key: cord-0689612-qfs8p13c
authors: Awosanya, Olatundun D.; Dalloul, Christopher E.; Blosser, Rachel J.; Dadwal, Ushashi C.; Carozza, Mariel; Boschen, Karen; Klemsz, Michael J.; Johnston, Nancy A.; Bruzzaniti, Angela; Robinson, Christopher M.; Srour, Edward F.; Kacena, Melissa A.
title: Osteoclast-Mediated Bone Loss Observed in a COVID-19 Mouse Model
date: 2021-10-02
journal: Bone
DOI: 10.1016/j.bone.2021.116227
sha: 0437f6fac4d35600d4847cf580dfbf3d46313602
doc_id: 689612
cord_uid: qfs8p13c

The consequences of SARS-CoV-2 infection on the musculoskeletal system represents 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, 1x10(3), 1x10(4), and 1x10(5) 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 1x10(5) PFU all experienced severe health declines that necessitated early euthanasia (6-7 dpi). For TG mice infected with 1x10(4) 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 1x10(3) 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.

On March 11, 2020 , the World Health Organization (WHO) classified the coronavirus disease 2019 (COVID-19) as a global pandemic. 1 Initially identified in December of 2019, the race for a vaccine and fight against the virus has come a long way. However, as of September 16, 2021, despite over 5.8 billion vaccine doses administered globally, the world-wide number of confirmed cases is over 226.9 million and the death count is nearing 4.7 million, with new infections occurring daily. 1 COVID-19 is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 infection severity ranges from asymptomatic to pneumonia and acute respiratory distress syndrome (ARDS). While the pulmonary consequences are now fairly wellestablished given the short duration of this disease, less is known about COVID-19 infection on extra-pulmonary systems, particularly its effects on bone. Therefore, this study sought to explore fundamental bone micro-structural and histomorphometric parameters in a mouse model of COVID-19 infection.

There are several mechanisms through which SARS-CoV-2 may impact the skeletal system.

For example, human angiotensin-converting enzyme 2 (hACE2), which SARS-CoV-2 utilizes as its entry receptor, is expressed in cortical and trabecular bone. 2, 3 Additionally, many inflammatory cytokines upregulated in the cytokine storm associated with COVID-19 infection have well-established roles in osteoclastogenesis and/or low bone mineral density including: IL-1β, IL-6, IL-17, CXCL10, and TNF-α. [3] [4] [5] [6] [7] [8] Further, hypocalcemia is common among COVID-19 patients and can be used to predict disease severity. 9 cells. [14] [15] [16] K18-hACE2 mice are susceptible to SARS-CoV-2, and intranasal inoculation can result in severe disease that mimics the severe form of the human disease, including interstitial pneumonia and pulmonary pathology, and upregulation of proinflammatory cytokines. 17, 18 In the current study, we investigated the consequences of SARS-CoV-2 infection on the skeletal system of K18-hACE2 mice. 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.

All animal studies were approved by the Indiana University School of Medicine Institutional 19 Mice were administered a viral concentration of 0 (mock), 1x10 3 , 1x10 4 , or 1x10 5 PFU (plaque-forming unit). Mice were weighed at baseline and daily postinfection. Several additional assessments were also completed each day including: body weight, activity score, posture score, and body condition score (BCS) as detailed below.

Vero-furin cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37˚C with 5% CO 2 . SARS-CoV-2 isolate USA-WA1/2020 was propagated in Vero-furin cells and progeny virions were collected in the supernatant 2-3 days post-infection. SARS-CoV-2 was quantified by a plaque assay in Vero-furin as previously described. 20 

Mice were assessed for activity level daily via cageside observation. The activity score was based on the degree of movement by the mice in the cage and gait as previously described. [21] [22] [23] As shown in Table 1 , a score of 0 indicated normal activity and that mice were bright and alert.

A score of 1 indicated slightly reduced activity or mild gait abnormality. A score of 2 indicated the very slow movement or a severely altered gait. A score of 3 indicated that movement was reluctant or did not take place at all.

