**4. Role of MM-enteric neuron communication in GI motility**

### **4.1 Intrinsic innervation and MMs**

The intrinsic and extrinsic sympathetic and parasympathetic neurons innervate the GI tract. The enteric nervous system (ENS) contains more than 100 million neurons and more than 400 million glial cells distributed in thousands of small ganglia that cooperate with the CNS, controlling digestive function [29]. However, the ENS can also control digestive function independently from the CNS. MMs share the space with cells contributing to GI motility, such as interstitial cells of Cajal (ICC), ENs, smooth muscle cells, PDGFRα-expressing cells, and glial cells [30]. In the last 5 years, multiple studies shed light on the functional interactions between MMs with all those different cell types to regulate GI motility. Here, we will describe the novel insights into the intimate communication MMs establish with ENs to control GI motility in health and disease.

From their first discovery, few morphological studies clearly showed the close anatomical association between MMs and ENs. Muller et al. described for the first time the interaction between MMs and ENs functionally [8]. Since then, multiple studies have been performed to elucidate the impact of this interaction on health and disease. This study showed that MMs express morphogenetic protein 2 (BMP2), while ENs express the BMP2 receptor (BMP2r). Functional interaction between BMP2 and BMP2r led to molecular pathway activations via the pSMAB1/5/8 pathway. Microbiome in this type of interaction was also playing a role. It was demonstrated that applying BMP2 to GI tissue in vitro promotes GI motility acceleration. In addition, the depletion of MMs led to colonic dysmotility in both ex vivo and in vivo models [8].

Neurohypophysis, known as the posterior portion of the pituitary gland, releases oxytocin into circulation. This hormone is essential during labor for inducing uterine contractions [31]. In addition, pro-inflammatory MMs can regulate the expression of oxytocin and its receptor. This has been shown in cell cultures of ENs, and the interaction is made possible via the STAT3 or NF-κΒ pathways. On the other side, anti-inflammatory MMs cause upregulation of oxytocin and its receptor via a TGF-β related mechanism [32]. Interestingly, lower concentrations of oxytocin and

#### *Muscularis Macrophages in Healthy and Diseased Gut DOI: http://dx.doi.org/10.5772/intechopen.109889*

its receptor have been associated with more pro-inflammatory cytokines in mouse models of dextran sulfate sodium (DSS) associated colitis [33].

A small population of MMs within the ganglia of the intestines was recently reported. These cells, also known as intra-ganglionic macrophages (IGMs), seem to have phagocytic properties [34]. Although IGMs interact with ENs in the same way as CX3CR1high MMs, there is not enough evidence to support critical phenotypic differences between these two cellular populations. In addition, experimental mice models of induced colitis have demonstrated loss of IGMs in association with increased pro-inflammatory MMs and enteric neural inflammation [35].

An interesting mouse model for the indirect study of MMs-ENs functional communication is Csf1op/op. These mice have a genetic lack mutation in the Csf1 gene that results in the absence of tissue macrophages [36]. This mouse model had an abnormal myenteric nervous plexus and more ENs than controls. Interestingly abnormal cellular changes are not confirmed for the other cell types, as both ICC and smooth muscle cells are not changed in the same animal model. MMs potentially also regulate ENs subtypes. For this reason, cholinergic neurons remained unchanged despite the increased numbers of nitrergic ENs [37]. This finding suggested that MMs may be capable of inducing different phenotypes of ENs. Furthermore, we used the same mouse model to find increased neuronal cells with shared cholinergic and nitrergic phenotypes, pointing to a more primitive population of ENs preserved in the adult muscularis propria [38]. Interestingly, this population is enriched during development in wild-type mice but is almost absent in adults. More studies are needed to show the possible contribution of MMs in the maturation of ENs to a specific adult subtype.

In the brain, microglia create an anatomical specialized somatic connection with neurons that facilitate their functional interaction. A concentration of organelles is associated with this connection, favoring the production of substances responsible for the functional interaction via the P2Y1R receptor. Recently a study has shown for the first time the presence of the same receptor on gut MMs and enteric glia, which must be studied further in the future. In addition, similar specialized anatomical connections between MMs, smooth muscle cells, and fibroblast-like cells have been described.

Although most of the studies were focused on the regulation of ENs by MMs, a few reports showed that also macrophage phenotype is shaped by ENs. For example, evidence from the study by Muller et al. showed that ENs supply Csf1 into the anatomic location of the muscularis propria, which in turn has an active role in the homeostasis of MMs, particularly in inducing an anti-inflammatory phenotype [8].

