Lung

response

to

Bordetella

pertussis

infection

in

mice

identified

by

gene-expression

profiling

Host

genetics

determines

the

course

of

Bordetella

pertussis

infection

in

mice

Previously

we

found

four

loci

and

three

novel

loci

designated

Bps

that

are

involved

in

the

control

of

B.

pertussis

infection

The

purpose

of

the

present

study

was

to

identify

candidate

genes

that

could

explain

genetic

differences

in

the

course

of

B.

pertussis

infection

assuming

that

such

genes

are

differentially

regulated

upon

infection

We

therefore

studied

the

course

of

mRNA

expression

in

the

lungs

after

B.

pertussis

infection

Of

the

genes

investigated

were

significantly

differentially

expressed

with

genes

upregulated

and

genes

downregulated

Upregulated

genes

were

involved

in

immune-related

processes

such

as

the

acute-phase

response

antigen

presentation

cytokine

production

inflammation

and

apoptosis

while

downregulated

genes

were

mainly

involved

in

nonimmune

processes

such

as

development

and

muscle

contraction

Pathway

analysis

revealed

the

involvement

of

granulocyte

function

toll-like

receptor

signaling

pathway

and

apoptosis

Nine

of

the

differentially

expressed

genes

were

located

in

were

located

in

and

were

located

in

We

conclude

that

B.

pertussis

infection

induces

a

wide

and

complex

response

which

appears

to

be

partly

specific

for

B.

pertussis

and

partly

nonspecific

We

envisage

that

these

data

will

be

helpful

in

identifying

polymorphic

genes

that

affect

the

susceptibility

and

course

of

B.

pertussis

infection

in

humans

Introduction

The

airway

pathogen

Bordetella

pertussis

can

cause

the

disease

known

as

whooping

cough

Despite

high

vaccination

coverage

pertussis

is

still

an

endemic

disease

with

peaks

in

incidence

every

years

during

the

last

decade

-LRB-

Crowcroft

and

Pebody

de

Greeff

et

al.

-RRB-

Susceptibility

to

and

severity

of

B.

pertussis

infection

in

infants

and

children

vary

widely

-LRB-

Kimman

-RRB-

The

spectrum

of

clinical

symptoms

ranges

from

subclinical

infection

to

mild

disease

severe

whooping

cough

and

death

In

previous

studies

we

examined

genetic

susceptibility

of

mice

to

B.

pertussis

and

identified

loci

in

the

mouse

genome

that

are

involved

in

restriction

of

colonization

and/or

clearance

of

B.

pertussis

We

and

others

have

provided

indications

for

the

role

of

several

polymorphic

host

genes

in

the

course

of

B.

pertussis

infection

including

the

interferon

gamma

receptor

-LRB-

Mahon

et

al.

-RRB-

toll-like

receptor

-LRB-

Higgins

et

al.

Mann

et

al.

-RRB-

and

three

putative

disease

loci

B.

pertussis

susceptibility

locus

and

-LRB-

and

Banus

et

al.

-RRB-

is

a

major

host

factor

involved

in

the

course

of

B.

pertussis

infection

mice

which

carry

a

nonfunctional

gene

exhibit

an

aberrant

course

of

infection

Functional

is

essential

for

an

efficient

TNF-α

and

IFN-γ

response

efficient

clearance

of

bacteria

from

the

lung

and

therewith

reduced

lung

pathology

-LRB-

Banus

et

al.

-RRB-

The

locus

is

located

on

chromosome

spanning

a

region

of

genes

and

has

a

dominant

positive

effect

on

the

clearance

of

B.

pertussis

from

the

lung

The

function

of

most

genes

in

this

locus

is

unknown

Two

other

loci

and

showed

genetic

interaction

and

are

located

on

chromosomes

and

respectively

-LRB-

Banus

et

al.

-RRB-

The

highest

linkage

between

the

number

of

colony-forming

units

-LRB-

CFU

-RRB-

and

the

locus

that

we

observed

showed

a

logarithm

of

the

odds

-LRB-

LOD

-RRB-

score

of

This

LOD

score

and

the

corresponding

P

value

-LRB-

P

-RRB-

are

statistically

significant

also

after

correction

for

multiple

comparisons

-LRB-

P

-RRB-

We

interpreted

these

data

as

suggestive

for

linkage

warranting

further

investigation

-LRB-

Banus

et

al.

-RRB-

In

the

present

study

we

aimed

to

enhance

the

insight

in

the

host

response

and

pathogenesis

of

B.

pertussis

infection

by

identifying

genes

that

are

differentially

expressed

upon

infection

In

particular

we

were

interested

in

identifying

candidate

genes

that

could

explain

genetic

differences

in

the

course

of

B.

pertussis

infection

assuming

that

such

genes

are

differentially

regulated

upon

infection

For

this

purpose

we

infected

mice

and

analyzed

gene-expression

profiles

in

the

lungs

on

days

and

after

inoculation

using

microarrays

We

were

especially

interested

if

genes

located

in

the

and

loci

were

differentially

expressed

after

B.

pertussis

infection

or

that

the

susceptibility

of

these

loci

was

regulated

alternately

Materials

and

methods

Experimental

design

Female

mice

-LRB-

eight

mice

per

group

total

of

-RRB-

were

intranasally

inoculated

with

CFU

of

the

streptomycin-resistant

Tohama

strain

of

B.

pertussis

-LRB-

-RRB-

in

μl

of

Verwey

medium

-LRB-

The

Netherlands

Vaccine

institute

Bilthoven

The

Netherlands

-RRB-

or

as

a

control

with

Verwey

Medium

only

One

and

days

after

inoculation

mice

were

euthanized

To

remove

blood

cells

from

the

lungs

mice

were

perfused

with

phosphate-buffered

saline

and

the

lungs

and

trachea

were

collected

-LRB-

King

et

al.

