In the intestinal myenteric plexus, the myenteric macrophages (MMs) are closely connected with the cell bodies of excitatory endogenous enteric neurons (8). MMs are the main source of bone morphogenetic protein 2 (BMP2), which stimulate intestinal neurons to regulate gastrointestinal motility. In turn, the development and reproduction of MMs are controlled by colony stimulating factor (CSF1) expressed by intestinal neurons (9). In the intestine of CSF^−/− mice, the number of NO⁺ neurons increases, and there is no expression of bone morphogenetic protein 2 in MMs, which leads to the immature development of neurons in the intestinal muscle layer. Macrophage depletion induced by anti-CSF1R treatment affects the differentiation of Paneth and other intestinal epithelial cells (10).

After intestinal bacterial infection, MMs is activated, which plays an important role in the protection of intestinal neurons. This is attributed to the rapid activation of external sympathetic neurons innervating the intestinal muscle layer, and the NE secreted by norepinephrine neurons binds to the β2-adrenergic receptor(β2AR) on MMs.

NE/β2 signal in MMs induces the expression of arginase-1 in vitro (8). Arginase 1 (Arg-1) mediates the production of neuroprotective polyamines, for example, spermine (11), Spermine can inhibit the activation of NLRP6 inflammatory bodies and inhibit NLRP6-Caspase-11-mediated neuronal injury (12). When spermine and DFMO (an inhibitor of spermine synthesis) were added to drinking water, it was found that the damage to intestinal neurons increased in the inhibition group. The protective effect of Arg-1 on neurons after intestinal infection was proved by hybridization between LysM^Cre mice and Arg-1^flox/flox mice (13). The macrophages of this selective activation program play an important role in the repair and protection of intestinal tissue, and this discovery lays a foundation for exploring MMs as a neuroprotective sentinel.

Furthermore, the effect of adrenergic neurons on MMs plays a central role in coordinating the tissue protective tolerance response after Salmonella enteric infection. It has been found that wild-type mice infected with an attenuated Salmonella typhimurium (spiB) can clear the infection within 7-10 days, but the result of infection is long-term (up to 4 months) impairment of gastrointestinal motility and reduction of intestinal neurons in a NLRP6/Caspase11-dependent manner. MMs deletion or impaired NE/β2 signaling can worsen spiB infection-induced enteric neuronal injury. Exogenous β2 receptor agonists enhance Arg-1 expression in MMs, protect enteric neurons, and limit the effects of spiB infection on gastrointestinal motility (13). One experiment used mice infected with S. commissioneri roundworms (Sv) followed by Salmonella infection. Mice showed reduced intestinal nerve loss compared to Salmonella infection alone. This suggests that the adrenergic signaling pathways in myeloid macrophages limit infection-induced neuronal loss by way of immune tolerance (6).

In a model of postoperative intestinal obstruction in mice and humans (characterized by a surgically induced inflammatory response in intestinal myenteric macrophages and impaired intestinal motility), vagal stimulation or pharmacological action on cholinergic enteric neurons prevented myenteric macrophage activation and reduced postoperative intestinal obstruction in a α7 nicotinic receptor (α7nAChR) dependent manner (14). In a mouse model of food allergy, vagus nerve stimulation ameliorates intestinal inflammation caused by food allergy in a 7nAChR-independent manner. This effect may be mediated by the vagal system through increased phagocytosis of allergenic substances by CX3CR1⁺ macrophages (15). Vagus nerve stimulation increased the phagocytic activity of CX3CR1+ macrophages, an effect that may be dependent on 4b2nAChR. Food antigen uptake and subsequent induction of food-specific Treg by anti-inflammatory CX3CR1+ macrophages is effective in preventing allergic reactions (16, 17).

