General role of intestinal macrophages

Tissue macrophages perform a variety of different functions and are an essential component of the mammalian organism. These functions depend on and are instructed by factors from the local environment and executed in response to specific functional demands [40]. These functions are mainly determined by the macrophage’s ability to sense their local environment. Under healthy conditions, these functions promote tissue homeostasis, but noxious challenges, i.e., infections, trauma, ischemia and metabolic stress, rapidly change the homeostatic functionality of macrophages to immunological mechanisms ensuring host defense and inflammation [2]. Tissue macrophages from different organs, even those with different ontogeny and development, may exhibit some common “classical” functions, i.e., phagocytosis and anti-microbial activity. However, each population is individually shaped by its local microenvironment and therefore tissue macrophages are extremely heterogeneous and their functions go far beyond the classical phagocytic and anti-microbial activities.

Excellent reviews are available focusing on tissue macrophage function in several organs including the lung, the liver, the spleen, and the intestine [6, 44, 103]. Intestinal macrophages are crucial in maintaining gut homeostasis [20, 42, 73]. Furthermore, these cells are numerous in the intestinal muscularis externa to regulate intestinal motility under homeostasis and during inflammation [3, 32, 75, 106]. Here, we focus on specialized resident macrophages which inhabit the gastrointestinal (GI) tract. As these cells consist of different subpopulations significantly differing in function, we compare macrophages of the muscularis externa to their proximate neighbors in the lamina propria mucosae and illuminate their functions under homeostasis and during acute inflammation. Given that the function and the phenotype overlap to dendritic cells, this review also discusses the recent advances to differentiate these myeloid cell subsets.

Historical view on muscularis macrophages

First evidence for the presence of macrophages in the muscularis externa was published by Taxi et al. who detected a macrophage-like interstitial cell type between the adjacent intestinal muscle layers that was able to take up trypan red [93]. These cells were mainly observed in three layers: in the subserosal layer, the myenteric plexus (between the circular and longitudinal muscle layer), and in association with the deep muscular plexus within the circular muscle layer (Fig. 1). Early microscopical analysis by Mikkelsen and colleagues demonstrated that cells in these three locations differ in their microanatomy, pattern formation, capability of dextran uptake, and express typical macrophage markers [69]. Subserosal macrophages showed a few longer slender processes, which are rather regularly distributed in rows. In contrast, cells of the myenteric plexus are more stellate shaped with shorter branches and are evenly distributed throughout the plexus (Fig. 1). Both cells were identified as specialized cells of the mononuclear phagocyte system [70]. Additional work from the same group demonstrated that these cells depend on the colony-stimulating factor 1 (CSF-1), because op/op mice, which are defective in the production of functional CSF-1 [113], lack resident muscularis macrophages with exception of some rare subserosal cells [68]. Further work demonstrated that these macrophages constitutively express MHC class II+ and outnumber F4/80+ cells indicating parallel presence of at least two different cell populations [72].

Fig. 1
figure 1

Neuroimmune interactions in the intestinal muscularis externa. Macrophages can be found throughout the complete GI tract. They differ in numbers, phenotypes, and functions. They are predominant in the mucosa but are also located in the submucosal plexus and form dense networks in the muscularis externa. In the latter, they appear in distinct dendritic morphologies reaching from a simple bipolar shape up to a stellate appearance with dendriform extensions connecting them with the surrounding tissue. Extrinsic and intrinsic neuronal processes innervate all intestinal layers, but intrinsic neuronal cell bodies are restricted to the submucosal and myenteric plexus. Close proximity and functional relationship between nerves and macrophages are particularly observed in the muscularis externa. Extrinsic parasympathetic (Nodose ganglion/Vagus nerve) and sympathetic (Superior/Celiac ganglia) innervation was shown to modulate macrophage immune function by acetylcholine or norepinephrine, respectively. In myenteric ganglia, macrophages also interact with enteric glia and can promote acute inflammation

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Macrophage subsets in the intestinal muscularis: ontogeny and specialization in the different layers of the intestine