Willems

et

al.

-RRB-

For

the

extraction

of

RNA

the

right

lung

was

collected

in

the

RNA

stabilization

reagent

-LRB-

RNAlater

Qiagen

Venlo

The

Netherlands

-RRB-

The

left

lung

was

fixed

intratracheally

using

formalin

for

histological

examination

The

number

of

viable

B.

pertussis

bacteria

was

determined

in

the

trachea

to

confirm

a

proper

infection

-LRB-

Verwey

et

al.

-RRB-

Therefore

approximately

cm

of

the

trachea

was

collected

in

μl

of

Verwey

medium

Bacterial

suspensions

were

diluted

in

Verwey

medium

and

the

number

of

CFU

was

determined

by

plating

on

Bordet

Gengou

agar

supplemented

with

sheep

blood

and

μg/ml

streptomycin

-LRB-

Tritium

Microbiology

Veldhoven

The

Netherlands

-RRB-

Plates

were

incubated

for

days

at

°C

before

counting

the

number

of

CFUs

using

a

ProtoCOL

Colony

counter

-LRB-

Synbiosis

Cambridge

UK

-RRB-

Animals

Female

mice

were

kindly

supplied

by

Dr.

Peter

Demant

-LRB-

Department

of

Molecular

and

Cellular

Biology

Roswell

Park

Cancer

Institute

Buffalo

NY

-RRB-

Mice

were

bred

to

the

appropriate

numbers

at

our

animal

testing

facility

in

Bilthoven

The

Netherlands

Mice

were

acclimatized

for

at

least

week

before

the

start

of

the

experiments

Mice

received

standard

laboratory

food

-LRB-

SRM-A

Hope

Farms

Woerden

The

Netherlands

-RRB-

and

tap

water

ad

libitum

All

animal

experiments

were

approved

by

the

Institute

's

Animal

Ethics

Committee

and

were

performed

according

to

the

National

Institutes

of

Health

-LRB-

-RRB-

guidelines

Clinical

and

pathological

examinations

Mice

were

weighed

before

inoculation

and

subsequently

every

day

after

inoculation

to

determine

the

relative

change

in

weight

Lung

weights

were

determined

postmortem

as

a

parameter

for

lung

inflammation

Formalin-fixed

lungs

were

embedded

in

paraplast

-LRB-

Monoject

St

Louis

MO

-RRB-

Sections

-LRB-

μm

-RRB-

were

stained

with

hematoxylin

eosin

Lung

lesions

were

examined

for

infiltration

of

inflammatory

cells

in

the

peribronchiolar

space

-LRB-

peribronchiolitis

-RRB-

infiltration

of

inflammatory

cells

in

the

alveoli

-LRB-

alveolitis

-RRB-

infiltration

of

inflammatory

cells

in

the

perivascular

space

-LRB-

perivasculitis

-RRB-

hypertrophy

of

mucus-producing

glands

free

protein

-LRB-

exudate

-RRB-

and

eosinophilia

Lung

lesions

were

scored

semiquantitatively

as

absent

minimal

slight

moderate

marked

or

strong

as

previously

described

-LRB-

Barends

et

al.

