www.nature.com/scientificreports Normal Gut Normal Gut H pylori tin 2+ PYY 2+ Leptin·PYY3 Lept Insulin Insulin· Ghrelin+ Insulin Chronic Inflammation Retardation Eosinophil Recruitment Eotaxin-1 Eot ystem development (including Reduced Neurogenesis slower brain and mental (usually associated with development) old age) Figure 3 Diagrammatic representation of the effect of normal gut microbiota and H pylori in mice. Normal gut microbiota plays an important role regulation of gut metabolic hormone homeostasis that affects the development of normal body mass and brain. In addition, normal gut microbiota also influences the outcome of H. pylori colonization by affecting the gut metabolic hormone changes and inflammation induced by H pylori. nutritional level has a direct impact on early developmental stages Therefore, it is possible that H. pylori (including brain and mentor development). Furthermore, ghrelin opmental stages will have long-term implication on brain develop- knockout mice had been shown to have increased anxiety in ment. Unfortunately, we did not carry out any behavioral study or se to a variety of stressors, such as acute restraint stress and motor control tests in this study for confirmation. ocial stress, in experimental settings. Higher ghrelin level in GF In conclusion, this study in SPF and GF mice with and without H mice might also explain the altered behavior in GF mice reported pylori imply that the gastric pathogen, H pylori, interacts with nor by Heitz et al. mal gut microbiota, which alters metabolic and inflammatory path Increased eotaxin-1 level in blood plasma is also associated with ways on SPFH mice compared to SPF mice(Figure 3).Our results aging in mice and humans. It has been demonstrated that exposing also suggest that there is an ongoing crosstalk between H pylori and oung mice to eotaxin-I or the blood plasma of older mice decreased the normal gut microbiota which associates with gut inflammation. eurogenesis and cognitive performance in behavioral tasks, Previous data have established causality between H pylori and gastri which are dependent on neurogenesis in the hippocampus. ulcer and stomach cancer. The data presented in this study, connect terestingly, a large population cohort retrospective study in ing H pylori to crosstalk with the normal gut microbiota, imply that Denmark has established a correlation between chronic H. Pylori the onset of H pylori mediated diseases in humans is more complex. and/or gastritis and Parkinson's disease The level of chemokine, Additional experiments are therefore highly warranted, in particula otaxin-I, was found to be highest in SPFH mice(p <0.01)at 16- in early life week post-colonization(Figure 2G). Eotaxin-1 is one of the chemo kines that mediates eosinophil migration, a prominent component of Method mice led us to hypothesize that these chemokines served as early H pylons train and inoculum Preparation. The 57B indication of inflammation and the interplay between H. Pylori sequenced s. Briefly, H pylori was cultured in Brain Heart Infusion Broth and normal gut microbiota may intensify chronic inflammation. supplemented with 0.4% yeast extract and 1%B-cyclodextran(BHYC)for 4 days at SCIENTIFIC REPORTS I 5: 8731 I DO1: 10.1038/ srep08731nutritional level has a direct impact on early developmental stages (including brain and mentor development). Furthermore, ghrelin knockout mice had been shown to have increased anxiety in response to a variety of stressors, such as acute restraint stress and social stress, in experimental settings31. Higher ghrelin level in GF mice might also explain the altered behavior in GF mice reported by Heijtz et al. Increased eotaxin-1 level in blood plasma is also associated with aging in mice and humans32. It has been demonstrated that exposing young mice to eotaxin-1 or the blood plasma of older mice decreased their neurogenesis and cognitive performance in behavioral tasks, which are dependent on neurogenesis in the hippocampus32. Interestingly, a large population cohort retrospective study in Denmark has established a correlation between chronic H. pylori and/or gastritis and Parkinson’s disease33. The level of chemokine, eotaxin-1, was found to be highest in SPFH mice (p , 0.01) at 16- week post-colonization (Figure 2G). Eotaxin-1 is one of the chemo￾kines that mediates eosinophil migration, a prominent component of H. pylori–induced gastritis34. Thus, high level of eotaxin-1 in SPFH mice led us to hypothesize that these chemokines served as early indication of inflammation and the interplay between H. pylori and normal gut microbiota may intensify chronic inflammation. Therefore, it is possible that H. pylori exposure during early devel￾opmental stages will have long-term implication on brain develop￾ment. Unfortunately, we did not carry out any behavioral study or motor control tests in this study for confirmation. In conclusion, this study in SPF and GF mice with and without H. pylori imply that the gastric pathogen, H. pylori, interacts with nor￾mal gut microbiota, which alters metabolic and inflammatory path￾ways on SPFH mice compared to SPF mice (Figure 3). Our results also suggest that there is an ongoing crosstalk between H. pylori and the normal gut microbiota which associates with gut inflammation. Previous data have established causality between H. pylori and gastric ulcer and stomach cancer. The data presented in this study, connect￾ing H. pylori to crosstalk with the normal gut microbiota, imply that the onset of H. pylori mediated diseases in humans is more complex. Additional experiments are therefore highly warranted, in particular in early life. Methods H. pylori Strain and Inoculum Preparation. The C57BL/6 mice-adapted H. pylori strain, 298, was used in this study. The genome of this strain has been fully sequenced35. Briefly, H. pylori was cultured in Brain Heart Infusion Broth supplemented with 0.4% yeast extract and 1% ß-cyclodextran (BHYC) for 4 days at Figure 3 | Diagrammatic representation of the effect of normal gut microbiota and H. pylori in mice. Normal gut microbiota plays an important role regulation of gut metabolic hormone homeostasis that affects the development of normal body mass and brain. In addition, normal gut microbiota also influences the outcome of H. pylori colonization by affecting the gut metabolic hormone changes and inflammation induced by H. pylori. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 8731 | DOI: 10.1038/srep08731 5