Fig. 4

Cold induced neuroimmune cells (CINCs) home in to inguinal scWAT and express Bdnf. Adult (12 week old) male C57BL/6 were either maintained at room temperature (RT) or cold exposed (5 °C) for 5 days, ATMs from inguinal scWAT depots were isolated using magnetic-activated cell sorting (MACS) by positive selection of CD11b⁺ followed by F4/80⁺ cells. Bdnf gene expression in doubly labeled CD11b⁺ F4/80⁺ macrophages was compared between RT and cold exposed animals, p = 0.5796 (a). Data were analyzed by two-tailed Student’s T-Test, N = 4 per group. Adult (12 week old) female control animals were either maintained at room temperature (RT) or cold exposed (5 °C) for 10 days, SVF from bilateral inguinal scWAT was isolated and FACS sorted using a 20 cell surface marker panel for myeloid lineage immune cells (b, c). Changes in M1/M2 polarity, RT versus cold M1 macrophages p = 0.8485, and RT versus cold M2 p = 0.3387 (b) and Ly6C⁺CCR2⁺Cx3CR1⁻ and Ly6C⁺CCR2⁺Cx3CR1⁺ macrophage precursors/monocytes (c, left panel) were measured between RT and cold exposed animals, p = 0.0401 and p = 0.0354, respectively. Adult (12–13 week old) male C57BL/6 were either maintained at room temperature (RT) or cold exposed (5 °C) for 14 days; SVF from bilateral inguinal scWAT was isolated and FACS sorted; Ly6C⁺CCR2⁺Cx3CR1⁻ and Ly6C⁺CCR2⁺Cx3CR1⁺ cells were compared between RT and cold exposed animals, p = 0.9525 and p = 0.0566, respectively (c, right panel). Data were analyzed by two-tailed Student’s T-Test, N = 3–4 per group for both sexes. Adult male (M) and female (F) control animals were either maintained at room temperature (RT) or cold exposed (5 °C) for 10 days, SVF from bilateral inguinal scWAT was isolated and FACS sorted using a 20 cell surface marker panel for myeloid lineage immune cells (N = 5 per group). t-Distributed Stochastic Neighbor Embedding (tSNE) analysis was performed to identify myeloid lineage cell population changes in response to cold exposure; Ly6C⁺CCR2⁺Cx3CR1⁺ were identified for both sexes as adipose CINCs (d). Bdnf gene expression measured in Ly6C⁺CCR2⁺Cx3CR1⁺ cells indicated that these infiltrating cells express Bdnf, p = 0.1529 (e). Data analyzed by two-tailed Student’s T-Test, N = 4 per group. For all error bars are SEMs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001