Articles in PresS. Am J Physiol Gastrointest Liver Physiol (November 17, 2005). doi:10.1152/ajpgi.00112.2005 -1-
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Differential cytokine response from dendritic cells to commensal and pathogenic bacteria in different lymphoid compartments in humans
Liam O’Mahony*, Louise O'Callaghan*, Jane McCarthy*, David Shilling†, Paul Scully*, Shomik Sibartie*, Eamon Kavanagh†, William O. Kirwan†, Henry.Paul Redmond†, John Kevin Collins*, Fergus Shanahan*.
Alimentary Pharmabiotic Centre* & Dept. of Surgery†, University College Cork, National University of Ireland, Cork.
Running head: Cytokine responses in different lymphoid tissues
Correspondence to: Prof. Fergus Shanahan, Alimentary Pharmabiotic Centre, Department of Medicine, Cork University Hospital, Cork, Ireland.
Tel: 353-21-4901226 Fax: 353-21-4345300 E-mail:
[email protected]
Copyright © 2005 by the American Physiological Society.
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Abstract Background: The resident host microflora condition and prime the immune system. However, systemic and mucosal immune responses to bacteria may be divergent. Aim: To compare, in vitro, cytokine production by human mononuclear and dendritic cells from mesenteric lymph nodes (MLN) and peripheral blood mononuclear cells (PBMCs) to defined microbial stimuli. Methods: Mononuclear cells and dendritic cells, isolated from the MLN (n=10) and peripheral blood (n=12) of patients with active colitis, were incubated in vitro with the probiotic bacteria, Lactobacillus salivarius UCC118 or Bifidobacterium infantis 35624, or the pathogenic organism Salmonella typhimurium UK1. IL-12, TNF- , TGF- and IL-10 cytokine levels were quantified by ELISA. Results: PBMCs and PBMC-derived DCs secreted TNF in response to the lactobacillus, bifidobacteria and salmonella strains, while MLNCs and MLN derived DCs secreted TNF- only in response to salmonella challenge. Cells from the systemic compartment secreted IL-12 following co-incubation with salmonella or lactobacilli, while MLN derived cells produced IL-12 only in response to salmonella. PBMCs secreted IL-10 in response to the bifidobacterium strain, but not in response to the lactobacillus or salmonella strain. However, MLNC secreted IL-10 in response to bifidobacteria and lactobacilli, but not in response to salmonella. Conclusion: Commensal bacteria induced regulatory cytokine production by MLNCs while pathogenic bacteria induce Th1 polarising cytokines. Commensal-pathogen divergence in cytokine responses are more marked in cells isolated from the mucosal immune system compared to peripheral blood mononuclear cells.
Key words: Human, Mucosa, Inflammation, Dendritic cells, Cytokines.
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Introduction
Scientific understanding of the gastrointestinal flora and its interaction with the host immune response has benefited recently from a convergence of interest from disparate traditional research disciplines. This reawakening of interest in intestinal bacteria has been due to several factors, including the discovery of Helicobacter pylori as a cause of peptic ulcer disease and gastric cancer, the role of the indigenous flora in the pathogenesis of inflammatory bowel disease, the development of cultureindependent molecular techniques to study enteric bacteria, and the increasing recognition of the role of commensal flora in the development and fine tuning of the mucosal immune response (2, 16, 25, 27, 28). Early studies with gnotobiotic mice which demonstrated the importance of bacterial colonisation in promoting the development of the immune response have been complemented by modern molecular techniques, including laser dissection microscopy, to identify the molecular signals and transduction pathways underpinning the regulatory influence of the enteric flora on the structure and function of the mucosal immune system (7, 8, 20). While the role of the intestinal epithelium as a sensor of the microbial environment within the gut is well established (15) and differential epithelial responses to commensal and pathogenic signals have been shown, regional specialisation of responses by gut-associated lymphoid cells to bacterial signals compared with systemic immune responses have received less attention. Although tissue-specific specialisation of immunologic responses to pathogens, including region-specific dendritic cell (DC) subsets, has been demonstrated in mice (11), evidence of this in humans is more sparse (31). Therefore, we performed a comparative study of cytokine responses from lymphoid populations including
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isolated DCs from the human mesenteric lymph node and peripheral blood. The results confirm a divergence of cytokine responses to commensal lactobacilli and bifidobacteria in comparison with pathogenic salmonella and demonstrate different patterns of response in the two lymphoid compartments.
