Macrophage activation as an archetype of mitochondrial repurposing

Abstract

Mitochondria are metabolic organelles essential not only for energy transduction, but also a range of other functions such as biosynthesis, ion and metal homeostasis, maintenance of redox balance, and cell signaling. A hallmark example of how mitochondria can rebalance these processes to adjust cell function is observed in macrophages. These innate immune cells are responsible for a remarkable breadth of processes including pathogen elimination, antigen presentation, debris clearance, and wound healing. These diverse, polarized functions often include similarly disparate alterations in the metabolic phenotype associated with their execution. In this chapter, mitochondrial bioenergetics and signaling are viewed through the lens of macrophage polarization: both classical, pro-inflammatory activation and alternative, anti-inflammatory activation are associated with substantive changes to mitochondrial metabolism. Emphasis is placed on recent evidence that aims to clarify the essential – rather than associative – mitochondrial alterations, as well as accumulating data suggesting a degree of plasticity within the metabolic phenotypes that can support pro- and anti-inflammatory functions.

Introduction

Mitochondria play essential roles in cell physiology including, and extending far beyond, serving as the sites of oxidative phosphorylation. In addition to synthesizing the majority of ATP in most cell types (Nicholls and Ferguson, 2013; Pagliarini and Rutter, 2013), mitochondria are also hubs for biosynthesis, Ca²⁺ handling, iron homeostasis, redox balance, and signal transduction (Chandel, 2014; Murphy and Hartley, 2018; Shadel, 2012). Mitochondrially derived signals can be as varied as protein release from the intermembrane space (Jiang and Wang, 2004), metabolite efflux (Ryan et al., 2019), redox signals [e.g. reactive oxygen species (ROS) production] (Finkel, 2011; Murphy, 2009; Reczek and Chandel, 2015), and release of damage-associated molecular patterns (DAMPs) (Grazioli and Pugin, 2018; Picca et al., 2017).

It is increasingly appreciated that the roles of mitochondria in energy metabolism and as a signaling organelle are not necessarily discrete. In fact, one purpose of mitochondria as a signaling organelle is as a communicator of metabolic health status, serving a checkpoint or feedback function to instruct nuclear gene expression and cellular function (Chandel, 2015). Perhaps reflective of this, some proteins with essential roles in energy transduction have dual functions in pathways associated with cell death and disease. For example, cytochrome c is an essential component of the electron transport chain, but also triggers caspase-mediated apoptosis when released to the cytoplasm upon mitochondrial outer membrane permeabilization (Jiang and Wang, 2004; Tait and Green, 2010). Similarly, the F_OF₁-ATP synthase that generates ATP during oxidative phosphorylation also dimerizes to form the mitochondrial permeability transition pore, a channel causing inner membrane permeability and causative of pathologies such as ischemic heart injury and muscular dystrophy (Bernardi, 2013). This mitochondrial plasticity in shifting from energy powerhouse to signaling platform often involves a classical definition of reduced or impaired mitochondrial function (lowered oxidative phosphorylation, redox imbalance, etc.) (Jazwinski, 2013). Remarkably, however, emerging evidence in murine bone marrow-derived macrophages (BMDMs) suggests this does not necessarily translate to reduced or impaired cellular function.

Macrophages are cells of the innate immune system responsible for a remarkable breadth of functions essential to human health including killing pathogens, clearing subcellular debris, and repairing tissue (Adams and Hamilton, 1984). In addition to their role in healthy physiology, targeting macrophage function may lead to improved therapies for a variety of pathologies including tissue fibrosis, cancer, and metabolic disease (Wynn and Vannella, 2016).

Macrophages were first described over 130 years ago by Metchnikoff, who suggested that the observed phagocytosis by ameboid cells could be a means of host defense rather than a deleterious consequence of tissue damage or infection (Gordon, 2016). Specificity for the functional role and activation state of a macrophage at any given time is set by coordinating intrinsic, extrinsic, and environmental cues (Murray, 2017). This allows for the broad and sometimes opposing range of functions (e.g. “kill or repair”) these cells must execute. This so-called polarization state is often simplified through the classification of pro-inflammatory “M1” and anti-inflammatory “M2” macrophages (Gordon and Taylor, 2005). The nomenclature is drawn from the response to cytokines secreted by subsets of CD4⁺ helper T (T_h) cells. A T_h1 response is associated with immunity to bacteria and infections as well as secretion of interferon-γ  (IFN-γ) and tumor necrosis factor-α (TNF-α), whereas a T_h2 response counteracts the microbicidal T_h1 response and is associated with allergy and helminth immunity along with production of interleukin-4 (IL-4), IL-10, and IL-13.

