pneumonia

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Vol7SpecialIssueforweb

EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72

as characterising levels of inflammation in the ears and lungs.

O4.4

The effect of macrophage polarisation and delayed apoptosis in the

innate immune response to Streptococcus pneumoniae infection

Lucy Morris, Helen Marriott, David Dockrell

University of Sheffield, Sheffield, UK

Shifts in macrophage polarisation are associated with COPD pathogenesis. Macrophages undergo apoptosis when infected

with Streptococcus pneumoniae, a common cause of COPD exacerbations, as part of the innate immune response. This

apoptosis is reduced in alveolar macrophages from COPD patients associated with increased expression of the antiapoptotic

protein Mcl-1. We hypothesise that macrophages from transgenic mice that over-express Mcl-1 (CD-68 Mcl-

1) will demonstrate altered polarisation resulting in reduced host defence to pneumococcal infection. Our objectives

were to determine any differences in basal polarisation of wild-type and CD68 Mcl-1 BMDMs; explore phagocytosis,

bacterial killing and apoptosis in polarised wild type and CD68 Mcl-1 BMDMs and MDMs after S. pneumoniae infection;

and understand how cytokine and chemokine repertoire changes in response to S. pneumoniae infection in polarised

wild-type and CD68 Mcl-1 BMDMs and MDMs. Bone marrow derived macrophages (BMDM) from Mcl-1-transgenic

and wild-type littermates were stimulated for 24 hours with cytokines; IL-4 (M2a), IL-10 (M2c) or IFNγ+LPS (M1) and

polarisation determined by western blotting, ELISA and RT-PCR. Polarised macrophages were infected with S. pneumoniae

for defined times and bacterial internalisation and survival assessed. Nuclear fragmentation of mock-infected versus

infected macrophages was quantified by staining with DAPI. Supernatants were collected and assessed for cytokine

expression by ELISA. Cell viability and hypodiploid DNA were assessed by MTT assays and PI staining, respectively. M1

polarisation caused increased levels of hyplodiploid DNA and decreased cell viability compared to other polarisation

states. Assessment of S. pneumoniae internalisation and intracellular survival over a 2–4 hour time course showed

increased bacterial clearance by M1 wild-type and transgenic macrophages. There was also increased apoptosis of M1

macrophages after S. pneumoniae infection 16 hours onwards. M1 polarisation increased proinflammatory cytokine

repertoire in BMDMs after S. pneumoniae infection, however this effect was not replicated in MDMs.

O4.5

Murine respiratory tract microbiome: important interactions with IL-17

and pneumococcal colonisation

Neil Ritchie, Tom Evans

University of Glasgow, Glasgow, UK

IL-17 is known to be important in the control of nasal colonisation with Streptococcus pneumoniae. The nasal microbiome

is a rich environment with a diverse range of bacterial species; the effect of IL-17 on the respiratory tract microbiome

is undefined. We characterised the respiratory tract microbiome of wild type and IL-17RAKO mice before and after

induction of colonisation with S. pneumoniae. Mice were inoculated with 5 x 10 5 cfu of serotype 3 S. pneumoniae.

Nasal and bronchoalveolar lavages were taken and S. pneumoniae quantified. DNA was extracted and V 1

-V 2

region

of the 16S rRNA gene was amplified using barcoded primers. Sequencing was carried out using the Illumina Mi-Seq

platform. On culture, S. pneumoniae achieved higher nasal colonisation density in IL-17RAKO mice. IL-17RAKO mice

had a lower abundance of neutrophils within nasal wash. The resting microbiome of IL-17RAKO mice was significantly

different from wild-type, with decreased diversity and increased abundance of proteobacteria. There was no difference

in the overall abundance of firmicutes (such as S. pneumoniae), although the diversity of firmicutes was significantly

lower in IL-17RAKO mice. The lower airways had much lower bacterial diversity than the nose and the microbiome

between IL-17RAKO and wild-type was similar. Nasal colonisation with S. pneumoniae was associated with a marked

loss of diversity in wild-type mice but little change in diversity among IL-17RAKO. This loss of diversity occurred even in

mice that did not have a high abundance of S. pneumoniae. Microbiome sequencing confirmed the higher abundance

of pneumococcus in nasal samples among IL17RAKO mice and showed a strong negative correlation with Ochrobactrum

anthropi, a nasopharyngeal commensal, suggesting intra-species competition for the same ecological niche. IL-17 is an

important cytokine in defining the resting nasal microbiome and contributes to control of pneumococcal colonisation.

pneumonia 2015 Volume 7

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