In the CNS, the microglia–neuron interaction happens early during development and is instrumental in setting up the adult brain. Recently, some studies have highlighted a possible role of MMs in the organization of ENS during development. A common finding is the independent intestine colonization by these two distinct cellular populations. In addition, MMs are directed towards specific niches, a particular localization that facilitates connection with the neural processes of ENs [21]. In addition, although Csf1r is mainly expressed in adulthood by ENs, it is primarily expressed by ICC and PDGF receptor alpha-positive cells during development. This result shows that during development, MMs may establish functional interactions with ENs independently of the Csf1 mechanism. Recent findings in a zebrafish irf8-deficient model showed that a lack of irf8 gene expression, typically expressed in MMs, can lead to MMs depletion and impaired gut motility [39].

By regulating membrane properties and ion exchange, ion channels respond to external changes with intracellular biochemical responses. Mounting evidence suggested the central role of ion channels in regulating tissue-resident macrophage functions. For example, in a variety of organs, ion channels contribute to macrophage phenotype, differentiation, and circulating monocyte extravasation. Although mounting evidence suggests the implication of those channels in regulating macrophage homeostasis and function in multiple organs, little is known about their contribution to MMs function.

TRP channels constitute a superfamily of Ca2 + −permeable, nonselective cation channels [40]. These channels can respond to temperature, pain, sound, and taste stimuli. Recent studies have highlighted an exciting novel role for these channels in regulating immune cells [41–43].

TRPV4 is expressed preferentially by MMs, and its activation leads to changes in GI motility by producing prostaglandin E2. The release of prostaglandin-2 from activated MMs produced a colonic contraction independently of neuronal activation. This channel may play a role in functional disease conditions. For example, an increase in TRPV4 expression has been reported in the colon of TNBS-treated mice, underlying a possible contribution of this channel to trigger inflammatory mediate the immune response. Notably, administering a selective channel antagonist reduces the severity of the inflammation. In line with this discovery, applying an agonist promotes the severity of inflammation. All this information about the TRPV4 channel is solid evidence of its implication in regulating homeostasis and inflammatory response. Although the mechanism by which this channel is implicated in regulating GI motility has been elucidated, further studies are needed to understand the mechanisms underlying TRPV4 implication in inflammation. The block of the P2X2 receptor channel reduced inflammation-related cellular damage in an IBD mouse model. Recently a study provided the expression of these channels on MMs and enteric glia. P2X2 MMs appeared to be mostly distributed within the myenteric plexus, where they anatomically establish a connection with ENs. Future studies are required to validate these studies and determine the role of this channel in immune-mediated GI function.

#### **4.2 Extrinsic innervation and MMs**

Gut-brain axis is made possible through the anatomic framework of visceral sensory (extrinsic afferent), sympathetic, and parasympathetic (efferent/autonomous) innervation. Visceral sensory nerve fibers do not directly regulate intestinal motility. However, they are extremely important for the gut-brain axis connection and regulation of several cells encountered within the ENS [44]. Several studies have suggested a possible physiologic relationship between MMs and peripheral nerves. One example is represented by CX3CR1-positive macrophages, which can be found in close association with nerve fibers of the sympathetic nervous system [45].

The interaction between MMs and visceral sensory fibers has recently been the subject of intensive investigations. More specifically, MMs affect catecholaminergic sympathetic signaling and its impact on systemic immunomodulation [46]. Recently, Gabanyi et al. proposed a role of β-adrenergic receptor 2 (β2ΑR) in this interaction. MMs expressing β2AR can be found in close anatomical relationship to the cell bodies of ENs. Indeed, MMs express higher levels of this receptor than other types of macrophages, including those of the lamina propria [7]. Furthermore, an effect of post-infectious neuronal loss mediated through adrenergic signaling by β2AR has also been demonstrated [47].

