Chronic, low-grade inflammation triggered by excess intake of dietary lipids has been proposed to contribute to the pathogenesis of metabolic disorders, such as obesity, insulin resistance, type 2 diabetes, and atherosclerosis. Although considerable evidence supports a causal association between inflammation and metabolic diseases, most tests of this link have been performed in cold-stressed mice that are housed below their thermoneutral zone. We report here that thermoneutral housing of mice has a profound effect on the development of metabolic inflammation, insulin resistance, and atherosclerosis. Mice housed at thermoneutrality develop metabolic inflammation in adipose tissue and in the vasculature at an accelerated rate. Unexpectedly, this increased inflammatory response contributes to the progression of atherosclerosis but not insulin resistance. These findings not only suggest that metabolic inflammation can be uncoupled from obesity-associated insulin resistance, but also point to how thermal stress might limit our ability to faithfully model human diseases in mice.

Here, we report that thermoneutral housing accelerates the onset of metabolic inflammation in C57BL/6J mice fed a high-fat diet (HFD) or Apoe −/− mice fed a high-fat/high-cholesterol Western diet (WD). Surprisingly, while an increase in vascular inflammation contributed to the progression of atherosclerotic lesions in Apoe −/− mice, the accelerated onset of metabolic inflammation in obese mice did not potentiate insulin resistance. These results reveal that ambient temperature is an important modifier of metabolic inflammation in mice that differentially impacts the development of metabolic diseases. They further suggest that metabolic inflammation is less likely to play a critical role in the pathogenesis of obesity-associated insulin resistance in humans, who, like thermoneutral mice, do not experience cold stress. We propose that thermoneutral housing might be one way to improve the predictive modeling of human diseases in mice.

Although these mechanistic studies have provided key insights into the pathogenesis of metabolic diseases, their direct relevance to human biology remains unclear for one simple reason: a majority of these studies have been carried out in cold-stressed mice. While humans live predominantly in their thermal comfort or neutral zone, a temperature at which they expend the least amount of energy for warmth or cooling, mice are normally housed at temperatures that put them under significant thermal and hemodynamic stress (). For example, at the normal vivarium temperature of 20°C –22°C, C57BL/6 mice exhibit an ∼2-fold increase in energy expenditure compared to those housed at thermoneutrality (30°C) (). A similar change in hemodynamic parameters is observed as the ambient temperature (T) of mice is shifted from 30°C to 20°C. In this case, heart rate of C57BL/6 mice increases from ∼300 to 600 beats/min, and their mean arterial pressure rises from ∼75 to 105 mmHg, reflecting an overall increase in adrenergic tone (). Because the ambient temperature has such a dramatic effect on the cardiovascular and metabolic systems of mice, we postulated that it might be a key determinant of the development of metabolic inflammation and disease.

Fueled by the discovery that obesity is associated with the onset of low-grade inflammation in white adipose tissue (WAT) via recruitment of macrophages (), similar links between inflammation and obesity-induced insulin resistance have been made. Among the panoply of immune cells that have been implicated, the recruitment and inflammatory activation of macrophages have been suggested to play a key role in the progression of obesity-associated insulin resistance (). In support of this idea, genetic manipulations that alter macrophage recruitment or activation have been linked to changes in insulin sensitivity. For example, a decrease in macrophage infiltration or their M1 inflammatory activation is associated with improvements in insulin action, whereas an increase in macrophage recruitment or an impairment in their ability to undergo anti-inflammatory M2 activation is generally associated with worsening of insulin resistance. Furthermore, studies with T and B cells have uncovered a similar dichotomy between the functions of inflammatory (CD4, CD8, and B cells) and anti-inflammatory (Tregs, innate-like B cells, and natural regulatory B cells) adaptive immune cells in the maintenance of metabolic homeostasis ().

