TLR2 and TLR4 Modulate Mouse Ileal Motility by the Interaction with Muscarinic and Nicotinic Receptors
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Muscle Contractility Studies
2.3. Gene Expression by Real-Time PCR
2.4. Western Blotting
2.5. Data Analysis and Statistics
2.6. Drugs and Solutions
3. Results
3.1. Effect of ACh on Ileal Motility of WT, TLR2−/− and TLR4−/− Mice
3.2. Effects of Muscarinic and Nicotinic ACh Receptors Antagonists on ACh-Evoked Response in Ileum from WT, TLR2−/− and TLR4−/− Mice
3.3. mRNA and Protein Expression Levels of Muscarinic and Nicotinic ACh Receptors in WT, TLR2−/− and TLR4−/− Mice
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Enck, P.; Aziz, Q.; Barbara, G.; Farmer, A.D.; Fukudo, S.; Mayer, E.A.; Niesler, B.; Quigley, E.M.; Rajilic-Stojanovic, M.; Schemann, M.; et al. Irritable bowel syndrome. Nat. Rev. Dis. Primers 2016, 2, 16014. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Guarino, M.P.; Cicala, M.; Putignani, L.; Severi, C. Gastrointestinal neuromuscular apparatus: An underestimated target of gut microbiota. World J. Gastroenterol. 2016, 22, 9871–9879. [Google Scholar] [CrossRef] [PubMed]
- Quigley, E.M. Microflora modulation of motility. J. Neurogastroenterol. Motil. 2011, 17, 140–147. [Google Scholar] [CrossRef] [PubMed]
- Hooper, L.V.; Wong, M.H.; Thelin, A.; Hansson, L.; Falk, P.G.; Gordon, J.I. Molecular analysis of commensal host-microbial relationships in the intestine. Science 2001, 291, 881–884. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Didari, T.; Mozaffari, S.; Nikfar, S.; Abdollahi, M. Effectiveness of probiotics in irritable bowel syndrome: Updated systematic review with meta-analysis. World J. Gastroenterol. 2015, 21, 3072–3084. [Google Scholar] [CrossRef] [PubMed]
- Dimidi, E.; Christodoulides, S.; Scott, S.M.; Whelan, K. Mechanisms of Action of Probiotics and the Gastrointestinal Microbiota on Gut Motility and Constipation. Adv. Nutr. 2017, 8, 484–494. [Google Scholar] [CrossRef] [Green Version]
- Bar, F.; Von Koschitzky, H.; Roblick, U.; Bruch, H.P.; Schulze, L.; Sonnenborn, U.; Bottner, M.; Wedel, T. Cell-free supernatants of Escherichia coli Nissle 1917 modulate human colonic motility: Evidence from an in vitro organ bath study. Neurogastroenterol. Motil. 2009, 21, 559-e17. [Google Scholar] [CrossRef]
- Guarino, M.P.; Altomare, A.; Stasi, E.; Marignani, M.; Severi, C.; Alloni, R.; Dicuonzo, G.; Morelli, L.; Coppola, R.; Cicala, M. Effect of acute mucosal exposure to Lactobacillus rhamnosus GG on human colonic smooth muscle cells. J. Clin. Gastroenterol. 2008, 42, S185–S190. [Google Scholar] [CrossRef]
- Kawasaki, T.; Kawai, T. Toll-like receptor signaling pathways. Front. Immunol. 2014, 5, 461. [Google Scholar] [CrossRef] [Green Version]
- Takeda, K.; Akira, S. Toll-like receptors. Curr. Protoc. Immunol. 2015, 109, 1–10. [Google Scholar] [CrossRef]
- Shea-Donohue, T.; Notari, L.; Sun, R.; Zhao, A. Mechanisms of smooth muscle responses to inflammation. Neurogastroenterol. Motil. 2012, 24, 802–811. [Google Scholar] [CrossRef] [PubMed]