J o u r n a l P r e -p r o o f Table 1 :

Mice were also assessed for posture through daily cageside observation. Posture scoring was assessed on a 0-3 range as previously detailed. 21 As shown in Table 2 , a score of 0 was the baseline and represented normal posture. A score of 1 represented a slightly hunched posture in a mouse. A score of 2 indicated a mouse was moderately hunched. A score of 3 indicated a mouse was severely hunched. 

Body condition scores (BCS) were performed as previously described 24 and ranged from 1 to 5. As detailed in Table 3 , the baseline or normal score is 3. A score of 4 represents an overconditioned and fleshy mouse. A score of 5 is an obese mouse. On the other hand, a score of 2 represents an underconditioned mouse showing signs of muscular wasting. A score of 1

represents an emaciated mouse with severe muscular wasting. 

Criteria for humane endpoints were established in consultation with IACUC and collaborating veterinarians. As described above, mice were observed daily for changes in activity, posture, body condition, and weight. The following combined measures triggered humane euthanasia. 1) Mice with a combined score for activity + posture equaling 5-6 (see Table 4 ). 2) Mice with a combined score for modified BCS + weight loss equaling 5-6 along with a ≥2 activity score for 2 consecutive days (see Table 5 ). 3) Mice with more than a 15% decline in body weight from baseline on 2 consecutive days. The scoring system above was developed with the goal of identifying mice that would likely die within 24 hours, allowing for the timely and humane euthanization of very sick animals. We believe that this system allowed us to predict death with accuracy. As some animals will recover after infection, this would also allow us to differentiate between those two populations. 

Body Condition Score (BCS) 1 Emaciated/severe muscular wasting 2

Underconditioned/modest muscular wasting 3 Normal 4

Overconditioned/well-fleshed 5 Obese J o u r n a l P r e -p r o o f 

At the time of euthanasia the right femur was dissected, stripped of soft tissue, and fixed in 10% neutral buffered formalin (NBF) for 3 days. Femurs were then transferred to 70% ethanol (EtOH) where they remained until processing for histological analyses as detailed below. Prior to histological processing femurs were imaged using a desktop SkyScan 1172 µCT imaging system (SkyScan, Kontich, Germany) with a 9.8 µm voxel size, 59 kV voltage, 167 µA current, and a 0.5-mm-thick aluminum filter. Image reconstructions were obtained via NRecon v.1.7.3 using a ring artifact correction of 5 and a beam hardening correction of 20%. The volume of interest (VOI) for metaphyseal analysis started 75 slices (or 0.738mm) proximal to the distal growth plate and extended 100 slices (total of 0.983mm) proximally. The trabecular and cortical bone were automatically segmented from each other using a custom task-list. The VOI for diaphyseal analysis was a 200-slice (total of 1.966mm) region centered at half the distance from the distal condyle to the proximal point of the femoral head.

After being scanned, femurs were decalcified in Immunocal Decalcifier (Statlab, Mckinney, TX) for 48 hours, processed, and embedded in paraffin. Femora were then cut into 5-µm-thick sections in the sagittal plane and mounted on glass slides. Slides were stained with tartrate resistant acid phosphatase (TRAP). Images were captured at 20x magnification using a Leica DM2700 M microscope (Leica Microsystems, Buffalo Grove, IL). Histomorphometric parameters were determined in an area 1000 pixels proximal to the growth plate with a 75-pixel border in a semi-automated manner using Bioquant OSTEO (Bioquant Image Analysis Co., Nashville, TN).

Statistical analyses were performed with GraphPad Prism (ver. 8, GraphPad Software, La Jolla, CA). Results are displayed as mean ± SD. To compare two groups, a two-tailed Student's t-test was used. To compare three or more groups, a one-way ANOVA with Tukey-Kramer post hoc test was performed. P-values < 0.05 were considered significant.

WT and TG mice were infected with 0, 1x10 3 , 1x10 4 , or 1x10 5 PFU of SARS-CoV-2 (n=4-6/group), as has been previously described. 12, 16, 25 All WT mice (mock-infected or infected) survived and were healthy for the study duration (12-14 days post-infection (dpi), Figures 2 and   3 ). Specifically, no significant differences in these mice from baseline were observed in any of the parameters examined. As also illustrated in Figure 2 PFU of SARS-CoV-2 that had to be humanely euthanized before the study endpoint.

