Tissue-resident macrophages are specialized immune cells inhabiting various tissues in our body, including the liver, lung, skin, brain, and intestine. These macrophages play pivotal roles in maintaining tissue homeostasis, immune surveillance, and tissue repair (Lee et al., 2021). In addition, they are crucial for development (Davies et al., 2013). For instance, microglia (i.e., brain-resident macrophage-like immune cells) colonize the entire central nervous system (CNS) during embryogenesis. During the neonatal period, microglia are key for synaptic pruning by engulfing unwanted or redundant synapses, a vital process for proper neuronal connectivity and circuit formation with a lifelong impact (Paolicelli et al., 2011). Disrupted synaptic pruning during neonatal development could lead to CNS disorders, such as autism spectrum disorders, schizophrenia, intellectual disability, and epilepsy.

The enteric nervous system (ENS) is a complex neuronal network primarily located in the gastrointestinal tract walls. These neurons are organized into two main plexuses: the myenteric plexus, situated between the layers of the muscularis externa, and the submucosal plexus, located within the submucosal layer. The ENS is responsible for digestion, nutrient absorption, and immune response, all modulated by environmental factors or CNS signals (Furness, 2012). A process similar to CNS development occurs in the intestine. After birth, enteric neurons continue to differentiate, innervate the intestine, and undergo synaptic maturation to facilitate the coordination of gut motility, secretion, and sensory function (Spencer and Hu, 2020). Defects in these processes lead to gastrointestinal diseases such as Hirschsprung’s disease and intestinal neuronal dysplasia (Obermayr et al., 2013).

Recently, Viola et al. (2023) demonstrated that resident macrophages of the muscularis externa (MMΦ) in the intestine are central to early postnatal ENS development through synaptic pruning and phagocytosis. The early postnatal period is critical for neural circuit development and maturation. During this early postnatal developmental period toward adulthood, the authors observed changes in the ENS architecture, revealing that the highest density of enteric neurons, ganglia, extraganglionic neurons, and interconnecting fibers was measured at postnatal day 10 (P10) and their density significantly decreased thereafter. Using ex vivo live imaging, the authors demonstrated a highly dynamic interaction between MMΦ and enteric neurons at P14. Moreover, depleting MMΦ before weaning using an intraperitoneal injection of a CSF1 receptor antibody (37.5 µg/g, twice per week, from P10 until P24) resulted in higher neuronal and synaptic density as well as disrupted gut motility in adulthood. These findings suggest that the early postnatal period is crucial for ENS refinement and optimal gut function development involving MMΦ. Interestingly, depleting MMΦ at P56 reduced neuronal density, indicating that MMΦ at that specific period might play a neurosupportive function in the ENS. This piece of evidence suggests that MMΦ affects ENS maturation or maintenance differently, depending on the developmental time point.

The authors also investigated the gene expression profile-related differences between MMΦ at two specific, distinct time points (P10 and P56), using single-cell RNA sequencing. MMΦ at P10 exhibited increased phagocytosis-, endocytosis-, and replication-associated gene expression. However, MMΦ at P56, referred to as neuron-associated (NA)-MMΦ in this study, exhibited increased immune surveillance-, antigen presentation-, and macrophage activation-related gene expression. These findings indicated that MMΦ at P10 and P56 possesses phagocytic and neurosupportive properties, respectively. These results suggested age-dependent changes in MMΦ functions and transcriptional identity.

To further determine the molecular mechanism underlying the interaction between the ENS and NA-MMΦ, the authors proposed the involvement of transforming growth factor-β (TGFβ) as TGFβ has been recognized as a neuro-surveillant microglia maturation signal with increasing expression between P10-P56 in the muscularis externa. Indeed, when bone marrow-derived macrophages were incubated with TGFβ, upregulated canonical NA-MMΦ marker expression could be observed. Furthermore, disrupting gut TGFβ signaling in mouse models using Tgfb knockout in enteric neurons or macrophages resulted in a reduced NA-MMΦ number and delayed gut transit time compared to the control, suggesting that TGFβ signaling is essential for the ENS–NA-MMΦ communication. In addition, the authors suggest a potential contribution of the microbiome to NA-MMΦ differentiation, as germ-free mice exhibited reduced NA-MMΦ differentiation compared to conventionally colonized mice.

The study of Viola et al. (2023) emphasizes the critical role of gut macrophages in ENS maturation and regulation. These findings underscore the intricate communication between neural and immune components within the gut, highlighting the significance of neuro-immune interactions in shaping the development and functionality of the gastrointestinal system. This study also suggests the possibility that early-life internal or environmental factors, including the gut microbiome, potentially affecting the gut macrophages might have a life-long-lasting effect on gut functions.