- Brun, P.; Giron, M.C.; Qesari, M.; Porzionato, A.; Caputi, V.; Zoppellaro, C.; Banzato, S.; Grillo, A.R.; Spagnol, L.; De Caro, R.; et al. Toll-like receptor 2 regulates intestinal inflammation by controlling integrity of the enteric nervous system. Gastroenterology 2013, 145, 1323–1333. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Barajon, I.; Serrao, G.; Arnaboldi, F.; Opizzi, E.; Ripamonti, G.; Balsari, A.; Rumio, C. Toll-like receptors 3, 4, and 7 are expressed in the enteric nervous system and dorsal root ganglia. J. Histochem. Cytochem. Off. J. Histochem. Soc. 2009, 57, 1013–1023. [Google Scholar] [CrossRef] [PubMed]
- Filippova, L.V.; Malyshev, F.S.; Bykova, A.A.; Nozdrachev, A.D. Expression of toll-like receptors 4 in nerve plexuses of the rat duodenum, jejunum, and colon. Dokl. Biol. Sci. 2012, 445, 215–217. [Google Scholar] [CrossRef] [PubMed]
- Caputi, V.; Marsilio, I.; Cerantola, S.; Roozfarakh, M.; Lante, I.; Galuppini, F.; Rugge, M.; Napoli, E.; Giulivi, C.; Orso, G.; et al. Toll-Like Receptor 4 Modulates Small Intestine Neuromuscular Function through Nitrergic and Purinergic Pathways. Front. Pharmacol. 2017, 8, 350. [Google Scholar] [CrossRef] [Green Version]
- Forcen, R.; Latorre, E.; Pardo, J.; Alcalde, A.I.; Murillo, M.D.; Grasa, L. Toll-like receptors 2 and 4 modulate the contractile response induced by serotonin in mouse ileum: Analysis of the serotonin receptors involved. Neurogastroenterol. Motil. 2015, 27, 1258–1266. [Google Scholar] [CrossRef]
- Forcen, R.; Latorre, E.; Pardo, J.; Alcalde, A.I.; Murillo, M.D.; Grasa, L. Toll-like receptors 2 and 4 exert opposite effects on the contractile response induced by serotonin in mouse colon: Role of serotonin receptors. Exp. Physiol. 2016, 101, 1064–1074. [Google Scholar] [CrossRef] [Green Version]
- Grasa, L.; Abecia, L.; Pena-Cearra, A.; Robles, S.; Layunta, E.; Latorre, E.; Mesonero, J.E.; Forcen, R. TLR2 and TLR4 interact with sulfide system in the modulation of mouse colonic motility. Neurogastroenterol. Motil. 2019, 31, e13648. [Google Scholar] [CrossRef]
- Tanahashi, Y.; Komori, S.; Matsuyama, H.; Kitazawa, T.; Unno, T. Functions of Muscarinic Receptor Subtypes in Gastrointestinal Smooth Muscle: A Review of Studies with Receptor-Knockout Mice. Int. J. Mol. Sci. 2021, 22, 20926. [Google Scholar] [CrossRef]
- Kondo, T.; Nakajima, M.; Teraoka, H.; Unno, T.; Komori, S.; Yamada, M.; Kitazawa, T. Muscarinic receptor subtypes involved in regulation of colonic motility in mice: Functional studies using muscarinic receptor-deficient mice. Eur. J. Pharmacol. 2011, 670, 236–243. [Google Scholar] [CrossRef]
- Harrington, A.M.; Peck, C.J.; Liu, L.; Burcher, E.; Hutson, J.M.; Southwell, B.R. Localization of muscarinic receptors M1R, M2R and M3R in the human colon. Neurogastroenterol. Motil. 2010, 22, 999–1008. [Google Scholar] [CrossRef] [PubMed]
- Papke, R.L. Merging old and new perspectives on nicotinic acetylcholine receptors. Biochem. Pharmacol. 2014, 89, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Foong, J.P.; Hirst, C.S.; Hao, M.M.; McKeown, S.J.; Boesmans, W.; Young, H.M.; Bornstein, J.C.; Vanden Berghe, P. Changes in Nicotinic Neurotransmission during Enteric Nervous System Development. J. Neurosci. 2015, 35, 7106–7115. [Google Scholar] [CrossRef] [PubMed]
