Abstract
The attenuation of hyper-inflammation in sepsis with the administration of anti-inflammatory macrophages is an interesting adjuvant therapy for sepsis. Because the induction of anti-inflammatory macrophages by microRNA (miR), a regulator of mRNA, has been mentioned, the exploration on miR-induced anti-inflammatory macrophages was performed. The over-expression of miR-223 and miR-146a in RAW264.7 induced M2 macrophage-polarization (anti-inflammatory macrophages) as evaluated by the enhanced expression of Arginase-1 and Fizz. However, miR-223 over-expressed cells demonstrated the more potent anti-inflammatory property against LPS stimulation as lesser iNOS expression, lower supernatant IL-6 and higher supernatant IL-10 compared with miR-146a over-expressed cells. Interestingly, LPS stimulation in miR-223 over-expressed cells, compared with LPS-stimulated control cells, demonstrated lower activity of glycolysis pathway and higher mitochondrial respiration, as evaluated by the extracellular flux analysis, and also down-regulated HIF-1α, an important enzyme of glycolysis pathway. In addition, the administration of miR-223 over-expressed macrophages with IL-4 pre-conditioning, but not IL-4 stimulated control cells, attenuated sepsis severity in LPS injected mice as evaluated by serum creatinine, liver enzymes, lung histology and serum cytokines. In conclusion, miR-223 interfered with the glycolysis pathway through the down-regulation of HIF-1α, resulting in the anti-inflammatory status. The over-expression of miR-223 in macrophages prevented the conversion into M1 macrophage polarization after LPS stimulation. The administration of miR-223 over-expressed macrophages, with IL-4 preconditioning, attenuated sepsis severity in LPS model. Hence, a proof of concept in the induction of anti-inflammatory macrophages through the cell-energy interference for sepsis treatment was proposed as a basis of cell-based therapy in sepsis.
Highlights
Sepsis, a syndrome of organ dysfunction due to the dys-regulation of host responses to systemic infection, is a major cause of death in patients with clinically ill conditions and has been recognized as an important world-wide healthcare problem [1]
The anti-inflammatory property of miR-223 was demonstrated by i) the reduction of supernatant IL-6, but not TNFα, after LPS activation (Fig 2G and 2H) and ii) the induction of supernatant IL-10 after IL-4 stimulation (Fig 2I)
While IL-4 stimulation alone induced very low expression of Inducible nitric oxide synthase (iNOS), Tumor necrosis factor α (TNF-α) and IL-1β compared with LPS stimulation (Fig 2A–2C), LPS activation in IL-4 pre-conditioning macrophages (IL-4/ LPS) demonstrated a similar level of these genes in comparison with LPS stimulation alone (Fig 3A– 3C)
Summary
A syndrome of organ dysfunction due to the dys-regulation of host responses to systemic infection, is a major cause of death in patients with clinically ill conditions and has been recognized as an important world-wide healthcare problem [1]. The imbalance of pro- and anti-inflammatory immune-responses is one of the important causes of death in sepsis and the anti-inflammation is an interesting adjunctive treatment of sepsis, especially during the proinflammatory state [2,3,4]. Macrophages, heterogeneous immune cells with pleiotropic functions [6], are fundamentally divided into classically M1 and alternative M2 polarization with pro- and anti-inflammatory property, respectively [7]. Macrophages in sepsis hyper-inflammation are predominant in M1 polarization and are responsible for the production of inflammatory mediators [8]. MiR-223 is essential for M2 polarization [15] and the down-regulation of miR-223 in patients with severe sepsis possibly induces predominant M1 polarization [16]. MiR-146a is well documented as an anti-inflammatory miR, preferentially augments M2 polarization [17], that attenuates sepsisinduced cardiac injury in a mouse model [18]
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