The initial molecular links between inflammation and metabolic disorders emerged from studies of atherosclerosis (). Here, the accumulation of the atherogenic low-density lipoprotein (LDL) in the subendothelium stimulates the release of chemokines by endothelial cells, which results in the recruitment of circulating monocytes. These recruited monocytes subsequently differentiate into macrophages or dendritic cells, phagocytose modified LDL, and initiate a complex inflammatory response that contributes to the progression of atherogenic lesions. In support of this model, impairment in monocyte recruitment, their differentiation into macrophages, their uptake of oxidatively modified LDL, or their secretion of inflammatory molecules has been shown to attenuate atherogenesis in the setting of hypercholesterolemia (). The situation is likely more complex in vivo because these proatherogenic functions of lesional macrophages are balanced by their antiatherogenic roles in limiting inflammation and promoting reverse cholesterol transport processes that are transcriptionally coordinated by the sterol-sensing liver X receptors (LXRs) ().

Inflammation is closely associated with, and perhaps contributes to, the pathogenesis of a number of human diseases, including obesity, type 2 diabetes, coronary artery disease, neurodegenerative disorders, and cancers (). Unlike the acute inflammatory responses that are elicited by infection and injury, inflammation in these disease states is low grade and chronic, leading some to suggest that it should be referred to as “metainflammation” (for metabolic inflammation) or more generally “parainflammation” (). While the nature of this metabolic inflammation is distinct (in terms of both duration and intensity), it engages many of the same cells, signals, and molecules that are involved in the classical inflammatory responses of infection and injury. Indeed, the cumulative evidence from a large number of studies suggests that the recruitment and inflammatory activation of macrophages is a key step in the progression of both obesity-associated insulin resistance and hypercholesterolemia-associated atherosclerosis ().

Based on these observations, we decided to more quantitatively assess the infiltration of myeloid cells into perivascular fat of Apoemice ( Figure S7 ). We found evidence for increased recruitment of total (∼68%), Ly6C(∼236%), and Ly6C(∼58%) macrophages ( Figures 6 G–6I) and decreased (∼50%) expression of CD301(encoded by Clec10a) by M2 macrophages in the thoracic perivascular fat of thermoneutral Apoemice ( Figure 6 J). These changes in the macrophage populations were independent of cellular proliferation, as evidenced by the comparable rates of BrdU incorporation in the perivascular macrophage subsets of Apoemice housed at a Tof 22°C and 30°C ( Figure 6 K). Overall, these results suggest that thermoneutral housing increases macrophage-mediated inflammation in the thoracic perivascular adipose tissue of Apoemice, which likely contributes to the progression of atherosclerotic plaques in the descending aortas of these animals.

Since the development of atherosclerotic plaques was exaggerated in the descending aortas of thermoneutral Apoemice, we investigated the contribution of thoracic perivascular fat to atherogenic inflammation in the vessel wall. Although the thermoneutral housing did not significantly alter the mass of thoracic perivascular fat ( Figure 6 A), it markedly affected its cellular composition and functional properties. For example, histological analyses revealed that perivascular fat of thermoneutral mice was paler and infiltrated by white adipocytes ( Figures 6 B and 6C). In agreement with these histological changes, qRT-PCR analysis revealed a 40%–99% reduction in the expression of brown adipocytes markers, such as Cidea, Cpt1b, Elovl3, and Ucp1, in the thoracic perivascular fat of thermoneutral mice ( Figure 6 D). Together, these findings suggested that the thoracic perivascular fat of thermoneutral Apoemice might have acquired the inflammatory properties of visceral white fat depots. Indeed, we observed that expression of mRNAs encoding inflammatory cytokines (Il1b, Tnf), chemokines (Ccl2), and chemokine receptors (Ccr2, Cx3cr1) was increased by ∼3.0- to 6.7-fold in the thoracic perivascular fat of thermoneutral Apoemice ( Figure 6 E). In contrast, the expression of M2 macrophage genes (Clec10a, Retnla, Mrc1, and Cd163) was not significantly different between Apoemice housed at a Tof 22°C and 30°C ( Figure 6 F).

(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.

