Fig 1: The inflammatory activation and osteoclastic differentiation of monocyte-macrophage lineage after SARS-CoV-2 infection.a The expression of osteoclastogenesis-related genes in the bone tissue of the hamsters at day 4 after treatment with PBS (Mock, n = 4) or SARS-CoV-2 infection (4 dpi, n = 5). b Representative immunofluorescence staining images showing the increase in the number of CD68+ and TRAP+ osteoclasts (indicated by arrowhead) after SARS-CoV-2 infection (n = 4). Tile scans (scale bars = 200 µm) and high-magnification of the boxed regions (scale bars = 50 µm), as well as corresponding quantification for the fluorescence intensity of CD68, are shown. c Representative immunofluorescence staining images and the corresponding quantification showing the upregulation of RANK-expressing cells at the trabecular bone surface after SARS-CoV-2 infection. Tile scans (scale bars = 200 µm) and high-magnification of the boxed regions (scale bars = 50 µm) are shown. d Representative multicolor immunohistochemical staining for TRAP, CD68, RANK, and IL-1β was performed at the distal metaphysis of the femur on day 4 after treatment with PBS (Mock) or SARS-CoV-2 infection (4 dpi). DAPI was used for nuclear counterstaining. Tile scans (scale bars = 200 µm) and high-magnification of the boxed regions (scale bars = 50 µm) are shown. Arrowheads indicate osteoclasts on the bone surface (n = 3). e The expression of IL-1β signaling-related genes in bone tissue at day 4 after treatment with PBS (Mock, n = 4) or SARS-CoV-2 infection (4 dpi, n = 5). Data were mean ± SD. ns: P > 0.05, *P < 0.05, **P < 0.01 by two-sided Student’s t-test.
Fig 2: The absence of SARS-CoV-2 infection in bone tissues.a Representative multicolor immunohistochemical staining for SARS-CoV-2 nucleocapsid protein (NP), angiotensin-converting enzyme 2 (ACE2), TRAP, CD68, and IL-1β was performed at the distal metaphysis of the femur after challenge with either PBS (Mock) or SARS-CoV-2 (4 dpi). Tile scans (scale bars = 200 µm) and high-magnification of the boxed regions (P periosteum, T trabeculae, S synovium; scale bars = 50 µm) are shown. Arrowheads indicate osteoclasts on the bone surface (n = 3). b Viral genome copies of the lung tissue (n = 5) and bone tissue (n = 4) harvested from SARS-CoV-2-infected hamsters. The viral genome copy was below the detection limit (dashed line) for all four bone samples. c The inflammatory cytokines, including IL-6, IL-1β, and TNF-α, in the serum of the hamsters treated with either PBS or SARS-CoV-2. d The expression of interferon signaling-related genes and e viral infection-associated inflammatory genes in bone tissue at day 4 after treatment with either PBS or SARS-CoV-2. Data were mean ± SD. ns: P > 0.05, *P < 0.05, **P < 0.01 by two-way ANOVA with Bonferroni’s post hoc test (c) or two-sided Student’s t-test (d, e).
Fig 3: Osteoclastogenesis in the femurs and tibias of the hamsters after SARS-CoV-2 infection.a–c Representative TRAP staining showing the increase in the number of TRAP+ osteoclasts at a the distal metaphysis of the femur (n = 4), b the proximal metaphysis of the tibia (n = 4), and c the lumbar vertebrae (n = 4) after the treatment with PBS (Mock) or SARS-CoV-2 infection (4 dpi). Lower images (scale bars = 100 µm) are high-resolution versions of the boxed regions in the upper images (scale bars = 500 µm). d Corresponding quantification of TRAP+ osteoclasts at the trabecular bone surface after treatment with PBS (Mock, n = 4) or SARS-CoV-2 infection (4 dpi, n = 8, 30 dpi, n = 5). e Representative immunofluorescence staining images and f the corresponding fluorescence intensity quantification showing the increase in the number of TRAP+ NFATc1+ osteoclasts at the distal metaphysis of the femur at 4 dpi (n = 4). Tile scans (scale bars = 200 µm) of the distal femoral metaphysis are shown along with high-magnification of the boxed regions (scale bars = 50 µm). Data were mean ± SD. ns: P > 0.05, *P < 0.05, **P < 0.01 by one-way ANOVA with Tukey’s post hoc test (d) or two-sided Student’s t-test (f).
Fig 4: Inflammatory cytokine IL-1β promotes osteoclastogenesis.a Representative microscopic images (scale bars = 500 µm) and b the corresponding quantifications (n = 12) showing the formation of TRAP+ multinuclear cells from bone marrow macrophage (BMM) isolated from young mice (3 months) and matured adult mice (6 months). Recombinant murine IL-1β or its neutralizing antibody (Neu-Ab) was added to the culture medium throughout the osteoclastic induction using M-CSF and RANKL. c The gene expression of IL-1R1 and IL-1RA in BMMs from young or matured adult mice with or without the presence of recombinant murine IL-1β or its neutralizing antibody (n = 3). d Representative microscopic images (scale bars = 500 µm) and e the correspond quantifications (n = 12) showing the formation of TRAP+ multinuclear cells when the BMMs were indirectly co-cultured with mesenchymal stem cells (MSCs) stimulated with recombinant murine IL-1β or its neutralizing antibody. f The gene expression of CSF1, OPG, and RANKL in MSC-treated with recombinant murine IL-1β or its neutralizing antibody (n = 3). Data were mean ± SD. ns: P > 0.05, *P < 0.05, **P < 0.01 by two-way ANOVA with Bonferroni’s post hoc test (b, c, e) or one-way ANOVA with Tukey’s post hoc test (f).
Supplier Page from Abcam for Anti-Tartrate Resistant Acid Phosphatase antibody [ACP5/1070]