Self-Maintaining Gut Macrophages Are Essential for Intestinal Homeostasis.

Macrophages are highly heterogeneous tissue-resident immune cells that perform a variety of tissue-supportive functions. The current paradigm dictates that intestinal macrophages are continuously replaced by incoming monocytes that acquire a pro-inflammatory or tissue-protective signature. Here, we identify a self-maintaining population of macrophages that arise from both embryonic precursors and adult bone marrow-derived monocytes and persists throughout adulthood. Gene expression and imaging studies of self-maintaining macrophages revealed distinct transcriptional profiles that reflect their unique localization (i.e., closely positioned to blood vessels, submucosal and myenteric plexus, Paneth cells, and Peyer's patches). Depletion of self-maintaining macrophages resulted in morphological abnormalities in the submucosal vasculature and loss of enteric neurons, leading to vascular leakage, impaired secretion, and reduced intestinal motility. These results provide critical insights in intestinal macrophage heterogeneity and demonstrate the strategic role of self-maintaining macrophages in gut homeostasis and intestinal physiology.

Neuronal programming by microbiota regulates intestinal physiology.

Neural control of the function of visceral organs is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence, and is often dysregulated in gastrointestinal disorders1. Luminal factors, such as diet and microbiota, regulate neurogenic programs of gut motility2-5, but the underlying molecular mechanisms remain unclear. Here we show that the transcription factor aryl hydrocarbon receptor (AHR) functions as a biosensor in intestinal neural circuits, linking their functional output to the microbial environment of the gut lumen. Using nuclear RNA sequencing of mouse enteric neurons that represent distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles that are controlled by the combined effects of host genetic programs and microbial colonization. Microbiota-induced expression of AHR in neurons of the distal gastrointestinal tract enables these neurons to respond to the luminal environment and to induce expression of neuron-specific effector mechanisms. Neuron-specific deletion of Ahr, or constitutive overexpression of its negative feedback regulator CYP1A1, results in reduced peristaltic activity of the colon, similar to that observed in microbiota-depleted mice. Finally, expression of Ahr in the enteric neurons of mice treated with antibiotics partially restores intestinal motility. Together, our experiments identify AHR signalling in enteric neurons as a regulatory node that integrates the luminal environment with the physiological output of intestinal neural circuits to maintain gut homeostasis and health.

Nano-positioning and tubulin conformation contribute to axonal transport regulation of mitochondria along microtubules.

Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules that fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin in neurons both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers like guanosine triphosphate (GTP)-tubulin. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin velocity and density on microtubules consisting of elongated dimers add to the increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin contributes to the regulation of mitochondrial motility and as such to the local distribution of mitochondria along axons.

Neuronal Distribution in Colorectal Cancer: Associations With Clinicopathological Parameters and Survival.

Over the past years, insights in the cancer neuroscience field increased rapidly, and a potential role for neurons in colorectal carcinogenesis has been recognized. However, knowledge on the neuronal distribution, subtypes, origin, and associations with clinicopathological characteristics in human studies is sparse. In this study, colorectal tumor tissues from the Netherlands Cohort Study on diet and cancer (n = 490) and an in-cohort validation population (n = 529) were immunohistochemically stained for the pan-neuronal markers neurofilament (NF) and protein gene product 9.5 (PGP9.5) to study the association between neuronal marker expression and clinicopathological characteristics. In addition, tumor and healthy colon tissues were stained for neuronal subtype markers, and their immunoreactivity in colorectal cancer (CRC) stroma was analyzed. NF-positive and PGP9.5-positive nerve fibers were found within the tumor stroma and mostly characterized by the neuronal subtype markers vasoactive intestinal peptide and neuronal nitric oxide synthase, suggesting that inhibitory neurons are the most prominent neuronal subtype in CRC. NF and PGP9.5 protein expression were not consistently associated with tumor stage, sublocation, differentiation grade, and median survival. NF immunoreactivity was associated with a worse CRC-specific survival in the study cohort (P = .025) independent of other prognostic factors (hazard ratio, 2.31; 95% CI, 1.33-4.03; P = .003), but these results were not observed in the in-cohort validation group. PGP9.5, in contrast, was associated with a worse CRC-specific survival in the in-cohort validation (P = .046) but not in the study population. This effect disappeared in multivariate analyses (hazard ratio, 0.81; 95% CI, 0.50-1.32; P = .393), indicating that this effect was dependent on other prognostic factors. This study demonstrates that the tumor stroma of CRC patients mainly harbors inhibitory neurons and that NF as a single marker is significantly associated with a poorer CRC-specific survival in the study cohort but necessitates future validation.

Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2.

The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4-/- ) CRC models and an indirect co-culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4-/- ENS cell secretome, which is enriched for Nidogen-1 (Nid1) and Fibulin-2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS-derived Nidogen-1 and Fibulin-2 enhance colorectal carcinogenesis.

Simultaneous whole-cell patch-clamp and calcium imaging on myenteric neurons.