Perivascular adipose tissue, which surrounds most blood vessels, provides mechanical support to the vasculature and participates in the maintenance of its functions (). Depending on its location and species, perivascular adipose tissue exhibits different characteristics. For example, thoracic perivascular fat in mice and humans has BAT-like properties, whereas the abdominal aorta is surrounded by a mixture of brown- and white-like adipose tissue. Although increased presence of thoracic perivascular adipose tissue is associated with higher prevalence of cardiovascular disease in humans (), murine perivascular adipose tissue has been implicated in both the promotion and attenuation of atherogenesis (). While the proatherogenic functions of perivascular fat have been attributed to the recruitment of macrophages and expression of inflammatory genes, cold-induced activation of perivascular fat has been suggested to attenuate atherogenesis via clearance of triglyceride-rich lipoproteins ().

Since changes in cholesterol homeostasis have been linked to alterations in myelopoiesis (), we next asked whether thermal neutral housing affected the steady-state numbers of myeloid or lymphoid cells. We found that Apoemice housed at a Tof 30°C had higher numbers of total, Ly6C, and Ly6Cmonocytes in blood and spleen, but not in the bone marrow ( Table S1 ). With the notable exception of blood neutrophils, steady-state numbers of other myeloid and lymphoid lineages were not different between blood, spleen, and bone marrow of Apoemice housed at a Tof 22°C and 30°C ( Table S1 ). These observations suggested that increased infiltration of the vessel wall by macrophages in thermoneutral Apoemice might result from the preferential recruitment of monocytes into the atherosclerotic lesions. In support of this postulate, we observed higher expression of the chemokines (Ccl2, Ccl8, and S100A8), which participate in the recruitment of monocytes to sites of inflammation, in the vessel wall of thermoneutral Apoemice ( Figure 5 L). This was accompanied by increased expression of markers for inflammatory M1 (Il1b and Il6) but not anti-inflammatory M2 macrophages ( Figures 5 L and 5M) (). Taken together, these data suggest that increased local inflammation in the vessel wall of thermoneutral Apoemice likely contributes to the progression of atherosclerotic plaques in these animals.

Because mice housed at thermoneutrality have accelerated metabolic inflammation, we next asked whether an increase in vessel wall inflammation contributed to the progression of atherosclerotic plaques in Apoemice housed at a Tof 30°C. We tested this hypothesis by quantifying the numbers of innate and adaptive immune cells present in the atherosclerotic lesions of Apoemice fed the WD. Flow cytometric analysis revealed a trend toward increased numbers of macrophages in lesions of thermoneutral Apoemice ( Figures 5 A and S6 A). Macrophage subtype analysis verified that lesions of thermoneutral Apoemice contained higher numbers of Ly6C(∼85%), Ly6C(∼65%), and Ly6C(∼65%) macrophages, whereas the numbers of resident macrophages did not change significantly ( Figures 5 B–5E and S6 A). This increase in macrophage recruitment was accompanied by an ∼55%–70% decrease in the median fluorescence intensity (MFI) of CD301 ( Figures 5 F and 5G), a marker of anti-inflammatory M2 macrophages that have been implicated in attenuating atherogenesis. We also observed higher numbers of CD103CD11b(∼55%) and CD103bCD11b(∼90%) dendritic cells in lesions of thermoneutral Apoemice ( Figures 5 H and 5I), both of which expressed substantially lower levels of CD301 ( Figures 5 J and 5K). These changes in innate immunity were associated with increased infiltration by CD4and CD8T cells, but not neutrophils or B cells in atherosclerotic plaques ( Figures S6 B–S6F).

(A–K) Quantification of immune cells present in aortas of Apoe −/− mice fed WD and housed at a T a of 22°C or 30°C (n = 6 per temperature). Macrophages (A), Ly6C hi macrophages (B), Ly6C mid macrophages (C), Ly6C lo macrophages (D), resident macrophages (E), CD301 MFI in macrophages (F), CD301 MFI in Ly6C lo 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).

