Differential Effects of Escherichia coli Nissle and Lactobacillus rhamnosus Strain GG on Human Rotavirus Binding, Infection, and B Cell Immunity This information is current as of October 1, 2021. Sukumar Kandasamy, Anastasia N. Vlasova, David Fischer, Anand Kumar, Kuldeep S. Chattha, Abdul Rauf, Lulu Shao, Stephanie N. Langel, Gireesh Rajashekara and Linda J. Saif J Immunol 2016; 196:1780-1789; Prepublished online 22 January 2016; Downloaded from doi: 10.4049/jimmunol.1501705 http://www.jimmunol.org/content/196/4/1780 Supplementary http://www.jimmunol.org/content/suppl/2016/01/22/jimmunol.150170 http://www.jimmunol.org/ Material 5.DCSupplemental References This article cites 66 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/196/4/1780.full#ref-list-1 Why The JI? Submit online. by guest on October 1, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Differential Effects of Escherichia coli Nissle and Lactobacillus rhamnosus Strain GG on Human Rotavirus Binding, Infection, and B Cell Immunity Sukumar Kandasamy, Anastasia N. Vlasova, David Fischer, Anand Kumar, Kuldeep S. Chattha, Abdul Rauf, Lulu Shao, Stephanie N. Langel, Gireesh Rajashekara, and Linda J. Saif Rotavirus (RV) causes significant morbidity and mortality in children worldwide. The intestinal microbiota plays an important role in modulating host–pathogen interactions, but little is known about the impact of commonly used probiotics on human RV (HRV) infection. In this study, we compared the immunomodulatory effects of Gram-positive (Lactobacillus rhamnosus strain GG [LGG]) and Gram-negative (Escherichia coli Nissle [EcN]) probiotic bacteria on virulent human rotavirus (VirHRV) infection and Downloaded from immunity using neonatal gnotobiotic piglets. Gnotobiotic piglets were colonized with EcN, LGG, or EcN+LGG or uncolonized and challenged with VirHRV. Mean peak virus shedding titers and mean cumulative fecal scores were significantly lower in EcN- colonized compared with LGG-colonized or uncolonized piglets. Reduced viral shedding titers were correlated with significantly reduced small intestinal HRV IgA Ab responses in EcN-colonized compared with uncolonized piglets post-VirHRV challenge. However the total IgA levels post-VirHRV challenge in the intestine and pre-VirHRV challenge in serum were significantly higher in EcN-colonized than in LGG-colonized piglets. In vitro treatment of mononuclear cells with these probiotics demonstrated that http://www.jimmunol.org/ EcN, but not LGG, induced IL-6, IL-10, and IgA, with the latter partially dependent on IL-10. However, addition of exogenous recombinant porcine IL-10 + IL-6 to mononuclear cells cocultured with LGG significantly enhanced IgA responses. The greater effectiveness of EcN in moderating HRV infection may also be explained by the binding of EcN but not LGG to Wa HRV particles or HRV 2/4/6 virus-like particles but not 2/6 virus-like particles. Results suggest that EcN and LGG differentially modulate RV infection and B cell responses. The Journal of Immunology, 2016, 196: 1780–1789. otavirus (RV) is a leading cause of diarrhea. It causes an shown a role for the intestinal microbiota in modulating enteric estimated 480,000 deaths in children ,5 years of age in viral infections and oral vaccine responses (6, 7). Ablation of the by guest on October 1, 2021 R developing countries (1). The efficacy of the available intestinal microbiota reduced the severity of RV infection and mod- RV vaccines is low in developing countries compared with de- ulated RV-induced adaptive immunity in mice (8). A higher abun- veloped countries (2). Many factors, such as malnutrition, dance of Clostridiales, Enterobacteriales, and Pseudomonadales micronutrient deficiencies, and breastfeeding (3–5), are implicated was associated with poor oral poliovirus vaccine responses in in- in the lower efficacy of enteric vaccines in impoverished countries. fants, whereas higher bifidobacteria abundance was positively cor- In addition to the aforementioned factors, recent studies have also related with greater oral poliovirus vaccine-specific T cell and Ab responses (9). Previous studies also showed a direct role of com- mensals in enhancing enteric viral infections, including poliovirus Food Animal Health Research Program, Ohio Agricultural Research and Develop- (10) and mouse mammary tumor virus (11) infections. Thus, the ment Center, Veterinary Preventive Medicine Department, Ohio State University, composition of the microbiota or certain members of commensal Wooster, OH 44691 microbial communities play a significant role in modulating viral ORCIDs: 0000-0003-1956-4503 (S.K.); 0000-0002-0917-2349 (D.F.); 0000-0002- 1441-4744 (L.S.). infections and host immunity to pathogens and vaccines. Probiotics are increasingly used to enhance oral vaccine re- Received for publication July 31, 2015. Accepted for publication December 14, 2015. sponses and to treat some enteric infections (12) as well as various This work was supported by National Institute of Allergy and Infectious Diseases/ National Institutes of Health Grant R01 A1099451 (to L.J.S.) and federal funds inflammatory diseases of the gastrointestinal (GI) tract in children appropriated to the Ohio Agricultural Research and Development Center of Ohio (13). Among probiotics, Gram-positive (G+) probiotics such as State University. Lactobacillus spp or Bifidobacteria spp have been administrated Address correspondence and reprint requests to Dr. Linda J. Saif and Dr. Sukumar in randomized human clinical trials (14, 15) and experimental Kandasamy, Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Veterinary Preventive Medicine Department, Ohio State Uni- studies (16–19) to reduce the severity of RV-induced diarrhea. versity, 1680 Madison Avenue, Wooster, OH 44691. E-mail addresses: saif.2@osu. Among G+ probiotics, Lactobacillus rhamnosus strain GG (LGG) edu (L.J.S.) and [email protected] (S.K.) has been extensively investigated for its beneficial health effects The online version of this article contains supplemental material. such as shortening the duration of human RV (HRV) diarrhea and Abbreviations used in this article: ASC, Ab-secreting cell; EcN, Escherichia coli enhancing HRV-specific immune responses in children (15, 20). Nissle; G2, Gram-negative; G+, Gram-positive; GI, gastrointestinal; Gn, gnotobi- otic; HRV, human rotavirus; LB, Luria–Bertani; LGG, Lactobacillus rhamnosus However, mechanisms of action of LGG on HRV infection and strain GG; MNC, mononuclear cell; PBCD, postbacterial colonization day; PCD, whether LGG has any superior probiotic effects on HRV infection postchallenge day; RV, rotavirus; VirHRV, virulent human rotavirus; VLP, virus- and immunity compared with a G- probiotic such as Escherichia like particle. coli Nissle (EcN) are largely unknown. G+ and G-negative (G2) Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 probiotics/commensals differ in microbe-associated molecular www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501705 The Journal of Immunology 1781 patterns and cell wall constituents, which may differentially Virus shedding, ELISA, ELISPOT, and flow cytometry analysis influence neonatal immune maturation and susceptibility to HRV A cell culture immunofluorescence assay was used to detect shedding of infections. In addition, E. coli is one of the first species to colonize HRV in rectal swab fluids as described previously (34). Ab-secreting cells newborn babies (21). EcN is widely used to treat inflammatory (ASCs) in duodenal MNCs were determined as described previously (36). disorders such as ulcerative colitis in humans (22). Beneficial HRV-specific Ab responses and total Ig levels were determined by ELISA + 2 2 effects of EcN are mediated through enhancing intestinal barrier (37). Flow cytometry staining to identify CD79b CD21 CD2 B cells was performed as described previously (18, 38). function (23) and moderating inflammatory disorders (24). Fur- Bacterial specific Ab responses were determined as previously described thermore, similar to other probiotics, EcN has antimicrobial and with some modifications (39, 40). EcN and LGG were prepared as pre- immunomodulatory properties, such as inhibition of pathogenic viously described for probiotic colonization. Wells of a 96-well ELISA 7 bacterial invasion of epithelial cells (25), induction of b-defensin plate were coated with 100 ml13 10 bacteria diluted in 0.05 M bicar- bonate buffer and incubated at 4˚C overnight. After overnight incubation, in epithelial cells (26), and modulation of T cell proliferation (27). bacteria were fixed to the plates by adding 100 ml of 80% acetone per well However, the role of EcN in the maturation of