Abstract

Abstract

Dendritic cells (DCs) are key regulators of both immunity and tolerance. Recent studies showed that DC activation is coupled to profound changes in DC metabolism that is characterized by increase in glycolysis. The molecular regulation of these metabolic changes remains poorly understood. MicroRNAs (miRs) have been linked to DC activation, but their role, if any, in critical regulation of DC activation by regulating metabolic reprogramming is unknown. Amongst miRs, one that can critically alters DC's response to stimulation is miR-142 (Sun et al., 2011 and 2013). Utilizing complementary loss and gain of function approaches, and through comprehensive gene arrays, metabolic and functional analyses, we found that miR-142 controls DC maturation and regulates their function. Upon stimulation, the miR-142 KO DCs showed reduced expression of surface molecules such as MHC class II, CD80, CD86 and CD40, and reduced expression of TNFa, but enhanced expression of IL-10 and IL-6 when compared to WT DCs. Priming capability for allogeneic reaction was significantly reduced in miR-142 KO DCs compared to WT DCs as demonstrated by significantly reduced T cell proliferation, expression of IL-2, IFNg and IL-17A. Both WT DCs and miR-142 KO DCs maintained glycolysis switch upon TLR stimulation as showed by similar contents of lactate production, Glut1 and HK2 induction and glucose uptake. These data are consistent with gene expression microarrays analyzed by IPA (Ingenuity Pathway Analysis) and GSEA(Gene Set Enrichment Analysis). Utilizing Seahorse technology, we observed that OCR/ECAR ratios and ATP-linked respiration in miR-142 KO DCs were significantly higher than those in WT DCs, along with an increased mitochondrial membrane potential (ΔΨM). These observations were consistent with our gene expression microarray which showed augmented OXPHOS in miR-142 KO DCs. Gene expression microarray also showed significant increase in fatty acid oxidation (FAO) pathway and enhanced expression of CPT1a, a rate-limiting enzyme of mitochondrial FAO by controlling FA mitochondrial transfer, in miR-142 KO DCs. Consistently, FAO assay showed significantly higher FAO rate in miR-142 KO DCs than that in WT DCs, which was partially, but significantly, reduced after specific knockdown of CPT1a in miR-142 KO DCs. Further investigation revealed that enhanced FAO in miR-142 KO DCs was associated with increase in FA uptake and electron transfer chain activity in miR-142 KO DCs, because of direct targeting CPT1a, FABP4, FABP5, Ndufs7 and Uqcrb. Importantly, restoration of miR-142 in miR-142 deficient DCs produced notable inhibition of protein expressions of its target genes including CPT1a, FABP4, Ndufs7 and UqcrB and induced further significant deduction in FAO, demonstrating that the miR-142 augments FAO in DCs by targeting multiple molecules to promote FA uptake (FABP4 and FABP5), FA mitochondrial transfer (CPT1a) and electron transfer chain activity (Ndufs7 and Uqcrb). Further functional analyses demonstrated that the enhanced OXPHOS and FAO in metabolic reprogramming of DCs that was controlled by miR-142 did not impair trained immunity. Finally, we evaluated the biological relevance of miR-142, and demonstrated that IL-10 expression was dual dependent on glycolysis and FAO in miR-142 KO DCs and that although expressions of IL-12 and TNFa were glycolysis-dependent in both WT and miR-142 KO DCs, FAO is negatively related to TNFa induction in miR-142 KO DCs. Our study demonstrated that targeting miR-142 in DCs may have therapeutic potential for clinic application to prevent autoimmune disease including GVHD and cancer therapy by mediating DC's activation through targeting metabolic reprogramming.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.