To test this postulate, we housed cohorts of Apoemice at a Tof 22°C or 30°C and fed them NC or Western diet (WD) for 16 weeks. En face analysis by Sudan IV staining revealed a marked increase in atherosclerotic lesion area in thermoneutral mice. For example, in Apoemice fed NC, we observed that thermoneutral housing increased lesion area by ∼46% ( Figures 4 A and 4B ). This increase in plaque burden was further accentuated when Apoemice were fed the WD. Compared to mice housed at a Tof 22°C, thermoneutral housing of Apoemice increased atherosclerotic lesion area by ∼94% ( Figure 4 A). The increase in lesion area in thermoneutral mice was most apparent in the descending aorta (∼3-fold increase), which develops atherosclerosis later in the course of the disease than the arch in en face analysis ( Figures 4 B and 4C) (). Staining of representative aortic root sections revealed that chow- or WD-fed Apoemice housed at a Tof 30°C had larger lesions, which were enriched in neutral lipids (oil red O), macrophages (CD68), and smooth muscle cells (SMA) ( Figure 4 D). This marked increase in plaque burden was associated a relatively modest ∼20% increase in serum cholesterol levels in thermoneutral Apoemice fed the WD, but not chow diet ( Figure 4 E). Effects on serum triglycerides were more variable ( Figure 4 F). These effects of ambient temperature on atherogenesis were independent of changes in body mass ( Figure 4 G), and thermoneutral housing did not potentiate obesity, glucose intolerance, and insulin resistance in C57BL/6J mice fed the WD ( Figures S4 E–S4G). These results together suggest that thermoneutral housing selectively modulates progression of atherogenesis but not insulin resistance.

(G) Body mass of Apoe −/− mice housed at a T a of 22°C or 30°C that were fed NC or WD for 16 weeks (n = 14–15 per group).

(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).

(D) Representative sections of aortic lesions from WD fed Apoe −/− mice housed at a T a 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).

(C) En face aorta preparations from Apoe −/− mice housed at a T a of 22°C or 30°C and fed NC or WD were stained with Sudan IV. Representative images are shown.

(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 T a 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).

Although initiated by the accumulation of modified low-density lipoproteins in the vessel wall, the progression of atherosclerosis is highly dependent on the recruitment of monocyte-derived macrophages, which exert both pro- and anti-atherogenic effects (). These recruited cells can scavenge oxidatively modified LDL, secrete cytokines, chemokines, and metalloproteinases, and present antigens to modulate adaptive immune responses in the vessel wall. The importance of inflammation in atherogenesis is well recognized in both mice and humans (), yet nearly all of the mechanistic studies linking innate and adaptive inflammation to atherogenesis have been performed with mice housed under thermal stress; i.e., a Tof 20°C–22°C. We thus asked whether thermoneutral housing might contribute to the progression of atherosclerosis via modulation of vessel wall inflammation.

Previous studies have demonstrated that genetic disruption or pharmacological inhibition of CCR2 protects mice from obesity-associated metabolic inflammation, glucose intolerance, and insulin resistance (). Since we observed that thermoneutral housing accelerates onset of innate and adaptive inflammation in adipose depots, we next asked whether this increase in adipose tissue inflammation was dependent on the chemokine receptor CCR2. For these studies, 6-week-old wild-type and Ccr2mice were acclimatized to a Tof 30°C for 2 weeks prior to initiation of HFD for 15 weeks. Under these conditions, we observed that Ccr2mice had a slightly higher body mass ( Figure 3 A) without significant changes in adipose tissue mass, including eWAT and scWAT ( Figure 3 B). In agreement with previous studies (), we found that loss of CCR2 markedly decreased the numbers of total (∼80%) and CD11c(∼78%) macrophages in the scWAT and eWAT of mice housed at thermoneutrality ( Figures 3 C, 3D, and S5A–S5C ). Similar reductions were also observed for total monocytes (∼79%–84%), Ly6Cinflammatory monocytes (∼90%–97%), and Ly6Cpatrolling monocytes (∼74%–84%) in the scWAT and eWAT of Ccr2mice housed at a Tof 30°C ( Figures 3 E–3G). This reduction in recruitment of inflammatory innate immune cells was accompanied by an ∼2- to 4-fold increase in the numbers of FoxP3regulatory T cells, but not CD4or CD8T cells ( Figures 3 H, S5 D, and S5E). Consequently, expression of inflammatory genes, including Tnf, Itgax, and Il6, was reduced by ∼50%–80% in the scWAT and eWAT of thermoneutral Ccr2mice ( Figures S5 F–S5H). Based on these findings, we predicted that thermoneutral housing would protect Ccr2mice from developing obesity-associated metabolic disease. Again, to our surprise, we failed to observe any improvements in glucose tolerance or insulin sensitivity in obese thermoneutral Ccr2mice, as assessed by fasting insulin levels, glucose and insulin tolerance tests, and insulin-stimulated phosphorylation of AKT in eWAT and liver ( Figures 3 I–3M). These data collectively suggest that adipose tissue inflammation does not measurably contribute to obesity-associated metabolic dysfunction in mice housed at thermoneutrality.

