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Host Lysozyme-Mediated Lysis of Lactococcus Lactis Facilitates Delivery of Colitis-Attenuating Superoxide Dismutase to Inflamed Colons

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Host Lysozyme-Mediated Lysis of Lactococcus Lactis Facilitates Delivery of Colitis-Attenuating Superoxide Dismutase to Inflamed Colons Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons Sonia A. Ballala,b,c,1, Patrick Veigab,c,d,1, Kathrin Fennb,c,1, Monia Michaudb,c, Jason H. Kimb,c, Carey Ann Gallinib,c, Jonathan N. Glickmane,f, Gaëlle Quéréd, Peggy Garaultd, Chloé Béald, Muriel Derriend, Pascal Courting,h, Saulius Kulakauskasg,h, Marie-Pierre Chapot-Chartierg,h, Johan van Hylckama Vliegd,2, and Wendy S. Garrettb,c,i,j,k,3 aDivision of Gastroenterology, Hepatology, and Nutrition, Boston Children’s Hospital, Boston, MA 02115; bDepartment of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA 02115; cDepartment of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, MA 02115; dDanone Nutricia Research, 91767 Palaiseau, France; eDepartment of Pathology, Harvard Medical School, Boston, MA 02115; fMiraca Life Sciences, Newton, MA 02464; gInstitut National de la Recherche Agronomique, UMR 1319 Micalis, 78352 Jouy-en-Josas, France; hAgroParisTech, UMR Micalis, 78352 Jouy-en-Josas, France; iDepartment of Medical Oncology, Dana–Farber Cancer Institute, Boston, MA 02115; jDepartment of Medicine, Harvard Medical School, Boston, MA 02115; and kBroad Institute of MIT and Harvard, Cambridge, MA 02142 Edited by Ralph R. Isberg, Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, MA, and approved May 11, 2015 (received for review February 3, 2015) Beneficial microbes that target molecules and pathways, such patient heterogeneity. Preclinical studies that use model systems as oxidative stress, which can negatively affect both host and that recapitulate key features of the human disease are needed microbiota, may hold promise as an inflammatory bowel disease to elucidate the mechanism of action of beneficial microbes on therapy. Prior work showed that a five-strain fermented milk hosts and their microbiota. Such information facilitates clinical product (FMP) improved colitis in T-bet−/− Rag2−/− mice. By vary- trial design by identifying patients with the host and microbial ing the number of strains used in the FMP, we found that Lacto- features most likely to benefit from the bioactivity of a beneficial coccus lactis I-1631 was sufficient to ameliorate colitis. Using microbe. Identifying microbes that target molecules and path- comparative genomic analyses, we identified genes unique to ways such as oxidative stress—which negatively affects both host L. lactis I-1631 involved in oxygen respiration. Respiration of oxygen and microbiota—affords opportunities for new IBD therapies. results in reactive oxygen species (ROS) generation. Also, ROS are Building on prior studies examining how a five-strain fermented produced at high levels during intestinal inflammation and cause milk product (FMP) affected the gut microbiome in a preclinical tissue damage. L. lactis I-1631 possesses genes encoding enzymes model of colitis and human subjects (10–12), herein we focused on that detoxify ROS, such as superoxide dismutase (SodA). Thus, we how individual bacterial strains in the FMP affected host response hypothesized that lactococcal SodA played a role in attenuating in several preclinical colitis models. One of the five strains, Lac- colitis. Inactivation of the sodA gene abolished L. lactis I-1631’s tococcus lactis subsp. lactis CNCM I-1631 (L. lactis I-1631), reduced beneficial effect in the T-bet−/− Rag2−/− model. Similar effects were obtained in two additional colonic inflammation models, − − Significance Il10 / mice and dextran sulfate sodium-treated mice. Efforts to understand how a lipophobic superoxide anion (O −) can be de- 2 Microbes hold promise as an inflammatory bowel disease (IBD) toxified by cytoplasmic lactoccocal SodA led to the finding that therapy. Lactococcus lactis, which has not been appreciated host antimicrobial-mediated lysis is a prerequisite for SodA release − as a beneficial microbe, attenuated colitis in three preclinical and SodA’s extracytoplasmic O scavenging. L. lactis I-1631 may 2 mouse IBD models. Neither colonization nor an intact bacte- represent a promising vehicle to deliver antioxidant, colitis-attenuat- rium throughout the colon per se was required. Rather, host ing SodA to the inflamed intestinal mucosa, and host antimicrobials MICROBIOLOGY lysozyme-mediated lysis in an inflamed colon led to L. lactis’s mayplayacriticalroleinmediatingSodA’s bioaccessibility. release of its superoxide dismutase, which was necessary for its colitis-attenuating and oxidative stress-reducing activity. Lactococcus lactis | oxidative stress | lysozyme | colitis | probiotics Overall, these findings unveil a mechanism by which a bacte- rium offers benefits to the host but requires the host for tar- nflammatory bowel disease (IBD) pathophysiology is driven by geted release of this beneficial activity. Furthermore, because Iboth host genetic mutations and the gut microbiota. Immune L. lactis is generally regarded as safe, it represents an oppor- dysregulation in IBD can result from deficiencies in acute in- tunity for rapid bench-to-bedside testing in IBD. flammatory response pathways (1) or impaired counterregulation of immune responsiveness (2). Host production of reactive oxygen Author contributions: S.A.B., P.V., K.F., J.v.H.V., and W.S.G. designed research; S.A.B., P.V., species (ROS) is an evolutionarily conserved response to microbes K.F., M.M., J.H.K., C.A.G., G.Q., P.G., C.B., P.C., and W.S.G. performed research; M.D. contributed new reagents/analytic tools; S.A.B., P.V., K.F., M.M., J.N.G., P.C., S.K., M.