20/10/2010

The mammary epithelial cell response to

infection is written in Milk Fat Globules.

Pauline Brenaut, Rama Bangera, Claudia Bevilacqua, Christelle Cebo, Patrice Martin

UMR Génétique Animale et Biologie Intégrative, Equipe Lait, Génome et Santé, Jouy-en-Josas

A recurrent problem for dairy production

Mastitis: an inflammation of mammary gland

Injury

Pathogens entry

inflammationnormal

causes significant economic losses to

the dairy industry (179$/year/cow)(Bar et al., 2008)-Milk production

-Milk income

-Milk quality bonuses

How to detect mastitis?

CXCL3

APP

analysis of MEC gene expression profiles

at early steps of infection

Find precocious markers secreted by MEC

PMN

Somatic Cell Count (SCC)

neutrophils (PMN) first cells

to arrive on inflammation site.

MEC first line of defense,

can play a major part in the

initiation of inflammation.(Chemokine, Acute Phase Protein)

(Pareek et al., 2005 ; Lahouassa et al., 2007)

Which methods are available to study a unique MEC population during the course of infection ?

methods kinetics Representativeness of MEC population

Biopsies

Laser captureMicrodissection(Bevilacqua et al., submitted)

MEC in milk (Boutinaud et al., 2008)

viability during

infection, milk quantity

Contamination by

other cell types

Specific selection of

MEC with laser

MFG a non invasive technique to study MEC

a novel approach first proposed by Maningat et

al., (2007) in human.

Is it possible for livestock species?

Is it really representative of MEC?

Milk fat globules in the alveolar lumen in a specimen

from rat mammary gland. (Heid and Keenan, 2005)

Objectives: Use RNA extracted from MFG

Characterize RNA extracted from MFG is it really representative of MEC?

Validate this technique to livestock

species a good model: goat (fat secretion

pathway similar with human)

Follow kinetics of infection Can we

follow the MEC response to infection using

RNA isolated from MFG?

(Brenaut et al., submitted)

Critical steps to obtain RNA of good quality

Step 1- sampling

Centrifuge milk (50ml) and

put quickly milk fat in TrizolTake a mammary gland

sample (250mg)

Step 2- Freezing at -80°C

Step 3- RNA extraction in the lab

Mammary

Gland

Milk fat

Skim milk

Cell pellet

Use of Trizol LS manufacturer’s

instructions

Use of Trizol manufacturer’s

instructions

Step 4- Quality analysis

Mammary Gland Milk Fat Globule

Presence of RNA in a sufficient amountand in a good quality. This material can beused for further analysis

Analysis of extracted RNA from MFG and MG

Quality and Quantity Bioanalyser AGILENT 2100

Yield: 12-15µg/250mg

RIN: 8 to 8.5

Mammary Gland Milk Fat Globule

Yield: 1-2µg/1ml fat

RIN: 6.4 to 8

Ability to reverse transcript

(on 1 µg of total RNA)

130 ng cDNA130 ng cDNA

Is this RNA representative of MEC RNA?

90% of transcripts are in commonbetween MFG and MG

Use Sheep Gene Expression Microarrays

8x15K (Agilent)

Cohybridization of Mammary Gland (RIN

8.5) and Milk Fat Globule (RIN 8.2)

Qualitative analysis derived from PMA call (Affymetrix) after Lowess normalization

(Feature Extraction Software 9.5.1.1)

comparison between MFG and MG transcriptome

5% 95% 88% 12%

8046419 1149

MG MFG

Number of transcripts

putatively associated with

MFG or MG (Venn diagram)

Expression of MEC specific genes in MFG

Analysis of milk protein gene expression in MFG compared with

MG and normalised with 2 references genes (24S and Cyclophilin)

No significant difference of milk proteingene expression between MFG and MG

validation of microarray results with qPCR

Expression

level in MG

0

0,5

1

1,5

2

2,5

CSN2 CSN3 Lalba

Expression of contaminant markers in MFG

No contamination by lymphocytes T andlittle contamination by macrophages.

Analysis of contaminants markers gene expression in MFG

compared with MG and normalised with 2 references genes (24S

and Cyclophilin)

validation of microarray results with qPCR

Expression

level in MG

0

0,2

0,4

0,6

0,8

1

1,2

Krt14

(negative control)

CD3e

(lymphocyte T)

CD68

(macrophages)

*

*** p<0,01

6h 12h 18h 24h 30h

Injection of Staphylococcus aureus in goats,

MFG sample collection at different times

post-infection

Test with a specific marker of infection

T1 T2 T3 T4 T50

1

2

3

4

6

5*

*

Expression level

in MFG T0

* p<0,01

Relative Expression (RQ) of Serum Amyloid A3, a

precocious marker of infection

Conclusion

RNA extracted from MFG are representative of

RNA from MEC

It is possible to extract RNA from

MFG in goat

A promising tool to follow time course of

infection

In progress…

Compare with microdissected cell on a higher

number of animals

Test in others species:

cow, mare, rabbit doe…

Make gene expression profiling analysis of

infection with more animals

Acknowledgements

équipe LGS-ICE (France)

Claudia Bevilacqua

Patrice Martin

Rama Bangera

Emmanuelle Rebours

Christelle Cebo

Léonardo Bianchi

Financial support by the

Centre Region (France),

CAPRICEL Contractéquipe VIM (France)

Luc Jounot (microarray analysis)

Università degli Studi di Milano (Italy)

Giuliano Pisoni

Paolo Moroni

Thank you for your attention