
==== Front
101573691
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Cell Rep
Cell Rep
Cell reports
2211-1247

38889006
10.1016/j.celrep.2024.114379
nihpa2011852
Article
The PP2A regulatory subunit PPP2R2A controls NAD+ biosynthesis to regulate T cell subset differentiation in systemic autoimmunity
Pan Wenliang 1*
Tsokos Maria G. 1
Scherlinger Marc 12
Li Wei 1
Tsokos George C. 13*
1 Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA
2 Rheumatology Department, Strasbourg University Hospital of Hautepierre, Strasbourg, France
3 Lead contact
AUTHOR CONTRIBUTIONS

W.P. designed and performed the experiments, analyzed the data, and wrote the manuscript. M.G.T. analyzed the data, provided critical expertise, and edited the manuscript. M.S. helped to perform the experiments with mice. W.L. performed the experiments involving human cells. G.C.T. conceived and supervised the study, interpreted the data, and wrote the manuscript.

* Correspondence: wpan2@bidmc.harvard.edu (W.P.), gtsokos@bidmc.harvard.edu (G.C.T.)
27 8 2024
23 7 2024
17 6 2024
20 9 2024
43 7 114379114379
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
SUMMARY

The protein phosphatase 2A (PP2A) regulatory subunit PPP2R2A is involved in the regulation of immune response. We report that lupus-prone mice with T cells deficient in PPP2R2A display less autoimmunity and nephritis. PPP2R2A deficiency promotes NAD+ biosynthesis through the nicotinamide riboside (NR)-directed salvage pathway in T cells. NR inhibits murine Th17 and promotes Treg cell differentiation, in vitro, by PARylating histone H1.2 and causing its reduced occupancy in the Foxp3 loci and increased occupancy in the Il17a loci, leading to increased Foxp3 and decreased Il17a transcription. NR treatment suppresses disease in MRL.lpr mice and restores NAD+-dependent poly [ADP-ribose] polymerase 1 (PARP1) activity in CD4 T cells from patients with systemic lupus erythematosus (SLE), while reducing interferon (IFN)-γ and interleukin (IL)-17 production. We conclude that PPP2R2A controls the level of NAD+ through the NR-directed salvage pathway and promotes systemic autoimmunity. Translationally, NR suppresses lupus nephritis in mice and limits the production of proinflammatory cytokines by SLE T cells.

Graphical abstract

In brief

Pan et al. report that PPP2R2A deficiency in T cells promotes NAD+ biosynthesis through the NR-directed salvage pathway and attenuates autoimmunity and nephritis in lupus-prone mice. NR inhibits Th17 and promotes Treg cell differentiation by PARylating histone H1.2. Translationally, NR suppresses lupus nephritis in mice and limits proinflammatory cytokine production by SLE T cells.
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pmcINTRODUCTION

The development of systemic autoimmunity and organ inflammation involves altered production of cytokines by T cells.1–3 Specifically, decreased interleukin (IL-2) production accounts for decreased cytotoxic T cell function and decreased function and numbers of regulatory T (Treg) cells,2 whereas the increased production of IL-17 and interferon (IFN)-γ may contribute directly to organ inflammation.4

T cell subset differentiation and function involves distinct metabolic reprogramming to support their unique energetic demands.5 For example, T helper 1 (Th1) and Th17 cells utilize mainly aerobic glycolysis and glutaminolysis, while naive CD4 and Treg cells use primarily fatty acid oxidation and oxidative phosphorylation.6 T cells from patients with systemic lupus erythematosus (SLE), the archetypic autoimmune disease, exhibit significant metabolic aberrations7 which can alter gene expression by modulating the epigenetic landscape8 and promote the differentiation of T cells toward a proinflammatory phenotype.8 Although some of them may reflect chronic activation, others may be genetically determined, as suggested by the association of SLE with risk alleles in genes that control mitochondrial ATP production (i.e., MT-ATP6 and UCP2).9,10 Inhibitors targeting the metabolic pathway have been used to treat mice with a lupus-like disease. For instance, the combination of metformin, an inhibitor of the mitochondrial electron transport chain complex I, and 2DG (2-deoxy-d-glucose), a glucose metabolism inhibitor, decreased autoimmunity and nephritis in lupus-prone mice.11 Additionally, treatment with an inhibitor of mitochondrial F1F0 ATP synthase, 1,4-benzodiazepine (Bz-423), led to the apoptosis of autoreactive CD4 T cells and the suppression of glomerulonephritis12, and pharmacologic inhibition of glutaminase 1, an enzyme that is in charge of converting glutamine to glutamate, reduced Th17 cell differentiation and disease activity in MRL.lpr mice.13,14

Protein phosphatase (PP) 2A is a ubiquitously expressed and highly conserved serine/threonine phosphatase that plays an essential role in multiple cellular processes, including cell division, cytoskeletal dynamics, and various signaling pathways.15 The PP2A core enzyme consists of the scaffold subunit A (PP2AA) and the catalytic subunit C (PP2AC). To form a functional holoenzyme, the core enzyme interacts with one of the many regulatory subunits (PP2AB) that define its function and tissue specificity.16 It has been shown that the mRNA and protein levels as well as the catalytic activity of PP2AC are increased in T cells from patients with SLE,17,18 and transgenic mice overexpressing PP2AC in T cells develop glomerulonephritis in an IL-17-dependent manner.19 Furthermore, PP2Ac is requisite for Treg cell function by regulating mammalian target of rapamycin complex 1 activity.20 PP2A regulatory subunits serve distinct T cell functions, with PPP2R2B being responsible for the IL-2 deprivation-induced T cell apoptosis,21 PPP2R2D (PR55δ) for limiting IL-2 production and Treg cell function,22 and PPP2R2A (PR55α) for promoting Th1 and Th17 differentiation.23

To further dissect the role of PPP2R2A in the development of lupus-like disease, we generated B6.lpr.dLckCreR2Afl/fl mice, which lack PPP2R2A only in T cells. Mice with T cell-specific PPP2R2A deficiency developed less systemic autoimmunity and nephritis. Metabolomics data revealed that PPP2R2A deficiency promoted nicotinamide (NAM) adenine dinucleotide (NAD+) production through the nicotinamide riboside (NR)-directed salvage biosynthesis pathway. Of translational value is our observation that NR supplementation significantly inhibited in vitro murine Th1 and Th17 differentiation, promoted Treg cell differentiation, and reduced significantly lupus-related pathology in MRL.lpr mice. Mechanistically, NR increased the levels of NAD+ and caused PARylation of histone H1.2, leading to reduced occupancy in the Foxp3 gene locus but increased occupancy in the Il17a gene locus, promoting Foxp3 gene transcription, and decreasing Il17a gene transcription. Finally, the NAD+-dependent poly [ADP-ribose] polymerase 1 (PARP1) enzymatic activity was found to be decreased in CD4 T cells from patients with SLE, and the addition of NR restored PARP1 activity and reduced IFN-γ and IL-17 expression in CD4 T cells from patients with SLE.

