Fig 2: A Representative Western blot (upper panel) and quantification of left atrial (LA) IL-6 (lower panel) in homogenates from normotensive Ctr (n = 9), SHR (n = 8), SHRob (n = 8) and SHRobRDN (n = 8). IL-6 in arbitrary units (AU) normalized to GAPDH. B Representative Western blot (upper panel) and quantification of left atrial TNFa (lower panel) in homogenates from normotensive Ctr (n = 9), SHR (n = 8), SHRob (n = 8) and SHRobRDN (n = 8). TNFa in arbitrary units (AU) normalized to GAPDH. *p < 0.05 versus Ctr; § p < 0.05 versus SHR; #p < 0.05 versus SHRob. Representative images of immunofluorescence stainings (TRITC red) for C F4/80 macrophages (upper panel; example target cells pointed to by white arrowheads; scale bar 200 µm) and quantification of F4/80 + cells per mm2 LA area (lower panel). Nuclei of cells were stained with DAPI (blue). Representative images of immunofluorescence stainings (TRITC red) for D Ly6G + neutrophils (upper panel; example target cells pointed to by white arrowheads; scale bar 200 µm) and quantification of Ly6G + cells per mm2 LA area (lower panel) in normotensive Ctr (n = 7), SHR (n = 7), SHRob (n = 8) and SHRobRDN (n = 7) rats. Nuclei of cells were stained with DAPI (blue). *p < 0.05 versus Ctr; §p < 0.05 versus SHR; #p < 0.05 versus SHRob

Fig 3: Effects of LIPUS on the inflammatory cell infiltration in the kidneys and spleens of an AKI mouse model. The LIPUS treatment was performed before IRI procedure and after IRI until the day of euthanization. The Ly6G (a neutrophil marker) and CD68 (a macrophage marker) protein levels in the kidneys (A-a) and spleens (A-b) were shown. The protein level was determined by Western blot and quantified by densitometry, which was normalized by GAPDH. The stainings of Ly6G (B-a) and CD68 (B-b) in the kidneys and spleens were determined by immunohistochemistry. The representative images of at least three independent experiments were shown. Scale bar = 100 µm. The IL-6 mRNA expression in the kidneys and spleens was determined by real-time PCR (C). Two reference genes of ß-actin and Gapdh were used. Data are presented as mean ± SEM (n = 4–6). *p < 0.05, versus Sham; #p < 0.05, versus IRI alone.

Fig 4: c-Myc controls the expression of pro-inflammatory cytokines in human keratinocytes. (A,B) Primary human keratinocytes were transfected with an siRNA for c-Myc (siMYC) or a control scramble siRNA (siCtrl) and 72 h after transfection, cells were collected for RT-PCR analysis of c-Myc mRNA expression (left panel, (A)), Western blot analysis of c-Myc protein expression (right panel, (A)) and RT-PCR analysis of mRNA expression levels of pro-inflammatory cytokines as indicated (B). (C) Primary human keratinocytes were treated with 0, 25 or 50 nM c-Myc inhibitor JQ1 and cells were collected at 24 h for RT-PCR analysis of mRNA expression levels of pro-inflammatory cytokines as indicated. (D) Primary human keratinocytes were infected with a c-Myc-expressing virus (c-Myc) or a control virus (empty), and 48 h after infection, cells were collected for RT-PCR analysis of c-Myc mRNA expression levels (left panel, (D)), Western blot analysis of c-Myc protein expression (right panel, (D)) and RT-PCR analysis of mRNA expression levels of pro-inflammatory cytokines as indicated (E). (F) Primary human keratinocytes were infected with a c-Myc-expressing virus (c-Myc) or a control virus (empty), and 12 h later, cells were treated with 1 mM phenformin for 48 h after infection and cells were collected for RT-PCR analysis of expression levels of c-Myc and pro-inflammatory cytokines as indicated. (G) Mouse ears were treated with following combinations: Left ears: Acetone (Negative control); Right ears: TPA (positive control) or TPA + JQ1. Ear thickness was measured at different time points as indicated. Two-way ANOVA with correction for multiple pairwise comparisons was used for statistical analysis of differences between two groups as indicated, n = 3, standard deviation bars are shown, * p < 0.05; ** p < 0.01; *** p < 0.005. (H) Histological analysis of ears of mice from (G), at 8 h after TPA treatment; (I,J) sections from (H) were analyzed for infiltration of inflammatory cells by IF analysis of Gr-1 ((I), green) and F4/80 ((I), green); DAPI was used as a counterstain for nuclei, red arrows indicate positive cells. Lower-magnification images of (I) are shown in Supplementary Figure S7. The corresponding quantification analysis of ((I) and Supplementary Figure S7) is shown in (J), Student’s t test analysis except (G) was used for all quantification data to compare two different concentration groups as indicated, n = 3, standard deviation bars are shown, * p < 0.05; ** p < 0.01; *** p < 0.005. Bars: 100 µm in (H); 50 µm in (I).

Fig 5: Il1b is driver of emergency hematopoiesis.(A) Granulocyte, monocyte, and lymphocyte numbers in the blood of Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and in uninfected controls (PBS), as determined using a hematology analyzer. ANOVA with Dunnett’s correction for multiple comparisons, n = 8 to 14 per genotype. **P < 0.01. (B) Neutrophil (CD45+ CD11b+ Ly6Cmed Ly6G+) and monocyte (CD45+ CD11b+ Ly6G- Ly6Chi) percentages (of living cells) in BM from Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and from uninfected controls (PBS), as determined flow cytometry. One-way ANOVA, 10 to 16 per genotype. ****P < 0.0001. (C) Lin- progenitor cells (CD5- CD45R- CD11b- Gr-1- 7-4- Ter-119-) and early lymphoid precursors (LSK) (Lin- Sca-1+ c-Kithi) in BM from Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and from uninfected mice (PBS), as determined by flow cytometry. ANOVA with Dunnett’s correction for multiple comparisons, n = 7 to 10 per genotype. (D) Common myeloid progenitors (CMPs) (Lin- Sca-1- c-Kithi CD16/32lo, CD34+) and granulocyte-macrophage progenitors (GMPs) (Lin- Sca-1- c-Kithi CD16/32hi, CD34lo) in BM from Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and from uninfected controls (PBS), as determined by flow cytometry. ANOVA with Dunnett’s correction for multiple comparisons, n = 7 to 10 per genotype. **P < 0.01. (E and F) G-CSF protein (encoded by the Csf3 gene) (E) and Csf3 mRNA levels (F) at the site of infection from Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and (for Csf3 mRNA) from uninfected controls (PBS), as determined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. Kruskal-Wallis with Dunn’s multiple comparisons (E), n = 22 to 24 per genotype. ***P < 0.001; ****P < 0.0001.