Phosphofructokinase P fine-tunes T regulatory cell metabolism, function, and stability in systemic autoimmunity

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by defective regulatory T (T_reg) cells. Here, we demonstrate that a T cell-specific deletion of calcium/calmodulin-dependent protein kinase 4 (CaMK4) improves disease in B6.lpr lupus-prone mice and expands T_reg cells. Mechanistically, CaMK4 phosphorylates the glycolysis rate-limiting enzyme 6-phosphofructokinase, platelet type (PFKP) and promotes aerobic glycolysis, while its end product fructose-1,6-biphosphate suppresses oxidative metabolism. In T_reg cells, a CRISPR-Cas9-enabled Pfkp deletion recapitulated the metabolism of Camk4^-/- T_reg cells and improved their function and stability in vitro and in vivo. In SLE CD4⁺ T cells, PFKP enzymatic activity correlated with SLE disease activity and pharmacologic inhibition of CaMK4-normalized PFKP activity, leading to enhanced T_reg cell function. In conclusion, we provide molecular insights in the defective metabolism and function of T_reg cells in SLE and identify PFKP as a target to fine-tune T_reg cell metabolism and thereby restore their function.

Fig. 1.. T cell–specific CaMK4 deficiency expands the T_reg cell compartment and ameliorates disease in lupus-prone mice.

Thirty-two–week-old C57Bl/6.lpr mice with (B6.lpr.Camk4^fl/fl.dlckCre, blue; n = 5) or without (C5B6.lpr.Camk4^fl/fl, red; n = 7) T cell–specific CaMK4 knockout were studied. Data are from two independent experiments. (A) Representative pictures of the spleen and cervical lymph node. (B and C) Spleen weight (B) and cellularity (C) at week 32. (D and E) Percentage of IL-17A⁺–producing cells (D) and CD25⁺FoxP3⁺ (T_reg) (E) cells among CD4⁺ T cells in the spleen and the peripheral blood of the mice. (F) Serum dsDNA antibodies were measured using enzyme-linked immunosorbent assay (ELISA). (G) Results from urine strip measuring proteinuria [results ranging from absent (0) to ++++ (4)]. (H) Representative periodic acid Schiff (PAS) staining of kidney tissues. (I and J) Glomeruli (I) and tubular pathology scores (J) were evaluated. Each point indicates the result from one mouse; bars represent means ± SEM. ns, not significant; *P < 0.05; **P < 0.01 using bilateral Student’s t test.

Fig. 2.. CaMK4 affects T cell glycolytic and oxidative metabolism during T_reg cell differentiation.

(A) CD62L⁺CD4⁺ T cells were isolated from the spleen of C57Bl/6 or Camk4^−/− mice and differentiated to iT_reg cells. The percentage of CD25⁺FoxP3⁺ iT_reg cells was evaluated using flow cytometry at day 3. Representative cytometry results (left) and the cumulative results (right; n = 4) are shown. (B) CD62L⁺CD4⁺ T cells of C57Bl/6 mice were differentiated to iT_reg cells and transfected at day 2 of culture with an empty vector or a CaMK4-OE vector. iT_reg cell differentiation was evaluated at day 3 using flow cytometry on viable transfected (GFP⁺) cells (n = 4). (C and D) CD62L⁺CD4⁺ T cells were isolated from the spleen of C57Bl/6 or Camk4^−/− mice were differentiated to iT_reg cells. Representative results of Seahorse XFe glycolysis stress test (C) or mitochondrial stress test (D) at 8, 24, or 72 hours. ECAR, extracellular acidification rate; 2-DG, 2-deoxyglucose; OCR, oxygen consumption rate. (E) Cumulative results of the basal respiration rate (left), the adenosine triphosphate (ATP)–linked respiration rate (middle), and the maximal respiration rate (right) at 8, 24, and 72 hours of wild-type (WT) and Camk4^−/− (KO) iT_reg cells (n = 3 independent experiments per time point with three technical replicates each). (F) WT and Camk4^−/− (KO) iT_reg cells were stained with MitoSOX (2 μM) and evaluated using flow cytometry. Representative (left) and cumulative data (right) of MitoSOX mean fluorescence intensity (MFI) are shown (n = 3 biological replicates per time point). (G) Total DNA from iT_reg from WT and Camk4^−/− mice were extracted, and a quantitative polymerase chain reaction (qPCR) was conducted using NADH dehydrogenase subunit 1 gene (ND1) probe to evaluate mitochondrial DNA (mtDNA). MtDNA copies per cell were calculated by the formula 2^−ΔCt. *P < 0.05 and **P < 0.01 using unpaired bilateral Student’s t test (A, F, and G) or paired bilateral Student’s t test (B and E).

Fig. 3.. CaMK4 controls the activity of the rate-limiting glycolysis enzyme PFKP at a posttranslational level.

