Prevention and Treatment of Colitis With Lactococcus lactis Secreting the Immunomodulatory Yersinia LcrV Protein
2007; Elsevier BV; Volume: 133; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2007.06.018
ISSN1528-0012
AutoresBenoît Foligné, Rodrigue Dessein, Michaël Marceau, Sabine Poiret, Mathias Chamaillard, Bruno Pot, Michel Simonet, Catherine Daniel,
Tópico(s)Pharmacological Effects of Natural Compounds
ResumoBackground & Aims: The low calcium response V (LcrV) protein synthesized by gram-negative, pathogenic yersiniae participates in bacterial evasion of the host's innate immune response by stimulating synthesis of the anti-inflammatory interleukin (IL)-10 and preventing the activation of proinflammatory cytokines. Methods: We genetically engineered the food-grade bacterium Lactococcus lactis to secrete the LcrV protein from the enteropathogenic species Yersinia pseudotuberculosis. The protective and therapeutic potential of orally administered LcrV-secreting L lactis was evaluated in 2 models of acute experimental colitis (induced by trinitrobenzene sulfonic acid [TNBS] and dextran sodium sulfate [DSS], respectively) in wild-type and knockout mice. Results: Oral administration of LcrV-secreting L lactis led to active delivery of LcrV and induction of IL-10 (via a Toll-like receptor 2–dependent pathway) in the colon and prevented TNBS-induced colitis, in contrast to the L lactis control not producing LcrV. Down-regulation of tissue inflammatory markers correlated well with the reduction in damage to the colonic mucosa. In contrast, TNBS-induced colitis was not prevented in IL-10−/− mice pretreated with LcrV-secreting L lactis, thus showing that IL-10 is required for LcrV protection. Administration of LcrV-secreting L lactis also proved to be very effective in preventing and treating acute DSS-induced colitis. Conclusions: LcrV-secreting L lactis decreased experimentally induced intestinal inflammation in 2 murine models of colitis. This novel approach highlights the potential of using pathogen-derived immunomodulating molecules in vivo as novel therapeutics for inflammatory bowel diseases. Background & Aims: The low calcium response V (LcrV) protein synthesized by gram-negative, pathogenic yersiniae participates in bacterial evasion of the host's innate immune response by stimulating synthesis of the anti-inflammatory interleukin (IL)-10 and preventing the activation of proinflammatory cytokines. Methods: We genetically engineered the food-grade bacterium Lactococcus lactis to secrete the LcrV protein from the enteropathogenic species Yersinia pseudotuberculosis. The protective and therapeutic potential of orally administered LcrV-secreting L lactis was evaluated in 2 models of acute experimental colitis (induced by trinitrobenzene sulfonic acid [TNBS] and dextran sodium sulfate [DSS], respectively) in wild-type and knockout mice. Results: Oral administration of LcrV-secreting L lactis led to active delivery of LcrV and induction of IL-10 (via a Toll-like receptor 2–dependent pathway) in the colon and prevented TNBS-induced colitis, in contrast to the L lactis control not producing LcrV. Down-regulation of tissue inflammatory markers correlated well with the reduction in damage to the colonic mucosa. In contrast, TNBS-induced colitis was not prevented in IL-10−/− mice pretreated with LcrV-secreting L lactis, thus showing that IL-10 is required for LcrV protection. Administration of LcrV-secreting L lactis also proved to be very effective in preventing and treating acute DSS-induced colitis. Conclusions: LcrV-secreting L lactis decreased experimentally induced intestinal inflammation in 2 murine models of colitis. This novel approach highlights the potential of using pathogen-derived immunomodulating molecules in vivo as novel therapeutics for inflammatory bowel diseases. Chronic inflammatory bowel diseases (including Crohn's disease and ulcerative colitis) have become a major public health concern in Western countries.1Podolsky D.K. Inflammatory bowel disease.N Engl J Med. 2002; 347: 417-429Crossref PubMed Scopus (3248) Google Scholar, 2Korzenik J.R. Podolsky D.K. Evolving knowledge and therapy of inflammatory bowel disease.Nat Rev Drug Discov. 