-RRB-

Transcription

profiling

Lungs

were

incubated

in

RNAlater

-LRB-

Qiagen

-RRB-

at

°C

for

day

after

which

the

tissues

were

transferred

to

a

fresh

tube

and

stored

at

°C

RNA

was

extracted

by

using

the

midi-RNA

isolation

kit

-LRB-

Qiagen

-RRB-

RNA

concentrations

were

measured

using

a

NanoDrop

Spectrophotometer

-LRB-

NanoDrop

Technologies

Wilmington

DE

-RRB-

RNA

quality

was

determined

using

the

Bioanalyzer

-LRB-

Agilent

Technologies

Santa

Clara

CA

-RRB-

and

was

expressed

as

the

RNA

Integrity

Number

-LRB-

RIN

-RRB-

as

defined

by

the

manufacturer

Only

samples

with

a

RIN

score

of

at

least

eight

-LRB-

on

a

scale

-RRB-

were

used

for

expression

profiling

Microarray

slides

were

spotted

at

the

Microarray

Department

of

the

University

of

Amsterdam

The

slides

contain

oligo

nucleotides

from

the

Sigma-Compugen

Mouse

oligonucleotide

library

additional

oligo

nucleotides

of

genes

of

which

were

located

in

the

region

spotted

in

duplicate

-LRB-

Isogen

Life

science

IJsselstein

The

Netherlands

-RRB-

as

well

as

appropriate

controls

and

blank

spots

Based

on

RNA

quality

and

yield

RNA

isolated

from

six

mice

per

group

was

selected

for

amplification

and

labeling

RNA

amplification

and

labeling

were

carried

out

using

the

Amino

Allyl

MessageAmp

II

aRNA

kit

-LRB-

Ambion

Austin

TX

-RRB-

using

μg

of

total

RNA

as

starting

material

RNA

samples

from

individual

mice

were

labeled

with

A

common

reference

containing

a

RNA

pool

of

all

samples

isolated

was

labeled

with

After

hybridization

and

washing

the

arrays

were

scanned

using

a

ScanArray

microarray

scanner

-LRB-

Perkin-Elmer

Wellesley

MA

-RRB-

Median

and

spot

signals

were

determined

using

ArrayVision

-LRB-

Imaging

Research

St.

Catharines

ON

Canada

-RRB-

Quality

control

was

performed

by

means

of

visual

inspection

of

the

scanned

images

raw

data

scatter

and

MA

plots

-LRB-

M

is

a

mnemonic

for

the

log-ratios

of

expression

A

is

a

mnemonic

for

the

log-intensity

of

each

spot

Smyth

and

Speed

-RRB-

as

well

as

a

normal

probability

plot

to

assess

signal

distribution

-LRB-

Pennings

and

Heisterkamp

-RRB-

If

more

than

of

the

spots

were

flagged

as

missing

data

slides

were

excluded

from

data

analysis

At

least

five

slides

per

group

were

used

for

further

analysis

Raw

microarray

signal

data

were

normalized

in

the

R

statistical

software

environment

-LRB-

R

Development

Core

Team

-RRB-

using

a

four-step

approach

of

-LRB-

-RRB-

natural

log

transformation

-LRB-

-RRB-

quantile

normalization

of

all

scans

-LRB-

-RRB-

correcting

for

differences

in

the

reference

signal

and

-LRB-

-RRB-

averaging

replicate

spot

data

Significance

of

differences

in

gene

expression

between

the

experimental

groups

was

calculated

in

R

with

analysis

of

variance

The

false

discovery

rate

-LRB-

FDR

-RRB-

was

calculated

according

to

Benjamini

and

Hochberg

-LRB-

-RRB-

The

FDR

is

the

expected

proportion

of

false

positives

among

the

genes

considered

significantly

differentially

expressed

-LRB-

Benjamini

et

al.

Benjamini

and

Hochberg

-RRB-

Gene-expression

data

were

visualized

by

hierarchical

clustering

-LRB-

using

Euclidian

distance

and

Ward

linkage

-RRB-

using

GeneMaths

-LRB-

Applied

Maths

St-Martens-Latem

Belgium

-RRB-

For

this

purpose

gene-expression

values

were

corrected

for

the

overall

average

to

allow

a

neutral

comparison

between

all

B.

pertussis

and

mock-inoculated

groups

Gene

categories

were

defined

by

Gene

Ontology

-LRB-

GO

-RRB-

Classification

and

enrichment

according

to

GO

categories

were

determined

using

DAVID/EASE

-LRB-

Dennis

et

al.

Hosack

et

al.

-RRB-

using

EASE

scores

as

P

values

for

enrichment

Additional

pathway

analysis

was

performed

using

MetaCore

-LRB-

GeneGo

-RRB-

Results

Bacteriologic

and

pathologic

findings

All

B.

pertussis-inoculated

mice

had

between

and

CFUs

in

the

trachea

during

the

first

days

after

inoculation

thereby

confirming

a

proper

infection

A

statistically

significant

decrease

in

the

number

of

bacteria

in

the

trachea

was

observed

over

time

during

the

first

days

postinoculation

-LRB-

P

linear

regression

SPSS

data

not

shown

-RRB-

Infected

mice

lost

up

to

of

their

body

weight

during

the

first

days

postinoculation

while

all

mock-inoculated

mice

gained

weight

up

to

during

these

days

This

difference

in

relative

gain

of

weight

between

mock

and

B.

pertussis-inoculated

mice

is

significant

from

day

to

postinoculation

-LRB-

Supplementary

data

Figure

-RRB-

We

determined

lung

weights

in

relation

to

body

weight

as

a

marker

for

inflammation

All

mock-inoculated

mice

had

relative

lung

weights

of

approximately

during

the

first

days

after

inoculation

while

B.

pertussis-infected

mice

had

relative

lung

weights

of

up

to

of

their

total

body

weight

This

difference

in

relative

lung

weights

between

mock

and

B.

pertussis-inoculated

mice

is

significant

from

day

to

postinoculation

-LRB-

Supplementary

data

Figure

-RRB-

A

summary

of

histopathological

findings

is

presented

in

Table

Intranasal

inoculation

of

B.

