Characterization of the expression and putative role of Mas-related gene receptors in the
neuroimmune interaction during intestinal inflammation
Thesis summary (for a general audience) by Leela Rani AVULA
Promotor: Prof. Dr. Jean-Pierre Timmermans
Co-promotor: Dr. Luc Van Nassauw
Begeleider: Dr. Roeland Buckinx
The gastrointestinal (GI) tract is constantly exposed to a multitude of dietary, commensal and
environmental antigenic loads, both harmless and potentially pathogenic. Normal GI functioning and
homeostasis is maintained by a balanced and finely orchestrated interplay between various cell systems
located within the gut wall, such as the immune cells, components of the intrinsic and extrinsic
innervations, epithelial, vascular and smooth muscle systems. Disruption of this dynamic balance may
lead to GI inflammation and to the further development of a number of debilitating intestinal pathologies,
including inflammatory bowel diseases (IBD), celiac disease as well as to the development of food
allergies. Moreover, chronic low-grade inflammation is also thought to underlie the pathogenesis of some
of the major functional GI disorders, such as the irritable bowel syndrome (IBS). Despite this extensive
knowledge of the importance of gut inflammation in a multitude of GI disorders, many questions as to the
exact underlying pathophysiological mechanisms remain unanswered, and effective treatment strategies
are hence scarce.
G protein-coupled receptors (GPCRs) are among the key players in inflammation in that they are
expressed by many inflammatory cells and that they tansduce extracellular signals activating cellular
responses. As such, GPCRs have become a major target class of therapeutic interest. One such group of
recently identified GPCRs is the Mas-related gene (Mrg) receptor family. The Mrg family is complex in
that it comprises over 50 rodent and human Mrg members (including a few comparable orthologs) that are
closely related to the Mas oncogene. Given the expression of Mrgs in specific subsets of spinal sensory
neurons, and the knowledge of some ligands that activate these receptors, some Mrg members have been
suggested to play important roles in a variety of (patho)physiological processes, including pain
sensation/modulation, mast cell activation, and mast cell-sensory neuron communication, mainly in
relation to the skin. Despite the possibility of relevance of the same processes (atleast to a certain extent)
in other organ systems such as the gut, in health and disease, conclusive information on Mrg presence and
function in these systems is virtually lacking. This PhD study therefore aimed to unravel the presence and
putative function of all cloned (n=22) Mrg members in the GI tract by comparing the ileum and the
ileum-innervating spinal afferents of healthy mice with those of two diverse inflamed conditions, i.e., a
trematode parasite (Schistosoma mansoni)-infected model and a chemical (2,4,6-trinitrobenzene sulfonate
(TNBS))-treated model. Several techniques, including combined multiple immunofluorescence, RT-PCR,
qPCR and intestinal motility analysis, were employed to achieve this goal. Since commercial antibodies
directed against most Mrg members are lacking, custom-developed antibodies against some of the most
interesting members were raised and applied in immunohistochemical studies. The quality of these
custom-developed antibodies was verified by immunoblot and ELISA experiments.
The results indicate that in the healthy mouse ileum, most Mrg members were either not or only
moderately expressed, showing a neuron-specific expression pattern. Interestingly, in the inflamed mouse
ileum, significant changes in the expression patterns of some Mrg members were observed, for instance,
expression of MrgA4, MrgB2 and MrgB8 was increased in enteric sensory neurons, expression of MrgE
and MrgF was decreased in enteric cholinergic secretomotor neurons, and MrgB10 and MrgD showed de
novo expression in enteric sensory neurons and in the newly recruited mucosal mast cells (MMCs).
Significant changes with respect to the ileum-innervating spinal afferents were only observed for MrgA4,
MrgE and MrgF during intestinal inflammation. The obtained results in mice provide clear evidence that
specific Mrg members are involved in the inflammatory response during intestinal inflammation,
particularly through their participation in primary afferent and MMC responses.
MrgD was considered as one of the most interesting Mrg members, since a human ortholog exists, and
since we found it to be de novo expressed in the mouse intestine following inflammation. Therefore,
experiments were repeated in MrgD-/- mice in order to further elaborate its role. We observed in this
knockout strain that lack of the MrgD gene and its corresponding receptor leads to increased expression
of the sensory neuropeptide (calcitonin gene-related peptide (CGRP)) in enteric sensory neurons in both
inflammation models, as well to increased numbers of MMC in the S. mansoni-infected mouse ileum
compared to the wild-type animals. Moreover, certain parameters such as the villous diameter and crypt
depth were also increased in this knockout strain during intestinal schistosomiasis. These findings clearly
indicate role(s) for MrgD in the regulation of mastocytosis and/or sensory responses in intestinal
inflammation.
Based on the above findings, we further explored the role of MrgD in mast cell and sensory neuronassociated responses using immunohistochemistry, PCR and intestinal motility analysis.
Immunohistochemical stainings showed that the MrgD-expressing MMCs and the CGRP-expressing
nerve fibres in the lamina propria are in close association in the S. mansoni-infected mouse ileum, adding
further support to our hypothesis of Mrg receptor involvement in the mast cell-sensory neuron cross-talk.
β-alanine, a ligand for MrgD, could be a possible candidate mediator in the neuroimmune interactions. In
this regard, it would be interesting to further explore the MrgD-mediated interaction between MMC and
neurons. To investigate the activation of other Mrgs present on neurons and/or MMCs and to unravel their
signal transduction pathways are also other interesting future research perspectives. When we compared
the GI motility between healthy and S. mansoni-infected wild-type and MrgD-/- mice, we found no
changes between the groups, indicating that MrgD-induced neuroimmune changes would primarily
influence the sensory pathways during intestinal inflammation. In parallel, we identified MrgD expression
in neurons and mast cells in the human intestine, suggesting possible translational potential and clinical
relevance.
This PhD study has unraveled the expression (and distribution) of all cloned Mrg receptors in the healthy
and inflamed mouse ileum and in the ileum-innervating spinal ganglia. The expression of some of the
relevant Mrg members in the human intestine has also been described. The study has clearly demonstrated
the involvement of specific Mrg subtypes in intestinal inflammation, in particular in sensory neuronal and
mast cell-associated responses. Together, our findings provide a strong basis for the further exploration of
Mrg members, especially MrgD, as experimental targets in intestinal inflammatory pathologies, focusing
on sensory perception and neuroimmune interactions.