Immunomodulatory e ects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells

FEMS Immunology and Medical Microbiology 38 (2003) 165^172 www.fems-microbiology.org Immunomodulatory e ects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells Karen Manon Lammers a;, Patrizia Brigidi b, Beatrice Vitali b, Paolo Gionchetti a, Fernando Rizzello a, Elisabetta Caramelli c, Diego Matteuzzi b, Massimo Campieri a a Department of Internal Medicine and Gastroenterology, University of Bologna, Policlinic S. Orsola, Via Massarenti 9, 40138 Bologna, Italy b Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy c Department of Histology and General Embryology, University of Bologna, Bologna, Italy Received 27 February 2003; received in revised form 3 April 2003; accepted 10 April 2003 First published online 17 May 2003 Abstract A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1l and an increase of interleukin-10. ß 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Bi dobacterium; Lactobacillus; Bacterial DNA; In ammatory bowel disease; Immune modulation 1. Introduction In ammatory bowel diseases (IBD) are characterized clinically by chronic in ammation in the intestine. The etiology of IBD remains unclear, even if human and experimental studies indicate that genetic host susceptibility, gut mucosal immune response and enteric bacteria may contribute to the pathogenesis of IBD [1]. The importance of bacteria in sustaining in ammation in IBD is further supported by the clinical experience that antibiotics reduce disease activity. Evidence exists that in patients with active IBD there is a breakdown of normal tolerance to the resident enteric bacteria ora which leads to an excessive * Correspondence author. Tel.: +39 (051) 6364122; Fax: +39 (051) 392538. E-mail address: karenmanon@hotmail.com (K.M. Lammers). mucosal immune response against enteric bacteria [2]. Interleukin 10 (IL-10) is a cytokine of particular therapeutic interest in IBD since it plays a key role in the control of in ammatory responses to intestinal antigens and can restore tolerance of T cells to resident intestinal bacteria. Therapeutic bene t of IL-10 administration has been reported from experimental studies with IL-10-de cient mice [3] and in clinical trials with IBD patients [4] albeit that the clinical usefulness of IL-10 is limited for technical reasons related to the organ-speci c delivery [5]. Thus, a therapeutic approach based on IL-10 which overcomes these limitations would provide great perspectives. The important role of bacteria in the pathogenesis of IBD has suggested the possibility of preventing or treating these disorders by manipulating the intestinal micro ora with probiotic treatment. Members of the genera Lactobacillus and Bi dobacterium are amongst the most common microorganisms in the human gastrointestinal tract. Evidence exists that these bacteria exert health-promoting activity because they play an important role in the control of 0928-8244 / 03 / $22.00 ß 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/s0928-8244(03)00144-5

166 K.M. Lammers et al. / FEMS Immunology and Medical Microbiology 38 (2003) 165^172 the intestinal micro ora and in maintenance of its normal state [6]. Because of their bene cial properties Lactobacillus and Bi dobacterium strains are commonly used in dairy and pharmaceutical probiotic preparations. VSL#3 (VSL Pharmaceuticals, Ft. Lauderdale, FL, USA) contains 450 billion per sachet of a viable lyophilized bacterial mixture, including three strains of bi dobacteria (Bi dobacterium longum, Bi dobacterium breve, Bi dobacterium infantis), four strains of lactobacilli (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus plantarum) and one strain of Streptococcus salivarius subsp. thermophilus. In recent clinical studies, its e cacy was shown as maintenance treatment and prophylactic therapy in patients with IBD [7,8]. In addition, Ulisse et al. [9] showed an increased tissue level of IL-10 in pouch patients treated with this