Fig 2: Cold exposure induces VAT remolding in obese mice. HFD-fed mice were maintained at 30°C (Warm) or exposed to 4°C for 10 days (Cold) as indicated in Fig. 1A. Representative IF in HFD-fed SCAT (A) and VAT (A–H) were shown. (A) CD206 (green) and F4/80 (red) IF. CLS are indicated by triangles. (B) Collagen I (red) and Arg1 (green) IF. (C) Picro Sirius Red staining imaged with polarized light microscopy, visualizing collagen fibers type I (thick fibers, yellow-orange) and type III (Thin fibers, green). (D) CD206 (red) and Lyve1 (green) IF. Red blood cells (arrow), Lyve1+ ATM2 (Mac), Lyve1+ lymphatic vessels (LV) are indicated. (E) Hyaluronan fibers (HABP-stain, red) and Lyve1 (green) IF. Arrows indicate ATM2 (F) aSMA (red) and Lyve (green) IF. (G) TUBB3 (red) IF. Sympathetic nerves (SN) are indicated by triangles, surrounding blood vessel (BV). Higher magnification image is shown at side. (H) Lyve1 (green) and TH (left) or UCP1 (right) (red) IF in adjacent sections (right two). Higher magnification images of the inlets (yellow dashed boxes) are also shown at left side. Note TH+ nerves (triangles) were closely associated with beige adipocytes (red) and blood vessels (BV). NB, nerve bundles. DAPI (blue) stains cell nuclei. For each IF analysis, at least 10 independent mice per group were examined, and representative images are shown. (I) Western blot analysis for TH, UCP1, and HSP90 (loading control). At right, quantified TH and UCP1 expression are plotted to show correlation.

Fig 3: Foz-/- mice show activation of liver macrophages with the appearance of crown-like structures. Liver haematoxylin and eosin staining (H&E) and F4/80 macrophage marker in mice fed a normal diet (ND) or a high fat diet for 12 (HFD12W) or 30 weeks (HFD30W). The insets in the image show enlargements centred on the macrophages (A). Quantification of liver positive F4/80 macrophage surface on liver slides (B) and of crown-like structures or lipogranulomas (C). Liver mRNA expression of F4/80 (D), CD11b (E), CX3C chemokine receptor 1 (CX3CR1) (F), and tumor necrosis factor alpha (TNF-a) (G). n = 4–8/group. * p < 0.05, ** p < 0.01.

Fig 4: Clodronate liposome mediated macrophage depletion in Col4a3KO mice.(A) KO male mice were dosed intraperitonally with CL (KO+CL) or PBSL (KO+PBSL) as control. Animals were entered in the study at the age of 4 weeks and were continually dosed until the age of 8 weeks. Injections were repeated every second day, except for the first two doses, which were injected on consecutive days. (B-D) CL significantly reduced F4/80 positive macrophage infiltrates in KO+CL kidney as compared to KO+PBS or KO mice at the age of 8 weeks. (B, C) Representative images and quantitative assessment of F4/80 stained macrophages in kidney sections from KO+CL and KO+PBSL reveal marked reduction in macrophage infiltrates following CL dosing. Scale bar: 100 µm. (D) Real-time PCR analysis of F4/80 mRNA expression in KO+CL, KO+PBSL, KO, and WT littermates at the age of 8 weeks. Significant reduction in F4/80 mRNA expression following CL dosing was observed. PBSL did not affect endogenous F4/80 mRNA expression as shown by similar F4/80 mRNA expression in KO and KO+PBSL mice. (E) Real-time PCR analysis of CD204 and CD206 mRNA showed significant reduction in the expression of both genes in KO kidney upon CL treatment. (F) Representative images and quantitative assessment of F4/80 and CD206 stained kidney sections from KO+CL and KO+PBSL revealed marked reduction in M1 (F4/80+CD206-) and M2 (F4/80+CD206+) macrophages. Asterisk: F4/80+CD206- M1 macrophages, arrow: F4/80+CD206+ M2 macrophages. Scale bar: 20 µm. Welch corrected ANOVA followed by posthoc pairwise t-test with a Bonferroni correction (C,D,E) and unpaired t-test (F) was used to evaluate differences between groups. (C,D,E,F) n = 5 mice per group. CL: clodronate liposomes; PBSL: PBS liposomes.

Fig 5: Histopathological analysis of testis in UPEC-elicited mouse epididymo-orchitis. Representative micrographs of (A,B,D,E) HE-stained and (G,H,K,L) PCNA-stained testicular sections from sham-treated controls and UPEC-infected mice 10 and 31 days after infection, respectively. (B) Sham (10 days 1A,G; 31 days 1D,K), UPEC (10 days 1B,H, 31 days 1E,L). Scale bar in panel A applies to 1A–L. (C,F) An adaptation of the classical Johnsen scoring system (Johnsen, 1970) was applied: 200 seminiferous tubule cross-sections per animal were examined and the most advanced germ cell type per tubule documented. Results are presented as the percentage of seminiferous tubule cross-sections containing the indicated germ cell type as the most advanced stage. Analyses were performed on tissue sections of sham and UPEC mice, 10 days (C) and 31 days (F) after infection, respectively. ES = elongated spermatids, RS = round spermatids, PSc = pachytene spermatocytes, SG = spermatogonia, SCO = Sertoli-cell-only. (J,M) Quantification of testicular PCNA-positive cells in sham and UPEC-infected mice 10 days (J) and 31 days (M) after infection. Two hundred tubule cross-sections were assessed for the presence (+) or absence (-) of PCNA-positive cells. Results are demonstrated as the percentage of seminiferous tubule cross-sections containing or not containing PCNA-positive cells. (N,0) Representative immunofluorescence image of 8-µm-thick acetone-fixed testicular cryosections from sham (N) and UPEC-infected mice (0) 10 days after infection, stained with an anti-F4/80 antibody (1200 dilution, product no. MCA497G, Bio-Rad, Munich, Germany), counterstained with DAPI. (C,F) Mann–Whitney U test for each category, comparing sham to UPEC-infected mice (####P < 0.0001, ###P < 0.001, ##P < 0.01, #P < 0.05). (J,M) Unpaired t test for each time-point, comparing sham to UPEC-infected mice (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05). Sham 10 days, n = 3; UPEC 10 days, n = 4; sham 31 days, n = 3, UPEC 31 days, n = 4.