E.coli JM83 Damages Mucosal Barrier Inducing Hirschsprung-Associated Enterocolitis via Activated TLR4 / NF-κB / P-p38 Signaling


 Purpose

Hirschsprung-associated enterocolitis (HAEC) is characterized by intestinal mucosal damage and unbalance of intestinal microbiota. Recent studies have shown that the TLR4/NF-κB/p-p38 signaling in the intestine is of great importance to intestinal mucosal integrity. This study aimed to investigate the role of TLR4/NF-κB/p-p38 signaling in the pathogenesis of HAEC in Escherichia coli (E. coli) JM83 infected Endothelin receptor B (Ednrb)−/− mice.
Methods

 Ednrb −/− mice were administered with E. coli JM83 by oral gavage to establish the HAEC model, mice were randomly divided into WT group, Ednrb−/− group and Ednrb−/−+ E. coli JM83 group. The role of TLR4/NF-κB/p-p38 signaling was evaluated by vivo study.
Results

The activation of the TLR4/NF-κB/p-p38 signaling induced by E. coli JM83 caused HAEC in Ednrb−/− mice, which was evidenced by a significantly increased expression of TNF-α, TGF-β and IL-10, decreased density of F-actin protein. While TLR4 knockdown improved the degree of enterocolitis and attenuated the expression of IL-10, TNF-α, TGF-β and increased the density of F-actin protein in Ednrb−/− mice after E. coli infection.
Conclusions

These results indicate that E. coli JM83 activates TLR4/NF-κB/p-p38 signaling to promote the development of HAEC. However, inhibition of this signaling may be benefit to the treatment and prevention of HAEC.


Introduction
Hirschsprung-associated enterocolitis (HAEC) is the most common complication of Hirschsprung disease (HSCR) [1,2], which occurred in the preoperative or postoperative stages even after de nitive pull-through surgery. Accumulating clinical evidence suggests that abnormalities in the intestinal microbiome, impaired intestinal mucosal barrier function, an altered systemic immune system and bacterial translocation are all possible causes of HAEC [3][4][5]. Intestinal tract is the most active immune organ in human body, which is constantly challenged by a large number of antigens. Clostridium di cile, Escherichia coli (E. coli) and certain viruses have been suggested as causative organisms of enterocolitis development [6]. The method in which gut microbes in uence the mucosal barrier and the development of pathogenic bacteria-mediated intestinal in ammatory disease remain unclear, but the de ciency of intestinal repair of in ammatory-driven injury may play an important role in the pathogenesis of HAEC.
Previous studies reported that the disorder of TLR4 signaling transduction led to the uncontrolled colitis, which was associated with the loss of mucosal integrity, development of ulcerations, colonic bleeding [7], these clinical features were in consistent with HAEC patients. Emerging evidence shows that TLR4 expression is increased in several intestinal in ammatory diseases, including in ammatory bowel colitis [8] and necrotizing enterocolitis [9]. When TLR4 is engaged by its ligands, the downstream signaling pathways, including the nuclear factor-κB (NF-κB) and MAPK p38 (p-p38) pathways, are activated; this activation is essential for the initiation of an in ammatory response by promoting and/or modulating the transcription and translation of in ammation-related genes, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-1β [10]. One literature reveals that the inhibition of NF-κB can protect the colonic epithelium from damage and/or promote epithelial repair [11,12].
We hypothesized that the pathogenic organism E. coli JM83 promoted HAEC development through TLR4/NF-κB/p-p38 signaling, in uencing intestinal mucosal barrier integrity. In this study, it was investigated whether TLR4/NF-κB/p-p38 signaling participated in the pathogenesis of HAEC during the invasive infection of Ednrb −/− mice with E. coli JM83 (used as an HAEC model).

