The Functional Redundancy of Neddylation E2s and E3s in Modulating the Fitness of Regulatory T Cells

Abstract

Neddylation is necessary for activation of Cullin-RING ligases (CRLs), which degrade various immune regulatory proteins. Our recent study showed that while depletion of neddylation E2–E3 pair Ube2f-Sag in regulatory T (T_reg) cells had no obvious phenotype, the same depletion of either Ube2m or Rbx1 caused inflammation disorders with different severity. Whether these E2s or E3s compensate each other in functional regulations of T_reg cells is, however, previously unknown. In this report, we generated Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl or Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl double-null mice by simultaneous deletion of both neddylation E2s or E3s in T_reg cells, respectively. Remarkably, Ube2m&Ube2f double-null mice developed much severe autoimmune phenotypes than did Ube2m-null mice, indicating that Ube2m markedly compensates Ube2f in T_reg cells. The minor worsened autoimmune phenotypes seen at the very early stage in Rbx1&Sag double-null than Rbx1-null mice is likely due to already severe phenotypes of the later, indicating a minor compensation of Rbx1 for Sag. The RNA profiling-based analyses revealed that up- and down-regulations of few signaling pathways in T_reg cells are associated with the severity of autoimmune phenotypes. Finally, severer inflammation phenotypes seen in mice with double E3-null than with double E2-null T_reg cells indicate a neddylation-independent mechanism of 2 E3s, also known to serve as the RING component of CRLs in regulation of T_reg cell fitness.

Introduction

Neddylation, a ubiquitination-like process, is catalyzed by an enzyme cascade, consisting of NEDD8 E1 activation enzyme, NEDD8 E2 conjugating enzyme, and NEDD8 E3 ligases. In mammalian cells, the heterodimer of NEDD8 activating enzyme E1 subunit 1 (NAE1/APPBP1) and ubiquitin like modifier activating enzyme 3 (UBA3/NAEβ) is the only one E1 found; ubiquitin conjugating enzyme E2 M (UBE2M, also known as UBC12) and ubiquitin conjugating enzyme E2 F (UBE2F) are 2 known E2s. Among over a dozen of E3s, Ring-box 1 (RBX1) and Ring-box 2 (RBX2, also known as SAG/RNF7/ROC2) are most well studied for cullin neddylation [1,2].

Cullin-RING ligases (CRLs) are the largest E3 ubiquitin ligase family, consisting of 4 subunits: (a) scaffold cullins, (b) adaptor proteins, (c) RING component (RBX1/2), and (d) substrate-recognizing receptor. There are 8 members of cullin family, including cullin-1 to cullin-3, cullin-4A and cullin-4B, cullin-5, cullin-7, and cullin-9, with cullin-1 to cullin-5 being most well studied, which are physiological substrates of neddylation. Cullin neddylation triggers the conformation change, leading to activation of CRLs [3]. The UBE2M-RBX1 E2–E3 pair catalyzes neddylation of cullin-1 to cullin-4, while the UBE2F-RBX2/SAG axis catalyzes cullin-5 neddylation [4]. In addition, RBX1 and RBX2/SAG also act as the RING component of cullin-1 to cullin-4 and cullin-5, respectively, for targeted ubiquitylation and subsequent proteasome degradation of numerous substrates [5–7]. Thus, RBX1 and RBX2/SAG are dual E3s for both neddylation and ubiquitylation mediated by CRLs.

Our previous studies revealed that during mouse embryonic development, Rbx1 and Sag/Rbx2 are functional nonredundant, since in mice total knockout of Rbx1 or Sag leads to embryonic lethality at different stage with different mechanism [8,9]. Although whether Ube2m or Ube2f is also functionally redundant during development is currently unknown, the fact that neddylation of cullin-1 to cullin-4 versus cullin-5 by the Ube2m-Rbx1 pair versus the Ube2f-Sag pair [4] strongly suggests their functional independency. On the other hand, we recently showed a cross-talk between UBE2M and UBE2F, 2 neddylation E2s, in which UBE2M serves as a ubiquitylation E2 to promote ubiquitylation of UBE2F for proteasome degradation [10]. Whether there is a cross-talk between RBX1 and SAG remains unknown.

Regulatory T (T_reg) cells are a suppressive subpopulation of CD4⁺ T lymphocytes, and transcription factor Foxp3 is the master marker of T_reg cells [11–13]. T_reg cells are essential for immune homeostasis, which is illustrated by the early-onset fatal inflammation caused by depletion of T_reg cells [14]. We recently investigated the role of neddylation-CRL system in T_reg cells using Foxp3^Cre-LoxP system by generating 4 conditional knockout mouse models with deletion in T_reg cells of 2 neddylation E2s, Ube2f or Ube2m, or 2 dual E3s, Sag or Rbx1, individually [15]. Interestingly, mice with the deletion of Ube2f or Sag in T_reg cells had no visible phenotype, indicating that the Ube2f-Sag axis plays little, if any, role in regulation of T_reg cells under physiological condition. Meanwhile, mice with the deletion of Ube2m or Rbx1 in T_reg cells suffered from severe autoimmune inflammatory phenotypes, indicating the functional requirement of the Ube2m-Rbx1 axis in T_reg cells [15]. The fact that much severer phenotypes are observed in Rbx1-null mice than in Ube2m-null mice indicates a neddylation-independent role of Rbx1 in T_reg cells. However, it is still unknown whether 2 neddylation E2s or E3s are redundant in functional regulation of T_reg cells.