RESULTS

PPP2R2A deficiency in T cells alleviates disease in lupus-prone mice

We have reported that PPP2R2A is upregulated in T cells from patients with SLE, and that genetic PPP2R2A deficiency in murine T cells reduces Th1 and Th17 differentiation and experimental autoimmune encephalomyelitis (EAE).23 To explore further the contribution of PPP2R2A in the development of systemic autoimmune disease, we crossed B6.lpr (lupus prone) with dLckCreR2Afl/fl mice to generate B6.lpr.R2Afl/fl (wild type) and B6.lpr.dLckCreR2Afl/fl mice in which PPP2R2A is deleted only in T cells (Figure S1A). As shown in Figures S1B–S1D, B6.lpr.dLckCreR2Afl/fl mice showed reduced size of thymi, decreased percentage of double-negative (DN) cells, and increased - percentage of double-positive cells in thymi as compared to those of B6.lpr.R2Afl/fl mice. Moreover, B6.lpr.dLckCreR2Afl/fl mice showed significantly reduced spleen (SPL) weight, as well as reduced size and total cell numbers in the SPL and cervical lymph nodes (CLNs) compared with B6.lpr.R2Afl/fl mice (Figures 1A and S1E). The percentages and numbers of T cells (CD45+Thy1.2+) in the SPLs and CLNs of B6.lpr.dLckCreR2Afl/fl mice were also significantly decreased compared to those of B6.lpr.R2Afl/fl mice (Figures 1B and 1C). Specifically, PPP2R2A deficiency in T cells significantly down-regulated the numbers of all T cell subsets, including CD4 (Thy1.2+CD4+CD8−), CD8 (Thy1.2+CD4−CD8+), and DN (Thy1.2+CD4−CD8−) T cells in the SPLs and CLNs of B6.lpr. mice (Figure 1D). In addition, IFN-γ-(Thy1.2+CD4+IFN-γ+, Thy1.2+CD8+IFN-γ+, and Thy1.2+CD4−CD8−IFN-γ+; Figure 1E) and IL-17A- (Thy1.2+CD4+IL-17+, Thy1.2+CD8+IL-17+ and Thy1.2+CD4−CD8−IL-17+; Figure 1F) producing cells were significantly decreased in the SPLs and CLNs of B6.lpr.dLckCreR2Afl/fl mice compared to those of B6.lpr.R2Afl/fl mice. In contrast, the percentages of the Thy1.2+CD4+ Foxp3+CD25hi cells (Treg cells; Figure 1G) were significantly increased in the SPLs and CLNs of B6.lpr.dLckCreR2Afl/fl mice in comparison with B6.lpr.R2Afl/fl mice. Moreover, B6.lpr. dLckCreR2Afl/fl mice developed less lupus-like disease, as evidenced by decreased serum anti-double-stranded DNA (dsDNA) immunoglobulin G (IgG) levels (Figure 1H), proteinuria (Figure 1I), and histopathologic signs of glomerular lesions and perivascular inflammatory cell accumulation (Figures 1J and 1K) compared to those in B6.lpr. R2Afl/fl mice. Collectively, PPP2R2A deficiency in T cells protects against hallmark features of disease in lupus-prone mice.

PPP2R2A deficiency in T cells enhances NAD+ production through the NR-directed salvage biosynthesis pathway

We had previously shown that PPP2R2A enhances Th1 and Th17 differentiation.23 To identify mechanisms responsible for the reduction in IFN-γ- or IL-17A-producing cells in B6.lpr mice with T cell-specific deletion of PPP2R2A, we characterized the global metabolic consequences of PPP2R2A deficiency in naive CD4 T cells isolated from the SPLs of B6.R2Afl/fl and B6.dLckCre R2Afl/fl mice cultured under Th1 and Th17 polarization conditions for 24 h. Principal-component analysis (PCA) of the metabolomics data revealed that the metabolite profile of R2Afl/fl cells was clearly separated from that of dLckCreR2Afl/fl cells under both Th1 and Th17 polarization conditions (Figure 2A). Statistical analysis revealed that the levels of 41 and 25 metabolites were significantly (p < 0.1) altered in PPP2R2A-deficient Th1 or Th17 cells, respectively (Figure 2B). Metabolite set enrichment analysis of significantly altered metabolites identified significantly (p < 0.05 and impact >0.1) impacted biochemical pathways, including those of glutathione metabolism, glycolysis/gluconeogenesis, nicotinate and NAM metabolism, pyruvate metabolism and terpenoid backbone biosynthesis in PPP2R2A-deficient Th1 cells, and pyrimidine metabolism, nicotinate and NAM metabolism, lysine degradation, and synthesis and degradation of ketone bodies in PPP2R2A-deficient Th17 cells (Figure 2C).

Nicotinate and NAM metabolism, which is important in the production of NAD+, emerged in both PPP2R2A-deficient Th1 and Th17 cells (Figure 2C). Mammalian cells transport the extracellular NAD+ precursors tryptophan (Try), nicotinic acid (NA), and NR/NAM into cells for NAD+ biosynthesis through de novo synthesis, Preiss-Handler, and salvage pathways, respectively.24–27 We made a closer examination of the metabolites involved in NAD+ biosynthesis. As presented in Figures 3A and 3B, the levels of Try and NA were decreased in Th1, but increased in Th17 PPP2R2A-deficient cells. In addition, the level of NAM was increased in Th1, but decreased in Th17 PPP2R2A-deficient cells (Figures 3A and 3B). However, the levels of NR and its downstream metabolite NAM mononucleotide (NMN) were increased in both Th1 and Th17 PPP2R2A-deficient cells (Figures 3A and 3B). Moreover, the levels of NAD+ or its reduced form NADH were decreased or increased in PPP2R2A-deficient Th1 cells, while increased or decreased in PPP2R2A-deficient Th17 cells (Figures 3A and 3B). Because NAD+ and NADH are inter-converted and the concentrations measured by mass spectrometry are relative, it is difficult to conclude whether the intracellular NAD+ levels are increased or decreased. Thus, to determine the absolute concentrations of total intracellular NAD+ (NAD+ and NADH) level, we used the NAD/NADH quantification kit to measure the concentration of total NAD+ (NAD+total), including NAD+ and NADH in R2Afl/fl or dLckCreR2Afl/fl naive CD4 T cells after culture under Th1 and Th17 polarization conditions for 24 h. As shown in Figure 3C, PPP2R2A deficiency increased the NAD+total levels in both Th1 and Th17 cells.

Since the level of NR was increased in both Th1 and Th17 PPP2R2A-deficient cells (Figures 3A and 3B), we further determined whether NR-directed NAD+ biosynthesis through the salvage pathway plays a role in NAD+ production in PPP2R2A-deficient or PPP2R2A-sufficient Th1 and Th17 cells. We applied vehicle (DMSO) or S-(4-nitrobenzyl)-6-thioinosine (NBTI), a small drug inhibitor of equilibrative nucleoside transporters (ENTs) that block the transport of NR from extracellular into intracellular space28,29 during R2Afl/fl and dLckCreR2Afl/fl Th1 or Th17 cell differentiation. As shown in Figure 3D, NBTI did not significantly affect the levels of NAD+total in R2Afl/fl Th1 or Th17 cells compared to DMSO-treated R2Afl/fl Th1 or Th17 cells, suggesting that the NR-directed salvage pathway is negligible in PPP2R2A-sufficient cells. On the contrary, NBTI treatment dramatically decreased the levels of NADtotal in dLckCreR2Afl/fl Th1 or Th17 cells when compared to DMSO-treated dLckCreR2Afl/fl counterparts (Figure 3D). Metabolomics analysis of extracellular metabolites showed that NBTI treatment blocked the uptake of NR as evidenced by the increased extracellular levels of NR when compared to DMSO-treated PPP2R2A-deficient T cells (Figure S2). These data signify the importance of the NR-directed salvage pathway in the biosynthesis of NAD+ in PPP2R2A-deficient T cells. We further performed qPCR analysis to determine the levels of NAD+ biosynthetic enzymes to determine whether PPP2R2A deficiency affects their expression. PPP2R2A deficiency significantly enhanced the mRNA expression levels of Nmrk1 (NRK1), Nmrk2 (NRK2), Nmnat1, Nmnat2, and Nmnat3 (Figure 3E), which are the key enzymes involved in the NR-directed NAD+ biosynthesis salvage pathway (Figure 3B) in both Th1 and Th17 cells. However, PPP2R2A deficiency did not significantly affect the level of Nampt, which is the rate-limiting enzyme in the NAM-mediated NAD+ salvage pathway (Figure 3B). Moreover, the mRNA levels of Nadsyn1 (NADS), which catalyzes the conversion of nicotinic acid adenine dinucleotide (NAAD) to NAD+ in de novo synthesis and the Preiss-Handler pathway (Figure 3B) was down-regulated in PPP2R2A-deficient Th1 and Th17 cells compared to PPP2R2A-sufficient counterparts (Figure 3E). Collectively, these results confirm that PPP2R2A deficiency increases NAD+ levels through the NR-directed NAD+ biosynthesis salvage pathway in both Th1 and Th17 cells.