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Materials and Methods
Study population This study was approved by the Ethics Committee of Cork University Hospital and informed consent was obtained from all volunteers. The study population involved patients with inflammatory bowel disease undergoing colectomy or small bowel resection. Mesenteric lymph nodes (MLN) were obtained from ten patients with active IBD (n=10: age range 19-41 (mean 32.2), 3 women and 7 men: 5 patients with CD; 5 patients with UC). All patients were prescribed and taking systemic steroids (5'- amino salicylic acid). All patients required surgical section because of progressive disease and failure to respond to steroids. Peripheral blood mononuclear cells (PBMC) were obtained from patients with active inflammatory bowel disease (n=12). The age range was 20-51 (mean 28). Five women and seven men were studied, 5 patients with CD; 7 patients with UC. None of the MLN and PBMC donors were the same subject.
Mesenteric lymph node cell (MLNC) isolation Mesenteric lymph nodes were selected for this study with careful regard to their anatomical location relative to areas of inflammation in the bowel. MLN selected directly drained an inflamed area of the bowel. From three patients within this study population, it was possible to obtain MLN draining segments of inflamed and non-inflamed bowel. Single cell suspensions were generated from MLN by gentle extrusion of the tissue through a 180µ mesh wire screen. Cells were washed and resuspended in DMEM (Dulbecco's modified eagle medium) containing 10% FCS (Invitrogen, Paisley, UK). Mononuclear cells were isolated by Ficoll-Hypaque density
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centrifugation (3) and resuspended at 1x106 cells/ml in complete media - DMEM containing 25mM glucose, 10% FCS, 1% nonessential amino acids, 50U/ml penicillin and 50µg/ml streptomycin (Invitrogen, Paisley, UK). These mononuclear cells are termed mesenteric lymph node cells (MLNCs).
Peripheral blood mononuclear cell (PBMC) isolation Peripheral blood was taken directly into sterile EDTA containing vacutainers. Mononuclear cells were isolated from blood by Ficoll-Hypaque density gradient centrifugation. Following a washing step, PBMCs were resuspended in complete media at 1 x 106 cells /ml.
Dendritic cell isolation DCs from MLN and peripheral blood were isolated using identical procedures. Cells were resuspended at 5 x 107 cells/ ml in PBS (without Ca2+ or Mg2+) with 4% FCS. For optimal recovery of DCs, 1mM EDTA was added to all media and cell suspensions were blocked with anti-CD32 antibodies (StemCell, Meylan, France). DCs were isolated from this cell suspension, according to the manufacturer's protocol, using a DC negative isolation kit (StemSep
depletion cocktail, StemCell, Meylan,
France). DCs were resuspended in Stemspan serum-free media (StemCell, Meylan, France) at 1x106 cells/ ml. Viability, determined by trypan blue exclusion, was consistently
98%. Purity of DC preparations were assessed using flow cytometry.
Cells which were HLA-DR positive and CD3/CD14/CD16/CD19/CD20/CD56 negative were termed DCs. All antibodies were obtained from BD Biosciences, (Oxford, UK).
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Bacterial strains We have previously reported the selection criteria for isolation of Lactobacillus salivarius UCC118 (L. salivarius) and Bifidobacterium infantis 35624 (B. infantis) (5). L. salivarius and B. infantis were routinely cultured anaerobically for 24-48 hours in deMann, Rogosa and Sharpe medium, MRS, (Oxoid, Basingstoke, UK) and MRS supplemented with 0.05% cysteine (Sigma, Dublin, Ireland), respectively. Salmonella typhimurium UK1 (S. typhimurium) was cultured aerobically for 18-24 hours in tryptic soya broth (Oxoid, Basingstoke, UK). Bacterial cultures were harvested by centrifugation (3,000g x 15 mins), washed with PBS and subsequently diluted to final cell densities of 1x107, 1x105 and 1x103 colony forming units (cfu)/ ml in DMEM.