In this study, we addressed this functionally redundant question by generating T_reg double knockout mouse models of 2 E2s, Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl, or 2 E3s, Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl, for simultaneous depletion of Ube2m and Ube2f or Rbx1 and Sag in T_reg cells, respectively. Both T_reg cell double knockout mice developed severe autoimmune disorders with an early-onset fatality. Given that Ube2f deletion in T_reg cells had no phenotype, much severe autoimmune phenotypes in Ube2m&Ube2f double deletion than in Ube2m single deletion in T_reg cells indicate that Ube2m compensates the function of Ube2f in T_reg cells. On the other hand, minor increased severity in T_reg Rbx1&Sag double deletion than Rbx1 single deletion suggests a major role of Rbx1 in regulation of T_reg cell function. Furthermore, a greater severity in autoimmune phenotypes of mice with Rbx1&Sag deficiency than Ube2m&Ube2f deficiency in T_reg cells suggests a neddylation-independent function of Rbx1/Sag. The comparison of RNA profiling in T_reg cells with paired genotypes revealed a positive correlation of up- or down-regulation of few key signaling pathways with the severity of autoimmune phenotypes of T_reg cell knockout mice.

Results

Fatal inflammation in Foxp3^CreUbe2m^fl/fl;Ube2f^fl/fl mice

We have previously reported the phenotypical changes caused by individual deletion of Ube2m or Ube2f in T_reg cells, and found that while the Foxp3^Cre;Ube2f^fl/fl mice had no visible phenotypic changes, the Foxp3^Cre;Ube2m^fl/fl mice developed severe inflammation disorders, where ~50% of mice died at about 4 months [15]. To study possible functional redundancy of 2 neddylation E2s in T_reg cells, we intercrossed the single-null mice [15] and Foxp3^YFP-Cre (Foxp3^Cre) mice [16] to generate conditional knockout mice with deletion of both Ube2m and Ube2f in T_reg cells simultaneously (designated as “Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl”). Strikingly, the Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice suffered from an early-onset alterations of appearance, including reduced body size, collapsed ears, festered skin (Fig. 1, A and B), and a significantly shortened life span with 100% of death rate at p55 (Fig. 1C). In Foxp3^Cre;Ube2m^fl/fl;Ube2f ^fl/fl mice at ~p20, the organ hematoxylin and eosin (H&E) staining revealed lymphocyte infiltration in liver, lung, kidney, stomach, colon, and skin (Fig. 1D). The autopsy revealed swollen peripheral immune organs, including lymph nodes and spleens, although the cellularity did not reach the statistically significant level (Fig. 1E and Fig. S1A). A more detailed characterization revealed a robust activation of immune cells from Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (~p20), as evidenced by a decreased ratio of CD4⁺/CD8⁺ T cells (Fig. S1B) and increased proportion of effector/memory T cells (CD44^hiCD62L^lo, T_eff/mem cells) among conventional T cells (CD4⁺Foxp3⁻, T_con cells) (Fig. 1F and Fig. S1C). Such fatal inflammatory disorders recaptured the phenotypes observed in mice with depleted T_reg cells [14] in severity, indicating a pivotal role of neddylation E2s in the T_reg cells.

Fig. 1.

Deletion of Ube2m and Ube2f in T_reg cells leads to an early-onset fatal inflammatory disorder. (A) Representative images of Foxp3^Cre and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (p20, scale bar =1 cm). (B) Gross body weight of Foxp3^Cre and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (n = 4). (C) Survival curves of Foxp3^Cre and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (n = 13). (D) H&E staining of multiple organs from Foxp3^Cre and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (p19 to p20, scale bar = 200 μm). (E) Representative images of the peripheral lymph nodes (top) and spleen (bottom) from Foxp3^Cre and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (p22, scale bar = 1 cm). (F) Expression of CD44 and CD62L in T_con cells from peripheral lymph nodes of Foxp3^Cre and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (p21).

Impaired suppressive functions and altered transcriptome of Ube2m&Ube2f-deficient T_reg cells

Given that both the T_reg/CD4⁺ ratios (Fig. 2, A and B) and absolute numbers of T_reg cells (Fig. 2C) are largely similar in peripheral lymph nodes derived from either Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl or Foxp3^Cre control mice around p20, we hypothesized that the severe inflammation disorders observed in Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice at the same age is most likely attributable to the loss of the suppressive function of T_reg cells. To test this hypothesis, we performed an in vivo suppression assay in immunodeficient Rag1^−/− mice. Transfer of naive T (T_nai) cells (CD4⁺Foxp3⁻CD44^loCD62L^hi) into Rag1^−/− recipients led to a wasting disease with colitis within 4 weeks. We found that such colitis was prevented by simultaneous transfer of T_reg cells from wild-type mice, but not from Ube2m&Ube2f-deficient mice (Fig. 2D), demonstrating that double deletion of neddylation E2s impairs the suppressive function of T_reg cells.

Fig. 2.

Impaired suppressive function of Ube2m&Ube2f-deficient T_reg cells. (A) Expression of Foxp3 in CD4⁺ T cells in peripheral lymph nodes from Foxp3^Cre and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice at p20. (B) T_reg/CD4⁺ ratios in peripheral lymph nodes from Foxp3^Cre and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (p19 to p21, n = 4). (C) T_reg cell numbers in peripheral lymph nodes from Foxp3^Cre and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (p19 to p21, n = 4). (D) Representative images of distal colon after H&E staining (scale bar = 200 μm). (E) Unsupervised cluster analysis of the transcriptional alterations in Ube2m&Ube2f-deficient T_reg cells compared to the Foxp3^Cre control T_reg cells with Fc > 1.5 and P < 0.05. (F) Differentially expressed genes related to T_reg cell function in CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt and Foxp3^Cre/wt;Ube2m^fl/fl;Ube2f^fl/fl mice, determined by transcriptional profiling with Fc > 1.5 and P < 0.05. (G) Gene set enrichment analysis (GSEA) of cytokine–cytokine receptor interaction genes in CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt and Foxp3^Cre/wt;Ube2m^fl/fl;Ube2f^fl/fl mice, determined by transcriptional profiling with Fc > 1.5 and P < 0.05. (H) GSEA of cellular senescence genes in CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt and Foxp3^Cre/wt;Ube2m^fl/fl;Ube2f^fl/fl mice, determined by transcriptional profiling with Fc > 1.5 and P < 0.05.

