Abstract
Neuroinflammation contributes to many neurologic disorders including Alzheimer’s disease, multiple sclerosis, and stroke. Microglia is brain resident myeloid cells and have emerged as a key driver of the neuroinflammatory responses. MicroRNAs (miRNAs) provide a novel layer of gene regulation and play a critical role in regulating the inflammatory response of peripheral macrophages. However, little is known about the miRNA in inflammatory activation of microglia. To elucidate the role that miRNAs have on microglial phenotypes under classical (M1) or alternative (M2) activation under lipopolysaccharide (‘M1’-skewing) and interleukin-4 (‘M2a’-skewing) stimulation conditions, we performed microarray expression profiling and bioinformatics analysis of both mRNA and miRNA using primary cultured murine microglia. miR-689, miR-124, and miR-155 were the most strongly associated miRNAs predicted to mediate pro-inflammatory pathways and M1-like activation phenotype. miR-155, the most strongly up-regulated miRNA, regulates the signal transducer and activator of transcription 3 signaling pathway enabling the late phase response to M1-skewing stimulation. Reduced expression in miR-689 and miR-124 are associated with dis-inhibition of many canonical inflammatory pathways. miR-124, miR-711, miR-145 are the strongly associated miRNAs predicted to mediate anti-inflammatory pathways and M2-like activation phenotype. Reductions in miR-711 and miR-124 may regulate inflammatory signaling pathways and peroxisome proliferator-activated receptor-gamma pathway. miR-145 potentially regulate peripheral monocyte/macrophage differentiation and faciliate the M2-skewing phenotype. Overall, through combined miRNA and mRNA expression profiling and bioinformatics analysis we have identified six miRNAs and their putative roles in M1 and M2-skewing of microglial activation through different signaling pathways.
Highlights
Inflammation is a dynamic and complex process triggered by tissue insult in any region of the body including the central nervous system (CNS) [1,2]
A differential mRNA expression profile is presented as heat map, highlighting 63 genes that were differentially regulated to a significance determined by a moderated FDR q, 161027 as compared against resting microglia (Fig. 1A)
To validate our microarray results we performed mRNA reverse transcription Polymerase Chain Reaction (RT-PCR) using total RNA isolated from the same samples on select M1-marker genes, and confirmed that expression of IL-6, IL-1b, and NOS2 genes were significantly induced upon LPS stimulation (Fig. 1D)
Summary
Inflammation is a dynamic and complex process triggered by tissue insult in any region of the body including the central nervous system (CNS) [1,2]. The innate immune system activates adaptive immunity through antigen presentation to boost the response to a specific target [3]. The CNS relies much more on innate immunity mediated through microglia, monocytes and macrophages while recruitment of neutrophils and T-cells, which mediate adaptive immunity, is limited [3,4,5]. Peripheral macrophages display plasticity characterized by their phenotypic response to pathogenic stimuli, such as ‘‘classical activation’’ (M1) phenotype characterized by both pro-recruitment and formation of cytotoxic reactive oxygen/nitrogen species. Pre-treatment of macrophages with various M2-promoting cytokines can influence the responsiveness to various M2 subtypes such as a pro-cleanup phenotype (M2a) with enhanced phagocytic capability of apoptotic bodies by interleukin-4 (IL-4)/IL-13 stimulation, or woundhealing phenotype (M2c) by IL-10, transforming growth factor-b (TGF-b) or corticosteroid stimulation [6,7]
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