NR inhibits Th1 and Th17 cell differentiation but augments Treg cell differentiation in vitro

To investigate whether NAD+ is essential for in vitro T cell differentiation, we isolated naive CD4+ T cells from the SPLs of R2Afl/fl mice and cultured them under Th1-, Th17-, and Treg-polarizing conditions in the presence of PBS or NR (500 μM) for 72 h in vitro. As presented in Figures 4A and 4B, NR treatment significantly reduced the percentages of IFN-γ (Th1)- and IL-17 (Th17)-producing cells. Surprisingly, NR treatment enhanced Foxp3-expressing cells (Treg cells; Figures 4A and 4B). As expected, NR treatment significantly up-regulated intracellular NAD+ levels in these cell subsets (Figure 4C), and NBTI significantly blunted NR-induced intracellular NAD+ levels (Figure 4D). Furthermore, NR treatment significantly down-regulated the mRNA expression level of Ifng and Tbx21 (T-bet) in Th1 cells (Figure 4E), suggesting that NR inhibited Th1 cell differentiation by down-regulating the Th1 master transcription factor T-bet. However, NR treatment significantly reduced the mRNA expression levels of Il17a, but not Rorc and Stat3 in Th17 cells (Figure 4F), and increased the levels of Foxp3, but not Cd25 and Stat5a in Treg cells (Figure 4G). Moreover, NR treatment did not induce significant changes in the levels of p-Stat3 in Th17 cells (Figure 4H) and p-Stat5 in Treg cells (Figure 4I). These results indicate that NR treatment inhibited Th17 cell polarization but augmented Foxp3 expression independently of the Stat3/Stat5 pathway.

NR treatment alters the occupancy of histone H1 in Il17a and Foxp3 loci

We next interrogated the mechanism whereby NR treatment inhibited IL-17 but augmented Foxp3 expression. NAD+ can directly participate in protein modification by serving as a substrate of NAD+-consuming enzymes including two major families, sirtuins and poly (ADP-ribose) polymerases (PARPs).24–27 Sirtuins use NAD+ as a co-substrate to remove acetyl moieties from lysine residues, resulting in protein deacetylation.24–27 Active PARPs (mainly PARP1) catalyze the transfer of ADP-ribose subunits from NAD+ to protein acceptors, a process called poly-ADP-ribosylation (PAR) or PARylation.24–27 Interestingly, NR treatment dramatically increased the levels of PARylated proteins (Figures 5A and S3B), while it did not significantly affect the levels of acetylated proteins (Figures S3A and S3B) in Th17 and Treg cells, implying that protein PARylation may play an important role in the NR-induced inhibition of IL-17 expression and augmentation of Foxp3 expression. In addition, PARP1 autoPARylation, which enhances PARP1 enzymatic activity,30 was increased in both NR-treated Th17 and Treg cells as compared to PBS-treated counterparts (Figure 5A). Moreover, NBTI blunted NR-induced PAR in both Th17 and Treg cells (Figure S3C), consistent with the finding presented in Figure 4D. Interestingly, PPP2R2A deficiency increased the level of PAR, which was blocked by NBTI in CD4 T cells stimulated with anti-CD3/CD28 (Figure S3D). To determine which proteins were extensively PARylated, we performed immunoprecipitation (IP)-mass spectrometry (MS) analysis of PARylated proteins from naive CD4 T cells after culture under Treg cell differentiation conditions for 24 h in the presence of PBS or NR (Figure S3E). This analysis detected 2,563 PARylated proteins in PBS- and NR-treated Treg cells, and statistical evaluation identified significantly (|log2 fold change| > 2) altered PARylated levels in 24 proteins with increased and 26 proteins with decreased PARylation in NR-treated Treg cells compared to PBS-treated Treg cells (Figure 5B). Gene Ontology (GO) pathway analysis of these significantly altered PARylated proteins showed that negative regulation of DNA recombination, chromosome condensation, nucleosome organization, and chromatin organization biological processes was impacted significantly (Figure 5C). Specifically, three subtypes (H1.4, H1.2, and H1.5) of the histone H1 protein family were involved in these processes, and their PARylation was up-regulated in NR-treated Treg cells (Figures 5B and 5C). Histone H1, especially the H1.2 subtype, regulates the epigenetic landscape by local chromatin compaction,31 and PARylation changes its occupancy of chromatin domains.32,33To further confirm H1.2 PARylation in Th17 and Treg cells, we pulled down H1.2 using an antibody against H1.2 and detected the PARylated H1.2 by western blot analysis using a PAR antibody, because a PARylated H1.2 antibody is not available. As presented in Figures 5D, S3F, and S3G, NR treatment increased the level of PARylated H1.2 in both Th17 and Treg cells, which is consistent with the finding by MS analysis. Intriguingly, PARylated H1.2 was increased in PPP2R2A-deficient as compared to PPP2R2A-sufficient CD4 T cells stimulated by anti-CD3/CD28 (Figure S3H). It is reported that the promoter-defined conserved noncoding sequences (CNS) 1 and CNS2 in the Foxp3 gene locus are required for Foxp3 gene transcription in Treg cells.34 In addition, CNS2 in the Il17a gene locus is sufficient and necessary for Il17a gene transcription in Th17 cells.35 We next asked whether NR treatment affects the occupancy of H1.2 in these regions. Chromatin IP (ChIP)-qPCR experiments revealed that NR treatment decreased the binding of H1.2 to the promoter CNS1 and CNS2 regions in the Foxp3 gene locus but that they increased the enrichment of H1.2 in the CNS2 region within the Il17a gene locus in both Treg and Th17 cells (Figure 5E). In contrast, NR treatment led to the accumulation of H3K36m2, which is associated with increased gene transcription in the CNS1 and CNS2 regions within the Foxp3 gene locus in Treg cells, but reduced the binding of H3K36m2 to the CNS2 region within the Il17a gene locus in Th17 cells (Figure 5F). Collectively, NR treatment reduced the occupancy of histone H1 protein in the Foxp3 gene locus but increased the occupancy in the Il17a gene locus, promoting Foxp3 gene transcription but decreasing Il17a gene transcription.

NR treatment suppresses lupus-related disease in MRL.lpr mice

To determine the role of NR in the regulation of lupus-associated pathology, we injected intraperitoneally PBS or NR Cl (NR chloride, 500 mg/kg body weight/day) into 8-week-old MRL.lpr (lupus prone) mice daily for 9 weeks (Figure S4A). MRL.mpj mice injected with PBS were used as the control. As shown in Figures 6A and S4B, NR Cl-treated MRL.lpr mice showed significantly reduced SPL weight and SPL and CLN size, and the cell numbers compared with PBS-treated MRL.lpr mice. The percentages and numbers of T cells (CD45+Thy1.2+) in SPLs and CLNs of NR-treated MRL.lpr mice were significantly decreased compared to those of PBS-treated MRL.lpr mice (Figures 6B and 6C). Specifically, NR Cl treatment down-regulated significantly the numbers of all T cell subsets, including CD4 (Thy1.2+CD4+CD8−), CD8 (Thy1.2+CD4−CD8+), and DN (Thy1.2+CD4−CD8−) T cells in the SPLs and CLNs of MRL.lpr mice (Figure 6D). Ki67 expression of T cells (CD45+Thy1.2+) in the SPLs and CLNs of NR Cl-treated MRL.lpr mice was significantly lower than in PBS-treated MRL.lpr mice, implying that NR Cl treatment significantly reduced T cell proliferation (Figures S4C and S4D). NR Cl treatment, however, did not affect the body weight of MRL.lpr mice compared to PBS-treated MRL.lpr mice (Figure S4E). Treatment significantly decreased IFN-γ-producing (Thy1.2+CD4+IFN-γ+, Thy1.2+CD8+IFN-γ+, and Thy1.2+CD4−CD8−IFN-γ+; Figure 6E) and IL-17A-producing (Thy1.2+CD4+IL-17+, Thy1.2+CD8+IL-17+, and Thy1.2+CD4−CD8−IL-17+; Figure 6F) cells in the SPLs and CLNs of NR-treated MRL.lpr mice compared to PBS-treated MRL.lpr mice. In contrast, the percentages of the Thy1.2+CD4+Foxp3+CD25hi Treg cells (Figure 6G) were significantly increased in the NR Cl-treated MRL.lpr mice in comparison with the PBS-treated MRL.lpr mice. Moreover, NR Cl-treated MRL.lpr mice developed less nephritis, as evidenced by the decreased anti-dsDNA IgG levels in the serum (Figure 6H), proteinuria (Figure 6I), and histopathologic signs of glomerular and tubular lesions and perivascular cell accumulation (Figures 6J and 6K) when compared with PBS-treated MRL.lpr mice. Western blot analysis of protein lysates extracted from the splenocytes of mice showed that treatment with NR Cl increased PARylated PARP1 but decreased the cleaved PARP1 in the splenocytes of MRL.lpr mice (Figure S4F), signifying that PARP1 was activated in MRL.lpr mice after NR Cl treatment. Collectively, NR Cl treatment suppresses disease in lupus-prone mice.

