1. Volume 44, Issue 6, Pages 1422-1433 (June 2016)
Adiponectin Enhances Antibacterial Activity of Hematopoietic Cells by Suppressing Bone Marrow Inflammation 
Yosuke Masamoto, Shunya Arai, Tomohiko Sato, Akihide Yoshimi, Naoto Kubota, Iseki Takamoto, Yoichiro Iwakura, Akihiko Yoshimura, Takashi Kadowaki, Mineo Kurokawa 
Immunity 
Volume 44, Issue 6, Pages (June 2016)
DOI: /j.immuni
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2. Immunity 2016 44, 1422-1433DOI: (10.1016/j.immuni.2016.05.010)
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3. Figure 1
Emergency Granulopoiesis Is Diminished in Obese Mice and Adipoq−/− Mice
(A) Number of myeloid CFUs in each femur from HFD or LFD mice with treatment by vehicle (Veh) or G-CSF (125 μg/kg q12h for 4 days) (n = 4–7).
(B) Myeloid colony-forming units (CFUs) in PB from G-CSF-treated HFD or LFD mice (n = 7).
(C) Concentration of adiponectin in diluted BM lavage fluid of HFD or LFD mice (ng/mL). Each femur was flushed out with 400 μl of phosphate-buffered saline (PBS) (n = 9).
(D) Myeloid CFUs in each femur from Adipoq+/+ or Adipoq−/− mice treated with G-CSF (n = 4–11).
(E) Proportion of BrdU+ cells in BM LSK fraction. Adipoq+/+ or Adipoq−/− mice were treated with G-CSF for 4 days followed by a single injection of BrdU 18 hr before analysis (n = 3–6). Representative flow cytometric data are shown in left.
Data are shown as mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Please see Figure S1 and Table S1.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
Antibacterial Response Is Severely Impaired in Obese and Adipoq−/− Mice
(A) Neutrophils in PB from HFD or LFD mice before and after L. monocytogenes infection (n = 6).
(B) Bacterial counts in liver from infected HFD or LFD mice at day 7 post-infection (n = 11). The dotted line shows the measurable limit.
(C) Neutrophils in PB from infected Adipoq+/+ or Adipoq−/− mice (n = 8).
(D) Neutrophils in peritoneal cavity of infected Adipoq+/+ or Adipoq−/− mice (n = 7).
(E) Bacterial counts in liver from infected Adipoq+/+ or Adipoq−/− mice (n = 18).
(F and G) HFD mice were allocated to intravenous full-length adiponectin (fAd; 50 μg/body) or vehicle treatment twice daily starting from a day before G-CSF treatment or infection throughout the experimental period. (F) Leukocytes in peritoneal cavity from infected HFD or LFD mice treated with fAd (n = 5 for HFD mice). Comparison was done between fAd- and vehicle-treated HFD mice. (G) Bacterial counts in liver at day 7 from infected HFD or LFD mice treated with fAd (n = 5 for HFD mice).
(H) Bacterial counts in liver at day 7 from infected Adipoq−/− mice treated with AdipoRon (50 mg/kg) or vehicle (n = 8).
Data are shown as mean ± SEM of two independent experiments in (H) and three in the other. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Please see Figure S2.
Immunity  , DOI: ( /j.immuni )
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5. Figure 3
Adiponectin is required for efficient STAT3 activation in hematopoietic cells
(A) Phosphorylated STAT3 (pSTAT3) immunoblotting of Adipoq+/+ or Adipoq−/− BM cells treated with G-CSF (50 ng/mL) in vitro (n = 3). STAT3 was used as a loading control.
(B) pSTAT3 immunoblotting of Lineage- BM cells from Adipoq+/+ or Adipoq−/− mice treated with a single dose of G-CSF (125 μg/kg). BM cells were harvested 4 hr after in vivo G-CSF administration (n = 4).
(C) Immunoblotting for pSTAT3 and C/EBPβ. BM neutrophils were harvested from Adipoq−/− mice with fAd treatment combined with G-CSF (n = 7).
(D) pSTAT3 immunoblotting of BM neutrophils from Adipoq+/+ or Adipoq−/− mice with or without L. monocytogenes (LM) infection (24 hr after infection) (n = 5).
(E) pSTAT3 immunoblotting of Lineage− progenitor cells from HFD or LFD mice treated with a single dose of G-CSF (125 μg/kg) (n = 9). BM cells were harvested 4 hr after in vivo G-CSF treatment.
(F) pSTAT3 immunoblotting of BM neutrophils from HFD or LFD mice with or without L. monocytogenes (LM) infection (24 hr after infection) (n = 7). HFD mice were treated with intravenous fAd twice daily from day −1 to day 1 post-infection.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Please see Figure S3.
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6. Figure 4 Adiponectin regulates SOCS3 expression in HSPCs.
(A) pSTAT3 immunoblotting of LSK and CMP cells from Adipoq+/+ or Adipoq−/− mice treated with a single dose of G-CSF (LSK: n = 3, CMP: n = 4). Cells were harvested 4 hr after in vivo G-CSF administration. STAT3 was used as a loading control.
(B) SOCS3 immunoblotting of LSK and CMP cells from Adipoq+/+ or Adipoq−/− mice (n = 3). β-Actin was used as a loading control.
(C) Socs3 mRNA in LSK and CMP cells from Adipoq+/+ mice or Adipoq−/− mice treated with fAd for 2 days (n = 4–7). Values relative to Adipoq+/+ cells are shown.