To define the underlying mechanism, we performed RNA profiling and analyzed the transcriptome alterations caused by Ube2m&Ube2f deletion in T_reg cells. Considering that the inflammation in Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice would affect the transcript signature of T_reg cells, we generated the inflammation-free female mice, in which one X chromosome expresses wild-type Foxp3 allele (Foxp3^wt) and the other expresses Foxp3^Cre mutant (designated as Foxp3^Cre/wt). The random inactivation of one X chromosome in each somatic cell caused about half of T_reg cells to express Foxp3^Cre mutant, so Ube2m and Ube2f genes were deleted in these T_reg cells; meanwhile, the other T_reg cells expressed normal Foxp3^wt allele and were kept absolutely normal. Due to the protection of the normal T_reg cells, the Foxp3^Cre/wt;Ube2m^fl/fl;Ube2f^fl/fl female mice are inflammation-free and healthy, so the CD4⁺YFP⁺ T_reg cells in Foxp3^Cre/wt;Ube2m^fl/fl;Ube2f^fl/fl mice were deficient of Ube2m&Ube2f and avoided the suffering from inflammation at the same time.

We then sorted the CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt; Ube2m^fl/fl;Ube2f^fl/fl and Foxp3^Cre/wt mice, respectively, followed by transcriptome analysis. The Ube2m&Ube2f-deficient T_reg cells (Fig. S2A) showed significant alterations in transcriptome, with 714 genes up-regulated and 1,289 genes down-regulated with fold change (Fc) > 1.5 and P value < 0.05 (Fig 2E and Fig. S2, B and C). Among them, many T_reg cell function-related genes were down-regulated, including Il10 [16] and Cst7 [17] (suppressive cytokines); Entpd1, Nt5e [18], and Il2r [19] (regulators of immune cell metabolism); Icos [20], Ctla4 [21], Pdcd1 [22], Lag3 [23], Nrp1 [24], and Tigit [25] (inhibitor via dendritic cells); and Gzmb [26] and Lgals3 [27] (apoptosis inducer) (Fig. 2F). Altered expression in migration-associated surface molecules was also observed in Ube2m&Ube2f-deficient T_reg cells, such as down-regulation of Cxcr3, Cxcr5, Cxcr6, and Cd44 [28] and up-regulation of Sell [29] (Fig. 2F). On the other hand, among the top 20 genes up-regulated in Ube2m&Ube2f-deficienct T_reg cells (Fig. S2D), none of them are previously known to negatively regulate T_reg cells upon induction, which is certainly an interesting subject for future investigation.

Gene set enrichment analysis (GSEA) of the altered genes revealed dramatic changes in multiple pathways upon Ube2m&Ube2f deficiency (Fig. S2E), specially down-regulation of T_reg-related pathways, such as cytokine–cytokine receptor interaction [30] (Fig. 2G) and T helper 17 (T_H17) cell differentiation [31] (Fig. S2F). Some up-regulated pathways were also seen in Ube2m&Ubef-deficienct T_reg cells, including DNA replication and repair, cell cycle, and metabolisms of amino acids tyrosine, phenylalanine, and tryptophan, as well as cellular senescence (Fig. S2E and Fig. 2H). How these pathways, upon double knockout of neddylation E2s, are up-regulated and how they functionally regulate T_reg cells are the open and interesting questions for future investigation, particularly for possible involvement of senescence pathway.

Phenotype and transcriptome comparison between Foxp3^Cre;Ube2m^fl/fl and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice

A direct life-span comparison between Foxp3^Cre;Ube2m^fl/fl [15] and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice revealed a much shortened life span in double E2-null mice (Fig. 3A), indicating a significant contribution of Ube2f in the maintenance of T_reg cells to the overall survival of mice. On the other hand, nondetectable phenotypic change in Ube2f T_reg-depleted mice [15] indicates that the function of Ube2f in T_reg cells is fully compensated by Ube2m. Thus, 2 neddylation E2s are functionally redundant in the regulation of T_reg cells. While the main functions of Ube2f in T_reg cells are compensated by Ube2m, Ube2f fails to functionally compensate Ube2m.

Fig. 3.

Enhanced inflammation phenotype and transcription alteration caused by Ube2m&Ube2f deficiency compared to Ube2m deficiency in T_reg cells. (A) Survival curves of Foxp3^Cre, Foxp3^Cre;Ube2m^fl/fl [15], and Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice. (B) Unsupervised cluster analysis of the transcriptional alterations in Ube2m&Ube2f- and Ube2m-deficient T_reg cells compared to the Foxp3^Cre control T_reg cells. (C) Genes related with T_reg cell function or regulation, and also down-regulated in Ube2m&Ube2f-deficient, but not Ube2m-deficient, T_reg cells. (D) GSEA of cytokine–cytokine receptor interaction genes altered in Ube2m&Ube2f-deficient, but not Ube2m-deficient, T_reg cells.

We next compared the transcriptome alterations between Ube2m&Ube2f-deficient and Ube2m-deficient T_reg cells [15], along with the Foxp3^Cre control. We normalized 2 sets of data (each from 3 individual mice, run at 2 different time periods), followed by unsupervised cluster analysis. The results showed that the transcriptional profile patterns were largely comparable in Foxp3^Cre control T_reg cells from 2 batches of experiments (Fig. 3B), suggesting a reproducibility of the experiments. The profiling comparison revealed 5 clusters (groups) of genes with altered expressions among 3 groups with an overall greater change seen in Ube2m&Ube2f double-deficient T_reg cells (Fig. 3B).