Macrophages are a heterogeneous population of phagocytic immune cells, which are present in almost all tissues [42]. In the dermis, macrophages are established prenatally but also after birth from blood-derived Ly6Chi monocytes. However, epidermal macrophages, also called Langerhans cells, seem to replenish independently of blood precursors [62]. Comparably, macrophages of the brain and the liver are long-living cells, which are derived from earlier yolk sac and fetal liver progenitors. These cells maintain themselves during life with little contribution of circulating Ly6C+ monocytes [1, 45, 112]. In contrast, intestinal lamina propria macrophages have an exceptionally short half-life of 3 weeks [50], which requires constant de novo generation by recruitment and differentiation of myeloid bone marrow-derived progenitors [8, 12, 101, 102, 115]. Fate mapping experiments established that short-lived Ly6C+ monocytes constitute homeostatic precursors of intestinal macrophages in the lamina propria [112]. These data demonstrated that neonatal intestinal macrophages are not maintained through adulthood [7, 8] and that lamina propria macrophages in the intestine represent a continuum of cells with an important role in regulating intestinal immunity [111]. However, these studies about lamina propria macrophages are mostly based on digests of the whole intestine, which include the population of macrophages in the intestinal muscularis. Hence, muscularis macrophages might share the functionality and the origin of lamina propria macrophages, but their specific characteristics might have also been overlooked.

Little is known about the specific characteristics of muscularis macrophages. The distribution of these cells in adult mice seems to be phenocopied in embryos, newborns and germ-free mice indicating that these cells develop independently of foreign antigens, and their turnover may be slower in comparison to lamina propria macrophages [71]. Macrophages can exhibit a high degree of specialization, and the local microenvironment shapes the functional adaptations of macrophages in space and time. This process of local tissue “imprinting” and the mechanisms that regulate the spatial functionality of macrophage have been reviewed recently [2]. Tissue-dependent adaptations might also contribute to the differential expression of several surface molecules within the different layers of the intestine [57]. A recent study demonstrated a specific functionality of two macrophage populations with distinct localization in the intestinal tissue [38]. By employing imaging and transcriptional profiling, lamina propria macrophages expressed proinflammatory genes, while muscularis macrophages showed genes encoding tissue-protective factors which resembled M2 macrophages and these intra-tissue adaptations seemed to be related to the high density of neuronal processes in the muscularis layer [38, 104]. Also, specific isolation techniques revealed that macrophages in the different intestinal layers also differ in surface molecule expression [57]. These data demonstrate unique intra-tissue adaptation of macrophages and a high degree of specialization within intestinal layers. Furthermore, these findings indicate that lamina propria and muscularis macrophages may develop independently from a common progenitor. As mentioned previously, lamina propria macrophages are constantly replaced by infiltrating Ly6C+ blood monocytes, which acquire a tolerogenic phenotype after tissue entry [12, 101, 102, 115]. In contrast, muscularis macrophages resemble a phenotype of tissue macrophages, which are non-inflammatory and protective to the tissue. Very recently, a reservoir of macrophages in the peritoneal cavity that invade visceral organs to affect tissue repair has been identified [105]. Following injury, these fully mature tissue macrophages infiltrated the liver via a non-vascular route to contribute to tissue repair [105]. These cells might also be capable to infiltrate the intestinal muscularis to populate this intestinal layer with tissue macrophages. However, further experiments are required to elucidate the origin and the functional properties of muscularis macrophages during health and disease.

Demarcation of muscularis macrophages to dendritic cells

In the intestinal lamina propria, macrophages and dendritic cells (DCs) form a continuous network of phagocytic cells [20]. Early studies demonstrated the presence of a phagocyte network also in the intestinal muscularis, and these cells were initially termed muscularis macrophages [69]. These cells are the main phagocyte subset in the healthy intestinal muscularis [57], which express low levels of CD11c but high levels of MHC class II and the CSF-1 receptor (CSF-1R) [75]. The important role of the CSF-1R is supported by the finding that CSF-1R-deficient mice showed dramatically reduced numbers of the resident muscularis CD11clow MHCIIhi macrophages [57]. It remains to be determined whether muscularis macrophages originate from early progenitors or whether there is constant replacement by the hematopoietic compartment as seen in the lamina propria [8].

