Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jul 23;5(16):e126457.
doi: 10.1172/jci.insight.126457.

PPP2R2B hypermethylation causes acquired apoptosis deficiency in systemic autoimmune diseases

Iris K Madera-Salcedo  1 , Beatriz E Sánchez-Hernández  2 , Yevgeniya Svyryd  2 , Marcela Esquivel-Velázquez  1 , Noé Rodríguez-Rodríguez  1 , María Isabel Trejo-Zambrano  1 , H Benjamín García-González  1 , Gabriela Hernández-Molina  1 , Osvaldo M Mutchinick  2 , Jorge Alcocer-Varela  1 , Florencia Rosetti  1 , José C Crispín  1
Affiliations

PPP2R2B hypermethylation causes acquired apoptosis deficiency in systemic autoimmune diseases

Iris K Madera-Salcedo et al. JCI Insight. .

Abstract

Chronic inflammation causes target organ damage in patients with systemic autoimmune diseases. The factors that allow this protracted response are poorly understood. We analyzed the transcriptional regulation of PPP2R2B (B55ß), a molecule necessary for the termination of the immune response, in patients with autoimmune diseases. Altered expression of B55ß conditioned resistance to cytokine withdrawal-induced death (CWID) in patients with autoimmune diseases. The impaired upregulation of B55ß was caused by inflammation-driven hypermethylation of specific cytosines located within a regulatory element of PPP2R2B preventing CTCF binding. This phenotype could be induced in healthy T cells by exposure to TNF-α. Our results reveal a gene whose expression is affected by an acquired defect, through an epigenetic mechanism, in the setting of systemic autoimmunity. Because failure to remove activated T cells through CWID could contribute to autoimmune pathology, this mechanism illustrates a vicious cycle through which autoimmune inflammation contributes to its own perpetuation.