Among the genes selectively down-regulated in double E2-deficient T_reg cells (group 1), many genes are known to be related with functional regulation of T_reg cells, including Ccr4 [32], Egr2 [33], Il10rb [34], Tccam1 [35], Runx3 [36], IL10 [16], Cd3g [37], Tnfrsf4 [38], Pglyrp1 [39], and Malt1 [40] (Fig. 3C). Among top 20 genes up-regulated in double E2-deficient T_reg cells (group 4) (Fig. S3A), Smad7 is the only one known to be involved in regulation of T_reg cells, whose up-regulation by the EZH2-FOXP3-RUNX1 axis inhibited T_reg cell differentiation in rheumatoid arthritis [41].

GSEA analysis of the genes specially altered in double-null T_reg cells revealed the changes in multiple pathways (Fig. S3B), and some down-regulated pathways are known to be involved in T_reg cell regulation, such as cytokine–cytokine receptor interaction [30] (Fig. 3D) and T_H17 cell differentiation [31] (Fig. S3C), whereas lysosome pathway, which is negatively correlated with function of T_reg cells [42], along with cell cycle and phosphatidylinositol 3-kinase/Akt pathways, is up-regulated in Ube2m&Ube2f double-deficient T_reg cells (Fig. S3, B and D). Thus, these greater alterations in gene expression are associated with and likely contributed to severer inflammatory disorders in Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice than in Foxp3^Cre;Ube2m^fl/fl mice.

Early-onset fatal inflammatory disorders in Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice

We next determined potential functional redundancy in T_reg cells between 2 E3s, Rbx1 and Sag/Rbx2, dual for both neddylation and ubiquitylation by CRLs. We crossed the Rbx1-flox, Sag-flox, and Foxp3^YFP-Cre (Foxp3^Cre) mice [16] to generate the Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice, with simultaneous deletion of both Rbx1 and Sag, the only 2 known catalytic subunits of CRLs, in T_reg cells. The Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice phenocopied the mice ablated of T_reg cells in vivo [14], with smaller body size (Fig. 4, A and B), much shortened life span (Fig. 4C), inflammatory changes in multiple organs (Fig. 4D), swollen peripheral immune organs (Fig. 4E and Fig. S4A), and robust activation of immune cells (Fig. 4F and Fig. S7, B and C). Thus, Rbx1 and Sag are absolutely essential for the maintenance of T_reg cell fitness.

Fig. 4.

Deletion of Rbx1 and Sag in T_reg cells leads to an early-onset fatal inflammatory disorder. (A) Representative images of Foxp3^Cre and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (p19, scale bar =1 cm). (B) Gross body weight of Foxp3^Cre and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (n = 4). (C) Survival curves of Foxp3^Cre and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (n = 19). (D) H&E staining of multiple organs from Foxp3^Cre and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (p20 to p21, scale bar = 200 μm). (E) Representative images of the peripheral lymph nodes (top) and spleen (bottom) from Foxp3^Cre and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (p19, scale bar = 1 cm). (F) Expression of CD44 and CD62L in T_con cells from peripheral lymph nodes of Foxp3^Cre and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (p20).

We then performed the in vivo suppression assay in Rag1^−/− immunodeficient mice to confirm the role of Rbx1&Sag deficiency in the suppressive function of T_reg cells and found that the Rbx1&Sag-deficient T_reg cells failed to prevent the colitis induced by T_nai cells (Fig. 5A), indicating impaired suppressive function. Thus, despite that the ratios and number of Rbx1&Sag-deficient T_reg cells were dropped dramatically around p20 in vivo (Fig. 5, B to D), the severe inflammation disorder in Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice was not the simple consequence caused by the decreased number of T_reg cells.

Fig. 5.

Impaired suppressive function of Rbx1&Sag-deficient T_reg cells. (A) Representative images of distal colon after H&E staining (scale bar = 200 μm). (B) Expression of Foxp3 in CD4⁺ T cells in peripheral lymph nodes from Foxp3^Cre and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice at p20. (C) T_reg/CD4⁺ ratios in peripheral lymph nodes from Foxp3^Cre and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (p19 to p20, n = 4). (D) T_reg cell numbers in peripheral lymph nodes from Foxp3^Cre and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (p19 to p20, n = 4). (E) Unsupervised cluster analysis of the transcriptional alterations in Rbx1&Sag-deficient T_reg cells compared to the Foxp3^Cre control T_reg cells with Fc > 1.5 and P < 0.05. (F) Differentially expressed genes related to T_reg cell function in CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt and Foxp3^Cre/wt;Rbx1^fl/fl;Sag^fl/fl mice, determined by transcriptional profiling with Fc > 1.5 and P < 0.05. (G) Relative abundance of Bach2 mRNA in CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt and Foxp3^Cre/wt;Rbx1^fl/fl;Sag^fl/fl mice, determined by transcriptional profiling. (H) GSEA of cytokine–cytokine receptor interaction genes in CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt and Foxp3^Cre/wt;Rbx1^fl/fl;Sag^fl/fl mice, determined by transcriptional profiling with Fc > 1.5 and P < 0.05. (I) GSEA of olfactory transduction genes in CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt and Foxp3^Cre/wt;Rbx1^fl/fl;Sag^fl/fl mice, determined by transcriptional profiling with Fc > 1.5 and P < 0.05.

