Administration of Protein Kinase D1 Induces a Protective Effect on Lipopolysaccharide-Induced Intestinal Inflammation in a Co-Culture Model of Intestinal Epithelial Caco-2 Cells and RAW264.7 Macrophage Cells.

Ditte Søvsø Gundelund Nielsen,Vibeke Andersen,Stig Purup,Marlene Fredborg

doi:10.1155/2017/9273640

Abstract

Inflammatory bowel diseases (IBD) are chronic inflammatory diseases involving all or part of the gastrointestinal tract. The stress-activated serine-threonine protein kinase D1 (PKD1) protein has previously been implicated in intestinal immune regulation. The objective of this study was to evaluate the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction of inflammation induced downregulation of TNF-α expression in RAW264.7 cells. In addition, PKD1 administered for 3 h prior to LPS stimulation reduced the subsequent inflammatory response through downregulation of TNF-α, IL-1β, and IL-6 in RAW264.7 cells. These results demonstrate a potential role of PKD1 in the intercellular communication between intestinal epithelial and immune cells, proposing a protective effect of PKD1 on the induction of an inflammatory response in macrophages, an important aspect during the pathogenesis of IBD.

Highlights

Inflammatory bowel diseases (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC) are characterized by an impaired intestinal barrier function and enhanced intestinal inflammation
Various concentrations of protein kinase D1 (PKD1) protein were administered apically to Caco-2 cells either before or after stimulation of RAW264.7 cells with LPS. This enabled us to study the ability of PKD1 to ameliorate an already established inflammatory response as well as the ability to suppress initiation of an inflammatory response subsequently induced by LPS
We observed LPS-induced secretion of TNF-α (11.5 ng/ml) in RAW264.7 cells when RAW264.7 cells were stimulated with 1 μg/ml of LPS (E. coli O127, Figure 2)

Summary

Introduction

IBD including Crohn’s disease (CD) and ulcerative colitis (UC) are characterized by an impaired intestinal barrier function and enhanced intestinal inflammation. An impaired intestinal barrier function is associated with disease severity in IBD patients [2] as microbes infiltrate the underlying tissue, inducing inappropriate and excessive activation of the mucosal immune system [3]. Reducing inflammation is of utmost importance in IBD patients and proinflammatory mediators such as IL-6 and TNF-α are enhanced in both CD and UC [5]. The immune-regulatory nuclear transcription factor-kappaB (NF-κB) is strongly activated in IECs and macrophages during the course of IBD, augmenting the secretion of proinflammatory cytokines as TNF-α, IL-1, IL-6, IL-12, and IL-23 [6]. Beneficial effects of the proinflammatory cytokines such as IL-1β, IL-6, and TNFα are observed in relation to both inflammation and reepithelialization of wounded intestinal tissue, emphasizing

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