pertussis

causes

an

acute

inflammatory

response

that

is

characterized

by

influx

of

polymorphonuclear

leukocytes

-LRB-

PMNs

-RRB-

and

macrophages

starting

in

perivascular

and

peribronchiolar

areas

on

day

and

extending

to

alveolar

walls

and

lumina

on

days

and

Table

evaluation

of

lung

changes

after

B.

pertussis

inoculationParameter

and

gradeNo

of

animalsaMockB

pertussisDay

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aEight

mice

per

group

were

inoculated

with

medium

-LRB-

mock

-RRB-

or

B.

pertussis.bHematoxylin

eosin-stained

slides

were

examined

for

peribronchiolitis

alveolitis

perivasculitis

hypertrophy

of

mucus-producing

glands

and

free

protein

-LRB-

exudate

-RRB-

Lung

lesions

were

scored

semiquantitatively

as

absent

-LRB-

not

included

in

table

-RRB-

minimal

slight

moderate

marked

or

strong.cTotal

percentage

of

animals

with

at

least

minimal

scoring

in

the

specified

category

Expression

profiling

Gene-expression

profiles

were

determined

in

at

least

five

animals

per

group

The

three

mock-inoculated

groups

euthanized

at

days

and

were

statistically

indistinguishable

-LRB-

i.e.

there

were

no

genes

with

FDR

-RRB-

Of

the

approximately

genes

tested

genes

were

significantly

differentially

expressed

in

B.

pertussis-inoculated

lungs

-LRB-

FDR

corresponding

to

P

-RRB-

Expression

levels

of

all

genes

as

well

as

comparisons

to

other

studies

are

presented

in

the

supplementary

data

Table

Hierarchical

clustering

showed

time-dependent

changes

in

gene

expression

with

upregulated

genes

and

downregulated

genes

Of

these

genes

were

more

than

twofold

upregulated

and

were

more

than

twofold

repressed

All

genes

followed

a

similar

trend

in

time

One

day

postinoculation

differential

gene

expression

was

already

observed

but

the

effect

was

larger

on

days

and

postinoculation

-LRB-

Fig.

-RRB-

There

was

no

identifiable

subset

of

genes

that

displayed

a

different

kinetic

trend

in

gene

expression

Fig.

of

microarray

data

from

mock

and

B.

pertussis-inoculated

mice

Analysis

was

performed

on

genes

whose

expression

was

found

to

be

significant

at

an

FDR

a

Hierarchical

clustering

shows

the

segmentation

of

the

genes

clustered

by

gene

expression

Relatively

increased

gene

expression

is

presented

in

red

while

relatively

decreased

gene

expression

is

presented

in

green

The

color

scale

shows

log

-LRB-

-RRB-

ratios

of

gene

expression

levels

compared

to

the

global

average

b

Course

of

B.

pertussis-regulated

gene

expression

diamonds

represent

the

average

of

upregulated

genes

and

squares

represent

the

average

of

downregulated

genes

Error

bars

show

the

standard

deviation

-LRB-

SD

-RRB-

of

the

average

gene

expression

To

assign

functions

to

differentially

expressed

genes

we

classified

them

according

to

GO

categories

In

addition

we

determined

enrichment

for

GO

categories

to

identify

pathophysiological

processes

involved

in

response

to

B.

pertussis

The

most

important

GO

categories

i.e.

with

the

highest

percentage

differentially

expressed

genes

are

summarized

in

Table

Most

upregulated

genes

are

involved

in

immune

response-related

processes

such

as

the

acute

phase

response

antigen

presentation

cytokine

activity

inflammation

and

apoptosis

whereas

downregulated

genes

are

involved

in

nonimmune

processes

such

as

development

muscle

contraction

cytoskeleton

and

general

processes

like

metabolism

Table

of

genes

induced

upon

B.

pertussis

infectiona

UpDownbTotal

on

array

P

valuecAcute-phase

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

up

-RRB-

Antigen

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

up

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

up

-RRB-

Cell

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

up

-RRB-

Chemokine

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

up

-RRB-

Complement

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

up

-RRB-

Cytokine

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

up

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

down

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

down

-RRB-

Immune

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

up

-RRB-

Inflammatory

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

up

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

down

-RRB-

Muscle

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

down

-RRB-

Listed

in

any

of

the

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aClassification

according

to

gene-ontology

-LRB-

GO

-RRB-

categoriesbAnalysis

was

performed

on

regulated

genes.cEase

score

for

GO

category

enrichment

in

the

specified

set

of

genesdSome

genes

are

classified

into

multiple

categories

To

reveal

intergene

relationships

we

performed

MetaCore

analysis

on

the

regulated

genes

in

addition

to

the

GO-based

analysis

presented

above

The

most

significant

pathways

are

presented

in

Table

This

analysis

revealed

the

significance

of

several

pathways

such

as

regulation

of

granulocyte

function

the

toll-like

receptor

-LRB-

TLR

-RRB-

signaling

pathway

the

classic

complement

pathway

and

apoptosis

Table

analysis

of

genes

induced

upon

B.