To explore possible underlying mechanism(s) that mediated the function of Rbx1&Sag in T_reg cells, we sorted the CD4⁺YFP⁺ T_reg cells from inflammation-free female Foxp3^Cre/wt;Rbx1^fl/fl;Sag^fl/fl and Foxp3^Cre/wt control mice and performed transcription profiling. Deletion of Rbx1&Sag (Fig. S5A) led to a comprehensive change of the levels of multiple genes in T_reg cells, with 641 genes up-regulated and 2,296 genes down-regulated with Fc > 2 and P < 0.05 (Fig. 5E and Fig. S5, B and C). Among down-regulated genes, many are reported to be related to the function of T_reg cells, including Il10 [16] and Cst7 [17] (suppressive cytokines); Entpd1 and Nt5e [18] (regulators of immune cell metabolism); Icos [20], Pdcd1 [22], Lag3 [23], Nrp1 [24], and Tigit [25] (inhibitor via dendritic cells); and Lgals3 [27] (apoptosis inducer), and impairment of migration-associated surface molecules, including down-regulation of Cxcr3, Cxcr5, and Cd44 [28] (Fig. 5F). Although none of the top 20 up-regulated genes, including Plk2, Sik1, Nr4a2, and Dusp10 (Fig. S5D), are known to negatively regulate T_reg cell functions upon induction, one of the up-regulated genes, Bach2 (up-regulated with Fc = 1.866 and P = 0.013) (Fig. 5G), is known as a negative regulator of T_reg cell function [43].

The GSEA pathway analysis of the altered genes (Fc > 1.5 and P < 0.05) also revealed the changes of many pathways (Fig. S5E), including down-regulation of T_reg-related pathways, such as cytokine–cytokine receptor interaction [30] (Fig. 5H), T_H17 cell differentiation [31] (Fig. S5F), and up-regulation of pathways involving olfactory transduction (Fig. 5I) and metabolisms of retinol and arachidonic acid (Fig. S5E).

Earlier occurrence of inflammatory disorder in Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice than in Foxp3^Cre;Rbx1^fl/fl mice

We next addressed the question of functional redundancy between 2 E3s in T_reg cells. Since mice with Rbx1-deficient T_reg cells already had an early-onset fatal phenotype, whereas mice with Sag-deficient T_reg cells had no phenotype [15], we first compared the survival of mice with 2 genotypes, Rbx1&Sag double deletion versus Rbx1 single deletion [15], in T_reg cells and found no difference, both having early-onset fatal death (Fig. S6A). Moreover, no difference was found in the activation of immune response when mice were near moribund at age of ~20 days (Fig. S6B). We then focused on potential difference in inflammatory responses occurring at the very early stage of 8 days after the birth between 2 types of mice. Compared to the Foxp3^Cre control, the ratio of T_eff/mem cells (CD4⁺Foxp3⁻CD44^hiCD62L^lo) among T_con cells (CD4⁺Foxp3⁻) is elevated dramatically in Rbx1-null or Rbx1&Sag double-null mice (Fig. 6A), indicating a robust immune activation. A substantially higher level was seen in double-null mice (Fig.A and B), demonstrating a more severe immune overactivation in vivo. Thus, like the Ube2m-Ube2f E2 pair, the Rbx1-Sag E3 pair also showed a functional redundancy in T_reg cells, although it is minor due to the dominant effect of Rbx1 to mask the Sag effect. Collectively, the Sag function is being fully compensated by Rbx1, whereas the Rbx1 function cannot be compensated by Sag in T_reg cells.

Fig. 6.

Enhanced inflammation phenotype and transcription alteration caused by Rbx1&Sag deficiency compared to Rbx1 deficiency in T_reg cells. (A) Expression of CD44 and CD62L in T_con cells from peripheral lymph nodes of infant Foxp3^Cre, Foxp3^Cre;Rbx1^fl/fl, and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (p8). The upper panel was cited from our previous study [15] for comparison purpose. (B) Proportion of CD44^hiCD62^lo effector/memory cells among T_con cells in peripheral lymph nodes from infant Foxp3^Cre, Foxp3^Cre;Rbx1^fl/fl, and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (p8). (C) Unsupervised cluster analysis of the transcriptional alterations in Rbx1&Sag- and Rbx1-deficient T_reg cells compared to the Foxp3^Cre control T_reg cells. (D) Genes related with T_reg cell function or regulation, and also down-regulated in Rbx1&Sag-deficient, but not Rbx1-deficient, T_reg cells. (E) Relative abundance of Bach2 mRNA in CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt, Foxp3^Cre/wt;Rbx1^fl/fl, and Foxp3^Cre/wt;Rbx1^fl/fl;Sag^fl/fl mice, determined by transcriptional profiling. (F) GSEA of cytokine–cytokine receptor interaction genes altered in Rbx1&Sag-deficient, but not Rbx1-deficient, T_reg cells. (G) GSEA of olfactory transduction genes altered in Rbx1&Sag-deficient, but not Rbx1-deficient, T_reg cells.

Enhanced transcription alterations caused by Rbx1&Sag double deficiency in comparison to Rbx1 single deficiency in T_reg cells

We further compared the transcriptome alterations between Rbx1&Sag- and Rbx1-deficient T_reg cells, along with Foxp3^Cre control T_reg cells, generated previously [15] and in this study, which showed large reproducibility (Fig. 6C, first 6 columns). Unsupervised cluster analysis of the transcriptome data revealed 4 groups of alterations: (a) genes down-regulated in both Rbx1&Sag double-deficient and Rbx1 single-deficient T_reg cells; (b) genes selectively down-regulated in Rbx1&Sag double-deficient T_reg cells; (c) genes selectively up-regulated in Rbx1&Sag double-deficient T_reg cells; and (d) genes up-regulated in both Rbx1&Sag double-deficient and Rbx1 single-deficient T_reg cells (Fig. 6C). Many group 2 genes are related with T_reg cell regulation, including Cxcr3 [44], Itch [45], Il7rb [46], and Il10rb [34] (Fig. 6D). Among the top 20 up-regulated genes in group 3 (Fig. S7A), none of them are known to negatively regulate T_reg cell function upon induction, and Bach2 in group 3 (not in the list of top 20 genes) appears to be the only gene known as a negative regulator of T_reg cells [43] (Fig. 6E).

GSEA analysis of the genes specially altered in Rbx1&Sag-deficient T_reg cells revealed changes of multiple pathways (Fig. S7B), where cytokine–cytokine receptor interaction was down-regulated (Fig. 6F), which are involved in T_reg cell regulation [30] and up-regulation of multiple pathways, such as the olfactory transduction pathway (Fig. 6G). Taken together, it appears that many genes are associated with and may contribute to severer inflammatory disorder seen in Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice.