In vitro cell stimulations All cells were seeded in 24 well tissue culture plates (Costar, Schiphol-Rijk, Netherlands) at 1x106 cells/ml. MLNCs were stimulated for 72 hours with L. salivarius, B. infantis or S. typhimurium at 3 different bacterial concentrations, 1x107 CFU, 1x105 CFU and 1x103 CFU. As 1x107 bacteria resulted in significant stimulation of cytokine production, this bacterial concentration was used in subsequent experiments. MLNCs isolated from non-inflamed bowel and PBMCs were stimulated for 72 hours with these bacteria at 1x107 cfu. MLN-derived and peripheral blood derived DCs were stimulated with L. salivarius, B. infantis or S. typhimurium. MLNC and PBMC cultures remained viable over the 72 hour culture period (>95% viable at 72 hours). Dendritic cell viability decreased rapidly after 24 hours incubation so all dendritic cell stimulations were terminated at 24 hours (mean viability at 24 hours >94%; mean viability at 72 hours = 31%). Non-stimulated cells
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were present to assess spontaneous levels of cytokine secretion. Plates were incubated in a 5% CO2 and 37oC humidified atmosphere after which supernatants were harvested for cytokine analysis. Cytokine production was measured, according to the manufacturer's instructions, using commercially available ELISA kits (R&D Systems, Abington, UK). Cytokines measured included TNF- , IL-12 p40, IL-10 and TGF- .
Intracellular cytokine staining Following stimulation of PBMCs with L. salivarius, B. infantis or S. typhimurium, cells were examined by flow cytometry for intracellular cytokine expression. PBMC incubations were performed for 3, 6 and 12 hours in the presence of Golgistop (BD BioSciences). Cells were stained with FITC conjugated anti-CD3 antibodies for the identification of T cells while dendritic cells were identified as being CD3/CD14/CD16/CD19/CD20/CD56 negative (FITC conjugated) and Cy5HLA-DR positive. Cells were fixed and permeabilised using a Cytofix/Cytoperm kit (BD BioSciences) followed by co-incubation with PE conjugated anti-IL-10 or antiTNF- antibodies. All antibodies were obtained from BD BioSciences. Flow cytometric analysis of the frequency of cytokine positive cells was performed using a BD FacsCalibur and Cell Quest Pro software.
Statistical Analysis Results were analysed using ANOVA analysis. Values are illustrated as the mean ± standard error mean. Statistically significant differences in cytokine production between non-stimulated cells (control) and cells stimulated with bacteria were accepted at p< 0.05.
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Results
Divergent immune responses of MLNCs to commensal and pathogenic bacteria Strain-strain divergence in cytokine responses (i.e commensal bacteria versus pathogenic bacteria) was observed in MLNCs draining inflamed bowel (Figure 1). L. salivarius and B. infantis induced IL-10 production by MLNCs at the highest bacterial dose (107 CFU). B. infantis also significantly induced TGF- production while L. salivarius induced TGF- production did not reach statistical significance. L. salivarius and B. infantis did not induce TNF- or IL-12. In contrast, S. typhimurium significantly induced IL-12 and TNF- production by MLNCs but did not induce IL10 or TGF- . To confirm that results were not skewed by the presence of inflammation in the resected specimens, MLNCs were isolated from two different drainage fields (from inflamed and non-inflamed segments), which were available in three patients with restricted disease distribution. The patterns of response of MLNCs to commensals and pathogens were similar irrespective of whether they were from sites draining inflamed or non-inflamed segments of bowel (Figure 2). Thus, the lactobacillus and bifidobacterium strains induced IL-10 and TGF- production while the salmonella induced IL-12 and TNF- production. However, while the cytokine pattern was similar between MLNCs from both sites, the quantity of S. typhimurium stimulated IL-12 and TNF- produced by MLNCs draining inflamed bowel was greater than the levels produced by MLNCs draining non-inflamed bowel.
Comparison between MLNC and PBMC cytokine responses
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The cytokine response of cells derived from the mucosal immune compartment to commensal bacteria was not reflected in the response of cells derived from the systemic compartment (Figure 3). While L. salivarius significantly induced IL-12 and TNF- production when co-incubated with PBMCs, these cytokines were not secreted by MLNCs. Similarly, B. infantis induced TNF- production by PBMCs but not by MLNCs. In addition, differences were also evident with regulatory cytokines; L. salivarius induced IL-10 production by MLNCs but not by PBMCs. B. infantis induced IL-10 production by both MLNCs and PBMCs. TGF- was not induced over control for any bacterial stimulated PBMC cultures (result not shown) while B. infantis induced TGF- production in MLNC cultures. In contrast to the responses to commensals, S. typhimurium induced a similar cytokine profile for both MLNCs and PBMCs.
