miRNA-Mediated Priming of Macrophage M1 Differentiation Differs in Gram-Positive and Gram-Negative Settings

Abstract

A proper regulation of macrophage polarization is essential for the organism’s health and pathogen control. Differentiation control is known to occur at the transcriptional as well as the posttranscriptional levels. The mechanisms involved, however, have not yet been fully elucidated. In this study, we co-cultured macrophages with viable Gram-positive and Gram-negative bacteria to mimic macrophage differentiation to the M1-like type in an inflammatory milieu. We found that Gram-positive stimulation resulted in increased expressions of miR-7a-5p, miR-148a-3p, miR-155-5p, and miR-351-5p. Of note, these miRNAs were found to target inhibitory mediators of the Rac1-PI3K-Akt pathway and the MyD88-dependent pathway. In contrast, Gram-negative stimulation-induced downregulation of miR-9-5p, miR-27b-3p, miR-93-5p, and miR-106b-5p is known to target key members of the Rac1-PI3K-Akt pathway and the MyD88-dependent pathway. These results, taken together, point to a fine-tuning of macrophage polarization by TLR-induced changes in macrophage miRNA profiles. Here, the miRNA-mediated priming of M1 differentiation seems to differ in the Gram-positive and Gram-negative settings in terms of the mechanism and miRNAs involved.

miRNA-Mediated Fine Regulation of TLR-Induced M1 Polarization

“…We observed that Akt1 mRNA levels are downregulated following TLR4 or TLR2 stimulation [94]. This is in line with the description of Akt1 as a target gene of the miRNA miR-155-5p [66,97], whose expression has been consistently reported to be upregulated by various members of the TLR family [4,5,7,11,12,20,24,25,34,42,47,58,[70][71][72][73]83,[90][91][92][98][99][100][101][102][103]. In addition, Akt1 activity is subject to post-transcriptional modulation.…”

“…It has been described that TRAF6 is ubiquitinated and proteosomally degraded in an IRAK1-dependent manner in the context of negative feedback [28]. In addition, our own studies show a downregulation of TRAF6 mRNA levels after TLR4 or TLR2 stimulation, which is accompanied by an upregulation of the miRNA miR-351-5p [66,94]. TRAF6 has been described as a target gene of miR-351-5p [95].…”

“…It should be noted, however, that the miRNAs identified as having altered expression levels vary from one study to another (Table 2). Only an increased expression of miR-155-5p in M1 polarization is consistently reported (Table 2; [11,58,[66][67][68][69][70][71][72][73]). The interstudy variability, in spite of the analysis of polarized macrophages of the same phenotype, points to the existence of additional influencing factors.…”

“…The lack of data is worrisome as comparative studies of macrophages stimulated by Gram-positive (i.e., LTA, R. equi) or Gram-negative (i.e., LPS, P. aeruginosa) agents support the hypothesis that the miRNA expression profile of M1 macrophages may vary, at least in part, depending on the activation of the TLR4 and TLR2 cascades, respectively [66]. The miRNAs associated with TLR2 activation in our study include miR-7a-5p, miR-148a-3p, miR-155-5p, and miR-351-5p [66]. An increase in miR-155-5p expression after TLR2 stimulation has also been reported in other studies [73].…”

“…It is interesting to note that most miRNA genes that are associated with M1 polarization have binding sites for TLR-induced transcription factors. These include subunits of AP-1 such as c-Fos, Fosl2, c-Jun, JunB, and JunD [84], transcription factors cooperating with AP-1 such as ATF4, NFATc1, and NFATc3 [43,85,86], the transcription factors Bcl6 and Bcl6B, which mediate the suppression of AP-1 activity [87], as well as the transcription factors Max and Myc, whose expression is under the influence of AP-1 [66,88]. The mutual interaction of the TLR signaling cascade and miRNA expression results in a complex regulatory process based on positive and negative feedback loops.…”