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Chronic cigarette smoke exposure alters

the murine gut microbiome


Liesbeth Allais ,
Frederiek-Maarten
1 Stephanie Kerckhof ,
Ken 2 Verschuere ,
1 Bracke ,
3

Rebecca De Smet , Debby Laukens , Martine De Vos , Nico Boon , Guy Brusselle ,
1 4 4 2 3

Tom Van de Wiele , Claude Cuvelier


2 1

1Department of Pathology, Ghent University, Ghent, Belgium


2Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent

University, Ghent, Belgium


3Laboratory for Translational Research in Obstructive Pulmonary Diseases, Department of

Respiratory Medicine, Ghent University Hospital, Ghent, Belgium


4Department of Gastro-enterology, Ghent University Hospital, Ghent, Belgium

Smoking kills slowly. I’m not in a hurry..

Background Results
The microbiome plays a crucial role in maintaining intestinal Analysis of the microbiome in smoke- and air-exposed mice revealed that
homeostasis. Disruption of this homeostatic environment microbial community structures changed significantly after smoke
leads to destabilisation of the gut immune system and exposure in all parts of the gut. In addition, the abundance of specific
aberrant immune responses against harmless microbiota, species, in particular Bifidobacterium sp. and Clostridium sp., tended to
which may be involved in the development of Crohn’s disease decrease in response to cigarette smoke in both colonic and ileal samples.
(CD). The most prominent environmental risk factor for CD is (a)
(b)

smoking. Therefore, the present study aims to investigate the


influence of cigarette smoke on the microbiome, in particular
the mucosa-adherent microbiota, and how this is linked to
changes in mucin production.

Methods 0.02026
0.02385
IIa4DC
0.01568
C57BL/6 mice were exposed to cigarette smoke (n = 6) or air (c)
0.02783 0.00818
0.01568
IIb4CC
IIb4DC
(n = 6) according to a well-established protocol. After 24 0.01080
IIa4IL

weeks, the animals were sacrificed and multiple parts of the 0.04145 0.03331
0.01080

0.01021
IIb4IL

gut (ileum, proximal and distal colon) were collected. 0.04308


0.01021
Ia4CC
Ia4DC
0.01110
Daily smoke exposure 0.01865
0.01110
Ib4CC
0.04219 Ib4DC

7 min cigarette smoke exposure 5 days/week 0.11339


Ia4IL

(5 cigarettes) 0.02

Figure 1. (a) Taxonomic distribution of species on genus level, based on 454 pyrosequencing. (b and c)
30 min rest NMDS plot and abundance-based Jaccard-based distance tree showing significantly different community
structures between the smoking and non-smoking group. I: non-smoking. II: smoking. DC: distal colon. IL:
ileum. CC: caecum.
7 min cigarette smoke exposure
(5 cigarettes)
A general analysis of mucin expression, using AB/PAS and HID/AB, could
not demonstrate an altered expression of acidic and neutral mucins, nor
30 min rest
changes in sulphated and sialylated mucins after cigarette smoke
exposure.
7 min cigarette smoke exposure (c)
(a) (b)
(5 cigarettes)

30 min rest

7 min cigarette smoke exposure


(5 cigarettes)

AB/PAS HID/AB
The microbial composition was analysed using denaturing
Figure 2. Protein expression of mucins in ileum and distal colon after air and smoke exposure.
gradient gel electrophoresis (DGGE) and 454 pyrosequencing. (a) Quantitative scoring showed no significant differences in general mucin expression. (b) AB/PAS:
Furthermore, the expression of mucins was determined at Acidic mucins are stained in blue, neutral mucins are stained in purple-pink. (c) HID/AB: Sulfomucins are
stained in brown-black, sialomucins are stained in blue.
both the mRNA level (real-time PCR) and the protein level
(Alcian Blue (AB)/Periodic Acid Schiff (PAS), High Iron Diamine However, in the ileum, mRNA expression of MUC2 and MUC3
(HID)/AB staining). significantly increased after cigarette smoke exposure (p = 0,04 and p =
0,03 respectively). In the colon, an increased expression of MUC4 was
Conclusion observed (p = 0,04).
Comparative microbial analysis in mice showed a general shift in the
mucosa-adherent bacterial population, as well as specific changes in
NS NS NS *

NS * * NS

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 Figure 3. mRNA expression of mucins in the ileum and distal colon after air and smoke exposure.
homeostasis. P-values lower than 0,05 were considered significant. Data are represented as mean±s.e.m.
NS: nonsignificant N = 6 in each group. *: p < 0,05

Contact
Liesbeth Allais
Department of Pathology
5Blok A, De Pintelaan 185, 9000 Gent, Belgium
Liesbeth.allais@ugent.be