Interactions Between Commensal Bacteria and Enteric Neurons, via FPR1 Induction of ROS, Increase Gastrointestinal Motility in Mice

Abstract

Background & aims: Reduced gastrointestinal (GI) motility is a feature of disorders associated with intestinal dysbiosis and loss of beneficial microbes. It is not clear how consumption of beneficial commensal microbes, marketed as probiotics, affects the enteric nervous system (ENS). We studied the effects of the widely used probiotic and the commensal Lactobacillus rhamnosus GG (LGG) on ENS and GI motility in mice.

Methods: Conventional and germ free C57B6 mice were gavaged with LGG and intestinal tissues were collected; changes in the enteric neuronal subtypes were assessed by real-time polymerase chain reaction, immunoblots, and immunostaining. Production of reactive oxygen species (ROS) in the jejunal myenteric plexi and phosphorylation (p) of mitogen-activated protein kinase 1 (MAPK1) in the enteric ganglia were assessed by immunoblots and immunostaining. Fluorescence in situ hybridization was performed on jejunal cryosections with probes to detect formyl peptide receptor 1 (FPR1). GI motility in conventional mice was assessed after daily gavage of LGG for 1 week.

Results: Feeding of LGG to mice stimulated myenteric production of ROS, increased levels of phosphorylated MAPK1, and increased expression of choline acetyl transferase by neurons (P < .001). These effects were not observed in mice given N-acetyl cysteine (a ROS inhibitor) or LGGΩSpaC (an adhesion-mutant strain of LGG) or FPR1-knockout mice. Gavage of mice with LGG for 1 week significantly increased stool frequency, reduced total GI transit time, and increased contractions of ileal circular muscle strips in ex vivo experiments (P < .05).

Conclusions: Using mouse models, we found that LGG-mediated signaling in the ENS requires bacterial adhesion, redox mechanisms, and FPR1. This pathway might be activated to increase GI motility in patients.

Keywords: Digestion; ERK; Microbiome; Signal Transduction.

Figure 1.. Lactobacillus rhamnosus GG (LGG) induces reactive oxygen species (ROS) and triggers p44/42 MAPK (Erk 1/2) phosphorylation in the enteric ganglia of GF mice.

GF mice were intra peritoneally injected with hydro-Cy-3 (20 uM) for 15 minutes before they were gavaged with Hank’s balanced salt solution (HBSS) or LGG @ 10¹⁰ colony forming units (cfu). After 2h, mice were sacrificed and the longitudinal muscle myenteric plexi (LMMP) from the jejunum was micro dissected and imaged by confocal microscopy. A separate piece of jejunum was cryosectioned and immunostained with antibodies for phospho-Erk 1/2 and detected with AF 488/green (pErk 1/2) and AF 594/red (Peripherin), along with DAPI as the nuclear stain. (A) Confocal images of LMMP showing ROS generation (B) Number of ROS-positive cells per ganglion in GF mice treated with HBSS and LGG. Representative confocal sections of jejunum showing (C) phospho-Erk ½ (green) and peripherin (red) in the myenteric ganglia, and (D) Graphical representation of the intensity of p-Erk (green) fluorescence in the myenteric ganglia as assessed by Image J. Magnification 40X, Scale bar 50 μm, Mean +/− SEM, n =3 per group, * P < 0.05 by t-test.

Figure 2.. Lactobacillus rhamnosus GG (LGG) induces enteric neuronal remodeling in GF mice.

GF mice were gavaged with HBSS or LGG (@ 10¹⁰ cfu) for 2 h and sacrificed. RNA was extracted from the ileum and real time PCR (q-PCR) was done to assess the changes in peripherin, Hand-2 and neuronal subtypes like ChAT, SERT, NPY, nNOS and TH. Immunostaining for ChAT was performed on cryofixed sections, and green fluorescence in the myenteric ganglia (arrows) representing ChAT immunoreactivity assessed by Image J. (A) Gene expression of Peripherin, Hand-2, and various neuronal subtypes viz ChAT, SERT, NPY, nNOS and TH. (B & B*) Representative Western blot for Hand-2, ChAT SERT and the graphical representation (C) Representative images of ChAT (green) and peripherin (red) immunostaining in the myenteric ganglia (arrows), DAPI (blue). (C*) Graphical representation of ChAT (green) staining intensity in arbitrary fluorescence units using Image J (n = 4 per group). Mean +/− SEM, n = 3 per group, * P < 0.05, ** P < 0.01, *** P < 0.001 by t-test. Magnification 40X, Scale bar B-50 μm & C-100 μm.

