Fig 1: IL‐37b suppressed RANKL‐induced osteoclast differentiation without inducing cytotoxicity. (a) RAW264.7 cells were seeded onto 96‐well plates and incubated for 24 or 72 hr with different concentrations of IL‐37b. The CCK‐8 assay was used to detect cell proliferation. (b) Precursor cells were cultured with various concentrations of IL‐37b followed by M‐CSF (50 ng/ml) and RANKL (50 ng/ml) stimulation for 72 hr. These cells were simultaneously exposed to all of these factors. The steps for TRAP staining are described in the methods. Scale bar = 200 μm. (c) TRAP‐positive multinucleated cells ( ≥ 3 nuclei) were identified as osteoclasts and were counted. (d) BMMs were treated with M‐CSF (50 ng/ml), RANKL (50 ng/ml), and IL‐37 (200 ng/ml) for 5 days. (e) RAW264.7 cells were plated on the Osteo Assay Surface and were cultured with RANKL and M‐CSF for 6 days in the presence or absence of 200 ng/ml IL‐37b. Scale bar = 200 μm. Quantification of the bone resorption area on the Osteo Assay Surface. N = 4. The data are presented as the means ± SD. *p < 0.05, **p < 0.01. CCK‐8: cell counting kit‐8; IL‐37b: interleukin‐37b; M‐CSF: macrophage colony stimulating factor; RANKL: receptor activator of nuclear factor‐κB ligand; TRAP: tartrate‐resistant acid phosphatase [Color figure can be viewed at wileyonlinelibrary.com]
Fig 2: The level of the IL‐37 protein was detected in clinical specimens from patients with bone infections. The exogenous administration of IL‐37 attenuated LPS‐induced inflammatory bone destruction in mice. (a) Four groups of samples were obtained noninfectious fractures and traumatic osteomyelitis‐infected samples. Samples (Birt et al., 2017; Mbalaviele et al., 2017; Takayanagi, 2009; Wojdasiewicz et al., 2014) were extracted from normal cancellous bone, infected cancellous bone, normal connective tissue, and infected connective tissue, respectively. Levels of IL‐37 (IL‐37b) and GAPDH were detected by immunoblotting. (b) Representative images of H&E staining and immunohistochemical staining. Arrows indicate IL‐37‐positive cells. Scale bar = 200 μm. (c) TRAP staining, immunohistochemical staining, and H&E staining in sections of calvarial bones from 6‐week‐old C57BL/6 mice treated with PBS, LPS, or LPS + IL‐37b. Arrows indicate TRAP‐positive cells. Scale bar = 400 μm; N = 4. (d) The 3D images, images of longitudinal sections, images of cross‐sections, and trabecular images were reconstructed at the distal femur. Quantification of Tb.Sp. and BV/TV in each group. Scale bar = 1 mm; N = 4. BV/TV: bone volume per tissue volume; Ctrl: control; ES/BS: eroded surface per bone surface; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; H&E: hematoxylin and eosin; IL‐37: interleukin‐37; LPS: lipopolysaccharide; PBS: phosphate‐buffered saline; Tb.Sp.: trabecular spacing; TRAP: tartrate‐resistant acid phosphatase [Color figure can be viewed at wileyonlinelibrary.com]
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