Osteoclast-mediated bone loss observed in a COVID-19 mouse model

Abstract

The consequences of SARS-CoV-2 infection on the musculoskeletal system represent a dangerous knowledge gap. Aging patients are at added risk for SARS-CoV-2 infection; therefore, a greater understanding of the resulting musculoskeletal sequelae of SARS-CoV-2 infection may help guide clinical strategies. This study examined fundamental bone parameters among mice treated with escalating viral loads. Male C57BL/6J (WT, n = 17) and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (K18-hACE2 transgenic mice, n = 21) expressing human ACE2 (TG) were divided into eight groups (n = 4-6/group) and subjected to intranasal dosing of 0, 1 × 10³, 1 × 10⁴, and 1 × 10⁵ PFU (plaque forming units) of human SARS-CoV-2. Animal health was assessed daily by veterinary staff using established and validated scoring criteria (activity, posture, body condition scores and body weight). We report here that mock and WT infected mice were healthy and completed the study, surviving until 12-14 days post infection (dpi). In contrast, the TG mice infected with 1 × 10⁵ PFU all experienced severe health declines that necessitated early euthanasia (6-7 dpi). For TG mice infected with 1 × 10⁴ PFU, 2 mice were also euthanized after 7 dpi, while 3 mice showed signs of moderate disease at day 6 dpi, but recovered fully by day 11 dpi. Four of the 5 TG mice that were infected with 1 × 10³ PFU remained healthy throughout the study. This suggests that our study mimics what is seen during human disease, where some patients develop severe disease resulting in death, while others have moderate to severe disease but recover, and others are asymptomatic. At necropsy, femurs were extracted and analyzed by μCT. No difference was found in μCT determined bone parameters among the WT groups. There was, however, a significant 24.4% decrease in trabecular bone volume fraction (p = 0.0009), 19.0% decrease in trabecular number (p = 0.004), 6.2% decrease in trabecular thickness (p = 0.04), and a 9.8% increase in trabecular separation (p = 0.04) among surviving TG mice receiving any viral load compared to non-infected controls. No differences in cortical bone parameters were detected. TRAP staining revealed surviving infected mice had a significant 64% increase in osteoclast number, a 27% increase in osteoclast surface, and a 38% increase in osteoclasts per bone surface. While more studies are needed to investigate the long-term consequences of SARS-CoV-2 infection on skeletal health, this study demonstrates a significant reduction in several bone parameters and corresponding robust increases in osteoclast number observed within 2 weeks post-infection in surviving asymptomatic and moderately affected mice.

Keywords: Bone mass; COVID-19; Infection; Musculoskeletal health; Osteoclasts; SARS-CoV-2.

Graphical abstract

Fig. 1

Overview of experimental design. Wild-type (WT) and K18-hACE2 (TG) mice were infected with 0, 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU of SARS-CoV-2. Mice underwent baseline and daily health assessments and were humanely euthanized if criteria were met or were allowed to complete the entire study (12–14 days post-infection). At necropsy, femurs were collected and evaluated by μCT and histological assessments.