probiotic therapy. The therapeutic bene t of VSL#3 was also shown by experimental studies in IL-10 knock-out mice [10,11]. Madsen et al. [11] found that an unknown, soluble factor secreted by the bacteria of this probiotic preparation enhanced epithelial barrier function and resistance to pathogenic bacterial invasion in T84 epithelial cells. In addition, sonicates ^ containing soluble cytoplasmic fraction ^ of each Lactobacillus and Bi dobacterium VSL#3 strain did not induce IL-8 production in HT29 epithelial cells [12], but stimulated IL-10 secretion in peripheral blood mononuclear cells (PBMC) (U. Helwig, personal communication). In the past few years, immune e ects of unmethylated DNA sequences (CpG motifs), predominantly present in bacteria, have been described [13]. Considering the high GC content of Bi dobacterium and ^ to a lesser extent ^ Lactobacillus chromosomal DNA, we decided to examine the e ect of VSL#3 bacteria DNA on IL-1L, IL-6 and IL-10 secretion and to evaluate the response induced by total bacterial DNA isolated from feces of healthy subjects before and after 2 weeks of VSL#3 treatment. 2. Materials and methods 2.1. Bacterial strains and culture conditions The VSL#3 bacterial strains used in this study are the following: B. infantis BI07, B. breve BBSF, B. longum BL04, L. acidophilus LA14, L. delbrueckii subsp. bulgaricus LB31, L. casei LC10, L. plantarum LPT and S. salivarius subsp. thermophilus TA061, designated S. thermophilus throughout this paper. Bi dobacterium and Lactobacillus strains were grown anaerobically (Anaerobic System, Model 2028, Forma Scienti c, Marietta, OH, USA) in MRS medium (Difco, Detroit, MI, USA) supplemented with 0.05% L-cysteine, at 37 C. S. thermophilus was cultured anaerobically in M17 medium (Difco) at 37 C. Enumeration of probiotic bacteria in fecal samples was carried out as previously reported [14]. Counting of the probiotic bacterial groups was performed by spreading dilutions onto plates containing the following selective agar media: LAMVAB [15] for Lactobacillus, RB [16] for Bi dobacterium and ST [17] slightly modi ed by adding bromocresol purple (30 mg l 31 ), bromocresol green (100 mg l 31 ) and nalidixic acid (30 mg l 31 ) for S. thermophilus. Plates were anaerobically incubated at 37 C for 24^ 48 h. Bacterial concentrations were expressed as CFU g 31 dry feces. Speci c identi cation of VSL#3 B. infantis and B. breve strains was performed by polymerase chain reaction (PCR)-mediated ampli cation using the strain-speci c primers and experimental conditions previously described [14]. Quanti cation was carried out by direct ampli cation of 30^50 colonies randomly selected from the highest dilution plates of RB medium. The ratio between the number of colonies which gave positive amplicons with the strain-speci c primers and the total number of colonies analyzed by PCR was calculated, and successively related to the total number of colonies grown on the highest dilution plate. 2.2. DNA preparation Isolation of genomic DNA from pure cultures of the probiotic bacteria was performed as previously described [18]. In order to obtain complete cell disruption, the method was slightly modi ed by prolonging the enzymatic lysis from 1 to 3 h and grinding with glass beads (150^212 Wm, Sigma, St. Louis, MO, USA). Puri cation of total bacterial DNA from feces of two healthy donors, before (t 0 ) and after 2 weeks of probiotic ingestion (t 1 ), was performed using the QIAamp DNA stool kit (Qiagen, Milan, Italy). Concentration and purity of all DNA preparations were determined by measuring OD 260 absorbance and OD 260=280 ratio, respectively. Only DNAs with an OD 260=280 ratio s 1.8 were used. DNAs were also assayed for lipopolysaccharide (LPS) content using the Limulus amebocyte assay (QCL-1000, BioWhittaker, Walkersville, MD, USA). LPS content was less than 0.01 U endotoxin Wg 31 DNA. 2.3. Cell preparation and cell culture For each group of experiments, PBMC were isolated from peripheral blood of four healthy volunteers by density gradient centrifugation (1.077 g ml 31 ) (Lymphoprep, Nycomed Pharma, Oslo, Norway). Cells were resuspended in RPMI 1640 culture medium (Life Technologies, Paisley, UK) supplemented with 10% (v/v) heat-inactivated (56 C, 1 h) fetal bovine serum, gentamicin (50 Wg ml 31 ) (Sigma), penicillin^streptomycin (1%) and sodium pyruvate solution (0.23 mmol l 31 ) (Sigma) (complete medium). All compounds were purchased endotoxin tested. Cells were cultured in complete medium in a concentration of 1U10 6 cells ml 31 in 96-well plates (Nunc, Roskilde, Denmark) in

