Gut Microbial Products Regulate Murine Gastrointestinal Motility via Toll-Like Receptor 4 Signaling

doi:10.1053/j.gastro.2012.06.034

Gastroenterology

Volume 143, Issue 4, October 2012, Pages 1006-1016.e4

https://doi.org/10.1053/j.gastro.2012.06.034 Get rights and content

Background & Aims

Altered gastrointestinal motility is associated with significant morbidity and health care costs. Toll-like receptors (TLR) regulate intestinal homeostasis. We examined the roles of TLR4 signaling in survival of enteric neurons and gastrointestinal motility.

Methods

We assessed changes in intestinal motility by assessing stool frequency, bead expulsion, and isometric muscle recordings of colonic longitudinal muscle strips from mice that do not express TLR4 (Tlr4^Lps-d or TLR4^−/−) or Myd88 (Myd88^−/−), in wild-type germ-free mice or wild-type mice depleted of the microbiota, and in mice with neural crest-specific deletion of Myd88 (Wnt1Cre^+/−/Myd88^fl/fl). We studied the effects of the TLR4 agonist lipopolysaccharide (LPS) on survival of cultured, immortalized fetal enteric neurons and enteric neuronal cells isolated from wild-type and Tlr4^Lps-d mice at embryonic day 13.5.

Results

There was a significant delay in gastrointestinal motility and reduced numbers of nitrergic neurons in TLR4^Lps-d, TLR4^−/−, and Myd88^−/− mice compared with wild-type mice. A similar phenotype was observed in germ-free mice, mice depleted of intestinal microbiota, and Wnt1Cre^+/−/Myd88^fl/fl mice. Incubation of enteric neuronal cells with LPS led to activation of the transcription factor nuclear factor (NF)-κB and increased cell survival.

Conclusions

Interactions between enteric neurons and microbes increases neuron survival and gastrointestinal motility in mice. LPS activation of TLR4 and NF-κB appears to promote survival of enteric neurons. Factors that regulate TLR4 signaling in neurons might be developed to alter gastrointestinal motility.

Section snippets

Animals

C3H/HeJ (spontaneous mutation in TLR4 gene, Tlr4^lps-d), C3H/HeOuJ mice (controls for TLR4 mutant mice), and TLR4^−/− mice on BL/10 background were obtained from Jackson Laboratories (Bar Harbor, ME). Myd88^−/− mice on BL/6 background and their controls were bred in the Emory animal facility. Swiss Webster germ-free mice and conventional mice were obtained from Taconic (Hudson, NY). Hemizygous for transgenic (Tg) (Wnt1-cre) male mice were bred with noncarrier (wild type) female mice to obtain

Tlr4^Lps-d Mice Exhibit Delayed Intestinal Motility

We first examined the role of TLR4 in modulating gastrointestinal motility in vivo. We used the C3H/HeJ mice, which have a spontaneous mutation at the LPS response locus (mutation in Toll-like receptor 4 gene, Tlr4^Lps-d) making them hyporesponsive to LPS. We observed that, even though the body weight of Tlr4^Lps-d mice was significantly lower than controls (C3H/HeOuJ, WT) (Supplementary Figure 1A), there was no difference in their food intake (Supplementary Figure 1B). The frequency of bowel

Discussion

We have demonstrated a role for enteric neuronal TLR4 in the regulation of intestinal motility. Lack of TLR4 signaling led to delayed gastrointestinal motility as demonstrated by reduced pellet frequency and delayed intestinal transit. In addition, in the absence of TLR4 signaling, we found a reduction in nitrergic neurons and resultant reduced nitrergic relaxation. Reduction of the microbiota with antibiotics led to a delayed intestinal transit in WT mice but not in the TLR4^Lps-d mice,

Acknowledgments

The authors thank Duke Geem, Jesse Aitken, Gayathri Srinivasan, and Benoit Chassaing for technical assistance.

References (36)

S. Rakoff-Nahoum et al.
Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis
Cell
(2004)
J.M. van Noort et al.
Toll-like receptors in the CNS: implications for neurodegeneration and repair
Prog Brain Res
(2009)
E. Okun et al.
Toll-like receptors in neurodegeneration
Brain Res Rev
(2009)
S.C. Tang et al.
Toll-like receptor-4 mediates neuronal apoptosis induced by amyloid β-peptide and the membrane lipid peroxidation product 4-hydroxynonenal
Exp Neurol
(2008)
F. Hua et al.
Activation of Toll-like receptor 4 signaling contributes to hippocampal neuronal death following global cerebral ischemia/reperfusion
J Neuroimmunol
(2007)
M.B. Arciszewski et al.
Vasoactive intestinal peptide rescues cultured rat myenteric neurons from lipopolysaccharide induced cell death
Regul Pept
(2008)
M. Anitha et al.
Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function
Gastroenterology
(2008)
M.S. Miller et al.
Accurate measurement of intestinal transit in the rat
J Pharmacol Methods
(1981)
Z.X. Li et al.
Granulocyte-colony stimulating factor is involved in low-dose LPS-induced neuroprotection
Neuroscience letters
(2009)
R. Medzhitov et al.
A human homologue of the Drosophila Toll protein signals activation of adaptive immunity
Nature
(1997)

I. Barajon et al.

Toll-like receptors 3, 4, and 7 are expressed in the enteric nervous system and dorsal root ganglia

J Histochem Cytochem

(2009)

M. Lafon et al.

The innate immune facet of brain: human neurons express TLR-3 and sense viral dsRNA

J Mol Neurosci

(2006)

A.C. Jackson et al.