Intracellular Cytokine Staining In stimulated PBMC cultures, both T cells and dendritic cells secreted IL-10 and TNF- in reponse to co-incubation with each of the bacterial strains examined (Figure 4). However, it is evident from these histograms that a greater percentage of the dendritic cell population (HLA-DR positive) was manufacturing cytokine compared to T cells within the same cultures. This suggests that dendritic cells are responsible for the bulk of cytokine released into culture supernatants and measured using ELISA.
Dendritic cell cytokine responses to microbial challenge Isolated DC preparations were consistently > 90% HLA-DR positive and CD3/CD14/CD16/CD19/CD20/CD56 negative (mean=93.2% +/- 2.9%). L. salivarius
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induced IL-10, TNF- and IL-12 secretion by peripheral blood derived DCs but only induced IL-10 secretion from MLN derived DCs (Figure 5). B. infantis induced IL-10 and TNF- , but not IL-12, production by peripheral blood derived DCs. B. infantis stimulated the production of IL-10 alone by MLN derived DCs. S. typhimurium significantly induced IL-12 and TNF- production by both MLN and peripheral blood derived DCs but only induced IL-10 secretion from PBMC derived DCs. It is important to note that the absolute levels of cytokines released from DCs was substantially less than that released from mononuclear cell populations. This may reflect the different culture periods for DCs (24 hours) and mononuclear populations (72 hours) but also suggests that individual DC subpopulations require co-operation with other mononuclear cells in order to achieve the optimal response to bacterial challenge.
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Discussion
Discriminatory responses to pathogens and commensals are expected, irrespective of the lymphoid compartment and this is evident in the present study. The expected TH1 polarised cytokine response to salmonella was found for both the mesenteric lymph node and peripheral blood. However, in vitro responses to the commensal bacteria were divergent in the two lymphoid compartments. While significant levels of TNF- were produced in response to the lactobacillus and bifidobacterium strains by peripheral blood mononuclear cells and by DCs derived from that source, mesenteric lymph node cells and their associated DCs did not elaborate this cytokine in the presence of the same microbial stimuli. In addition, the ratio of IL-12/IL10 production by cells from the periphery in response to L. salivarius is reversed in the mesenteric lymph node. The murine mesenteric lymph node has been shown to restrict access of mucosal dendritic cells bearing commensal bacteria to the systemic immune compartment (17). However, dendritic cell cytokine responses to commensals and pathogens within the human mesenteric lymph node have not previously been explored. The human mesenteric lymph node is a rich repository of mucosalassociated lymphoid cells and has the advantage of enabling purification of DC and other cells without introducing the potentially confounding variable of artifactual distortion due to enzymatic tissue digestion which is required for cell isolation from the intestinal lamina propria (12, 13, 26). However, access to human mesenteric lymph nodes is limited and largely dependent on availability of surgically resected material from patients undergoing colectomy for inflammatory bowel disease or colorectal cancer. To avoid unnecessary confounding variables, the present study was
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restricted to material from patients with inflammatory bowel disease because reports by us and by others of local immunosuppression in tumor-draining lymph nodes (24). Mesenteric nodes in the drainage field of gastrointestinal tumors are subject not only to the immunosuppressive effects of occult micrometastases that are undetected by conventional histology but are also exposed to immunosuppressive factors from the primary tumor. However, the divergent results between the mesenteric and peripheral lymphoid compartments were not disease-related as the samples from both sites were taken from patients with active disease. More importantly, we took advantage of the availability of lymph nodes draining inflamed and non-inflamed segments of colonic mucosa in the case of three individuals undergoing colectomy for sub-total colonic involvement with ulcerative colitis. The results showed that the pattern of cytokine responses to the different microbial stimuli were the same irrespective of whether the nodes were in the field of drainage of inflamed or non-inflamed colonic segments. Striking plasticity of DC responses to different microbial stimuli has previously been reported (9, 14). Cytokine responses by DCs may also vary for different species of bacteria within the same genus (4). Discriminatory cytokine responses to different microbes enables the host to interpret the local environment and sense danger. This signalling is mediated by a series of pattern recognition receptors including toll-like receptors and C-type lectins on the surface of DCs (1, 6, 19). The expression of these receptors is probably variant in different tissues. Another factor which may influence cytokine production in the different lymphoid compartments is the dose of bacteria exposed to the system. Variability of cytokine responses may also reflect heterogeneity within DC populations. Thus, murine DCs from the intestinal Peyer’s patch promote IL-10 rather than IL-12p40 responses, whereas the latter are more characteristic of splenic DCs (10). The results of the present study