The ganglion-free colon of HSCR patients lacked intrinsic myenteric and submucosal plexuses, but cholinergic nerve fibers were significantly thickened, and the incidence and prognostic level of postoperative HAEC correlated with the proliferation of cholinergic nerve fibers. Some researchers divided the intestinal mucosa into high-fiber tissue and low-fiber tissue according to the degree of fibrous hyperplasia, and by immunofluorescence co-localization they found that macrophages from high-fiber tissue were associated with Tubulin+AChE+ neurons, and macrophages from the mucosal lamina propria of ganglion descending colon tissue from HSCR and control patients appeared to be associated with Tubulin+AChE- neurons. The physical proximity of cholinergic neurons to macrophages facilitates the interaction between the two (18). Analysis of the transcriptome of myeloid macrophages from high-fiber tissue and low-fiber tissue corresponding colonic segments, they found a trend of elevated levels of nicotinic and muscarinic receptors, high expression of CX3CR1, CD14+ (M2 macrophage marker) in macrophages isolated from high-fiber tissue. The percentage of Treg cells was significantly elevated. Macrophages exhibited an anti-inflammatory, tissue-resident phenotype. In contrast, in low-fiber tissue, the expression of cellular inflammatory factors such as IL-17, IL-6, and IL-1b was significantly increased, CCR2+ was highly expressed in macrophages, and the ratio of Th17/Treg was significantly elevated (18).

Under inflammatory conditions, monocyte-derived CCR2+ macrophages infiltrate the intestinal mucosa and initiate Th17 cell responses. Under steady-state conditions, CX3CR1⁺ macrophages secrete anti-inflammatory cytokines and maintain Treg numbers. CX3CR1⁺ macrophages are essential for epithelial cell integrity, control of bacterial translocation, and commensal tolerance (16). Muscarinic acetylcholine receptor activates ALDH gene expression in macrophages (antigen presenting cells) of CX3CR1+, which produces retinoic acid and promotes the expansion of intestinal Treg cells (19). In addition, Treg cells are able to inhibit the action of Th17 cells (20), Proliferative cholinergic nerve fibers influence the differentiation of CD4+ helper cells, especially the proportion of Treg, through their regulatory effects on macrophages, thereby regulating intestinal inflammation.

In the follow-up of 42 HSCR patients 1 year after surgery, 9 patients developed HAEC, of which 7 showed a low-fiber phenotype and 2 showed a high-fiber phenotype; these observations suggest that HSCR patients with a low-fiber phenotype are at a higher risk of developing HAEC after surgery (18), This study identified and explained the relationship between the high-fiber phenotype of the intestinal mucosa and the incidence of HAEC after the surgery of HSCR from an immunological perspective, providing a theoretical basis for the prophylactic treatment of HAEC.

In the further, intestinal serotonergic neurons are also cholinergic neurons. Tryptophan hydroxylase 2 (THP2), a marker of 5-HT expression in serotonergic neurons, is significantly decreased in intestinal neurons of HAEC patients and is restored postoperatively. Although only 5% of intestinal 5-HT is stored in neurons, only neuronal-derived 5-HT plays a role in regulating intestinal motility compared to intestinal epithelial-derived 5-HT. HAEC-mediated 5-hydroxytryptaminergic neuronal damage may lead to colonic dysfunction and recurrent enterocolitis (21). Another study shows that cholinergic innervation in rectosigmoid colon in HSCR patients is associated with secretion of pro-inflammatory IL-8, which increases the risk of HAEC (22).

Patients with HSCR typically present with exogenous cholinergic nerve fibers throughout the aganglionic rectosigmoid. Studies have shown that cholinergic signaling can reduce inflammatory responses and α7nAChR plays an important role in the neuroimmune signaling pathways. Thus, sparse exogenous cholinergic innervation in the sigmoid mucosa and antagonism of α7nAChR are associated with increased frequency of inflammatory immune cells and higher incidence of HAEC in patients with HSCR (22).

Neurokinin P is also called substance P (SP). Endocrine neurons are the main source of intestinal SP. SP acts through interaction with cell surface receptors NK1R, NK2R and NK3R. SP induces peripheral blood mononuclear cells (including lymphocyte proliferation and immunoglobulin production) to stimulate the production of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α (23–25). This may be due to the positive regulation of NK1R, as inflammatory factors such as IL-12, IL-18 and TNF-α induce NK1R expression in T cells (26), while IL-10 and TGF-β decreased the expression of NK1R (27). NK1R has the greatest affinity for SP, which is primarily associated with inflammatory processes (28). NK1R receptor density is significantly increased in patients with Crohn’s disease and ulcerative colitis (29). Expression of substance P was also shown to correlate directly with the severity of Trypanosoma cruzi megacolon. It was higher in submucosal and myenteric plexus neurons in the dilated portion of the megacolon compared to the undilated portion and the uninfected population, and this might be related to the preferential destruction of inhibitory motor neurons (VIP and NOS immunoreactivity) in the intestine of chagasic patients with megacolon by Trypanosoma cruzi and inflammatory processes (30). However, another study described lower myenteric and submucosal plexus SP concentrations in rectal samples from trypanosomatid patients, which may be associated with reduced intestinal peptidergic neuronal damage (31).

