Regulation of Inflammatory Macrophage Activation by a miRNA Tandem

Inflammatory monocytes are recruited to the inflamed endothelium of the artery wall where they differentiate into macrophages. By the ingestion of modified low- density lipoproteins (LDLs), macrophages produce free cholesterol that is further exported to the liver in HDL form (Cuchel and Rader 2006). However, excessive lipid accumulation leads to a chronic, unresolved inflammatory response of macrophages that exacerbate atherosclerosis.

Macrophages undergo specific differentiation depending on the local tissue environment. Two distinct states of polarized activation of macrophages have been defined: the classically activated (M1) macrophage phenotype and the alternatively (M2) macrophage phenotype. Both phenotypes display different roles, and whereas activated M1 macrophages act as effector cells during the immune response, M2-activated macrophages are rather involved in immunosuppression. Several M1-M2 intermediates have been described and identified in atherosclerosis (Wolfs et al. 2011; Murray et al. 2014; Locati et al. 2013). Inflammatory activation of macrophages induces signaling cascades such as TLR/Myd88 and NF-кр, transcription factors, inflammatory mediators, and miRNAs (Tedgui and Mallat 2006). During this M1-type activation, numerous miRNAs are downregulated, whereas a small set of miRNAs are upregulated such as miR-147 and miR-155 (Curtale et al. 2013; Ma et al. 2011; O’Connell et al. 2010; Nazari-Jahantigh et al. 2012a; Graff et al. 2012).

miR-155 is encoded by exon 3 of B-cell receptor inducible (BIC) gene and controls several aspects of macrophage function such as inflammatory response, lipid uptake, and apoptosis (Nazari-Jahantigh et al. 2012a; Koch et al. 2012). miR-155 expression is reduced during macrophage differentiation while it is strongly induced upon Ml polarization through MyD88- and TRIF-dependent signaling cascades (Nazari-Jahantigh et al. 2012a; O’Connell et al. 2007). In addition, LDL and mildly oxidized LDL can induce the expression of miR-155 in macrophages (Nazari- Jahantigh et al. 2012a). miR-155 expression is negatively regulated in macrophages by the transcription factors Ets2 and YY1 (Quinn et al. 2014; Tian et al. 2014) and by the protein kinase AKT1, which is activated by TLR signaling (Androulidaki et al. 2009). PI3K-AKT signaling limits the expression of inflammatory mediators in macrophages, and the suppression of AKT1 promotes the inflammatory M1-type phenotype by upregulation of miR-155 (Guha and Mackman 2002; Arranz et al. 2012; Androulidaki et al. 2009).

Beyond that, miR-155 can also be indirectly regulated by other miRNA, and so miR-342-5p can increase the expression of miR-155 in macrophages by targeting AKT1 thereby triggering the expression of pro-inflammatory mediators such as NO, TNFa, and IL6 (Wei et al. 2013a). Although the expression of miR-342-5p is not regulated during TLR4-mediated macrophage activation, its effect on AKT1 expression is only detectable in inflammatory macrophages (Wei et al. 2013a). The effects of miR-342-5p on AKT1 levels are based on the relative abundance of two of its targets, Bmpr2 and AKT1 (Wei et al. 2013a). In unstimulated macrophages, miR-342-5p preferentially targets Bmpr2 that is highly expressed and which appears to have a more efficient binding site for miR-342-5p than AKT1 (Wei et al. 2013a). During inflammatory activation, however, the levels of Bmpr2 are downregulated and due to the lack of its preferential target miR-342-5p switch to target also AKT1 expression leading finally to an increase of miR-155 and subsequently high expression of inflammatory mediators (Fig. 1.2) (Wei et al. 2013a). The expression of Bmpr2 mRNA in unstimulated macrophages inhibits their inflammatory activation by preventing the targeting of AKT1 by miR-342-5p. This represents a beautiful example of ceRNA-based regulatory system controlling macrophage polarization and also illustrates how two miRNAs in tandem, miR-342-5p and miR-155, orchestrate the inflammatory macrophage activation.

The process by which miR-155 upregulates the expression of the proinflammatory mediators, like the chemokine Ccl2, in macrophages is by targeting the transcriptional repressor Bcl6. Bcl6 is upregulated during macrophage differentiation by the activation of NF-kB signaling, and counter-regulates the NF-кр- mediated transcriptional upregulation of many inflammatory genes. In addition to Bcl6, miR-155 can target other transcripts, such as Socs1 and Sfpi1, which can contribute to the regulation of macrophage inflammatory responses (Nazari- Jahantigh et al. 2012b). However, in contrast to Bcl6, Socs1 and Sfpil1 are not involved in the inflammatory activation of macrophages mediated by miR-155, and their functional role is not fully understood (Nazari-Jahantigh et al. 2012a). Whether the different miR-155 targets act as ceRNAs and thus regulate the targeting of Bcl6 by miR-155 is currently unclear.