- Zhou, X.; Ren, J.; Brown, E.; Schneider, D.; Caraballo-Lopez, Y.; Galligan, J.J. Pharmacological properties of nicotinic acetylcholine receptors expressed by guinea pig small intestinal myenteric neurons. J. Pharmacol. Exp. Ther. 2002, 302, 889–897. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pardo, J.; Wallich, R.; Martin, P.; Urban, C.; Rongvaux, A.; Flavell, R.A.; Mullbacher, A.; Borner, C.; Simon, M.M. Granzyme B-induced cell death exerted by ex vivo CTL: Discriminating requirements for cell death and some of its signs. Cell Death Differ. 2008, 15, 567–579. [Google Scholar] [CrossRef]
- Gordon, R.K.; Gray, R.R.; Reaves, C.B.; Butler, D.L.; Chiang, P.K. Induced release of acetylcholine from guinea pig ileum longitudinal muscle-myenteric plexus by anatoxin-a. J. Pharmacol. Exp. Ther. 1992, 263, 997–1002. [Google Scholar]
- Honda, K.; Takano, Y.; Kamiya, H. Pharmacological profiles of muscarinic receptors in the longitudinal smooth muscle of guinea pig ileum. Jpn. J. Pharmacol. 1993, 62, 43–47. [Google Scholar] [CrossRef] [Green Version]
- Gericke, A.; Sniatecki, J.J.; Mayer, V.G.; Goloborodko, E.; Patzak, A.; Wess, J.; Pfeiffer, N. Role of M1, M3, and M5 muscarinic acetylcholine receptors in cholinergic dilation of small arteries studied with gene-targeted mice. Am. J. Physiol. Heart Circ. Physiol. 2011, 300, H1602–H1608. [Google Scholar] [CrossRef] [Green Version]
- Kedmi, M.; Beaudet, A.L.; Orr-Urtreger, A. Mice lacking neuronal nicotinic acetylcholine receptor β4-subunit and mice lacking both α5- and β4-subunits are highly resistant to nicotine-induced seizures. Physiol. Genom. 2004, 17, 221–229. [Google Scholar] [CrossRef]
- Somm, E.; Guerardel, A.; Maouche, K.; Toulotte, A.; Veyrat-Durebex, C.; Rohner-Jeanrenaud, F.; Maskos, U.; Huppi, P.S.; Schwitzgebel, V.M. Concomitant α7 and β2 nicotinic AChR subunit deficiency leads to impaired energy homeostasis and increased physical activity in mice. Mol. Genet. Metab. 2014, 112, 64–72. [Google Scholar] [CrossRef] [Green Version]
- Girard, B.M.; Merriam, L.A.; Tompkins, J.D.; Vizzard, M.A.; Parsons, R.L. Decrease in neuronal nicotinic acetylcholine receptor subunit and PSD-93 transcript levels in the male mouse MPG after cavernous nerve injury or explant culture. Am. J. Physiol. Ren. Physiol. 2013, 305, F1504–F1512. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Buchholz, B.M.; Chanthaphavong, R.S.; Bauer, A.J. Nonhemopoietic cell TLR4 signaling is critical in causing early lipopolysaccharide-induced ileus. J. Immunol. 2009, 183, 6744–6753. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Killoran, K.E.; Miller, A.D.; Uray, K.S.; Weisbrodt, N.W.; Pautler, R.G.; Goyert, S.M.; van Rooijen, N.; Conner, M.E. Role of innate immunity and altered intestinal motility in LPS- and MnCl2-induced intestinal intussusception in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 2014, 306, G445–G453. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tattoli, I.; Petitta, C.; Scirocco, A.; Ammoscato, F.; Cicenia, A.; Severi, C. Microbiota, innate immune system, and gastrointestinal muscle: Ongoing studies. J. Clin. Gastroenterol. 2012, 46, S6–S11. [Google Scholar] [CrossRef]