pertussis

infectionName

of

Pathway

involvedaMain

processP

valueNo

of

genes

in

pathwayExpressedTotalTranscription

regulation

of

granulocyte

developmentImmune

-LRB-

-RRB-

ligands

and

common

TLR

signalling

pathwayImmune

-LRB-

-RRB-

-LRB-

-RRB-

of

IAP

proteins

in

-LRB-

-RRB-

and

HPETE

diosynthesis

and

-LRB-

-RRB-

signaling

via

JAK-STAT

pathwayCell

-LRB-

-RRB-

biosynthesis

and

-LRB-

-RRB-

regulation

pathway

-LRB-

extension

GEFs/GAPs

-RRB-

Immune

-LRB-

-RRB-

in

innate

immunity

responseImmune

-LRB-

-RRB-

-LRB-

-RRB-

complement

pathwayComplement

-LRB-

-RRB-

erythropoietin

signaling

pathway

-LRB-

part

-RRB-

Immune

-LRB-

-RRB-

cascadeCell

-LRB-

-RRB-

NO

antiapoptotic

-LRB-

-RRB-

signaling

pathwayCytokine

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

outside-in

signalingCell

-LRB-

-RRB-

and

adhesionChemokine

-LRB-

-RRB-

was

performed

on

genes

whose

expression

was

found

to

be

significant

at

an

FDR

The

list

is

presented

by

descending

P

values

P

values

represent

the

probability

of

particular

mapping

arising

by

chance

given

the

numbers

of

genes

in

all

Metacore

pathways

genes

involved

in

the

specified

pathway

and

genes

regulated

upon

B.

pertussis

inoculation.ECM

Extracellular

matrix

GAP

GTPase-accelerating

protein

GEF

guanine

nucleotide

exchange

factor

HPETE

hydroperoxyeicosatetraenoic

acid

HETE

hydroxyeicosatetraenoic

acid

IAP

inhibitor

of

apoptosis

interleukin

JAK

Janus

tyrosine

kinase

MIF

macrophage

migration

inhibitory

factor

MAPK

mitogen-activated

protein

kinase

NO

nitric

oxide

RhoA

ras

homolog

gene

family

member

A

STAT

signal

transducers

and

activators

of

transcriptionaClassification

according

to

main

gene-ontology

-LRB-

GO

-RRB-

categories

Genes

located

in

that

were

differentially

regulated

upon

B.

pertussis

inoculation

and

their

function

according

to

the

GO

database

are

presented

in

Table

contains

genes

of

which

nine

were

differentially

expressed

The

strongest

upregulated

genes

in

this

locus

were

tryptophanyl-tRNA

synthetase

-LRB-

Wars

fourfold

-RRB-

and

tumor

necrosis

factor

alpha-induced

protein

-LRB-

-RRB-

Zinc

finger

FYVE

domain

containing

-LRB-

-RRB-

and

kinesin

family

member

-LRB-

-RRB-

were

the

strongest

downregulated

genes

Table

located

in

that

are

differentially

regulated

upon

B.

pertussis

inoculationGenBank

accessionGene

symbolLocationaFold

ratio

changeP

valueDescriptionPathwaybDay

Vaccinia-related

kinase

kinase

Tryptophanyl-tRNA

synthetaseAminoacyl-tRNA

ligase

Heat

shock

protein

alphaProtein

Tumor

necrosis

factor

alpha-induced

protein

Gene

model

-LRB-

NCBI

-RRB-

G-protein

coupled

receptor

Zinc

finger

FYVE

domain

containing

ion

Kinesin

family

member

motor

Phospholipase

D

family

member

Similar

to

hypothetical

protein

is

located

on

chromosome

location

is

presented

as

distance

from

centromere

-LRB-

Mb

-RRB-

bClassification

according

to

gene-ontology

-LRB-

GO

-RRB-

categories

Genes

located

in

and

that

were

differentially

regulated

upon

B.

pertussis

inoculation

are

presented

in

Supplementary

data

Table

is

located

on

chromosome

and

contains

genes

of

which

were

differentially

expressed

-LRB-

-RRB-

was

the

strongest

upregulated

gene

-LRB-

-RRB-

in

this

locus

and

general

transcription

factor

IIIC

polypeptide

beta

was

the

strongest

downregulated

gene

-LRB-

twofold

-RRB-

in

this

locus

is

located

on

chromosome

and

contains

genes

of

which

were

differentially

expressed

Chemokine

-LRB-

C

C

motif

-RRB-

ligand

was

the

strongest

upregulated

gene

-LRB-

-RRB-

in

this

locus

and

RIKEN

cDNA

gene

was

the

strongest

downregulated

gene

-LRB-

-RRB-

in

this

locus

Discussion

We

have

recently

shown

that

genetic

differences

of

the

host

play

an

important

role

in

the

restriction

of

colonization

and/or

clearance

of

B.

pertussis

from

the

lungs

of

mice

-LRB-

Banus

et

al.

