1. Volume 23, Issue 1, Pages 165-178 (January 2016)
Thermoneutral Housing Accelerates Metabolic Inflammation to Potentiate Atherosclerosis but Not Insulin Resistance 
Xiao Yu Tian, Kirthana Ganeshan, Cynthia Hong, Khoa D. Nguyen, Yifu Qiu, Jason Kim, Rajendra K. Tangirala, Peter Tontonoz, Ajay Chawla 
Cell Metabolism 
Volume 23, Issue 1, Pages (January 2016)
DOI: /j.cmet
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Cell Metabolism 2016 23, 165-178DOI: (10.1016/j.cmet.2015.10.003)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Thermoneutral Housing Accelerates Onset of Metabolic Inflammation in Adipose Tissue
Kinetics of infiltration of epididymal WAT by immune cells in C57BL/6J mice fed normal chow (NC) or high-fat diet (HFD) and housed at a Ta of 22°C or 30°C (n = 4–5 per temperature/diet/time point).
(A and B) Total numbers of CD45+ hematopoietic cells at 22°C (A) and 30°C (B).
(C and D) Macrophages at 22°C (C) and 30°C (D).
(E and F) CD11c+ CD206− macrophages at 22°C (E) and 30°C (F).
(G and H) CD169+ macrophages at 22°C (G) and 30°C (H).
(I and J) CD11c− CD206+ cells at 22°C (I) and 30°C (J).
(K and L) CD301+ cells at 22°C (K) and 30°C (L).
Data are represented as mean ± SEM.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 2
Increase in Metabolic Inflammation Does Not Potentiate Insulin Resistance in Thermoneutral Mice
(A and B) Quantification of infiltrating neutrophils (A) and monocytes (B) in the model of zymosan-induced peritonitis in C57BL/6J mice housed at a Ta 22°C or 30°C (n = 10 per group).
(C) Quantification of monocytes following zymosan-induced peritonitis in WT and Ccr2−/− mice housed at a Ta of 30°C (n = 4–5 per genotype).
(D) Changes in body mass of C57BL/6J mice housed at a Ta of 22°C or 30°C and fed normal chow (NC) or high-fat diet (HFD) starting at 6 weeks of age.
(E–G) Mass of adipose tissues of C57BL/6J mice housed at a Ta of 22°C or 30°C and fed NC or HFD; eWAT (E), scWAT (F), and BAT (G).
(H–L) Assessment of glucose homeostasis in C57BL/6J mice housed at a Ta of 22°C or 30°C and fed NC or HFD. Glucose (H) and insulin (I) tolerance tests were performed 9 and 10 weeks after initiation of HFD, respectively (n = 5 per temperature and diet). Serum insulin (J) and insulin-stimulated AKT signaling in eWAT (K) and liver (L) in C57BL/6J mice housed at a Ta of 22°C or 30°C and fed HFD.
Data are represented as mean ± SEM.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 3
CCR2 Contributes to Adipose Tissue Inflammation, but Not Insulin Resistance, in Thermoneutral Mice
(A) Changes in total body mass of WT and Ccr2−/− fed HFD for 15 weeks at a Ta of 30°C.
(B) Adipose tissue mass of WT and Ccr2−/− fed a HFD for 15 weeks at 30°C.
(C–H) Quantification of macrophages (C), CD11c+ cells (D), monocytes (E), Ly6Chi monocytes (F), Ly6Clo monocytes (G), and FoxP3+ (H) in scWAT and eWAT of thermoneutral WT and Ccr2−/− mice fed HFD.
(I–M) Assessment of glucose homeostasis in thermoneutral WT and Ccr2−/− mice fed HFD. Serum insulin (I), glucose (J), and insulin (K) tolerance tests, and insulin-stimulated AKT signaling in eWAT (L) and liver (M).
Data are represented as mean ± SEM; n = 4–5 mice per genotype.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 4 Thermoneutral Housing Exacerbates Atherosclerosis
(A and B) Quantification of en face atherosclerotic lesions in Apoe−/− mice fed normal chow (NC) or Western diet (WD) for 16 weeks and housed at a Ta of 22°C or 30°C (n = 17–20 per temperature and diet). Total lesion area is quantified in (A), whereas atherosclerotic lesions in arch and descending aorta are quantified in (B).
(C) En face aorta preparations from Apoe−/− mice housed at a Ta of 22°C or 30°C and fed NC or WD were stained with Sudan IV. Representative images are shown.
(D) Representative sections of aortic lesions from WD fed Apoe−/− mice housed at a Ta of 22°C (left panels) and 30°C (right panels) were stained with oil red O for neutral lipids (top), CD68 for macrophages (middle), and smooth muscle actin (SMA) for smooth muscle cells (bottom).
(E and F) Quantification of serum cholesterol (E) and triglycerides (F) Apoe−/− mice fed NC or WD at the two different ambient temperatures (n = 5–19 per diet and temperature).
(G) Body mass of Apoe−/− mice housed at a Ta of 22°C or 30°C that were fed NC or WD for 16 weeks (n = 14–15 per group).
Data are represented as mean ± SEM.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 5 Increased Vascular Wall Inflammation in Thermoneutral Mice
(A–K) Quantification of immune cells present in aortas of Apoe−/− mice fed WD and housed at a Ta of 22°C or 30°C (n = 6 per temperature). Macrophages (A), Ly6Chi macrophages (B), Ly6Cmid macrophages (C), Ly6Clo macrophages (D), resident macrophages (E), CD301 MFI in macrophages (F), CD301 MFI in Ly6Clo macrophages (G), CD103−CD11b+ DCs (H), CD103+CD11b− DCs (I), CD301 MFI in CD103−CD11b+ DCs (J), and CD301 MFI in CD103+CD11b− DCs (K); (n = 10–12 per group for A–E, H, and I; n = 5–6 per group for F, G, J, and K).
(L and M) qRT-PCR analysis of mRNA markers of M1 (L) and M2 (M) macrophages in aortas of Apoe−/− mice fed the WD.
Data are represented as mean ± SEM.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 6
Thermoneutral Housing Increases Inflammation in Thoracic Perivascular Fat
(A) Thoracic perivascular fat mass of Apoe−/− mice fed WD at 22°C and 30°C.
(B and C) Gross histology (B) and H&E staining (C) of thoracic perivascular fat sections from Apoe−/− mice fed WD at 22°C and 30°C.
(D–F) qRT-PCR analysis of brown fat (D), M1 inflammation (E), and M2 genes (F) in the thoracic perivascular fat of Apoe−/− mice fed WD at 22°C and 30°C.
(G–I) Flow cytometric quantification of macrophages (G), Ly6chi macrophages (H), and Ly6Clo macrophages (I) in perivascular fat of Apoe−/− mice fed WD at 22°C and 30°C.
(J) CD301 MFI of thoracic perivascular fat macrophages from Apoe−/− mice fed WD at 22°C and 30°C.
(K) BrdU incorporation in thoracic perivascular fat macrophages of Apoe−/− mice fed WD at 22°C and 30°C.
Data are represented as mean ± SEM, n = 6 per temperature.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

9. Figure 7
Model for Differential Effects of Metabolic Inflammation in Thermoneutral Mice
Thermoneutral housing accelerates the onset of metabolic inflammation in white adipose tissue and the vasculature. While this increase in vascular inflammation contributes to progression of atherosclerosis, it does not contribute to worsening of obesity-associated insulin resistance in thermoneutral mice.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions