the mammary epithelial cell response to infection is ...the mammary epithelial cell response to...
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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
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