Expression of Toll-like receptor 3 in the human cerebellar cortex in rabies, herpes simplex encephalitis, and other neurological diseases

J Neurovirol

(2006)

S.C. Tang et al.

Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits

Proc Natl Acad Sci U S A

(2007)

R. Wadachi et al.

Trigeminal nociceptors express TLR-4 and CD14: a mechanism for pain due to infection

J Dent Res

(2006)

C. Rumio et al.

Activation of smooth muscle and myenteric plexus cells of jejunum via Toll-like receptor 4

J Cell Physiol

(2006)

T.B. Clarke et al.

Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity

Nat Med

(2010)

C.B. Forsyth et al.

Increased intestinal permeability correlates with sigmoid mucosa α-synuclein staining and endotoxin exposure markers in early Parkinson's disease

PloS One

(2011)

Cited by (295)

Ameliorating effects of transcutaneous auricular vagus nerve stimulation on a mouse model of constipation-predominant irritable bowel syndrome
2024, Neurobiology of Disease
Limited treatment options have been shown to alter the natural course of constipation-predominant irritable bowel syndrome (IBS-C). Therefore, safer and more effective approaches are urgently needed. We investigated the effects of transcutaneous auricular vagus nerve stimulation (taVNS) in a mouse model of IBS-C. In the current study, C57BL/6 mice were randomly divided into normal control, IBS-C model control, sham-electrostimulation (sham-ES), taVNS, and drug treatment groups. The effects of taVNS on fecal pellet number, fecal water content, and gastrointestinal transit were evaluated in IBS-C model mice. We assessed the effect of taVNS on visceral hypersensitivity using the colorectal distention test. 16S rRNA sequencing was used to analyze the fecal microbiota of the experimental groups. First, we found that taVNS increased fecal pellet number, fecal water content, and gastrointestinal transit in IBS-C model mice compared with the sham-ES group. Second, taVNS significantly decreased the abdominal withdrawal reflex (AWR) score compared with the sham-ES group, thus relieving visceral hyperalgesia. Third, the gut microbiota outcomes showed that taVNS restored Lactobacillus abundance while increasing Bifidobacterium probiotic abundance at the genus level. Notably, taVNS increased the number of c-kit-positive interstitial cells of Cajal (ICC) in the myenteric plexus region in IBS-C mice compared with the sham-ES group. Therefore, our study indicated that taVNS effectively ameliorated IBS-C in the gut microbiota and ICC.
Role reversals: non-canonical roles for immune and non-immune cells in the gut
2024, Mucosal Immunology
The intestine is home to an intertwined network of epithelial, immune, and neuronal cells as well as the microbiome, with implications for immunity, systemic metabolism, and behavior. While the complexity of this microenvironment has long since been acknowledged, recent technological advances have propelled our understanding to an unprecedented level. Notably, the microbiota and non-immune or structural cells have emerged as important conductors of intestinal immunity, and by contrast, cells of both the innate and adaptive immune systems have demonstrated non-canonical roles in tissue repair and metabolism. This review highlights recent works in the following two streams: non-immune cells of the intestine performing immunological functions; and traditional immune cells exhibiting non-immune functions in the gut.
The many faces of microbiota-gut-brain axis in autism spectrum disorder
2024, Life Sciences
The gut-brain axis is gaining more attention in neurodevelopmental disorders, especially autism spectrum disorder (ASD). Many factors can influence microbiota in early life, including host genetics and perinatal events (infections, mode of birth/delivery, medications, nutritional supply, and environmental stressors). The gut microbiome can influence blood-brain barrier (BBB) permeability, drug bioavailability, and social behaviors. Developing microbiota-based interventions such as probiotics, gastrointestinal (GI) microbiota transplantation, or metabolite supplementation may offer an exciting approach to treating ASD. This review highlights that RNA sequencing, metabolomics, and transcriptomics data are needed to understand how microbial modulators can influence ASD pathophysiology. Due to the substantial clinical heterogeneity of ASD, medical caretakers may be unlikely to develop a broad and effective general gut microbiota modulator. However, dietary modulation followed by administration of microbiota modulators is a promising option for treating ASD-related behavioral and gastrointestinal symptoms. Future work should focus on the accuracy of biomarker tests and developing specific psychobiotic agents tailored towards the gut microbiota seen in ASD patients, which may include developing individualized treatment options.
Germ-Free Animals: A Key Tool in Unraveling How the Microbiota Affects the Brain and Behavior
2024, The Gut-Brain Axis, Second Edition
Numerous studies have implicated the gut microbiota in the development, function, and maintenance of brain health and behavior. Indeed, research utilizing germ-free animals (animals raised devoid of all microorganisms) has been instrumental in providing evidence supporting a role for gut microbes in gut–brain axis communication. Key findings have highlighted the role of the gut microbiota in a multitude of neurophysiological processes including neurogenesis, myelination, blood–brain barrier permeability, neuroplasticity, neurotransmission, microglial activation, and neuroinflammation. Further, the molecular mechanisms underlying germ-free behavior are being elucidated and implicate gut microbes in a myriad of complex behaviors including stress responsivity, anxiety-like behavior, sociability, and cognition. Though the germ-free mouse has been the model employed by most studies linking the gut microbiota to the brain, alternative models such as zebrafish, Drosophila, Caenorhabditis elegans, and the pig are also emerging, further advancing our mechanistic understanding of microbiota–gut–brain communication. The principal advantage of the germ-free model is in proof-of-principle studies, whereby researchers can selectively introduce a specific bacterium or consortium and investigate its mechanistic underpinnings during specific windows throughout the host's lifespan. However, a germ-free upbringing significantly influences neurodevelopmental outcomes that may deem them unsuitable for certain investigations. Further, limited translatability to humans is another key consideration in the use of germ-free models, highlighting the need for alternative and complementary strategies. Increasing our understanding of the impact of the gut microbiota on brain health and behavior using a variety of approaches will enable the development of novel therapeutics targeting disorders associated with perturbed microbiota–gut–brain axis signaling.
Influence of the Microbiota on the Development and Function of the “Second Brain”—The Enteric Nervous System
2024, The Gut-Brain Axis, Second Edition
The enteric nervous system (ENS) is the intrinsic nervous system of the gut, made up of an extensive network of neurons that lines the walls of the gastrointestinal (GI) tract. The framework of the ENS is laid during the first and second gestational trimesters, but the network continues to undergo modifications throughout later gestation and into postnatal life. Colonization of the GI tract by trillions of microorganisms during the early postnatal period represents a significant change from the prenatal condition that undoubtedly affects the developing ENS. The specific changes to the ENS brought about by microbial colonization of the gut are only beginning to be uncovered. In this chapter, we highlight a growing field of research elucidating the role of the intestinal microbiota in shaping the developing ENS. We conclude the review with perspectives regarding areas of potential clinical relevance.
The Microbiota Conducts the Vasoactive Intestinal Polypeptide Orchestra in the Small Intestine
2024, Cellular and Molecular Gastroenterology and Hepatology