K.M. Lammers et al. / FEMS Immunology and Medical Microbiology 38 (2003) 165^172 167 a5%co 2 -humidi ed incubator at 37 C. All experiments were performed in duplicate. 2.4. Stimulation experiments Genomic DNAs from the probiotic strains were applied to PBMC at the following concentrations: 3, 6.25, 12.5, 25, 50, and 70 Wg ml 31 for 24 h. For kinetics, genomic DNA of two strains, L. casei and B. breve, was applied at a concentration of 20 Wg ml 31 over a period of 24, 48 and 72 h. Total bacterial genomic DNAs, isolated from feces at times t 0 and t 1, were applied to PBMC from three donors for 24 h. As a positive control, LPS (Sigma) at a concentration of 20 ng ml 31 was used. As a negative control, methylated DNA from sh sperm (Roche, Italy) was applied at the same concentrations used for bacterial DNA. At the indicated time points, supernatants were harvested for cytokine measurement, centrifuged and stored at 320 C until assay. 2.5. Enzyme-linked immunosorbent assay (ELISA) Culture supernatants were analyzed for IL-1L, IL-6 and IL-10 with commercially available anti-human monoclonal capture antibodies to IL-1L (MAB601) (RpD Systems, Abingdon, UK), IL-6 (18871D) and IL-10 (18551A) (Pharmingen, San Diego, CA, USA), and speci c biotinylated anti-human monoclonal detection antibodies to IL-1L (BAF201) (RpD systems), IL-6 (18882D) and IL-10 (18562D) (Pharmingen). o-phenylenediamine bu - er/h 2 O 2 (Sigma, Steinheim, Germany) was used as a substrate. Extravidin-peroxidase (Sigma) was applied at a concentration of 1:1000. Human recombinant IL-1L (201-LB-005) (RpD systems), human recombinant IL-6 (1966T) and IL-10 (19701V) (Pharmingen) were used as standards. The optical density values of the samples were read at 490 nm on an ELISA plate reader. The detection limit of the assay was 15.7 pg ml 31 for IL-1L and 31 pg ml 31 for IL-6 and IL-10. 2.6. Statistical analysis Experimental data are expressed as mean þ S.E.M. The statistical signi cance of di erences in mean values was determined with Student s t-test. Di erences were considered to be signi cant at P 6 0.05. 3. Results 3.1. Probiotic bacterial DNA elicits IL-1, IL-6 and IL-10production We investigated the implication of pure genomic DNAs from VSL#3 Bi dobacterium, Lactobacillus and S. thermophilus strains in the release of the cytokines IL-1L, IL-6 Fig. 1. PBMC from four healthy donors were cultured with 70 Wg ml 31 of each genomic DNA extracted from VSL#3 bacteria. IL-1L, IL-6 and IL-10 production was measured in supernatants. The cytokine response to bacterial DNAs was compared to that obtained with LPS (20 ng ml 31 ): *P 6 0.05, **P 6 0.01. and IL-10. The rst encouraging results were obtained by challenging PBMC from healthy human subjects with 70 Wg ml 31 of bacterial DNA (Fig. 1). This dose was chosen accordingly to previous experimental results which established 50^100 Wg ml 31 as the maximal stimulatory dose range of Escherichia coli DNA [19]. To avoid stimulatory e ects due to contamination of bacterial proteins or LPSs, all DNA preparations used in the experiments had purity values (OD 260=280 s 1.8) and LPS contents ( 6 0.01 U endotoxin Wg 31 DNA) previously demonstrated