NR supplementation restores PARP1 activity and reduces IFN-γ and IL-17 expression in CD4 T cells from patients with SLE

Low PARP1 enzymatic activity has been reported in peripheral blood mononuclear cells (PBMCs) from patients with SLE.36 PARP1 is involved in DNA repair, inflammation, and cell death, and it enhances its enzymatic activity in autoPARylation,30 whereas it loses its activity once cleaved.37,38 To determine the translational value of our findings, we compared the PARP1 activity of CD4 T cells from healthy control subjects and patients with SLE (Table 1) by determining the levels of PARylated and cleaved PARP1. As presented in Figures 7A and 7B, PARP1 autoPARylation was significantly decreased, whereas the cleavage of PARP1 into multiple fragments was at significantly higher levels in CD4 T cells from patients with SLE as compared to those in CD4 T cells from race-, age-, and sex-matched healthy subjects. This finding suggests that CD4 T cells from patients with SLE have low PARP1 enzymatic activity. Moreover, culture of SLE CD4+ T cells in the presence of NR led to a significant increase in PARylated PARP1 and PAR, but it did not affect the cleaved PARP1 (Figures 7C and 7D), suggesting that PARP1 activity was enhanced. Moreover, the mRNA expression levels of IFNG and IL17A were decreased after supplementation with NR (Figure 7E). Collectively, these results demonstrate that NR supplementation can restore PARP1 enzymatic activity and reduce IFN-γ and IL-17 expression in CD4 T cells from patients with SLE.

DISCUSSION

In this communication, we present evidence that PPP2R2A, a regulatory subunit of PP2A, is needed for the development of systemic autoimmunity, and its genetic absence in T cells limits disease expression. While deciphering the involved mechanisms, we fortuitously found that this regulatory subunit controls the entry of NR into the cells and NAD+ biosynthesis through the salvage pathway, and it regulates T cell differentiation by PARylating histone H1.2, leading to its differential occupancy of regulatory regions of the Foxp3 and Il17a loci. At the translational level, NR treatment prevented systemic autoimmunity in MRL.lpr mice and decreased IFN-γ and IL-17 production in CD4 T cells from patients with SLE.

SLE is an autoimmune disorder that is characterized by diverse T effector cell dysfunction involving increased cytokine production such as IL-17 and IFN-γ, which may contribute directly to organ inflammation.1–3,4 IL-17, a proinflammatory cytokine, is produced rapidly and in large amounts by CD4+, γδ+, and CD4−CD8− (DN) TCRαβ+ T cells39–41 that are present in the periphery and in the kidney tissue of patients with lupus nephritis.41 We had previously found that PPP2R2A was upregulated in T cells from patients with SLE and EAE, and genetic PPP2R2A deficiency in murine T cells reduced Th1 and Th17 differentiation.23 In the present study, using a lupus-prone mouse, we demonstrate that mice with PPP2R2A deletion only in T cells produced less IL-17- and IFN-γ-producing T cells and developed less lupus-like glomerulonephritis, signifying that PPP2R2A is a promising therapeutic target for lupus-related organ inflammation.

T cells reprogram their metabolism to support their energetic demands according to their activation status.5 For example, Th1 and Th17 cells utilize mainly aerobic glycolysis and glutaminolysis, while naive CD4 T cells and Treg cells primarily use fatty acid oxidation and oxidative phosphorylation.6 T cells from patients with SLE exhibit significant metabolic abnormalities7 that can alter gene expression by modulating the epigenetic landscape8 and promote the differentiation of T cells toward proinflammatory subsets such as Th1 and Th17 cells.8 In the present study, we have identified that NAD+ metabolism is significantly impacted in T cells subjected to Th1 and Th17 cell polarization conditions when PPP2R2A expression is absent. Specifically, PPP2R2A deficiency increased NAD+ levels when T cells were cultured under proinflammatory conditions. This finding implies that the increased NAD+ levels in PPP2R2A-deficient cells probably account for the reduced ability of PPP2R2A-deficient naive CD4 T cells to differentiate into Th1 and Th17 cells.23 Indeed, Th1 and Th17 cell differentiation was significantly reduced after we supplemented the cell medium with NR to boost intracellular NAD+ levels. Mammalian cells use NAD+ precursors Try, NA, and NR/NAM to synthesize NAD+ through de novo synthesis, Preiss-Handler, and salvage pathways, respectively.24–27 Our data reveal that blockage of the NR-directed pathway did not significantly affect intracellular NAD+ levels in PPP2R2A-sufficient cells but caused a dramatic down-regulation of NAD+ levels in PPP2R2A-deficient cells, suggesting that the NR-directed salvage pathway is negligible in PPP2R2A-sufficient cells, while PPP2R2A-deficient cells rewire the NR-directed salvage pathway to boost intracellular NAD+. The fact that PPP2R2A deficiency inhibited the metabolic enzyme involved in de novo synthesis and Preiss-Handler pathways while it enhanced the expression of enzymes that contribute to the NR-mediated salvage pathway indicates that PPP2R2A is vital in the regulation of NAD+ metabolism in T cells. Thus, PPP2R2A, which was found up-regulated in SLE T cells,23 represents one of the factors that drive metabolic abnormalities in T cells.

NAD+ is not only an essential cofactor for multiple cellular redox processes linked to fuel utilization and energy metabolism but it is also a substrate for multiple enzymes such as sirtuins and PARPs to regulate signaling pathways, post-translational modifications, epigenetic changes, and RNA stability and function.24–27 NAD+ levels decline during aging, and alterations in NAD+ homeostasis can be found in virtually all age-related diseases, including neuro-muscular, cardiometabolic, liver, and kidney diseases.24,25 For this reason, the therapeutic and preventive potential of NAD+ boosting has been scrutinized in a variety of preclinical models and disease settings.24,25 In patients with SLE, cellular NAD+ levels were found to be low in CD8 T cells due to the increased expression of CD3842 and high IFN signaling.43 Replenishment of cellular NAD+ levels using either a CD38 inhibitor44 or NMN43 restored the cytotoxicity of CD8 T cells. NR has been used widely to boost intracellular NAD+ levels 45 and is transported into the cell via SLC29A1.28 NR can be either directly administered or from the conversion of NAD+, NAM, or NMN by CD73 extracellularly.24–27 Previously, NR was found to reduce autophagy and type I IFN signaling in monocytes from patients with SLE and can be used as a potential adjunct to treat SLE patients.46 In this study, we provide evidence that the administration of NR in MRL.lpr mice decreased the numbers of IL-17- and IFN-γ-producing T cells, increased the percentage of Treg cell population, and reduced the development of lupus nephritis. In addition, low PARP1 enzymatic activity has been reported in PBMCs from patients with SLE.36 In the present report, we demonstrate that PARP1 autoPARylation, which promotes its enzymatic activity,30 was decreased in CD4 T cells from patients with SLE, while supplementation with NR increased the level of PARylated PARP1 and reduced IFN-γ and IL-17 production. These findings provide rationale to use NR to treat patients with SLE.

