Periodic Reporting for period 4 - NEUMACS (Neuron-associated macrophages in the gut as novel target for the treatment of enteric neuropathies)
Periodo di rendicontazione: 2024-04-01 al 2024-09-30
The gastrointestinal tract has the vital task to digest and absorb ingested food, a complex process requiring coordinated integration of motility, secretion, vascularization and absorption. Thereto the gastrointestinal tract is equipped with its own nervous system, the enteric nervous system (ENS), capable of controlling gut function independently of input from brain or spinal cord. Reduction in number or dysfunction of the neurons within the gut wall, also referred to as enteric neuropathy, significantly impacts on gut function, resulting in stasis of luminal contents and malabsorption, chronic pain, vomiting, bloating and severe constipation. Enteric neuropathies are common in prevalent disorders such as obesity, diabetes, and ageing, all major contributors to the health burden. Despite the continuous global increase in incidence of these disorders, the insight in the mechanisms leading to the reduction or dysfunction of enteric neurons is limited and most importantly, adequate treatment is lacking. Recently, we collected evidence that survival of enteric neurons is guaranteed by a unique subpopulation of resident macrophages closely associated to the ENS and expressing a typical neuroprotective / -supportive transcriptome. In line, depletion of these neuron-associated macrophages (NA-MF) results in apoptosis and a reduction in number of enteric neurons leading to severely impaired gastrointestinal motility. We pose the provocative hypothesis that enteric neuropathy results from impaired support to the ENS by NA-MF, leading to neural distress and apoptosis. Using state-of-the-art methods, we will first characterize in depth the NA-MF population to subsequently unravel the mechanisms leading to failure of NA-MF to support and protect the ENS in animal models and in patients. These ground-breaking insights will allow us to identify therapeutic targets for the treatment of enteric neuropathies, representing an exponentially growing health problem of the 21st century.
In the past 5 years, we have studied in depth the intestinal macrophage population with a special interest in the subpopulation that closely interacts with the enteric nervous system, both in mice and humans. In mice, we introduced the concept that these so-called neuron-associated macrophages (NA-MF) closely resemble microglia, the resident macrophages of the brain. We showed that NA-MF have a similar gene signature, morphology and above all a similar function as brain microglia. An unexpected but exciting finding was that resident macrophages in the gut, like microglia, play a key role in shaping the enteric nervous system early in life. Before weaning, enteric neurons are rather disorganized with too many neurons and too many interconnecting nerve tracts compared to the adult and mature enteric nervous system. We showed that around the time of weaning, intestinal macrophages start to engulf neurons and synapses and transform the immature enteric nervous system into its mature state with well-organized ganglia elegantly interconnected with nerve tracts. Notably, we demonstrated that once macrophages have finalized this vital task, they completely switch their function and start protecting and nourishing the enteric neurons. Surprisingly, this switch is instructed by the enteric neurons themselves via the release of TGF-beta, which interestingly imprints macrophages to become neuroprotective and obtain a microglia-like transcriptome. Finally, we were able to identify a membrane marker characteristic for NA-MF, i.e. F11r, which allowed us to investigate the NA-MF in more detail in intestinal disease models, but also in a variety of other organs. Not only in the gut, but also in lung, liver, pancreas, peripheral nerves, etc we were able to demonstrate the presence of NA-MF, again characterized by the expression of microglia-like genes and closely interacting with nerve fibres. Also in the human gut, we identified the NA-MF population and demonstrated the role of TGF-beta to obtain the microglia-like phenotype. So taken together, the concept of NA-MF is not restricted to the gut but is biologically conserved and consistent throughout the entire nervous system, both in mice and humans.
In the second part of the project, the potential role of NA-MF in intestinal neurodegenerative disorders such as ageing, diabetes and obesity was evaluated. We introduced and optimized preclinical models of these three conditions and developed new methods, such as a novel method to measure gastric emptying in mice using MRI. We showed that the number of enteric neurons indeed decreases (=neurodegeneration) in all three models, a finding that was associated with impaired intestinal function. In aged mice, we identified a gene potentially contributing to the loss of enteric neurons, a finding that is currently further explored. In diabetic mice, we have identified two macrophage populations which protect diabetic mice from developing delayed gastric emptying, a finding of which the therapeutic potential is further explored.
Finally, we have succeeded to develop a co-culture platform derived from human inducible pluripotent stem cells (iPSCs). iPSC-derived enteric neurons are co-cultured with iPSC-derived macrophages up to 60 days revealing enteric ganglia interconnected with nerve tracts and covered with mature macrophages. This co-culture is unique and will allow us to further study in great depth how macrophages interact with enteric neurons and will serve as a platform to validate identified therapeutic targets, opening new avenues for the treatment of intestinal neurodegenerative disorders.
In the second part of the project, the potential role of NA-MF in intestinal neurodegenerative disorders such as ageing, diabetes and obesity was evaluated. We introduced and optimized preclinical models of these three conditions and developed new methods, such as a novel method to measure gastric emptying in mice using MRI. We showed that the number of enteric neurons indeed decreases (=neurodegeneration) in all three models, a finding that was associated with impaired intestinal function. In aged mice, we identified a gene potentially contributing to the loss of enteric neurons, a finding that is currently further explored. In diabetic mice, we have identified two macrophage populations which protect diabetic mice from developing delayed gastric emptying, a finding of which the therapeutic potential is further explored.
Finally, we have succeeded to develop a co-culture platform derived from human inducible pluripotent stem cells (iPSCs). iPSC-derived enteric neurons are co-cultured with iPSC-derived macrophages up to 60 days revealing enteric ganglia interconnected with nerve tracts and covered with mature macrophages. This co-culture is unique and will allow us to further study in great depth how macrophages interact with enteric neurons and will serve as a platform to validate identified therapeutic targets, opening new avenues for the treatment of intestinal neurodegenerative disorders.
Unexpectedly we identified a new role for intestinal macrophages, namely that early in life, they are key players in the maturation of the enteric nervous system. Moreover, in collaboration with our colleagues from hepatology, we further explored the role of another subpopulation of macrophages of which we previously demonstrated to be long-lived and involved in the maintenance of blood vessel integrity. Using the know-how obtained in the past few years, we have collected interesting novel insights in the role of these blood vessel associated macrophages in bacterial translocation in a mouse model of liver disease. This work is groundbreaking and will have a major impact on our understanding of liver disease.
In the coming years, we expect to obtain major novel insights in the cross-talk between enteric neurons and macrophages by studying these two cell populations while they are maturing and interacting in the co-culture system. These experiments will identify signalling molecules that support neurons and increase the survival of neurons, which is crucial knowledge to ultimately develop drugs that will increase the success rate of neuronal stem cell transplantation, an approach currently evaluated as treatment of neurodegenerative disorders such as achalasia and Hirschsprung’s disease.
In the coming years, we expect to obtain major novel insights in the cross-talk between enteric neurons and macrophages by studying these two cell populations while they are maturing and interacting in the co-culture system. These experiments will identify signalling molecules that support neurons and increase the survival of neurons, which is crucial knowledge to ultimately develop drugs that will increase the success rate of neuronal stem cell transplantation, an approach currently evaluated as treatment of neurodegenerative disorders such as achalasia and Hirschsprung’s disease.