mice were used for plotting a standard curve, and the highest standard was set at 100 AU. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by Student's t ‐test.

Given the previous demonstration that CDDO‐Me suppresses cell proliferation ( 19 , 20 ), we investigated whether CDDO‐Me suppressed the development of splenomegaly in mice with lupus. To address this, 2‐month‐old female B6. Sle1.Sle3 mice were treated for 60 days with CDDO‐Me or placebo, and then spleen size and cellularity were assessed in both groups. Notably, the mean spleen weights in the CDDO‐Me–treated group were decreased almost 50% compared to the placebo‐treated group (Figure 1 A). Consistent with these findings, the mean number of splenocytes was also decreased in the CDDO‐Me–treated group compared to the placebo‐treated group (Figure 1 B).

DISCUSSION

Lupus is a highly complex autoimmune disease, in which B cells, T cells, and even myeloid cells are hyperproliferative and hyperactive. These hyperactivated immune cells can infiltrate organs, causing tissue damage, resulting in end‐organ problems such as nephritis. Previous studies have demonstrated that despite the distinct genetic backgrounds of mouse models used for studying spontaneous lupus, these different strains share the up‐regulation of similar cell signaling pathways involving PI3K/Akt/mammalian target of rapamycin (mTOR), MAP kinases, STAT‐3/STAT‐5, NF‐κB, multiple Bcl‐2 family members, and various cell cycle molecules in B cells (4). Several key signaling molecules, including NF‐κB (22), STAT‐3 (23), Ca2+/calmodulin‐dependent protein kinase IV (24), and Syk (25), have also been observed to be altered in lupus T cells. Furthermore, several of these signaling intermediates are positive regulators of a number of inflammatory cytokines and chemokines. Hence, intervention in leukocyte signaling pathways might be beneficial in the treatment of lupus.

Triterpenoids are natural plant products generated by the cyclization of squalene and are used for medicinal purposes in many Asian countries, since they have been reported to have anticarcinogenic activity (26-29). Because the biologic activities of some of the natural triterpenoids are not strong enough, new analogs of these molecules have been chemically synthesized in an attempt to produce more potent agents. One of these analogs, CDDO, was found to inhibit the proliferation of many human cancer cells and to suppress the ability of various inflammatory cytokines such as interferon‐γ (IFNγ), interleukin‐1 (IL‐1), and tumor necrosis factor α (TNFα). CDDO‐Me is a methyl derivative of CDDO that was found to be as active as CDDO in suppressing the increased production of nitric oxide by IFNγ in mouse macrophages (30). Furthermore, there are a number of studies showing that CDDO‐Me can block selected signaling pathways. For example, other investigators have identified CDDO‐Me as a potent caspase‐mediated apoptosis inducer in human lung cancer in acute myelogenous leukemia (12, 31). CDDO‐Me has also been shown to directly inhibit both JAK‐1 and STAT‐3 (7) and to inhibit the NF‐κB pathway through direct inhibition of IKKβ on Cys179 (10). This compound has also been shown to inhibit IκB kinase and to enhance apoptosis induced by TNF and chemotherapeutic agents through down‐regulation of NF‐κB–regulated gene products in human leukemia cells (32).

Our findings are consistent with those described above, since CDDO‐Me treatment diminished the activation of MEK‐1/2 in B cells, and of ERK, MEK, and STAT‐3 in T cells. In both T cells and B cells, NF‐κB showed a trend toward reduced activation following CDDO‐Me treatment, but these differences did not reach statistical significance. These findings suggest that CDDO‐Me can suppress cell activation and inflammatory signals mediated via multiple signaling axes not only in cancer cells, but also in immune cells and possibly in other tissues (including renal cells).

Indeed, this is the first study to demonstrate that CDDO‐Me is beneficial in suppressing hyperactivation of immune cells, particularly CD4+ T cells (Figure 1 and Table 1). In a murine acute graft‐versus‐host disease model, CDDO‐Me exhibited an increased ability to inhibit allogeneic T cell responses and induce cell death of alloreactive T cells in vitro (33). In a transgenic adenocarcinoma of the mouse prostate (TRAMP) cancer model, CDDO‐Me induced apoptosis in TRAMP C1 cells, as revealed by the increased expression of annexin V and cleavage of procaspases 3, 8, and 9; CDDO‐Me also inhibited NF‐κB–regulated antiapoptotic Bcl‐2, Bcl‐x L , and X‐linked inhibitor of apoptosis protein (19, 20). Additionally, CDDO‐Me participates in the induction of apoptosis in acute myeloid leukemia (34). In this study, both splenic B cells and T cells were decreased in the CDDO‐Me–treated mice compared to the placebo‐treated controls, suggesting that CDDO‐Me might induce apoptosis in splenic B cells and T cells, thereby subduing autoimmunity.