Live calcium imaging is often used as a proxy for electrophysiological measurements and has been a valuable tool that allows simultaneous analysis of neuronal activity in multiple cells at the population level. In the enteric nervous system, there are two main electrophysiological classes of neurons, after-hyperpolarizing (AH)- and synaptic (S)-neurons, which have been shown to have different calcium handling mechanisms. However, they are rarely considered separately in calcium imaging experiments. A handful of studies have shown that in guinea pig, a calcium transient will accompany a single action potential in AH-neurons, but multiple action potentials are required to generate a calcium transient in S-neurons. How this translates to different modes of cellular depolarization and whether this is consistent across species is unknown. In this study, we used simultaneous whole-cell patch-clamp electrophysiology together with calcium imaging to investigate how enteric neurons respond to different modes of depolarization. Using both traditional (4 Hz) and also high-speed (1,000 Hz) imaging techniques, we found that single action potentials elicit calcium transients in both AH-neurons and S-neurons. Subthreshold membrane depolarizations were also able to elicit calcium transients, although calcium responses were generally amplified if an action potential was present. Furthermore, we identified that responses to nicotinic acetylcholine receptor stimulation can be used to distinguish between AH- and S-neurons in calcium imaging.NEW & NOTEWORTHY Live calcium imaging is an important tool for investigating enteric nervous system (ENS) function. Previous studies have shown that multiple action potentials are needed to generate a calcium response in S-neurons, which has important implications for the interpretation of calcium imaging data. Here, we show that in mouse myenteric neurons, calcium transients are elicited by single action potentials in both AH- and S-neurons. In addition, nicotinic acetylcholine receptor stimulation can be used to distinguish between these two classes.

The enteric nervous system in gastrointestinal disease etiology.

A highly conserved but convoluted network of neurons and glial cells, the enteric nervous system (ENS), is positioned along the wall of the gut to coordinate digestive processes and gastrointestinal homeostasis. Because ENS components are in charge of the autonomous regulation of gut function, it is inevitable that their dysfunction is central to the pathophysiology and symptom generation of gastrointestinal disease. While for neurodevelopmental disorders such as Hirschsprung, ENS pathogenesis appears to be clear-cut, the role for impaired ENS activity in the etiology of other gastrointestinal disorders is less established and is often deemed secondary to other insults like intestinal inflammation. However, mounting experimental evidence in recent years indicates that gastrointestinal homeostasis hinges on multifaceted connections between the ENS, and other cellular networks such as the intestinal epithelium, the immune system, and the intestinal microbiome. Derangement of these interactions could underlie gastrointestinal disease onset and elicit variable degrees of abnormal gut function, pinpointing, perhaps unexpectedly, the ENS as a diligent participant in idiopathic but also in inflammatory and cancerous diseases of the gut. In this review, we discuss the latest evidence on the role of the ENS in the pathogenesis of enteric neuropathies, disorders of gut-brain interaction, inflammatory bowel diseases, and colorectal cancer.

Development of the intrinsic innervation of the small bowel mucosa and villi.

Detection of nutritional and noxious food components in the gut is a crucial component of gastrointestinal function. Contents in the gut lumen interact with enteroendocrine cells dispersed throughout the gut epithelium. Enteroendocrine cells release many different hormones, neuropeptides, and neurotransmitters that communicate either directly or indirectly with the central nervous system and the enteric nervous system, a network of neurons and glia located within the gut wall. Several populations of enteric neurons extend processes that innervate the gastrointestinal lamina propria; however, how these processes develop and begin to transmit information from the mucosa is not fully understood. In this study, we found that Tuj1-immunoreactive neurites begin to project out of the myenteric plexus at embryonic day (E)13.5 in the mouse small intestine, even before the formation of villi. Using live calcium imaging, we discovered that neurites were capable of transmitting electrical information from stimulated villi to the plexus by E15.5. In unpeeled gut preparations where all layers were left intact, we also mimicked the basolateral release of 5-HT from enteroendocrine cells, which triggered responses in myenteric cell bodies at postnatal day (P)0. Altogether, our results show that enteric neurons extend neurites out of the myenteric plexus early during mouse enteric nervous system development, innervating the gastrointestinal mucosa, even before villus formation in mice of either sex. Neurites are already able to conduct electrical information at E15.5, and responses to 5-HT develop postnatally.NEW & NOTEWORTHY How enteric neurons project into the gut mucosa and begin to communicate with the epithelium during development is not known. Our study shows that enteric neurites project into the lamina propria as early as E13.5 in the mouse, before development of the submucous plexus and before formation of intestinal villi. These neurites are capable of transmitting electrical signals back to their cell bodies by E15.5 and respond to serotonin applied to neurite terminals by birth.

Morphogenesis and motility of the Astyanax mexicanus gastrointestinal tract.

Through the course of evolution, the gastrointestinal (GI) tract has been modified to maximize nutrient absorption, forming specialized segments that are morphologically and functionally distinct. Here we show that the GI tract of the Mexican tetra, Astyanax mexicanus, has distinct regions, exhibiting differences in morphology, motility, and absorption. We found that A. mexicanus populations adapted for life in subterranean caves exhibit differences in the GI segments compared to those adapted to surface rivers. Cave-adapted fish exhibit bi-directional churning motility in the stomach region that is largely absent in river-adapted fish. We investigated how this motility pattern influences intestinal transit of powdered food and live prey. We found that powdered food is more readily emptied from the cavefish GI tract. In contrast, the transit of live rotifers from the stomach region to the midgut occurs more slowly in cavefish compared to surface fish, consistent with the presence of churning motility. Differences in intestinal motility and transit likely reflect adaptation to unique food sources available to post-larval A. mexicanus in the cave and river environments. We found that cavefish grow more quickly than surface fish when fed ad libitum, suggesting that altered GI function may aid in nutrient consumption or absorption. We did not observe differences in enteric neuron density or smooth muscle organization between cavefish and surface fish. Altered intestinal motility in cavefish could instead be due to changes in the activity or patterning of the enteric nervous system. Exploring this avenue will lead to a better understanding of how the GI tract evolves to maximize energy assimilation from novel food sources.