Pathophysiological Roles of Neuro-Immune Interactions between Enteric Neurons and Mucosal Mast Cells in the Gut of Food Allergy Mice

Recently, the involvement of the nervous system in the pathology of allergic diseases has attracted increasing interest. However, the precise pathophysiological role of enteric neurons in food allergies has not been elucidated. We report the presence of functional high-affinity IgE receptors (FcεRIs) in enteric neurons. FcεRI immunoreactivities were observed in approximately 70% of cholinergic myenteric neurons from choline acetyltransferase-eGFP mice. Furthermore, stimulation by IgE-antigen elevated intracellular Ca2+ concentration in isolated myenteric neurons from normal mice, suggesting that FcεRIs are capable of activating myenteric neurons. Additionally, the morphological investigation revealed that the majority of mucosal mast cells were in close proximity to enteric nerve fibers in the colonic mucosa of food allergy mice. Next, using a newly developed coculture system of isolated myenteric neurons and mucosal-type bone-marrow-derived mast cells (mBMMCs) with a calcium imaging system, we demonstrated that the stimulation of isolated myenteric neurons by veratridine caused the activation of mBMMCs, which was suppressed by the adenosine A3 receptor antagonist MRE 3008F20. Moreover, the expression of the adenosine A3 receptor gene was detected in mBMMCs. Therefore, in conclusion, it is suggested that, through interaction with mucosal mast cells, IgE-antigen-activated myenteric neurons play a pathological role in further exacerbating the pathology of food allergy.

Neuroprotective Roles of the Adenosine A3 Receptor Agonist AST-004 in Mouse Model of Traumatic Brain Injury

Background: Traumatic brain injury (TBI) remains one of the greatest public health concerns with increasing morbidity and mortality rates worldwide. Our group reported stimulation of astrocyte mitochondrial metabolism by P2Y1 receptor agonists significantly reduced cerebral edema and reactive gliosis in a TBI model. Subsequent data on the pharmacokinetics (PK) and rapid metabolism of these compounds suggested neuroprotection was likely mediated by a metabolite, AST-004, which binding data indicated was an adenosine A3 receptor (A3R) agonist. Methods: The neuroprotective efficacy of AST-004 was tested in a controlled closed cortical injury (CCCI) model of TBI in mice. Results: Twenty-four (24) hours post-injury, mice subjected to CCCI and treated with AST-004 (0.22mg/kg) exhibited significantly less secondary brain injury. These effects were quantified with less cell death (PSVue794 fluorescence) and loss of blood brain barrier breakdown (Evans Blue extravasation assay), compared to vehicle treated TBI mice. TBI treated mice also exhibited significantly reduced neuroinflammatory markers, glial-fibrillary acidic protein (GFAP, astrogliosis) and ionized Ca2+ binding adaptor molecule 1 (Iba1, microgliosis), both at the mRNA (gRT-PCR) and protein (western blot and immunofluorescence) levels, respectively. Four (4) weeks post-injury, AST-004 treated TBI mice presented significantly reduced impairment of long-term memory. Spatial memory was assessed with a contextual fear conditioning behavior assay (freezing behavior after shock). Finally, AST-004 treatments were found to increase in vivo ATP production in astrocytes (gfap-targeted luciferase activity), consistent with the proposed mechanism of action. Conclusions: These data reveal AST-004 as a novel A3R agonist that increases astrocyte energy production and enhances their neuroprotective efficacy after brain injury.

ADENOSINE A3 RECEPTOR INTERACTS WITH GASDERMIN D TO MODULATE INTESTINAL EPITHELIAL CELL PYROPTOSIS IN ULCERATIVE COLITIS

Background Adenosine A3 receptor (A3AR) plays a role in intestinal inflammation, but little is known about its mechnisms in intestinal inflammation such as ulcerative colitis (UC). Pyroptosis, characterized by Gasdermin D (GSDMD) activation, is implicated in the pathogenesis of UC. We investigated the role of A3AR in GSDMD-mediated pyroptosis in UC and its underlying molecular mechanisms. Methods The expression of A3AR in colonic mucosa of patients with UC were examined. A3AR agonist was used to study the role of A3AR in ex vivo colonic explants of UC patients. In addition, human intestinal epithelial cells Caco-2 were used to further verify the effect of A3AR on pyroptosis induced by LPS+ATP. RT-qPCR and western blotting were used to detect the expression levels of pyroptosis-associated factors including NLRP3, caspase-1, gasdermin-D N-terminal domain (GSDMD-NT), IL-1β and IL-18 in colonic tissues and Caco-2 cells. Immunofluorescence was used to detect the protein expression in tissues and cells. Enzyme-linked immunosorbent assay was used to determine the levels of IL-1β and IL-18 in tissue and cell culture supernatants. Lactate dehydrogenase (LDH) release assay and propidium iodide (PI) staining were used to measure cell pyroptosis. Molecular interactions beween A3AR and GSDMD were investigated using Co-IP and GST pull-down assays. Results A3AR expression was significantly reduced in colonic mucosa of patients with active UC, and colonic epithelial cells were the main cell subpopulation with down-regulated A3AR expression. Pyroptosis-associated factors, including Caspase-1, NLRP3, and GSDMD-NT, were upregulated in UC colonic tissues. The expression of A3AR and GSDMD-NT was negatively correlated. A3AR agonist reduced the production of cytokines (IL-1β and IL-18) and attenuates the expression levels of NLRP3 and GSDMD-NT in the colonic tissues of patients with UC. Furthermore, A3AR overexpression alleviated pyroptosis with reduced LDH release, PI-stained cell number and decreased expressions of GSDMD-NT, NLRP3, caspase-1, IL-1β, and IL-18 in the LPS+ATP-stimulated Caco-2 cells, whereas the opposite occurred in cells treated with small interfeing RNA (siRNA) targeting A3AR. Knockdown of GSDMD in Caco-2 cells significantly blocked the effects of A3AR overexpression or down-regulation on pyroptosis, suggesting that A3AR acts through the GSDMD-mediated pyroptosis pathway. Co-IP and GST pull-down assays showed that A3AR interacted with GSDMD. Conclusion A3AR modulates intestinal inflammation in UC through GSDMD-mediated intestinal epithelial cell pyroptosis. We propose a novel mechanism by which A3AR-GSDMD interaction affects UC through pyroptosis, suggesting that A3AR is a potential target for the treatment of UC.