168 K.M. Lammers et al. / FEMS Immunology and Medical Microbiology 38 (2003) 165^172 to be inactive in immune stimulation [20]. Fish sperm DNA did not have stimulatory e ects on PBMC (data not shown). Genomic DNA induced strain-speci c immune e ects: by comparison with LPS, genomic DNAs of VSL#3 B. infantis, S. thermophilus, L. plantarum and L. bulgaricus stimulated a pronounced IL-1L secretion, whereas B. longum, L. acidophilus and L. casei DNAs induced a signi cantly lower IL-1L production. DNAs of B. breve, B. infantis and S. thermophilus elicited IL-6 levels comparable to that obtained by LPS, while genomic DNAs of B. longum and Lactobacillus strains induced signi cantly lower IL-6 concentrations. Interestingly, DNA isolated from B. breve, B. infantis and S. thermophilus stimulated high secretion of IL-10 which exceeded the LPS-induced IL-10 level up to three-fold. Subsequently, genomic DNAs of L. casei and B. breve were applied to PBMC at a concentration of 20 Wg ml 31 for 24, 48 and 72 h (Fig. 2). The DNAs of these strains were selected as representatives for the three Bi dobacterium and four Lactobacillus strains present in the VSL#3 mixture. The magnitude of IL-1L, IL-6 and IL-10 secretion reached maximal levels after 24 h of stimulation and then remained constant. Genomic DNA of B. breve was con rmed to be a more potent inducer of interleukins than L. casei DNA and was responsible for the highest IL-10 release. 3.2. Dose-dependent secretion of interleukins by bacterial DNA Fig. 2. PBMC from four healthy donors were incubated with 20 Wg ml 31 of genomic DNA of L. casei (closed squares) and B. breve (open squares) for 72 h. As controls medium (open circles) and LPS (20 ng ml 31 ) (closed circles) were used. Production of cytokines, assayed at intervals of 24 h, was compared to that induced by LPS: *P 6 0.05, **P 6 0.01. Genomic DNA of each VSL#3 strain was then applied to the PBMC culture system at di erent concentrations, ranging from 3 to 70 Wg ml 31, to determine which dose induced the maximum cytokine secretion (Fig. 3). This dose interval was chosen on the basis of previously described experimental results showing that cytokine production by immune cells could be detected after application of at least 3 Wg ml 31 E. coli DNA, and optimal stimulation required a bacterial DNA concentration s 50 Wg ml 31 [19]. Two di erent patterns of cytokine response could be clearly distinguished: genomic DNAs of B. longum, S. thermophilus and Lactobacillus strains induced the highest levels of IL-1L at a concentration of 3 Wg ml 31, and lower stationary levels when applied at the concentration range of 6.25^70 Wg ml 31. In contrast, B. breve and B. infantis genomic DNAs showed a dose-dependent enhancement of IL-1L, reaching a plateau at concentrations of 12.5 Wg ml 31. It is noteworthy that genomic DNAs of L. acidophilus, L. casei and S. thermophilus induced signi cantly lower levels of IL-1L than did DNAs of the other strains. The IL-10 production pattern was similar to that of IL-1 showing the highest values after challenge with 3 Wg ml 31 of Lactobacillus strains, B. longum and S. thermophilus genomic DNAs, and lower stationary levels at the other doses applied. Genomic DNAs of B. breve and B. infantis induced a dose-dependent IL-10 secretion, reaching a plateau at a concentration of 12.5 Wg ml 31.In comparison with the other genomic DNAs, B. breve and B. infantis DNAs appeared to be the most potent IL-10 inducers, provoking a three-fold higher IL-10 secretion. Furthermore, IL-10 production stimulated by DNAs of these two strains exceeded IL-1L production over the whole dose range. Incubation with L. casei and S. thermophilus DNAs showed a weaker but analogous response. Conversely, B. longum and L. acidophilus DNAs induced signi cantly higher levels of IL-10 than IL-1L only at the concentration of 3 Wg ml 31. Challenge with genomic

K.M. Lammers et al. / FEMS Immunology and Medical Microbiology 38 (2003) 165^172 169 Fig. 3. PBMC from four healthy volunteers were challenged with di erent doses (3^70 Wg ml 31 ) of bacterial DNAs. IL-10 (closed circles) and IL-1L (open circles) secretion was determined after 24 h of incubation. IL-1L and IL-10 values obtained at each dose were compared: *P 6 0.05, **P 6 0.01.

