**5. Macrophages**

#### *5.1. Macrophage Origin, Polarization, and Function*

Naïve macrophages are widely distributed in all tissues via circulation through the bloodstream [82]. These cells remove apoptotic cells and foreign material via phagocytosis and participate in various processes, such as wound healing and tissue repair [83]. Macrophages are derived from bone marrow hematopoietic stem cells [84]. When stimulated by cytokines, bone marrow-derived macrophages develop into monocytes that then differentiate into pre-macrophages [35,85]. Finally, they become mature macrophages that can be released into the bloodstream [35,86]. Macrophages respond to current conditions to form a heterogeneous cell population [87]. Under the influence of various stimuli, they usually differentiate into one of two phenotypes (polarization) [88]: proinflammatory type (M1) and anti-inflammatory or reparative type (M2) [89]. M1 macrophages are proinflammatory and secrete cytokines, while M2 macrophages are anti-inflammatory and promote tissue repair to resolve inflammation [87]. M2 macrophages can be further sub-classified as M2a, M2b, M2c, and M2d based on transcriptional changes that result from exposure to different stimuli [90]. Lipopolysaccharides (LPS), TNF-<sup>α</sup>, and interferon gamma (IFN)-γ are used to convert macrophages to M1, IL-4 and IL-13 to M2a, immune complexes (IC) and toll-like receptors (TLR) to M2b, IL-10 to M2c, and adenosine A2A receptor (A2AR) agonist to M2d (Figure 3B) [29]. Therefore, stimulus-dependent polarization controls the specific functions and phenotypes of macrophages.

#### *5.2. Relationships between Macrophages and MaRs*

Macrophages participate in the biosynthesis of SPMs with both anti-inflammatory and pro-resolving properties [34]. SPMs are enzymatically biosynthesized from essential fatty acids with different stereochemistry [74,80,91]. MaRs—a new family of macrophage-derived mediators—are synthesized from DHA by macrophages and are potent in the resolution of inflammation [33]. Most importantly, MaR1 directly enhances neutrophil activation and the switch from macrophage M1 to M2 phenotype [92], both of which promotes anti-inflammatory and pro-resolving actions, inhibiting neutrophil infiltration and stimulating macrophage phagocytosis and efferocytosis to enhance the clearance of inflammation without affecting the innate response [27,49].

#### **6. Role of MaRs in Inflammation Resolution**

The level of this potent leukocyte agonist decreases in the later stages of the self-limited inflammatory response [93]. It is possible that other signals regulate leukocyte responses to promote tissue repair and regeneration. Given the pivotal roles of chemical signals in infections, it has been revealed that new mediators, within self-resolving infections, can regulate tissue repair and regeneration without immune suppression [34]. MaR-1 exhibits potent pro-resolving and tissue regenerative activity and is involved in self-limited infections that regulate tissue regeneration [34,94]. New pathways and mediators in planaria promote recovery and regeneration during infection [27]. The recruitment of leukocytes into the lesioned spinal cord is regulated by various proinflammatory mediators [95,96]. Cytokines mediate inflammation by acting on specific receptors that activate different intracellular inflammatory cascades [97]. Additionally, MaR-1 downregulates cytokine expression in mouse models of colitis and acute respiratory distress syndrome [41,49,92]. However, little is known about the intracellular cascades regulated by MaR-1.

