Mitochondrial protein CMPK2 regulates IFN alpha-enhanced foam cell formation, potentially contributing to premature atherosclerosis in SLE

Abstract

Background: Premature atherosclerosis occurs in patients with SLE; however, the mechanisms remain unclear. Both mitochondrial machinery and proinflammatory cytokine interferon alpha (IFN-α) potentially contribute to atherogenic processes in SLE. Here, we explore the roles of the mitochondrial protein cytidine/uridine monophosphate kinase 2 (CMPK2) in IFN-α-mediated pro-atherogenic events.

Methods: Foam cell measurements were performed by oil red O staining, Dil-oxLDL uptake and the BODIPY approach. The mRNA and protein levels were measured by qPCR and Western blotting, respectively. Isolation of CD4+ T cells and monocytes was performed with monoclonal antibodies conjugated with microbeads. Manipulation of protein expression was conducted by either small interference RNA (siRNA) knockdown or CRISPR/Cas9 knockout. The expression of mitochondrial reactive oxygen species (mtROS) was determined by flow cytometry and confocal microscopy.

Results: IFN-α enhanced oxLDL-induced foam cell formation and Dil-oxLDL uptake by macrophages. In addition to IFN-α, several triggers of atherosclerosis, including thrombin and IFN-γ, can induce CMPK2 expression, which was elevated in CD4+ T cells and CD14+ monocytes isolated from SLE patients compared to those isolated from controls. The analysis of cellular subfractions revealed that CMPK2 was present in both mitochondrial and cytosolic fractions. IFN-α-induced CMPK2 expression was inhibited by Janus kinase (JAK)1/2 and tyrosine kinase 2 (Tyk2) inhibitors. Both the knockdown and knockout of CMPK2 attenuated IFN-α-mediated foam cell formation, which involved the reduction of scavenger receptor class A (SR-A) expression. CMPK2 also regulated IFN-α-enhanced mtROS production and inflammasome activation.

Conclusions: The study suggests that CMPK2 plays contributing roles in the pro-atherogenic effects of IFN-α.

Keywords: Atherosclerosis; CMPK2; Foam cell formation; Interferon alpha; Macrophages; Mitochondria.

Fig. 1

Induction of CMPK2 by immune cells from SLE patients. Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats according to the “Materials and methods” section. Cells (3 × 10⁶ cells/ml) were treated in the presence or absence of immune complexes formed by mixing U937 cellular extract (1%) and serum (1:1000 dilution) from three different SLE donors for 24 h. The cells were collected, and total RNA was prepared. Then, the levels of CMPK2 mRNA were analyzed with qPCR. The data are presented as the means ± SD (a). PBMCs, CD14+ monocytes, and CD4+ T lymphocytes were prepared from the whole blood of SLE patients or healthy controls. The levels of CMPK2 were determined by qPCR (b). CMPK2, cytidine/uridine monophosphate kinase 2

Fig. 2

IFN-α enhanced foam cell formation. THP-1-derived macrophages (TDMs), Raw 264.7 cells, and mouse bone marrow-derived macrophages (BMDMs) were stimulated with oxLDL in the presence or absence of IFN-α, and foam cell formation was measured by oil red O staining (a, left). The cells were examined by light microscopy and the percentages of oil red O positive cells in 5 microscopic fields for each independent experiment were counted and calculated (a, right). One dot stands for an independent experiment. The extent of Dil-oxLDL uptake by BMDMs was determined and statistically analyzed and the results are shown (b, n = 6). Similar to the finding shown in b, the extent of Dil-oxLDL uptake by TDMs was determined (c, n = 4). Foam cell formation induced by different stimuli, including LDL and cholesterol crystals (CCs), was measured (d). Asterisks indicate values that are significantly different from the relevant control (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001). n, sample numbers of independent experiments