VIP⁺ neurons mediate VIP secretion, and VIP not only monitors intestinal epithelial status by regulating lymphocytes, but also regulates mononuclear phagocytes (MNPs) and the cytokines they secrete, tilting immunity toward the Th2 type (32). VIP-sensitized dendritic cells induce Treg production and restore immune tolerance. VIP-producing neurons in the intrinsic layer are very close to ILC3, which selectively expresses type 2 VIP receptors.IL-22 secretion is inhibited by ILC3 binding to VIP, which results in reduced levels of AMP, an epithelial-derived antimicrobial peptide, and rhythmic changes in VIP levels promote normal function of the intestinal barrier (33).

Using a mouse model of intestinal helminth infection, it has been shown that neuromedin U increases protective immune responses and induces parasite clearance through activation of ILC2 (34, 35). Conversely, signals from sympathetic adrenergic neurons attenuate diminished ILC2 effector function and prevent chronic pathological type 2 inflammation by activating ILC2-specific β2-adrenergic receptors (36).

Enteric-specific IL-18 neurons drive the production of the antimicrobial peptide AMP in goblet cells and protect the intestine during Salmonella typhimurium infection, which is important for the mucosal barrier and coordinated homeostasis of the intestine (37).

Neuropeptides have innate host defense enhancing and direct antimicrobial effects and some neurons distributed in the intestine are one of the main sources. Neuropeptides regulate intestinal immunity in endocrine and paracrine ways. In patients with sepsis, the intestinal barrier is damaged, intestinal permeability is increased, and bacterial cell wall components (e.g. lipopolysaccharide) can stimulate nociceptive receptors located in the lamina propria of the intestine and induce increased synthesis and release of calcitonin gene related peptide (CGRP), substance P, neuropeptide Y, vasoactive intestinal peptide, VIP and other neuropeptides. CGRP can directly act on lymphocytes, dendritic cells and macrophages in the intestinal tract, significantly improving the host’s intestinal defense ability (38).

The release of SP from peptidergic neurons activates mast cells and promotes their degranulation, followed by the release of pro-inflammatory mediators from mast cells, such as TNF-α, IL-1β and trypsin. They strengthen the relationship among mast cells, trypsin, neuroinflammation and neuronal death (39–41). After co-culture of intestinal secretory neurons with mast cells, mast cells release histamine and protease through degranulation resulting in increased intra-neuronal Ca2+ concentration and significantly increased neuronal sensitivity, but the function of mast cells themselves is inhibited (42).

In a study using RET deficient mice, the authors demonstrated that microorganism-induced glial cell-derived GDNF (ligand) could can activate intestinal ILC3 subsets to express neuroregulatory receptor tyrosine kinase (RET) (53).Activated RET could induce IL3 to secrete IL-22 and increase the expression of tight junction proteins in epithelial cells, which could effectively promote intestinal homeostasis (53). When EGCs are exposed to pro-inflammatory cytokines (IL-1 β and/or TNF-α), IL-7 can also be up-regulated. T lymphocyte function analysis shows that the induced expression of classical IL-7 can protect T cells from cell death (54). In patients with megacolon caused by Trypanosoma cruzi, EGCs express II-type HLA-DR and B7 costimulatory molecules. Therefore, EGCs also act as an antigen-presenting cell on the activation of lymphocytes, thus affecting the progress of chronic digestive trypanosomiasis (55).

Pro-inflammatory signal pathways induce Cx43-dependent M-CSF production in glial cells through protein kinase C (PKC) and tumor necrosis factor invertase (TACE), and M-CSF promotes the transformation of intestinal myometrial macrophages to M1. Connexin-43 is needed for communication between glial cells in this process. Knockout of Connexin-43 in glial cells can prevent the development of visceral hypersensitivity after chronic colitis (43).

The interaction between EGCs and mast cells may inhibit intestinal inflammation. The activation of mast cells can promote the activation of enteric glial cells and macrophages, resulting in intestinal mucosal injury and neuronal reduction (56). GDNF from EGCs can also significantly inhibit mast cell degranulation and reduce inflammation (57).