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Team : IALTAUnder the supervision of Pr Gilles Foucras
UMR INRAE ENVT 1225 – IHAP
Blandine GausseresLaurence Guzylack
Chervin Hassel
REIDSOCS project: Description of lactating mammary gland immune cells using single-cell
RNA-Seq approach.
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Milk quality decreases 300 000 somatic cells (CCS)/mL
EC 2004/853 standard, April 29th 2004 400 000 cells/mL
Milk : Positive trade balance of 3.5 billions €
Problematic of REIDSOCS project: milk production
E.coliS.aureusS.uberis
Mastitis: mammary gland infections
Mammary gland:
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SNP SOCS-2 (suppressor of cytokine signaling 2)
Resistant Susceptible
Rupp Et.al 2015
Bullock AN, […], Knapp S, PNAS 2006
SOCS2-WT
SOCS2-R96C
ArgCGGTGT Cyst
Non functionnal SOCS-2 protein
GH
PRL
SOCS 2
IFN
Croissance
Immunité
Production de lait
SOCS-2: negative regulator of multiple signaling pathways:
Problematic of REIDSOCS project: ewe genetic mutation
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Aims of the REIDSOCS project
Determine the immune cells composition of lactatingmammary gland using unprior approach: scRNA-Seq
Compare the immune cells composition between WT and SOCS-2 R96C KI mice using flow cytometry and scRNA-Seq
Identify the immune function alteration duringStaphylococcus aureus infection associated withSOCS-2 R96C phenotype
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WT
H T
R 9 6C
2 0
2 5
3 0
3 5
4 0
4 5
m a le
Mic
e b
od
y w
eig
ht
WT
H T
R 9 6C
1 5
2 0
2 5
3 0
fe m a le
Mic
e b
od
y w
eig
ht
5
NS
********
**
********
SOCS2R96C mice
N=15 N=9 N=33 N=22 N=12 N=28
SOCS-2 invalidation: phenotype confirmation (gigantism)
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SOCS2R96C vs WT mice (n = 3)
Lactatingmammary gland (without LN) cells
extraction
Systemicinjection of
CD45 antibody
Multiplexing using CD45 TotalSeq antibody
(biolegend)
FACS sort of CD45+ viable cells
scRNA-SEQ libraries (10X genomics) + NGS Illumina
sequencingData analysis Cellular composition
Gene expression levels
Mammary gland immune cells transcriptome by scRNA-Seq
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1) FACS sort
7AAD
SSC
FITC
SSC
84.4
VioBlueSS
C
7.2687.7
11.9
FSC
SSC 50.1
FSCFSC-
W
99.2
SSCSSC-
W
100
Viability Blood cell exclusion CD45 cells selection
Thanks to Alexia Zakaroff-Girard and Elodie Riant
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2) Librairies preparation
8
We inject 18000 cells > almost 6000 sequencing cells
(catch rate of 33%) in 2 injection wells.
Thanks to Frederic Martins
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3) Analysis pipeline of scRNASEQ data analysis
Cell ranger
TotalSeq demultiplexing Normalization Clustering and UMAP Cell type attribution Clusters
MONOCLE 3 Trajectory inferrencehttps://hoohm.github.io/CITE-seq-Count/
https://satijalab.org/seurat/https://cole-trapnell-lab.github.io/monocle3/
CITE-seq-Count (HTO libraries)
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Lane A Cellranger results
Lane B Cellranger results
Aggregation of A & B lanes
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1112051 cells218 cells2078 cells
Demultiplexing with hashtag oligos and doublet exclusion under Seurat
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Ridge plots indicated the enrichment for selected HTOs
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UMAP representation of the cell clusters (n=19) of all WT and KI mices
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CD45 expression level in the cell clusters - All mices
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152913 cells 560 cells 597 cells 449 cells 3624 cells 523 cells
Heterogeneity of CD45 positive cells between micesKI1 KI2 KI3 WT1 WT2 WT3
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UMAP representation of CD45 positive cells – Without WT2 (LN)
± 5000 cells = 13 cell clusters
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Mouse CellAtlas
Tabula muris
Immgen_ref rna_seq_ref
https://github.com/rnabioco/clustifyrdata
Clusters cell type automatic attribution: clustifyrdata
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UMAP representation of CD45 positive cells – Without WT2 (LN)
13 identified cell clusters
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Specific macrophages supopulations in lactating mammary gland?