(C–H) Quantification of macrophages (C), CD11c + cells (D), monocytes (E), Ly6C hi monocytes (F), Ly6C lo monocytes (G), and FoxP3 + (H) in scWAT and eWAT of thermoneutral WT and Ccr2 −/− mice fed HFD.

A large number of preclinical studies with genetically modified mice have linked adipose tissue inflammation to the development of obesity-associated insulin resistance (). Since we observed earlier recruitment of innate and adaptive immune cells into the eWAT of thermoneutral mice, we postulated that these animals might be more susceptible to development of obesity-associated metabolic disease. To test this hypothesis, we initially assessed glucose homeostasis in mice 3–4 weeks after the initiation of HFD. We chose this time point because we observed a robust increase in the numbers of innate and adaptive immune cells in the eWAT of mice housed at a Tof 30°C but not 22°C ( Figures 1 and S1 ). To our surprise, tests of glucose and insulin tolerance in thermoneutral mice revealed no differences from those housed at a Tof 22°C, which lacked evidence of metabolic inflammation ( Figures S4 A, S4B, and 1 ). Since it has been suggested that obesity-induced adipose tissue inflammation primarily contributes to the establishment of a state of chronic insulin resistance (), we next asked whether prolonged feeding of HFD potentiated glucose intolerance and insulin resistance in thermoneutral mice. Over a 10-week period of HFD feeding, C57BL/6J mice housed at a Tof 22°C gained slightly more weight than those housed at a Tof 30°C ( Figure 2 D). This increase in body mass was not associated with significant changes in adipose tissue mass, including eWAT, subcutaneous WAT (scWAT), or BAT mass ( Figures 2 E–2G). Despite increased recruitment of inflammatory cells into adipose depots ( Figures 1 and S1 ), thermoneutral mice did not exhibit evidence of impaired glucose tolerance and insulin sensitivity, as quantitatively assessed by serum levels of insulin, insulin tolerance tests, and insulin-stimulated phosphorylation of AKT in eWAT and liver ( Figures 2 H–2L). Analysis of serum lipids revealed that HFD-fed mice housed at a Tof 30°C had higher levels of total cholesterol and triglycerides ( Figures S4 C and S4D). In aggregate, these data suggest that thermoneutral housing of mice uncouples the onset of metabolic inflammation in adipose depots from the development of acute and chronic insulin resistance.

Since chronic low-grade inflammation has been implicated in the pathogenesis of a number of metabolic and degenerative diseases, we next asked whether thermoneutral housing leads to a generalized increase in innate inflammatory responses in mice. We tested this hypothesis using zymosan-induced peritonitis, a self-resolving model of acute inflammation. Previous studies have shown that zymosan-induced sterile peritonitis results in rapid (peaking at 4 hr) extravasation of neutrophils into the peritoneal cavity, followed by the recruitment of monocytes (peaking at 16–24 hr) (). Compared to mice housed at a Tof 22°C, thermoneutral housing of mice potentiated the acute inflammatory response to zymosan, as evidenced by the increase in neutrophil (∼70%) and monocyte (∼80%) recruitment into the peritoneal cavity ( Figures 2 A and 2B ). Moreover, deletion of the chemokine receptor CCR2, which mediates recruitment of Ly6cmonocytes to sites of inflammation (), decreased monocytic infiltration of the peritoneal cavity by ∼64% ( Figure 2 C), suggesting that signaling via CCR2 might also contribute to the development of increased innate inflammation in obese thermoneutral mice.

(H–L) Assessment of glucose homeostasis in C57BL/6J mice housed at a T a 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 T a of 22°C or 30°C and fed HFD.