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with human mesenteric lymph node support the murine evidence for regional specialisation of DCs. Notwithstanding, the degree to which one may extrapolate across species is limited because normal murine responses to intestinal antigens are biased toward a Th2 profile whereas human mucosal immune responses appear to have a Th-1 bias (22). Translocation of bacteria is an important issue in clinical medicine and translocation was traditionally measured by culturing bacteria from the mesenteric lymph node. More recently, mesenteric lymph nodes have been demonstrated to be important gatekeepers limiting bacterial access to the systemic circulation (17). The results of the present study have clinical implications because commensal bacteria, including those studied here, have been used as probiotics in animal models of inflammatory bowel disease (18, 23), and are in human clinical trials (www.proeuhealth.vtt.fi). Whether the numbers of bacteria used in our studies are clinically relevant is unknown but are likely to be relevant in the context of clinical conditions with increased gut permeability and a high rate of sepsis (e.g. IBD). Moreover, numbers of bacteria recovered from the periphery in experimentally infected animals are in the range of bacteria tested in this study (30). Lastly, we have previously demonstrated that probiotics administered parenterally have beneficial effects – therefore it is important to understand how the immune system perceives them in different locations (29). In this respect, it is noteworthy that while lactobacilli are known to stimulate IL-12 responses from human peripheral blood (21), the more relevant responses are probably those from gut-associated lymphoid tissue. In contrast to peripheral blood, regulatory cytokines such as IL-10 and TGF- were produced in preference to TNF- and IL-12 after exposure in vitro to lactobacilli and bifidobacteria.
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In conclusion, regional specialisation of immune responses to microbial stimuli is evident in humans. In addition to polarised responses to commensals and pathogens, there are divergent responses in peripheral blood and mesenteric lymphoid cells to microbial challenge. This should be considered in the interpretation of immunological responses to commensal or probiotic organisms in humans.
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Grants
The authors are supported in part by Science Foundation Ireland in the form of a centre grant (Alimentary Pharmabiotic Centre), by the Health Research Board (HRB) of Ireland, the Higher Education Authority (HEA) of Ireland, and the European Union (PROGID QLK-2000-00563).
Disclosures The authors (LO'M, JKC, FS) have been affiliated with a multi-departmental university campus-based research company (Alimentary Health Ltd), which investigates host-flora interactions and the therapeutic manipulation of these interactions in various human and animal disorders. The content of this article was neither influenced nor constrained by this fact.
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Figure Legends. Figure 1. MLNC cytokine profiles. MLNC cytokine secretion is dependant on the type and dose of bacterial strain being tested. L. salivarius stimulated IL-10 production while B. infantis induced IL-10 and TGF secretion by MLNCs. In contrast, S. typhimurium induced IL-12 and TNF production. Results are expressed as mean cytokine level +/- standard error following stimulation with 0, 1x103, 1x105 or 1x107 bacterial cells, n=10 MLNC stimulations. * (P 0.05), significant difference between non-stimulated cells (control) to cells stimulated with bacteria. (A) IL-12 (B) TNF- (C) IL-10 and (D) TGF- .
Figure 2. Inflammed versus non-inflammed MLNC. Bacterial stimulated cytokine production by MLNC draining non-inflamed bowel (n=3) is similar to cytokine production by MLNC draining inflamed bowel (n=10) in response to stimulation with 1x107 L. salivarius, B. infantis or S .typhimurium. L. salivarius and B. infantis induced IL-10 and TGF production while S. typhimurium induced IL-12 and TNF production. However, S. typhimurium induced greater amounts of TNF and IL-12 production in MLNC draining inflamed versus noninflamed bowel. * (P 0.05), significant difference between nonstimulated cells (control) to cells stimulated with bacteria. (A) IL-12, (B) TNF- , (C) IL-10 and (D) TGF- .