With respect to the percent change from baseline in body weight at the time of euthanasia, as shown in Figure 2B , collectively, infected mice exhibited a significant 14.3% reduction in body weight compared to the 4.9% increase in body weight observed in non-infected mice (p=0.0008). As shown in Figure 2B , the majority of the mice that were euthanized prior to the study endpoint exhibited larger changes in body weight than that observed in those that survived. Indeed, comparing non-infected mice to those that were euthanized prior to the study endpoint showed a significant 21.8% reduction in body weight (p=0.0003), whereas comparing non-infected mice to those that survived to the study endpoint showed no significant difference in body weight change (p=0.2). Overall this suggests, that surviving infected mice exhibit body weight changes that were not significantly different from non-infected controls, while infected mice with severe illness requiring early humane euthanasia experienced signicant reductions in body weight. 

A primary goal of this study was to examine whether infection with SARS-CoV-2 resulted in changes in bone parameters. Following euthanasia, femurs were collected and µCT analysis was completed. To examine whether mice with SARS-CoV-2 infection had different trabecular bone parameters compared to mice which were not infected, we combined the mice as detailed above. Briefly, infected mice were TG mice infected with 1x10 3 , 1x10 4 , or 1x10 5 PFU of SARS-CoV-2 virus, and non-infected mice were mock-infected (0 PFU) TG mice as well as all WT mice (infected with 0, 1x10 3 , 1x10 4 , or 1x10 5 PFU of SARS-CoV-2 virus). Figure 4A shows representative µCT images of the trabecular bone within the distal metaphyseal region of femurs from non-infected and infected mice used for these analyses. As detailed in Figure 4B , TG mice receiving any amount of virus (infected mice) showed a significant decrease in trabecular bone volume fraction (BV/TV) compared to non-infected mice (-15.4%, p=0.004).

Although a significant difference in BV/TV was detected when all infected mice are grouped together and compared to all non-infected mice, mice that were euthanized at 6-7 dpi did not show a reduction in bone mass (BV/TV: -7.6%, p=0.2), whereas infected mice that survived to the study endpoint (12-14 dpi) showed a significant reduction in bone mass (BV/TV: -24.4%, p=0.0009). This suggests that bone loss is likely occurring during the latter stages of infection.

When examining the parameters associated with the reduction in BV/TV, as shown in Figure   4C , no significant difference was detected in trabecular thickness (Tb.Th) when all infected TG mice were compared to all non-infected mice (p=0.4). However, when comparing infected mice that survived to the study endpoint compared with those that did not survive, it was determined that surviving mice exhibited a significant reduction in Tb.Th (-6.2%, p=0.04), whereas mice humanely euthanized did not exhibit a change in Tb.Th (+1.5%, p=0.6), compared to all noninfected mice. With regard to trabecular number (Tb.N), as illustrated in Figure 4D , combined all infected TG mice had a significant reduction in Tb.N compared to all non-infected mice (-13.7%, p=0.004). Surviving mice exhibited a significant reduction in Tb.N (-19.0%, p=0.004), while mice J o u r n a l P r e -p r o o f Journal Pre-proof euthanized 6-7 dpi exhibited no change inTb.N (-9.0%, p=0.1) compared to all non-infected mice. Finally, with respect to trabecular separation (Tb.Sp), as shown in Figure 4E , combined all infected TG mice experienced a significant increase in Tb.Sp compared to non-infected controls (+8.5%, p=0.01). For Tb.Sp, surviving mice showed a significant increase (+9.8% p=0.03), but mice euthanized 6-7 dpi showed a non-significant increase (+7.2% p=0.1) compared to noninfected mice.

J o u r n a l P r e -p r o o f 

We then examined whether SARS-CoV-2 infection resulted in changes to cortical bone parameters. Femoral cortical porosity was examined at both the distal metaphyseal region and the diaphyseal midshaft. No significant differences were observed between infected and noninfected mice in either bone region ( Figure 5 ). We also found no significant differences among 

Here we report that in a mouse model of COVID-19, infection of K18-hACE2 TG mice with SARS-CoV-2 represents 3 important classes of COVID patients: those who die (1x10 5 PFU), those with moderate to severe disease who may recover (1x10 4 PFU), and those who are asymptomatic (1x10 3 PFU). All of the infected TG mice experienced a robust loss of trabecular bone volume fraction (Figure 4) , likely owing to the significant 64% increase in TRAP+ osteoclasts ( Figure 6 ). Importantly, when we examined only mice that survived to the study endpoint (approximately 1 week longer survival duration), an even more dramatic change in trabecular bone parameters was observed. While additional studies would be required to test this idea, it is is not an unreasonable hypothesis as it takes time for new osteoclasts to form and begin breaking down bone.