(E–G) Mass of adipose tissues of C57BL/6J mice housed at a T a of 22°C or 30°C and fed NC or HFD; eWAT (E), scWAT (F), and BAT (G).

(D) Changes in body mass of C57BL/6J mice housed at a T a of 22°C or 30°C and fed normal chow (NC) or high-fat diet (HFD) starting at 6 weeks of age.

(C) Quantification of monocytes following zymosan-induced peritonitis in WT and Ccr2 −/− mice housed at a T a of 30°C (n = 4–5 per genotype).

(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 T a 22°C or 30°C (n = 10 per group).

Because HFD feeding and thermoneutral housing promote increased lipid accumulation in brown adipocytes ( Figures S2 K and S2L), we also quantified the kinetics of immune cell infiltration in this thermogenic adipose depot. Analogous to eWAT, HFD feeding gradually resulted in the accumulation of innate (M1 and M2 macrophages, eosinophils, neutrophils) and adaptive (CD4and CD8T cells, B cells) immune cells in the BAT of mice housed at a Tof 22°C ( Figures S3 A, S3C, S3E, S3G, S3I, S3K, S3M, S3O, S3Q, S3S, S3U, and S3W). Again, the kinetics of immune cell recruitment were left shifted in thermoneutral mice, as we observed much earlier accumulation (at 3–6 weeks after initiation of HFD diet) of innate and adaptive immune cells in the BAT of mice housed at a Tof 30°C ( Figures S3 B, S3D, S3F, S3H, S3J, S3L, S3N, S3P, S3R, S3T, S3V, and S3X). Together, these data suggest that housing C57BL/6J mice at a Tof 30°C accelerates the onset of HFD-induced metabolic inflammation in adipose depots.

Adipose tissue contains at least two distinct populations of macrophages, termed M1 and M2, which exert differential effects on insulin sensitivity (). M1 macrophages, which are lipid laden and express CD11c, accumulate during obesity and have been implicated in the promotion of obesity-associated insulin resistance. In contrast, M2 macrophages express CD301 and CD206 and have been implicated in the maintenance of insulin sensitivity in lean and obese animals. We thus investigated whether thermoneutral housing altered the recruitment kinetics of these two adipose tissue macrophage (ATM) subsets. We found that the numbers of M1 (CD11cCD206) and M2 (CD11cCD206, CD11cCD206, and CD301) ATMs increased 12 weeks after initiation of HFD in mice housed at a Tof 22°C ( Figures 1 E, 1I, 1K, and S1 L). In contrast, we observed much earlier (at 3–6 weeks after HFD feeding) accumulation of both M1 and M2 ATMs in thermoneutral mice ( Figures 1 F, 1J, 1L, and S1 M). Since in a separate screen we identified high expression of CD169 on lipid-laden macrophages, we monitored its expression in ATMs of lean and obese mice. We found that the increase in the numbers of CD169ATMs paralleled that of CD11cCD206M1 ATMs and was most prominently observed in mice housed at a Tof 30°C ( Figures 1 G and 1H). qRT-PCR analysis provided independent verification of accelerated accumulation of macrophages in the eWAT of thermoneutral mice ( Figures S2 A–S2J).

To investigate the relationship between ambient temperature and onset of metabolic inflammation, we housed cohorts of C57BL/6J mice at 22°C or 30°C and fed them normal chow (NC) or high-fat diet (HFD, 60% of calories from lipids) for various time periods. Previous studies have demonstrated that C57BL/6J mice fed HFD recruit innate and adaptive immune cells in their epididymal white adipose tissue (eWAT) as they gain weight (). We thus employed fluorescence-activated cell sorting (FACS) to quantitatively assess the kinetics of immune cell infiltration of eWAT in mice housed at a Tof 22°C or 30°C ( Figure S1 A). 12 weeks after the initiation of HFD, we observed an increase in the numbers of hematopoietic (CD45), innate (macrophages), and adaptive (CD4and B cells) immune cells in eWAT of mice housed at a Tof 22°C ( Figures 1 A, 1C, S1 B, and S1D), findings that are consistent with previous reports examining the patterns of immune cell infiltration in obese eWAT (). This recruitment of innate and adaptive immune cells was accelerated in mice housed at a Tof 30°C, as we could detect significant immune cell infiltration in eWAT as early as 3 weeks after initiation of HFD ( Figures 1 B, 1D, S1 C, and S1E). Thermoneutral housing also accelerated the recruitment of neutrophils and CD8T cells ( Figures S1 F–S1I), which have been pathogenically linked to obesity-induced insulin resistance. In contrast, we did not observe significant differences in the numbers of eosinophils present in the eWAT of mice housed at a Tof 22°C or 30°C ( Figures S1 J and S1K).