Some of these muscularis phagocytes expressed high levels of DEC205 and were equipped with the activation markers CD80, CD86, and CD95 to function as antigen-presenting cells suggesting that some phagocytes in the intestinal muscularis resemble a DC-like phenotype [37]. These DCs in the intestinal muscularis were also able to activate Th1 memory cells in a murine model of postoperative ileus, a frequent disease after intestinal surgery [32]. However, further studies are required to better characterize these cells during homeostasis and inflammation.

In the lamina propria, DCs can be distinguished from macrophages by the differential expression of CX3CR1, F4/80, CD64, and CD103 [20, 44]. Several studies have demonstrated that although DCs express CD103, macrophages are devoid of CD103 expression but express high levels of CX3CR1, F4/80, and CD64 [7, 19, 85]. CD103+CD11b− DCs depend on the transcription factors Batf3 and Irf8 [31, 46, 56], cross-present antigens to CD8+ T cells [60], induce regulatory T cells and modulate T cell responses [35, 43, 44]. In contrast, CD103+CD11b− DCs develop from the common Flt3L-dependent pre-cDC progenitor [12, 102] by mechanisms involving the transcription factors Irf4 [80, 83]. They are unique to the intestinal tract and contribute to intestinal immunity [12, 19, 86]. However, little is known about these DC subtypes in the intestinal muscularis and further studies are required to describe the precise role of these DCs and to clearly differentiate these cells from muscularis macrophages.

Functionality of intestinal macrophages in health and disease

Under healthy conditions, blood-derived Ly6C+ monocytes are constantly recruited to populate the intestine with macrophages [8]. After tissue entry, these Ly6C+ monocytes possess a tolerogenic signature, which maintains homeostasis [12, 101, 102, 115]. Exposure to the luminal microbiota does not induce proinflammatory responses by these cells [7, 75]. Accordingly, muscularis macrophage polarization has also been shown to be affected by luminal bacterial infection and attributed to norepinephrine signaling to ß2 adrenergic receptors [38]. Hence, extrinsic signals by sympathetic neurons can skew muscularis macrophages to a tissue-protective phenotype identifying a protective neuroimmune communication between neurons and macrophages [38]. This neuroimmune axis also contributes to maintenance of homeostasis by regulating intestinal peristalsis [75]. A distinct macrophage subset in the intestinal muscularis regulates peristaltic activity in the colon and secreting BMP2, which activates the corresponding receptor expressed on enteric neurons [75]. However, this tolerogenic program is impaired in acute inflammation so that these cells acquire a proinflammatory signature and induce intestinal diseases [7, 32, 96, 106, 116].

A major reason for the activation of lamina propria macrophages is the continuous presence of foreign antigens, which prevents tolerance induction in macrophages during inflammation and tissue damage. The activation of muscularis macrophages under inflammatory circumstances might also depend on pathogen-associated molecular patterns (PAMPs), because translocation of bacteria from the luminal site has been observed in the murine model of postoperative Ileus and after surgical procedures in human subjects [54, 90]. However, it was recently shown that the induction of postoperative ileus was independent of Toll-like receptor signaling [92]. Instead, the upstream receptor for IL-1 (IL-1R1) was identified as a critical component for the development of postoperative ileus (POI) [92]. Bone marrow chimeric animals identified the important role of IL-1R1 expression on enteric glia cells, and these cells secreted the proinflammatory molecules MCP-1 and IL-6 after stimulation with IL1α or IL1β. Antagonism of IL-1R1 by anakinra reduced the inflammatory response and prevented POI [92]. Monocytes and macrophages are also required to maintain IFNγ and IL-17-dependent T cell responses in the mucosa during Citrobacter rodentium infection [84]. Also, in the intestinal muscularis, IL-17A induces inducible nitric oxide synthase (iNOS) expression in muscularis macrophages, which reduced intestinal motility [74]. The finding of hypomotility of the intestine is surprising, because IL-17 responses are mostly related with acute bacterial gastroenteritis, which cause hypermotility and diarrhea. Although the causative agent can be identified in some cases (e.g., bacterial gastroenteritis), non-specific viral agents and/or psychogenic factors can also induce hypermotility of the gastrointestinal tract. In rodent models of IBD, there is emerging evidence that pathogenic macrophages—from an unknown origin—can be found within the myenteric plexus [55, 59]. These cells communicate with the nervous system to induce this inflammatory disease [10, 28]. However, studies about muscularis macrophages in regulating intestinal hypermotility are limited and their role in the induction of this disease is largely unknown. In contrast, their role in inducing hypomotility has been demonstrated in several rodent models, particularly in postoperative ileus.