Fig. 2

Effects of SARS-CoV-2 infection on survival and body weights in mice. C57BL/6J (WT) and K18-hACE2 (TG) mice were infected with 0, 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU of SARS-CoV-2 and survival (A) and the percent change in body weight at euthanasia from baseline (B) were assessed at baseline and daily until humane euthanasia occurred or 12–14 days post infection (n = 4–6/genotype/viral load). A) Kaplan-Meier survival curves. It should be noted that all WT mice (0, 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU) and TG mice infected with 0 PFU survived for the entire study and are overlapping the gray line at 100%. Different colored lines represent the different viral infection doses (gray = 0 PFU, red = 1 × 10³ PFU, green = 1 × 10⁴ PFU, blue = 1 × 10⁵ PFU). B) Percent change in body weight at the time of euthanasia from baseline body weight measurements for non-infected (n = 22) and infected mice (n = 16). Data from individual mice are shown. ***p < 0.005 compared to non-infected mice. Statistical analysis was performed using a Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Effects of SARS-CoV-2 infection on activity, posture, and body condition scores in mice. C57BL/6 J (WT) and K18-hACE2 (TG) mice were infected with 0, 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU of SARS-CoV-2 and several common, validated veterinary health measures were scored at baseline and daily until humane euthanasia occurred or 12–14 days post infection (n = 4–6/genotype/viral load). A) Average activity score (range 0–3, normal = 0). B) Average posture score (range 0–3, normal = 0). C) Average body condition score (range 1–5, normal = 3). Data are presented as the mean ± standard deviation. Dashed lines represent WT mice and solid lines represent TG mice. Different colored lines represent the different viral infection doses gray = 0 PFU, red = 1 × 10³ PFU, green = 1 × 10⁴ PFU, blue = 1 × 10⁵ PFU). Note, activity and posture scores increase from 0 when mice exhibit declines in health, whereas BCS drops below 3 when mice are underconditioned or rises above 3 when mice are overconditioned. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Effects of SARS-CoV-2 infection on trabecular bone parameters in mice. C57BL/6J (WT) and K18-hACE2 (TG) mice were infected with 0, 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU of SARS-CoV-2. Non-infected mice included mock-infected (0 PFU) TG mice and all WT mice (infected with 0, 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU) (n = 22). Infected mice included TG mice infected with 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU of SARS-CoV-2 virus (n = 15, due to a femur being broken during removal from a TG mouse infected with 1 × 10⁴ PFU, early endpoint). A) Representative μCT images of the distal metaphyseal femur of surviving TG mice infected with 0 and 1 × 10⁴ PFU. B–E) Quantitative microstructural parameters determined by μCT. B) Bone volume fraction (BV/TV). C) Trabecular thickness (Tb.Th). D) Trabecular number (Tb.N). E) Trabecular separation (Tb.S). Data are presented as the mean ± standard deviation. Mice which were humanely euthanized prior to the study endpoint are denoted by pink circles. *p < 0.05 and ***p < 0.005 compared to non-infected mice. Statistical analysis was performed using a Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

SARS-CoV-2 infection does not impact cortical bone parameters. C57BL/6J (WT) and K18-hACE2 (TG) mice were infected with 0, 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU of SARS-CoV-2. Non-infected mice included mock-infected (0 PFU) TG mice and all WT mice (infected with 0, 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU) (n = 22). Infected mice included TG mice infected with 1 × 10³, 1 × 10⁴, or 1 × 10⁵ PFU of SARS-CoV-2 virus (n = 15, due to a femur being broken during removal from a TG mouse infected with 1 × 10⁴ PFU, early endpoint). A) Representative μCT images of the cross-sectional area at the midshaft of the femur of surviving TG mice infected with 0 and 1 × 10⁴ PFU. B–G) Quantitative microstructural parameters determined by μCT. B) Cortical bone porosity (Ct.Po, distal). C) Cortical bone porosity (Ct.Po, midshaft). D) Tissue area (T.Ar, midshaft). E) Bone area (B.Ar, midshaft). F) Marrow area (M.Ar, midshaft). G) Polar moment of intertia (PMOI, midshaft). Data are presented as the mean ± standard deviation. Mice which were humanely euthanized prior to the study endpoint are denoted by pink circles. No significant differences were detected. Statistical analysis was performed using a Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Osteoclast parameters are elevated in mice infected with SARS-CoV-2. A) Representative micrographs of TRAP staining in femurs from non-infected (n = 8) and infected mice (n = 7). Osteoclasts stain red (red arrows). Quantitation of B) Number of osteoclasts (N.Oc); C) number of osteoclasts per bone surface (N.Oc/BS); D) osteoclast surface (Oc.S); and E) osteoclast surface per bone surface (Oc.S/BS). *p < 0.05 or ***p < 0.005 compared to non-infected mice. Statistical analysis was performed using a Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Schematic demonstrating our hypothetical model describing how the cytokine storm associated with SARS-CoV-2 infection may result in osteoclast-mediated bone loss.