2006; 5: 197-209Crossref PubMed Scopus (159) Google Scholar Although the etiology of these conditions remains unclear, it is believed that dysregulation of the immune responsiveness of the intestinal mucosa to commensal enteric bacteria plays an important role in the induction and perpetuation of digestive tract inflammation. Reduction and prevention of mucosal inflammation by aminosalicylates, corticosteroids, immunosuppressive compounds, and antibiotics are the primary goals in the treatment of patients with inflammatory bowel disease; however, given that the aforementioned therapeutic agents fail to induce or maintain illness remission in about 30% of all patients,2Korzenik J.R. Podolsky D.K. Evolving knowledge and therapy of inflammatory bowel disease.Nat Rev Drug Discov. 2006; 5: 197-209Crossref PubMed Scopus (159) Google Scholar, 3Isaacs K.L. Lewis J.D. Sandborn W.J. et al.State of the art: IBD therapy and clinical trials in IBD.Inflamm Bowel Dis. 2005; 11: S3-S12Crossref PubMed Scopus (84) Google Scholar there is a need for new anti-inflammatory drugs. The coevolution of humans and infectious agents has exerted selective pressure on the immune system so that it maintains its control over potentially lethal infections. 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Yersinia's stratagem: targeting innate and adaptive immune defense.Curr Opin Microbiol. 2006; 9: 55-61Crossref PubMed Scopus (92) Google Scholar). LcrV injection in mice inhibits synthesis of proinflammatory tumor necrosis factor (TNF)-α and the interferon gamma cytokine and enhances anti-inflammatory interleukin (IL)-10 cytokine production in the spleen following infection with LcrV-deficient Yersinia pestis. Recent studies have shown that LcrV acts through the Toll-like receptor 2 signaling pathway to stimulate IL-10 synthesis in macrophages.15Sing A. Roggenkamp A. Geiger A.M. et al.Yersinia enterocolitica evasion of the host innate immune response by V antigen-induced IL-10 production of macrophages is abrogated in IL-10-deficient mice.J Immunol. 2002; 168: 1315-1321Crossref PubMed Scopus (126) Google Scholar, 16Sing A. Rost D. 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We then used 2 murine models of acute colitis to assess the ability of the orally administered recombinant strain to prevent and treat mucosal inflammation caused by either intrarectal instillation of trinitrobenzene sulfonic acid (TNBS) or administration of dextran sulfate sodium (DSS) in the animals' drinking water. The TNBS hapten induces acute colitis displaying Crohn's disease–like features, notably transmural inflammatory cellular infiltration associated with a Th1-dominated cytokine profile.18Neurath M. Fuss I. Strober W. TNBS-colitis.Int Rev Immunol. 2000; 19: 51-62Crossref PubMed Scopus (182) Google Scholar DSS-induced colitis is an extensively studied model for murine ulcerative colitis, which causes injury to the base of the crypts with subsequent inflammation, apoptosis, and ultimately epithelial ulceration.19Okayasu I. Hatakeyama S. Yamada M. et al.A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.Gastroenterology. 1990; 98: 694-702Abstract PubMed Google Scholar, 20Vetuschi A. Latella G. Sferra R. et al.Increased proliferation and apoptosis of colonic epithelial cells in dextran sulfate sodium-induced colitis in rats.Dig Dis Sci. 2002; 47: 1447-1457Crossref PubMed Scopus (109) Google Scholar Inasmuch as the LcrV protein induces anti-inflammatory IL-10 biogenesis, we also constructed an IL-10–secreting lactococcal strain to concomitantly compare its protective effect with that of LcrV-producing L lactis. Bacterial strains and plasmids are listed in Table 1.Escherichia coli and Y pseudotuberculosis were cultured in Luria–Bertani broth at optimal growth temperatures. L lactis was grown at 30°C in M17 medium (Difco; Becton Dickinson, Sparks, MD) supplemented with 0.5% of glucose. Chloramphenicol (Sigma-Aldrich, St Quentin Fallavier, France) was added to culture media for bacterial selection when necessary, at a final concentration of 20 μg/mL for Escherichia coli and 10 μg/mL for L lactis strains.Table 1Bacterial Strains and PlasmidsStrains and plasmidsRelevant characteristicsSourceStrains L lactis MG1363 (Ll)L lactis subsp cremoris, plasmid-freeWells et al22Wells J.M. Wilson P.W. Le Page R.W. Improved cloning vectors and transformation procedure for Lactococcus lactis.J Appl Bacteriol. 