- Barona, I.; Fagundes, D.S.; Gonzalo, S.; Grasa, L.; Arruebo, M.P.; Plaza, M.A.; Murillo, M.D. Role of TLR4 and MAPK in the local effect of LPS on intestinal contractility. J. Pharm. Pharmacol. 2011, 63, 657–662. [Google Scholar] [CrossRef]
- Gonzalo, S.; Valero, M.S.; Martinez de Salinas, F.; Vergara, C.; Arruebo, M.P.; Plaza, M.A.; Murillo, M.D.; Grasa, L. Roles of Toll-Like Receptor 4, IkappaB Kinase, and the Proteasome in the Intestinal Alterations Caused by Sepsis. Dig. Dis. Sci. 2015, 60, 1223–1231. [Google Scholar] [CrossRef]
- Sawyer, G.W.; Ehlert, F.J. Contractile roles of the M2 and M3 muscarinic receptors in the guinea pig colon. J. Pharmacol. Exp. Ther. 1998, 284, 269–277. [Google Scholar]
- Papke, R.L.; Dwoskin, L.P.; Crooks, P.A.; Zheng, G.; Zhang, Z.; McIntosh, J.M.; Stokes, C. Extending the analysis of nicotinic receptor antagonists with the study of α6 nicotinic receptor subunit chimeras. Neuropharmacology 2008, 54, 1189–1200. [Google Scholar] [CrossRef] [Green Version]
- Thomsen, M.S.; Zwart, R.; Ursu, D.; Jensen, M.M.; Pinborg, L.H.; Gilmour, G.; Wu, J.; Sher, E.; Mikkelsen, J.D. α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties. PLoS ONE 2015, 10, e0130572. [Google Scholar] [CrossRef]
- Galligan, J.J. Nerve terminal nicotinic cholinergic receptors on excitatory motoneurons in the myenteric plexus of guinea pig intestine. J. Pharmacol. Exp. Ther. 1999, 291, 92–98. [Google Scholar]
- Obaid, A.L.; Nelson, M.E.; Lindstrom, J.; Salzberg, B.M. Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system. J. Exp. Biol. 2005, 208, 2981–3001. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hayashi, E.; Yamada, S.; Mori, M. Comparative studies on anti-nicotinic action of hexamethonium, mecamylamine and adenosine in the guinea pig isolated ileum. Jpn. J. Pharmacol. 1977, 27, 659–665. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pauwelyn, V.; Lefebvre, R.A. 5-HT4 receptors facilitate cholinergic neurotransmission throughout the murine gastrointestinal tract. Neurogastroenterol. Motil. 2017, 29, e13064. [Google Scholar] [CrossRef] [PubMed]
- Alfonzo-Mendez, M.A.; Alcantara-Hernandez, R.; Garcia-Sainz, J.A. Novel Structural Approaches to Study GPCR Regulation. Int. J. Mol. Sci. 2016, 18, 27. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Romero-Fernandez, W.; Borroto-Escuela, D.O.; Alea, M.P.; Garcia-Mesa, Y.; Garriga, P. Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation. Glycobiology 2011, 21, 1663–1672. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Albuquerque, E.X.; Pereira, E.F.; Alkondon, M.; Rogers, S.W. Mammalian nicotinic acetylcholine receptors: From structure to function. Physiol. Rev. 2009, 89, 73–120. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Grasa, L.; Abecia, L.; Forcen, R.; Castro, M.; de Jalon, J.A.; Latorre, E.; Alcalde, A.I.; Murillo, M.D. Antibiotic-Induced Depletion of Murine Microbiota Induces Mild Inflammation and Changes in Toll-like Receptor Patterns and Intestinal Motility. Microb. Ecol. 2015, 70, 835–848. [Google Scholar] [CrossRef]
- Reardon, C.; Duncan, G.S.; Brustle, A.; Brenner, D.; Tusche, M.W.; Olofsson, P.S.; Rosas-Ballina, M.; Tracey, K.J.; Mak, T.W. Lymphocyte-derived ACh regulates local innate but not adaptive immunity. Proc. Natl. Acad. Sci. USA 2013, 110, 1410–1415. [Google Scholar] [CrossRef] [Green Version]