The primary neurotransmitter secreted by the vagus nerve, specifically from its preganglionic fibers, is acetylcholine (ACh). Besides its neurotransmitting role, ACh has essential parts in the inflammation process. This has been demonstrated

#### *Muscularis Macrophages in Healthy and Diseased Gut DOI: http://dx.doi.org/10.5772/intechopen.109889*

by experimental models of endotoxin administration, where the subjects showed a reduced inflammatory response after ACh stimulation [48]. In addition, stimulation of the vagus nerve, which is a primary source of ACh signaling increase, has a positive impact on reducing inflammation by promoting an anti-inflammatory MMs activation through the alpha-7-nACh receptor (α7nAChR) [49]. Vagal nerve stimulation is important in the pathophysiology of gastroparesis, enhancing a pro-inflammatory response [50]. The vagus nerve, also known as the tenth cranial nerve, or cranial nerve X, is the longest nerve in the body and one of the major suppliers of parasympathetic innervation to the gut. The vagus nerve originates from two distinct regions of the CNS: the ambiguous nucleus and the dorsal motor nucleus [51]. The multiple effects of vagal innervation on the gut have been well investigated in various studies.

Stimulation of the vagal nerve induces an anti-inflammatory phenotype in MMs. This has been studied in an experimental model of mechanical mucosal stimulation, which reduces overall inflammation. This effect is independent of splenic vagal stimulation since vagal splenic denervation does not hinder MMs activation. It seems that α7nAChR is extremely important in this process since MMs extracted from mice deficient in α7nAChR are unresponsive to vagal nerve stimulation [52–54].

In addition, extrinsic vagus nerve innervation participates in gastric motility regulation. According to preclinical studies, the vagus nerve plays a significant role in ameliorating inflammatory response in Inflammatory Bowel Disease (IBD). Mice with resected vagal nerves can develop a severe form of colitis, resulting in a surge of pro-inflammatory cytokines such as TNF-α, interleukin-1β, and interleukin-6 [55]. MMs from experimental models of genetic or pharmacological sympathetic nerve deprivation display pro-inflammatory phenotype. This MMs phenotypic activation in sympathetic innervation-deprived mice depends partially on monocyte transmigration into the intestinal muscularis propria [56].

As a consequence of an overall increase in inflammation, the same mouse models experienced an acceleration of GI transit. As discussed below, manual manipulation of the gut during surgery is implicated in the induction of postoperative ileus, a condition associated with increased levels of macrophages with anti-inflammatory phenotype [57]. It seems that severe forms of IBD frequently arise in patients with clinical depression or a setting of severe psychological stress. Although most research has been performed in humans, experimental animal models of depression exist, and it has been shown that they are more susceptible to developing severe colitis [58]. Notably, a post-vagotomy status can diminish any benefit from administering antidepressant medications. Through an unclear mechanism, transferring macrophages from experimental animal models of depression induces a trait in the recipient mice, which can become much more susceptible to severe forms of colitis [59].

Stimulation of the vagal nerve has important implications in gastroparesis, a disease we will discuss in detail below. Briefly, gastroparesis is characterized by reduced gastric motility and an enhanced pro-inflammatory phenotype in MMs. In addition, vagal nerve stimulation induces anti-inflammatory MMs activation, which improves overall clinical symptoms [60].

The induction of anti-inflammatory MMs underlines the preventive role of vagal nerve stimulation in gastroparesis by the STAT3-JAK2 molecular signaling pathway [61]. In contrast, pro-inflammatory MMs induction occurs during and after abdominal surgery, leading to increased inflammation of muscularis propria and reduced gastric and intestinal mobility. This situation can be regulated by performing vagal nerve stimulation [50]. Furthermore, optogenetic manipulation of colonic sympathetic nerves reduces leukocyte recruitment, favoring a recovery from induced experimental colitis.

### **5. MMs and GI diseases**

Because of the intimate interaction which MMs establish with ENs and other cells required for GI motility, it comes without surprise that these cells are implicated in various pathologic conditions impairing GI motility. Therefore, we will describe below different pathological conditions where an implication of MMs has been described.

#### **5.1 Gastrointestinal inflammation**

Inflammatory bowel disease (IBD) is a disorder characterized by inflammation of the GI tract. Because of this inflammation, the muscularis propria undergoes tissue changes, such as smooth muscle hypertrophy and plexitis of the ENs. The gut microbiota functions as a continuous reserve of bacterial antigens. Due to their anatomical association, these microorganisms continuously activate lamina propria macrophages, preventing them from becoming tolerant during sustained inflammatory conditions [62]. MMs activation depends on pathogen-associate molecular patterns (PAMPs) in the post-operated gut [63]. Although Toll-like receptors usually recognize PAMPs, an event which is crucial in further mediation of the innate immune response [64], postoperative gut hypomotility, also known as postoperative ileus, does not directly depend on Toll-like receptor pathways, but on interleukin-1 upstream receptor (IL-1R1) [65].