1993; 74: 629-636Crossref PubMed Scopus (167) Google Scholar E coli MC1061Cloning hostSambrook et al23Sambrook J. Fritsch E.F. Maniatis T. Molecular cloning: a laboratory manual.2nd ed. Cold Spring Harbor Laboratory, New York, NY1989Google Scholar Y pseudotuberculosis IP32777Template for lcrV amplificationSebbane et al56Sebbane F. Devalckenaere A. Foulon J. et al.Silencing and reactivation of urease in Yersinia pestis is determined by one G residue at a specific position in the ureD gene.Infect Immun. 2001; 69: 170-176Crossref PubMed Scopus (57) Google ScholarPlasmids pZero-2Cloning vectorInvitrogen pNZYRL lactis pSH71 replicon, allows translational fusion to the usp45 signal sequence downstream of the usp45 promoter, CmrDaniel et al57Daniel C. Poiret S. Goudercourt D. et al.Selecting lactic acid bacteria for their safety and functionality by use of mouse colitis model.Appl Environ Microbiol. 2006; 72: 5799-5805Crossref PubMed Scopus (106) Google Scholar pMEC237lcrV gene fused to usp45 signal sequence in pNZYRThis study pMEC243mIL-10 (murine) fused to usp45 signal sequence in pNZYRThis studyCmr, chloramphenicol resistance. Open table in a new tab Cmr, chloramphenicol resistance. To generate plasmid pMEC237, a polymerase chain reaction (PCR) was performed on total genomic DNA from Y pseudotuberculosis strain 32777 using the specific OMEC268 (5′CAGTTGACATGATTAGAGCCTACGAAC3′) sense primer and OMEC269 (5′CCCAAGCTTTCATTTACCAGACGTGTCATC3′) antisense primer, which hybridize with the 5′ and 3′ ends of the LcrV-encoding region, respectively. The forward and reverse primers contained HincII and HindIII restriction sites, respectively (underlined in the previously mentioned sequences). The resulting amplicon was subcloned into the pZero-2 plasmid, and the construction was verified by DNA sequencing. Subsequently, the insert was digested with the HincII/HindIII endonucleases and subcloned into the HincII/HindIII-digested plasmid pNZYR. The resulting construct (bearing the LcrV-coding sequence fused to the Usp45 secretion signal downstream of the lactococcal Usp45 promoter21van Asseldonk M. Rutten G. Oteman M. et al.Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp lactis MG1363.Gene. 1990; 95: 155-160Crossref PubMed Scopus (184) Google Scholar) was subsequently introduced into L lactis MG1363 by electrotransformation, as described elsewhere.22Wells J.M. Wilson P.W. Le Page R.W. Improved cloning vectors and transformation procedure for Lactococcus lactis.J Appl Bacteriol. 1993; 74: 629-636Crossref PubMed Scopus (167) Google Scholar A similar strategy was used to construct plasmid pMEC243. Mature murine IL-10–encoding complementary DNA (cDNA) was generated and amplified from total RNA isolated from stimulated murine bone marrow–derived dendritic cells, using reverse PCR with the specific OMEC286 (5′GCGTCGACAGCAGGGGCCAGTACAGC3′) sense primer and OMEC287 (5′CCCAAGCTTAGCTTTTCATTTTGATC3′) antisense primer. Molecular biology procedures were performed as described by Sambrook et al.23Sambrook J. Fritsch E.F. Maniatis T. Molecular cloning: a laboratory manual.2nd ed. Cold Spring Harbor Laboratory, New York, NY1989Google ScholarL lactis MG1363 containing the empty vector pNZYR (referred to as "Ll") served as a control. Hence, lactococcal strains harboring pMEC237 and pMEC243 were designated Ll-LcrV and Ll-IL-10, respectively. Bacterial culture supernatants were filtered through 0.22-μm Millex HA membranes (Millipore, Cork, Ireland), and proteins were precipitated with 100% trichloracetic acid. After washing with ice-cold ethanol, pellets were solubilized in Laemmli buffer with β-mercaptoethanol. Proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose blots. LcrV was detected by using a mouse monoclonal specific anti-LcrV antibody (1:50,000)24Motin V.L. Nakajima R. Smirnov G.B. et al.Passive immunity to yersiniae mediated by anti-recombinant V antigen and protein A-V antigen fusion peptide.Infect Immun. 1994; 62: 4192-4201PubMed Google Scholar and then a horseradish peroxidase–conjugated rabbit anti-mouse immunoglobulin G (1:100,000; Sigma-Aldrich). Immune complexes were revealed using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Purified recombinant Y pestis LcrV25Gomes-Solecki M.J. Savitt A.G. Rowehl R. et al.LcrV capture enzyme-linked immunosorbent assay for detection of Yersinia pestis from human samples.Clin Diagn Lab Immunol. 