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Figure 3. MLNC and PBMC cytokine profiles. Bacterial induced cytokine production by MLNCs (n=10) and PBMCs (n=12) were divergent. L. salivarius induced IL-12 and TNF- production by PBMCs, which were not induced in MLNCs. In addition, L. salivarius did not induce IL-10 production by PBMCs. B. infantis induced TNF production by PBMCs which was not induced in MLNCs. S. typhimurium induced a similar cytokine profile from both MLNCs and PBMCs. Results are expressed as mean cytokine level +/- standard error following stimulation with 0 (non-stimulated) or 1x107 bacterial cells. * (P 0.05), significant difference between non-stimulated cells to cells stimulated with bacteria.
Figure 4. Intracellular cytokine staining. These representative histograms illustrate that T cells (CD3 positive) and dendritic cells (CD3/CD14/CD16/CD19/CD20/CD56 negative and HLA-DR positive) stain for intracellular TNF- and IL-10 following stimulation with L. salivarius, B. infantis or S. typhimurium. The percentage of dendritic cells positive for intracellular cytokine was greater than the percentage of cytokine positive T cells. The percent values illustrated represent the percent positive for each cell type.
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Figure 5. Dendritic cell cytokine profiles. Cytokine production by DCs, in response to commensal bacteria, depends on the site from which the DCs were isolated. L. salivarius and B. infantis induced TNFproduction by peripheral blood derived DCs (n=3) which was not induced in MLNC derived DCs (n=5). In addition, L. salivarius induced IL-12 production by PBMC derived DCs but not MLNC derived DCs. In contrast, S. typhimurium induced TNFand IL-12 production by both PBMC and MLNC derived DCs. IL-10 was secreted by PBMC and MLNC derived DCs upon co-incubation with L. salivarius and B. infantis while PBMC derived DCs alone secreted IL-10 in response to S. typhimurium. Results are expressed as mean cytokine level +/- standard error following stimulation with 0 (non-stimulated) or 1x107 bacterial cells. * (P 0.05), significant difference between nonstimulated cells to cells stimulated with bacteria.
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Figure 1. 800
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Figure 2.
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Figure 3. 3000 PBMC
MLNC
*
IL-12 pg/ml
2400
1800
*
1200
*
600
0 Non-stimulated
4000
TNF-alpha pg/ml
3500
PBMC
MLNC
L. salivarius
B. infantis
S. typhimurium
* *
*
3000 2500 2000 1500 1000
*
500 0 Non-stimulated
L. salivarius
B. infantis
S. typhimurium
3500 PBMC
MLNC
3000
*
IL-10 pg/ml
2500 2000 1500
*
1000
*
500 0 Non-stimulated
L. salivarius
B. infantis
S. typhimurium
- 27 -
G-00112-2005.R2
Figure 4.
5%
1%
IgG
6%
IL10
TNF 10
0
10
1
10
2
10
3
10
4
10
IgG
0
10
1
10
2
10
3
10
10
1
10
2
10
1
10
3
10
4
22%
TNF
IgG
0
26%
IgG 0
10
CD3
2%
10
4
10
2
10
3
10
4
10
0
10
1
10
HLA-DR
2
10
3
10
4
IL10 10
0
10
1
10
2
10
3
10
4
- 28 -
G-00112-2005.R2
Figure 5. 120 PBMC-DC MLNC-DC
*
IL-12 pg/ml
90
* 60
* 30
0 Non-stimulated
L. salivarius
B. infantis
1000
*
PBMC-DC
TNF-alpha pg/ml
800
MLNC-DC
S. typhimurium
*
600
400
*
200
*
0 Non-stimulated
L. salivarius
B. infantis
S. typhimurium
1000
*
PBMC-DC MLNC-DC
*
IL-10 pg/ml
750
500
250
*
*
*
L. salivarius
B. infantis
0 Non-stimulated
S. typhimurium