Phenotypes and transcriptome comparisons of mice and T_reg cells with double E2 versus double E3 deficiency

It is well established that cullin neddylation is essential for activation of CRLs, and Rbx1 and Sag serve as dual E3 for both neddylation and CRL-medicated ubiquitylation [47,48]. To verify the functional relationship between neddylation and CRLs in T_reg cells, we compare the degree of autoimmune disorders and transcriptional alterations caused by Ube2m&Ube2f versus Rbx1&Sag deficiency in T_reg cells.

Although both Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice and Foxp3^Cre; Rbx1^fl/fl;Sag^fl/fl mice suffered from severe autoimmune disorders with much shortened life span, the degree of severity still differs. In fact, the survival of Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice is significantly shorter than that of Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice, with P = 0.0350 (Fig. 7A). In the peripheral lymph nodes from mice at p19 to p21, the ratios of T_eff/mem cells (CD4⁺Foxp3⁻CD44^hiCD62L^lo) among T_con cells (CD4⁺Foxp3⁻) are higher in Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice than in Foxp3^Cre; Ube2m^fl/fl; Ube2f^fl/fl mice, although the difference is not statistically significant due to variations among mice (Fig. 7B). Thus, double E3-null mice had more dramatic immune overactivation than double E2-null mice.

Fig. 7.

Enhanced inflammation phenotype and transcription alteration caused by Rbx1&Sag deficiency compared to Ube2m&Ube2f deficiency in T_reg cells. (A) Survival curves of Foxp3^Cre, Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl, and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice; P = 0.0350 between the survivals of Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice. (B) Proportion of CD44^hiCD62^lo effector/memory cells among T_con cells in peripheral lymph nodes from Foxp3^Cre, Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl, and Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice (p19 to p23). (C) Unsupervised cluster analysis of the transcriptional alterations in Ube2m&Ube2f- and Rbx1&Sag-deficient T_reg cells compared to the Foxp3^Cre control T_reg cells. (D) Genes related with T_reg cell function or regulation, and also down-regulated in Rbx1&Sag-deficient, but not in Ube2m&Ube2f-deficient, T_reg cells. (E) GSEA of Hippo signaling pathway altered in Rbx1&Sag-deficient, but not Ube2m&Ube2f-deficient, T_reg cells.

The comparison of transcriptome data of CD4⁺YFP⁺ T_reg cells from Foxp3^Cre/wt, Foxp3^Cre/wt;Ube2m^fl/fl;Ube2f^fl/fl, and Foxp3^Cre/wt;Rbx1^fl/fl;Sag^fl/fl mice revealed 6 groups of altered genes (up or down), unique to either double E2-deficient or double E3-deficient or both deficient T_reg cells (Fig. 7C). We mainly focused on genes uniquely altered in double E3-null or double E2-null T_reg cells. Among selectively down-regulated genes in double E3-null T_reg cells, many are related to function or regulation of T_reg cells (Fig. 7D), whereas among up-regulated genes (Fig. S8A), none of them are known to negatively regulate T_reg cells upon induction. Similarly, among selectively altered genes (down and up) in double E2-null T_reg cells (Fig. S9, A and B), few down-regulated, but not up-regulated, genes were known to regulate T_reg cell functions. Nevertheless, the changes unique in double E3-null T_reg cells likely represent CRL-dependent mechanism, whereas the changes unique in double E2-null T_reg cells likely represent CRL-independent, but neddylation-dependent, mechanisms, consistent with our recent studies [49–51].

Similar GSEA pathway analysis of 2 groups of genes unique to double E3-deficient T_reg cells revealed remarkable alterations in many pathways (Fig. S8B), including down-regulation of Hippo signaling pathway (Fig. 7E), which plays an important role in the maintenance of T_reg cell function [52], and of pathways related to lysosome, as well as up-regulation of olfactory transduction and metabolisms of arachidonic acid and retinol (Fig. S8, B and C), which are previously unknown in regulation of the T_reg cell functions.

GSEA pathway analysis of 2 groups of genes unique to double E2-deficient T_reg cells also revealed alterations in a number of pathways (Fig. S9C), including down-regulation of ubiquitin-mediated proteolysis and up-regulation of DNA replication pathways (Fig. S9, D and E). Notably, the number of genes unique to double E2-deficient T_reg cells is relatively smaller than in double E3-deficient T_reg cells, which is consistent with the observation that Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice suffer more dramatic autoimmune disorders than do the Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice.

Collectively, the early-onset fatal disorders seen in both double E2- and double E3-deficient mice indicate functional similarity of neddylation and CRLs in regulation of T_reg cells. More severe inflammatory disorders in double E3-deficient mice suggest a neddylation-independent function of CRLs in T_reg cells.

Discussion

In this study, we investigated the role of the neddylation-CRL axis in functional regulation of T_reg cells using several in vivo conditional knockout mouse models. Double deletion of neddylation E2s Ube2m&Ube2f or E3s Rbx1&Sag in T_reg cells leads to the impairment of suppressive function of T_reg cells and results in severe autoimmune disorders, fully demonstrating the pivotal role of the neddylation-CRL axis in the maintenance of T_reg cell fitness. Thus, the neddylation-CRL axis is essential for T_reg cell functions as the new key regulators of T_reg cells beyond Foxp3 [11–13].

The functional independence of the Ube2m-Rbx1 and Ube2f-Sag axes was previously demonstrated both biochemically [4,53] and biologically [8,9]. Specifically, our recent study revealed that the Ube2m-Rbx1 axis plays a pivotal role in functional regulation of T_reg cells, while the Ube2f-Sag axis is dispensable in T_reg cells at the steady status [15]. This T_reg-based functional study also supports the notion of functional independence between the Ube2m-Rbx1 and Ube2f-Sag axes.

