Early systemic inflammatory response in mice after a single oral gavage with live Escherichia coli is evidenced by increased TNF-alpha and nitric oxide production Ana Nemec a,⇑ , Aleš Jerin b , Irena Zdovc c , Tomaz ˇ Budefeld d , Frank J.M. Verstraete e , Damijan Erz ˇen f , Marjeta Šentjurc g , Milan Petelin h , Tina Hitti a , Zlatko Pavlica a a Veterinary Faculty Small Animal Clinic, University of Ljubljana, Gerbic ˇeva 60, 1115 Ljubljana, Slovenia b University Medical Centre Ljubljana, Institute of Clinical Chemistry and Biochemistry, Njegoševa 4, Ljubljana, Slovenia c Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbic ˇeva 60, 1115 Ljubljana, Slovenia d Centre for Animal Genomics, Veterinary Faculty University of Ljubljana, Gerbic ˇeva 60, 1115 Ljubljana, Slovenia e Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616, USA f University Clinic of Respiratory and Allergic Diseases Golnik, 4240 Golnik, Slovenia g Joz ˇef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia h Department for Oral Medicine and Periodontology, Faculty of Medicine, University of Ljubljana, Hrvatski trg 1, 1000 Ljubljana, Slovenia article info Article history: Received 26 October 2010 Accepted 22 March 2011 Available online xxxx Keywords: Bacteremia Escherichia coli ATCC 25922 Endotoxemia Nitric oxide Oral inoculation abstract Twenty-four female BALB/c mice were orally inoculated with 10 8 CFU Escherichia coli ATCC 25922 and euthanized 2.5, 7, 13 and 25 h post-inoculation. The levels of organ nitric oxide (NO) and plasma endo- toxin, TNF-alpha and nitrite/nitrate (NO x ) were compared to those found in sham-inoculated mice, to evaluate systemic host-response to a low-level oral exposure to Gram-negative bacteria. Organ bacterial culture and immunohistochemistry for iNOS were performed on lungs, liver, kidneys and brain from all mice. Organ NO and plasma TNF-alpha levels were higher in E. coli-inoculated animals, but no differences were detected in plasma endotoxin levels, NO x or iNOS immunostaining for any of the animal groups. Sin- gle oral gavage with live E. coli stimulates an early systemic immune response in clinically healthy mice as evidenced by increased plasma TNF-alpha and organ NO levels, but bacteremia and endotoxemia are not related to this inflammatory response. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Mucosal epithelia form a major interface between the body and the environment and comprise an extensive vulnerable barrier that is reinforced by numerous innate defense mechanisms cooperating intimately with adaptive immunity to maintain appropriate im- mune responses to entering antigens (Garg et al., 1999; Stokes and Waly, 2006; Brandtzaeg, 2007). Importance of the mucosal im- mune response to (harmful) bacteria is particularly well estab- lished in vaccine research, where live mucosal vaccines, like natural infections, give rise to local (mucosal) and systemic im- mune responses without eliciting disease (Brandtzaeg, 2007; Liu et al., 2009). While the importance of systemic immune response and in particular nitric oxide (NO) production in response to Gram-negative bacteria like Escherichia coli after parenteral inocu- lation is established (Lai and Komarov, 1994; Plonka et al., 2003; Berliner and Fujii, 2004), the NO response following infection through more natural peroral route (especially with bacteria that are usually transmitted orally, like E. coli or a known periodonto- pathogen Porphyromonas gingivalis) is much less investigated. We have previously found that low-level oral infections with some Gram-negative bacteria, i.e. E. coli or P. gingivalis, can stimu- late systemic immune response in the acute (E. coli) and chronic (P. gingivalis) model of infection, as evidenced by stimulation of inducible nitric oxide synthase (iNOS) leading to NO production, without signs of illness in experimental animals (Nemec et al., 2009a,b). Similarly, we have previously reported that lung infec- tion with P. gingivalis also stimulates systemic increase in some cytokines (IL-1beta) before any pneumonia is clinically or histolog- ically detected (Nemec et al., 2009c), which suggests that the whole body is alerted when these bacteria enter the body in low numbers through mucosal surfaces of the gastrointestinal or respiratory tract before any obvious local disease develops. In this study, therefore, we used a previously described murine model of single peroral inoculation with live E. coli, a standard experimental bacterium with a typical bacterial endotoxin (Diya 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.03.021 ⇑ Corresponding author. Present address: Dentistry & Oral Surgery Service, Veterinary Medical Teaching Hospital, University of California, Davis, Davis, CA 95616-8747, USA. Tel.: +1 530 752 2470; fax: +1 530 752 9620. E-mail addresses: anemec@ucdavis.edu (A. Nemec), ales.jerin@kclj.si (A. Jerin), irena.zdovc@vf.uni-lj.si (I. Zdovc), tomaz.budefeld@vf.uni-lj.si (T. Budefeld), fjverstraete@ucdavis.edu (F.J.M. Verstraete), damjan.erzen@klinika-golnik.si (D. Erz ˇen), marjeta.sentjurc@ijs.si (M. Šentjurc), milan.petelin@mf.uni-lj.si (M. Petelin), tina.hitti@gmail.com (T. Hitti), zlatko.pavlica@vf.uni-lj.si (Z. Pavlica). Research in Veterinary Science xxx (2011) xxx–xxx Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc Please cite this article in press as: Nemec, A., et al. Early systemic inflammatory response in mice after a single oral gavage with live Escherichia coli is evidenced by increased TNF-alpha and nitric oxide production. Res. Vet. Sci. (2011), doi:10.1016/j.rvsc.2011.03.021