Fig 2: Increased infiltration of M2-like DARC+ macrophages following HFD and interleukin 22 (IL-22)-induced DARC expression. (A,B) Infiltrating monocyte-derived DARC+ cells show characteristics of M2-like macrophages. Frequency of DARC+ cells in CCR7+CD11b+F4/80+ (classical inflammatory M1) or CD206+CD11b+F4/80+ (alternative tissue repair M2) monocytes from CD11b+F4/80+ subset among the SVCs of wild-type adipose tissue (A) and these population were quantified (B). (C) Augmentation of the DARC+CD206+ subset, but not the DARC+CCR7+ subset, was identified in obese SVCs as shown by FACS analysis (n = 7~8 mice per group). * p < 0.05 versus the corresponding control. Data are shown as a mean ± SEM. Comparisons were performed using t-tests (two groups). Data were combined from at least three independent experiments. (D) Mean fluorescence intensity (MFI) of M2 DARC+ macrophages were increased after HFD consumption. MFI of DARC was determined in CCR7+CD11b+F4/80+ or CD206+CD11b+F4/80+ subset of chow- and HFD-fed mice. (E) Identification of IL22Ra+ cells in the DARC+ subset of splenic monocytes. The gating and staining strategies used in the FACS quantification of an IL-22Ra1+ subset in DARC+CD11b+Ly6Clo or DARC+CD11b+Ly6hi cells are shown. Numbers (black outline at left) indicate the percentage of CD11b+ cells among the splenocytes. (F) DARC expression was increased by IL-22 treatment of monocyte/macrophages. BM cells isolated from WT mice were treated with or without mIL-22 (20 ng/mL) for 24 h and the frequency of DARC+ cells among the CD11b+ or F4/80+ monocytes was then analyzed by FACS as described in the materials and methods. ** p < 0.01, *** p < 0.001 versus the control. Data were combined from at least three independent experiments and are shown as the mean ± SEM. Comparisons were performed using t-tests (two groups).

Fig 3: DARC+ macrophages show regulatory M2 characteristics under obese conditions. (A) Body weights of WT and db/db mice were monitored (0–15 weeks, n = 8 mice per group). (B) Frequency of CD206+DARC+ subsets in the monocyte/macrophage populations in SVCs from eWATs of WT and db/db mice. FACS plots identified CD206+DARC+ subsets in populations of myeloid cells with F4/80+ marker and the graph (right) shows the proportions of CD206+DARC+ in monocytes/macrophages of SVCs defined by F4/80+. (C) MFI of DARC+F4/80+ cells and ratio of CD206/CCR7 in DARC+F4/80+ cells were analyzed by FACS in WT and db/db mice. (D) The DARC+ subset is enhanced by an HFD corresponding to an increase in the Th22+ subset. Distribution of the DARC+ cells was analyzed among the SVCs of eWATs of WT and IL-22-/-mice subjected to a chow diet or an HFD. * p < 0.05 versus the corresponding control. (E) An IL-22 deficiency skews the monocytes/macrophages toward an inflammatory state. BMMs from WT and IL-22 KO mice were stimulated with either 25 ng/mL IFN-? and 100 ng/mL LPS or 10 ng/mL IL-4 to derive M1 or M2 macrophages, respectively. qPCR was used to analyze the mRNA levels of TNF-a, IL-1ß, IL-12ß, and iNOS (M1 markers) and Arg1, Fizz1, Ym1, IL-4, and IL-10 (M2 markers) before (E, left) and after (E, right) stimulation. All samples were normalized to the GAPDH expression level. The results represent the mean ± SEM of at least three independent experiments. Comparisons were performed using Tukey post hoc comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the corresponding control.

Fig 4: DARC expression in monocytes is dependent on an IL-22/IL-22Ra1-STAT5b signaling axis. (A–D) THP1 cells were incubated with 20 ng/mL IL-22 in the absence or presence of the indicated concentrations of the STAT5b inhibitor, CAS285989, for 24 h and DARC protein expression was determined by immunoblotting assay (A, B) and FACS analysis (C, D). Western blot was quantified and expressed as the ratio of DARC and ß-actin intensity. Data are representative of three independent experiments with similar results. (E) RT-PCR ChiP assay results showing the occupancy of p-STAT5 over the atypical chemokine receptor 1 (Ackr1) promoter. Three predicted STAT5b binding regions were located downstream from the core promoter region of ackr1 (−10,000 to −4500) (E, upper). Interactions between STAT5b and the ackr1 (DARC) promoter sequence with or without IL-22 treatment in THP-1 cells were determined by ChiP assay using specific PCR primers (1~3) (E, lower). (F) Firefly luciferase constructs containing the core promoter region of ackr1 (−1001 to −1), PCR 3 primer-selective fragment (−5524 to −4564), and luciferase coding region in pGL3 enhancer vector. +1 indicates the transcriptional start site. (G) HEK 293 cells were co-transfected with the firefly luciferase constructs (WT, MT1, MT2, or MT3 of STAT5b binding sites), incubated for 24 h, and then treated with 20 ng/mL IL-22. A Renilla luciferase control was included in the dual-luciferase assay. The relative luciferase activity represents the dual luciferase activity ratio (firefly/Renilla luciferase). The results represent the mean ± SEM of at least three independent experiments. Comparisons were performed using ANOVA (multiple groups). ** p < 0.01, *** p < 0.001 corresponding to control group versus WT control.

Fig 5: Presence of DARC+IL-22R1 cells in human peripheral blood. (A) The presence of a DARC+ subset among the monocytes or macrophages of human blood. The relative proportions of IL-22Ra1+- or DARC+- subset in monocytes/macrophages, defined by CD14+, CD11b+, or CD16+, were estimated in human PBMCs. (B) DARC+ cells are present among CD16+CD14+ macrophages. Myeloid monocyte subsets of human blood were gated as (1) CD16hiCD14lo-non-classical, (2) CD16+CD14+-intermediate, and (2) CD16loCD14hi-classical monocytes (B, upper). The DARC+ subset frequency in cell subpopulations sorted using an inflammatory M1 maker, CD86+, or alternative M2 marker, CD206+, was quantified from selective populations of (1), (2) and (3) (C, middle and lower) using FACS analysis. * p < 0.05 versus the corresponding control. (C) IL-22 signaling enhances M2-like macrophage potential. PBMCs isolated from human healthy donors (n = 3~4) were treated with 20 ng/mL rIL-22 for 24 h and then classical inflammatory M1- or alternative M2-related genes were identified by qPCR. * p < 0.05, ** p < 0.01 versus the corresponding control. Values are expressed as a mean ± SEM. Comparisons were performed using t-tests (two groups). (D) Intracellular FACS analysis of cytokine-producing monocytes following IL-22 stimuli. Isolated PBMCs were stimulated with IL-22, incubated in the presence of brefeldin A, and then analyzed by FACS. Representative flow cytometry histograms of IL-10- and TGF-ß-producing monocytes from gated DARC+ monocytes in blood are shown (healthy donors, n = 4). (E) STAT5 signaling is necessary for DARC expression via IL-22 in human primary monocytes. Flow cytometry was used to analyze DARC expression in peripheral blood mononuclear cells (PBMCs) isolated from three donors. Non-stimulated cells as resting PBMCs served as a control. Cells were pretreated with STAT5i for 30 min and incubated with or without 20 ng/mL of IL-22 for 24 h, followed by DARC expression analysis by FACS. *** p < 0.001 versus the corresponding control. Data are the mean ± SEM. Comparisons were performed using t-tests (two groups) or ANOVA (multiple groups). Data were combined from at least three independent experiments.