Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

47 Pages·2017·5.78 MB·English

MCB Accepted Manuscript Posted Online 15 May 2017
Mol. Cell. Biol. doi:10.1128/MCB.00063-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
Suzuki et al.
1 Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation
2 in Scurfy Mice
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4 Running Title: NRF2 Alleviates Inflammation in Scurfy Mice
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6 Takuma Suzukia,b, Shohei Murakamia, Shyam S. Biswalc, Shimon Sakaguchid, Hideo Harigaeb,
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7 Masayuki Yamamotoe and Hozumi Motohashia# o
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9 Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, a
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10 Tohoku University, Sendai, Japana; Department of Hematology and Rheumatologyb and e
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11 Department of Medical Biochemistrye, Tohoku University Graduate School of Medicine, fr
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12 Sendai, Japan; Department of Environmental Health Sciences, Bloomberg School of Public

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13 Health, Johns Hopkins University, Baltimore, Maryland, USAc; Experimental Immunology, tt
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14 World Premier International Research Center, Immunology Frontier Research Center, //
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15 Graduate School of Medicine, Osaka University, Osaka, Japand c
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17 T.S. and S.M. contributed equally to this work.
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19 #CORRESPONDING AUTHOR: Hozumi Motohashi /
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20 Hozumi Motohashi
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21 Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, p
22 Tohoku University, ril
23 4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan 8
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24 TEL: [phone], 2
25 FAX: [phone], 0
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26 E-MAIL: [email protected] 9

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29 Word count for the Materials and Methods; 845 words u
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31 Combined word count for the Introduction, Results, and Discussion; 3,497 words
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35 Abstract
36 The transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2) plays crucial roles
37 in the defense mechanisms against oxidative stress and mediates anti-inflammatory actions in
38 various pathological conditions. Recent studies have shown that the dysfunction of regulatory
39 T cells (Tregs) is directly linked to the initiation and progression of various autoimmune
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40 diseases. To determine the Treg-independent impact of NRF2 activation on autoimmune w
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41 inflammation, we examined Scurfy (Sf) mice that are deficient in Tregs and succumb to a
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42 severe multi-organ inflammation by 4 weeks of age. We found that systemic activation of d

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43 NRF2 by Keap1 (kelch-like ECH-associated protein 1) knockdown ameliorated tissue m

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44 inflammation and lethality in Sf mice. Activated T cells and their cytokine production were tt
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45 accordingly decreased by Keap1 knockdown. In contrast, NRF2 activation through cell /m
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46 lineage-specific Keap1 disruption (i.e., in T cells, myeloid cells or dendritic cells) only .
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47 achieved partial or no improvement in the inflammatory status of Sf mice. Our results indicate
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48 that systemic activation of NRF2 suppresses effector T-cell activities independently of Tregs g
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49 and that NRF2 activation in multiple cell lineages appears to be required for sufficient n

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50 anti-inflammatory effects. This study emphasizes the possible therapeutic application of r
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51 NRF2 inducers in autoimmune diseases that are accompanied by Treg dysfunction. ,
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52 9

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53 Keywords: KEAP1, NRF2, Scurfy mice, inflammation, autoimmune diseases y
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55 Introduction
56 The KEAP1-NRF2 system plays important roles in the antioxidant response and contributes
57 to cytoprotection from various redox disturbances (1, 2). Under normal conditions, the
58 transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2) is negatively regulated
59 by KEAP1 (kelch-like ECH-associated protein 1) via polyubiquitination and subsequent
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60 degradation. When cells are exposed to oxidative stress, NRF2 escapes from w
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61 KEAP1-mediated degradation, translocates into the nucleus, and enhances the expression of a
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62 various genes encoding antioxidant proteins and detoxifying enzymes. d

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63 In addition to this antioxidant function, we recently showed that NRF2 exerts m