170 K.M. Lammers et al. / FEMS Immunology and Medical Microbiology 38 (2003) 165^172 of total bacterial DNA extracted from feces at time t 0 and t 1 (Fig. 4). Fecal concentrations of lactobacilli, bi dobacteria and S. thermophilus, determined by culture techniques, showed an increase up to 3 logs after the probiotic treatment. At time t 0, bi dobacterial titer was 2.5U10 7 CFU g 31 and increased to 4U10 9 CFU g 31 at time t 1. t 0 and t 1 counts of lactobacilli were 3U10 5 CFU g 31 and 1.1U10 6 CFU g 31, while for S. thermophilus values of 7U10 3 CFU g 31 and 8U10 6 CFU g 31 were found. The availability of strain-speci c primers for VSL#3 B. breve and B. infantis [14] allowed their PCR detection and quanti cation in the fecal samples. B. breve and B. infantis strains were detected only after probiotic treatment at concentrations of 3U10 8 CFU g 31 and 1U10 8 CFU g 31, respectively, demonstrating that the increased fecal concentration of Bi dobacterium is related to the presence of these exogenous strains. A pronounced di erence in the cytokine response was observed after stimulation with t 0 and t 1 total bacterial DNAs extracted from feces of the two volunteers treated with VSL#3. DNA isolated from feces before the probiotic administration induced higher levels of IL-1L than IL-10, while DNA from feces after probiotic treatment enhanced the IL-10 secretion but reduced the IL-1L secretion. IL-6 production was not altered. 4. Discussion Fig. 4. PBMC from three healthy donors were cultured with 5 Wg ml 31 of total bacterial DNA extracted from feces of a healthy subject before and after 2 weeks of VSL#3 treatment. IL-1L (open circles) and IL-10 (closed circles) production was determined after 24 h of incubation. DNAs of L. plantarum and L. bulgaricus showed comparable levels of IL-1L and IL-10 at 3 Wg ml 31, but at higher doses (6.25^70 Wg ml 31 ) IL-1L exceeded signi cantly IL-10. The strain-related patterns, previously described, were also shown for IL-6. DNAs of B. breve and B. infantis induced in a dose-dependent way, and the other DNAs elicited the highest production at the lowest concentration (data not shown). 3.3. Stimulatory e ects of fecal bacterial DNA on interleukin production Two healthy volunteers ingested for 2 weeks two sachets daily of VSL#3 (equivalent to 900 billion viable microorganisms), and feces were collected before (t 0 ) and after the probiotic administration (t 1 ). PBMC from three donors were challenged with a concentration of 5 Wg ml 31 The gut represents a complex ecosystem in which the intestinal micro ora and the host interact in order to maintain an immunologically balanced intestinal in ammatory response ( physiologic in ammation ). Several clinical and experimental studies indicate that the gut in ammation may be associated with an imbalance in intestinal micro ora, with a relative predominance of aggressive bacteria and an insu cient concentration of protective species. A recent therapeutic strategy in IBD involves manipulation of the local microenvironment by oral administration of probiotic bacteria, in order to restore the microbial balance [21]. The encouraging clinical results in IBD patients treated with the probiotic preparation VSL#3 have prompted us to elucidate the interaction between intestinal mucosa and microorganisms. Of particular interest are the results concerning the ability of VSL#3 probiotic bacteria to increase signi cantly the pouch tissue level of IL-10, and to reduce the proin ammatory cytokine levels [9]. Furthermore, a recent study demonstrated that the protective e ect of probiotic bacteria could be mediated by release of a soluble factor(s) that alters epithelial permeability and protects against pathogenic bacterial invasion [11]. These data raise the question whether this soluble proteinaceous factor or another di erent cytoplasmic bacterial component(s), such as DNA, could be involved in cytokine induction and modulation. Actually, substantial evidence exists that bacterial CpG motifs in-