Figure 3:. LGG-induced redox signaling and Erk 1/2 phosphorylation in the enteric neurons is contact/adhesion-dependent.

Conventional mice received intra peritoneal injections of hydro-Cy-3 15 minutes before gavage with HBSS or LGG (@ 10¹⁰ cfu), LGGΩSpaC or LGG along with NAC (@ 150 mg/kg body weight). After 2h, mice were sacrificed, and the jejunal LMMP micro dissected for confocal imaging. The jejunal cryosections were immunostained for pErk 1/2 or total Erk 1/2 along with DAPI (nuclear stain, blue). (A) Confocal images of LMMP showing ROS production in cell bodies (arrows), (B) Representative cryosections of jejunum showing pErk (green) immunostaining, (C) p-Erk 1/2 (green) fluorescence in LMMP & (C*) submucosal plexi from mice treated with HBSS or LGG for 2 h. (D & D* ) Total Erk 1/2 (green) in the myenteric ganglia (arrows), (E & E*) Representative Western blot for pErk/Total Erk 1/2 and the graphical representation. Mean +/− SEM from 3 to 4 independent experiments (n =3 - 4 mice per group per experiment), ANOVA followed by Tukey’s test, *P < 0.05, ** P < 0.01, *** P < 0.001. Magnification 40X. Scale bar 50 μm.

Figure 3:. LGG-induced redox signaling and Erk 1/2 phosphorylation in the enteric neurons is contact/adhesion-dependent.

Conventional mice received intra peritoneal injections of hydro-Cy-3 15 minutes before gavage with HBSS or LGG (@ 10¹⁰ cfu), LGGΩSpaC or LGG along with NAC (@ 150 mg/kg body weight). After 2h, mice were sacrificed, and the jejunal LMMP micro dissected for confocal imaging. The jejunal cryosections were immunostained for pErk 1/2 or total Erk 1/2 along with DAPI (nuclear stain, blue). (A) Confocal images of LMMP showing ROS production in cell bodies (arrows), (B) Representative cryosections of jejunum showing pErk (green) immunostaining, (C) p-Erk 1/2 (green) fluorescence in LMMP & (C*) submucosal plexi from mice treated with HBSS or LGG for 2 h. (D & D* ) Total Erk 1/2 (green) in the myenteric ganglia (arrows), (E & E*) Representative Western blot for pErk/Total Erk 1/2 and the graphical representation. Mean +/− SEM from 3 to 4 independent experiments (n =3 - 4 mice per group per experiment), ANOVA followed by Tukey’s test, *P < 0.05, ** P < 0.01, *** P < 0.001. Magnification 40X. Scale bar 50 μm.

Figure 4:. LGG-induced ENS remodeling in conventional mice is sensitive to inhibition by the ROS inhibitor N-acetyl L-cysteine (NAC).

Conventional mice were gavaged with HBSS, LGG @ 10¹⁰ cfu/ml) or LGGΩSpaC (@ 10¹⁰ cfu) or LGG along with NAC (@ 150 mg/kg body weight). After 2h, the mice were sacrificed, RNA extracted from the ileum and q-PCR was performed to assess the changes in Hand-2, ChAT neurons and SERT transporter. Immunostaining for ChAT/Peripherin was performed on cryofixed sections and green fluorescence in the myenteric ganglia representing ChAT was assessed by Image J. Further LMMP were peeled from jejunum, fixed and immunostained for ChAT/DAPI. (A) q-PCR (B & B*) Representative Western blot for Hand-2, ChAT, SERT and Peripherin and the graphical representation (C) ChAT staining of jejunal cryosections with myenteric ganglia (arrows) (D) ChAT staining in jejunal LMMP and (E) Representative 3D confocal images of ChAT (green)/Peripherin (red) immunostaining in whole mouse ileum by CLARITY immunostaining. Corresponding staining data in B, C & D are also represented by histograms. Magnification 40x. Scale bar 50 um. Mean +/− SEM, n = 4 per group, ANOVA followed by Tukey’s test, * P < 0.05, *** P < 0.001.