Another important observation is that the bone loss observed following SARS-CoV-2 infection occurs even in the surviving TG mice infected with 1x10 3 PFU, which had no clinical signs of illness as assessed by measuring changes in body weights, activity scores, posture scores, and BCS (Figures 2 and 3) . The finding that all surviving mice in the 1x10 3 PFU group were as active as the controls, is important as if a mouse is sick and not moving, we may expect significant bone loss. Indeed, perhaps the most dramatic case scenario for inactivity and lack of mobility would be with unloading such as that observed with spaceflight or hindlimb unloading.

In a hindlimb unloading study completed in the same background strain of mice at approximately the same age for the same study duration (4 month old C57BL/6 mice subjected to 2 weeks of unloading) investigators observed a 22% reduction in distal femur bone volume fraction. 26 In comparison, our surviving TG mice infected with 1x10 3 PFU of SARS-CoV-2 experienced bone loss and remained load bearing throughout the study.

Although limitations of our study include low sample size (n=4-6/genotype/viral dose), only one sex (male), and one age (approximately 19 weeks at the time of infection), as we are still in the midst of the COVID-19 pandemic, it was important to report on a statistically sound J o u r n a l P r e -p r o o f observation that has not been yet examined as a sequalae of this disease. This is especially true because as of September 16, 2021 the number of confirmed COVID-19+ cases reached over 226.9 million, while the death count was nearly 4.7 million. 1 Thus, there are millions of people who have already suffered from COVID-19 and bone loss may be yet another critically important secondary complication associated with having been infected with SARS-CoV-2.

Bone loss is a common side effect of aging and post-menopausal osteoporosis. Indeed, studies demonstrate that approximately 1 in 2 women, and 1 in 4 men over the age of 50 will suffer a fractured bone as a result of osteoporosis. 27 Therefore, it is formally possible that those who contracted COVID-19 may be more likely to be diagnosed with osteoporosis and/or may be at higher risk of developing fractures. However, it is important to note that these studies do not demonstrate whether the bone loss observed can be recovered over time. While it would be important to determine this for all individuals, it may be particularly important to understand this for the aged population, where they are already at risk for age-associated osteoporosis.

It is critically important to understand the long-term consequences of this dramatic bone loss. However, it is equally important to understand the mechanisms of action. While not specifically tested in this study, it is well known that patients with severe forms of COVID-19 have upregulated expression of numerous cytokines and growth factors which together lead to the inflammatory "cytokine storm". [28] [29] [30] [31] [32] [33] Many of these cytokines, including IL-1β, IL-6, IL-17, CXCL10, and TNF-α are known to regulate osteoclastogenesis and/or bone resorption, [3] [4] [5] [6] [7] [8] suggesting these factors may contribute to the SARS-CoV-2 stimulated bone loss observed in our K18-hACE2 mouse model. We recognize that the cytokine storm is just one possible mechanism by which bone loss occurs and much more work is needed to fully elucidate the mechanisms of action. For example, a recent study found that SARS-CoV-2 could directly infect bone marrow macrophages to a higher extent than mature osteoclasts. 34 Further, their study suggests that infection may alter the ability of bone marrow macrophages to differentiate into osteoclasts.

Regardless of the mechanism of action, infection with SARS-CoV-2 results in a dramatic upregulation in osteoclastogenesis and a robust loss of bone within 2 weeks post-infection in a COVID-19 mouse model. Whether these findings translate into humans remains to be determined, but if they do, an increase in fractures may be observed secondary to COVID-19 disease in the not too distant future.

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We would like to thank Ms. Jennifer Sickles and Ms. Chelsea Roberts for their assistance with caring for the mice. This project was supported, in part, by the Cooperative