K.M. Lammers et al. / FEMS Immunology and Medical Microbiology 38 (2003) 165^172 171 duce the immune response against challenge with a wide variety of pathogens and have therapeutic activity in murine models [22] and in human clinical trials [23]. In this perspective, we rst investigated the implication of pure genomic DNAs from VSL#3 Bi dobacterium, Lactobacillus and S. thermophilus strains in the release of the cytokines IL-1L, IL-6 and IL-10 using a human culture model. The results show that bacterial genomic DNA induces a remarkable strain-speci c immune response. A second series of experiments was designed to study whether the bene cial immune e ects observed during the VSL#3 treatment could be partly due to changes of the total bacterial DNA released by a modi ed micro ora. In this respect, bacterial DNA extracted from feces of healthy subjects collected before and after the probiotic administration was applied to the PBMC culture model. We consider this approach more appropriate to mimic the in vivo situation than investigating the stimulatory capacity of the DNA recovered from the VSL#3 mixture, as the intestinal colonization of exogenous bacteria is not strictly related to their concentrations in the pharmaceutical formulation but strain- and host-dependent. Interestingly, these experiments showed a diverse IL-1L and IL-10 cytokine production after stimulation with bacterial DNA isolated from feces before and after probiotic treatment. Bacterial DNA from micro ora before probiotic ingestion induced higher IL-1L than IL-10 secretion, contrarily, total bacterial DNA from a probiotics-enriched micro ora led to higher production of IL-10 than IL-1L. Our data suggest that genomic DNA released by exogenous bi dobacteria could provide a stimulus for mucosal IL-10 production. Taking into account that the manufacturing process and the gastroenteric passage a ect the bacterial viability, the results obtained in this study may indicate a potential therapeutic e ect of genomic DNA released by the dead bacteria ingested during the probiotic administration. Several articles describe di erent cytokine responses to CpG motifs present in bacterial DNA, some of these motifs exerting a more pronounced immunomodulatory e ect than others [13]. It would be interesting to know whether the strain-speci c cytokine release demonstrated in this study is related to the genomic sequence of each strain. However, the genome sequences of the VSL#3 probiotic strains have not yet been characterized and thus it is not possible to predict the redundancy of the di erent CpG motifs. At present, the only useful information available is the GC content. The high GC percentage reported in the literature for the Bi dobacterium species present in VSL#3 (58^61%) strongly suggests that the DNA composition may account for the cytokine response, and in particular that a high GC content favors IL-10 secretion. To our knowledge this is the rst report to show the immunomodulatory e ects of genomic DNA of probiotic bacteria. The observed di erences in kinetics and magnitude of IL-1L and IL-10 release in response to bacterial DNAs provide interesting information about the in uence of gut micro ora and/or bacterial components on the intestinal mucosal immune response. Acknowledgements K.M.L. is supported by a grant from the Fondazione del Monte di Bologna e Ravenna. References [1] Shanahan, F. (2002) Crohn s disease. Lancet 359, 62^69. [2] Duchmann, R., Kaiser, I. and Hermann, E. et al. (1995) Tolerance exists towards resident intestinal ora but is broken in active in ammatory bowel disease (IBD). Clin. Exp. Immunol. 102, 448^455. 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