Keywords: Autoimmunity; Immunology; Lupus.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Expression and induction of B55β is impaired in T cells from patients with systemic autoimmune diseases.
(A) Abundance of the B55β transcript was quantified in activated T cell blasts from healthy donors (HDs) and patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren’s syndrome (SS), before (basal) and 48 hours after IL-2 withdrawal. Data are presented as median and quartiles (25th to 75th). Whiskers represent the 10th and 90th percentiles, and outliers are indicated by circles (HD basal vs. SLE basal P = 0.024; HD 48 hours vs. SLE 48 hours P = 0.002; HD 48 hours vs. SS 48 hours P = 0.012; Mann-Whitney U test) (HD n = 25; SLE n = 32; RA n = 28; SS n = 21). (B) Induction of B55β induced by IL-2 withdrawal (24 hours) is shown (HD basal vs. 24 hours P = 0.005; SLE basal vs. 24 hours P = 0.066; RA basal vs. 24 hours P = 0.064; SS basal vs. 24 hours P = 0.007, paired two-tailed t test) (HD n = 20; SLE n = 20; RA n = 21; SS n = 16). (C) The proportion of controls and patients with increased B55β expression after IL-2 withdrawal is shown. The number in the pie chart indicates the number of controls or patients included in the analysis. Under the corresponding pie chart, χ2 of the distribution ratio (increase/no change/decrease) of HDs versus each autoimmune disease is indicated. IL-2 w, IL-2 withdrawal.
Figure 2
Figure 2. CWID is impaired in T cells that fail to upregulate B55β.
(A) Apoptosis was quantified by flow cytometry in activated T lymphoblasts from patients with AID and HDs, before (basal) and after IL-2 withdrawal (24 and 48 hours). Results are expressed as mean + SEM of annexin V+ DAPI− cells. *P < 0.05, two-tailed t test (HD n = 19; AID n = 59). (B) Apoptosis was compared in T cell subsets from HDs (n = 19) and from patients with AID with normal (n = 30) or defective (n = 25) B55β upregulation at 24 hours of IL-2 w. +P < 0.05 vs. HDs; ++P < 0.01 vs. HDs; ***P < 0.001 vs. AID with normal B55β induction; 2-way ANOVA with Bonferroni’s posttest. (C) Correlation between B55β induction at 24 hours (fold change over basal) and T cell apoptosis at 48 hours (fold change over basal). Spearman’s r = 0.39, and P = 0.004.
Figure 3
Figure 3. Defects in B55β upregulation are not caused by expansion or contraction of the CAG trinucleotide repeat in PPP2R2B.
(A) Schematic representation of the genomic location of the first exon and 5′ untranslated region (UTR) of PPP2R2B. The location of the CAG repeats and of the primers used to quantify the number of repeats in each individual are indicated by an inverted triangle and by arrows, respectively. (B) The distribution of allele length in HDs and patients with SLE and RA is shown (HD n = 20; SLE n = 20; RA n = 21). (C) CAG repeat length in individuals who exhibited normal versus abolished upregulation of B55β during cytokine withdrawal was compared (normal B55β induction n = 21; deficient B55β induction n = 26).
Figure 4
Figure 4. Local CpG DNA methylation is abnormally increased in patients with SLE and RA at the PPP2R2B locus.
(A) Schematic representation of PPP2R2B, indicating the location of DNAse I hypersensitivity sites detected in human naive CD4+ T cells and Th1-differentiated CD4+ T cells (16). Also, the location of a large CpG island, composed of 97 CpG dinucleotides, that encompasses the first exon and the 5′ UTR of the gene is shown. (B) Methylation-specific PCR was performed in T cell genomic DNA after bisulfite conversion. Shown are representative results of 3 HDs, 3 patients with SLE, and 3 patients with RA. M, methylated; U, unmethylated. Band density was quantified and the M/U ratio of each sample is shown. (C) Cumulative data from B presented as CpG methylation index: (M band + U band)/M band. HD n = 20; SLE n = 11; RA n = 12. **P < 0.01; ***P < 0.001; 1-way ANOVA with Tukey’s multiple-comparisons test.
Figure 5
Figure 5. Methylation of discrete cytosines regulates B55β expression in patients with SLE and RA.
(A) Schematic representation of PPP2R2B, indicating the location of the CpG dinucleotides examined by pyrosequencing. (B) Methylation (percentage) of specific CpG dinucleotides in T cells isolated from HDs, patients with SLE, and patients with RA (HD n = 9–20; SLE n = 12–20; RA n = 8–20). RA vs. HD, *P < 0.05; SLE vs. HD, +P < 0.05; 2-way ANOVA with Tukey’s multiple-comparisons test. (C) Differences in the DNA methylation between patients and controls. The diameters of each circle represent the difference between methylation in HDs and the indicated population. The color of each circle indicates the P value of the corresponding comparison. Large differences were found in Amp 1. In addition, significant methylation differences were found in Amp 2 in patients with RA and in Amp 3 in patients with SLE. (D) Heatmap showing relative methylation (fold change over the mean of HDs) of the CpG dinucleotides from Amp 1. Samples were ordered by unsupervised clustering. The colored boxes on the right side of the heatmap indicate whether the sample corresponds to an HD or a patient; the black and white boxes indicate B55β induction after cytokine withdrawal. (E) Segregation of HDs and patients in clusters 1 and 2 (upper) and segregation of individuals with normal or defective B55β induction (lower). P values were calculated using χ2 test.