Th17 cells that produce IL-17, IL-22, and IL-23 and Treg cells that produce anti-inflammatory cytokines play opposite roles in the regulation of the inflammatory immune response.47 Patients with SLE have increased numbers of Th17 cells41,48 but decreased numbers of Treg cells2 in both peripheral blood and tissues. Stat3/RORyt signaling is critical for Th17 cell differentiation,49,50 while development and differentiation of Treg cells and expression of Foxp3 depend on another transcription factor, Stat5.51,52 We found that NR treatment inhibits Th17 cell differentiation and IL-17 expression independently of Stat3/RORyt signaling, but enhanced Treg cell differentiation and Foxp3 expression without affecting the expression of the transcription factor Stat5. NAD+ can participate directly in protein modification, and modulate chromatin structure and gene transcription through PARP1.53 In agreement with this thesis, we identified many PARylated proteins that are involved in the chromatin organization process after NR treatment, including increased levels of PARylated histone 1.2. Specifically, we found that histone H1 displayed increased occupancy in the Il17a locus and decreased occupancy in the Foxp3 locus after NR treatment. It is reported that H1 histones can regulate gene transcription by controlling the epigenetic landscape in T cells.31 Here, we provide evidence that modulation of histone H1.2 by NR alters its occupancy in Il17a and Foxp3 loci to control their transcription.

In summary, we have shown that PPP2R2A, a regulatory subunit of PP2A, regulates NAD+ metabolism, and its specific targeting increases NAD+ production and decreases Th1/Th17 cell differentiation in autoimmune diseases. Importantly, administration of NR to lupus-prone mice increased NAD+ levels and suppressed autoimmunity and nephritis, whereas culture of CD4 T cells from patients with SLE in the presence of NR decreased IFN-γ and IL-17 production. NR is orally bioavailable54 and reduces levels of circulating inflammatory cytokines in humans,55 and accordingly, patients with autoimmune diseases should benefit from NR supplementation.

Limitations of the study

Our study has several limitations. For instance, in the polar metabolomics profiling, only 303 metabolites were examined. Although these 303 metabolites participated in the bulk of the metabolic processes, the measured metabolites in each pathway were limited. Although we are able to assign metabolites into specific pathways, it is possible that others belonging to other pathways were not measured. Another limitation has to do with the experiments in mice. Although we used MRL.lpr mice to treat with NR Cl, genetic deletion of Ppp2r2a in T cells was performed in B6.lpr mice. It is known that MRL.lpr mice display more severe autoimmunity and pathology than B6.lpr mice.56,57 Thus, we may have recorded a differential effect between PPP2R2A deletion specific in T cells and NR treatment on lupus pathology possibly because the B6.lpr mice develop milder disease than do the MRL.lpr mice. In addition, we used LckCre to delete PPP2R2A expression only in T cells rather than other cell types. However, NR Cl administration into mice by intraperitoneal injection could affect not only T cells but also other cell subsets. It has been reported46 that NR reduces autophagy and type I IFN signaling in monocytes from patients with SLE. Finally, the effect of NR on other immune cell subsets is currently largely unknown.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, George C. Tsokos (gtsokos@bidmc.harvard.edu).

Materials availability

R2Afl/fl or dLckCreR2Afl/fl mice in B6, or B6.lpr background are available from the lead contact upon request.

Data and code availability

All data reported in the paper are available from the lead contact upon request.

This paper does not report original code.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Mice

PPP2R2A-flox (R2Afl/fl) and dLckCreR2Afl/fl mice in which PPP2R2A is deleted in T cells were generated as previously described.23 In the current study, we crossed B6.MRL-Faslpr/J (B6.lpr, The Jackson Laboratory, Stock No: 000482) with LckCreR2Afl/fl mice to generate B6.lpr.R2Afl/fl (wild-type) and B6.lpr.LckCreR2Afl/fl mice where PPP2R2A is deleted only in T cells. To dissect the role of PPP2R2A in the development of systemic lupus erythematosus-like autoimmune disease, twenty-six-weeks-old B6.lpr.R2Afl/fl and B6.lpr.dLckCreR2Afl/fl sex-matched littermates were used to study lymphoproliferation. For renal pathology, thirty-five-weeks-old sex-matched littermates were used. MRL/MpJ-Faslpr/J (MRL.lpr, Stock No: 000485) and MRL/MpJ (Stock No: 000485) mice were purchased from The Jackson Laboratory. To determine the role of nicotinamide riboside chloride (NR Cl, TruNiagen, ChromaDex) in the regulation of lupus-related pathology, we injected intraperitoneally PBS or NR (500 mg/kg body weight/day) into 8-weeks-old MRL.lpr mice daily for 9 weeks. MRL.mpj mice injected with PBS were used as lupus-related disease-free control. All mice were bred and housed in a specific pathogen-free environment in a barrier facility in accordance with the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee (IACUC).

Patients enrolled in the study and CD4 T cell isolation and stimulation

A total of 24 patients with systemic lupus erythematosus and age-, sex-, and ethnicity-matched 14 healthy donors were enrolled in this study. Primary CD4 T cells were purified from peripheral venous blood obtained from healthy volunteers as well as patients. The blood was incubated for 30 min with a rosette CD4 T cell purification kit (Stem Cell Technologies). Lymphocyte separation medium (17-829E, Lonza) was subsequently used to separate these complexes from CD4 T cells. For stimulation, CD4 T cells were cultured in RPMI 1640 medium, (10-040-CVR, Corning), 10% heat-inactivated FBS (SH3007003HI, Hyclone), 2 mM glutamine (25-030-081, Gibco, Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin (15140122, Thermo Fisher Scientific) at 37°C, 5% CO2. CD4 T cells were stimulated with plate-bound OKT3/anti-CD3 (5 μg/mL, 317325, BioLegend) and anti-CD28 (1 μg/mL, CD28.2, BioLegend) in the presence or absence of NR (500 μM) for the indicated time.Studies were approved by the Institutional Review Board of Beth Israel Deaconess Medical Center (BIDMC).

Study approval

Human samples study (protocol 2006-P-0298) was approved by BIDMC IRB. Informed consent was obtained from all study subjects. All animal procedures were approved by the IACUC of BIDMC, Harvard Medical School. All mice were maintained in an SPF animal facility (BIDMC). All mice were genotyped to validate claimed strain.

METHOD DETAILS

In vitro T cell differentiation

Naive CD4+ T cells were purified by mouse CD4+CD62L+ T cell Isolation Kit II (Miltenyi Biotec). Purified naive T cells were stimulated with plate-bound goat anti-hamster IgG (SKU 0856984, MP Biomedicals), soluble anti-CD3 (0.25 μg/mL,145-2C11; BioLegend), and anti-CD28 (0.5 μg/mL, 37.51; BioLegend) for Th0-nonpolarized condition culture. In addition to Th0-nonpolarized condition, the following stimulation was used for each polarized condition: IL-12 (20 ng/mL; R&D Systems) and anti-IL-4 (10 μg/mL, C17.8; BioLegend) for Th1; IL-6 (3 ng/mL; R&D Systems), TGF-β1 (0.3 ng/mL; R&D Systems), anti–IL-4 (10 μg/mL, C17.8; BioLegend), and anti–IFN-γ (10 μg/mL; XMG1.2; BioLegend) for Th17; and IL-2 (20 ng/mL; R&D Systems), TGF-β1 (3 ng/mL), anti–IL-4 (10 μg/mL), and anti–IFN-γ (10 μg/mL) for Treg. In addition to antibodies and cytokines, in some experiments, cells were cultured in the presence of PBS or NR (500 μM, HB-5832, Combi-Blocks).