(D) Neutrophils in PB from Listeria-infected HFD or LFD mice on Vav1-cre; Socs3fl/+ or Socs3+/+ background (n = 4–5). p value represents differences between Socs3+/+ HFD and Socs3+/+ LFD mice.
(E) Bacterial counts in liver at day 7 from Listeria-infected HFD or LFD mice on Vav1-cre; Socs3fl/+ or Socs3+/+ background (n = 4–5).
(F) Neutrophils in PB from Listeria-infected HFD or LFD mice on Lyz2-cre; Socs3fl/+ or Socs3+/+ background (n = 4-6).
(G) Bacterial counts in liver at day 7 from Listeria-infected HFD or LFD mice on Lyz2-cre; Socs3fl/+ or Socs3+/+ background (n = 4–6).
Data are shown as mean ± SEM of two independent experiments in (D–G) and three in the other. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Please see Figure S4.
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7. Figure 5
TNF is critical for attenuated emergency granulopoiesis in obese mice.
(A) TNF (pg/mL) in BM lavage fluid or plasma from Adipoq+/+ mice or Adipoq−/− mice with or without fAd treatment (50 μg q12h for 2 days) (n = 4–5).
(B) TNF in culture supernatants of macrophages (F4/80+ CD115− Gr-1− SSClo-mid) from Adipoq+/+ or Adipoq−/− BM (n = 6). Cells were cultured in serum-free media (RPMI-1640).
(C) SOCS3 immunoblotting of Adipoq+/+ Lineage− cells incubated with 10 ng/mL of TNF for the indicated period (n = 3). β-Actin was used as a loading control.
(D) Socs3 mRNA in LSK cells from HFD or LFD mice on Tnf+/+, Tnf+/−, or Tnf−/− background (n = 3–4). Values relative to Tnf+/+ LFD mice are shown.
(E) Myeloid CFUs in each femur from HFD or LFD mice on Tnf+/+ or Tnf−/− background with treatment by G-CSF (n = 2 for vehicle-treated, n = 4 for G-CSF treated mice).
Data are shown as mean ± SEM of two independent experiments in (B), (D), and (E). The mean ± SEM from a representative experiment of three are shown in (A) and (B). ∗p < 0.05, ∗∗∗p < Please see Figure S5.
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8. Figure 6
Adiponectin Suppresses the Production of TNF-Expressing M1-Like Macrophages in the BM
(A) Tnf mRNA in BM macrophages from Adipoq−/− mice after in vitro treatment by fAd with or without LPS (1 μg/mL) for 24 hr (n = 8–10: vehicle, n = 3: LPS).
(B) MHC-II+ BM macrophages in each femur from Adipoq+/+ LFD, Adipoq−/− LFD, and Adipoq+/+ HFD mice (n = 10).
(C) Proportions of Ly6C+ and Ly6C− subsets within BM monocytes (CD115+) from Adipoq+/+ or Adipoq−/− mice (n = 12).
(D) Nr4a1 mRNA in BM Ly6C+ monocytes treated with fAd in vitro (n = 4). Values relative to vehicle-treated monocytes are shown.
(E) Socs3 mRNA in LSKs and CMPs from Adipoq−/− mice treated with or without cytosporone B (25 mg/kg/day) for 2 days (n = 4). Values relative to vehicle-treated mice are shown.
(F) Bacterial counts in liver at day 7 from Listeria-infected recipient mice, reconstituted with hematopoietic cells expressing shRNAs against Adipor1, Adipor2 or Luciferase (Luc) (n = 5–6).
Data are shown as mean ± SEM of two independent experiments in (E) and (F), four in (D), and three in the other. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Please see Figure S6.
Immunity  , DOI: ( /j.immuni )
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9. Figure 7
Adiponectin Derived from Non-Hematopoietic Cells Is Essential for Emergency Granulopoiesis
(A) Scheme of BM chimeric mice. Lethally irradiated (10.5 Gy) Adipoq+/+ or Adipoq−/− recipient mice were reconstituted with BM cells from Adipoq+/+ or Adipoq−/− donor mice.
(B) BM myeloid CFUs in each femur from Adipoq+/+ or Adipoq−/− recipient mice transplanted with Adipoq+/+ or Adipoq−/− donor BM cells followed by G-CSF (n = 4–8).
(C) pSTAT3 immunoblotting of BM cells treated with G-CSF (50 ng/mL) in vitro for the indicated period (n = 3). Adipoq+/+ and Adipoq−/− recipient mice reconstituted with Adipoq+/+ BM cells were compared in the upper panel. Adipoq+/+ or Adipoq−/− cells from Adipoq+/+ recipient mice were compared in the lower panel. STAT3 was used as a loading control.
(D) Socs3 mRNA in LSK cells from G-CSF-treated Adipoq+/+ or Adipoq−/− recipient mice transplanted with Adipoq+/+ or Adipoq−/− donor BM cells (n = 4). Values relative to Adipoq+/+ cells from Adipoq+/+ recipient mice are shown.
(E) TNF (pg/mL) in BM lavage fluid from G-CSF-treated Adipoq+/+ or Adipoq−/− recipient mice with Adipoq+/+ or Adipoq−/− donor BM cells (n = 4).
Data are shown as mean ± SEM of two independent experiments in (B), (D), and (E), and three in (C). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Please see Figure S7.
Immunity  , DOI: ( /j.immuni )
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