Session Type: ACR Abstract Session
Session Time: 4:30PM-6:00PM
Background/Purpose: Systemic lupus erythematosus (SLE) is disease characterized by an imbalance between pro-inflammatory (such as Th1 and Th17) and regulatory cells (Tregs). Th1 and Th17 T cells preferentially rely on glucose as opposed to Treg that utilize fatty acids as an energy source. Treatment of lupus-prone mice with 2DG, a glycolysis inhibitor, reduced disease activity by normalizing T cell metabolism. Our previous work showed that calcium/calmodulin-dependent protein kinase IV (CaMK4) is overexpressed in SLE CD4 T cells reprogramming T cells towards a proinflammatory phenotype. Based on mass spectrometry data, we hypothesized that CaMK4 affects SLE T cell phenotype by regulating the expression of phosphofructokinase (PFKP), a key regulatory enzyme of glycolysis.
Methods: CD4+ cells were isolated from healthy donors (HD), patients with SLE (SLEDAI 0-26) or mice (control, Camk4-/-, MRL-lpr). RNA-sequencing was performed by BGI; CUFFDIFF2 was used for analysis. PFKP was detected by RT-PCR and Western blotting. Glycolysis was measured by ECAR on Seahorse XFp analyzer. Proteins bound to CaMK4 were detected by mass-spectrometry after performing a pull-down assay with Flag-tagged human CaMK4 overexpressed in HEK293T cells.
Results: To address whether CaMK4 is involved in glycolysis we measured ECAR in CD3/CD28-ionomycin stimulated CaMK4-deficient and -sufficient CD4+ cells. Cells lacking CaMK4 had significantly decreased glycolysis (n=4; p< 0.05) suggesting that CaMK4 promotes glycolysis. To elucidate the mechanistic link between CaMK4 and metabolic reprogramming we performed a pull-down assay followed by mass-spectrometry that identified PFKP as a binding partner. Next, we examined whether the CaMK4-PFKP interaction is important in T cell differentiation. Naïve CD4 cells were cultured under Th1 or Treg-polarizing conditions. We observed 4.8-fold decrease of PFKP mRNA expression in wild type Tregs which was further reduced by 58% in Tregs lacking CaMK4. Moreover, the differentiation potential toward Tregs and IL-2 production was by 18% greater in CD4 T cells lacking CaMK4. This was in contrast to wild type CD4 T cells in which PFKP was overexpressed. We analyzed PFKP expression in CD4 T cells obtained from patients with SLE and found a statistically significant upregulation (vs. HD; n=14; p< 0.01). PFKP mRNA expression was higher in patients with active disease as measured by SLEDAI (p=0.003) even after adjusting for age or sex and degree of immunosuppression (p=0.01). PFKP upregulation in T cells obtained from SLE patients, compared to HD, was confirmed at the protein level as well (p< 0.05). Similarly, PFKP protein was significantly upregulated in MRL.lpr CD4 T cells compared to cells from control mice (n=8; p< 0.05).
Conclusion: We have generated evidence that CaMK4 interacts with PFKP to promote glycolysis and limit regulatory potential of Tregs by suppressing their differentiation and IL-2 production. In addition, we found that PFKP is significantly overexpressed in CD4 T cells from patients with SLE and lupus-prone mice. Our data identify PFKP as a novel therapeutic target to restore Treg functional homeostasis.
To cite this abstract in AMA style:Vukelic M, Yoshida N, Umeda M, Orite S, Bhargava R, Kono M, Gavanescu-Stockton I, Hisada R, Tsokos G. Calcium/ Calmodulin – Dependent Protein Kinase IV Associates with Phosphofructokinase to Promote Glycolysis and Limit IL-2 Production [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/calcium-calmodulin-dependent-protein-kinase-iv-associates-with-phosphofructokinase-to-promote-glycolysis-and-limit-il-2-production/. Accessed June 21, 2021.
« Back to 2019 ACR/ARP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/calcium-calmodulin-dependent-protein-kinase-iv-associates-with-phosphofructokinase-to-promote-glycolysis-and-limit-il-2-production/