Animals
Wild-type (WT) (n=20) and Ednrb ex3/ ex3 (n=8) mice were purchased from the Institute of Model Animals of Wuhan University (Wuhan, China). Endothelin receptor B-null (Ednrb ex3/ ex3 ) was used on the C57/B6J background. Brie y, after mating Ednrb ex3/+ mice with Ednrb ex3/ ex3 , the homozygotes (referred to herein as Ednrb −/− ) were easily distinguished from the WT and heterozygote littermates (referred to herein as Ednrb +/+ and Ednrb +/− , respectively) by the white fur color and gradually enlarging abdomens (due to absence of ganglion cells at the end of rectum). Ednrb +/+ and Ednrb +/− were normal phenotypes and didn't develope aganglionosis. Therefore, 20 Ednrb +/+ or Ednrb +/ − animals were randomly assigned to WT group. Total 20 Ednrb −/− animals were obtained in this study which displayed distal colonic aganglionosis involving 5-10 mm of the colon. Naive mice were de ned as WT and Ednrb −/− mice without any intervention. According to the manufacturer's protocols. Brie y, after anesthetized with ether (2-4 %), a 2 mm diameter anal canal was inserted into the anus of the experimental mice, the 5 mice were randomly selected form WT and Ednrb −/− groups were transfected with small interfering RNA (siRNA) duplexes (100 nM) into colon targeting TLR4. TLR4 forward, sense strand: 5'-UUCGAGACUGGACAAGCC -3' and an antisense strand: 5'-UGGCUUGUCCAGUCACGA -3' (20 nM; Guangzhou RiboBio Co., Ltd.) to generate WT + TLR4 siRNA (n=5) and Ednrb −/− + TLR4 siRNA(n=5) mice groups [13]. The knockdown of TLR4 gene was con rmed at protein level by western blot. The whole experiment lasted for 40 days after mice birth, all experiments involving animals were performed in a speci c pathogen-free environment, in accordance with the Zunyi Medical University (Zunyi, China) Guidelines for Animal Care. Animals were kept under a 12-h light/dark cycle with access to food and water.

Ethic Statements
All animal experimental protocols were complied with the Guide for the Care and Use of Laboratory

Establishment Of Haec Model
The colon samples from Ednrb −/− + E. coli mice were stored in 4% buffered formalin and embedded with para n for subsequent hematoxylin and eosin and immuno uorescence staining analysis to verify the HAEC model. In ammatory cell in ltration of the crypts (cryptitis and crypt abscesses) was lighter in HAEC mice than in human beings. The severity of HAEC was evaluated according to the modi ed grading system reported by Porokuokka et al [14] to recently re ect HAEC mice epithelial pathology. Parts of the small bowel (jejunum and ileum) or large bowel (cecum and colon) were excised by separation from the mesentery to prepare a single intestinal cell suspension.

Hematoxylin &eosin (He) And Immunohistochemistry (Ihc)
The colon sections were removed following euthanasia and cut to a thickness of 3 mm, stained with HE and imaged by light microscopy (Nikon Corporation, magni cation, x20). The stained section assigned an in ammation score in a blinded manner, as previously described [14]. TLR4 protein expression levels were detected by IHC. The IHC sections were incubated pass the night at 4˚C in primary antibody solution containing anti-human TLR4 antibody (cat. no. A0456; dilution, 2 µg/l, 1:200; OriGene Technologies, Inc.) and a biotin-streptavidin HRP detection system for 12 h, followed by detection with HRP-conjugated goat anti-rabbit IgG secondary antibody (cat. no. 2019629, 1:200; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). Negative controls were treated with PBS instead of primary antibodies. All sections were observed by an optical microscope (OLYMPUS BH-2; Olympus Corporation, magni cation, x100).

Immuno uorescence
For immuno uorescence staining, colon tissue was separated from mice. Colon tissue was xed in PBS/4% PFA and 10% sucrose solution at 4˚C for 1 h, followed by overnight cryoprotection in PBS/30% sucrose (cat no. 7124; Merck KGaA) for 3 days at 4˚C. Tissue sections were cut into 20-mm sections using a Leica Cryostat Microtome and blocked using a Streptavidin/Biotin Blocking kit (Vector Laboratories, Inc.) and stored at -80˚C until processing. Colon tissue samples were stained with either mouse or rabbit anti-F-actin (cat. no.8927, 1:2,000; Merck KGaA), with the addition of DAPI (cat no. 6982; BioLegend, Inc.). Sections were mounted in a FV1000 laser-scanning confocal microscope (Olympus Corporation, magni cation, x20).