Specific focus on macrophages and DCs in postoperative ileus

A first interpretation of resident muscularis macrophage exhibiting immunogenic functions came from Mikkelsen and colleagues in 1995 [66]. The authors demonstrated that these cells were devoid of several enzymes that were described as macrophage activation markers indicating that these cells are not capable in activating an immunogenic cascade. However, Bauer identified resident muscularis macrophages as immunocompetent cells that become rapidly activated by a surgical trauma, by an extra-abdominal surgery and during sepsis [33, 51]. The resulting consequence is a POI or—in case of bacterial translocation—a septic ileus, both describing a motility disturbance of the GI tract. Clinical symptoms are nausea, vomiting as well as abdominal distension and pain. In consequence, aspiration of stomach contents into the lung, pneumonia, and even bowel perforation and sepsis can occur. The medico-economic burden of POI outnumbers 1.4 billion dollars annually, alone in the USA [49].

Research in the twentieth century primarily focused on investigating the neuronal mechanisms causing POI [11]. First evidence for an inflammatory impact in POI development was shown in an experimental rat model by showing increased expression of the lymphocyte-associated antigen-1 in resident muscularis macrophages after surgical manipulation of the GI tract [51]. Several subsequent studies demonstrated that resident macrophages release cytokines and chemokines during POI which in turn recruited further leukocytes from the blood circulation [11, 110]. Strong evidence for their role in the pathogenesis of POI came from depletion studies showing that either a pharmacological or a genetic depletion approach of resident macrophages prevented POI [106, 107, 109]. Importantly, some resident muscularis macrophages express markers of dendritic cells (see previous section), which complicate the specific conclusions about macrophages and DCs in regulating POI. As only a few studies distinguish between both cell types in the pathogenesis of POI, the reader should be aware that the term “macrophage” may also describe functions of DC.

The first evidence that muscularis “macrophages” also express markers and exhibit functions of dendritic cells was shown in colonic resections from human pediatric patients [88]. The authors identified increased presence of CD11c+ CD83+, and CD11c+ CD83− DCs in the muscularis externa of patients exhibiting a severe inflammation. This increase was accompanied by a reduction in CD11c+ CD83+ lamina propria DCs indicating relocation of DCs within the intestinal layers. One year later, dendritic cells were also described in the intestinal muscularis externa in mice and these cells responded to microbial stimuli, such as LPS and oral live bacteria, by upregulation of CD80, CD86, and DC-205 [37]. However, some of these F4/80+ cells did not express DC-specific surface molecules suggesting that both macrophages and DCs coexist in the intestinal muscularis externa [66, 79, 106]. Such coexistence is further supported by findings from our group demonstrating that DC-derived IL-12 subsequently activates macrophages to induce the expression of the iNOS [32]. This in turn leads to NO release from the stellate-shaped resident macrophages which directly inhibit smooth muscle cells [99]. Together, these findings established a molecular cascade linking intestinal DCs—which sense local injury—to intestinal macrophages, which can modulate intestinal peristalsis.

Besides its effects on smooth muscle cells itself, NO can also act on other surrounding cells, i.e., intrinsic or extrinsic neurons. We recently demonstrated that enteric glial cells (EGC), another cell type of the enteric nervous system (ENS), become activated and release IL-6 and MCP-1 upon IL-1 binding during POI [92]. Although the initial activation of the resident macrophage during POI is still a conundrum, IL-1 might be derived from these cells. Previous work indicated that activation of macrophages is a very immediate and transient step in the POI cascade and involves activation of p38 MAP kinase and JNK kinase [107]. Given that some of the kinases respond in minutes after onset of the surgical trauma, one could postulate that a quick, but active release of prestored mediators, i.e., neurotransmitters, may be responsible for the immediate activation. Indeed, we demonstrated that calcitonin gene-related peptide (CGRP)+ nerve endings within the muscularis release CGRP in response to the surgical trauma and CGRP in turn activated the resident muscularis macrophages to transcribe IL-1 [41], which in turn may activate EGC. Further experiments are required to elucidate the crosstalk between these cells in POI.