-P.C.-C., (3). However, chronic and excessive ROS up-regulate host in- J.v.H.V.,andW.S.G.analyzeddata;andS.A.B.,P.V.,K.F.,andW.S.G.wrote flammatory pathways (4, 5) and result in oxidative stress. Chronic the paper. intestinal inflammation and oxidative stress affect not only the host Conflict of interest statement: P.V., G.Q., P.G., C.B., M.D., and J.v.H.V. are employees of but also the microbiota. Oxidative stress within the lumen is a fitness and hold equity in Danone Nutricia Research. This study was supported by a grant from Danone Nutricia Research, R01CA154426 (National Cancer Institute), K08AI078942 (Na- challenge for gut anaerobic bacteria. IBD patient fecal microbiomes tional Institute of Allergy and Infectious Diseases), a Burroughs Wellcome Career in Med- reflect a pattern of response to oxidative stress with enrichments in ical Sciences Award, and a Searle Scholars Award. genes for sulfate transport and cysteine and glutathione metabolism This article is a PNAS Direct Submission. (6). In IBD, oxidative stress contributes to chronic inflammation Freely available online through the PNAS open access option. and dysbiosis, and modulating oxidative stress may help to restore 1S.A.B., P.V., and K.F. contributed equally to this work. intestinal homeostasis. 2Present address: Chr. Hansen A/S, 2970 Hørsholm, Denmark. Beneficial microbes hold promise for IBD inflammation and 3To whom correspondence should be addressed. Email: [email protected]. dysbiosis (7). However, human clinical trials have shown mixed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. results (8, 9) because of variations in microbes under study and 1073/pnas.1501897112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1501897112 PNAS | June 23, 2015 | vol. 112 | no. 25 | 7803–7808 Downloaded by guest on September 29, 2021 gut oxidative stress and attenuated colitis in three mouse models −/− −/− A T-bet-/- Rag2 -/- C Il10-/- E WT-DSS of colonic inflammation: BALB/c T-bet Rag2 mice, BALB/c 10 10 10 − − ** 12* 12*** / *** * *** Il10 mice, and BALB/c wild-type mice treated with dextran sulfate 8 ** 8 8 sodium (DSS), a colitogenic mucosal disruptant. The colitis-attenu- 6 6 6 L. lactis L. lactis ating activity of I-1631 was dependent on I-1631 4 4 4 superoxide dismutase A (SodA), which reduced colonic epithelial 2 2 2 Histologic Colitis Score Histologic Colitis Score ROS. Our data also support that host factors, increased at sites of Histologic Colitis Score 0 0 0 inflammation, facilitated targeted delivery of L. lactis I-1631 effects. sham milk fermented milk products sham milk L. lactis sham milk L. lactis B. animalis --- ++-- FMP FMP Specifically, lysis of L. lactis I-1631 by the host peptidoglycan hy- L. delbruckeii -- + + + - - L. delbruckeii -- + + + -- drolase and antimicrobial lysozyme-1 appeared to mediate L. lactis L. lactis -- + + -- + I-1631 SodA release and reduction in host oxidative stress. S. thermophilus --- +++ - T-bet-/- Rag2 -/- Il10 -/- WT-DSS B sham L. lactis FMP D sham L. lactis FMP F sham L. lactis FMP Results An L. lactis FMP Attenuates Colitis in T-bet−/− Rag2−/−, Il10−/−, and DSS-Treated Mice. Unraveling the specific bioactivities that ben- eficial microbes afford to a host is challenging. Prior work sug- gested that a five-strain FMP attenuated colitis in an innate immune model of colitis (10) and furnished the opportunity to determine the contributions of these strains to attenuating co- Fig. 1. L. lactis FMP attenuates colitis in T-bet−/− Rag2−/−, Il10−/−, and DSS- litis. We began by varying the number of strains present in the −/− −/− −/− −/− treated BALB/c wild-type mice. (A) Histologic colitis scores from T-bet Rag2 five-strain FMP fed to T-bet Rag2 mice (13). Two single- mice treated as labeled. Symbols represent data from individual mice from strain FMPs prepared with either Bifidobacterium animalis subsp. three experiments. (B) Endoscopic distal colon images (Upper) and H&E − − − − lactis I-2494 (B. animalis I-2494) or L. lactis I-1631 ameliorated section photomicrographs from distal colons (Lower)ofT-bet / Rag2 / mice − − colitis to a level comparable to the five-strain FMP (Fig. 1A). treated as labeled. (C) Histologic colitis scores from Il10 / mice, treated as This was not unexpected for B. animalis I-2494, because this labeled. Symbols represent data from individual mice from three experiments. probiotic species has antiinflammatory activities in various (D) Endoscopic distal colon images and H&E section photomicrographs from − − animal models (14, 15).
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