Inhibition of Salmonella-induced IL-8 synthesis and expression of Hsp70 in enterocyte-like Caco-2 cells after exposure to non-starter lactobacilli

Abstract

Oral administration of lactobacilli as probiotics is gaining importance in the treatment of intestinal inflammations. We investigated the effect of non-starter lactobacilli Lactobacillus casei subsp casei 2756, Lactobacillus curvatus 2775, and Lactobacillus plantarum 2142 as well as their spent culture supernatants (SCS) on Salmonella enteritidis 857 growth, interleukin (IL)-8 and heat shock protein 70 (Hsp70) synthesis in undifferentiated crypt-like and differentiated villus-like Caco-2 cells. The cells were infected with graded numbers of non-starter lactobacilli or S. enteritidis 857 for 1 h and allowed to recover for 24 h or exposed to 200 bacteria/cell for 1 h and allowed to recover for different periods of time. In another experiment S. enteritidis 857 was first pre-treated with SCS-lactobacilli for 1 h before infecting the cells. The levels of IL-8 and Hsp70 were assessed using sandwich ELISA and immunostaining of Western blots, respectively. The effect of SCS-lactobacilli on S. enteritidis 857 growth was evaluated by agar plate diffusion test.

The non-starter lactobacilli induced a significant increase in the levels of both IL-8 and Hsp70. However, compared with the S. enteritidis 857 induced IL-8 synthesis, the levels of IL-8 induced by the lactobacilli at any equivalent bacterial number were far lower. After exposure of Caco-2 cells to S. enteritidis 857 pre-treated with SCS-lactobacilli, it appeared that their SCS inhibited the S. enteritidis 857 growth and IL-8 synthesis and in addition induced the expression of Hsp70. The differences in response of crypt- and villus-like Caco-2 cells are merely a reflection of their differentiation status.

Our data suggest that the beneficial effect of non-starter lactobacilli to the intestinal inflammations might be associated with a decrease of the IL-8 levels. This effect could be mediated, at least in part, by the bacteria themselves or via a secreted antimicrobial product(s) either directly against the pathogens or indirectly through the synthesis of Hsp70.

Introduction

Lactobacillus casei subsp casei 2756, L. curvatus 2775, and L. plantarum 2142 are non-starter lactic acid bacteria that constitute considerably the microflora of mature cheese. Their inclusion in cheese manufacturing increases the level of free amino acids, peptides, and free fatty acids, which leads to enhanced flavour intensity and accelerates cheese ripening (Corsettti et al., 1998, Franklin and Sharpe, 1963, McSweeney et al., 1993). A considerable number of studies on non-starter lactic acid bacteria have focused on the effect of the microbes to the dairy products with little or no emphasis on the consumer. It has been shown however, that several Lactobacillus strains including L. casei subsp casei and L. plantarum exert a positive effect upon the consumer following oral administration. They improve the intestinal microbial balance, confer protection against potential enteropathogenic bacteria, and prevent and cure intestinal diseases (Fuller, 1989, Brassart and Schiffrin, 1997, Sugita and Togawa, 1994, Campieri et al., 2000, Gionchetti et al., 2000). These effects are mediated through production of various acids, hydrogen peroxide or bacteriocins, competition for nutrients or adhesion receptors, and anti-toxin actions. The lactobacilli have also been shown to stimulate the immune system and regulate cytokine production. They suppress synthesis of interleukin (IL)-8, transforming growth factor (TGF-β), and tumour necrosis factor (TNF-α) by the intestinal epithelial cells (Reid et al., 2001, Wallace et al., 2003). Further, when administered intravenously, lactobacilli cultivation products induce the expression of a putative protective protein (Hsp70) in the rat heart that subsequently protects the heart against ischaemia and reperfusion tachyarrhythmia (Oxman et al., 2000). Since heat shock proteins suppress the synthesis of inflammatory cytokines like IL-8 (Malago et al., 2002) and lactobacilli induce these proteins in the rat heart, it is reasonable to suggest that lactobacilli also exert a beneficial effect upon the gut following oral uptake.

Many intestinal pathogens including S. enteritidis 857 induce an instant innate immune response following their invasion. This localized response involves the rapid expression and up-regulation of an array of pro-inflammatory cytokines, predominantly IL-8. The IL-8 attracts and directs neutrophil granulocytes to the site of inflammation, a response that is vital to the inflammatory diarrhoea caused by Salmonella infection (McCormick et al., 1998, Lee et al., 2000, Greenberg et al., 2002). Although this response is triggered to eliminate the pathogen, the persistent production of IL-8 often causes chronic inflammation that usually leads to tissue damage. Such an inflammation is characterised by high levels of IL-8 and is observed in several intestinal disorders like ulcerative colitis, pouchitis, and Crohn's disease (Hecht and Savkovic, 1997, Hata et al., 2001). Interventions that decrease these levels have shown to significantly alleviate the conditions (Harig et al., 1989, O'Morain et al., 1982, Sanderson, 1997, Casellas et al., 1998). Although some lactobacilli inhibit IL-8 production by intestinal epithelial cells and some are implicated in the treatment and prevention of such intestinal diseases (Campieri et al., 2000, Gionchetti et al., 2000), the direct link between suppression of IL-8 and alleviation of these diseases imparted by lactobacilli has not been established. Furthermore, the capacity of lactobacilli to induce Hsp70 that is known to inhibit IL-8 production by intestinal epithelial cells (Oxman et al., 2000, Malago et al., 2002), suggests a positive correlation between induction of Hsps, inhibition of IL-8 and improvement of intestinal disorders in the gut inhabited by lactobacilli. It is therefore compelling to hypothesize that part of the mechanism imparted by lactobacilli to the welfare of the intestinal epithelium involves regulation of IL-8 production either direct or indirect via induction of Hsp synthesis.

To gain more insight into the role of probiotics to the infected gut, we evaluated the positive effect of non-starter lactobacilli to the intestinal epithelium against the pathogenic S. enteritidis 857. This bacterium induces synthesis of IL-8 and subsequent intestinal inflammations manifested as diarrhoeagenic illnesses in human (McCormick et al., 1998, Lee et al., 2000, Greenberg et al., 2002). We first assessed whether the non-starter lactobacilli L. casei subsp casei 2756, L. curvatus 2775, and L. plantarum 2142 and their microbial products can alter the S. enteritidis 857 induced IL-8 production. Secondly, we investigated whether these bacteria and their microbial products induce the expression of Hsp70 that can account for their beneficial effects to the gut. We used enterocyte-like Caco-2 cells, an in vitro model of the human intestinal epithelium that differentiate in culture and acquire characteristics both structurally and functionally of either crypt cells (5-day-old Caco-2 cells) or villus cells (19-day-old Caco-2 cells) of the small intestine (Koninkx et al., 1992, Koninkx, 1995, Ovelgönne et al., 2000, Malago et al., 2003).