The current in vitro findings on the miR-155-mediated regulatory mechanisms in macrophages have been validated also in models of atherosclerosis in vivo. miR- 155 expression is upregulated in mouse and human atherosclerotic lesions and is increased in CD14+ monocytes from patient with coronary heart disease


V. Egea et al.

Tandem mechanism of miR-342-5p and miR-155 in inflammatory macrophage activation

Fig. 1.2 Tandem mechanism of miR-342-5p and miR-155 in inflammatory macrophage activation. Under resting conditions (left side of the illustration), AKT1 and Bmpr2 compete for the binding to miR-342-5p in macrophages. The preferential binding of miR-342-5p to Bmpr2 facilitates basal translation of AKT1, which inhibits the expression of miR-155. The lack of miR-155 enables the expression of Bcl6 and HBP1 thereby impairing the production of inflammatory factors and the uptake of modified LDL (mLDL). Under inflammatory conditions (right side of the illustration), the transcription level of Bmpr2 is downregulated increasing the availability of miR- 342-5p for the binding to AKT1 mRNA. This suppression of AKT1 results in upregulation of miR-155, which targets Bcl6 and HBP1 unleashing inflammatory gene expression and mLDL uptake

(Nazari-Jahantigh et al. 2012a; Du et al. 2014). The increased expression of the prototypical M1 macrophage miRNA, miR-155, indicates that primarily M1-type macrophages contribute to the coronary heart diseases and atherosclerosis (Nazari- Jahantigh et al. 2012a). In contrast to vascular cells, miR-155 deficiency in macrophages attenuates atherosclerosis due to the increased Bcl6 expression, which impairs Ccl2 production and thereby limits the lesional macrophage accumulation (Nazari-Jahantigh et al. 2012a; Aiello et al. 1999; Wei et al. 2013c). In line with this data, miR-155 deficiency in hematopoietic cells and whole body attenuates atherosclerosis by an increment of Bcl6 and SOCS1 levels in lesional macrophages, subsequently leading to a reduction of inflammatory genes including Ccl2, TNFa, IL6, and IL1b (Du et al. 2014). Therefore, overexpression of miR-155 in inflammatory macrophages within atherosclerotic lesions amplifies the vascular inflammation exacerbating the progress of atherosclerosis (Nazari-Jahantigh et al. 2012a; Du et al. 2014). Notably, miR-342-5p levels are also increased in mouse atherosclerotic lesions after 3 and 10 months of high-fat diet feeding (Wei et al. 2013a). miR- 342-5p suppression attenuates atherosclerosis in Apoe-/- mice (Wei et al. 2013a). Inhibition of miR-342-5p enhances the AKT1 expression in lesional macrophages, which presumably results in reduced atherosclerosis by downregulating miR-155 expression (Wei et al. 2013a). This indicates that miR-342-5p and miR-155 regulatory tandem in inflammatory macrophages indeed exacerbate atherosclerosis.

Conversely, in hematopoietic cells, miR-155 deficiency increases atherosclerosis in LDLR-/- mice without significant change in lesional macrophage content (Donners et al. 2012). This discrepancy could be due to distinct expression levels of miR-155 targets at the different stages of atherosclerosis and possible switching of the targets in macrophages during the development of lesions. Therefore, miR-155 probably mediates opposing effects at different stages of atherosclerosis by switching the target transcript.

Besides its role in inflammatory activation, miR-155 regulates additional macrophage functions as, for instance, the lipid uptake. By the targeting of HMG box- transcription protein1 (HBP1), a negative regulator of MIF, miR-155, increases the uptake of oxLDL by macrophages (Tian et al. 2014). A systemic delivery of antagomirs of miR-155 in Apoe-/- mice thereby reduces the expression of HBP1 and decreases atherosclerosis presumably by limiting not only the expression of IL6 and TNFa but also the lesional macrophage lipid accumulation (Tian et al. 2014). Similarly, miR-155 deficiency in macrophages reduced the formation of macrophage- derived foam cells in atherosclerotic lesions (Nazari-Jahantigh et al. 2012a). These data illustrates the crucial role of miR-155 at the interface between inflammatory activation and lipid handling in macrophages.

Taken together, the regulatory tandem of miR-342-5p and miR-155 enhances the inflammatory activation of macrophages in atherosclerosis. The functional studies clearly demonstrate that Bcl6 is targeted by miR-155 mediating an enhanced inflammatory response and thereby increase in lesional macrophage accumulation exacerbating the progress of atherosclerosis. However, the functional role of other miR-155 targets such as Socs1 in atherosclerosis remains to be further determined. Finally, the ceRNA-based regulation of miR-342-5p activity thereby modulating the miR- 155-mediated macrophage inflammatory response beautifully illustrates a safeguard mechanism to avoid inappropriate activation of macrophages.

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