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64 anti-inflammatory effects by decreasing the mRNA levels of pro-inflammatory cytokine genes tt
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65 including Il6 and Il1b (3). Indeed, the critical contributions of NRF2 to reduced inflammation /m
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66 have been demonstrated in mouse models of various pathological conditions, such as .
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67 elastase-induced emphysema (4), cecal ligation and puncture-induced sepsis (5), dextran
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68 sulfate sodium-induced colitis (6), allergen-driven airway inflammation (7), and g
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69 dystrophin-deficient muscular dystrophy (8). n

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70 NRF2 has also been shown to mitigate autoimmune-mediated inflammation. NRF2 r
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71 deficiency exacerbates rheumatoid arthritis (RA) (9) and systemic lupus erythematosus (SLE) ,
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72 (10, 11), while NRF2 activation ameliorates experimental autoimmune encephalomyelitis (3). 9

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73 These studies exploited experimentally induced autoimmunity in mice, which does not y
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74 completely mimic the pathogenesis of autoimmune diseases in human. Currently, decreased e
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75 numbers and/or functional impairments of regulatory T cells (Tregs), which enable the
76 aberrant activation of autoreactive T cells (12-15), have been shown to contribute to
77 autoimmune conditions in human patients, such as those with RA, SLE, primary Sjögren’s
78 syndrome, and multiple sclerosis (MS) (16–18). In autoimmune-mediated inflammatory
79 conditions, Tregs are suppressed due to the high levels of pro-inflammatory cytokines that are
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80 produced by other immune cells and/or tissue cells. To correct autoimmunity by reactivating
81 Tregs and subsequently restoring self-tolerance, the activities of the cells that produce
82 pro-inflammatory cytokines need to be controlled (17–19). Thus, in addition to the direct
83 modulation of Tregs to increase their beneficial activities, appropriate control of inflammatory
84 cells other than Tregs is important for the improvement of autoimmune diseases.
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85 A recent study demonstrated that T cell-specific activation of NRF2 increases the w
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86 number of Tregs (20), which suggests that NRF2 inhibits the inflammatory response by lo
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87 potentiating Treg-mediated immune suppression. However, it remains unknown whether d

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88 NRF2 has any inhibitory effects on autoimmune-mediated inflammation in a o
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89 Treg-independent manner. t
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90 To determine whether NRF2 activation exerts Treg-independent suppression of //
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91 autoimmune-mediated inflammation, we used Scurfy (Sf) mice, which possess a missense b
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92 mutation in the Foxp3 gene on the X chromosome (21). Because the development and m
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93 maintenance of Tregs largely depends on the transcription factor FOXP3 (22, 23), Sf mice are rg
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94 almost completely deficient in functional Tregs and thereby develop severe multi-organ n

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95 inflammation with hyperactivation of autoreactive effector T cells, which results in lethality pr
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96 by 4 weeks of age (24). Thus, we used Sf mice to investigate the Treg-independent ,

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97 suppressive effects of NRF2 on the activation status of inflammatory cells, especially effector 1
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98 T cells. y

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99 We found that systemic activation of NRF2 induced by Keap1 knockdown mitigated e
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100 tissue inflammation and improved the survival of Sf mice. NRF2 also suppressed the
101 activation of effector T cells and reduced their cytokine production. Almost comparable but
102 modest outcomes were observed following the pharmacological activation of NRF2 in Sf
103 mice by administrating an NRF2-inducer, CDDO-Im (oleanolic triterpenoid
104 1-[2-cyano-3,12-dioxooleane-1, 9(11)-dien-28-oyl] imidazole). In contrast, NRF2 activation
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105 by Keap1 disruption in a cell lineage-specific manner, including T cells, myeloid cells or
106 dendritic cells, induced only partial or no improvement in the inflammatory status of Sf mice.
107 These results indicate that systemic activation of NRF2 suppresses the autoimmune response
108 in a Treg-independent manner and suggest that coordination of multiple cell lineages is
109 essential for the NRF2-mediated anti-inflammatory effects in autoimmune diseases with Treg
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110 dysfunction. w
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112 Results
113 Systemic NRF2 activation by Keap1 knockdown alleviates multiple organ inflammation
114 and improves the survival rate of Sf mice. To investigate the effects of NRF2 activation on
115 the inflammatory milieu resulting from autoimmunity, we genetically activated NRF2 by
116 reducing Keap1 expression in Sf (Foxp3sf/Y) mice. Foxp3sf/X female mice were crossed with
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117 male mice possessing knockout (Keap1–) (25) and knockdown floxed (Keap1FA) alleles of w
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118 Keap1 (26, 27) to generate Sf::Keap1FA/– (Foxp3sf/Y::Keap1FA/–) mice. We confirmed the a
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119 decreased expression of Keap1 in representative tissues of mice in a Keap1FA/– background, d