Here, we used double knockout of either neddylation E2s or E3s in T_reg cells and showed that both E2s and E3s are functionally redundant in T_reg cells, as evidenced by the following: (a) The phenotypes of autoimmune disorders in Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl (double E2 knockout in T_reg cells) mice are much more severe than in Foxp3^Cre;Ube2m^fl/fl (single E2 knockout in T_reg cells) mice with much shortened life span (Fig. 3A). (b) The same autoimmune phenotypes are severer in Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl (double E3s knockout in T_reg cells) mice than in Foxp3^Cre;Rbx1^fl/fl (single E3 knockout in T_reg cells) mice at the very early age only (Fig. 6A and B). We further found that the autoimmune phenotypes were much severer in Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl mice than in Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl mice (Fig. 7A).

We conclude from these results that (a) the Ube2f-Sag axis plays a role in T_reg cells, which is, however, compensated by the Ube2m-Rbx1 axis. More specifically, the autoimmune phenotypes by Ube2f depletion were largely compensated by Ube2m, whereas the same autoimmune phenotypes by Sag depletion were minimally compensated by Rbx1, due to the dominant role of Rbx1 in T_reg cells. (b) The functional redundancy in T_reg cells is in one-way direction from the Ube2m-Rbx1 axis to the Ube2f-Sag axis, indicating much more significant role of CRL1 to CRL4 than of CRL5. (c) Rbx1 and Sag play neddylation-dependent and neddylation-independent roles in regulating T_reg cell fitness.

To pursue the possible underlying mechanism, we performed the comprehensive transcriptome profiling analyses of T_reg cells derived from the wild-type control or double knockout mice, and compared between single-null versus double-null T_reg cells for both E2s or E3s, respectively. Compared to the wild-type control T_reg cells, T_reg cell deficiency of Ube2m&Ube2f caused dramatic alterations in mRNA transcriptome. Many genes (such as Il10, Cst7, and Cxcr3 to Cxcr6) and pathways (e.g., cytokine–cytokine receptor interaction and T_H17 cell differentiation), previously known to regulate T_reg cells, were down-regulated (Fig. 2 and Fig. S2), supporting the lost-of-function phenotypes. On the other hand, interestingly, many up-regulated genes (none of the top 20 genes) (Fig. S2D), with the exception of Sell (Fig. 2F) [29], or pathways (Fig. S2E) are previously unknown to negatively regulate T_reg cell function upon activation. Nevertheless, some up-regulated pathways, such as cellular senescence (Fig. 2H), could contribute to the loss-of-function phenotype and deserve detailed future investigation.

Likewise, compared to the wild-type control T_reg cells, T_reg cell deficiency of Rbx1&Sag also caused dramatic alterations in mRNA transcriptome. Many genes (such as Il10, Cst7, Entpd1, and Nt5e) and pathways (again cytokine–cytokine receptor interaction and T_H17 cell differentiation), previously known to regulate T_reg cells, were down-regulated (Fig. 5 and Fig. S5), further supporting the lost-of-function phenotypes. On the other hand, interestingly, many up-regulated genes (again none of the top 20 genes) or pathways are previously unknown to regulate T_reg cell function upon activation. One exception is Bach2, a known negative regulator of T_reg cell functions [43], which is up-regulated in Rbx1&Sag-deficienct T_reg cells (Fig. 5G). However, it is completely unknown how some up-regulated pathways, such as those involving olfactory transduction (Fig. 5I and Fig. S5E), negatively regulate T_reg cell functions.

Transcriptome comparison between T_reg cell deficiency of Ube2m&Ube2f versus Ube2m also showed many alterations. A list of 11 genes (e.g., Ccr4, Egr2, and Il10), uniquely down-regulated in double E2 null (Fig. 3C), are all known to regulate T_reg cell functions. Again, the pathways associated with cytokine–cytokine receptor interaction and T_H17 cell differentiation were also down-regulated uniquely to double E2 null T_reg cells (Fig. 3D and Fig. S3C). Among top 20 up-regulated genes (Fig. S3A), Smad7 is the only one reported to inhibit T_reg cell differentiation in rheumatoid arthritis upon up-regulation by the EZH2-FOXP3-RUNX1 axis [41]. The other up-regulated genes, along with up-regulated pathways (Fig. S3B), unknown to negatively regulate T_reg cell functions, open a new window to study neddylation involvement in T_reg cell functions, for example, how up-regulation of lysosome pathway (Fig. S3B and D) contributes to the loss-of-function phenotype of T_reg cells.

Transcriptome comparison between T_reg cell deficiency of Rbx1&Sag versus Rbx1 also showed many alterations, but to a lesser extent (Fig. 6C and Fig. S7). Down-regulation of Cxcr3, Fas, Egr2, and Itch and of cytokine–cytokine receptor interaction pathway appeared to be unique to double E3-null T_reg cells (Fig. 6, D and F). Among up-regulated genes and pathways (Fig. S7), Bach2 and olfactory transduction (Fig. 6, E and G) appeared to be unique. Given a minor phenotypic difference between Rbx1/Sag double-null and Rbx1 single-null mice, these alterations are unlikely to play a major role in the loss-of-function phenotypes.