Western Blot Analysis
Western blot analysis was performed following the manufacturer's protocol and concentration were measured by the Enhanced Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). Forty µl Proteins were loaded in each well. extracts (120 µl) were mixed with SDS-PAGE (Beyotime Institute of Biotechnology), heated at 90˚C for 5 min. And then transferred to polyvinylidene uoride membranes. The membranes were blocked with 5% fat-free milk in Tris-buffered saline for 45 min at room temperature. The membrane was the wash with TBST, and then incubated with primary rabbit anti-mouse antibodies anti-TLR4 (1: Table 1). The qPCR cycle program was: initial denaturation 50˚C for 2 min, 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 60 sec. Data analysis was calculated by the 2 −ΔΔCq method.  Normally distributed data were presented as the mean ± standard deviation (SD). And statistical analysis were performed using Student's t-test and one-way ANOVA test. Statistical comparisons of two or more groups with two independent variables were analyzed by two-way ANOVA and a Bonferroni's post-hoc test. P value <0.05 was considered statistically signi cant.
We employed the endothelin receptor B targeted-null mouse (Figure 1a, 1b) to exhibit the pathological features of HSCR, such as aganglionosis in the rectum and distal colon, as well as enterocolitis [13]. First, WT and Ednrb −/− mice were infected with E. coli JM83, and in ammation scores in the colon were histologically assessed. As shown in Figure 2, naive Ednrb −/− mice developed enterocolitis spontaneously at 5 weeks (Figure 2c), whereas Ednrb −/− mice infected with E. coli succumbed to lethal enterocolitis at 3 weeks (Figure 2d). By contrast, WT mice infected with E. coli only developed mild in ammation in the colon (Figure 2b).

TLR4 knockdown reverses intestinal in ammation
To determine the importance of TLR4/NF-κB/p-p38signaling in E. coli JM83 infection-induced HAEC, the severity of enterocolitis and cytoskeletal F-actin expression, a critical structure for preserving the integrity of the epithelial barrier, was assessed by TLR4 knockdown in WT and Ednrb -/mice. A marked interruption in mucosal structures was observed, and large numbers of in ammatory cells and abscesses were found to have in ltrated the mucosa and sub-mucosa upon E. coli infection in Ednrb -/mice (Figure 3c), whereas the severity of E. coli infection-induced enterocolitis was markedly alleviated in Ednrb -/mice following TLR4 knockdown (Figure 3d). In addition, the mild degree of in ammation in the colon observed in E. coliinfected WT mice was almost completely reversed by TLR4 knockdown (Figure 3b).
F-actin expression was increased in the cytoplasm of intestinal epithelial cells, alongside increased tight junction integrity in the intestinal mucosal barrier in both WT and TLR4 siRNA-transfected WT mice 3 weeks after E. coli infection (Figure 4a, 4b). By contrast, Ednrb -/mice exhibited a substantially decreased density of F-actin protein and severely disordered tight junction structures in response to E. coli infection ( Figure 4c). Of note, TLR4 knockdown in Ednrb -/mice gradually increased the density of F-actin and partly reversed tight junction integrity (Figure 4d).

