Epigenetic Mechanisms in Immune Disease: The Significance of Toll-Like Receptor-Binding Extracellular Vesicle-Encapsulated microRNA

Abstract

Immune and inflammatory diseases arise from a complex combination of genetic and environmental factors (David et al., 2018; Surace and Hedrich, 2019). MicroRNA are a class of non-coding single-stranded RNA molecules of 19–23 nucleotides in length. In response to environmental triggers, microRNA mediate epigenetic cell fate decisions critical in immune homeostasis by driving cellular activation, polarization, and immunological memory cell development (Mehta and Baltimore, 2016; Curtale et al., 2019). Pattern recognition receptors (PRR) recognize conserved molecular components of pathogens and respond by secreting reactive oxygen species and cytokines that alert the immune system about infection (Medzhitov et al., 1997). They can also interact with various endogenous ligands i.e., lipids, glycans, proteins, and nucleic acids, when released under sterile conditions of cellular stress, tissue injury, and transplantation. As activators of PRR-signaling, endogenous ligands initiate immune cell recruitment and tissue repair. However, sustained PRR-signaling may result in an exacerbated inflammatory response, which can have lethal effects or lead to autoimmunity (reviewed in Yu et al., 2010). In addition to their well-documented canonical function regulating gene expression through RNA interference in the cytoplasm (Bartel, 2004), specific GU-rich microRNA sequences can activate pro-inflammatory signaling pathways by direct interaction with the ribonucleic-acid binding Toll-like receptor 7/8 (TLR-7/8) of innate immunity located in cellular endosomes (Heil et al., 2004). Extracellular vesicles are a heterogeneous population of membrane vesicles naturally secreted by living cells that facilitate intercellular exchanges (Valadi et al., 2007; Raposo and Stoorvogel, 2013). Exported inside extracellular vesicles, Toll-like receptor-binding microRNA released by cells from injured or stressed tissues can reach the endosomal compartment and propagate inflammatory signals in distant recipient cells (Figure 1). The contributions of a dozen of TLR-7/8-binding microRNA (let-7b/c, miR-7a, miR-21, miR-29a/b, miR-34a, miR-122, miR-133a, miR-142, miR-145, miR-146a, miR-208a, and miR-210) to inflammation have been described to date in settings of cancer, sepsis, neurological, autoimmune, and graft-vs.-host diseases (Fabbri et al., 2012; Lehmann et al., 2012; He et al., 2014; Park et al., 2014; Salama et al., 2014; Liu et al., 2015; Yelamanchili et al., 2015; Kim et al., 2016; Coleman et al., 2017; Feng et al., 2017; Ranganathan et al., 2017; Young et al., 2017; Salvi et al., 2018; Xu et al., 2018; Wang et al., 2019). Using confocal microscopy co-localization, co-precipitation, and TLR inhibitors, these studies demonstrate direct binding of these microRNA to TLR-7 in mouse and TLR-8 in human, independently of RNA interference. Furthermore, transgenic TLR-7^−/− mice are protected against the degenerative and inflammation-related effects of TLR-binding microRNA (Fabbri et al., 2012; Lehmann et al., 2012; Yelamanchili et al., 2015; Liang et al., 2019). Since their discovery in 2012, the significance of microRNA as endogenous ligands of innate immunity in health and disease is still a matter of debate (Chen et al., 2013; Fabbri et al., 2013; He, X. et al., 2014; Bayraktar et al., 2019). As part of the dynamic continuum of the endocytic intercellular communication pathway, TLR-binding microRNA transported via extracellular vesicles likely serve both adaptive and maladaptive stress responses in cells expressing TLR-7/8. Figure 1. As part of the intercellular endocytic communication pathway, TLR-binding microRNA transmitted via extracellular vesicles serve adaptive and maladaptive stress responses. Environmental stress (1) promotes secretion of extracellular vesicles and microRNA, (self-) antigen and danger-associated molecule release (2). After uptake by innate immune cells, specific GU-rich extracellular vesicle-encapsulated microRNA sequences can stimulate TLR-7/8 signaling in the endosome of recipient cells. Subsequent activation of the NF-κB pathway exacerbates inflammation through cytokine secretion, expression of co-stimulatory molecules (3) and self-induction of TLR-binding microRNA expression and extracellular vesicle secretion (4). So far, unconventional TLR-binding activity has been observed solely for extracellular microRNA and, out of 14 studies, 11 ascertain transfer in association with extracellular vesicles. The effects of danger-associated molecular patterns depend on their detection, a truism applicable to TLR-7-binding microRNA: they can act as such if and only if they reach the endosomal compartment. Encapsulation within extracellular vesicles constitutes a means for microRNA to enter the endocytic pathway where they may directly engage TLR-7/8 signaling (Mulcahy et al., 2014). In contrast, for RNA-interference activity, internalized microRNA have to escape from the endosome (Montecalvo et al., 2012), a rate-limiting step identified in the delivery of therapeutic short interference RNA (Johannes and Lucchino, 2018) and viral infection (Staring et al., 2018). It is conceivable that TLR-binding microRNA are conducive to exerting RNA interference-mediated effects in donor cells and TLR-binding effects or combinations of both after transfer via extracellular vesicles in recipient immune cells, i.e., major sites of TLR-7/8 expression (Lin et al., 2020; Sun et al., 2020). The relative proportion of free and particulate microRNA in biofluids still raises controversy, which is in part linked to technical pitfalls in the proper assessment of RNA concentrations in extracellular vesicles and biofluids (Arroyo et al., 2011; Turchinovich et al., 2011; Gallo et al., 2012; Crossland et al., 2016; Jeppesen et al., 2019). While free soluble RNA are short-lived due to high physiological levels of ribonuclease activity, microRNA chaperone protein complexes, or...