1. Supplementary Figures and Figure Captions
Title：Forkhead Box O1 Regulates Macrophage Polarization Following Staphylococcus aureus Infection in Mouse Liver
Journal name：Clinical Reviews in Allergy & Immunology
Author names：Yu-Chen Wang1, Hong-Di Ma1, Xue-Ying Yin1, Yin-Hu Wang1, Qing-Zhi Liu1, Jing-Bo Yang1, Qing-Hua Shi2, Baolin Sun2, M. Eric Gershwin3, and Zhe-Xiong Lian1,4
1Liver Immunology Laboratory, Institute of Immunology and The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui , China; 2The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China, Hefei , China; 3Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, Davis, CA USA; 4Innovation Center for Cell Signaling Network, Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui , China
Correspondence to: Zhe-Xiong Lian, M.D., Ph.D., Liver Immunology Laboratory, Institute of Immunology and The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei , China; Phone: ; Fax: ;

2. Supplementary Fig. 1 IL-10 (pg/ml) (b) IL-4 (pg/ml) IL-6 (pg/ml) *
TNF-α (pg/ml)
MCP-1 (pg/ml)
CLO-lip
PBS-lip
(a)
25.1
18.9
45.7
35.4
18.5
33.3
63.9
2.4
2.3
56.9
F4/80
CD11b
Ly6G
Ly6C

3. Supplementary Fig. 2 FoxO1 p-Akt p-Erk p-Mek Akt GAPDH
BMDM WT TLR2-/- TLR4-/- WT TLR2-/- TLR4-/- WT TLR2-/- TLR4-/-
S. aureus
PGN
p-FoxO1
(a)

4. Supplementary Fig. 3 (a) IL-10 (pg/ml) * IL-6 (pg/ml) MCP-1 (pg/ml)
TNF-α (pg/ml)
FoxO1fl/fl
LysCre/+FoxO1fl/fl
IFN-γ (pg/m)
(b)
IL-17A
IFN-γ
9.4
14.5
0.6
0.3
CD4+ T cell (%)
IFN-γ+ of
1.5
1.9
2.4
3.5
IL-10+ of
IL-10
IL-4
IL-17A+ of
IL-4+ of
9.9
10.1
Liver
Spleen
CTLA4
Foxp3+
(c)
12.9
14.3

5. Supplementary Fig. 4 CD80 (a) Isotype LysCre/+FoxO1fl/fl FoxO1fl/fl
MHC-II
CD11c
CD86
MFI *
(b)
(c)
LV-Control
LV-FoxO1
FoxO1
β-actin
(d)
Cxcl10
Relative expression
Ccr2
Tnf
Il6
Ccl2

6. Supplementary Fig. 5 Hoechst 33342 CFSE-labeled S. aureus (b) CD68
CX3CR1
Isotype
LysCre/+FoxO1fl/fl
FoxO1fl/fl
MFI
FoxO1f/f
LyzCre/+FoxO1f/f
(c)
(a)
CFSE-labeled S. aureus
Hoechst 33342

7. Supplementary Figure Captions
Supplementary Fig. 1 T cell infiltration requires macrophages following S. aureus infection in the liver.
(a) Clodronate liposomes (n=5) or PBS liposomes (n=5) were i.p injected into C57BL/6 mice before infection. At 72 hr after S. aureus i.v infection, cells were isolated by liver perfusion as described. Cells were stained with anti-CD45.2, anti-CD11b, anti-F4/80, anti-Ly6C and anti-Ly6G. Total CD45.2+ cells are shown on the left and gated cells shown on the right. Kupffer cells are gated on F4/80hi CD11blo, other myeloid cells are gated on CD11bhi F4/80lo. In these myeloid cells, monocyte are Ly6Chi Ly6Glo, neutrophils are Ly6Ghi Ly6Cint and monocyte-derived macrophages are Ly6Clow Ly6G-.
(b) Mouse serum in (A) were collected 6 hours after infection, and measured by BD CBA Soluble Protein Flex Set System (BD Bioscience).
Supplementary Fig. 2 Decreased FoxO1 in macrophages during S. aureus infection.
(a) BMDMs from WT, TLR2-/- and TLR4-/- mice were treated with PGN (10ug/ml) or heat-inactivated S. aureus (5x107/ml). Cells were harvested 30 mins after treatment for immunoblotting.
Supplementary Fig. 3 Macrophage FoxO1 knockout impaired Th1 and Th17 responses in mice.
(a) FoxO1fl/fl mice (n=4) and LysCre/+FoxO1fl/fl mice (n=4) were i.v infected with S. aureus. Serum was collected 6 hr after infection and measured by BD CBA Soluble Protein Flex Set System.
(b) S. aureus were i.v injected into FoxO1fl/fl mice (n=5) and LysCre/+FoxO1fl/fl (n=5) mice. At 72 hr after infection, mononuclear cells from spleen were analyzed for cytokine expression after treatment with PMA and ionomycin for 4 hr. Flow cytometry was gated on CD19-Nk1.1-CD3+CD4+ cell subsets. Statistical analysis is shown on the right.
(c) At 72 hr after S. aureus i.v infection, mononuclear cells from the spleen and liver were stained with anti-CD3, anti-NK1.1, anti-CD4, anti-CD8α and anti-CTLA4, intracellular staining with anti-Foxp3 was performed after fixation and permeabilization. Flow cytometry pictures were gated on CD19-Nk1.1-CD3+CD4+ cell subsets.

8. Supplementary Fig. 4 Pro-inflammatory activation of macrophages upon TLR2 signal requires FoxO1.
(a) FoxO1fl/fl mice (n=4) and LysCre/+FoxO1fl/fl mice (n=4) were i.v infected with S. aureus. At 72 hr after infection, cells from the peritoneal cavity were collected and stained with fluorescence targeted antibody anti-CD19, anti-CD11b, anti-F4/80, anti-Ly6C, anti-MHC-II, anti-CD11c, anti-CD80 and anti-CD86. Flow cytometry pictures were gated on CD19-CD11bhighF4/80high cell subsets.
(b) Statistical analysis in (A) is shown.
(c) Primary BMDMs from C57BL/6 wild type mice were transduced with either lentivirus-based control vector (LV-ctrl) or LV-FoxO1 expression plasmids. Immunoblotting was performed for monitoring transduction efficiency.
(d) Primary BMDMs from C57BL/6 wild type mice were transduced with either lentivirus-based control vector (LV-ctrl) or LV-FoxO1 expression plasmids. BMDMs were treated with inactivated S. aureus (5x107/ml) for 6 hr and underwent quantitative real-time PCR analysis.
Supplementary Fig. 5 FoxO1 represses M2 polarization and immune suppressive function in macrophages.
(a) S. aureus were labeled with CFSE (Green) for 5 mins and then co-cultured with BMDMs from FoxO1fl/fl mice (n=5) and LysCre/+FoxO1fl/fl mice (n=5) for 90 mins. Extracellular S. aureus were eliminated by incubation with lysostaphin 10 U/ml at 37 ℃ for 30 mins. Hoechst 33342 (10mg/ml, Blue) were add to label all cell nuclei. The cells were washed and visualized using confocal microscopy.
(b) BMDMs from FoxO1fl/fl mice (n=4) and LysCre/+FoxO1fl/fl mice (n=4) were labeled with fluorescence targeted antibody 24 hr after IL-4 (10 ng/ml) and IL-13 (10 ng/ml) treatment.
(c) Statistical analysis in (C) is shown.