Mass spectrometry

Polar metabolomics profiling

Metabolites were extracted from cells or culture medium using 80% methanol (v/v). Polar metabolomics profiling (303 metabolites) was performed by using liquid chromatography-tandem mass spectroscopy (LC/MS) in the BIDMC mass spectrometry core according to the protocol.58 Once the SRM data were acquired, peaks were integrated in order to generate chromatographic peak areas used for quantification across the sample set. Metabolomics data were analyzed using MetaboAnalyst 5.0 (http://www.metaboanalyst.ca/).59 Briefly, missing metabolite raw intensity values were filled in with the lowest detectable intensity of the respective metabolites, and all raw intensities were normalized to the sum intensity of the respective replicate, mean-centered and divided by the standard deviation of each variable. Metabolites between groups were identified and subjected to principal component analysis (PCA), statistical analysis (t test) and enrichment analysis.

Protein identification

In order to identify PolyADP-ribosylated protein, we performed immunoprecipitation-mass spectrometry (IP-MS) analysis. Briefly, protein lysate was digested by TPCK modified trypsin (1 μg/μL) overnight at 37°C with shaking 250 rpm. Peptides were then purified by loading samples onto reversed-phase C18 cartridges (SepPak Classic, 360 mg sorbent, 0.85 mL, Waters) and lyophilized. The dried peptides were then dissolved with IP buffer and incubated with antibody against PAR/pADPr (Clone # 10HA, 4335-MC-100, R&D Systems) for IP experiments using Dynabeads Protein G Immunoprecipitation Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. The antibody-bound peptides were then purified and subjected to LC/MS analysis for identification of peptide/protein in the BIDMC mass spectrometry.

NAD/NADH quantification

The total intracellular NAD levels (NAD+ and NADH) were determined using NAD/NADH Quantitation Kit (MAK037, Sigma-Aldrich) following manufacture’s instructions.

Quantitative (q) PCR

Total RNA was isolated from purified T cells with RNeasy Plus Micro Kit (QIAGEN). The isolated RNA was transcribed into cDNA using the RNA to cDNA premix (Clontech) according to the manufacturer’s instructions. SYBR green was purchased from Roche, and the assays were performed on 96-well reaction plates (Invitrogen). The real-time PCR was performed on LightCycler 480 device (Roche). In all experiments, β-actin was used as reference gene to normalize gene expression and ΔΔCT method was used to calculate the fold change of target gene expression. The primers used are shown in Table S1.

Western blotting

Cell lysates were prepared using RIPA buffer (Boston BioProducts) containing protease cocktail inhibitor (cOmplete Mini EDTA-free, Roche) and phosphatase cocktail inhibitor (PhosSTOP, Roche). Protein concentration was determined by Coomassie protein assay reagent (MilliporeSigma). Total protein (30 μg) was resolved by a NuPAGE 4%–12% Bis-Tris gel (Invitrogen), and transferred to PVDF membrane (Thermo Fisher Scientific). After blocking with 5% nonfat milk (M-0841, LabScientific), the membrane was incubated with primary antibody overnight at 4°C. Subsequently, the membrane was incubated with secondary antibody for 90 min at room temperature. Western ECL substrate (1705061, Bio-Rad) was used to develop the immunoblot. The picture was captured and analyzed by Image Lab (Version 5.2.1) using ChemiDoc XRS+ System (Bio-Rad). Primary antibodies against PARP1 (sc-7150, Santa Cruz Biotechnology), PAR (4335-MC-100, R&D Systems), and β-actin (A5316, Sigma-Aldrich), and goat anti-mouse IgG (H + L) secondary antibody (31430, Thermo Fisher Scientific) and goat anti-rabbit IgG (H + L) secondary antibody (31460, Thermo Fisher Scientific) were used. The results were quantified by plotting the intensity of the band. β-actin was used as the loading control.

Flow cytometry

Cells were stained with fluorescence-tagged antibodies purchased from eBioscience, BD Biosciences, Tonbo Bioscience, or BioLegend (Table S2), and analyzed using Cytoflex flow cytometer. Flow cytometry data were analyzed using CytExpert version 2.0 of Flowjo. For intracellular cytokine staining, cells were stimulated with 50 ng/mL of PMA, 1 μM of ionomycin, and 1 μg/mL of brefeldin A for 4 h in the presence of brefeldin A; they were then harvested, fixed, and stained with BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit. For cell sorting, FACSAria (BD) was used and the purity of the sorted population was over 95%.

Chromatin immunoprecipitation (ChIP)-qPCR

ChIP experiments were performed with the MAGnify Chromatin Immunoprecipitation System (49–2024; Life Technologies) following the manufacturer’s instructions. Briefly, 1 × 106 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The reaction was stopped with glycine for 5 min, and the samples were lysed and sonicated to obtain 200- to 500-bp fragments. Immunoprecipitation was performed with an H1.2 antibody (JU43-48, NBP2-75932, Novus Biologicals), H3K36m2 (07–274, Millipore-Sigma) or an IgG control (49–2024; Life Technologies). Cross-linking was reversed, and DNA was eluted and purified using DNA-purification magnetic beads. Enrichment for the mouse Foxp3 promoter, CNS1 and CNS2, and Il17a CNS2 genomic sequence in the samples was quantified by real-time qPCR and was normalized with the input samples. The primers used are shown in Table S1.

Renal histopathology

Hematoxylin-Eosin- and Periodic acid-Schiff (PAS)-stained paraffin sections of formalin-fixed kidneys from each mouse were assessed blindly for glomerular lesions (hypercellularity, exudates, hyalinosis, necrosis, crescent formation), tubular lesions (atrophy, dilation, necrosis, casts) and perivascular mononuclear cell accumulation. Glomerular lesions and perivascular cell accumulations were scored on a scale of 0–3, and tubular lesions were scored as percentage of cortical tubule involvement, as previously described.60

Enzyme-linked immunosorbent assay (ELISA)

dsDNA antibody levels were measured using a homemade enzyme-linked immunosorbent assay as previously described.61 Briefly, sera were diluted 1:50 with 1% PBS bovine serum albumin and incubated at room temperature with shaking for 2 h on 96-well plates precoated with l-lysin (0.05 mg/mL) for 2 h at room temperature, then coated with Calf thymus DNA (0.1 mg/mL, Sigma-Aldrich) at 4°C overnight. Standard was made using serial dilution of the serum of one 16-week-old MRL/lpr mouse. After washing, the wells were incubated with an alkaline phosphatase–conjugated goat anti-mouse immunoglobulin G antibody (1:5000; Jackson ImmunoResearch) for 1 h, and colorimetric reaction was provoked by adding diethanolamine substrate buffer (Thermo Fisher Scientific) and PNPP (p-Nitrophenyl Phosphate)-phosphatase substrate (Sigma-Aldrich) and read at 405 nm. The results are expressed as units per volume.

Immunoprecipitation (IP)

IP experiments were conducted using Dynabeads Protein G Immunoprecipitation Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Briefly, protein lysates were prepared using ice-cold IP lysis buffer containing protease cocktail inhibitor (cOmplete Mini EDTA-free, Roche) and phosphatase cocktail inhibitor (PhosSTOP, Roche). Then, the mixture magnetic beads/antibody either against H1.2 (JU43-48, NBP2-75932, Novus Biologicals) or normal rabbit IgG (s49-2024; Life Technologies) as indicated was made by 10 min at room temperature under continuous mixing. Then beads/antibody mixture was then incubated with protein lysate overnight at 4°C. The beads were then collected on a magnetic stand and washed, and the protein complex was eluted from the beads for Western blot analysis.