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 T a of 22°C or 30°C (n = 4–5 per temperature/diet/time point).

Although it is well appreciated that housing mice at the normal vivarium temperature of 20°C–22°C poses significant cold stress (), there is a paucity of literature that has studied the development of metabolic disease in mice housed at thermoneutrality (28°C–30°C). As some prior reports have found alterations in innate and adaptive immune responses in thermoneutral mice (), we decided to systematically investigate how thermoneutral housing modulates the onset of metabolic inflammation and disease.

Discussion

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Schellen L. Cold exposure--an approach to increasing energy expenditure in humans. Figure 7 Model for Differential Effects of Metabolic Inflammation in Thermoneutral Mice Show full caption 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. There is increasing evidence that inflammation is associated with modern human diseases, such as obesity, insulin resistance, type 2 diabetes mellitus, and atherosclerosis (). Over the last two decades, mechanistic studies performed in mice have supported a causal link between inflammation in metabolic and vascular tissues, and the progression of obesity-associated insulin resistance and atherosclerosis, respectively (). However, nearly all of these studies have been carried out in mice that are housed in environmental conditions (Tof 20°C–22°C) that impose significant stress on their metabolic, immune, and cardiovascular systems. In contrast, most humans are thermally unstressed as they live in their thermal comfort or neutral zone (), making it unclear whether these rodent studies can be directly translated to human disease. In order to begin to address this gap in our knowledge, we asked whether thermoneutral housing modulated the development of metabolic inflammation, insulin resistance, and atherogenesis in mice. We found that although the onset of metabolic inflammation occurred earlier in thermoneutral mice, it did not translate equally into severity of metabolic disease. While increased inflammation in the vessel wall was associated with accelerated atherosclerosis, earlier onset of metabolic inflammation in adipose tissues did not potentiate obesity-induced insulin resistance or glucose intolerance ( Figure 7 ). Our studies with thermoneutral mice support the targeting of inflammation for the treatment of atherosclerosis but raise the possibility that this approach might be less efficacious in the treatment of insulin resistance or type 2 diabetes mellitus in thermoneutral humans.

a of 20°C–22°C). In contrast, we found that the housing of mice at thermoneutrality completely uncoupled the adipose tissue inflammatory response from the development of insulin resistance and glucose intolerance ( A central tenet for the emerging field of “immunometabolism” has been that obesity causes infiltration of adipose tissue by macrophages, which promote adipose tissue inflammation and insulin resistance. While evidence in support of this hypothesis is strong, much of it comes from studies performed in cold-stressed mice (since most published studies do not document the ambient temperature of their vivarium, we assume that mice were housed at the normal Tof 20°C–22°C). In contrast, we found that the housing of mice at thermoneutrality completely uncoupled the adipose tissue inflammatory response from the development of insulin resistance and glucose intolerance ( Figure 7 ). For example, using mice that lacked Ccr2, we found that while inflammation in eWAT and scWAT was reduced by ∼80%, glucose homeostasis was unaffected, suggesting that the contribution of inflammation to insulin resistance in thermoneutral humans might be limited.

a of 20°C–22°C imposes significant cold stress, resulting in the secretion of glucocorticoids and the activation of sympathetic nervous system ( Gordon, 1993 Gordon C.J. Temperature regulation in laboratory rodents. Leduc, 1961 Leduc J. Catecholamine production and release in exposure and acclimation to cold. Shum et al., 1969 Shum A.

