Thermoneutrality but Not UCP1 Deficiency Suppresses Monocyte Mobilization Into Blood

Abstract

Rationale: Ambient temperature is a risk factor for cardiovascular disease. Cold weather increases cardiovascular events, but paradoxically, cold exposure is metabolically protective because of UCP1 (uncoupling protein 1)-dependent thermogenesis.

Objective: We sought to determine the differential effects of ambient environmental temperature challenge and UCP1 activation in relation to cardiovascular disease progression.

Methods and results: Using mouse models of atherosclerosis housed at 3 different ambient temperatures, we observed that cold temperature enhanced, whereas thermoneutral housing temperature inhibited atherosclerotic plaque growth, as did deficiency in UCP1. However, whereas UCP1 deficiency promoted poor glucose tolerance, thermoneutral housing enhanced glucose tolerance, and this effect held even in the context of UCP1 deficiency. In conditions of thermoneutrality, but not UCP1 deficiency, circulating monocyte counts were reduced, likely accounting for fewer monocytes entering plaques. Reductions in circulating blood monocytes were also found in a large human cohort in correlation with environmental temperature. By contrast, reduced plaque growth in mice lacking UCP1 was linked to lower cholesterol. Through application of a positron emission tomographic tracer to track CCR2⁺ cell localization and intravital 2-photon imaging of bone marrow, we associated thermoneutrality with an increased monocyte retention in bone marrow. Pharmacological activation of β3-adrenergic receptors applied to mice housed at thermoneutrality induced UCP1 in beige fat pads but failed to promote monocyte egress from the marrow.

Conclusions: Warm ambient temperature is, like UCP1 deficiency, atheroprotective, but the mechanisms of action differ. Thermoneutrality associates with reduced monocyte egress from the bone marrow in a UCP1-dependent manner in mice and likewise may also suppress blood monocyte counts in man.

Keywords: atherosclerosis; bone marrow; housing; macrophages; thermogenesis.

Figure 1. Ambient temperature regulates atherosclerosis progression

Male (top) and female (bottom) Ldlr^−/− mice were housed at the indicated ambient temperature and fed HFD for four weeks, then assayed for (A) food intake (left), rectal temperature (right), and (B) UCP1 mRNA expression in brown adipose pads. C) Atherosclerotic plaque area in the aortic sinus of Ldlr^−/− mice was measured following 8 weeks HFD treatment at the indicated environmental temperature. D) Representative oil red o staining to highlight regions of lipid deposition. E) Representative en face plaque staining of aortic arches and quantification following 8 weeks HFD. Triacylglycerides (F), nonesterified free fatty acids (G), and total cholesterol levels (H) were measured in the plasma from Ldlr^−/− mice following 8 weeks HFD at the indicated temperatures. I) Corticosterone levels in the plasma of Ldlr^−/− mice fed HFD for 8 weeks. J) Kinetic analysis of plasma lipoproteins HDL, LDL, and vLDL at zero, four, and eight weeks of HFD feeding at indicated temperatures. All experiments are representative experiments with 5–10 animals per group, repeated in two or three independent experiments. Statistical analysis was performed by unpaired student T-test, * p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 2. Monocyte recruitment into plaque is reduced in mice housed at thermoneutral environment

A) CD68 staining of aortic sinus of Ldlr^−/− mice following 8 weeks HFD at 22°C or 30°C ambient temperature challenge, with quantification (B). C) Following clodronate liposome and bead labeling of Ly6C⁺ monocytes, Ldlr^−/− mice on HFD were assayed for bead deposition into plaque regions of the aortic sinus, and (E) quantification of beads recruited per section. E) Total blood and bone marrow cellularity was measured following bead labeling. C) Percentage of blood and bone marrow monocytes were calculated by flow cytometry. (A-D) Data presented are combined between 3 independent experiments and include n≥10 animals per group. (E-F) Data include 4–5 animals per group and are representative of three independent experiments. Statistical analysis was performed by unpaired student T-test, * p≤0.05, **p≤0.01.

Figure 3. Thermoneutral housing reduces circulating blood monocytes

A) Ldlr^−/− mice were housed at indicated temperatures and measured for total blood leukocytes on chow or HFD. B) Circulating Ly6C⁺ and Ly6C⁻ monocyte number on chow or after 6 weeks of HFD treatment. C) Apoe^−/− fed HFD for four weeks and assayed for total blood cell and monocyte subset numbers. D) Representative flow histogram of BrdU incorporation in Ly6C⁺ monocytes in the blood 24 hours following i.p. injection of 1 μg BrdU. E) BrdU incorporation in circulating Ly6C⁺ blood monocytes 24 hrs post BrdU injection i.p, in strains Ldlr^−/− (left) and Apoe^−/− (right). A-C) Data presented are combined between ≥3 independent experiments and include n≥10 animals per group. D-E) are representative experiments containing n≥4 animals per group and repeated two times each. Statistical analysis was performed by unpaired student T-test, * p≤0.05, **p≤0.01.