Figure 4:. LGG-induced ENS remodeling in conventional mice is sensitive to inhibition by the ROS inhibitor N-acetyl L-cysteine (NAC).

Conventional mice were gavaged with HBSS, LGG @ 10¹⁰ cfu/ml) or LGGΩSpaC (@ 10¹⁰ cfu) or LGG along with NAC (@ 150 mg/kg body weight). After 2h, the mice were sacrificed, RNA extracted from the ileum and q-PCR was performed to assess the changes in Hand-2, ChAT neurons and SERT transporter. Immunostaining for ChAT/Peripherin was performed on cryofixed sections and green fluorescence in the myenteric ganglia representing ChAT was assessed by Image J. Further LMMP were peeled from jejunum, fixed and immunostained for ChAT/DAPI. (A) q-PCR (B & B*) Representative Western blot for Hand-2, ChAT, SERT and Peripherin and the graphical representation (C) ChAT staining of jejunal cryosections with myenteric ganglia (arrows) (D) ChAT staining in jejunal LMMP and (E) Representative 3D confocal images of ChAT (green)/Peripherin (red) immunostaining in whole mouse ileum by CLARITY immunostaining. Corresponding staining data in B, C & D are also represented by histograms. Magnification 40x. Scale bar 50 um. Mean +/− SEM, n = 4 per group, ANOVA followed by Tukey’s test, * P < 0.05, *** P < 0.001.

Figure 5.. Formyl peptide receptor (FPR1) is critical to the LGG-mediated effects of ROS activation and pErk 1/2 phosphorylation in the enteric ganglia.

Conventional mice were gavaged with HBSS or fMLF (0.5 uM/100 gm body weight). In a separate experiment, WT, FPR1 and FPR2 KO mice were gavaged with HBSS or LGG (@ 10¹⁰ cfu/ml). After 2h, mice were sacrificed and the jejunal LMMP was micro dissected and imaged for ROS. The jejunum was cryo sectioned and immunostained for pErk 1/2 and total Erk 1/2 along with DAPI (nuclear stain, blue). RNA was extracted from the ileum of WT, FPR1 and FPR2 KO mice and q-PCR was performed with primers for Peripherin, ChAT and SERT. Immunostaining for ChAT was performed by CLARITY on whole ileum. Confocal images of LMMP showing ROS production (arrows) in conventional mice gavaged HBSS or fMLF in (A), and representative sections of jejunum showing pErk (green) and Total Erk 1/2 (green) in the myenteric ganglia (arrows) in (B) Confocal images of ROS production in WT FPR1 KO and FPR2 KO mice shown in C with the corresponding histogram. Representative sections of jejunum showing pErk (green) staining in the myenteric ganglia (arrows) in WT, FPR1 KO and FPR2 KO mice in D, with the histogram showing intensity of p-Erk 1/2 (green) fluorescence. Fold change in ChAT and SERT mRNA shown in (E). Mean +/− SEM, n = 4 per group, ANOVA followed by Tukey’s test, ** P < 0.01, *** P < 0.001. Magnification 40x, Scale bar 50 μm.

Figure 5.. Formyl peptide receptor (FPR1) is critical to the LGG-mediated effects of ROS activation and pErk 1/2 phosphorylation in the enteric ganglia.

Conventional mice were gavaged with HBSS or fMLF (0.5 uM/100 gm body weight). In a separate experiment, WT, FPR1 and FPR2 KO mice were gavaged with HBSS or LGG (@ 10¹⁰ cfu/ml). After 2h, mice were sacrificed and the jejunal LMMP was micro dissected and imaged for ROS. The jejunum was cryo sectioned and immunostained for pErk 1/2 and total Erk 1/2 along with DAPI (nuclear stain, blue). RNA was extracted from the ileum of WT, FPR1 and FPR2 KO mice and q-PCR was performed with primers for Peripherin, ChAT and SERT. Immunostaining for ChAT was performed by CLARITY on whole ileum. Confocal images of LMMP showing ROS production (arrows) in conventional mice gavaged HBSS or fMLF in (A), and representative sections of jejunum showing pErk (green) and Total Erk 1/2 (green) in the myenteric ganglia (arrows) in (B) Confocal images of ROS production in WT FPR1 KO and FPR2 KO mice shown in C with the corresponding histogram. Representative sections of jejunum showing pErk (green) staining in the myenteric ganglia (arrows) in WT, FPR1 KO and FPR2 KO mice in D, with the histogram showing intensity of p-Erk 1/2 (green) fluorescence. Fold change in ChAT and SERT mRNA shown in (E). Mean +/− SEM, n = 4 per group, ANOVA followed by Tukey’s test, ** P < 0.01, *** P < 0.001. Magnification 40x, Scale bar 50 μm.