-RRB-

In

the

present

study

we

wanted

to

extend

the

understanding

of

the

host

response

to

B.

pertussis

and

identify

genes

and

pathways

that

play

a

role

in

the

pathophysiological

response

to

B.

pertussis

infection

In

particular

we

were

interested

in

identifying

candidate

genes

that

could

explain

genetic

differences

in

the

course

of

B.

pertussis

infection

assuming

that

such

genes

are

differentially

regulated

upon

infection

For

these

purposes

we

studied

the

time

course

of

mRNA-expression

in

the

lungs

of

mice

after

a

B.

pertussis

infection

by

microarray

analysis

We

were

especially

interested

if

genes

located

in

the

and

loci

were

differentially

expressed

after

B.

pertussis

infection

or

that

the

susceptibility

conferred

by

these

loci

was

regulated

alternately

Of

the

approximately

genes

investigated

we

found

-LRB-

-RRB-

genes

to

be

significantly

differentially

expressed

Differential

gene

expression

was

already

observed

on

day

postinoculation

but

the

maximum

effect

was

observed

on

day

postinoculation

This

effect

remained

constant

till

at

least

day

postinoculation

The

observed

course

in

gene

expression

after

B.

pertussis

inoculation

was

similar

to

that

induced

by

another

gram-negative

lung

pathogen

Francisella

tularensis

-LRB-

Andersson

et

al.

-RRB-

In

a

study

of

expression

profiles

in

the

lungs

of

F.

tularensis-infected

mice

-LRB-

-RRB-

genes

out

of

the

genes

investigated

were

upregulated

Genes

involved

in

immune

responses

were

activated

strongly

on

day

postinoculation

but

hardly

or

not

at

all

on

days

and

-LRB-

Andersson

et

al.

-RRB-

As

expected

most

upregulated

genes

are

involved

in

immune

and

inflammation-related

processes

such

as

antigen

presentation

complement

cytokine

and

chemokine

activity

Other

processes

involved

include

the

acute

phase

response

and

metabolism

Most

downregulated

genes

are

involved

in

nonimmune

processes

such

as

metabolism

development

cell

cycle

or

muscle

contraction

We

speculate

that

by

downregulating

these

genes

the

energy

balance

is

shifted

in

favor

of

the

immune

response

Evidently

cellular

influx

in

the

lungs

is

likely

to

influence

the

gene-expression

profile

Our

data

provide

more

detailed

information

on

the

central

role

of

PMNs

in

the

pathogenesis

of

B.

pertussis

infection

Pathogenesis

of

B.

pertussis

infection

is

characterized

by

colonization

and

proliferation

of

the

ciliated

mucosal

cells

resulting

in

damage

of

the

respiratory

epithelium

and

an

acute

increase

in

the

levels

of

inflammatory

cytokines

resulting

in

cellular

infiltrate

in

the

alveolar

spaces

-LRB-

Mattoo

and

Cherry

Mills

Preston

-RRB-

Circulating

PMNs

are

rapidly

recruited

to

the

lungs

to

bind

and

ingest

B.

pertussis

subsequently

killing

the

bacteria

by

a

combination

of

reactive

oxygen

and

granule

components

Finally

the

PMNs

undergo

apoptosis

-LRB-

Kobayashi

et

al.

-RRB-

TLR

ligands

such

as

lipopolysaccharides

-LRB-

LPS

-RRB-

are

critical

components

for

the

recruitment

and

priming

of

PMNs

Serum

antibody-mediated

clearance

of

B.

pertussis

also

requires

a

TLR-induced

early

recruitment

of

PMNs

However

pertussis

toxin

limits

this

rapid

serum

antibody-mediated

clearance

by

inhibiting

PMN

recruitment

-LRB-

Kirimanjeswara

et

al.

-RRB-

As

determined

by

pathway

analysis

in

this

study

the

most

significant

pathways

involved

in

the

response

to

B.

pertussis

infection

are

regulation

of

PMN

function

TLR

signaling

pathway

extracellular

matrix

remodeling

and

apoptosis

It

has

been

described

that

apoptosis

of

PMNs

in

which

plays

an

important

role

-LRB-

Jablonska

et

al.

-RRB-

is

accelerated

after

phagocytosis

of

bacteria

-LRB-

Kobayashi

et

al.

b

-RRB-

Therefore

the

involvement

of

three

out

of

four

pathways

may

at

least

partly

be

explained

by

the

influx

of

PMNs

Our

data

thus

further

illustrate

the

central

role

of

PMNs

in

the

course

of

B.

pertussis

infection

and

the

involved

mechanisms

in

PMN

function

We

found

the

response

to

B.

pertussis

to

be

wide

and

complex

Some

remarkable

findings

were

the

involvement

of

mucin

genes

intracellular

pathogen

resistance

gene

and

the

excessive

upregulation

of

serum

amyloid

A

-LRB-

SAA

-RRB-

genes

We

found

the

mucin

-LRB-

Muc

-RRB-

genes

and

to

be

significantly

upregulated

Expression

of

mucin

in

the

respiratory

tract

is

one

important

innate

host

defense

mechanism

that

respiratory

pathogens

must

overcome

-LRB-

Belcher

et

al.

-RRB-

which

encodes

a

respiratory

tract

mucin

glycoprotein

was

expressed

at

day

upon

B.

pertussis

inoculation

which

encodes

mucin

subtype

B

protein

in

tracheobronchial

epithelial

cells

was

expressed

days

upon

inoculation

Belcher

et

al.

