The mitochondrial gene-CMPK2 functions as a rheostat for macrophage homeostasis

Abstract

In addition to their role in cellular energy production, mitochondria are increasingly recognized as regulators of the innate immune response of phagocytes. Here, we demonstrate that altering expression levels of the mitochondria-associated enzyme, cytidine monophosphate kinase 2 (CMPK2), disrupts mitochondrial physiology and significantly deregulates the resting immune homeostasis of macrophages. Both CMPK2 silenced and constitutively overexpressing macrophage lines portray mitochondrial stress with marked depolarization of their membrane potential, enhanced reactive oxygen species (ROS), and disturbed architecture culminating in the enhanced expression of the pro-inflammatory genes IL1β, TNFα, and IL8. Interestingly, the long-term modulation of CMPK2 expression resulted in an increased glycolytic flux of macrophages akin to the altered physiological state of activated M1 macrophages. While infection-induced inflammation for restricting pathogens is regulated, our observation of a total dysregulation of basal inflammation by bidirectional alteration of CMPK2 expression only highlights the critical role of this gene in mitochondria-mediated control of inflammation.

Keywords: CMPK2; M1 macrophage; immuno-metabolism; infection; mitochondria.

Figure 1

CMPK2 expression is induced following TLR4 stimulation of infection. (A–F) Analysis of CMPK2 expression in THP1 macrophages following: infection with Mtb at a MOI of 5 (A), stimulation with various TLR ligands—c, untreated cells; L, LPS (10 ng/ml); P, Pam3CSK4 (20 ng/ml); Z, Zymosan (10 µg/ml); I, poly IC (2 µg/ml), (B) LPS stimulation by immunoblotting with CMPK2-specific antibody, (C) LPS stimulation with and without TLR4 inhibitor CLI095, (D) Infection with STM, (E) Infection with STM in the presence of CLI095, (F) Expression of CMPK2 was quantitated by qPCR. Values are normalized with GAPDH and mean fold change compared to control cells ± SEM from N = 3 independent experiments. Mtb, Mycobacterium tuberculosis; MOI, multiplicity of infection; LPS, lipopolysaccharide; STM, Salmonella enterica subsp. enterica serovar Typhimurium. *p<0.05, ***p<0.001.

Figure 2

CMPK2 regulates the basal inflammation of macrophages. (A) Expression of CMPK2 in CMPK2 silenced macrophages by immunoblotting with specific antibodies. For immunoblotting, the expression of α-TUBULIN was used as control. One representative blot of three independent experiments is shown. The levels of CMPK2 expression are depicted as mean fold change ± SEM of N = 3 experiments by densitometry. (B) Expression of the IL1β and TNFα transcripts following infection with Mtb for 6 h was evaluated in NT or KD macrophages by qPCR. (C) Basal level expression of transcripts CMPK2 and cytokine genes in KD macrophages was estimated by qPCR with specific primers. Values are normalized with GAPDH and mean fold change compared to control cells ± SEM from N = 3 independent experiments. (D) Levels of TNFα and IL1β in the culture supernatants of THP1 stably expressing non-targeting (NT) or siRNA specific to CMPK2 (KD) estimated by ELISA. Values are represented as mean pg/ml of the cytokine in triplicate assays from N = 3 experiments. (E) Expression of CMPK2 in the CMPK2 silenced cells (KD) with stable expression of CMPK2 (KD-OE) or empty vector (KD-VC). The levels in the corresponding NT and KD cells are also shown. Values are represented as mean ± SEM of the triplicate assays from N = 3 experiments. (F) Expression of TNFα and IL1β in the CMPK2 silenced cells with stable expression of CMPK2 (KD-OE) or empty vector (KD-VC). The levels in the corresponding NT and KD cells are also shown. Values are represented as mean ± SEM of the triplicate assays from N = 3 experiments. (G) Expression of TNFα and IL1β in THP1 cells after stable expression of CMPK2. Values are represented as mean ± SEM of the triplicate assays from N = 3 experiments. (H) Analysis of activation of the ERK (p42/44) and NFκB (p65) signaling pathways in NT, KD, VC, and OE macrophages by immunoblotting with antibodies specific for the phosphorylated (active) and non-phosphorylated forms of the proteins. Representative blot of one experiment out of three individual assays is shown. Expression of α-TUBULIN was used as control. (I) ERK phosphorylation in macrophages with and without treatment with ERK specific inhibitor U0126. Antibodies specific for the phosphorylated (active) and non-phosphorylated forms of the proteins were used to probe cell extracts. Expression of α-TUBULIN was used as control. Blots are representative of two independent experiments. Expression of IL1β (J) and TNFα (K) in the four types of macrophages after treatment with U0126 was analyzed by qPCR. Cells left untreated were used as control. The relative gene expression fold in triplicate assay wells is represented with respect to GAPDH as mean ± SEM for N = 3. (L) The schematic of mutated catalytic site also depicted (D330A). Immunoblot analysis of CMPK2-mCherry fusion protein using an antibody specific for mCherry protein in protein lysates of VC, OE, and D330A THP1 cells with mCherry specific antibody. GAPDH was used as control. (M) Expression of TNFα and IL1β in THP1 cells after stable expression of vector alone (VC), full-length CMPK2 (OE), and catalytic mutant of CMPK2 (D330A). The relative gene expression fold in triplicate assay wells is represented with respect to GAPDH as mean ± SEM from N = 4. Mtb, Mycobacterium tuberculosis. *p<0.05, **p<0.01, ***p<0.001. nd, not detected.