Pro-inflammatory activation can occur via detection of pathogens by pattern recognition receptors (PRRs), proteins present at both the plasma and endosomal membranes (O'Neill et al., 2013). PRRs bind subsets of molecular scaffolds typically associated with microbes [pathogen associated molecular patterns (PAMPs)] or tissue damage [damage associated molecular patterns (DAMPS)]. Innate PRRs are broadly classified in two groups: membrane-bound, including Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), and cytoplasmic, such as NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) (Akira and Takeda, 2004). Regardless of their localization, PRRs function by receptor-mediated activation of broad transcriptional programs, resulting in an orchestrated inflammatory response to eliminate pathogens or damaged cells (Barton and Medzhitov, 2003).

In addition to activation by PAMPs and DAMPs, macrophages can also be polarized to a range of activation states by secreted cytokines. This can occur in both paracrine [e.g. IFN-γ or anti-inflammatory IL-4 secreted by lymphocytes] and autocrine [e.g. pro-inflammatory interleukin-1β (IL-1β) or anti-inflammatory transforming growth factor-β (TGF-β)] manners upon receptor binding (Mosser and Edwards, 2008). In some cases, activation of PRR- and cytokine receptor-mediated activation integrate to amplify the inflammatory response. In the hallmark example, activation of TLR4, which recognizes the gram-negative bacteria membrane component lipopolysaccharide (LPS), synergizes with IFN-γ to create a powerful, composite pro-inflammatory signal (Held et al., 1999).

It is often accepted that the M1/M2 framework is based on rigidly prescribed conditions that rarely, if ever, occur physiologically, and do not necessarily reflect the spectral nature of macrophage activation (Martinez and Gordon, 2014). Nonetheless, the use of tightly controlled, in vitro polarization assays coupled with modern metabolic techniques has revealed profound and unexpected roles for several aspects of mitochondrial function in the control of the innate immune response.

An essential role for metabolism in macrophage function may be somewhat obvious in the traditional definition, as activation-induced phagocytosis, motility, and cytokine synthesis are all ATP-consuming processes that require commensurate changes in ATP production. Indeed, early biochemical studies showed that glucose consumption in naïve, unstimulated macrophages is far below its enzymatic capacity, suggesting an ability to quickly upregulate metabolic pathways in response to external stimuli (Newsholme et al., 1986). Abundant genetic and pharmacologic evidence now exists to demonstrate several essential roles for metabolism in shaping macrophage function, and these are surveyed in multiple comprehensive reviews (Arts et al., 2016a; Benmoussa et al., 2018; Caputa et al., 2019; Geeraerts et al., 2017; Langston et al., 2017; O'Neill and Pearce, 2016; Odegaard and Chawla, 2011; Russell et al., 2019; Van den Bossche et al., 2017; Viola et al., 2019; Weinberg et al., 2015). Such roles include increased and/or rerouted metabolic flux through specific pathways, post-translational modifications by metabolic intermediates, and signaling initiated by metabolites, redox triggers, or nucleic acids.

In the present manuscript, we focus on mitochondria as a hub for both energy transduction as well as metabolite and signal generation, and discuss how these different modes are used to shape macrophage function. It is unquestioned that, in murine macrophages, oxidative metabolism is associated with anti-inflammatory activation (stimulation with IL-4 ± IL-13), and classical inflammatory activation (LPS ± IFN-γ) involves the collapse of oxidative phosphorylation and repurposing of mitochondria towards accumulation of metabolites and other pro-inflammatory triggers. However, the essential, targetable metabolic requirements to adjust macrophage function may not be as black-or-white as initially thought. In fact, a crude analogy could be drawn to macrophage polarization itself. Similarly to how the M1/M2 paradigm is perhaps too bifurcated and discrete to reflect the graded nature of macrophage function (Martinez and Gordon, 2014; Murray, 2017), so too may be a one-size-fits-all approach that suggests ‘polarized’ metabolic phenotypes are indispensable for pro- or anti-inflammatory macrophage activation. Rather, accumulating evidence suggests bioenergetic and signaling roles for mitochondria in both pro- and anti-inflammatory macrophage activation, although these manifest in different ways.