View all citing articles on Scopus

: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by the NIH-RO1 (DK080684, to S.S.), VA-MERIT award (to S.S.), DK06411 (to S.V.S.), and DDRDC (DK064399).

View full text

Original ResearchBasic and Translational—Alimentary TractGut Microbial Products Regulate Murine Gastrointestinal Motility via Toll-Like Receptor 4 Signaling

Background & Aims

Methods

Results

Conclusions

Section snippets

Animals

Tlr4Lps-d Mice Exhibit Delayed Intestinal Motility

Discussion

Acknowledgments

Cell

Prog Brain Res

Brain Res Rev

Exp Neurol

J Neuroimmunol

Regul Pept

Gastroenterology

J Pharmacol Methods

Neuroscience letters

A human homologue of the Drosophila Toll protein signals activation of adaptive immunity

Nature

Toll-like receptors 3, 4, and 7 are expressed in the enteric nervous system and dorsal root ganglia

J Histochem Cytochem

The innate immune facet of brain: human neurons express TLR-3 and sense viral dsRNA

J Mol Neurosci

Expression of Toll-like receptor 3 in the human cerebellar cortex in rabies, herpes simplex encephalitis, and other neurological diseases

J Neurovirol

Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits

Proc Natl Acad Sci U S A

Trigeminal nociceptors express TLR-4 and CD14: a mechanism for pain due to infection

J Dent Res

Activation of smooth muscle and myenteric plexus cells of jejunum via Toll-like receptor 4

J Cell Physiol

Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity

Nat Med

Increased intestinal permeability correlates with sigmoid mucosa α-synuclein staining and endotoxin exposure markers in early Parkinson's disease

PloS One

Original Research
Basic and Translational—Alimentary Tract
Gut Microbial Products Regulate Murine Gastrointestinal Motility via Toll-Like Receptor 4 Signaling

Tlr4^Lps-d Mice Exhibit Delayed Intestinal Motility