Immune/Inflammatory Response and Hypocontractility of Rabbit Colonic Smooth Muscle After TNBS-Induced Colitis

Abstract

Background: The contractility of colonic smooth muscle is dysregulated due to immune/inflammatory responses in inflammatory bowel diseases. Inflammation in vitro induces up-regulation of regulator of G-protein signaling 4 (RGS4) expression in colonic smooth muscle cells.

Aims: To characterize the immune/inflammatory responses and RGS4 expression pattern in colonic smooth muscle after induction of colitis.

Methods: Colitis was induced in rabbits by intrarectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Innate/adaptive immune response RT-qPCR array was performed using colonic circular muscle strips. At 1-9 weeks after colonic intramuscular microinjection of lentivirus, the distal and proximal colons were collected, and muscle strips and dispersed muscle cells were prepared from circular muscle layer. Expression levels of RGS4 and NFκB signaling components were determined by Western blot analysis. The biological consequences of RGS4 knockdown were assessed by measurement of muscle contraction and phospholipase C (PLC)-β activity in response to acetylcholine (ACh).

Results: Contraction in response to ACh was significantly inhibited in the inflamed colonic circular smooth muscle cells. RGS4, IL-1, IL-6, IL-8, CCL3, CD1D, and ITGB2 were significantly up-regulated, while IL-18, CXCR4, CD86, and C3 were significantly down-regulated in the inflamed muscle strips. RGS4 protein expression in the inflamed smooth muscles was dramatically increased. RGS4 stable knockdown in vivo augmented ACh-stimulated PLC-β activity and contraction in colonic smooth muscle cells.

Conclusion: Inflamed smooth muscle exhibits up-regulation of IL-1-related signaling components, Th1 cytokines and RGS4, and inhibition of contraction. Stable knockdown of endogenous RGS4 in colonic smooth muscle increases PLC-β activity and contractile responses.

Keywords: Colitis; Rabbit; Regulators of G-protein signaling; Smooth muscle cells; shRNA.

Fig. 1

Representative macroscopic and histopathological changes in rabbit colon at 5 days after intrarectal instillation of ethanol (a, c, e) or TNBS (b, d, f). a–d Macroscopic examination showing mucosal ulceration, hyperemia, bleeding, inflammation, and wall thickening (white arrow) in distal colon after TNBS. A piece of tissue was missing due to quick snap frozen for RNA and protein extraction. The inflamed region was shown in the entire colon. e, f Hematoxylin/eosin staining showing extensive mucosal damage, crypt loss, smooth muscle thickening, and intensive inflammatory infiltration in distal colon after TNBS (f) in contrast to ethanol control (e). cm and lm circular and longitudinal muscles, mu mucosal layers, smm submucosal muscles. Scale bars 50 μm

Fig. 2

Statistically significant changes in mRNA expression of IL-1 signaling, Th1 cytokines, and inflammatory mediators in colonic smooth muscle after rabbit TNBS-induced colitis. a The 3D profile graphs showing the fold difference in expression of each gene between the TNBP and ethanol groups in the 96-well format of the PCR array. Columns pointing up (with z-axis values >1) indicate an up-regulation of gene expression, and columns pointing down (with z-axis values <1) indicate a down-regulation of gene expression in the TNBS group relative to the ethanol group. b The Volcano plot graphs the log2 of the fold change in each gene’s expression between the samples versus its p value from the Student’s t test. The black vertical line indicates fold changes of 1. The pink vertical lines the 3-fold changes in gene expression. The blue horizontal line indicates p < 0.05 by unpaired two-tailed Student’s t test. c, d Diagram illustration of representative genes using relative gene expression (2^ΛC_^t) for statistical analysis by the Student’s t test. *p < 0.05 indicates significant changes of each gene at day 5 (n = 5) and day 7 (n = 3) after TNBS treatment as compared with the corresponding ethanol control

Fig. 3

Inhibition of colonic smooth muscle contraction (a) and up-regulation of RGS4 mRNA (b) and protein (c) 5 days after rabbit TNBS-induced colitis. a Contraction of freshly dispersed colonic circular smooth muscle cells (SMCs) after treatment (30 s) with acetylcholine (0.1 μM) plus M2 receptor antagonist methoctramine (0.1 μM) was measured by scanning microscopy. Contraction was expressed as percent decrease in cell length from before acetylcholine treatment. b, c The levels of RGS4 mRNA and protein in colonic circular muscle strips were determined by RT-qPCR and Western blot, respectively. GAPDH was used as internal normalization control. Values are mean ± SE of 3–4 experiments. **p < 0.01 indicates significant difference from corresponding vehicle control. The number in c indicates individual animal. The ratio indicates the relative density over corresponding GAPDH. The fold change represents the average expression levels in TNBS group over the average levels in ethanol group

Fig. 4

Effective knockdown of endogenous RGS4 in rabbit colonic smooth muscles after colonic intramuscular microinjection of RGS4A shRNA-expressing lentivirus. a–d Direct microscopic observation of EGFP (green) showing efficient transduction of lentivirus in circular SMCs (arrows) and myenteric plexus (mp, stars) at 2 (a, c) and 4 weeks (b, d) after infection. Scale bars 40 μm. The cm and lm indicate circular and longitudinal smooth muscle layers, respectively. The nucleus is stained with DAI (blue). e, f RT-qPCR and Western blot analysis at 2 weeks after intramuscular infection showing dramatic reduction of RGS4 mRNA and protein by RGS4 shRNA in circular muscle strips of distal colon. Values are mean ± SE of 3 experiments. *p < 0.05 indicates significant difference from corresponding empty vector control

Fig. 5

Significant increase in acetylcholine-stimulated PLC-β activation (a) and initial contraction (b) in the colonic SMCs of distal colon after colonic intramuscular microinjection of RGS4 shRNA-expressing lentivirus. SMCs were isolated from circular colonic muscle strips of distal and proximal colon, labeled with myo-[³H]inositol in serum-free medium for 1 day. The hydrolysis of the phosphoinositide (PI) was stimulated with ACh (0.1 μM) plus M2 receptor antagonist methoctramine (0.1 μM) or DPDPE (1 μM, a specific δ-opioid receptor agonist) for 0.5 min. The level of [³H]inositol phosphate was determined with liquid scintillation and expressed as count per minute (cpm). The dose-dependent contractile responses at 30 s after stimulation with ACh in the presence of M2 receptor antagonist methoctramine (0.1 μM) was measured by scanning microscopy. Contraction was expressed as the percent decrease in cell length from before ACh treatment. Values are mean ± SE of 3 experiments. ^#p < 0.05 indicates a significant increase in ACh-induced initial contraction compared with the corresponding empty shRNA vector control. Values are mean ± SE of 3 experiments. *p < 0.05 indicates a significant increase in agonist-stimulated PLC-β activity compared with vehicle control. ^#p < 0.05 indicates a significant increase in ACh-induced PLC-β activity compared with the corresponding empty shRNA vector control

Fig. 6

Up-regulation of the classical IKK2/p65 pathway of NFκB activation in inflamed colonic circular smooth muscles. a Western blot analysis was performed with antibodies against indicated component of classical and non-classical NFκB signaling. The number indicates individual animal. The ratio indicates the relative density over corresponding GAPDH. b The fold changes represent the average expression levels in TNBS group over the average levels in control–ethanol group. *p <0.05 indicates a statistical significance by one-tailed Student’s t test