-LRB-

-RRB-

described

that

B.

pertussis

induces

mucin

gene

transcription

by

cells

Evidently

gene

expression

studies

do

not

allow

to

define

the

functional

role

of

regulated

genes

Further

studies

should

define

whether

the

mucin

response

is

favorable

to

the

host

the

pathogen

or

both

In

our

study

we

found

a

threefold

upregulation

of

the

intracellular

pathogen

resistance

gene

-LRB-

-RRB-

mediates

innate

immunity

to

mycobacterium

tuberculosis

-LRB-

Pan

et

al.

-RRB-

Pan

et

al.

-LRB-

-RRB-

speculated

that

the

gene

product

might

function

in

integrating

signals

generated

by

intracellular

pathogens

with

mechanisms

controlling

innate

immunity

cell

death

and

pathogenesis

Our

data

therefore

suggest

that

may

be

involved

in

intracellular

presence

of

B.

pertussis

Interestingly

B.

pertussis

was

shown

to

survive

intracellularly

in

macrophages

PMNs

and

bronchoalveolar

lavage

-LRB-

BAL

-RRB-

cells

of

mice

-LRB-

Hellwig

et

al.

Mills

-RRB-

Among

the

upregulated

genes

upon

B.

pertussis

inoculation

SAA

genes

were

upregulated

to

a

remarkably

high

extent

SAA

is

an

acute

phase

protein

whose

expression

is

markedly

upregulated

during

inflammation

and

infection

-LRB-

Uhlar

and

Whitehead

-RRB-

Among

other

functions

this

protein

plays

an

important

role

in

high

density

lipoprotein

cholesterol

metabolism

by

promoting

cellular

cholesterol

efflux

through

a

number

of

different

efflux

pathways

-LRB-

van

der

Westhuyzen

et

al.

-RRB-

In

our

study

and

were

induced

and

respectively

after

B.

pertussis

infection

again

underlining

the

important

role

for

the

acute

phase

response

after

B.

pertussis

infection

Although

strongly

upregulated

it

is

clear

that

the

acute-phase

response

is

not

specific

for

B.

pertussis

infection

In

a

previous

study

we

observed

a

upregulation

of

the

membrane-receptor

and

a

fivefold

upregulation

of

the

downstream

cytokine

gene

Tnf-α

days

post-B

pertussis

inoculation

by

means

of

quantitative

real-time

polymerase

chain

reaction

-LRB-

PCR

-RRB-

Thus

a

slight

upregulation

-LRB-

-RRB-

of

this

receptor

induces

a

more

pronounced

-LRB-

fivefold

-RRB-

induction

of

this

downstream

proinflammatory

cytokine

-LRB-

Banus

et

al.

-RRB-

In

the

present

study

we

obtained

similar

results

by

using

microarray

analysis

expression

was

increased

while

the

expression

of

Tnfaip

was

increased

days

postinoculation

The

results

of

the

present

study

hereby

confirm

our

previous

observations

In

a

recent

study

a

high

correlation

between

gene-expression

values

determined

by

quantitative

real-time

PCR

and

microarray

was

observed

-LRB-

Canales

et

al.

-RRB-

Given

these

results

and

our

observations

we

conclude

that

the

results

at

least

for

the

two

given

genes

are

similar

for

both

techniques

It

is

very

likely

that

the

expression

profile

to

B.

pertussis

is

partly

unique

to

B.

pertussis

and

partly

nonspecific

To

examine

the

specificity

of

B.

pertussis-regulated

gene

expression

we

compared

our

data

to

other

studies

In

a

recent

review

by

Jenner

and

Young

-LRB-

-RRB-

a

comparison

was

made

between

published

transcriptional

profiling

data

from

studies

that

involved

different

host

pathogen

interactions

A

set

of

genes

that

comprise

a

common

host

transcriptional

response

was

defined

We

found

that

genes

affected

by

B.

pertussis

infection

were

listed

in

this

common

host

pathogen

response

further

illustrating

that

many

genes

regulated

by

B.

pertussis

are

indeed

not

specific

for

B.

pertussis

infection

but

are

involved

in

a

common

response

to

pathogens

One

explanation

for

the

broad

response

in

expression

profiles

is

the

complexity

of

the

bacterium

It

is

for

instance

known

that

LPS

is

a

very

potent

immune

stimulus

which

induces

a

strong

inflammatory

response

-LRB-

Kawai

et

al.

Qin

et

al.

Wells

et

al.

-RRB-

Transcriptional

profiling

of

LPS-induced

acute

lung

injury

in

mice

resulted

in

immune-related

upregulated

genes

-LRB-

Jeyaseelan

et

al.

-RRB-

Of

these

genes

were

also

regulated

upon

B.

pertussis

inoculation

in

this

study

Previously

we

have

demonstrated

that

the

locus

has

a

dominant

positive

effect

on

the

clearance

of

B.

pertussis

from

the

lung

Of

the

genes

in

this

locus

nine

were

differentially

expressed

Some

of

these

genes

could

explain

the

linkage

of

this

locus

with

susceptibility

to

B.

pertussis

infection

Tumor

necrosis

factor

alpha-induced

protein

-LRB-

-RRB-

has

been

described

as

a

tumor

necrosis

factor-alpha-inducible

primary

response

gene

in

endothelial

cells

-LRB-

Sarma

et

al.