In experimental mouse models, expression of IL-1R1 by enteric glial cells is associated with co-expression, among others, of the proinflammatory cytokine interleukin-6. Therefore, it is not surprising that the administration of anakinra, a potent IL-1R1 antagonist, reduced inflammation and the occurrence of postoperative ileus in these animals [65]. Given that interleukin-17 positively regulates inducible nitric oxide synthase (iNOS) expression by MMs, it seems reasonable that gut motility can be impaired following an interleukin-17 surplus in the gut microenvironment [66]. A resulting hypomotility contrasts with clinical symptoms of bacterial GI infections, which normally are intestinal hypermotility and episodes of diarrhea. In rat animal models of IBD, MMs within the myenteric nervous plexus are responsible for the persistent inflammatory condition underlying the pathogenesis of the disease [67].

Inflammation is generally associated with increased monocyte recruitment onto the muscularis propria. In one recent study, during inflammation, monocyte recruitment is promoted by enteric glia that expresses multiple genes potentially responsible for monocyte engraftment onto the muscularis. In addition, in the presence of enteric glia supernatant, bone marrow-derived macrophages induce macrophage activation to an anti-inflammatory phenotype. This finding was confirmed in-vivo, where tamoxifen-induced enteric glial removal reduced monocyte recruitment responsible for the anti-inflammatory protective CD206 MMs. However, this leads to an increased overall level of inflammation in the tissue. Also, a functional interaction in the opposite direction seems true. IL1B, a proinflammatory marker, induces the activation of enteric glia to an "activated state" called gliosis. Importantly conditional removal of Il1BR in ECC prevents MMs activation in POI.

Interestingly, this is in line with similar observations on astrocytes in the CNS, cells that have a similar function to EGC. Recently, another group proposed a new route for monocyte recruitment during inflammation. After damage, large peritoneal macrophages are recruited from the serosal side and participate in tissue repair via ATP. Until now, the immediate and unique access for monocytes onto the muscularis propria was considered the submucosa region where big blood vessels are present.

#### **5.2 Gastroparesis**

Gastroparesis is a significant motility GI disorder characterized by delayed emptying without obvious etiopathogenic factors [13]. Gastroparesis is commonly seen in patients with diabetes mellitus (DM), with a prevalence of approximately 40% in type 1 DM and 20% in type 2 DM. Given the prevalence of DM in the general population, gastroparesis is a prevalent condition, leading to increased patient morbidity and socioeconomic costs [68]. Other conditions predisposing to the development of gastroparesis are surgical operations of the stomach or esophagus. However, in a significant subset of patients, the condition can be idiopathic, meaning no apparent predisposing factor can be identified [13].

The clinical signs and symptoms of gastroparesis vary. Patients often present with GI symptoms such as bloating, postprandial fullness, reduced food intake, nausea and vomiting, and weight loss, which can be evident at later stages [69]. The condition is frustrating for patients, severely affecting their quality of life, and is associated with concomitant anxiety or clinical depression symptoms in as many as half of them [70]. Gastroparesis has also been associated with reduced survival of patients. In a significant cohort of patients with gastroparesis, the 5-year overall survival of patients with DG, adjusted for age and gender, was 67%, in contrast to 81% in the non-gastroparesis population [70]. Patients with idiopathic gastroparesis had slightly better outcomes than patients with diabetic gastroparesis (DG). This finding can be explained by the increased co-morbidities that can be seen in patients suffering from DM [70].

The underlying mechanisms responsible for DG have long been unclear until recent evidence suggested a significant role of MMs in the pathophysiology of this condition. In mouse models and patients with DG, a reduction of CD206, antiinflammatory MMs has been observed compared to controls. In addition to reduced anti-inflammatory MMs, DG has been associated with more pro-inflammatory markers, normally absent in controls. Therefore, this series of studies highlighted the possible role of MMs activation in the pathophysiology of DG. It is crucial to notice that in vitro experiments identified MMs activation via oxidative stress as one of the possible causes. Increased oxidative stress levels, generally associated with DG-induced activation of MMs to a pro-inflammatory phenotype and combination of IL6 and TNF-alpha, lead to ICC reduction in vitro.

