Fig 2: Immunohistochemical renal fibrotic changes in hypertensive (H) and hypertensive animals treated with daily oral spironolactone (H + SP) following 4 or 12 weeks when compared to normotensive (N) animals. Immunohistochemical stains showing expression of connective tissue growth factor (CTGF), alpha smooth muscle actin (aSMA), and phosphorylated SMAD2 (pSMAD2). Macrophage infiltration is shown by positive expression of CD68 (indicated by arrows). Positive expression of each antibody is shown by brown stain. Scale bars 50 µm. Below: Histograms show quantification of each antibody. Significant difference between N and H is indicated by * (*p < .05, ***p < .001). Significant difference between H and H + SP is indicated by # (# p < .05, ## p < .01, ### p < .001)

Fig 3: Prkaa1 upregulation is associated with macrophage metabolism in mice fed a high-fat diet. (A) Schema illustrating leukocyte sorting from adipose tissues by CD45-positive staining. (B) Real-time PCR analysis and quantification of mRNA levels of Prkaa1 and Prkaa2 in leukocytes sorted from stromal vascular cells in HFD- and CD-fed mice. n = 8. (C,D) Real-time PCR analysis and quantification of mRNA levels of Slc2a1, Pfkfb3, Cd36, and Cpt1 in leukocytes sorted from stromal vascular cells in HFD- and CD-fed mice. n = 8. (E,F) Representative images and quantification data of Prkaa1, Slc2a1, and CD36 co-staining with F4/80 or CD68 (macrophage markers) in adipose tissues from HFD- and CD-fed mice. n = 4, Scale bars, 20 µm. All data were expressed as mean ± SEM. Statistical significance was determined by unpaired Student’s t-test. *p < 0.05 was considered significant, **p < 0.01, ***p < 0.001.

Fig 4: Effect of blocking mTOR pathway using rapamycin in the murine model. (A) Protocol of the murine model and rapamycin was administered intraperitoneally. (B) Number of polypoid lesions. (C and D) IL-17A+ and CD68+ cell counts. (E) Number of elastase+ neutrophils.Subjects were divided into 3 groups; (-) Con: negative control group, (+) Con, positive control (nasal polyp) group, and Rapamycin: rapamycin treatment group.mTOR, mechanistic target of rapamycin; IL, interleukin; OVA, ovalbumin; i.n., intranasal; i.p., intraperitoneal; ALM, aluminum hydroxide; SEB, Staphylococcus aureus enterotoxin B.*P < 0.01, and †P < 0.001.

Fig 5: Macrophages are temporally recruited to participate in incorporation of the biological cover into host tissue during expansion. Representative images of (A) Trichrome staining of ADM attached to host tissue and (B) double immunofluorescence staining of CD31 and ACTA2 of ADM at 10 weeks of expansion. Dashed line and black arrows in (A) mark attachment of ADM to host tissue and cells infiltrating ADM, respectively. (C,D) Representative images and (E) quantitative analysis of double immunofluorescence staining of CD68 and iNOS of ADM attached to host tissue at (C) 2 weeks and (D) 10 weeks of expansion. The yellow arrows indicate double positive CD68+iNOS+ cells. In (A) slides were counterstained with hematoxylin and in (B–D) slides were counterstained with DAPI to mark nuclei (blue stain). (F) Relative expression of pro-inflammatory markers in control (Ctr) and expanded skin (TE and ADM model) assessed by qRT-PCR at 2 and 10 weeks of expansion compared to corresponding unexpanded control. The values were normalized to the geometric mean calculated for 2 reference genes: RLPL0 and RPS27A and are presented as the average for 3 biopsies. Magnification in (A) 2×, in (B) 10×, in (C,D) 20×. Error bars represent SD. Statistical significance calculated in (E) with unpaired Student t-test and in (F) with one-way ANOVA with Bonferroni correction for multiple comparison is shown as * p-value < 0.01, **** p-value = 0.0001, ns—not statistically significant.