-RRB-

Aminoacyl-tRNA

synthetases

catalyze

the

aminoacylation

of

tRNA

with

their

cognate

amino

acid

Tryptophanyl-tRNA

synthetase

-LRB-

Wars

-RRB-

catalyzes

the

aminoacylation

of

tRNA

-LRB-

trp

-RRB-

with

tryptophan

and

is

induced

by

interferon

-LRB-

Ibba

and

Soll

-RRB-

The

role

of

the

nine

genes

in

the

B.

pertussis

infection

process

is

unknown

and

will

be

investigated

in

future

experiments

Thus

by

using

a

microarray

approach

we

selected

nine

possible

candidate

genes

from

the

present

in

However

by

this

approach

we

focused

on

transcriptionally

regulated

genes

and

the

possibility

remains

that

susceptibility

to

B.

pertussis

is

regulated

in

a

different

way

such

as

phosphorylation

or

protein

binding

In

comparison

to

the

region

the

and

regions

contain

considerably

more

genes

namely

and

genes

respectively

of

which

and

were

differentially

expressed

post-B

pertussis

inoculation

Among

these

differentially

expressed

genes

are

several

interesting

candidate

genes

that

may

explain

the

linkage

of

these

loci

with

susceptibility

to

B.

pertussis

infection

was

the

most

strongly

induced

gene

in

the

region

-LRB-

-RRB-

is

a

proinflammatory

cytokine

that

can

be

secreted

by

macrophages

after

activation

-LRB-

Akira

and

Takeda

-RRB-

Among

the

genes

differentially

expressed

in

the

region

was

a

large

group

of

chemokines

of

which

Chemokine

-LRB-

C

C

motif

-RRB-

ligand

was

the

strongest

upregulated

gene

-LRB-

-RRB-

Upon

B.

pertussis

infection

this

large

group

of

chemokines

is

induced

to

promote

immune

cells

to

migrate

to

the

site

of

infection

The

role

of

the

other

B.

pertussis-induced

genes

in

these

loci

is

still

unknown

and

will

be

investigated

in

future

experiments

In

conclusion

we

found

genes

in

the

lungs

of

mice

that

are

differentially

expressed

after

B.

pertussis

inoculation

Most

upregulated

genes

are

involved

in

immune

and

inflammation-related

processes

or

in

nonspecific

processes

Most

downregulated

genes

are

involved

in

nonimmune

processes

A

substantial

number

of

genes

and

pathways

provide

more

detailed

information

on

the

central

role

of

PMNs

in

the

pathogenesis

of

B.

pertussis

In

particular

the

transcriptional

profiles

indicate

the

significance

of

genes

involved

in

TLR

activation

the

recruitment

and

activation

of

PMNs

and

apoptosis

Electronic

supplementary

material

Raw

data

as

well

as

the

detailed

description

of

the

experiment

will

be

uploaded

to

the

freely

accessible

online

database

ArrayExpress

-LRB-

-RRB-

upon

acceptance

of

the

manuscript

Expression

levels

of

all

genes

as

well

as

comparisons

to

other

studies

are

presented

in

the

supplementary

data

Table

Supplementary

data

Fig.

Gain

of

body

weight

after

infection

Relative

gain

of

body

weight

at

different

days

postinoculation

with

medium

-LRB-

mock

-RRB-

or

B.

pertussis

Boxes

represent

the

mean

of

the

relative

weights

of

B.

pertussis

infected

mice

diamonds

represent

the

mean

of

the

relative

weights

of

mock

inoculated

mice

-LRB-

GIF

kb

-RRB-

High

resolution

image

file

-LRB-

EPS

kb

-RRB-

Supplementary

data

Fig.

Lung

weight

after

infection

Relative

lung

weight

of

mice

after

infection

with

B.

pertussis

at

several

days

postinoculation

Solid

bars

represent

the

mean

relative

weight

of

B.

pertussis

infected

mice

grey

bars

represent

the

mean

relative

lung

weight

of

mock

inoculated

mice

-LRB-

GIF

kb

-RRB-

High

resolution

image

file

-LRB-

EPS

kb

-RRB-

Supplementary

data

Table

All

B.

pertussis

induced

genes

and

overlapped

with

other

studies

genes

induced

upon

B.

pertussis

inoculation

whose

expression

was

found

to

be

significant

at

an

FDR

of

which

had

an

FR

higher

then

bJenner

and

Young

-LRB-

-RRB-

cPalmer

et

al.

-LRB-

-RRB-

BMC

Genomics

dB

cells

and

following

columns

are

subsets

described

by

Palmer

et

al.

-LRB-

-RRB-

-LRB-

XLS

kb

-RRB-

Supplementary

data

Table

Genes

located

in

and

that

are

differentially

regulated

upon

B.

pertussis

inoculation

-LRB-

XLS

kb

-RRB-

O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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