Figure 5.. Formyl peptide receptor (FPR1) is critical to the LGG-mediated effects of ROS activation and pErk 1/2 phosphorylation in the enteric ganglia.

Conventional mice were gavaged with HBSS or fMLF (0.5 uM/100 gm body weight). In a separate experiment, WT, FPR1 and FPR2 KO mice were gavaged with HBSS or LGG (@ 10¹⁰ cfu/ml). After 2h, mice were sacrificed and the jejunal LMMP was micro dissected and imaged for ROS. The jejunum was cryo sectioned and immunostained for pErk 1/2 and total Erk 1/2 along with DAPI (nuclear stain, blue). RNA was extracted from the ileum of WT, FPR1 and FPR2 KO mice and q-PCR was performed with primers for Peripherin, ChAT and SERT. Immunostaining for ChAT was performed by CLARITY on whole ileum. Confocal images of LMMP showing ROS production (arrows) in conventional mice gavaged HBSS or fMLF in (A), and representative sections of jejunum showing pErk (green) and Total Erk 1/2 (green) in the myenteric ganglia (arrows) in (B) Confocal images of ROS production in WT FPR1 KO and FPR2 KO mice shown in C with the corresponding histogram. Representative sections of jejunum showing pErk (green) staining in the myenteric ganglia (arrows) in WT, FPR1 KO and FPR2 KO mice in D, with the histogram showing intensity of p-Erk 1/2 (green) fluorescence. Fold change in ChAT and SERT mRNA shown in (E). Mean +/− SEM, n = 4 per group, ANOVA followed by Tukey’s test, ** P < 0.01, *** P < 0.001. Magnification 40x, Scale bar 50 μm.

Figure 6.. Localization of FPR1 receptors on enteric neuronal cells in mouse intestine by q PCR and Fluorescence in situ hybridization (FISH).

RNA was extracted from the myenteric plexi of jejunum, ileum and colon of mice and q-PCR performed using murine FPR1, FPR2 primers, and normalized with peripherin. Jejunal cryosections were subjected to FISH using FPR1 RNA probes along with pan neuronal marker peripherin probes. (A) Relative expression of FPR 1 & 2 in jejunum, ileum & colon (B) Localization of FPR1 on epithelial cells and enteric ganglia (white arrows), (C) Zoomed image of myenteric ganglia showing localization of FPR1 on peripherin positive neurons (D) CLARITY staining for peripherin (green) and DAPI on whole ileum and (E) 3D CLARITY image created from Z stack of image in D showing neuronal processes (green) extending into the gut lumen. Magnification 40x, Scale bar 50 um.

Figure 7:. Daily LGG gavage improves gastrointestinal motility in Conventional mice.

Conventional mice were gavaged daily with HBSS, LGG, or LGGΩSpaC @ 10¹⁰ cfu/ml for 1 or 2 weeks, and gastrointestinal motility was assessed by stool frequency (number of stool pellets per hour per mouse), total gastrointestinal (GI) transit time (time for expulsion of the first red stool pellet after gavage of the red carmine dye) stool wet and dry weights, and stool water content. A separate group of conventional mice were gavaged daily with HBSS or LGG @ 10¹⁰ cfu/ml for 2 weeks, and GI motility was assessed by isometric muscle recording from distal ileal circular muscle strips after incubation with L-Nitro-Arginine Methyl Ester (L-NAME) followed by electrical field stimulation (EFS) at 24 V, 10 Hz, 0.3 milliseconds for 20 seconds. The y axis represents force, millinewton (mN); x axis represents time. Contraction was expressed as a percentage change from baseline muscle tone. (A) Stool frequency (B) GI transit time (in minutes) C) Stool wet weight and (D) Stool water content after 1 week of LGG gavage, (E) Representative tracings from circular ileal muscle strips of mice gavaged with HBSS or LGG for 2 weeks, (F) Percent contraction. Mean +/− SEM, ANOVA followed by Tukey’s test or t test, n = 5, * P < 0.05.