The reduced activation of lymphocytes was associated with a reduction in the production of autoantibodies such as anti‐dsDNA, anti‐ssDNA, antihistone, and anti–glomerular basement membrane in B6.Sle1.Sle3 mice following CDDO‐Me treatment (Figure 1). Importantly, the most prominent benefit of this drug lies in its effective prevention of renal damage, as marked by the dramatic reduction in proteinuria, BUN, GN score, and other renal pathology measures (Figure 1). A recent clinical trial of bardoxolone methyl (another name for CDDO‐Me) carried out in patients with advanced chronic kidney disease and type 2 diabetes mellitus has demonstrated its capacity in sustaining an increase in the estimated glomerular filtration rate (15). Our findings are consistent with the results of that study, and suggest that CDDO‐Me might be of therapeutic benefit in chronic renal disease arising from multiple initial triggers.

Besides dampening cell signaling, triterpenoids may also improve disease outcomes through other mechanisms. CDDO and its derivatives have been found to induce Nrf2 signaling, which in turn induces cytoprotective and antioxidative genes (35, 36). The transcription factor Nrf2 binds and activates the antioxidant response element (37), a cis‐acting sequence found in the 5′‐flanking region of genes encoding many cytoprotective enzymes, including NQO‐1 (38-40). It has been shown that ROS are present at higher levels during lupus nephritis (41). Therefore, antioxidant molecules such as Nrf2 and NQO‐1 might be beneficial in protecting against ROS‐induced kidney damage. In this study, we have shown that Nrf2 and its partner NQO‐1 were significantly induced in the kidneys of B6.Sle1.Sle3 mice after CDDO‐Me treatment (Figure 3). Our results suggest that renal damage and potentially other tissue damage may be ameliorated by CDDO‐Me, in part via the activation of the antioxidant pathway.

The importance of Nrf2 in protecting against lupus nephritis has been reported previously. Interestingly, Nrf2‐deficient female mice develop lupus‐like autoimmune nephritis (42). Similar to the findings of the present study, other natural agents that are beneficial in lupus nephritis have also been associated with the elevation of renal Nrf2. Antroquinonol, a purified compound and major effective component of Antrodia camphorate, inhibited the production of ROS and nitric oxide, but increased the activation of Nrf2 within the kidneys in an accelerated mouse model of severe lupus nephritis. This was associated with significantly reduced infiltrating T cell proliferation and renal lesions (43). Epigallocatechin‐3‐gallate, the major bioactive polyphenol in green tea, has also been shown to increase Nrf2 and ameliorate renal disease in (NZB × NZW)F1 mice (44).

The present study showed that CDDO‐Me abrogates ROS both in vivo and in vitro (Figure 5) (additional results are available from the corresponding author upon request). However, our findings suggest that the impact of CDDO‐Me on ROS levels and lymphocyte signaling may be independent events (results are available from the corresponding author upon request), though this needs to be examined more carefully. There is some evidence in the literature linking both of these molecular phenomena. Thus, the inactivation of STAT‐3 has been observed to suppress load‐driven mitochondrial activity, leading to elevated levels of ROS in cultured primary osteoblasts (45). Conversely, ROS activates STAT‐3 and induces IL‐6 production in cancer cells (46). Also, treating rat sympathetic neurons with an MEK‐1 inhibitor greatly decreased cellular concentrations of glutathione, a major cellular antioxidant (47). Clearly, the mechanistic links between ROS production and lymphocyte signaling warrant further study, particularly in the context of autoimmunity.

There is a very interesting relationship between oxidative stress and one particular cell signaling pathway in lupus. The antioxidant N‐acetylcysteine (NAC) has been shown to inhibit mTOR activity in vitro, and also confer therapeutic benefit in murine lupus (48, 49). Consistent with the results of those earlier studies, more recent work by Lai and coworkers has also demonstrated that NAC confers therapeutic benefit in patients with SLE, once again associated with mTOR inhibition and enhanced lymphocyte apoptosis (50). Although mTOR was not directly assessed in the present study, our finding that Akt phosphorylation is reduced in T cells following CDDO‐Me treatment is consistent with the findings of the studies described above. Although further mechanistic studies are warranted, taken together, these findings suggest that one important mechanism of action of antioxidants in lupus might be reduced signaling via the Akt/mTOR axis coupled with elevated apoptosis of immune effector cells. Indeed, there is recent evidence that mTOR is a direct target of CDDO‐Me (51). The relationship between oxidative stress and mTOR, and its implications for the pathogenesis and treatment of SLE, are elegantly discussed in a recent review by Perl (52).

In summary, CDDO‐Me, a drug known to inhibit cell growth, has a reproducible impact on suppressing murine lupus and lupus nephritis when administered before disease develops, and more importantly, after the onset of disease. This agent appears to be operating by suppressing multiple cell signaling axes in leukocytes (and possibly other tissue) and countering oxidative stress. Given the efficacy of this agent in modulating immune cell signaling as well as lupus nephritis, this may be an attractive option to pursue in the context of human lupus therapies.