Macrophage gene regulatory networks of miR-146 and miR-155

The differential association of miR-146 and miR-155 with macrophage inflammatory gene expression downstream of TLR stimulation suggested that these miRNAs may have different functions and target different mRNAs. To dissect their target-profiles, known and predicted targets of miR-146 and miR-155 in macrophage inflammatory signal transduction were tested for repression by both miRNAs. For miR-155 these were TAB2 (involved in TLR and cytokine receptor triggered NFκB activation) and IKKε (involved in anti-viral cytokine production downstream of TLRs), (Kawai and Akira, 2006), (Fig. 22A, B). A manual search for miR-155 seed-pairing sites within the 3’UTRs of genes associated with macrophage inflammatory signal transduction additionally revealed a 7-mer miR-155 seed-complementary site within the 3’UTR of NIK kinase (Fig. 22B). NIK has been implicated in NFκB activation downstream of TLR4 (Beutler, 2000), (Fig. 22A).

Importantly, the miR-155 target site in NIK mRNA is deeply conserved among vertebrates (Fig. 22C). Therefore, NIK was included as a potential target of miR-155.

Furthermore, the presently known miR-146 targets in the macrophage inflammatory response, TRAF6 and IRAK1, which constitute essential TLR4 signal transduction components (section 4.3.2, Fig. 22A), were tested for repression by both miR-146 and miR-155 (Fig. 25).

Figure 22: Known and putative miRNA targets in the macrophage TLR4 response. (A) Scheme of miR-146 and miR-155 targets in macrophage signaling pathways downstream of TLR4 activation (adapted from Kawai and Akira, 2006). Continuous arrow-lines indicate intracellular signaling pathways triggering pro-inflammatory transcription downstream of TLR4; dashed arrow-line indicates secondary autocrine/paracrine stimulation of pro-inflammatory transcription via TNFα release. (B) Binding sites of miR-155 within the 3’UTRs of the murine messengers of NIK, IKKε and TAB2, predicted by the RNAhybrid algorithm. Lines indicate Watson-Crick base pairings, dots indicate wobble base pairs. Crosses denote positions of mutations introduced into the 3’UTR sequences in order to abrogate regulation of the luciferase 3’UTR reporters tested in Fig. 23. (C) Section of the alignment of NIK 3’UTR sequences of the indicated vertebrate species. The black frame highlights the conserved miR-155 seed-complementary site.

The respective 3’UTRs were fused to a Renilla luciferase reporter gene which was over-expressed in MEF cells along with the respective miRNA mimics, as described above (section 6.3). Compared to a control miRNA mimic, delivery of a miR-155 mimic significantly reduced the activity of the TAB2, IKKε and NIK reporters (Fig. 23A-C). Two consecutive point-mutations in the predicted miR-155 seed-pairing sites of the TAB2, IKKε and NIK reporters, respectively (Fig. 22B), abrogated regulation, which demonstrates direct targeting by miR-155 (Fig. 23A-C). Importantly, no specific suppression of the TAB2, IKKε and NIK reporters was observed upon delivery of a miR-146 mimic (Fig. 23A-C). It remained to be determined whether endogenous miR-155, induced upon LPS challenge of macrophages, impacts the activities of the TAB2, IKKε and NIK reporters. To this end, macrophages transfected with the intact or miR-155 binding-site deficient reporters were stimulated with 1 ng LPS / ml (miR-146 inducing stimulus; Fig. 18E, F) or 1000 ng LPS / ml (miR-155 inducing stimulus; Fig. 18D). Upon

24 h of LPS treatment reporter activity relative to control-treatment (no LPS) was determined. A significant repression of the wild-type TAB2, IKKε and NIK reporter constructs compared to the respective miR-155 binding-site mutant constructs was observed only at the miR-155 inducing stimulus of 1000 ng LPS per ml but not at the miR-146 inducing stimulus of 1 ng LPS per ml (Fig. 23D-F). These results suggest that the pro-inflammatory signal transducers TAB2, IKKε and NIK are selectively controlled by miR-155 (and not miR-146) during the macrophage inflammatory response.

Figure 23: MiR-155 but not miR-146 targets NIK, IKKε and TAB2. (A-C) Activities of the indicated wild-type (wt) or miR-155 target-site mutant (mut) reporters were determined in MEF cells upon co-transfection of miR-146a or miR-155 mimics, compared to control mimic co-co-transfection. (D-F) The activities of the reporters introduced in (A-C) were tested in RAW264.7 macrophages upon stimulation with 1 or 1000 ng LPS / ml for 24 h compared to control-treatment (0 ng LPS / ml). Asterisks denote significant differences between the indicated pairs of samples (P-value < 0.05).

Figure 24:MiR-146 but not miR-155 targets TRAF6 and IRAK1 3’ UTR reporters. (A, B) Binding sites of miR-146 within the 3’UTRs of the murine messengers of TRAF6 and IRAK1, predicted by the RNAhybrid algorithm. Lines indicate Watson-Crick base pairings, dots indicate wobble base pairs. Crosses denote positions of mutations introduced in order to abrogate regulation of luciferase 3’UTR reporters. (D-E) Activities of the indicated wild-type (wt) or miR-146 target-site mutant (mut) reporters were determined in MEF-cells upon transfection of miR-146a or miR-155 mimics, compared to control mimic co-transfection.. Asterisks denote significant differences between the indicated pairs of samples (P-value <

0.05).

To determine whether miR-155 and miR-146 act redundant or control distinct target profiles in the macrophage inflammatory response it remained to be addressed whether the known 146 targets IRAK1 and TRAF6 may also be repressed by miR-155. To this end IRAK1 and TRAF6 3’UTR reporters (Fig. 24A, B) were transfected into MEF cells. As expected (Taganov, et al., 2006) both reporters were readily repressed upon delivery of a miR-146 mimic; yet, the miR-155 mimic did not impact the reporter actvities (Fig. 24C, D). Introduction of two consecutive point-mutations into the predicted miR-146 target sites of IRAK1 and TRAF6 (Fig. 24A, B) rendered the reporters non-responsive to miR-146 (Fig. 24C, D).

Collectively, these results demonstrate that miR-146 and miR-155 control distinct targets in macrophage innate immune pathways. The selective control of signaling components involved in various pro-inflammatory pathways suggests that miR-155 constitutes a pervasive regulator of pro-inflammatory signaling, while miR-146 seems to be limited to control of TLR signal transducers (see Fig. 22A).