Further studies are required to understand the underlying mechanisms driving immune-mediated ICC loss in DG, which represent the main cellular changes observed in DG. Neshatian et al. showed that the phenotype of MMs could be altered in response to DM-induced tissue oxidative stress [68]. More specifically, activated MMs produce heme oxygenase-1 (HO1), which has an important anti-oxidative role, protecting against the development of gastroparesis in experimental models of diabetic mice. In contrast, neuromuscular depletion can occur secondary to the activation of those MMs that cannot produce HO1 [68]. This can severely impact gastric motility, acting as a prerequisite for developing gastroparesis [68]. Further evidence has shown that in an experimental model of diabetic mice, the depletion of MMs reduced the incidence of the development of DG [71].

#### **5.3 Post operative ileus**

Post operative ileus (POI) is a very common condition, which can be described as a transient decreased GI motility condition following abdominal surgery. This results in prolonged hospitalization and recovery time, reducing patient quality of life and increasing healthcare expenditure. Although the pathophysiology of POI is complex, it seems to be arising in a background of neurogenic and inflammatory deregulation, mediated by corticotropin-releasing factor, which promotes central and autonomic nervous system response [72]. The resulting inflammatory response is characterized by sustained expression of intercellular adhesion molecule-1 (ICAM-1) and P-selectin, which both facilitate circulating monocyte extravasation [73]**.** This is further supported by experimental evidence, demonstrating that targeting of the adhesion molecules by monoclonal antibodies leads to reduction of transmigrating white blood cells and attenuating muscle contractility dysfunction [74]. Neurogenic dysregulation can be experimentally prevented by activation of 5-hydroxytryptamine receptor-4 (5HT4R) and reduction of nicotinic receptor activation. This happens mostly during vagus nerve stimulation, since activation of cannabinoid receptors (CB) in cholinergic neurons inhibits acetylcholine release and reduces gut motility, leading to delayed gastric emptying. These findings are supported by experimental findings using CB1 −/− mice. Although POI and systemic inflammation were noted both in wild-type and CB1 −/− mice, the latter had higher plasma levels of interleukin-6 (IL-6) and cytokine-induced neutrophil chemoattractant-1 (KC/CINC1) in gut mucosal and submucosal tissues [75]. Mast cells also contribute to the development of POI with a variety of mechanisms, including degranulation induced by neurotransmitters released in response to gut surgical manipulation and mechanical stretch. The exacerbation of gastroparesis by mast cell degranulation can be partially alleviated by mast-cell stabilizing treatments [76]. As mast cells express Kit, experiments in mast cell deficient Kit / Kitv mice showed that gut manipulation did not result in significant increase in transmigration of white blood cells [77]. Moreover, mice with abnormal Kit also have deficits in the ICC, which explains the impaired gut mobility even without surgical manipulation [78]. The above findings demonstrate the multifactorial background of development of POI, warranting further investigation on the role of MPMs in POI.

#### **5.4 Aging-associated dysmotility**

Life expectancy is increasing progressively, and some GI diseases are more prevalent in the elderly. Old people have a slower gastric emptying that can affect appetite regulation. This usually represents an underrecognized clinical problem that may lead to adverse life quality and increased mortality. Most GI dysmotility problems in the elderly happen in the colon, where region-specific changes to enteric neuron numbers have been observed in both mice and humans. This is in line with changes observed in neuronal-mediated smooth muscle contractility. Since a smooth muscle contractility pattern is required for an effective GI transit, those changes may reflect gastrointestinal motility disorders in the elderly. Like many other different cell types, macrophages are affected by time by changing their transcriptome, functions, and phenotype. Changes to the macrophage population observed in aging may underline the tissue changes associated with diseases. For example, multiple reports show the central role of microglia in brain tissue changes related to Alzheimer's disease and other aging associated brain diseases [79]. Also, in the gut, recent changes to MMs have been reported in humans and mice that could be underlying GI motility changes in the elderly. A recent characterization of MMs phenotype in old mice showed an increased MMs subpopulation that expresses pro-inflammatory genes [80]. Notably, the accumulation of this population is contracted within the myenteric plexus, where they co-localized with the A-Synuclein marker, suggesting that they may play a role in *Muscularis Macrophages in Healthy and Diseased Gut DOI: http://dx.doi.org/10.5772/intechopen.109889*

phagocyting. Lineage tracing experiments to study the origin of tissue-resident MMs revealed that also, with age, the number of protective CX3CR1 MMs are reduced. The remaining CX3CR1 are accumulated within the myenteric plexus where they continue to interact with ENs. Further studies are needed to understand the underlying mechanism responsible for this type of reduction, given the role of embryonic MMs in preserving GI homeostasis.