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120 which was accompanied by increased expression of Nqo1, one of the typical NRF2 target m

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121 genes, and elevated accumulation of NRF2 protein (Fig. 1A, B). These results indicated tt
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122 successful activation of NRF2 in Sf::Keap1FA/– mice. /m
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123 We first compared the severity of systemic inflammation in Sf::Keap1FA/– mice and b.
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124 Sf mice at the age of 15-19 days, which is generally before Sf mice succumb to the disease.
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125 The anemia and thrombocytopenia typically found in Sf mice were almost completely g
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126 improved in Sf::Keap1FA/– mice (Table 1), and the inflammatory skin lesions found on the n

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127 pinnae and tail of Sf mice were also improved in Sf::Keap1FA/– mice (Fig. 2A). Histological r
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128 analysis further showed that the infiltration of immune cells was decreased in the skin, liver ,
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129 and lungs of Sf::Keap1FA/– mice (Fig. 2B). Accordingly, Sf::Keap1FA/– mice exhibited an 19

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130 improved survival rate compared with that of Sf mice (Fig. 2C). However, approximately half y
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131 of the Sf::Keap1FA/– mice died by 25 days of age, which is very similar to that of Sf mice, e
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132 despite the apparent inhibition of the inflammatory response. This early-phase lethality in
133 Sf::Keap1FA/– mice was likely caused by feeding difficulties due to hyperkeratotic lesions
134 within the upper digestive tract, which is inherent to Keap1FA/– mice (27). And recent report
135 has suggested that severe KEAP1 insufficiency causes other complex abnormalities (28),
136 which might have lead to the early lethality of Sf::Keap1FA/– mice. Consistently, Sf::Keap1FA/–
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137 mice were smaller than WT and Sf mice at the same age (Fig. 2D, E). It should be noted that
138 all Sf::Keap1FA/– mice that avoided the early onset of lethality survived thereafter.
139 To further confirm the anti-inflammatory effect of NRF2 in Sf mice, we used
140 additional Keap1 knockdown mice, Keap1FA/+ and Keap1FA/FA, which have higher Keap1
141 expression than Keap1FA/– mice (Fig. 3A and 3B) and do not suffer from feeding difficulties or
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142 other abnormalities. All Sf::Keap1FA/+ and Sf::Keap1FA/FA mice exhibited increased survival w
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143 rates compared with that of the Sf mice (Fig. 3C). Importantly, Sf, Sf::Keap1FA/+ and lo
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144 Sf::Keap1FA/FA mice showed a decreasing order of Keap1 expression and an increasing order ed

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145 of NRF2 protein and Nqo1 mRNA (Fig. 3A and 3B). In a good agreement with the graded o
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146 NRF2 activity, Sf, Sf::Keap1FA/+ and Sf::Keap1FA/FA mice tended to show an increasing order ht
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147 of survival, implying a correlation between NRF2 activation and improved survival, although //
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148 the difference between Sf::Keap1FA/+ and Sf::Keap1FA/FA mice did not reach statistical b
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149 significance (Fig. 3C). Collectively, these results suggest that systemic activation of NRF2 by m
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150 Keap1 knockdown effectively alleviates the chronic inflammation caused by autoimmunity in rg
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151 Sf mice. n