F4/80 CD11c double positivemacrophages
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Csn3 macro
P2ry6 macro
Mx1 macro
Birc5 macro Proliferation
+++
Antigen presentation
+++
Neutrophilchemotaxis
InnateImmune
response
Monocle analysis on F4/80 CD11c macrophages supopulations
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Macro DP; 82%
macro F480; 3%
LT; 6%NK; 5%
B cells; 0% Neutro; 3%
Macro DP; 68%
macro F480; 6%
LT; 7%
NK; 6%
B cells; 5%Neutro; 7%
WT
KI
WT vs KI immune cell composition difference? – scRNA-Seq and HTO
Macrophages and neutrophils altered in KI phenotype?
N = 2 WT
N = 3 KI
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Phenotying analysis strategy by flow cytometry (Miltenyibiotec MACSQ10)
Lactating MG (d7) of females WT (n = 10) vs KI (n = 10)
4) Immunophenotyping of SOCS2R96C mice by flow cytometry
FSC-A
SS
C-A
FSC-H
FSC
-A
SSC-H
SS
C-A
Viab
SS
C
CD45
SS
C
CD11c
F4/8
0
CD3
CD
19
NKp46
Ly6G
50.0
2.3
18.127.1
76.0
6.6
16.2
9.9
11.876.4
68.3
91.4 94.6
77.6
12.3
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0,010,020,030,040,050,060,070,080,090,0
LB LT Neutro NK F4-80+ F4-80+ CD11c+ CD11c+ F4/80- Autres Cell
% (v
s vCD
45+)
Proportion of CD45+ leucocytes
WTKI
0
1000
2000
3000
4000
5000
6000
7000
LB LT Neutro NK F4-80+ F4-80hiCD11chi
CD11c+F4/80-
Autres Cell vCD45+
Number of cells (/mg of MG N°4)
N = 10 WT / 10 KI
Significant differences
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Macro DP; 82%
macro F480; 3%
LT; 6%
NK; 5%B cells; 0% Neutro; 3%
Macro DP; 68%
macro F480; 6%
LT; 7%
NK; 6%
B cells; 5%Neutro; 7%
WT
KI
Macro DP; 78
macro F480;
2,1
LT; 9,5
NK; 2,9
B cells; 2,3 Neutro; 0,9 Other cells; 4,3
Macro DP; 68macro
F480; 2,6
LT; 18,5
NK; 4,1B cells; 1,7
Neutro; 0,8 Other cells; 4,3
scRNA-Seq Flow cytometry
Significant differences
N = 10 WT
N = 10 KI
N = 2 WT
N = 3 KI
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Conclusions
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F4/80+ CD11c+ macrophages
Neutrophils
T lymphocyte
NK cells
B lymphocyte
Cristea S, Polyak K. Dissecting the mammary gland one cell at a time. Nat Commun. 2018 Jun 26;9(1):2473.
Lactatingmammary gland
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Determine the transcriptome of both immune and non immune lactating
mammary gland cells during bacterial infection (Staphylococcus aureus) with
spatial information
> Lactating females (7-10 days)
Intramammary injection ofStaphylococcus aureus
(103 CFU/20 µL) Inclusion in OCTLactating mammary
gland dissection
Spatial transcriptomic
Tissue section of lactatingmammary gland
Collaboration with Spatial Transcriptomics Pilot Facility (Annelie Mollbrink), Sweden
Identification of SOCS-2 function during Staphylococcus aureus infection in mice lactating mammary gland
Prospects on REIDSOCS project
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Gilles FOUCRAS, PR Guillaume TABOURET, CR Laurence GUZYLACK-PIRIOU, CR Chervin HASSEL, IR post doc Nathan CEBRON, Doctorant Blandine GAUSSERES, IE Julie COURNET, TR Christian TASCA, TR
Team : IALTATeam leader : Pr Gilles FoucrasUMR INRA ENVT 1225 – IHAP
AL AERNATIVES
IMM
UN
ITE
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32N = 5 WT / 8 KI
0
200
400
600
800
1000
1200
WT KI
Poid
s (m
g)Poids des 2 glandes mammaires N°4
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332913 cells 560 cells 597 cells 449 cells 523 cells
CD45 positive cells – Without WT2 (LN)