Figure 4. PET/CT imaging identified differential CCR2⁺ cells in aortic arch and bone marrow of mice at thermoneutral housing

A) ⁶⁴Cu-DOTA-ECL1i PET/CT images showing specific uptake at aortic arch of Ldlr^−/− mice on HFD and housed at indicated ambient temperature. B) Quantification of tracer uptake in the aortic arch of Ldlr^−/− mice housed in the indicated temperature. Biodistribution of C) ⁶⁴Cu-DOTA-ECL1i and (D) CXCR4 tracer ⁶⁴Cu-AMD3100 in Ldlr^−/− mice fed HFD and housed at the indicated ambient temperature. All experiments were performed on 4–8 animals for each group and repeated in two independent experiments. Statistical analysis was performed by unpaired student T-test, * p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 5. Accumulation of bone marrow monocytes in mice housed at thermoneutrality

A) C57bl/6 mice were housed at 22°C or 30°C environment for seven days, then assayed for circulating monocytes (Ly6C⁺ and Ly6C⁻). B) C57bl/6 mice housed at indicated temperatures were treated with BrdU i.p. for 24hrs, then assayed for incorporation of BrdU in Ly6C⁺ monocytes to measure bone marrow egress C) Snapshot from 2-photon imaging approach of bone marrow in Cx3cr1^gfp reporter mice crossed with Cxcl12^dsred to examine monocytes within the bone marrow. D) Measurement of Cx3cr1^gfp expression per frame, Cxcl12^dsred expression per frame, and ratio of GFP/Dsred in bone marrow from animals housed at indicated temperature. E) Flow cytometeric analysis of Cx3cr1^gfp/+ bone marrow total cellularity and GFP⁺ monocytes housed at indicated ambient temperature. F) CCR2 MFI for bone marrow monocytes from Cx3cr1^gfp mice housed at 22°C or 30°C. (A-B, E-F) Data are representative experiments from three independent experiments with n≥5 per group. C) Image is a representative image of greater than 5 independent experiments. D) Data are combined from 5 independent experiments for each group. Statistical analysis was performed by unpaired student T-test, * p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 6. Environmental temperature correlates with circulating monocyte levels in humans

A) Peripheral blood was assayed for complete blood counts (CBC) with automated differentials for monocyte levels through a 12-month period and plotted as monthly averages. B) Monthly environmental temperature with mean high, average, and low temperatures reported for each month that peripheral blood was collected. C) Regression plot of average monthly cell numbers against average monthly temperature for monocytes. E) Regression plots for Neutrophils, Eosinophils, Basophils, and Lymphocytes numbers against average monthly temperature. Data in A and C are mean ± SEM, and represent a total cohort of n=15,516 patients, with approximately 1,200 measurements per month. Pearson linear regression analysis was performed to test correlations between monthly cell counts and environmental temperatures, p-values and R² are reported.

Figure 7. Impact of UCP1 deficiency on atherosclerosis and glucose tolerance in comparison to Thermoneutrality

A) Oil red O lipid staining of aortic sinus from the indicated mice following HFD treatment for 8 weeks at 22°C or 30°C housing, which quantification (right). B) Total body weight in mice after HFD. C) Ucp1^−/− Ldlr^−/− mice fed HFD for 12 weeks and were housed at 22°C or 30°C and assayed for plaque development. D) Glucose tolerance tests (GTT) were performed on 22°C or 30°C housed Ldlr^−/− and Apoe^−/− mice following 8 weeks HFD. E) Ucp1^−/− Ldlr^−/− and Ldlr^−/− mice fed HFD for 8 weeks and housed at 22°C were assayed by GTT. F) Ucp1^−/− Ldlr^−/− mice fed HFD were housed at 22°C or 30°C and assayed for GTT. All data are representative experiments, containing n≥5 animals per group and repeated in two or three independent times. Statistical analysis was performed by unpaired student T-test, * p≤0.05, **p≤0.01, ***p≤0.001.

Figure 8. UCP1-deficiency does not drive changes in monocyte distribution following thermoneutral challenge

WT or UCP1^−/− mice housed at 22°C ambient temperature were assayed for blood monocyte numbers (A) or biodistribution of ⁶⁴Cu-DOTA-ECL1i probe (B). C) Ldlr^−/− or UCP1^−/− Ldlr^−/− mice were housed at 22°C ambient temperature on HFD for 8 weeks, and assayed for blood leukocyte numbers. D) Ldlr^−/− or Ucp1^−/− Ldlr^−/− mice housed at 22°C and fed HFD were assayed for ⁶⁴Cu-DOTA-ECL1i biodistribution. A, C) are representative experiments n≥5 animals per group and repeated two independent times. B, D) Data are 5–8 animals combined per group. Statistical analysis was performed by unpaired student T-test, * p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.