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152 pr
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153 Systemic NRF2 activation by Keap1 knockdown suppresses the activation of effector T ,

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154 cells and their cytokine production. Abnormal activation and proliferation of CD4+ effector 1
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155 T cells have been shown to be a major pathogenic factor in Sf mice (29). We next examined y

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156 the effect of NRF2 activation on the status of effector T cells and their cytokine expression e
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157 levels. As an initial analysis, the spleen/body weight and lymph node (LN)/body weight ratios
158 were calculated to evaluate the proliferation of leukocytes (Fig. 4A, B). The spleen/body
159 weight and LN/body weight ratios were markedly elevated in Sf mice compared with those of
160 the WT mice. Although the spleen/body weight ratio was not different between the groups, the
161 LN/body weight ratio was significantly lower in Sf::Keap1FA/– mice compared with those in
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162 the Sf mice, implying that NRF2 activation is effective at suppressing lymphocyte
163 proliferation in LNs.
164 We then examined the activation status of CD4+CD8– and CD4–CD8+ T cells
165 (hereafter referred to as CD4+ and CD8+ T cells, respectively) in LNs. The frequencies of
166 CD4+ and CD8+ T cells were not different between Sf and Sf::Keap1FA/– mice (Fig. 4C, left
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167 panel). Among CD4+ T cells, activated populations of CD25+, CD44+ and CD69+ cells were w
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168 markedly increased in Sf mice compared with those in the WT mice, which is consistent with lo
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169 previous findings (29, 30). Moreover, this aberrant increase was mitigated in Sf::Keap1FA/– ed

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170 mice (Fig. 4C, right panel and 4D), suggesting that NRF2 activation inhibits the activation of o
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171 CD4+ T cells. ht
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172 This observation was verified by assessing the number of cytokine-positive CD4+ //
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173 effector T cells, which differentiate from naïve CD4+ T cells when they are activated by T cell b
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174 receptor stimulation. Consistent with a previous study (31), Sf mice exhibited a dramatic m
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175 increase in Th1, Th2 and Th17 type effector CD4+ T cells that are marked by the production rg
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176 of IFN-γ, IL-4 and IL-17A, respectively (Fig. 5A, B). This increase in cytokine-producing T n

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177 cells was mitigated in Sf::Keap1FA/– mice. In particular, Sf::Keap1FA/– mice exhibited a pr
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178 dramatic decrease in the frequency of IFN-γ+ CD4+ T cells by more than 80% compared with ,

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179 that in Sf mice, and IFN-γ+ CD8+ T cells were also similarly decreased in Sf::Keap1FA/– mice. 1
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180 These results suggest that systemic NRF2 activation suppresses the activation of effector T y

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181 cells and their cytokine production in autoimmune diseases induced by Treg deficiency. e
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182 Previous studies showed that there are Foxp3-independent T cell populations, such as
183 type 1 regulatory T (Tr1) cells, which suppress autoimmunity through secretion of high levels
184 of IL-10 and TGF-β (32, 33). Because IL-10-producing T cells do not rely on Foxp3
185 expression for their differentiation and function (34, 35), these cells may contribute to the
186 NRF2-mediated suppression of autoimmune responses in Sf mice that are deficient in Foxp3.
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187 To examine this possibility, we measured the frequency of IL-10+ CD4+ T cells. However, it
188 was not increased but rather decreased in Sf::Keap1FA/– mice compared with that in Sf mice
189 (Fig. 5A, B). Thus, the anti-inflammatory effect of NRF2 activation in Sf mice is not due to
190 the expansion of IL-10-producing suppressive T cells but to the inhibition of effector T cells,
191 particularly those producing IFN-γ.
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192 w
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193 IFN-γ production is dramatically suppressed by systemic NRF2 activation. The lo
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194 inflammatory symptoms in Sf mice are highly dependent on the types of cytokines secreted in d

