CCR2-/- male mice display reduced inguinal fat pad, and increased inguinal lymph node size.

a) In situ orientation of inguinal fat pads with embedded inguinal lymph nodes in WT male, CCR2-/- male and CCR2-/- female mice. Scale bar: 1cm. b) i) Excised inguinal fat pads from WT male (top) and CCR2-/- male (bottom). Scale bar: 1cm. ii) Inguinal fat pad weight (in grams) of WT and CCR2-/- mice, both male and female, at 8-10 weeks and 16+ weeks of age. Each point represents the combined weight of both inguinal fat pads of a single mouse. c) i) Excised inguinal lymph nodes from WT male (top) and CCR2-/- male (bottom). Scale bar: 0.5cm. ii) Live cell counts of inguinal lymph nodes, expressed as fold of WT and CCR2-/- mice, both male and female, at 8-10 weeks and 16+ weeks of age. Each point represents the combined weight of both inguinal lymph nodes of a single mouse. The number of live cells was determined via dye exclusion of trypan blue and normalised to the average of WT counts (age and gender matched) to obtain values expressed as fold of WT. Unpaired t test with Welch’s correction was performed to determine statistical significance, with a p value of 0.05 determined as significant. *p<0.05, **p<0.01, ***p<0.001. d) Correlation of inguinal fat pad (x-axis) and lymph node (y-axis) weight, both expressed in grams, showing a considerable degree of dispersion and variability at the resting state in CCR2-/- males. Each point corresponds to the combined weight of both fat pads and both lymph nodes of a single CCR2-/- mouse. Simple linear regression analysis was conducted, yielding an equation of Y=-0.03174x+0.01662 (plotted, black), with an R2 value of 0.6898. The slope coefficient was found to be significantly non-zero, as indicated by a p value of <0.0001. N= 63. e) Overall mouse weight, in grams, of WT and CCR2-/- mice, both male and female, at 8-10 weeks and 16+ weeks of age.

The observed phenotype is associated with skin wounding.

a) Examples of more severe fighting induced injuries observed in CCR2-/- males housed together in groups of 5 from the age of 8 weeks compared to CCR2-/- males housed alone. Of note, some CCR2-/- males in the ‘together’ group had to be culled to before the agreed timepoint due to the extent of their fighting injuries (third panel image showing necrotic tail tip). b) In situ and excised (top=alone, bottom=together) inguinal fat pads and lymph nodes from CCR2-/- males housed alone or together in groups of 5. Scale bar= 1cm. c) Inguinal fat pad weight, in grams, of 14-16 weeks old CCR2-/- mice, either housed together in groups of 5 or alone, from the age of 8 weeks. Both inguinal fat pads of each mouse are pooled for each individual measurement. d) Inguinal lymph node weight, in grams, of 14-16 weeks old CCR2-/- mice, either housed together in groups of 5 or alone, from the age of 8 weeks. Both inguinal lymph nodes of each mouse are pooled for each individual measurement. e) Correlation of inguinal fat pad (x-axis) and lymph node (y-axis) weight, both expressed in grams, of i) CCR2-/- males housed alone (dark blue), ii) WT males (light blue), iii) CCR2-/- females (purple) and iv) CCR2-/- males housed together (red). Simple linear regression for CCR2-/- alone, WT and CCR2-/- females was non-significant, with R2 values of 0.2203, 0.3046 and 0.05 respectively. The slope coefficient of the simple linear regression on CCR2-/- housed together was significantly non-zero (p=0.004), with an R2 value of 0.8477. f) Combined linear regressions of the correlation between Lymph node weight and fat pad weight of CCR2-/- housed alone (blue), CCR2-/- housed together (red), WT (blue), CCR2-/- females (purple) and previously calculated linear regression on 63 CCR2-/- males (black, Figure 1D) g g) Inguinal lymph node weight, in grams, of WT males at 14 weeks housed either alone or in groups of 5 from the age of 8 weeks. Each dot represents the combined weight of both inguinal lymph nodes of each mouse. Red dots indicate mice with obvious signs of fighting injuries. h) Fat pad weight, in grams, of WT males at 14 weeks housed either alone or in groups of 5 from the age of 8 weeks. Each dot represents the combined weight of both inguinal fat pads of each mouse. Red dots indicate mice with obvious signs of fighting injuries. i) Representative image of fighting induced skin lesions observed in WT males (one of the mice marked as red dot in graphs 2ai and 2bi) housed together in groups of 5 per cage. Scale bar=2cm. Unpaired t test with Welch’s correction was performed in 2A, 2D-F, to determine statistical significance, with a p value of 0.05 determined as significant. *p<0.05 ***p<0.001

CCR2-/- mice display extensive wounding-related skin inflammation.

