Immunopharmacology and Inflammation
Inhibition of NF-κB by a PXR-dependent pathway mediates counter-regulatory activities of rifaximin on innate immunity in intestinal epithelial cells

https://doi.org/10.1016/j.ejphar.2011.06.058Get rights and content

Abstract

A dysregulated interaction between intestinal epithelial cells (IEC) and components of innate immunity is a hallmark of inflammatory bowel diseases. Rifaximin is a poorly absorbed oral antimicrobial agent increasingly used in the treatment of inflammatory bowel diseases that has been demonstrated to act as a gut-specific ligand for the human nuclear receptor pregnane-X receptor (PXR). In the present study we investigated, whether activation of PXR in IEC by rifaximin, emanates counter-regulatory signals and modulates the expression of cytokines or chemokines mechanistically involved in dysregulated intestinal immune homeostasis documented in inflammatory bowel diseases. Our results demonstrate that primary IEC express PXR that regulate the pattern of cytokines and chemokines expressed. PXR silencing decreases TGF-β and IP-10 while increases the expression of TNF-α, IL-8, Rantes and increase the production of PGE2. This pattern is further exacerbated by treating anti-PXR siRNA cells with bacterial endotoxin (LPS). Exposure to rifaximin caused a robust attenuation of generation of inflammatory mediators caused by LPS and increased the generation of TGF-β. PXR silencing completely abrogated these anti-inflammatory effects of rifaximin. By Western blot analysis we found that rifaximin abrogates the binding of NF-κB caused by LPS. Finally, exposure of human colon biopsies from inflammatory bowel diseases patients to rifaximin reduced mRNA levels of IL-8, Rantes, MIP-3α and TNFα induced by LPS. Collectively, these data establish that rifaximin exerts counter-regulatory activities at the interface between enteric bacteria and intestinal epithelial cells. The ability of rifaximin to activate PXR contributes to the maintenance of the intestinal immune homeostasis.

Introduction

The gut mucosa and associated microbiota are key elements in the maintenance of the steady-state equilibrium essential for gut physiology and protection against damage and invasion. The homeostatic control of gut interaction with beneficial bacteria occurs in an actively controlled non-inflammatory environment, dictated both by signals from the microbiota to intestinal epithelial cells (IEC) and by IEC-conditioning components of the immune system (Artis, 2008). IEC are a dynamic partner in mucosal immune responses through the regulated production of chemokines, cytokines, growth factors, and antimicrobial molecules essential to mucosal inflammation and host defense (Artis, 2008). The inflammatory bowel diseases are a family of self destructive disorders of intestine for which defects in barrier and immunological functions of IEC play a pathogenic role (Papadakis and Targan, 2000, Podolsky, 1999). IEC secrete many mediators such as cytokines or chemokines in response to bacterial triggering, in part via the Toll like receptor (TLR)-4 and NF-kappa B (NF-κB) pathway (Hornef et al., 2002).

Several chemokines are known to be prominently regulated by inflammatory mediators and have dysregulated expression patterns in inflammatory bowel diseases as well as colorectal carcinoma (Papadakis and Targan, 2000). In chronic intestinal inflammation, expression of TLR4 on IEC is abnormal and could contribute to the sustained mucosal inflammation in inflammatory bowel diseases (Cario and Podolsky, 2000). Avoiding inappropriate activation of TLR4 via NF-κB inhibition, during inflammatory bowel diseases, could be crucial in the gastrointestinal tract, where epithelial cells are in constant contact with a dense and complex milieu of commensal microorganisms. At least 27 nuclear receptors (NRs) are expressed in the colon and intestine (Bookout et al., 2006) including steroid and xenobiotic receptor (SXR), also known as a pregnane-X-receptor (PXR), a transcription factor important for xenobiotic metabolism. Rifaximin, is a broad-spectrum semi-synthetic rifamycin derivative that shows good antimicrobial activity and negligibly absorbed after oral administration (Hoover et al., 1993, Jiang et al., 2010, Scarpignato and Pelosini, 2006). Controlled trials have shown that rifaximin is safe and effective for treating bacterial intestinal infections (Jiang and DuPont, 2005, DuPont et al., 2007), irritable bowel syndrome (Pimentel et al. 2011) and hepatic encephalopathy (Bass et al. 2010). Results from recent clinical studies have shown that rifaximin at the dose of 800 mg bid, is effective in inducing clinical remission of moderately active Crohn's disease (Prantera et al., 2006, Prantera et al., 2010). Recent studies have shown that rifaximin acts as a gut-specific ligand for human PXR (Ma et al. 2007). We have previously shown that rifaximin could contribute to the maintenance of the intestinal barrier integrity by regulating the metabolism of xenobiotics by increasing the expression and activity of PXR and PXR-regulated genes (Mencarelli et al., 2010). The preventive and therapeutic role of rifaximin has been demonstrated in rodent models using transgenic mice that express the human PXR (Cheng et al., 2010).

Here we have demonstrated that PXR is an essential component of the network of genes that regulates intestinal innate immunity. Activation of PXR by rifaximin in human IEC contributes to the regulation of innate immunity responses by IEC.

Section snippets

Material

Rifaximin-α (Alfa Wassermann, Bologna, Italy), batch number 2008001312, was dissolved in DMSO at the final concentration of 10 mM and subsequently serial dilutions were made each time in complete medium. Control cells were treated with DMSO alone. The final concentration of DMSO was 0.5%. TNFα was from Invitrogen (Milan, Italy) while all other reagents were from Sigma-Aldrich (Milan, Italy). PGE2 assay kit was from Cayman Chemical Company (Michigan, USA).

Cells treatment

The experiments were performed in primary

Evaluation of PXR expression in naive intestinal epithelial cells and PXR silencing

We have first evaluated the expression of PXR in CRL-1831, a normal human colon epithelial cell line and compared it to HepG2, a hepatocarcinoma cell line that expresses high levels of PXR. Results shown in Fig. 1A and B, demonstrate that colon epithelial cells express PXR mRNA, even if its expression was lower than that of HepG2 cells. To explore the mechanistic involvement of PXR in IEC immune regulation, PXR gene expression was silenced by an anti-PXR small interfering RNA (siRNA). As

Discussion

In the present study we have provided evidence that rifaximin counter-regulates effector functions of IEC triggered by engagement of TLR4 by bacterial endotoxin. It is well established that pattern-recognition molecules, such as the TLR4, “sense” the luminal content and become activated in response to change in the composition of intestinal lumen or epithelial damage (Franchimont et al., 2004). TLR4 responds to LPS by regulating the expression/activity of NF-κB, a master regulator of

Contributors

AM participated in the design of the study, contributed in cell culture experiments, data analysis and in drafting the manuscript. MB contributed to the interpretation of data, drafting and critical revision of the manuscript. CD and GP contributed to RT-PCR data in colon biopsy. BR contributed to RT-PCR co-IP and EMSA data in cells. ED and FB participated in the data interpretation and manuscript drafting. PR provided colon biopsies. SF performed conception and design of the study, contributed

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