(A) The metabolites from WT or Camk4^−/− T cells after 8 hours of differentiation under iT_reg cell–polarizing conditions were extracted and semiquantitatively measured using liquid chromatography–mass spectrometry (LC-MS; n = 3 biological replicates per genotype). Heatmap showing the average normalized level of metabolites from the glycolysis, pentose phosphate pathway, and the cell energy balance from three biological replicates. *P < 0.10, **P < 0.05, and ***P < 0.01 using multiple unpaired Student’s t test with single pooled variance. (B) Metabolites from the glycolysis pathway are shown in a color reflecting their relative level in each genotype: increased in Camk4^−/− (red, P < 0.10), decreased in Camk4^−/− (blue, P < 0.10), or unchanged (black). (C) The relative expression of the three PFK isoforms were measured gene using reverse transcription qPCR (RT-qPCR) in WT and Camk4^−/− iT_reg cells (n = 3). Expressions of PFK isoforms are relative to the housekeeping gene. (D) PFKP expression was compared between WT and Camk4^−/− iT_reg cells using the ΔΔCt method (n = 5). (E) Western blot of WT and Camk4^−/− iTreg cells showing PFKP and β-actin (left) and cumulative densitometry results (right; n = 4). (F) PFK enzymatic activity of WT and Camk4^−/− iT_reg cell lysates was measured using a colorimetric assay (n = 5). (G) PFK enzymatic activity was measured in the lysates of Camk4^−/− iT_reg cells transfected with either an empty vector or a CaMK4-OE vector (n = 5). *P < 0.05 and **P < 0.01 using unpaired bilateral Student’s t test. ****P < 0.0001 using one-way ANOVA with Holm-Sidak’s correction.

Fig. 4.. CaMK4 phosphorylates serine-539 of PFKP and affects iT_reg cell differentiation.

(A) CD62L⁺ CD4⁺ T cells from Camk4^−/− mice were differentiated in iT_reg cells and transfected with FLAG-tagged CaMK4-OE vector on day 2. At day 3, proteins were extracted from cell lysate and immunoprecipitated with an anti-FLAG antibody. Western blot showing CaMK4 and PFKP. IgG, immunoglobulin G; IB, ImmunoBlot. (B) Cell lysates from WT and Camk4^−/− iT_reg cells were immunoprecipitated with a PFKP antibody. Western blot of the immunoprecipitates revealing phospho-serine residues (top) and PFKP (bottom). (C) Schematic of the phosphosproteomics experiment. Cell lysates from WT and Camk4^−/− iT_reg cells were immunoprecipitated with a PFKP antibody and migrated on a NuPAGE bis-tris gel. The band corresponding to PFKP was cut and processed by LC-MS to identify phosphorylated residues on PFKP. Data are from two independent experiments. (D) Site-directed mutagenesis was conducted on a PFKP-OE vector to substitute S162 or S539 by an alanine residue (PFKP S162A and PFKP S539A, respectively). CD62L⁺ CD4⁺ T cells from C57Bl/6.FoxP3.Gfp mice were transfected at day 2 of culture with an empty vector or a PFKP-OE vector, and iT_reg cell differentiation was measured at day 3 on DsRed⁺ (transfected) viable cells using flow cytometry. Representative of cell differentiation (left) and cumulative data of three independent experiments (right). **P < 0.01 and ***P < 0.001 using one-way ANOVA test with Holm-Sidak’s correction.

Fig. 5.. The CaMK4/PFKP axis affects iT_reg cell metabolic rheostat and differentiation through PFKP end product F-1,6P.

(A) iT_reg cells were differentiated from WT or Camk4^−/− CD62L⁺ CD4⁺ T cells. After 24 hours of differentiation, protein lysates were extracted, and Western blot was conducted. The left panel shows the stained membrane, and the right panel indicates the dosimetry measurement of Thr¹⁷²-phosphorylated AMPK (n = 5). (B) iT_reg cells were differentiated from Camk4^−/− CD62L⁺ CD4⁺ T cells and transfected with an empty vector or a CaMK4-OE vector. Representative Western blot of iT_reg cell lysates at 24 hours after transfection. (C to F) iT_reg cells were differentiated from Camk4^−/− CD62L⁺ CD4⁺ T cells with 1 mM F-1,6P or F-6P. (C) AMPK phosphorylation status (n = 4). (D) Mean fluorescence intensity of MitoSOX among living cells (n = 4). (E) Mitochondrial content was measured using qPCR (n = 4). (F) Percentage of CD25⁺FoxP3^hi iT_reg cells at day 3 of differentiation. *P < 0.05, **P < 0.01, and ***P < 0.001 using unpaired Student’s t test (B), paired one-way ANOVA with Holm-Sidak’s correction (C to E), or unpaired one-way ANOVA with Holm-Sidak’s correction (F).

Fig. 6.. CRISPR-Cas9–mediated PFKP knockdown alters the metabolism of iT_reg cells and affects their in vitro immunosuppressive function.

