Liesbeth Allais. 1. , Frederiek-Maarten Kerckhof. 2. , Stephanie Verschuere. 1. , Ken Bracke. 3. ,. Rebecca De Smet. 1. , Debby Laukens. 4. , Martine Devos. 4.
Chronic cigarette smoke exposure alters the murine gut microbiome Liesbeth Allais1, Frederiek-Maarten Kerckhof2, Stephanie Verschuere1, Ken Bracke3, Rebecca De Smet1, Debby Laukens4, Martine Devos4, Nico Boon2, Guy Brusselle3, Tom Van de Wiele2, Claude Cuvelier1 1
Department of Pathology, Ghent University, Ghent, Belgium
2
Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium 3
Laboratory for Translational Research in Obstructive Pulmonary diseases, Department of Respiratory Medicine, Ghent University Hospital, Ghent, Belgium 4
Department of Gastroenterology, Ghent University Hospital, Ghent, Belgium
Background The microbiome plays a crucial role in maintaining intestinal homeostasis. Disruption of this homeostatic environment leads to destabilisation of the gut immune system and aberrant immune responses against harmless microbiota, which may be involved in the development of Crohn’s disease (CD). The most prominent environmental risk factor for CD is smoking. Therefore, the present study aims to investigate the influence of cigarette smoke on the microbiome, in particular the mucosaadherent microbiota, and how this is linked to changes in mucin production. Methods C57BL/6 mice were exposed to cigarette smoke (n = 6) or air (n = 6) according to a well-established protocol. After 24 weeks, the animals were sacrificed and multiple parts of the gut (ileum, proximal and distal colon) were collected. The microbial composition was analysed using denaturing gradient gel electrophoresis (DGGE) and 454 pyrosequencing. Furthermore, the expression of mucins was determined at both the mRNA level (real-time PCR) and the protein level (Alcian Blue (AB)/Periodic Acid Schiff (PAS), High Iron Diamine (HID)/AB staining). Results Analysis of the microbiome in smoke- and air-exposed mice revealed that microbial community structures changed significantly after smoke exposure in all parts of the gut. In addition, the abundance of specific species, in particular Bifidobacterium sp. and Clostridium sp., tended to decrease in response to cigarette smoke in both colonic and ileal samples. A general analysis of mucin expression, using AB/PAS and HID/AB, could not demonstrate an altered expression of acidic and neutral mucins, nor changes in sulphated and sialylated mucins after cigarette smoke exposure. However, in the ileum, mRNA expression of MUC2 and MUC3 significantly increased after cigarette smoke exposure (p = 0,04 and p = 0,03 respectively). In contrast, colonic expression of MUC2 and MUC3 was unaltered, but an increased expression of MUC4 was observed (p = 0,04). Conclusion Comparative microbial analysis in mice showed a general shift in the mucosa-adherent bacterial population, as well as specific changes in Bifidobacterium sp. and Clostridium sp., in response to cigarette smoke. In addition, smoking alters intestinal mucins, which play an important role in the gut barrier, but also in the colonization efficiency of specific gut microbiota. These findings may point to a role for altered interactions between the microbiome and intestinal mucosa contributing to the effect of smoking on intestinal homeostasis.