Crosstalk of resident muscularis macrophages with neuronal cells

Tissue-associated neurons are classified as intrinsic or extrinsic neurons. The extrinsic innervation of the gut includes the sympathetic or parasympathetic autonomous nervous system. The intrinsic innervation consists of the ENS which contains enteric neurons and EGC. The different neuronal cell types fulfill several functions by use of different neurotransmitters and are predominantly organized within the intestinal plexus. The most prominent plexus of the muscularis externa are the myenteric and the deep myenteric plexus wherein the majority of intestinal muscularis macrophages are embedded as a regularly distributed network (Fig. 1). A recent immunohistochemical study confirmed interdigitating connections between macrophage processes and dendrites of enteric neurons [81], which enable the communication between the macrophages and the enteric nerves, predominantly those located in the myenteric ganglia [69]. Another work demonstrated that macrophages in the muscularis externa are CX3CR1+ cells that are in close proximity to βIII tubulin+ nerves [75]. In general, those nerve-macrophage associations were observed in the small intestine, the colon and the stomach of rodents and humans [9]. Additionally, resident macrophages are also distributed in the serosal plexus (containing rather fine nerve bundles than ganglia) and in the deep myenteric plexus. Of note, macrophages within the different plexuses significantly differ in morphology [65].

A general association of nerves and macrophages is known for a long time, but little is known whether muscularis macrophages interact with special subtypes of neurons. However, myenteric neurons, which consist of cholinergic and nitrergic neurons, regularly appeared to be contacted to macrophages [81]. Interestingly, reduced ChAT and nNOS expression in myenteric neurons were observed in a murine model of POI [36] and op/op mice, which lack resident macrophages due to a mutation in the CSF-1 gene [68], exhibit more myenteric neurons than wild-type mice [75]. This indicates that the neuronal environment shapes macrophage function (and vice versa). In the following paragraphs, we give further insight into nerve-macrophage interactions and speculate about other putative communications by other neurotransmitters.

Neuroimmunological features of the extrinsic parasympathetic innervation

A role of the extrinsic parasympathetic innervation in regulating peripheral immune modulation has been known for decades. More recent studies provided further mechanistic insights into the mechanisms that regulate the neuroimmune axis. Tracey and colleagues demonstrated that the vagus nerve is able to dampen inflammation by the release of acetylcholine (ACh), the principal parasympathetic neurotransmitter [13]. In a series of subsequent experiments, the concept of the so-called “cholinergic anti-inflammatory pathway” was developed. The pathways are the efferent part of a reflex which is triggered by sensory afferents in the periphery, i.e., at a site of trauma or infection [97]. In response to a systemic inflammation, this pathway turned out to be more complex and involves ACh-producing ChAT+ splenic T cells which become activated by the vagus nerve and in turn dampen macrophages via cholinergic and or adrenergic pathways [63]. In the intestinal muscularis, the anti-inflammatory effects of the vagus nerve are independent of the spleen [23]. However, vagally released ACh does not act directly on the resident macrophages but intermediately targets cholinergic enteric neurons which in turn release ACh [17, 64]. The close proximity of cholinergic nerves (that have not been further identified as extrinsic or intrinsic nerves) was also shown before [25]. The responsible cholinergic receptor that mediated macrophage suppression was identified as the α7 nicotinic ACh receptor [25, 64]. Although ACh is the principal vagal neurotransmitter, there may be other mediators coreleased from vagal inputs that can also modulate macrophage immune function. Furthermore, other nicotinic or muscularis ACh receptors could also be involved in macrophage silencing.

