Effect of Vibrio Cholerae on Chemokine Gene Expression in HT29 Cells and Its Modulation By
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BASIC IMMUNOLOGY doi: 10.1111/j.1365-3083.2008.02214.x .................................................................................................................................................................. CORE Effect of Vibrio cholerae on Chemokine Gene Expression in HT29 cells and its Modulation by brought to you by Publications of the IAS Fellows Lactobacillus GG N. S. Nandakumar, S. Pugazhendhi, K. Madhu Mohan, K. Jayakanthan & B. S. Ramakrishna provided by Abstract Department of Gastrointestinal Sciences, Epithelial cells participate in the innate immune response to pathogenic bacte- Christian Medical College, Vellore, India ria by elaborating chemokines. This study examined the effect of Vibrio cholerae and Lactobacillus rhamnosus GG on inflammatory chemokine gene expression in the HT29 human intestinal epithelial cell line. HT29 cells were exposed to Received 27 July 2008; Accepted in revised form 3 November 2008 V. cholerae 0139, Lactobacillus or both for 2 h and cultured further thereafter for 4 h. RNA was extracted from the cells and expression of genes for chemokines Correspondence to: Dr B. S. Ramakrishna, and related molecules was quantitated by real time PCR using a pathway- Department of Gastrointestinal Sciences, focused PCR array. TLR4 was silenced using shRNA and output of interleu- Christian Medical College, Vellore 632 004, kin-8 (IL-8) into the media quantitated with and without V. cholerae exposure. India. E-mail: [email protected] NFjB and p38 MAP kinase activation were determined by immunoblotting for IjBa and phosphorylated p38. Vibrio cholerae significantly upregulated gene expression for the neutrophil chemoattractant CXCL chemokines, IL-8, CXCL and CXCL in HT29 cells, while downregulating the expression of macro- phage-attracting C-C chemokines. TLR4 silencing did not reduce IL-8 output from HT29 cells in response to V. cholerae.IjBa degradation was noted in the HT29 cells soon after exposure to V. cholerae and this recovered over time after removal of bacteria. p38 MAP kinase activation was not noted. Vibrio cholerae upregulated the expression of neutrophil attractant chemokines, most promi- nently IL-8, in HT29 cells, but downregulated macrophage-attracting chemo- kines. Probiotic lactobacilli modulated the IL-8, but not the other chemokine gene changes, in response to V. cholerae. interleukin-8 (IL-8), a potent chemokine and chemoattr- Introduction actant of polymorphonuclear leucocytes, when they con- Cholera, caused by the bacterium Vibrio cholerae remains a tact pathogenic bacteria [6]. Chemokine secretion is significant public health problem characterized by pan- probably not dependent on the presence of cholera toxin, demics originating from areas of endemicity [1]. Vibrio as volunteers given toxin-deficient strains also developed cholerae induces diarrhoea through the elaboration of chol- significantly increased titres of lactoferrin and CXCL8 [7]. era toxin which stimulates adenyl cyclase in intestinal epi- Chemokines are subdivided into four subfamilies on the thelial cells and leads to fluid secretion [2]. Although basis of the position of either one or two cysteine residues considered to be the paradigm of non-inflammatory secre- located near the amino terminus of the protein (CXCL, tory diarrhoea, increases in inflammatory factors such as CCL, CL and CX3CL) [8]. Studies in mice suggest that View metadata, citation and similar papers at core.ac.uk myeloperoxidase, lactoferrin, nitric oxide and eicosanoids, V. cholerae deficient in toxin production can elicit a major are reported in cholera [3]. The presence of congested pro-inflammatory response [9]. The pro-inflammatory blood vessels and polymorphonuclear leucocyte infiltration response induced by the bacterium is probably responsible of the lamina propria have been reported in the acute for containment of the infection. Inoculation of Vibrio into stage of cholera in both adults and children [4]. Polymor- mice with a mutation in Toll-like receptor 4 results in phonuclear leucocytes can induce chloride secretion simi- more severe disease and in attenuation of the cytokine lar to that seen with enterotoxins [5] and this could play responses, suggesting that the pro-inflammatory response an important role in diarrhoea. Epithelial cells secrete is mediated at least in part by TLR4 [9]. Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 69, 181–187 181 182 Vibrio Cholerae and Epithelial Cell Chemokine Gene Expression N. S. Nandakumar et al. .................................................................................................................................................................. Probiotic bacteria are commonly used in the treatment twice with appropriate medium without serum and anti- of diarrhoeal disease. Lactobacillus rhamnosus GG (LGG), biotics and maintained in serum free medium for at least one of the best-studied probiotic bacteria, has been 2 h. Following this, cells were overlaid with medium shown to be effective in the treatment of acute diarrhoea alone (control), V. cholerae (100 bacteria per epithelial in children [10]. In the present study, we sought to study cell) and ⁄ or Lactobacillus GG (1000 bacteria per epithelial the pattern of activation by V. cholerae of chemokines in cell) for 2 h. The ratios of bacteria per epithelial cell intestinal epithelial cells, and to determine whether were determined in earlier studies examining the dose Lactobacillus GG would modulate these innate inflamma- effect. After 2 h, the extracellular bacteria were removed tory responses. To this purpose we used an inflammatory by washing and further incubated for 4 h in the presence cytokine pathway-focused PCR array and also examined of 50 lg ⁄ ml gentamicin to kill the remaining extracellu- the role of TLR4 and of NFjB and p38 MAP kinase lar bacteria. At the end of this period, culture superna- activation in the Vibrio-epithelial cell interaction. tant was removed and stored at )20 °C for quantitation of IL-8 secretion, while cells were removed and stored at )80 °C for total RNA isolation. Materials and methods ELISA for IL-8. Interleukin-8 was assayed in culture Cell culture. HT29 cells (National Center for Cell Sci- supernatants using ELISA (BD Opt EIA SET, Catalog ences, Pune, India) were grown in culture flasks in high No. 555, BD Biosciences, San Jose, CA, USA) after cen- glucose Dulbecco’s Modified Eagle’s Medium (DMEM) trifugation at 9000 g for 10 min to remove bacterial (Gibco BRL, Paisley, UK) containing 100 U of penicil- debris. Assays followed the standard protocol recom- lin ⁄ ml, 100 lg of streptomycin ⁄ ml and 20 lg of ampho- mended by the manufacturer. Substrate solution tetram- tericin B supplemented with 10% fetal bovine serum ethyl benzidine ⁄ H202 (BD Biosciences) was added for (Gibco BRL) and incubated at 37 °C with 5% CO2. colour development and plates were read at 450 nm in Bacterial protocols. A clinical isolate of V. cholerae an ELISA plate reader (Victor 3; Perkin Elmer, Waltham, O139, kindly provided by the Clinical Microbiology MA, USA). Department, Christian Medical College, Vellore, was used Chemokine and chemokine receptors array. Total RNA was for these studies. Disease with either the O139 strain or isolated from infected cells using Trizol reagent (Sigma, St the O1 strain is known to produce equivalent increases Louis, MO, USA, Cat. No. T9424) as per manufacturer’s in inflammatory mediators [4]. Lactobacillus GG (Cultu- instructions. Reverse transcription of RNA was performed relle) was grown in de Man Rogosa Sharpe broth (Hime- in a final volume of 20 ll containing 0.5 mM of each dia, Mumbai, India) under anaerobic conditions for 16 h nucleotide triphosphate, 40 U of RNAase inhibitor at 37 °C on the day before the experiments. (Ambion, Foster City, CA, USA), 50–100 ng of random Bacterial culture and infection of HT29 cells. Bacteria hexamers (Amersham Pharmacia, Little Chalfont, Buckin- were grown standing overnight at 37 °C in Luria–Bertani ghamshire, UK), 200 U of Moloney Murine Leukaemia broth before use. They were pelleted by centrifugation at virus RT (Finnzymes) and 15 ll of the extracted RNA or 800 g and washed three times with sterile phosphate-buf- H20. The sample was then incubated at 42 °C for 1 h. The fered saline (PH 7.4). The bacterial concentration in the Human Chemokines and Receptors RT2 ProfilerÔ PCR suspension medium was determined by measurement of Array (SuperArray Inc., Frederick, MD, USA) was used for absorbance at 540–600 nm and comparing with standard these studies. This is an inflammatory pathway-focused barium chloride optical density. The bacteria were then array containing primers targeting members of the C-C re-suspended in antibiotic free medium. and C-X-C motif subfamilies of small inducible cytokines HT29 cells were cultured in 24-well plates (Falcon and their receptors as well as other related genes. The array Milliwell; BD, Franklin Lakes, NJ, USA) until they profiles the expression of 84 genes that encode chemokines reached confluence. Prior to infection, cells were washed and their receptors (Table 1). In addition, it profiles five Table 1 List of gene products profiled by the inflammatory chemokine pathway-specific PCR array. Family Genes involved CC motif ligands CCL1, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8. C-X-C motif ligands CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL2, CXCL3, CXCL5, CXCL6, CXCL9. CC motif receptors: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,