CD62L⁺ CD4⁺ T cells from CRISPR-Cas9–expressing mice were differentiated into iT_reg cells and transfected at day 1 with control or Pfkp target sgRNAs. (A) Western blot showing PFKP expression in iT_reg cell lysates. (B) Phosphofructokinase activity of T_reg cell lysates (n = 3). (C) Representative of a Seahorse glycolysis stress test of control (red) or Pfkp target (blue) iT_reg cells. (D) Representative Western blot (left) and cumulative densitometry result (right; n = 4) showing phospho-AMPK and total AMPK in control and PFKP target iT_reg cells. (E) Mean florescence intensity of MitoSOX marker in control and Pfkp target iT_reg cells (n = 3). (F) Representative of a Seahorse mitochondrial stress test of control (red) and Pfkp target (blue) iT_reg cells. (G) Percentage of CD25⁺ FoxP3⁺ among control and Pfkp target iT_reg cells was measured using flow cytometry at day 3 of differentiation. Representative flow plot (left) and cumulative results (right) are shown (n = 4). (H) Control or Pfkp target iT_reg cells were cocultured for 3 days with CellTrace Violet–stained T_conv cells (T_reg/T_conv cells, ratio of 1:1 to 1:4) and antigen-presenting cells together with CD3/CD28 stimulation. Representative proliferation of T_conv cells under different coculture ratio (left) and cumulative results of iT_reg cell immunosuppression activity (right; n = 3 independent experiments of three replicates each). Each dot represents the value of one biological replicate, and bars indicate means and SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 using unpaired bilateral Student’s t test.

Fig. 7.. PFKP modulation improves the iT_reg cell in vivo function and stability in the adoptive transfer colitis model.

(A) Design of the in vivo experiment. Cas9-GFP expressing CD62L⁺ CD4⁺ T cells were transfected with control of Pfkp target sgRNA and differentiated into iT_reg cells. CD45RB^hi CD4⁺ T (T_conv) cells were sorted using fluorescence-activated cell sorting (FACS) from CD4⁺ T cells presorted from the splenocytes of C57Bl/6 mice. T cell–deficient RAG1^−/− mice were transferred with 4 × 10⁶ T_conv and 5 × 10⁵ iT_reg. Data are from three independent experiments of two to three mice per condition. (B) Weight of mice transferred with T_conv cells only (purple, n = 4), T_conv and control iT_reg cells (red, n = 8), or T_conv and Pfkp target iT_reg cells (blue, n = 8). (C) Representative colon pathology of RAG1^−/− mice at the end of the adoptive transfer experiment. Scale bars, 100 μm. (D) After sacrifice, the percentage of CD25⁺FoxP3⁺ T_reg cells was evaluated among initially transferred iT_reg identified as GFP⁺ CD4⁺ T cells, in the spleen and in mesenteric lymph nodes (mLNs; n = 8). (E) After sacrifice, the percentage of IL-17A–expressing cells was evaluated among initially transferred iT_reg identified as GFP⁺ CD4⁺ T cells, in the spleen and in mLNs (n = 8). (B) Points indicate the mean of the group, and bars show SEM. **P < 0.01 and ***P < 0.001 using two-way ANOVA with Holm-Sidak’s correction. (E and F) Each point indicates a biological replicate, and bars show means and SEM. ***P < 0.001 using one-way ANOVA test with Holm-Sidak’s correction.

Fig. 8.. CaMK4 regulates PFKP activity in SLE CD4⁺ T cells and impairs human T_reg cell immunosuppressive functions.

(A) RT-qPCR of the three PKF isoforms in CD4⁺ T cells of HDs (n = 6) and CD4⁺ T cells of patients with SLE (n = 6). (B) PFK activity was measured from the lysate of CD4⁺ T cells cultured during 24 hours with CD3/CD28 activation. Samples from HDs (n = 11) and patients with SLE (n = 22). (C) Spearman correlation of the CD4⁺ T cell PFK activity and the SLEDAI (n = 22).The delimited gray area indicates the 95% confidence interval of the linear regression. (D) PFK activity was measured from the lysate of CD4⁺ T cells cultured during 24 hours with CD3/CD28 activation with or without KN93 (10 μM; n = 22). (E and F) Immunosuppressive assay were conducted by sorting T_conv and T_reg cells using FACS from HD. T_conv cells stained with CellTrace Violet proliferation marker were cultured either alone (upper panel) or with T_reg cells at a 1:1 ratio (bottom), with CD3/CD28 activation ± KN93 (10 μM). Cells were stained for viability, and proliferation was assessed using flow cytometry at day 7. (E) Representative results of T_conv cells proliferation. (F) The percentage of immunosuppression was calculated by comparing T_reg/T_conv cell proliferation to T_conv cell proliferation from the corresponding condition (n = 4 biological replicates). Each point represents one donor, and lines and bars indicate means and SEM. **P < 0.01 using nonparametric Mann-Whitney test (B), nonparametric Wilcoxon test (D), or unpaired two-tailed Student’s t test (F). ****P < 0.0001 using paired one-way ANOVA with Holm-Sidak’s correction. DMSO, dimethyl sulfoxide.