Discussion
HAEC can occur at any time before, during and after endorectal pull-through surgery, which is the de nitive procedure for HSCR [15]. There exists a wide variation in the reported incidence of HAEC, which occurs in 2-33% of patients with common type and 50% of patients with long type HSCR. Clinically, HAEC is characterized by discomfort, decreased appetite, abdominal distention, loose foul-smelling stools, fever and sepsis [16]. Previous studies tended to believe that the postoperative HAEC were related to surgical factors, such as anastomotic stricture or leak, and bowel obstructions [1,4,[17][18][19][20]. At present, many results have indicated that the pathogenesis of HAEC are related to mucosal barrier, intestinal microbiota, and immune function [21][22][23]. At least partly due to the widely use of Ednrb −/− animal in HAEC researches, the order in which these HAEC-related etiology features change is gradually understood, which has promoted improvements in the treatment and prevention of HAEC [24]. A previous multicenter study has identi ed that the HAEC patients showed reduced abundance of the phyla Firmicutes and increased Bacteroidetes and Proteobacteria, by comparing the bacterial microbiome composition of children with HSCR to those who had a history of HAEC [25]. These results strongly indicate a dysequilibrium in the gut microbial ecosystem of HAEC patients, such that the dominance of bacteria (E. coli) predisposes patients to the development of HAEC.
In this study, E. coli JM83 was used as the pathogenic bacteria to infect the intestine of Ednrb −/− mice to establish an HAEC mouse model. A previous study reported that Ednrb −/− mice developed HAEC on postnatal days 24-26 and 100% mortality was recorded by day 28 after birth [26]. Clinical histopathologic features of HAEC patients mainly include the colon crypt dilatation, mucin retention, enterocyte adherence of bacteria, epithelial damage, leukocyte in ltration, ulceration, and in the terminal stages, transmural necrosis and perforation [27,28]. In our study, the histopathological results showed that a number of in ammatory cells in ltrated the mucosa and submucosa of the intestinal wall, even abscesses were observed in E. coli JM83-infected Ednrb −/− mice, which was in accordance with the human manifestation.
Ednrb −/− mice developed HAEC 3 weeks after E. coli JM83 infection, with a few mice dying of abdominal distention, diarrhea and dehydration.
A study showed the passive transport of mucosal barrier in Ednrb −/− mice, the E. coli transport was signi cantly reduced in proximal colon rather than in the distal colon [29]. Previous studies showed that the dysfunction of intestinal epithelial contributed to the reduction in expression and changes in distributions of these F-actin, in uencing the barrier function and increasing permeability [30,31]. The expression of the F-actin gradually decreased with activating TLR4/NF-κB/p-p38 signaling, and therefore eventually led to intestinal mucosal damage. Studies reported that TLR4 activated by bacteria may be a major mediator of activating intestinal mucosal immunity, advancement of intestinal in ammation and immune response promotion [32]. Therefore, our results match the fact that HAEC may happen even after the postoperative pull-through surgery.
TLR4/NF-κB/p-p38 signaling pathway is transmitted through adaptor proteins, and signaling through MyD88 may be necessary to drive phagocytosis [8, 33,34]. Studies have shown that the main function of TLR4 signaling in macrophages is to induce in ammatory response and protect host from pathogenic bacteria [35]. Whereas, the In vivo experiments of the present study revealed that the TLR4 protein receptors accumulated in the colon for 3 weeks following stimulation with E. coli JM83. TLR4 stimulated NF-κB through MyD88 in a mice model, and the level of NF-κB and p-p38/p38 was increased in the colon wall following stimulation with E. coli JM83. Likewise, NF-κB induced TNF-α and TGF-β increased with the degree of enterocolitis in Ednrb −/− mice, and therefore eventually led to intestinal mucosal damage.
The above results indicate that TLR4/NF-κB/p-p38 signaling pathway plays a central role in the initiation of innate cellular immune responses, in the development of subsequent adaptive immune responses to invading bacterial infection, and eventually promoting intestinal mucosal tissue damage in HAEC. This process is consistent with the pathogenesis of IBD in past adult studies [36]. By contrast, the mucosal barrier integrity was maintained without the development of enterocolitis following TLR4 knockdown. Former studies have indicated that enhanced TLR4 expression is related to mortality in sepsis model [37].
However, siRNA-transfected TLR4/NF-κB/p-p38 signaling could reverse the in ammatory effects caused by E. coli infection, indicating that TLR4/NF-κB/p-p38 signaling plays a central role in maintaining the balance of gut homeostasis during the pathogenesis of HAEC. Under certain conditions, this downregulation of TLR4 signaling would ameliorate the degree of immune-mediated enterocolitis, provides a new idea for the treatment and prevention of HAEC.
In conclusion, the present study highlights the response of the intestinal mucosal barrier to HAEC induced by the pathogenic bacteria of E. coli. In addition, the activation of TLR4/NF-κB/p-p38 signaling in Ednrb -/mice by E. coli JM83 lead to the development of in ammation, which is shown to be an underlying mechanism. Furthermore, inhibition of TLR4/NF-κB/p-p38 signaling may be bene t to the treatment and prevention of HAEC, contributing to the improvement of intestinal mucosal integrity.