2005; 12: 339-346PubMed Google Scholar was used as a reference for quantifying lactococcal LcrV production. Microtiter plates (Nunc-Immuno Plate, Roskilde, Denmark) were coated with a specific mouse anti-LcrV monoclonal antibody (1:10,000) and thereafter incubated with increasing concentrations of purified LcrV or serial dilutions of crude L lactis supernatants. Rabbit anti-LcrV polyclonal serum26Nilles M.L. Williams A.W. Skrzypek E. et al.Yersinia pestis LcrV forms a stable complex with LcrG and may have a secretion-related regulatory role in the low-Ca2+ response.J Bacteriol. 1997; 179: 1307-1316Crossref PubMed Google Scholar (1:10,000) and then peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:50,000) were used for detection. For quantification of LcrV secretion in vivo, the entire mouse colon (9 cm long) was homogenized in phosphate-buffered saline containing 1% bovine serum albumin and then sonicated. Peritoneal cells were collected by washing the peritoneal cavity with ice-cold RPMI 1640 (Life Technologies, Paisley, Scotland). After centrifugation, pelleted cells were resuspended at a final concentration of 2 × 106/mL in RPMI 1640 supplemented with 2 mmol/L l-glutamine, 50 μmol/L β-mercaptoethanol, 1 mmol/L sodium pyruvate, 150 μg/mL gentamycin, and 10% heat-inactivated fetal bovine serum. Subsequently, cells were plated in 24-well tissue culture dishes (Corning, NY) and incubated at 37°C in a humidified 5% co2 atmosphere for 24 hours. Nonadherent cells were removed by washing the cell culture with sterile, endotoxin-free phosphate-buffered saline. E coli lipopolysaccharide (LPS; Sigma-Aldrich) was used for macrophage stimulation. Macrophages (2 × 106 cells/well) were incubated for 3 hours with phosphate-buffered saline containing either stationary-phase L lactis (106 colony-forming units/well) or no bacteria at all, before a 24-hour contact period with 2.5 μg/mL LPS. Seven- to 9-week-old female BALB/c, C57BL/6, IL-10−/− (C57BL/6 background) mice were purchased from Charles River (St Germain sur l'Arbresle, France), and TLR2−/− mice (C57BL/6 background) were kindly provided by Dr S. Akira (University of Tokyo).27Takeuchi O. Hoshino K. Kawai T. et al.Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components.Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2845) Google Scholar We did not observe any spontaneous signs of inflammation in the colons of both IL-10−/− and TLR2−/− mice. Experiments were performed in an accredited establishment (no. A59107; Institut Pasteur de Lille) according to European guidelines (number 86/609/CEE), and animal protocols were approved by the local ethics committee. Groups of 10 mice were given either carbonate buffer (control mice) or 2 × 108 live L lactis (treated mice) daily for 5 consecutive days via the intragastric route. Acute colitis was triggered on day 5 by intrarectal administration of a 50-μL solution of TNBS (Sigma-Aldrich) in 50% ethanol; 100 mg/kg and 150 mg/kg yielded similar severities of intestinal inflammation in BALB/c and C57BL/6 mice, respectively.28Foligne B. Nutten S. Steidler L. et al.Recommendations for improved use of the murine TNBS-induced colitis model in evaluating anti-inflammatory properties of lactic acid bacteria: technical and microbiological aspects.Dig Dis Sci. 2006; 51: 390-400Crossref PubMed Scopus (73) Google Scholar Animals were subsequently monitored daily for loss of body weight. Three days after induction of colitis, mice were killed; blood samples were immediately taken and stored in heparinized tubes. After mouse dissection, 2 independent observers blindly scored the macroscopic inflammation of the colon on the Wallace scale.29Wallace J.L. MacNaughton W.K. Morris G.P. et al.Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease.Gastroenterology. 1989; 96: 29-36Abstract PubMed Scopus (0) Google Scholar The percent relative protection was calculated as described previously: 100 × (Average Wallace Score of Control Mice − Average Wallace Score of Treated Mice)/Average Wallace Score of Control Mice.28Foligne B. Nutten S. Steidler L. et al.Recommendations for improved use of the murine TNBS-induced colitis model in evaluating anti-inflammatory properties of lactic acid bacteria: technical and microbiological aspects.Dig Dis Sci. 2006; 51: 390-400Crossref PubMed Scopus (73) Google Scholar Two-centimeter-long fragments of the distal colon were collected and frozen at −80°C for histologic analysis; paraffin-embedded 