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Department

www.uu.nl/science/

Figure 1

Mice were treated with the TLR-9 agonist CPG-ODN 1826 by oropharyngeal aspiration only once (24h = acute), 5 times (5 days = subacute) or 5 times a week for 5 weeks (Chronic). CPG induces airway inflammation in WT mice but not in TLR-9 KO as LPS did. Moreover, chronic exposure to CPG induces lung emphysema and hypertrophy of the heart.

Microbes and microbial products in cigarette smoke.

Implications for chronic obstructive pulmonary disease.

Gert Folkerts1, Eric Jubinville2, Esmail Mortaz1, Aletta Kraneveld1, Johan Garssen1,3, Caroline Duchaine2, Gillina F.G. Bezemer1,2

1Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands. Email: G.Folkerts@uu.nl

2Département de biochimie, microbiologie et bioinformatique, Université Laval, Ste-Foy Québec, Canada.

3Nutricia Research, Utrecht, The Netherlands.

Introduction There is growing evidence that changes in the lung microbiome are involved in chronic obstructive pulmonary disease (COPD) exacerbations. Cigarette smoke is the best known risk factor for COPD. Health concerns of microbes in tobacco are starting to be acknowledged. Other groups showed that tobacco flakes inhaled from cigarettes could carry bacteria into the lungs. Moreover, bacterial products such as LPS remain in cigarette smoke and could contribute to inflammation. Our group is one of the first suggesting that Toll-like receptors (TLRs) are involved in the pathogenesis of COPD. TLRs are activated by microbial products. Our in vitro studies suggest that cigarette smoke induces inflammation partly via TLR9. TLR9 is activated by bacterial DNA. See Figure below.

Results Here we provided further evidence for a role of TLR9 in the development of COPD. First, we demonstrated that chronic (5 weeks) activation of pulmonary TLR9 in mice leads to lung inflammation, emphysema, heart hypertrophy (Figure 1). Moreover, it is demonstrated that not all bacteria are destroyed during tobacco burning. When smoke from burning cigarettes is passed over agar plates, colony-forming units clearly follow smoke deposition patterns unrelated to tobacco flake transmission. When cigarette smoke is bubbled trough a medium or directly measured in main or side stream viable bacteria can be detected (Figure 2).

Perspective Currently, a role for bioaerosol exposure in the development of COPD is still underappreciated. Our data emphasize that certain microorganisms and their products could become airborne during combustion. This information is relevant for (passive) smokers but also for women and children who use biomass for cooking indoors in developing countries. Better insight into cause could contribute to disease prevention and could open up new treatment strategies..

Bezemer et al, Pharm Rev 2012 (64): 337-358

CpG PBS

0.0

0.1

0.2

0.3

RV

(m

g)/

To

tal (m

g)

CONTROL

CPG

*

0

10

20

30

40

50

60

70

1

Lm

(u

m)

control

CpG

*

0.0

0.5

1.0

Mil

lio

ns

Mac Lym Neu

controlacutesubacutechronic

*

*

* *

*

Inefficient combustion of organic material that

is contaminated with micro-organisms creates an

aerosol containing bacterial fragments and/or

viable micro-organisms that could

subsequently be inhaled.

These micro-organisms and/or their products

contribute to COPD development.

Results Tobacco Extract Dilution

(1-100CFU)

genus Query

lenght

Query

cover

Max Identity

3R4F tobacco 10-3 Bacillus sp 971 100% 99-100%

γ-3R4F tobacco 100 Bacillus sp 950 100% 99%

smoke 2 cigarettes Bacillus sp 940 83% 63%

Working Hypothesis

Plate Large medium small

CS1 - 10 136

CS2 - 2 5

CS3 9 6 8

CS4 4 - 6

CS5 6 14 10

Control 1 - - -

Control 2 - - -

Control 3 - - -

Control 4 - - -

Control 5 - - -

Results Tobacco Smoke

Summery table: Culturable Bacillus species from cigarettes, irradiated cigarettes and cigarette smoke. Figure 2