- Favre, J.; Musette, P.; Douin-Echinard, V.; Laude, K.; Henry, J.P.; Arnal, J.F.; Thuillez, C.; Richard, V. Toll-like receptors 2-deficient mice are protected against postischemic coronary endothelial dysfunction. Arter. Thromb. Vasc. Biol. 2007, 27, 1064–1071. [Google Scholar] [CrossRef] [Green Version]
- Kishibe, M.; Griffin, T.M.; Radek, K.A. Keratinocyte nicotinic acetylcholine receptor activation modulates early TLR2-mediated wound healing responses. Int. Immunopharmacol. 2015, 29, 63–70. [Google Scholar] [CrossRef] [Green Version]
- Nakamura, Y.; Kimura, S.; Takada, N.; Takemura, M.; Iwamoto, M.; Hisaoka-Nakashima, K.; Nakata, Y.; Morioka, N. Stimulation of toll-like receptor 4 downregulates the expression of α7 nicotinic acetylcholine receptors via histone deacetylase in rodent microglia. Neurochem. Int. 2020, 138, 104751. [Google Scholar] [CrossRef] [PubMed]
Gene | Reference | GenBank Accession Number | Forward and Reverse Primers |
---|---|---|---|
mAChRs M2 | [28] | NM_203491.3 | CGGACCACAAAAATGGCAGGCAT CCATCACCACCAGGCATGTTGTTGT |
mAChRs M3 | [28] | NM_033269.4 | CCTCTTGAAGTGCTGCGTTCTGACC TGCCAGGAAGCCAGTCAAGAATGC |
nAChRs α3 | [29] | NM_145129.3 | GTGGAGTTCATGCGAGTCCCTG TAAAGATGGCCGGAGGGATCC |
nAChRs α7 | [30] | NM_007390.3 | CAGCAGCTATATCCCCAATGG GGCTCTTTGCAGCATTCATAGA |
nAChRs β4 | [31] | NM_148944.4 | TGTACAACAATGCCGATGGG CCTGTGGGTTCACTGTCCTT |
HPRT | [16] | NM_013556.2 | CTGGTGAAAAGGACCTCTCGAA CTGAAGTACTCATTATAGTCAAGGGCAT |
GAPDH | [16] | NM_008084 | AACGACCCCTTCATTGAC TCCACGACATACTCAGCAC |
WT | TLR2−/− | TLR4−/− | ||||
---|---|---|---|---|---|---|
LogEC50 | EC50 (µM) | LogEC50 | EC50 (µM) | LogEC50 | EC50 (µM) | |
(95% Confidence Intervals) | n | (95% Confidence Intervals) | n | (95% Confidence Intervals) | n | |
ACh | −6.234 | 0.58 | −6.123 | 0.75 | −6.560 | 0.27 |
(−6.944 to −5.524) | n = 16 | (−6.946 to −5.300) | n = 29 | (−7.336 to −5.784) | n = 25 | |
AF-DX 116 + ACh | −5.787 | 1.63 | −6.224 | 0.59 | −7.140 | 0.07 |
(−7.786 to −3.788) | n = 13 | (−6.963 to −5.484) | n = 11 | (−10.268 to −4.011) | n = 11 | |
4-DAMP + ACh | −6.106 | 0.78 | −6.727 | 0.18 | - | - |
(−7.238 to −4.974) | n = 9 | (−10.217 to −3.237) | n = 8 | n = 12 | ||
Mecamylamine + ACh | −6.063 | 0.86 | −5.949 | 1.12 | −7.178 | 0.07 |
(−7.330 to −4.797) | n = 8 | (−6.801 to −5.097) | n = 8 | (−9.097 to −5.259) | n = 11 | |
α-Bungarotoxin + ACh | −5.874 | 1.33 | −5.890 | 1.28 | −6.245 | 0.56 |
(−6.385 to −5.362) | n = 10 | (−6.612 to −5.169) | n = 11 | (−7.162 to −5.329) | n = 9 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Layunta, E.; Forcén, R.; Grasa, L. TLR2 and TLR4 Modulate Mouse Ileal Motility by the Interaction with Muscarinic and Nicotinic Receptors. Cells 2022, 11, 1791. https://doi.org/10.3390/cells11111791
Layunta E, Forcén R, Grasa L. TLR2 and TLR4 Modulate Mouse Ileal Motility by the Interaction with Muscarinic and Nicotinic Receptors. Cells. 2022; 11(11):1791. https://doi.org/10.3390/cells11111791
Chicago/Turabian StyleLayunta, Elena, Raquel Forcén, and Laura Grasa. 2022. "TLR2 and TLR4 Modulate Mouse Ileal Motility by the Interaction with Muscarinic and Nicotinic Receptors" Cells 11, no. 11: 1791. https://doi.org/10.3390/cells11111791