5-μm sections stained with May–Grünwald–Giemsa were examined under the microscope, and tissue lesions were scored according to the Ameho criteria.30Ameho C.K. Adjei A.A. Harrison E.K. et al.Prophylactic effect of dietary glutamine supplementation on interleukin 8 and tumour necrosis factor alpha production in trinitrobenzene sulphonic acid induced colitis.Gut. 1997; 41: 487-493Crossref PubMed Scopus (220) Google Scholar Additionally, the degree of polymorphonuclear neutrophil infiltration in the distal colon was assessed by quantifying myeloperoxidase (a granule enzyme), as reported previously.31Bradley P.P. Priebat D.A. Christensen R.D. et al.Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker.J Invest Dermatol. 1982; 78: 206-209Crossref PubMed Scopus (3148) Google Scholar Murine IL-6, IL-10, and serum amyloid protein A (SAA) levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN), BD Pharmingen (San Diego, CA), and Biosource International (Camarillo, CA), respectively, with a lower limit of sensitivity of 15 pg/mL for both IL-6 and IL-10 and of 30 ng/mL for SAA. For quantification of colonic murine IL-10 and IL-6 levels, the entire mouse colon was homogenized, as described previously. Total RNA from colonic biopsy specimens was isolated with the Nucleospin II Extraction Kit (Macheray Nagel, Duren, Germany). Messenger RNAs (mRNAs) were retrotranscribed from 1 μg nucleic acid by using the High-Capacity cDNA Archive Kit according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). The resulting cDNA was amplified by the SYBR Green Real-Time PCR Kit and detected on a Prism 7000 detection system (Applied Biosystems). The forward and reverse primers used were as follows: 5′GACCCTCACACTCAGATCATCTTCT3′ and 5′CCACTTGGTGGT-TTGCTACGA3′ for murine tnf-α (GenBank accession no. NM_013693.1), 5′AATCTATACCTGTCCTGTGTAATGAAAGAC3′ and 5′TGGGTATTGCTTGGGATCCA3′ for murine il-1β (GenBank accession no. NM_008361.2), 5′CATTTGAATTCCCTGGGTGAGA3′ and 5′TGCTCCACTGCCTTGCTCTT3′ for murine il-10 (GenBank accession no. NM_010548.1), 5′AGTTTGTTGAGTCATTCACCAGACA3′ and 5′CCACTGCTTGTACAGCAATTGG3′ for murine cox-2 (GenBank accession no. NM_011198.2), and 5′GAATGGGTCAGAAGGACTCCTATGT3′ and 5′CCATGTCGTCCCAGTTGGTAA3′ for the β-actin–encoding gene actb (GenBank accession no. NM_007393.1). β-actin mRNA was used for normalization. On completion of the PCR amplification, a DNA melting curve analysis was performed to confirm the presence of a single amplicon. Relative mRNA levels (2ΔΔC) were determined by comparing (1) the PCR cycle thresholds (Ct) for the gene of interest and actb (ΔC) and (2) ΔC values for treated and untreated animal groups (ΔΔC), as described previously.32Sierro F. Dubois B. Coste A. et al.Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells.Proc Natl Acad Sci U S A. 2001; 98: 13722-13727Crossref PubMed Scopus (313) Google Scholar Only increases in RNA levels by a factor of >3 were considered to be significant. Acute colitis was induced in BALB/c mice by administration of DSS (mol wt, 36,000–50,000; MP Biomedicals, Illkirch, France) dissolved in the animals' drinking water. Groups of 10 mice were used, and a healthy control group (ie, not exposed to DSS) was given distilled water. Animals were weighed daily throughout the experiment. After the mice were killed, colon lengths and weights were scored before processing for microscopic analysis. Briefly, cross-sectional rings of the midcolon were fixed in 4% formaldehyde and embedded in paraffin. Sections (4 μm) were stained with May–Grünwald–Giemsa, and histologic scoring was performed as described by Hartmann et al.33Hartmann G. Bidlingmaier C. Siegmund B. et al.Specific type IV phosphodiesterase inhibitor rolipram mitigates experimental colitis in mice.J Pharmacol Exp Ther. 2000; 292: 22-30PubMed Google Scholar Statistical analysis was performed using the nonparametric Mann–Whitney U test. Differences were considered to be statistically significant when the P value was <.05. In a first step, we transformed plasmid pMEC237 into L lactis MG1363. This recombinant expression vector (derived from plasmid pNZYR) bears the PCR-generated Y pseudotuberculosis lcrV gene fused to the secretion signal of the lactococcal Usp45 protein. Because the secretion mechanism of Y pseudotuberculosis LcrV has not yet been elucidated,34Skrzypek E. Straley S.C. Differential effects of deletions in lcrV on secretion of V antigen, regulation of the low-Ca2+ response, and virulence of Yersinia pestis.J Bacteriol. 