Although IL-10 has anti-inflammatory properties, its contribution to the healing process is not fully established. In a recent report, MaR1 increased the levels of IL-10 postoperatively for 14 days [98]. Notably, MaR-1 has been reported to interact with stem cells to reduce chronic inflammation and improve wound healing following SCI [77,99]. Interestingly, macrophages incubated with MaR-1 are polarized toward an anti-inflammatory phenotype and increase MRC1 mRNA expression (an M2 macrophage phenotype marker), implying a possible role of MaR-1 in M2 macrophage polarization [42]. TNFα is one of the earliest cytokines to appear following tissue damage and is associated with the production of many cytokines, including IL-1β and IL-6. MaR-1 attenuates the release of proinflammatory cytokines and TNFα in macrophages [49,100]. In addition, intracellular adhesion molecule 1 (ICAM-1) is an epithelial PMN ligand that promotes neutrophil migration through epithelial cells during inflammation [92]. MaR-1 inhibits the 10-fold upregulation of ICAM-1, suggesting that it contributes to the resolution of inflammation by a ffecting neutrophil clearance and e fferocytosis. Another possible avenue for treatment with MaRs is motor neuron disease, a fatal neurodegenerative disease that causes loss of motor neuron function and progressive degeneration [101]. However, the molecular mechanisms of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are not ye<sup>t</sup> full known. Many pathogenic changes occur in the a ffected motor neurons, including mitochondrial dysfunction, hyper excitability, glutamate excitotoxicity, and nitroxidative stress [101]. Superoxide dismutase 1 (SOD1) G93A and transactivation response DNA-binding protein (TDP)-43A315T cause oxidative stress, endoplasmic reticulum (ER) stress, and inflammation. MaR-1 possesses neuroprotective e ffects against stress-induced cell death induced by various factors, such as SOD1 G93AA315T and TDP-43A315T, inhibiting NF-κB activation [102]. Therefore, MaR-1 may also contribute to treatment options for motor neuron diseases, such as ALS and SMA (spinal muscular atrophy).

#### **7. Role of MaRs and Macrophages in Inflammation Resolution**

Macrophages are involved in the processes of homeostasis, tissue repair, and regeneration [29]. They are recruited to damaged nerve sites through the activation of M1 and M2 subtypes [29]: M1 macrophages exhibit a proinflammatory profile and mediate cytotoxic actions; and M2 macrophages have anti-inflammatory e ffects and promote tissue healing and recovery [31,32]. The balance of M1 and M2 macrophages regulates early events in local inflammation [103]. In this process, cytokine contributes to the recruitment of M1 macrophages [104]. It releases other proinflammatory cytokines, such as TNFα, IL-1 α, and IL-β, to promote further tissue damage [105]. To control this process, activated M2 macrophages release anti-inflammatory cytokines, IL-10 and TGF-β, which mediate tissue regeneration and inhibition of the proinflammatory function [106]. Also, an increased ratio of M2 macrophages significantly enhances nerve regeneration and wound healing [107]. DHA plays important roles in peripheral organs, as well as in the central nervous system, and is the precursor of various molecules that regulate the resolution of inflammation [50,108]. Macrophages derived from mice deficient of Elov12 (Elovl2-/-)—the main enzyme for DHA synthesis—demonstrate an increased expression of M1-like markers (iNOS and CD86), whereas M2 macrophages downregulate M2-like markers such as CD206 [50]. Similarly, the impairment of systemic DHA synthesis in activated macrophages results in an alteration of M1/M2 macrophages, supporting the important role played by DHA in regulating the balance between pro- and anti-inflammatory processes. Inflammation resolution is an active and highly regulated inflammatory process that is necessary to prevent the transition into chronic inflammation with the spread of tissue injury or exacerbated scarring [13]. However, di fferential leukocyte subpopulations reduce or otherwise impair the ability to resolve inflammation at the lesion site after acute experimental spinal cord injury (SCI) [95,103]. For example, after SCI, M2 macrophage function is shown to be defective in resolving inflammation, impairing tissue remodeling and healing [77]. Macrophages in SCI are not defined within the M1-M2 dichotomy. MaR1 is e ffective in enhancing several stages of inflammation resolution after SCI through the downregulation of cytokines, reduction of neutrophils and macrophages, shift in macrophage phenotype, and stimulation of macrophage phagocytosis [77]. Treatment with MaR1 after SCI reportedly enhances neutrophil clearance and reduces macrophage accumulation in the lesioned tissue [77]. Inappropriate biosynthesis of SPMs after SCI interferes with the resolution of inflammation and contributes to the pathophysiology of SCI. Abnormal production of SPMs is also reported in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease and multiple sclerosis [68,75]. Thus, the potential function of MaRs can be confirmed by their immunoresolvent effects on the phagocytosis of macrophages and their inhibitory functions on cytokine levels and inflammatory signaling pathways [77]. Chronic inflammation is the basis of the common pathology of age-related diseases, such as cardiovascular disease, diabetes, and Alzheimer's disease [2], involving alterations to the immune system that promote chronic inflammation. Macrophages are important in these age-associated changes that cause chronic inflammatory diseases [72]. Recent studies have shown that aging impairs macrophage phagocytosis, resulting in a failure to resolve damage-associated molecular patterns in aged animals [2,45,51].