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Description P-value FDR q-value Clusterimmune response 2.87E-4 1 P2ry6_macro
antigen processing and presentation of peptide or polysaccharide antigen via MHC class II 5.09E-4 1 P2ry6_macroantigen processing and presentation of peptide antigen via MHC class II 5.09E-4 7.79E-1 P2ry6_macro
antigen processing and presentation of peptide antigen 6.36E-4 7.3E-1 P2ry6_macroantigen processing and presentation 6.36E-4 5.84E-1 P2ry6_macro
cytokine-mediated signaling pathway 6.81E-5 2.26E-1 Csn3_macrosignal transduction 1.82E-4 3.02E-1 Csn3_macro
granulocyte chemotaxis 2.3E-4 2.55E-1 Csn3_macroneutrophil chemotaxis 2.3E-4 1.91E-1 Csn3_macroneutrophil migration 2.3E-4 1.53E-1 Csn3_macro
myeloid leukocyte migration 2.3E-4 1.27E-1 Csn3_macrocellular amide metabolic process 4.68E-6 8,00E-03 Socs3_macro
translation 1.14E-5 9.77E-3 Socs3_macroamide biosynthetic process 1.14E-5 6.51E-3 Socs3_macropeptide metabolic process 1.14E-5 4.88E-3 Socs3_macro
peptide biosynthetic process 1.14E-5 3.91E-3 Socs3_macronegative regulation of developmental process 1.66E-4 6.5E-1 CD206_macro
cell differentiation 3.14E-4 6.15E-1 CD206_macropositive regulation of chemotaxis 7.01E-4 9.15E-1 CD206_macro
regulation of anatomical structure morphogenesis 8.07E-4 7.9E-1 CD206_macronegative regulation of cell proliferation 9.68E-4 7.58E-1 CD206_macro
cell cycle 2.83E-20 8.48E-17 Birc5_macro
cell cycle process 5.36E-20 8.02E-17 Birc5_macro
cell division 3.25E-16 3.24E-13 Birc5_macro
mitotic cell cycle process 1.54E-14 1.15E-11 Birc5_macro
regulation of cell cycle 2.52E-13 1.51E-10 Birc5_macro
regulation of cell cycle process 5.52E-10 2.76E-7 Birc5_macro
defense response 9.87E-11 6.52E-8 Mx1_macro
response to other organism 5.48E-10 1.81E-7 Mx1_macrodefense response to virus 1.16E-9 3.29E-7 Mx1_macro
response to virus 5.79E-9 1.43E-6 Mx1_macrodefense response to other organism 1.57E-8 3.45E-6 Mx1_macro
innate immune response 2.33E-8 4.62E-6 Mx1_macroimmune effector process 3.23E-8 5.81E-6 Mx1_macro
immune response 3.3E-8 5.45E-6 Mx1_macroimmune system process 1.62E-7 2.47E-5 Mx1_macroresponse to bacterium 7.38E-5 9.14E-3 Mx1_macro
cellular response to interferon-alpha 1.16E-4 1.35E-2 Mx1_macroregulation of innate immune response 1.44E-4 1.59E-2 Mx1 macro
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WT1 WT3 KI1 KI2 KI3 WT KI
P2ry6_macro 28% 31% 20% 17% 27% 29% 21%
Csn3_macro 32% 7% 17% 21% 17% 20% 18%
Socs3_macro 25% 30% 12% 21% 21% 27% 18%
NK_cells 0% 9% 12% 5% 2% 5% 6%
Neutro 1% 5% 11% 7% 4% 3% 7%
CD206_macro_DC 2% 5% 5% 10% 4% 3% 6%
CD127_LT 4% 5% 6% 4% 1% 4% 4%
CD27_LT 0% 4% 7% 2% 1% 2% 3%
Birc5_macro 5% 0% 3% 3% 7% 3% 4%
B_cells 0% 0% 0% 7% 9% 0% 5%
Mx1_macro 2% 3% 2% 2% 2% 2% 2%
Cd3_Cd14_macro 0% 0% 4% 0% 0% 0% 1%
Tk1_macro 1% 1% 1% 1% 4% 1% 2%
CD45 positive cells – Without WT2 (LN)
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Fluorochome Target CellsV1 Pacific blue Ly-6G NeutrophilsV2 VioGreen CD45 Immune cellsB1 FITC Viobility Fixable Dye Dead cellsB2 PE Nkp46 (CD335) NK cellsB3 PE-Vio615 CD19 LBB4 PeCy7 CD11c Myeloid cells (DC / macro)R1 APC F4/80 macrophagesR2 APC-Vio770 CD3 LT
Phenotying analysis strategy by flow cytometry(Miltenyi MACSQUANT10)
Lactating MG (day=7)
Females WT (n = 10) vs KI (n = 10)
Immunophenotyping of SOCS2R96C mice by flow cytometry
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NanoScan PET/CT, Mediso(Service Exploration Non-Invasive, CREFRE)
SOCS2R96C mice: size alteration