Neuroimmunological features of the extrinsic sympathetic innervation

Tissue macrophages were also found in close proximity to tyrosinhydroxylase+ noradrenergic nerve fibers, which are mainly of extrinsic sympathetic nature. These nerves originate from the superior mesenteric and celiac ganglia and are present in the myenteric and deep muscular plexus but not in the serosa. Muscularis macrophages highly express the β2 adregnergic receptor, and its coding gene ADRB2 is among the most differentially expressed genes between lamina propria macrophages and muscularis macrophages [38]. Importantly, sympathetic neurons cover—together with the macrophages—the outer perimeter of blood vessels in the smooth muscle wall of the GI tract [81] indicating that this neuroimmune interaction may also affect the blood vessel function and its barrier integrity. Functionally, β2 adrenergic signaling seems to be involved in macrophage polarization in vitro and in vivo and drives alternative macrophage polarization [38]. Given that recent work showed numerous functional states of macrophages beyond the M1/M2 classification [76], the β-adrenergic stimulation of macrophages likely results in a unique β-adrenergic-induced status whose in vivo function remains to be determined. On the other hand, α2-adrenoceptors were shown to induce classical macrophage activation. The same CX3CR1+ muscularis macrophages that showed high levels of β2-adrenoceptor mRNA also transcribe the α2-adrenoceptor [38]. However, compared to the β2-adrenoceptor, the α2-adrenoceptor was not upregulated compared to CX3CR1+ laminal propria macrophages [38] (see also GEO Nr.:GSE74131). In contrast, in a murine model of postoperative ileus, resident muscularis macrophages failed to show α2-adrenergic receptors expression whereas infiltrating monocytes expresses high levels of this receptor [58]. Thus, the role of α2-adrenoceptor in shaping muscularis macrophages remains unclear.

Neuroimmunological features of the intrinsic innervation

As described previously, muscularis macrophages are not only associated with extrinsic nerve fibers but are also in close proximity to cells of the ENS. Although it was suggested that muscularis macrophages may interact with special subtypes of enteric neurons [81], strong evidence for this hypothesis is missing. However, muscularis macrophages were described to respond to several neurotransmitters that are release from enteric nerves. Some of these responses are described in the following section. However, these mediators are not exclusively produced by enteric neurons. Indeed, some are co-released by extrinsic parasympathetic and sympathetic nerve fibers besides the principal neurotransmitters ACh and NE, respectively.

The list of enteric neurotransmitters with proven peripheral immune modulatory actions is long and include vasoactive intestinal peptide (VIP), serotonin, NO, adenosine, ATP, ACh, NE, and many more (Table 1). Principally, the underlying mechanism of enteric neuron-derived neurotransmitters may not differ from extrinsically delivered neurotransmitters. There may be differences in the location, concentration, and dynamics of release though. Thus, the effects of ACh and NE, which are the primary neurotransmitters of the parasympathetic and sympathetic nervous system, respectively, will not be reviewed again.

Table 1 Intrinsic and extrinsic intestinal neurotransmitters which affect muscularis macrophage function
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VIP is a pleiotropic neuropeptide [29] known to exhibit anti-inflammatory effects in macrophages via its receptors VPAC-1 and VPAC-2 [18]. In mice, VIP-positive nerve fibers are found in close proximity to resident macrophages. VPAC-1 receptors have been identified on muscularis macrophages [17], but it is unknown if they mediate immunomodulatory signals.

Serotonin (5-hydroxytryptamin, 5-HT) is also known to trigger inflammation, but the expression of its receptors on muscularis macrophages is not well characterized. However, 5-HT seems to indirectly act on muscularis macrophages by triggering the release of ACh from cholinergic nerves via 5-HT4 receptors. In turn, this triggers an anti-inflammatory cascade in macrophages via α7-nACh receptors [98]. Existence of other 5-HT receptors that trigger alternative activation polarization of macrophages, namely 5-HT2 and 5-HT7, was recently shown in human peripheral blood-derived monocytes that underwent either GM-CSF or M-CSF stimulation to induce M1 or M2 macrophage polarization, respectively [27]. Nevertheless, existence of other 5-HT receptors and their function in muscularis macrophages remains so far elusive.