#### **8. Role of MaRs in Resolution of Inflammatory Pain**

MaR1 not only regulates the resolution of inflammation, but also plays a powerful role in preventing hyperalgesia sensitivity in inflammatory- and chemotherapy-induced chronic inflammatory pain [23,27]. MaR1 dramatically reduces vincristine-initiated neuropathic pain in a cancer chemotherapy model [27] and in temporomandibular joint pain [26]. In addition, MaR1 has played an important role in the prevention of postoperative pain in orthopedic surgery models [24]. Postoperative pain managemen<sup>t</sup> with MaR1 may help control the onset of neuroinflammation. Acute perioperative treatment with MaR1 delayed the development of mechanical and cold allodynia. Moreover, MaR1 has been shown to induce analgesia by regulating transient receptor potential vanilloid 1 (TRPV1) currents in neurons. [27]. Intrathecal treatment with MaR1 reduces inflammatory pain with a long-lasting analgesic profile through the inhibition of astrocytic and microglial activation [109].

In the periphery, various cytokines contribute to neutrophil recruitment into the tissue and, consequently, an increase in inflammatory processes and pain [28,110]. Intrathecal MaR1 treatment reduces recruitment and leukocyte count [94,100]. In addition, MaR1 reduces CFA-induced mRNA expression of Nav1.8 and Trpv1 channels [26]. MaR1 likely controls TRPV1 expression in DRG neurons during inflammation. Thus, targeting these channels is e ffective for reducing inflammatory pain [111,112]. Under noxious stimuli, nociceptor neurons release neuropeptides, such as CGRP and substance P, which control the recruitment of immune cells to the inflamed tissue [113,114]. MaR1 reduced the release of CGRP from DRG neurons, indicating a possible mechanism by which MaR1 reduces inflammatory pain through reduced recruitment of neutrophils and macrophages. In the spinal cord, TNFα and IL-1β contribute to spinal cord plasticity and, hence, central sensitization [97]. Cytokines improve the amplitude of AMPA- and glutamate-induced excitatory currents [97]. Indeed, the CFA model induces central sensitization with stronger activation of astrocytes when compared to microglia [111,115,116]. Central sensitization has been recognized as the main cause of pathological pain, resulting in plastic changes in the CNS [117]. Intrathecal treatment with MaR1 reduced CFA-induced astrocyte and microglial activation and decreased activation by TNFα, IL-1β, and NF-κB [26]. In addition, the interaction between glial cells and nociceptor neurons has been linked to these plastic changes in the spinal cord [26]. MaR1 reduces glial cell activation and blocks capsaicin-induced TRPV1 calcium influx as well as spontaneous EPSC frequency [26]. Thus, MaR1 can reduce spinal cord plastic changes and inhibit central sensitization via presynaptic and postsynaptic mechanisms [26]. MCTRs also rescue *Escherichia coli* infection-mediated delays in tissue regeneration of planaria, in addition to protecting mice from second-organ reflow injury and promoting repair by limiting neutrophil infiltration [41]. Furthermore, each MCTR promotes the resolution of *E. coli* infections by increasing bacterial phagocytosis and limiting neutrophil infiltration [25]. Phagocytosis is a main means by which macrophages resolve inflammation [2]. Increasing evidence suggests that MaRs produce potent anti-inflammatory and pro-resolution profiles, partially by enhancing macrophage activity [17,93]. However, it remains unclear how MaRs regulate macrophage phagocytosis.