QUANTIFICATION AND STATISTICAL ANALYSIS

Western blots were quantified by plotting the intensity of the band using Image Lab (Bio-rad). β-actin was used as the loading control. Flow cytometry data was analyzed by Flowjo or CytoExpert 2.0. SLE is an autoimmune disease with a complex pathogenesis. In addition, even healthy people of different age, sex, or ethnicity have variable T cell numbers and function. In order to mitigate this variability, we paired each SLE patient to age-, sex-, and ethnicity-matched healthy control in order to exclude the differences caused by age-, sex-, and ethnicity. All statistical analyses were conducted using GraphPad Prism 7 (GraphPad software Inc.). Data were presented as mean ± SD. Statistical differences between 2 populations were calculated by t test (2-tailed) including multiple t test, unpaired t test, or paired t test. For multiple populations’ comparison, two-way ANOVA with Turkey’s multiple-comparisons test was used. p < 0.05 was considered statistically significant.

Supplementary Material

1

ACKNOWLEDGMENTS

This work was supported by NIH grants R01 AI136924 and R01 AI085567.

Figure 1. PPP2R2A deficiency in T cells alleviates disease in lupus-prone mice

(A–F) B6.lpr.R2Afl/fl (wild type, n = 8) and B6.lpr.dLckCreR2Afl/fl (n = 10) mice (26 weeks old) were euthanized for collection of SPLs and CLNs and subsequently analyzed by fluorescence-activated cell sorting (FACS). (A) Representative photographs (left) and cumulative data of the cell numbers (right) in SPLs and CLNs. Percentages (B) and numbers (C) of T cells (CD45+Thy1.2+), and numbers of CD4 (Thy1.2+CD4+CD8−), CD8 (Thy1.2+CD4−CD8+) and DN (Thy1.2+CD4−CD8−) T cells (D) in SPLs and CLNs. Numbers of IFN-γ-producing (Thy1.2+CD4+IFN-γ+, Thy1.2+CD8+IFN-γ+, and Thy1.2+CD4−CD8−IFN-γ+; E) and IL-17A-producing (Thy1.2+CD4+IL-17+, Thy1.2+CD8+IL-17+ and Thy1.2+CD4−CD8−IL-17+; F) cells in SPLs and CLNs.

(G) Percentage of Treg cells (Thy1.2+CD4+Foxp3+CD25hi) in SPLs and CLNs.

(H–K) B6.lpr.R2Afl/fl (n = 6) and B6.lpr.dLckCreR2Afl/fl (n = 4) mice 35 weeks old were studied. (H) ELISA measurement of anti-dsDNA IgG levels in the serum. (I) Proteinuria by urine strip test. (J) Representative images of kidney sections. The arrow points to a small perivascular mononuclear cell accumulation. H&E; scale bar: 20 μm. (K) Cumulative data of the histopathologic scores in the kidneys of the indicated mice. Mean ± SD is shown.

*p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 using unpaired t test (A–C, G–I, and K) or multiple t test (D–F).

Figure 2. PPP2R2A deficiency in T cells alters metabolite profile under Th1 and Th17 polarization conditions

Metabolomics analysis was performed on PPP2R2A wild-type (R2Afl/fl) or PPP2R2A knockout (dLckCreR2Afl/fl) naive CD4 T cells after culture under Th1 and Th17 polarization conditions for 24 h (n = 3 mice/group).

(A) PCA of the metabolites of R2Afl/fl and dLckCreR2Afl/fl Th1 (left) or Th17 (right) cells.

(B) Heatmap showing the significantly (p < 0.1) altered metabolites in R2Afl/fl or dLckCreR2Afl/fl Th1 (up) or Th17 (down) cells.

(C) Significantly altered metabolites in PPP2R2A-deficient Th1 (up) or Th17 (down) cells were analyzed by metabolite set enrichment analysis.

Figure 3. PPP2R2A deficiency in T cells enhances NAD+ production through the NR-directed NAD+ biosynthesis pathway

(A) Metabolomics analysis was performed on PPP2R2A wild-type (R2Afl/fl) or PPP2R2A knockout (dLckCreR2Afl/fl) naive CD4 T cells after culture under Th1 or Th17 polarization condition for 24 h (n = 3 mice/group). Relative levels of metabolites involved in the NAD+ biosynthesis in R2Afl/fl and dLckCreR2Afl/fl Th1 or Th17 cells.

(B) Summary of the changes of metabolites in 3 NAD+ biosynthesis pathways in PPP2R2A-deficient Th1 or Th17 cells. The summary was based on the data from (A). NAMN, nicotinic acid mononucleotide; NMNAT1–3: NAM mononucleotide adenyl-transferases 1–3; NAMPT, NAM phosphoribosyltransferase.

(C and D) Quantification of NAD+total (NAD+ and NADH) in R2Afl/fl and dLckCreR2Afl/fl naive CD4 T cells cultured under Th1 or Th17 cell differentiation conditions and in the presence of DMSO or NBTI (10 μM) for 24 h (D). Total NAD+ (NAD+total) levels were measured using a NAD/NADH quantification kit. The data were generated from 3 independent experiments.

(E) R2Afl/fl and dLckCreR2Afl/fl naive CD4 T cells were cultured under Th1 or Th17 cell differentiation conditions for 24 h before RNA was extracted for qPCR analysis. The data were generated from 3 independent experiments. Mean ± SD is shown.

ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 using unpaired t test.

Figure 4. NR inhibits Th1 and Th17 cell differentiation but augments Treg cell differentiation in vitro

(A, B, and E–I) Naive CD4+ T cells isolated from the SPLs of R2Afl/fl mice were cultured under Th1-, Th17-, or Treg-polarizing conditions in the presence of PBS or NR (500 μM) for 72 h (A and B) or 24 h (C, E–I) in vitro.

(D) Naive CD4+ T cells isolated from the SPLs of R2Afl/fl mice were cultured under Th1-, Th17-, or Treg-polarizing conditions in the presence of PBS or NR (500 μM) with DMSO or NBTI (10 μM) for 24 h.

Cells were stained with the indicated antibodies before FACS analysis (A and B) or were lysed using an NAD extraction buffer (C and D). Representative flow cytometry plots are shown in (A). Cumulative data (n = 3 mice/group, duplicate) depicting the percentages of IFN-γ (Th1)- and IL-17A (Th17)-producing cells and Foxp3 (Treg)-expressing cells (B) and NAD+ levels measured by ELISA kit (C and D) are shown.

(E–G) qPCR analysis of indicated gene expression. The data were generated from 3 independent experiments.

(H and I) FACS analysis of p-Stat3 or p-Stat5 expression in Th17 (H) and Treg (I) cells with PBS or NR treatment. Representative flow plots (left) and cumulative data (n = 3 mice/group, duplicate) of mean fluorescence intensity (right) are shown. Mean ± SD is shown.

ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 using unpaired t test.

Figure 5. NR treatment alters the occupancy of histone H1 protein in Il17a and Foxp3 loci

(A) After CD4 naive T cells were cultured under Th17 and Treg cell polarization conditions in the presence or absence of NR for 24 h, protein lysates were extracted for western blot analysis.

(B) IP-MS analysis of PARylated proteins from naive CD4 T cells after culture under Treg cell differentiation conditions in the presence of PBS or NR for 24 h (n =2 mice/group). Significantly altered (|log2 fold change| > 2) PARylated proteins in NR-treated compared to PBS-treated cells are shown in the heatmap.

(C) GO pathway analysis of the PARylated proteins that were significantly altered in NR-treated cells.

(D) After Naive CD4 T cells were cultured under Th17 and Treg cell polarization conditions in the presence of PBS or NR for 24 h, protein lysates were immunoprecipitated with an antibody against H1.2 or IgG, and western blot analysis was applied using the indicated antibodies. Representative blots (left) and cumulative densitometry data (n = 3, right) are shown. In quantification, PARylated H1.2 was first normalized to total H1.2 in PBS- or NR-treated group. Then, PBS-treated group was normalized to 1.

(E and F) ChIP-qPCR assays were performed for H1.2 and H3K36m2 at promoterCNS1 and CNS2 regions in the Foxp3 gene locus and CNS2 region in the Il17a gene locus in CD4 naive T cells cultured under Th17 and Treg cell polarization conditions in the presence of PBS or NR for 24 h. The data are from 3 independent experiments. Mean ± SD is shown.

p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 using unpaired t test.