Another neurotransmitter, γ-amino butyric acid (GABA), has also been studied for its immune modulatory functions. GABA receptors are found throughout the GI tract, predominantly in the ENS and enteroendocrine cells [5]. Mikkelsen et al. found GABA immunoreactivity in the resident macrophages of different mammals [66]. Although it remains elusive if GABA signaling affects muscularis macrophages, the expression of the GABA receptors has been shown in microglia, the resident macrophages of the CNS, and GABA receptor agonisms exerted an immunosuppressive function [61].

Nitric oxide (NO) is an inhibitory neurotransmitter that suppresses excitability in neurons [21] and is continuously produced by nNOS in neurons. However, NO is also well known for its immunological and anti-microbial functions. The extent to which NO production from inhibitory motor neurons and interneurons affects local immunity is unclear. However, NO release significantly increases after iNOS activation which is a hallmark of muscularis macrophages activation during inflammation [32, 34, 47, 52]. The tremendous amounts of NO that can be released from leukocytes during inflammation exert profound changes on the ENS and on intestinal function.

The enteric nervous system also harbors a network of EGC which outnumbers enteric neurons [39]. EGC are found in all plexus and in interganglionic regions in the smooth muscle layers and the lamina propria with extensions projecting up to the tips of the villi. The immunological importance of EGC has been shown in two independent studies, which observed a fulminant jejunoileitis and enterocolitis after a severe intestinal barrier dysintegrity [16, 22]. However, the mechanisms remain elusive. Surprisingly, although the nerve-macrophage interactions are in focus of a current investigations, little is known about macrophage-EGC communication. A recent study showed that EGC of the muscularis externa responds to IL-1, which is released from resident macrophages and infiltrating monocytes during POI [92]. Whether EGC also affect muscularis macrophage function remains unclear, but their immunomodulatory role and the variety of factors released by EGC during inflammation [78] strongly indicates that they may affect muscularis macrophage function.

Crosstalk of resident muscularis macrophages with other non-immune cells

In humans and rodents, muscularis macrophages are also in close contact to smooth muscle cells and to interstitial cell of Cajal (ICC) in the myenteric and deep myenteric plexus [48]. ICCs are the pacemakers of the gut [67] and share a common precursor with smooth muscle cells [82]. While macrophage-nerve interactions are well known (see previous section), interactions of macrophages and ICC and smooth muscle cells are not well described. However, iNOS-dependent NO release by resident macrophages during LPS-induced endotoxemia diminished the amount of ICC and disturbed their tone-giving function and iNOS antagonism or macrophage inhibition by gadolinium chloride prevented this detrimental effect [100]. With regard to a smooth muscle cell-macrophage interaction, peritoneal macrophages were shown to release mediators that in turn activate smooth muscle cells and vice versa in responses a non-physiological mechanical stretch stimulus [108], which could occur also in the bowel wall. Nevertheless, interactions of muscularis macrophages with intestinal smooth muscle cells or ICC remain so far elusive.

Resident macrophages as therapeutic targets

A large number of publication demonstrated that POI originates from the activation of the resident macrophages or DCs, and this activation was also observed in surgical specimens in the human GI tract [53, 95]. These studies suggested that targeting macrophages and/or DCs might be a promising strategy for POI prevention. Targeting cytokines and chemokines contributing to POI pathogenesis may also be an effective therapy, but inappropriate pharmacokinetics and high treatment costs have limited further clinical development. A rather specific approach was achieved by administration of semapimod, a tetravalent guanylhydrazone that prevents activation of the c-Raf/p38 MAPK kinase pathway in macrophages and subsequently POI [107]. Importantly, higher peripheral doses of semapimod reached brain levels sufficiently to centrally trigger cholinergic anti-inflammatory pathways (described previously) and this may result in a cumulative inhibitory effect on muscularis macrophage [26]. Although this compound was not further developed, these data clearly indicate that selective immune modulatory drugs are effective approaches to target muscularis macrophages during inflammation.

