**2. Anatomic localization of MMs**

Histologically the GI tract is a complex organ consisting of different layers: the mucosa, the submucosal layer, the muscularis propria, and the serosa (**Figure 1**).

The mucosa, which consists of epithelium, the lamina propria, and the muscularis mucosa, is the innermost layer, and consequently, it is continuously exposed to digested food and microbiota. On the opposite side, the serosa is associated with the peritoneum and constitutes the gate for extrinsic fibers engraftment onto the GI tract from the central nervous system (CNS). The submucosa layer presents large blood vessels, lymphatics, and connective tissue.

Underneath, the muscularis propria consists of two muscle layers with different orientations separated by the myenteric plexus region, which houses enteric neurons' (ENs) cell bodies [10]. The primary function of the muscularis propria is to regulate the GI contraction needed for a proper movement of food.

Gut tissue-resident macrophages are encountered in all the different layers of the GI tract. However, most gut tissue macrophages are localized in the lamina propria, below the epithelial lining. These macrophages are in a close anatomical relationship with adult tissue stem cells of intestinal crypts, as well as Paneth cells, a specialized cellular population secreting antimicrobial substances to the gut lumen [11]. A second discrete population of macrophages is associated with the submucosal nervous plexus [7]. Because of the massive presence of blood vessels, this anatomical region also represents the door for circulating monocyte entrance onto the underneath muscularis propria.

MMs have a different distribution and morphology within the regions of the muscularis propria. MMs lying in the two muscular layers share an elongated morphology following the muscle orientation. Most MMs are distributed within the myenteric plexus, where they are closely associated with ENs. This population of MMs shares a characteristic morphology with multiple branches originating from the same cell body.

In comparison to the macrophages present in the mucosa, MMs have an overall anti-inflammatory, protective phenotype, as they express CD163, IL10, Mrc1, and Hmox1, all anti-inflammatory genes [7]. In addition, these cells have phagocytic properties and a distinct CD11clow / MHCIIhigh / CSF-1Rhigh phenotype [8]. In line with other tissue-resident macrophages, colony-stimulating factor-1 (Csf1–1) is critical for their survival and maintenance. In experimental mice models lacking CSF-1R, MMs with CD11clow / MHCIIhigh phenotype is virtually absent, supporting a primary role of CSF-1R in maintaining this macrophage population [12].

**Figure 1.** *Different layers of the gastrointestinal tract wall.*

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

Macrophages can also be found in the capillary-rich subserosal connective tissue [13], as well as the mucosa-associated lymphoid tissue (MALT), which includes Peyer's patches [14]. Finally, a layer of macrophages is present within the serosal layer. We have little information regarding these cells' role and function; further studies are needed to elucidate their function in GI homeostasis and diseases. New technologies, such as spatial transcriptomic, will fill the knowledge gap in understanding the phenotypic differences between MMs distributed within the different muscularis propria regions. This information will uncover the specific role of niche-specific macrophages on GI dysfunction and their possible contributions to functional diseases.

### **3. Origins and natural history of MMs**

Like many other tissue-resident macrophages, MMs are heterogeneous. Multiple sequencing approaches identified populations that share a distinct phenotype and function. One of the main factors contributing to such diversity is represented by their origin [2]. The classic hypothesis of macrophages originating from blood monocytes has been recently challenged by the so-called theory of "resident macrophages" [15]. The initial unified hypothesis about tissue macrophages was that monocytes freely circulate in the blood and transmigrate to the tissues under a suitable stimulus, where they acquire a macrophage phenotype [16]. However, we now know that part of tissue-resident macrophages also derives directly from progenitor cells in the fetal liver and yolk sac [2]. For this reason, in most organs, tissue-resident macrophages consist of both embryonic- and monocyte-derived cells. Embryonic macrophages engraft into the tissue during the development phase, and throughout life, these cells are maintained by self-renewing. The latter have a shorter life and continuously invade the tissue to maintain tissue-resident macrophages. The first information regarding a possible alternative origin to circulating monocytes was acquired in the CNS, showing that the tissue-resident macrophages of the CNS originate from precursors presumably located in the yolk sac. These precursors express the CSF-1 receptor and migrate to the liver during embryogenesis. Unlike other tissue macrophage populations [17, 18], the microglial population shares an embryonic origin exclusively.

With the progression of technologies, other studies have shown that in opposition to microglia, the whole pool of tissue-resident macrophages are characterized by the coexistence of monocyte- and embryonic-derived macrophages in other organs, such as the heart, liver, and dermis [19]. Only recently, studies shed light on the dual origin of MMs. Like microglia in the CNS, MMs highly expressed CX3CR1, a tissue-resident cell marker [20]. Using a lineage tracing mouse model, CX3CR1 MMs were followed during the evolutive stages (from embryonic to adulthood) [21]. This population represents tissue-resident MMs at the embryonic stage but rapidly decline in the first weeks after birth. With age, embryonic cells that remain in the tissue are named long-lived MMs and, in concert with circulating monocytes, form tissue-resident MMs [22]. Although a decline in this population during development was observed, the total number of MMs throughout the years is maintained due to the ongoing circulating monocytes' ingress.

It comes without surprise that embryonic and monocyte-derived MMs have different molecular transcriptional profiles. They have two distinct subsets, as demonstrated based on the expression of CX3CR1. The first subset is CX3CR1 high, and the second is CX3CR1 low. The latter also expresses C-C chemokine receptor 2 (CCR2), which significantly regulates monocytic inflammatory response [23]. Also, the close anatomical relationship of MMs with ENs is sustained by the expression of multiple

genes related to cellular adhesion, anchoring the cytoskeleton and neuronal development. Not surprisingly, these genes are not expressed by other MMs populations but are also enriched by microglia that are also closely communicating with neurons in the CNS. A non-exhaustive list of these genes includes Apolipoprotein E (ApoE), Fc receptor-like scavenger (FCRLS), Platelet factor 4 (PF4), Cystatin C (CST3), and Disabled-2 (Dab2) [24]. MMs are located within dedicated niches of the muscularis propria. The close interaction of MMs with ENs can be demonstrated by depleting MMs and observing the resulting depletion of ENs [25]. The same can be observed not only in animals but also in human subjects. Bajko et al. investigated the transcriptional molecular profile of macrophages, pointing towards two distinct populations of macrophages, the former deriving from the yolk sac and the latter from monocytes [26]. Those macrophages that survived after embryonic life showed localization into anatomical niches in the same way it had previously been demonstrated in mice [27]. Moreover, several investigators demonstrated tissue-resident macrophages in patients with monocyte deficiency, as in congenital monocytopenia [28].