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195 the serum; for instance, IFN-γ is abundantly secreted in Sf mice, and its suppression results in o
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196 prolonged survival and delayed inflammation in the pinnae, skin, lungs and liver (36). To t
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197 investigate the effects of NRF2 activation on systemic cytokine production, we assessed //
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198 serum cytokine levels. In Sf mice, the serum concentrations of pro-inflammatory cytokines b
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199 such as IFN-γ, TNF-α and IL-6 were elevated, while the levels of IFN-γ and TNF-α were m
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200 decreased to near basal levels in Sf::Keap1FA/– mice (Fig. 6A). However, there were no rg
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201 significant changes in the serum concentrations of IL-4 and IL-17A between Sf and n

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202 Sf::Keap1FA/– mice. We also attempted to measure the concentration of IL-10, but it was not pr
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203 detectable (data not shown). These results suggest that the reduction of the serum levels of ,

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204 IFN-γ and TNF-α is closely related to the suppression of autoimmune inflammation by NRF2. 1
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205 Because most of the serum IFN-γ is regarded to be derived from T cells and natural y

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206 killer cells (37), we quantified the mRNA levels of Ifng in lymphoid tissues, i.e., the LNs and e
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207 spleen, to determine whether the reduced production of IFN-γ in Sf::Keap1FA/– mice was
208 caused by a decrease in its mRNA. The Ifng mRNA level was significantly higher in Sf mice
209 than WT mice, whereas Sf::Keap1FA/– mice showed almost comparable level of Ifng mRNA to
210 that in WT mice, suggesting that NRF2 activation decreases Ifng mRNA in the LNs and
211 spleen (Fig. 6B).
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212
213 T cell-specific loss of Keap1 attenuates IFN-γ production in T cells and partially
214 improves inflammation. To determine whether NRF2 activation exerts anti-inflammatory
215 effects in a T cell-autonomous manner, we conducted T cell-specific Keap1 disruption in a Sf
216 background (Sf::Keap1FB/FB::Lck-Cre, Sf::Keap1-TKO) and compared these animals to
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217 Sf::Control mice (Sf::Keap1FB/FB) by utilizing another Keap1 floxed allele, Keap1FB, that w
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218 expresses a comparable amount of the Keap1 gene to the wild-type allele (38). We confirmed lo
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219 that the Keap1 gene was deleted in approximately 70% of T cells, judging from the reduction d

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220 of Keap1 expression, while the NRF2 target gene Nqo1 was upregulated in the T cells of o
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221 Keap1-TKO mice (Fig. 7A). From macroscopic observations, the skin inflammation in the t
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222 pinnae and tail of Keap1-TKO mice appeared to be ameliorated (Fig. 7B). Indeed, histological //
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223 analysis of pinna skin sections showed that the thickening and cellular infiltration observed in b
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224 Sf::Control mice were reduced in Keap1-TKO mice (Fig. 7C). However, the heavy infiltration m
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225 of inflammatory cells in the liver and lungs as well as anemia and thrombocytopenia were not rg
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226 improved in Sf::Keap1-TKO mice (Fig. 7C, Table S1). n

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227 Analysis of LN cells showed that the frequencies of CD4+ and CD8+ T cells were pr
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228 decreased in the Sf::Keap1-TKO mice compared with those in the Sf::Control mice (Fig. 8A, ,

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229 left panel). The frequencies of CD4+ T cells expressing activation markers were not different 1
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230 between Sf::Control and Sf::Keap1-TKO mice, although the frequency of CD25+CD4+ cells y

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231 were marginally decreased in the latter group (Fig. 8A, right panel and 8B), suggesting that e
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232 the activation of CD4+ T cells is not suppressed by T cell-specific Keap1 deficiency.
233 Despite the similar phenotypic activation of CD4+ T cells between Sf::Control and
234 Sf::Keap1-TKO mice, the frequencies of CD4+ and CD8+ T cells positive for IFN-γ were
235 significantly decreased in Sf::Keap1-TKO mice, while those of CD4+ T cells positive for IL-4
236 or IL-17A were unaffected (Fig. 9A, B). These results confirm that the reduction of IFNγ by
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