1995; 177: 2530-2542Crossref PubMed Google Scholar we decided to clone the whole protein-encoding region. The segregational and structural stability of pMEC237 in L lactis MG1363 was verified by growing bacteria in broth in the presence and absence of chloramphenicol, because this plasmid bears a chloramphenicol acetylase–encoding gene (see Table 1).35Pavan S. Hols P. Delcour J. et al.Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects.Appl Environ Microbiol. 2000; 66: 4427-4432Crossref PubMed Scopus (131) Google Scholar We confirmed that pMEC237 (like pNZYR) was stable after 100 generations of bacteria in the absence of antibiotic selective pressure (data not shown). LcrV release by the recombinant L lactis (Ll-LcrV) was assessed by Western blot analysis of the microbial culture supernatants (Figure 1). The secreted LcrV protein was detected in the early bacterial growth phase, and its concentration (as quantified by ELISA) in the cell supernatant from stationary phase grown cells was 28 ± 8 μg/mL (mean level ± SEM from 3 independent cultures). Constitutive secretion of recombinant LcrV had no impact on the growth rate of Ll-LcrV (data not shown). ELISA quantification of secreted LcrV in the entire colon of mice fed daily with 2 × 108 live Ll-LcrV for 5 consecutive days gave a per-colon value of 224 ± 30 ng of LcrV protein. The next step consisted in evaluating the in vitro and in vivo functionality of recombinant LcrV. Inflammatory IL-6 production by LPS-induced, BALB/c-derived macrophages was significantly lower when the cells were preincubated with L lactis Ll or Ll-LcrV, although the latter induced a stronger protective effect (Figure 2). In contrast, under the same experimental conditions, cellular production of the anti-inflammatory cytokine IL-10 was significantly enhanced by the LcrV-secreting L lactis strain only (a 5-fold increase). Quantification of IL-10 in the entire colon from BALB/c mice fed daily for 5 consecutive days with L lactis Ll, Ll-LcrV or buffer indicated that when compared with Ll or buffer, oral administration of Ll-LcrV enhanced IL-10 intestinal production whereas the colonic IL-6 concentration did not change (Figure 3). However, there were no significant differences in blood IL-6 or IL-10 levels when comparing the various groups of mice (data not shown). Similar findings were obtained in C57BL/6 mice (data not shown). In contrast, we found that the IL-10 level in the colon of TLR2−/− mice fed with Ll-LcrV or Ll did not significantly differ (mean value of 5 animals ± SEM, 365 ± 100 vs 382 ± 101 pg per colon, respectively), thus showing that IL-10 induction by LcrV-secreting L lactis is indeed TLR2 mediated. We then investigated the ability of the recombinant Lactococcus strain to prevent acute TNBS-induced colitis in BALB/c mice (Figure 4, Figure 5). Intrarectal instillation of TNBS triggered intense inflammation of the distal colon, which was highly infiltrated by polymorphonuclear neutrophils. Daily intragastric administration of 2 × 108 non–LcrV-producing L lactis (Ll) (resuspended in 0.2 mol/L NaHCO3 buffer plus 1% glucose to protect the bacteria against digestive acidity) for 5 consecutive days before TNBS administration did not influence the intensity of colitis. In contrast, Ll-LcrV administration resulted in a significant reduction in intestinal damage, including decreased loss of goblet cells and crypts and a reduction in inflammatory infiltrates (mainly neutrophils) and colon wall thickness. The 50% protection against colitis (calculated from the Wallace score; see Materials and Methods) conferred by this treatment decreased to values of 35% and 20% when the mice were fed with 10-fold and 100-fold less Ll-LcrV, respectively.Figure 5Histologic features of LcrV-mediated protection. The figure shows representative May–Grünwald–Giemsa—stained colon sections on day 3 after instillation of TNBS in BALB/c mice pretreated with lactococcal strains (A) Ll or (B) Ll-LcrV as compared with (C) healthy mice not given TNBS (original magnification 40×). A corresponds to an Ameho score of 5, showing transmural inflammation and important thickening of the colon wall, characterized by prominent i
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