Figure 6
Figure 6. Methylation of PPP2R2B is associated with systemic inflammation.
(A) Erythrocyte sedimentation rate (ESR), at the time of sample collection, in patients assigned to clusters 1 and 2. *P = 0.02, Mann-Whitney U test. (B) Correlation between ESR and CpG methylation (Amp 1) in patients with SLE and RA. Spearman’s r = 0.758, and P = 0.01. (C) Disease activity score in 28 joints (DAS28) in patients with RA assigned to clusters 1 and 2. ***P < 0.0001, unpaired two-tailed t test. (D) SLE Disease Activity Index (SLEDAI) in patients with SLE from clusters 1 and 2. NS, not significant (unpaired two-tailed t test).
Figure 7
Figure 7. TNF-α induces PPP2R2B methylation, abolishes B55β expression, and impairs CWID in healthy T cells.
(A) T cells from HDs were activated and expanded in the presence of IL-2 for 10 days. In addition to IL-2, at days 0, 2, 4, 6, and 8, the indicated cytokines (TNF-α, IFN-α, IL-6, IL-21, or IL-17) were added to the culture. At day 10, cells were counted, washed, and replated in the absence of IL-2 and proinflammatory cytokines. (B) Apoptosis (annexin V+ SYTOX Orange−) was quantified before (0 hours) and after IL-2 withdrawal (n = 3–6). *P < 0.05; **P < 0.01; 2-way ANOVA with Tukey’s multiple-comparisons test. (C) Expression of B55β was determined (qPCR) before and after (24 and 48 hours) IL-2 withdrawal. Results were normalized against ACTB (Ct) and then against cells expanded in the presence of IL-2 but in the absence of other cytokines (ΔΔCt). *P < 0.05; **P < 0.01; ***P < 0.001; 2-way ANOVA. (D) Methylation of the CpG dinucleotides from Amp 1 was determined by pyrosequencing in cells expanded in IL-2 (dotted line) and compared with the CpG DNA methylation of the same cells expanded in the presence of TNF-α or IL-21 (n = 8–11). *P < 0.05, paired two-tailed t test. (E) The relative change in methylation status of single CpG dinucleotides in response to TNF-α and IL-21 is shown (n = 8–11). *P < 0.05, paired two-tailed t test.
Figure 8
Figure 8. CTCF binding to PPP2R2B is decreased in T cells of patients with AID.
(A) Schematic representation of PPP2R2B, indicating the location of the region that was analyzed (Amp 1). (B) CTCF motif present in Amp 1. (C) Quantification (ChIP-qPCR) of CTCF binding at Amp 1 in healthy T cells activated and expanded (basal) versus T cells expanded in the presence of TNF-α, n = 3. (D) CTCF binding to Amp 1 in activated T cells for HDs and patients with AID, n = 4. Relative CTCF enrichment was normalized against basal in C or HDs in D. Results are expressed as mean + SEM. *P < 0.05, paired two-tailed t test.
Figure 9
Figure 9. Mechanism through which PPP2R2B modulates the perpetuation of T cells in patients with AID.
(A) After activation and expansion of healthy T cells, levels of IL-2 decrease, promoting clonal contraction by apoptosis. Low IL-2 concentration induces the expression of B55β through the binding of CTCF to a motif located within the CpG island of the PPP2R2B promoter. This mechanism promotes the termination of the immune response. (B) In the context of autoimmunity, where T cells are exposed to a proinflammatory environment, PPP2R2B becomes hypermethylated, preventing the binding of CTCF. This impairs the induction of B55β and apoptosis of T cells when IL-2 levels are low. In consequence, survival of self-reactive activated T cells is facilitated and the autoimmune response is perpetuated.
See this image and copyright information in PMC

References

    1. Flores-Mendoza G, Sansón SP, Rodríguez-Castro S, Crispín JC, Rosetti F. Mechanisms of tissue injury in lupus nephritis. Trends Mol Med. 2018;24(4):364–378. doi: 10.1016/j.molmed.2018.02.003. - DOI - PubMed
    1. Dominguez-Villar M, Hafler DA. Regulatory T cells in autoimmune disease. Nat Immunol. 2018;19(7):665–673. doi: 10.1038/s41590-018-0120-4. - DOI - PMC - PubMed
    1. McKinney EF, et al. A CD8+ T cell transcription signature predicts prognosis in autoimmune disease. Nat Med. 2010;16(5):586–91. doi: 10.1038/nm.2130. - DOI - PMC - PubMed
    1. McKinney EF, Lee JC, Jayne DR, Lyons PA, Smith KG. T-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection. Nature. 2015;523(7562):612–616. doi: 10.1038/nature14468. - DOI - PMC - PubMed
    1. Shi Y. Serine/threonine phosphatases: mechanism through structure. Cell. 2009;139(3):468–484. doi: 10.1016/j.cell.2009.10.006. - DOI - PubMed

Publication types

MeSH terms