a) CD45+ cell content of the skin, assessed via flow cytometry, in WT (black), CCR2-/- housed alone (blue) and CCR2-/- housed together (red) male mice, with representative FACS plots for each. b) Neutrophil and eosinophil numbers in the skin of WT (black), CCR2-/- housed alone (blue) and CCR2-/- housed together (red) males, with representative FACS plots for each (neutrophils= CD11b+Ly6G+ red box, eosinophils= CD11b+SiglecF+ blue box). c) T-cell numbers (CD11b+ CD3+ CD19-) in the skin of WT (black), CCR2-/- housed alone (blue) and CCR2-/- housed together (red) male mice. d) i) representative flow cytometry gating to define ii) Inflammatory monocyte (CD11b+Ly6C++ Ly6G-) and iii) macrophage (CD11b+ CD64+) numbers in in the skin of WT (black), CCR2-/- housed alone (blue) and CCR2-/- housed together (red) males, with representative FACS plots for CD11b/CD64 staining highlighting macrophages (red box). iv) Representative histograms showing CD206 staining, assessed via flow cytometry, on the CD11b+CD64+ macrophage population in the skin of WT males (black histogram) and CCR2-/- males housed together (red histogram). e) Correlation of fat pad weight (in grams, x-axis) and normalised CD45+ cell counts in the skin of CCR2-/- males (y-axis), alone (blue dots) and housed together (red dots). Fat pad weight values represent the pooled weight of both fat pads for each mouse. Simple linear regression analysis was conducted (black line), with an R2 value of 0.6759 and a slope coefficient significantly non-zero, as indicated by a p value of <0.0001. N=19 (6 resting, 13 inflamed). For 3b-d, Leukocyte numbers were normalised to the weight of the skin sampled. Unpaired t test with Welch’s correction was performed to determine statistical significance in 3A-D with a p value of 0.05 determined as significant. **p<0.01 ***p<0.001, ****p<0.0001.

Inguinal fat pads and lymph nodes in CCR2-/- mice display altered cellularity.

See also Figures S1 and S2. a) i) Representative FACS plots showing CD11b+ CD64+ macrophages in the inguinal fat pads of WT and CCR2-/- male mice (red oval). ii-iii) Inguinal Fat infiltrating monocyte (CD11b+CD64-Ly6C++Ly6G-) and macrophage (CD11b+CD64+) normalised counts in WT and CCR2-/- males. Both inguinal fat pads for each mouse were pooled to obtain one measurement, and counts were normalised to the combined weight of both fat pads. b) i) Representative FACS plots showing further CD11c/CD206 staining of the CD11b+CD64+ inguinal fat pad macrophage population found in WT and CCR2-/- mice, both male (upper panels) and females (lower panels). ii-iii) Number of pro-inflammatory (CD11c+) and anti-inflammatory (CD11c-CD206+) adipose tissue macrophages in WT and CCR2-/- inguinal fat pads, in both males and females, at 8-10 weeks and 16+ weeks of age. Each point represents the combined macrophage content of both inguinal fat pads of a single mouse. c) i) Representative FACS plots showing CD11b+ CD64-neutrophils (Ly6G+, red box) and eosinophils (SiglecF+, blue box) in the inguinal fat pads of WT and CCR2-/- mice, both male and female. ii-iii) Quantification of inguinal fat pad eosinophils and neutrophils in WT and CCR2-/- inguinal fat pads, in both males and females, at 8-10 weeks and 16+ weeks of age. Each point represents the combined macrophage content of weight of both inguinal fat pads of a single mouse. d) i) Representative FACS plots showing subcapsular macrophages (CD3-CD19-CD64+ CD11c-, red box) and dendritic cell (CD3-CD19-CD64-CD11c+, blue box) and ii) quantification of subcapsular macrophage numbers in the inguinal lymph node of WT and CCR2-/- mice, both male and female, at 8-10 weeks and 16+ weeks of age. Each point represents counts from both inguinal lymph nodes for each mouse. 4b-d: Values were normalised to the average of WT counts (age and gender matched) to obtain values expressed as fold of WT. Unpaired t test with Welch’s correction was performed to determine statistical significance in 4Ai, 4C-D, with a p value of 0.05 determined as significant. *p<0.05, **p<0.01, ***p<0.001.

Reduced inguinal fat pad weight and increased inguinal lymph node weight is independent of iCCRs.

a) i) Inguinal fat pad weight and ii) inguinal lymph node weight, in grams, of 14-16 weeks old male iCCR-/- mice compared to age and gender matched WT (with representative images showing in situ orientation for both, iii) WT top panel and iCCR-/- bottom panel, scale bar=0.5cm). Both inguinal fat pads and inguinal lymph nodes of each mouse are pooled for each individual measurement. b) Leukocyte subset frequencies in the inguinal fat pad of male mice of 16 weeks of age in WT, CCR2-/- and iCCR-/-. i) Frequencies expressed as % of CD45+, assessed via flow cytometry. ii) Quantification of leukocyte numbers in the fat pad of 20 week old iCCR-/- males compared to WT males, assessed via flow cytometry. c) Quantification of macrophage subsets (total, Type 1 CD11c+ and Type 2 CD11c-) in the fat pad of 20 week old iCCR-/- males compared to WT males, assessed via flow cytometry. d) Frequencies of Type 1 (CD11c+, orange) vs Type 2 (CD11c-, blue) adipose tissue macrophages in male WT, CCR2-/- and iCCR-/- male mice (as proportion of total CD64+ F480+ macrophages) at 8-10 weeks and at 16+ weeks. For 5B and C, Counts were normalised to the average of WT counts (age and gender matched) of each leukocyte subset to obtain values expressed as fold of WT. Unpaired t test with Welch’s correction was performed in 5A-C to determine statistical significance, with a p value of 0.05 determined as significant. *p<0.05, **p<0.01, ***p<0.001

The Fat Pad Phenotype can be induced in WT females in a model of skin inflammation.