Figure 6. NR treatment alleviates lupus-related manifestations in MRL.lpr mice

To determine the role of NR in the regulation of lupus-related pathology, we intraperitoneally injected PBS (n = 7) or NR Cl (500 mg/kg body weight/day, n = 8) into 8-week-old MRL.lpr mice daily for 8 weeks. MRL.mpj (n = 6) mice injected with PBS were used as controls.

(A) The representative picture (left) and cumulative data of the cell numbers (right) of SPLs and CLNs (A).

(B–D) The percentages (B) and cell numbers (C) of T cells (CD45+Thy1.2+) and the cell numbers of CD4 (Thy1.2+CD4+CD8−), CD8 (Thy1.2+CD4−CD8+), and DN (Thy1.2+CD4−CD8−) T cells (D) in SPLs and CLNs.

(E and F) The cell numbers of IFN-γ-producing (Thy1.2+CD4+IFN-γ+, Thy1.2+CD8+IFN-γ+, and Thy1.2+CD4−CD8−IFN-γ+; E) and IL-17A-producing (Thy1.2+CD4+IL-17+, Thy1.2+CD8+IL-17+, and Thy1.2+CD4−CD8−IL-17+; F) cells in SPLs and CLNs.

(G) The percentage of Treg cells (Thy1.2+CD4+Foxp3+CD25hi) in SPLs and CLNs.

(H) ELISA measurement of anti-dsDNA IgG levels in the serum.

(I) Proteinuria measured using urine strip.

(J) Representative images of glomerular, perivascular, and tubular histologic findings in kidney sections. The arrows point to a glomerular crescent (upper row) and to tubular epithelial cell vacuolization and flattening (bottom row). H&E; scale bar: 20 μm.

(K) Cumulative data of the histopathologic scores in the kidneys of mice from the indicated treatment groups. Mean ± SD is shown.

*p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 using unpaired t test (A–C, G–I, and K) or multiple t test (D–F).

Figure 7. NR supplement restores PARP1 activity and reduces IFNG and IL17A mRNA expression in CD4 T cells from patients with SLE

(A) Western blot analysis of the protein expression levels of PARP1 and β-actin in CD4 T cells from patients with SLE or matched healthy controls. Healthy controls 1 (H1) is age, sex, and ethnicity matched with SLE patient 1 (L1); H2 is age, sex, and ethnicity matched with L2, and so on.

(B) Cumulative data for quantification of the levels of PARylated PARP1 vs. total PARP1 (top) or cleaved PARP1 vs. full-length PARP1 (bottom) in the blots shown in (A). Healthy controls (HC), n = 14; SLE patients, n = 14.

(C–E) CD4 T cells from patients with SLE were stimulated with anti-CD3/CD28 in the presence of PBS or NR for 24 h. Then, protein lysates were extracted for western blot analysis (C and D), and total RNA was isolated for qPCR analysis (E). Representative blots (C) and cumulative densitometry data (n = 8, D) are shown. n = 12 in €. Mean ± SD is shown.

**p < 0.01 and ****p < 0.0001 using paired t test (B) or multiple t test (D and E).

Table 1. Demographic and clinical characteristics of the patients with SLE and healthy control subjects

	SLE patients (24)	Healthy controls (14)	
Age, y, mean (range)	32.7 (8–59)	20.8 (8–51)	
Sex, no. (%)			
 Female	24 (100)	14 (100)	
 Male	0	0	
Ethnicity, no. (%)			
 White	12 (57.9)	8 (57.1)	
 African American	5 (26.3)	4 (28.6)	
 Asian	4 (15.8)	1 (7.1)	
 Hispanic	3 (13.3)	1 (7.1)	
SLEDAI1, mean (range)	3.67 (0–14)	–	
1 SLEDAI: Systemic lupus erythematosus disease activity index.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies			
	
Antibodies used for flow cytometry are listed in Table S2	N/A	N/A	
Rabbit antibody against PARP1	Santa Cruz Biotechnology	sc-7150; RRID:AB_2160738	
Mouse antibody against PAR	R&D Systems	4335-MC-100; RRID:AB_2572318	
Mouse antibody against β-actin	Sigma-Aldrich	A5316; RRID:AB_476743	
Mouse antibody against PPP2R2A (2G9)	Cell Signaling Technology	#5689; RRID:AB_10827877	
Rabbit antibody against H1.2 (JU43-48)	Novus Biologicals	NBP2-75932; RRID:AB_2810088	
Rabbit antibody against H3K36m2	Millipore-Sigma	07-274; RRID:AB_310484	
Rabbit IgG control	Thermofisher Scientific	49-2024; RRID:AB_2148077	
Goat anti-rabbit IgG (H + L) secondary antibody, HRP	Thermo Fisher Scientific	Cat # 31460; RRID:AB_228341	
Goat anti-Mouse IgG (H + L) Secondary Antibody, HRP	Thermo Fisher Scientific	Cat # 31430; RRID:AB_228307	
anti-CD3	BioLegend	145-2C11; RRID:AB_2616673	
anti-CD28	BioLegend	Clone 37.51; RRID:AB_2810333	
anti–IL-4	BioLegend	QA19A66; RRID:AB_2910503	
anti–IFN-γ	BioLegend	XMG1.2; RRID:AB_2295769	
	
Biological samples			
	
Peripheral venous blood obtained from healthy volunteers as well as patients with SLE	This study	N/A	
	
Chemicals, peptides, and recombinant proteins			
	
Western ECL substrate	Bio-Rad	1705061	
Nicotinamide riboside chloride	ChromaDex	TruNiagen	
Nicotinamide riboside	Combi-Blocks	HB-5832	
IL-12	R&D Systems	419-ML-010	
TGF-β1	R&D Systems	7666-MB-005	
IL-2	R&D Systems	402-ML-020	
IL-6	R&D Systems	406-ML-005	
	
Critical commercial assays			
	
Mouse CD4+CD62L + T cell Isolation Kit.	Miltenyi Biotec	130-104-453	
NAD/NADH Quantitation Kit	Sigma-Aldrich	MAK037	
MAGnify Chromatin Immunoprecipitation System	Life Technologies	49-2024	
RosetteSep™ Human CD4+ T cell Enrichment Cocktail	StemCell Technologies	#15022	
RNeasy Plus Mini kit	QIAGEN	Cat# 74134	
Dynabeads Protein G Immunoprecipitation Kit	Thermo Fisher Scientific	10007D	
	
Experimental models: Organisms/strains			
	
B6.lpr.LckCreR2Afl/fl mice.	This paper	N/A	
B6.lpr.R2Afl/fl mice	This paper	N/A	
B6.LckCreR2Afl/fl mice	This paper	N/A	
B6.R2Afl/fl mice	This paper	N/A	
MRL.lpr mice	The Jackson Laboratory	Stock No: 000485	
MRL.mpj mice	The Jackson Laboratory	Stock No: 000485	
B6.lpr mice	The Jackson Laboratory	Stock No: 000482	
	
Oligonucleotides			
	
qPCR primers are in Table S1	N/A	N/A	
	
Software and algorithms			
	
Prism 7	Graphpad	Version 7.0; RRID:SCR_002798	
MetaboAnalyst 5.0	Online Platform	https://www.metaboanalyst.ca/; RRID:SCR_015539	
Image Lab (ChemiDoc XRS+ System)	Bio-Rad	Version 5.2.1; RRID:SCR_014210	
CytExpert version 2.0	Beckman Coulter	Version 2.0; RRID:SCR_017217	
FlowJo	FlowJo	Version 10.0; RRID:SCR_008520	

Highlights

PPP2R2A deficiency in T cells attenuates autoimmunity and nephritis in lupus-prone mice

PPP2R2A deficiency promotes NAD+ biosynthesis through NR-directed salvage pathway

NR inhibits Th17 and promotes Treg cell differentiation by PARylating histone H1.2

NR inhibits lupus nephritis in mice and limits the proinflammatory cytokines of SLE T cells

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114379.

DECLARATION OF INTERESTS

The authors declare no competing interests.
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