Finally, we compared the phenotypes of Foxp3^Cre;Ube2m^fl/fl; Ube2f^fl/fl (double E2-null) mice with Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl (double E3-null) mice and found that double E3-null mice suffer more severe autoimmune disorders with a shorter life span (Fig. 7A). Consistently, the transcriptional alterations caused by Rbx1&Sag deficiency in T_reg cells are more dramatic than those in Ube2m&Ube2f-deficient T_reg cells, with a more remarkable decrease of T_reg cell regulatory genes and down-regulation of Hippo signaling pathway (Fig. 7, D and E), which was known to play an important role in the maintenance of T_reg cell function [52]. Among up-regulated genes and pathways, such as olfactory transduction and metabolisms of retinol and arachidonic acid, which are unique to double E3-null (Fig. S8B and C), it is completely unknown whether and how they negatively regulate T_reg cell function, nor their contribution to severe phenotypes. In addition, we also identified genes and pathways unique to double E2 knockout T_reg cells (Fig. 7C and Fig. S9). These alterations are likely independent of inactivation of Rbx1/Sag-CRLs but involve neddylation modification of non-cullin substrates via other dual E3s for both neddylation and ubiquitylation [49–51].

It is not surprising that many changes in gene expression occur in these T_reg conditional knockout models, given the fact that neddylation E2/E3s are required for cullin neddylation to activate CRLs, which are responsible for ubiquitylation and degradation of ~20% cellular proteins doomed for proteasome degradation [54], thus regulating many biochemical and biological processes. Mechanistically, it is anticipated that inactivation of CRLs, resulting from depletion of neddylation E2/E3, will cause the accumulation of many cellular substrates, including (a) transcription factors and repressors, which would directly affect gene expression, and (b) other signal molecules and cell cycle regulators, which would indirectly affect gene expression. Unfortunately, given that T_reg cells are a rare population in vivo and no in vitro cell lines were established, it is technically challenging to define the detailed underlying mechanisms by these altered genes and pathways in our T_reg cell conditional knockout setting. Nevertheless, our study defined genes and pathways responsible for both neddylation-dependent and neddylation-independent roles for neddylation E2s and dual E3s.

In summary, in this study, we used mouse models of T_reg cell double knockout of neddylation E2s or E3s and made 3 major findings: (a) Neddylation enzymes play an absolute essential role in the maintenance of T_reg cell fitness and their dual disruption causes severer autoimmune phenotypes; (b) there is previously unknown functional redundancy between Ube2m and Ube2f, and to a lesser extent between Rbx1 and Sag; and (c) dual E3s Rbx1 and Sag have neddylation-dependent and neddylation-independent role. Finally, our study has sound physiological and pathological relevance to autoimmune diseases, given the fact that a variety of human autoimmune diseases, including systemic lupus erythematosus, inflammatory bowel disease, and rheumatoid arthritis, are subjected to fine regulation by CRLs [55], whose activation requires neddylation E2, UBE2M/UBE2F, and E3 RBX1/RBX2.

Materials and Methods

Mice

The Foxp3YFP-Cre mice (The Jackson Laboratory, no. 016959) were provided by X. Feng (Peking Union Medical College, China) [56]. The Ube2m-flox, Ube2f-flox, Rbx1-flox, and Sag-flox mice were generated previously [15,57]. The Rag1^−/− mice were obtained from GemPharmatech Co. Ltd., China (strain no. T004753).

Mice were fed in specific pathogen-free (SPF) conditions. All animal experiments were approved by the Animal Ethics Committee of Zhejiang University; animal care was provided in accordance with the principles and procedures by the regulatory standards at Zhejiang University Laboratory Animal Center.

Flow cytometry

Peripheral lymph nodes and spleens from indicated mice were grinded into single cells. Cells were washed in phosphate-buffered saline containing 2% (w/v) fetal bovine serum and then stained with indicated antibodies for analysis of surface proteins. For intracellular proteins, cells were fixed and permeabilized with Pharmingen Transcription Factor Buffer Set (BD Pharmingen, 562574). Flow cytometry was performed on CytoFLEX LX (Beckman).

Antibodies used were listed as follows: anti-CD4 (GK1.5, eBioscience), anti-CD8α (53-6.7, BD Pharmingen), anti-CD44 (IM7, BioLegend), anti-CD62L (MEL-14, BioLegend), and anti-Foxp3 (150D, BioLegend).

Cell sorting

The CD4⁺ T cells were isolated from the single-cell suspension of peripheral lymph nodes and spleens by Mouse CD4 T Lymphocyte Enrichment Set-DM (BD Biosciences, 558131), followed by fluorescence-activated cell sorting to purify T_reg cells (CD4⁺YFP⁺) and/or T_nai cells (CD4⁺YFP⁻CD44^loCD62L^hi) with purities >99%, performed on SONY Cell Sorter (SH800S).

In vivo suppression assay

T_nai cells (CD4⁺YFP⁻CD44^loCD62L^hi, 4 × 10⁵) and T_reg cells (CD4⁺YFP⁺, 2 × 10⁵), sorted from the peripheral lymph nodes and spleens of indicated mice (8 to 12 weeks old), were administrated into Rag1^−/− mice via intraperitoneal injection as described [58]. Four weeks after the intraperitoneal injection, mouse colons were harvested and fixed by formalin, followed by H&E staining.

Transcriptome profiling

CD4⁺YFP⁺ T_reg cells were sorted from the peripheral lymph nodes and spleens of indicated mice (8 to 12 weeks old). To generate enough materials, 2 to 3 mice were pooled for one sample. RNA was purified from the sorted cells with the miRNeasy Mini Kit (Qiagen, 21704). The RNAs were reverse-transcribed, amplified, and labeled (Affymetrix GeneChip Pico Kit, 703308) to achieve enough cDNAs. Then, the cDNA samples were hybridized to Clariom S Arrays, mouse (902931). The Applied Biosystems Expression Console Software 1.4 was employed to analyze the microarray datasets.

Statistical analysis

The P values were calculated by Mann–Whitney test, 2-tailed unpaired Student’s t test, using GraphPad Prism software. Mouse survival and respective P values were analyzed by the log-rank test. The statistical significance was evaluated by P < 0.05. All error bars represent SEM (n ≥ 3).

Data Availability

The microarray data generated in this study have been deposited in Gene Expression Omnibus under accession code GSE237499.

Supplementary Materials

Supplementary 1

Figs. S1 to S9