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Scherer P.E. Adipocyte inflammation is essential for healthy adipose tissue expansion and remodeling. A key question that emerges from our work is the following: how does one reconcile these new results with hundreds of previous reports that have suggested a critical role for adipose tissue inflammation in the promotion of insulin resistance? One possible explanation for this discrepancy might be that the contribution of inflammation to insulin resistance is limited to animals that are metabolically stressed. For example, housing mice at a Tof 20°C–22°C imposes significant cold stress, resulting in the secretion of glucocorticoids and the activation of sympathetic nervous system (). Because both glucocorticoids and catecholamines are catabolic, they interfere with the normal anabolic actions of insulin (). Thus, when these cold-stressed mice become obese, adipose tissue inflammation might be the additional hit that causes mice to metabolically decompensate. In contrast, the activation of these adaptive stress responses is markedly reduced in thermoneutral mice, and the inflammatory response to obesity is well tolerated in metabolic tissues. In fact, the observed accelerated onset of metabolic inflammation in thermoneutral mice might be a direct consequence of the systemic decrease in glucocorticoids and catecholamines, which are known to suppress the inflammatory functions of lymphoid and myeloid cells (). Lastly, the accelerated onset of metabolic inflammation in thermoneutral mice might facilitate the adipose tissue remodeling, thus enabling better storage of excess nutrients ().

−/− mice. A priori, it was unclear whether thermoneutral housing would be pro- or anti-atherogenic. On the one hand, mice at a T a of 30°C exhibit a reduction in their adrenergic tone ( Swoap et al., 2008 Swoap S.J.

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Zarins C.K. Retarding effect of lowered heart rate on coronary atherosclerosis. −/− mice (−/− mice fed either NC or WD. The magnitude of this effect is also significant because, despite the predicted improvements in hemodynamic parameters, thermoneutral Apoe−/− mice fed the WD had an ∼2-fold increase in total lesion area and ∼3-fold increase in lesion area in the descending aorta ( Bäck and Hansson, 2015 Bäck M.

Hansson G.K. Anti-inflammatory therapies for atherosclerosis. Tabas and Glass, 2013 Tabas I.

Glass C.K. Anti-inflammatory therapy in chronic disease: challenges and opportunities. −/− mice might be a better model for preclinical evaluation of these novel therapeutics. Unlike diet-induced obesity, thermoneutral housing accelerated the development of atherosclerotic lesions in Apoemice. A priori, it was unclear whether thermoneutral housing would be pro- or anti-atherogenic. On the one hand, mice at a Tof 30°C exhibit a reduction in their adrenergic tone (), which might be anti-atherogenic because it improves hemodynamics by decreasing heart rate and blood pressure (). On the other hand, we postulated that the accelerated onset of metabolic inflammation might stimulate the development of atherosclerotic lesions in thermoneutral animals. In support of the latter, we observed that atherosclerotic lesions were markedly increased in thermoneutral Apoemice ( Figure 7 ). It is important to note that the increase in lesion area was observed in thermoneutral Apoemice fed either NC or WD. The magnitude of this effect is also significant because, despite the predicted improvements in hemodynamic parameters, thermoneutral Apoemice fed the WD had an ∼2-fold increase in total lesion area and ∼3-fold increase in lesion area in the descending aorta ( Figures 4 A and 4B). Although we cannot exclude the possibility that the modest (∼20%) increase in serum cholesterol is contributing to the massive increase in atherosclerotic plaque area (∼200%–300%, depending on location), the observed increase in atherogenesis correlated well with an ∼2-fold increase in cellular inflammation in the vessel wall and thoracic perivascular fat. We also cannot exclude the possibility that thermoneutrality might influence the lipid composition of atherogenic lipoproteins or their rate of uptake into the vasculature. Nevertheless, since anti-inflammatory therapies are currently being considered for the treatment of human atherosclerosis (), our work suggests that thermoneutral Apoemice might be a better model for preclinical evaluation of these novel therapeutics.

Given the broad importance of ambient temperature in regulating metabolic, immune, cardiovascular, and behavioral responses in mice, we and others propose that more predictive modeling of human physiology and disease can be achieved in mice by paying closer attention to their housing temperature. Future investigations in thermoneutral mice not only might make these preclinical studies more translatable, but might also yield important insights and surprises in our understanding of mammalian physiology and the mechanisms by which it adapts to environmental challenges.