Fig 1: IL-1ß treatment increased IL-1R1 and p-IRAK-1 expression in RBECs (n=4). (A) Immunoreactive bands of IL-1R1 (80 kDa) and ß-actin (42 kDa). (B) The bar graph shows increased IL-1R1 expression in the IL-1ß group compared with that in the control group (**P<0.01). (C) Immunofluorescence images showing the expression of CD31+ RBECs (a and d, green), IL-1R1 (b and e, red), and the co-localization of IL-1R1 and RBECs (c and f). Enhanced IL-1R1 immunofluorescence was evident in the IL-1ß group compared with the control group. Scale bars (a-f): 10 µm. (D) Immunoreactive bands of p-RIAK-1 (77 kDa), t-IRAK-1 (77 kDa) and ß-actin (42 kDa). (E) The bar graph shows increased p-IRAK-1 expression in the IL-1ß group compared with the control group (**P<0.01). The protein expression of p-IRAK-1 was significantly suppressed with IL-1Ra treatment (*P<0.05). RBECs, rat brain capillary endothelial cells; IL-1ß, interleukin-1ß; IL-1R, interleukin-1 receptor; IL-1Ra, interleukin-1 receptor antagonist; HC, high concentration of carbon dioxide; ns, non-significant.
Fig 2: Impaired pregnancy outcomes are reverted by IL-1 axis genetic ablation and pharmacological blocking.Assessment of IL-1ß signaling impact in fetal by genetic ablation of Il1b and Il1r1 or by blocking the receptor with Anakinra, a recombinant of the IL-1Ra. (A and B) Weight reduction of fetuses born from infected pregnant mice expressed in percentage of their NI controls at G19. (C and D) Relative resorption rates of infected in relation to their NI controls. (B and D) Anakinra treatment was performed in WT (C57BL/6) mice, and vehicle represents an infected group treated with Anakinra denaturated in phosphate-buffered saline. Data are represented as means ± SD of 24 to 46 fetuses (n = 3 to 12 pregnant mice) (A and C) or 3 to 12 pregnant mice (B and D) per group and are cumulative of two or more independent experiments. The differences between each group were determined by one-way ANOVA with Bonferroni’s post hoc correction (A and C) or Student’s t test (B and D). *P < 0.05 and ***P < 0.001.
Fig 3: Hypercapnia increased IL-1R1 expression in the cerebrovascular endothelial cells of hypoxemic rats (n=4). (A) Immunoreactive bands of IL-1R1 (80 kDa) and ß-actin (42 kDa). (B) There was an interaction effect between hypoxia treatment and hypercapnia treatment (P<0.05). (C) Simple effects analyses revealed increased IL-1R1 expression in the hypoxemia group (*P<0.05), but not in the hypercapnia group (P>0.05) compared with the Sham group. The HH group exhibited the highest expression levels of IL-1R1 when compared with the hypoxemia group (**P<0.01) and the hypercapnia group (**P<0.01). (D) Immunofluorescence images showing the expression of CD31+ cerebrovascular endothelial cells (a, d, g and j, green), IL-1R1 (b, e, h and k, red), and the co-localization of IL-1R1 and cerebrovascular endothelial cells (c, f, i and l). Of note, enhanced IL-1R1 immunofluorescence was evident in the hypoxemia group, but not in the hypercapnia group, compared with the Sham group. The HH group emitted the strongest IL-1R1 fluorescence as compared with the hypoxemia and hypercapnia groups. Scale bars (a-l): 50 µm. The concentrations of O2 and CO2 in the air were 21 and 0.03%, respectively. IL-1R, interleukin-1 receptor; HH, hypercapnia + hypoxemia; ns, non-significant.
Fig 4: Synovial fluid and tissues isolated from patients undergoing revision total knee arthroplasty (TKA) are characterized by dramatic tissue remodeling and chronic inflammation. A: Representative images of Masson trichrome and IL-1R1 (green [fluorescein isothiocyanate (FITC)])/vimentin (red [tetrarhodamine isothiocyanate (TRITC)]) stained infrapatellar fat pad from patients undergoing primary TKA and patients undergoing revision TKA. There is a significant increase in collagen and IL-1R1 expression in revision TKA tissue compared with primary TKA tissue, and IL-1R1 is predominantly expressed on elongated bipolar mesenchymal cells in revision TKA tissue. Images acquired on a Nikon inverted microscope and a Nikon A1R point scanning confocal microscope, respectively. B: Venn diagram demonstrating change in protein expression in synovial fluid, infrapatellar fat pad, and synovial membrane isolated from patients undergoing revision TKA compared with patients undergoing primary TKA. Protein expression was quantified using human V-Plex electrochemiluminescence detection kits from MesoScaleDiscovery. Markers in black text are significantly elevated in revision TKA, markers in blue text are not significantly different or undetectable, and markers in red text are significantly decreased in revision TKA. Significance was taken as P < 0.05. C: Representative images of vimentin [green (FITC)]– and IL-6 [red (TRITC)]–stained infrapatellar fat pad from patients undergoing revision TKA. There are a significant number of vimentin and IL-6 dual-positive fibroblasts in post-TKA infrapatellar fat pad. Images acquired on a Nikon A1R point scanning confocal microscope. Original magnification, ×20 (A and C); ×40 (inset). CRP, C-reactive protein; FGF, fibroblast growth factor; Flt, Fms-like tyrosine kinase 1; GM-CSF, granulocyte-macrophage colony-stimulating factor; ICAM, intercellular adhesion molecule; IFN, interferon; IP-10, interferon gamma-induced protein 10; PIGF, placental growth factor; SAA, serum amyloid A; Tie, tyrosine kinase; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor.
Fig 5: IL-1ß and IL1R1 showed a higher expression level in the RHD patient mitral valve compared with the CHD patient. a-c IL-1ß and IL1R1 in the mitral valve were assayed by IHC as described in the Methods using IL-1ß or IL1R1 antibody b and c. Normal rabbit IgG was used to substitute antibodies as a negative control (a). The graph is representative of 6 CHD patients and 6 RHD patients. Bar = 50 µm. d The expression of IL-1ß and IL1R1 was assessed by Western blot as described in the Methods section. ß-actin was used as an internal control
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