See also Figure S3. a) i) In situ orientation of inguinal fat pad and inguinal lymph node in WT and CCR2-/- females at rest and after 3 consecutive days of Aldara application to the skin, with ii) fat pad and iii) lymph node weight quantification. Each point represents the combined macrophage content of weight of both inguinal fat pads of a single mouse. Unpaired t test was performed to determine statistical significance, with a p value of 0.05 determined as significant. ***p<0.001, ****p<0.0001. b) Correlation of inguinal fat pad (x-axis) and lymph node (y-axis) weight, both expressed in grams, of WT females receiving vehicle (blue) or Aldara cream (red) to the shaved back for 3 consecutive days and culled at Day 4. Simple linear regression was significantly non-zero (p<0.0001), with an R2 values of 0.69. The vehicle only group (blue) does not show any significant correlation, while the correlation is still significant in the Aldara only group (red). c i) CD45+ cell content of the inguinal fat pad after 3 consecutive days of Aldara application, assessed via flow cytometry, in WT and CCR2-/- females. Blue= vehicle control, Red= aldara cream. Leukocyte numbers were normalised to the weight of the inguinal fat sampled. Each point represents the combined inguinal fat pads of a single mouse. ii) Quantification of adipose tissue macrophages (CD64+ F480+) in the inguinal fat pad after 3 consecutive days of Aldara application, assessed via flow cytometry, in WT and CCR2-/- females. Blue= vehicle control, Red= aldara cream. Macrophage numbers were normalised to the weight of the inguinal fat sampled. Each point represents the combined inguinal fat pads of a single mouse. d) Quantification of live skin leukocytes (CD45+) in the skin of WT females after 3 consecutive days of Aldara application, assessed via flow cytometry. Blue= vehicle control, Red= aldara cream. Leukocyte numbers were normalised to the weight of the inguinal fat sampled. e) Correlation between inguinal fat pad weight (x-axis) and number of normalised CD45+ cells in skin (y-axis) of WT females receiving vehicle (blue) or Aldara cream (red) to the shaved back for 3 consecutive days and culled at Day 4. Simple linear regression was significantly non-zero (p<0.0062) only in females receiving Aldara, with an R2 values of 0.6294. The correlation was not significant in vehicle treated females (R2=0.00469). Unpaired t test with Welch’s correction was performed in 6C-D to determine statistical significance, with a p value of 0.05 determined as significant. ****p<0.0001.

The fat pad phenotype is at least partially dependent on macrophages.

a) Principal component analysis (PCA) plot illustrating distinct clustering of RNA samples from the inguinal fat pad of Aldara and vehicle treated female mice. Each point represents the pooled macrophage population of the inguinal fat pads of 4 mice. b) Heatmaps showing significantly upregulated and downregulated genes involved in macrophage function and lipid transport between inguinal fat pad macrophages from Aldara-treated or vehicle-treated mice. c) Expression of signature genes involved in changes in adipocyte function and phagocytosis in Aldara treated (red) and vehicle treated (blue) inguinal fat pad macrophages, measured as changes in log2fold expression. d) Overlap of the Aldara treated inguinal fat-pad macrophage RNA signature with the single cell RNA Sequencing data-set of visceral adipose tissue leukocytes from Weinstock et al, 2019. For each cell type, the markers were overlapped with the DEGs upregulated in Aldara. An overlap fold and p-value was calculated using a hypergeometric test. e) PASI (Psoriasis area and severity index) score of the skin of WT females and females treated with CSF1R inhibitor AZD7507, after 3 days of daily Aldara application to the skin. f) Representative images of Inguinal lymph node and fat pad weight of female mice before and after 3 days of Aldara induced inflammation g) i-ii) Graphs comparing Inguinal lymph node and fat pad weight of female mice before and after 3 days of Aldara induced inflammation, with and without pre-treatment with CSF1R inhibitor AZD7507. Unpaired t test was performed to determine statistical significance, with a p value of 0.05 determined as significant. *p<0.05, **p<0.01, ***p<0.001.

The fat pad phenotype is associated with lipolysis and free fatty acid availability.

a) list of transcripts encoding proteins involved in lipolysis which are upregulated in theadipose tissue macrophages in Aldara treated mice. b) exemplar free fatty acid analysis showing levels of i) FA 20:1; ii) FA 20:2; iii) FA 22:1; iv) FA 22:2. Statistics were analysed using one-way ANOVA with a p-value of 0.05 determined as significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.