Innervation in the cholinergic anti-inflammatory pathway also seems to be a promising target in dampening intestinal macrophage activation during inflammation, and muscularis macrophages were shown to express the target receptor of this pathway, the α7-nACh receptor [64]. Selective α7-nACh receptor agonists were developed and have been tested in experimental models of acute and chronic intestinal inflammation. One of these agonists, AR-R17779, was shown to reduce POI [94] but was also shown to worsen experimental colitis [89] Another α7-nACh receptor agonist, GTS-21, was recently shown to prevent septic ileus, but its selectivity for the α7-nACh receptor remains questionable [77]. Finally, clinical trials with use of α7-nACh agonists for treatment of immunological disorders have not pursued anymore. To this end, an electrical stimulation of the vagus nerve appears to be another promising approach [25, 64] to trigger the cholinergic anti-inflammatory pathway and data confirming effectiveness are expected from ongoing clinical trials. The electrical stimulation may have less side effects than pharmaceutical, but it is so far performed by invasive vagus nerve stimulation. Invasive treatment may not be an appropriate therapy in common clinical routine option, particularly in acute diseases like postoperative or septic ileus. However, non-invasive techniques are under development and results are expected in the near future.

In addition to the well-described vagal anti-inflammatory effects, immune modulation by sympathetic nerves is an equivalent in the (neuro)immune system. However, catecholamines, particularly NE released from sympathetic nerves, may have opposing roles. While α-adrenergic signaling is thought to boost inflammation (and this was also confirmed in the POI model [58]), β-adrenergic pathway suppresses innate and adaptive immunity. Recently, it was shown that β2-adrenergic signaling affects resident macrophage/DC polarization [38]. β2-adrenergic agonism by salbutamol increased M2 marker expression in a murine peritoneal macrophage cell line and in primary muscularis macrophages while β2-antagonism reduced it. A comparable effect was observed in an acute experimental lung injury model, and this indicates a broader anti-inflammatory effect on resident macrophages by β2-agonists [14]. Indeed, β-adrenergic innervation is disturbed during POI and also chronic inflammatory bowel diseases [30, 114]. Nevertheless, it remains unclear if β2-adrenoceptor agonism in muscularis macrophage affects outcome of intestinal diseases, but selective activation of β2-adrenergic signaling may help to counterregulate inflammation or accelerated resolution programs via resident macrophages.

Following the theory that a stimulation of endogenous immunomodulatory pathways or use of endogenous mediators may be less harmful than administration of pharmaceuticals, further research should pay special attention to those mechanisms. Besides the immunoregulatory neuronal pathways, several other endogenous mechanisms are able to effectively shut down inflammation. A new class of endogenous immune modulating molecules are poly-unsaturated-derived lipid mediators. These mediators are derived from cyclooxygenase and/or lipoxygenase metabolism and have been shown to exert powerful anti-inflammatory actions [15, 87], i.e., enhancement of efferocytosis in macrophages. During POI, docosahexaenoic acid (DHA)-derived metabolites, namely protectin DX and resolvin D2, were highly induced in the postoperative muscularis. However, only protectin DX prevented POI [91] indicating the high selectivity and specificity of these mediators. Although it remains unclear whether protectin DX directly targets an unknown protectin DX receptor on resident macrophages, resident macrophages in other tissues were shown to be sensitive to a variety of proresolving DHA-derived lipid mediators [4, 24, 87].

Conclusion

Taken together, macrophages in the intestinal muscularis externa share functional and phenotypical similarities to long-living tissue macrophages, which contrast macrophages in the neighboring lamina propria. The functionality of macrophages at both sites is shaped by the local microenvironment and muscularis macrophages in particular, which seem to tightly interact with the dense nervous system. Multi-parametric state of the art imaging and isolation techniques have allowed further insights into the localization and the different phenotypes of intestinal macrophages in health and disease. Given that the phenotype and the functionality drastically change under disease conditions, a more thorough analysis is required to delineate the role of these cells in the inflamed muscularis externa. Conclusively, open questions remain with regard to the localization, origin, and functionality of macrophages in the muscularis externa and the regulatory crosstalk with the enteric and the peripheral nervous system in inflammatory settings in particular. Future studies are required to understand the crosstalk between these functional units in the different intestinal muscle layers. Unraveling the cellular and molecular mechanisms underlying the tissue-specific control of macrophage development and activation in the muscularis externa will be crucial to understand disease-specific adaptations in muscularis macrophages and the disturbance of intestinal motility to develop novel therapeutic approaches regulating the functionality of macrophages in the intestinal muscularis externa.