Figure 3

Modulation of CMPK2 affects the mitochondrial physiology in macrophages. (A) Expression of CMPK2 in subcellular fractions of THP1 macrophages by immunoblotting with specific antibodies: T, total cell extract; C, cytoplasmic fraction; M, mitochondria. Expression of cytosolic protein α-TUBULIN and mitochondria resident protein VDAC is also represented. Blots are representative of three independent experiments. (B) Mitochondrial fraction was subjected to Proteinase K treatment in the presence or absence of Triton X-100 and given osmotic shock (OS) and analyzed by immunoblotting. Data are representative of three independent experiments. (C) Mitochondrial fraction was incubated with mitochondrial buffer with and without Na₂CO₃ and centrifuged at 13,000 rpm for 15 min. The pellet (P) and supernatant (S) fractions were immunoblotted. Data are representative of three independent experiments. (D) Kinetic profile of IL1β expression in NT and KD during differentiation of monocytes to macrophages. Expression was checked at different time intervals after PMA treatment. Values are mean fold change in expression with respect to GAPDH + SEM triplicate assays of N = 3 experiments. (E) Analysis of mitochondrial ROS in NT or KD macrophages. Cells were stained with MitoTracker Deep Red (as an internal control), and ROS-specific MitoSOX Red and specific populations were quantified by FACS. The histogram plots of a representative experiment of N = 4 are depicted. The extent of MitoSOX red mean fluorescence intensity (MFI) + SEM is represented graphically in the inset. (F) Expression of IL1β in the macrophages with the addition of a specific mitochondrial ROS inhibitor MQ (1 µM) on day 1 of PMA treatment was analyzed by qPCR. Values are mean fold change in expression with respect to GAPDH + SEM for triplicate assays of N = 3 experiments. (G) Analysis of mitochondrial membrane potential in NT, KD, VC, and OE macrophages by TMRE staining. Fluorescence values normalized to protein in samples are represented as mean fluorescent intensity + SEM for triplicate assays of N = 3 experiments. (H) Expression of total and phosphorylated (S616) DNM1L by immunoblotting with specific antibody was analyzed and is depicted along with the levels of α-TUBULIN as a control. Relative intensity values are depicted as mean+ SEM of N = 3. (I, J) Mitochondrial architecture in control (NT) and KD (I) or VC and OE (J) macrophages were analyzed by confocal microscopy. A representative image is depicted with the scale bar representing 10 µm, and the region is shown in higher magnification as depicted by the box. PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; FACS, fluorescence-activated cell sorting. *p<0.05, **p<0.01, ***p<0.001.

Figure 4

Macrophages with dysregulated CMPK2 display increased hypoxia. (A) Scatter plot of genes differentially expressed in KD or OE macrophages relative to the expression levels in the control macrophages (NT or VC), respectively. Change in expression is depicted as log₂ fold change in expression. The number of genes upregulated and downregulated in the macrophages is depicted as a Venn diagram (inset). (B) GSEA hallmark pathway enrichment analysis of the commonly upregulated and downregulated genes in the KD and OE macrophages are represented as a bubble plot. X axis is the number of genes of the pathway and size of the bubble depicts significance (−log p-value). (C) Heat map of the hypoxia response gene expression in the CMPK2 dysregulated macrophages KD and OE. The values represent log₂ fold change from the corresponding control cells. (D) Analysis of HIF1α in protein extracts of NT, KD, VC, and OE cells. Equal amounts of protein from the different extracts were probed with specific antibodies, and a representative blot of three independent experiments is depicted. The change in expression level of HIF1α with respect to α-TUBULIN was calculated by densitometric analysis and is graphically represented. GSEA, Gene Set Enrichment Analysis. **p<0.01.

Figure 5

Macrophages with dysregulated CMPK2 are metabolically similar to activated M1 macrophages with higher glycolytic flux. (A) The expression patterns of genes specific to M1 and M2 activated macrophages in the CMPK2 dysregulated macrophages KD and OE are represented as heat maps. The values represent log₂ fold change from the corresponding control cells. (B) Continual estimation of oxygen consumption in NT, KD, and VC, OE macrophages until 3 h with Oroboros oxygraph. The level of oxygen consumption was calculated, and mean OCR ± SEM of N = 3 experiments is shown. (C) Metabolite levels in the CMPK2 silenced (KD) and overexpression (OE) and their respective control macrophages were assayed from cellular extracts by MS. The levels of individual metabolites in the KD and OE macrophages are represented as relative abundance compared to control cells as mean ± SEM from three independent experiments (N = 3). Expression of genes involved in the glycolysis and serine-to-glycine biosynthetic pathway estimated by qRT-PCR is also shown. (D) Expression of IL1β and TNFα in THP1 cells treated with the serine biosynthesis inhibitor NCT-503 and its inactive form PHGDH-inactive. Expression values are mean fold change in expression with respect to GAPDH ± SEM triplicate assays of N = 3 experiments after 6 h of treatment. *p<0.05, **p<0.01, ***p<0.001.

Figure 6

CMPK2 regulates the bactericidal activity of macrophages. (A) Growth kinetics of Mtb in NT and KD macrophages at different times post-infection at a MOI of 5 for 6 h Values are mean CFU ± SEM values in triplicate assays of N = 4. (B) Growth kinetics of STM in NT and KD macrophages at different times post-infection at a MOI of 10 for 20 min. Values are mean CFU ± SEM values in triplicate assays of N = 4. Mtb, Mycobacterium tuberculosis; MOI, multiplicity of infection; CFU, colony-forming unit; STM, Salmonella enterica subsp. enterica serovar Typhimurium. *p<0.05, **p<0.01.