Fig. 3

Signaling pathways involved in the activation and regulation of CMPK2. BMDMs were treated with different doses of IFN-α, IFN-γ, thrombin, lipopolysaccharide (LPS), Pam3CSK4, Poly(I:C), IL-1β, CpG ODN1826, or CpG ODN1585, and the expression of CMPK2 mRNA was measured by qPCR (a). The mRNA expression of CMPK2, SR-A, and ABCG1 was determined in TDMs treated with different doses of IFN-α (b). TDMs treated with 100 U/ml IFN-α were collected at different time points, and the protein levels of CMPK2, SR-A, and actin were measured by Western blotting (c). TDMs pretreated for 2 h with different doses of chemical compounds, including LY294002, PD98059, SP600125, SB203580, AG490, ruxolitinib, BMS-986165, or DMSO, were also treated with IFN-α for 24 h, and then, the cells were collected for the determination of CMPK2 mRNA by qPCR (D, left part) and in part by Western blotting (d, right part), respectively. Asterisks indicate values that are significantly different from the relevant control (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001)

Fig. 4

Effects of CMPK2 knockdown on BMDMs and Raw 264.7 cells stimulated by IFN-α. Raw 264.7 cells were treated with oxLDL in the presence or absence of IFN-α for 24 h. The expression of CMPK2 mRNA and protein was measured by qPCR and Western blotting, respectively (a). Foam cell formation was measured by oil red O staining (b), and BODIPY analysis was also carried out according to the description in the “Materials and methods” section (c). Similar analyses were performed with BMDMs (d–f). Asterisks indicate values that are significantly different from the relevant control (*P < 0.05, **P < 0.01, and ***P < 0.001)

Fig. 5

Effect of CMPK2 knockout on TDMs. The CMPK2 gene was knocked out through the CRISPR/Cas9 approach in THP-1 cells, as explained in detail in the “Materials and methods” section, and two clones, #3-2 and #3-8, were obtained; their partial sequences are shown (a). The expression of CMPK2 induced by IFN-α was completely abolished in CMPK2-knockout (KO) cells, as shown by Western blotting (b). The level of IFN-α-mediated enhancement as measured in the oxLDL-treated TDMs was significantly lower in the CMPK2-KO cells, compared to the wild-type controls (c). Analysis with BODIPY revealed results consistent with the results from oil red O staining (d). The uptake of Dil-oxLDL induced by IFN-α treatment was reduced in the CMPK2-KO cells (e). IFN-α-induced expression of SR-A was determined by Western blotting (f). IFN-α-induced expression of CMPK2 in mitochondrial and cytosolic fractions of cellular lysates was determined by Western blotting (g, the results from 3 independent experiments were shown in Supplementary Figure 5). Asterisks indicate values that are significantly different from the relevant control (*P < 0.05)

Fig. 6

Effects of CMPK2-KO on mtROS production. Wild-type TDMs (#WT) and CMPK2-KO clones #3-2 and #3-8 were treated with IFN-α, oxLDL, or IFN-α+oxLDL for 24 h, and the generation of mtROS was detected by MitoSOX staining and then analyzed by flow cytometry (A-1). Representative flow cytometry results are shown in A-2. The unstimulated control is stained gray, and orange, blue and purple represent the conditions in the presence of different stimuli. In parallel, representative images of wild-type TDMs, clone #3-2 and clone #3-8 labeled with MitoSOX+DAPI (merged pictures) under different conditions are shown in A-3. The results (A) of the wild-type TDMs and clone #3-2 or clone #3-8 were further analyzed and compared (B). TDMs were transfected with GFP or CMPK2-GFP to induce the overexpression of CMPK2 and then stimulated or not with oxLDL, and the production of mtROS was measured by flow cytometry (C). The stimulation with rotenone served as a positive control. The results of the effects of CMPK2 knockdown on oxLDL-, IFN-α-, or oxLDL+IFN-α-induced mtROS production in BMDMs were determined (D). Scale bar = 25 μm in A-3. Asterisks indicate values that are significantly different from the relevant control (*P < 0.05, **P < 0.01, and ***P < 0.001)