TEXT
Introduction
Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2), leads to severe cases and long-term
complications, including multi-organ
dysfunction(1-7). Post-recovery focuses on
respiratory, cardiac, and neurological functions, neglecting potential
skeletal issues. Older patients with comorbidities and medication use
may suffer unnoticed impacts on bone health.
While the diverse clinical symptoms of COVID-19 are well-documented, the
degenerative changes in the skeletal system induced by SARS-CoV-2 remain
underexplored. Up to 24% of long-term COVID patients reported
persisting bone pain or burning for seven months
post-infection(8). Interestingly,
non-intensive care COVID-19 patients exhibited significantly higher bone
mineral density (BMD) than those in critical
care(9).
Bone remodeling is a pivotal and ongoing process for maintaining bone
balance, relying on the coordination of osteoblasts and osteoclasts
guided by osteocytes(10). Osteoclasts
(OC), specific to bone tissue, originate from
monocyte/macrophage-derived precursors influenced by macrophage
colony-stimulating factor (M-CSF) and receptor activator nuclear factor
kappa B ligand (RANKL). The formation of osteoclasts involves the fusion
of mononucleated precursors. The process of bone resorption, facilitated
by integrins such as the CD51/61 vitronectin receptor, encompasses
matrix metalloproteinases (MMPs) produced by
OC(10-12).
During viral infections, osteoclasts, targeted by microorganisms,
undergo significant activation, leading to bone loss. In the case of HIV
infection, bone resorption is promoted, with the extent influenced by
the infection load(13,
14). On the other hand, Zika virus
infection impedes bone remodeling in
osteoclasts(15). Research utilizing
animal models suggests that SARS-CoV-2 affects bone metabolism both
during acute infection and in the post-recovery
phases(16-18).
This study examines the direct effects of SARS-CoV-2 on
osteoclastogenesis, maturation, and resorption activity using osteoclast
precursors from human peripheral blood mononuclear cells. Two SARS-CoV-2
strains were evaluated for their differential effects on OC
differentiation and osteoclastogenesis. Results indicate that even in
the absence of productive infection, SARS-CoV-2 exposure accelerates
osteoclastogenesis, bone loss, and resorption at various stages of OC
differentiation.
Material and Methods
Monocytes-derived macrophage culture and differentiation to
osteoclasts . Human monocytes were isolated from the blood of anonymous
healthy donors and differentiated as monocyte-derived macrophages (MDM),
as described previously (14). Briefly,
monocytes were seeded on slides in 24-well plates at a density of
5×105 cells/mL in RPMI medium (Gibco) supplemented
with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich), 2
mM of L-glutamine (Gibco), 1 mM of sodium pyruvate (Gibco),
penicillin-streptomycin (Sigma-Aldrich) and M-CSF (10ng/mL) (StemCell
Technologies) for 6 days. Then, mature osteoclasts (OC) were obtained
from cultured MDM (osteoclast precursors) in alpha minimum essential
medium (α-MEM) (Gibco) supplemented with 10 % FBS, 2mM of L-glutamine
(Gibco), 1mM of sodium pyruvate (Gibco), and penicillin-streptomycin
(α-MEM complete medium), M-CSF (10ng/mL) and RANKL (30ng/mL) (StemCell
Technologies) for 9 days (mature osteoclast).
Ethical approval for this study was granted by the institutional review
board and local ethical committee (Number: RESCD-2023-872). Buffy coats
from healthy donors, aged 18 to 60 with a balanced gender ratio, were
sourced from Hospital de Clínicas “José de San Martín”, Facultad de
Medicina, Universidad de Buenos Aires. All human samples, obtained
regardless of this study, were provided without personally identifiable
information.
Virus. SARS-CoV-2 ancestral (Wh) strain was provided by Dr.
Sandra Gallego (Universidad Nacional de Córdoba, Argentina), and the
Omicron (BA.5) strain was obtained from a nasopharyngeal swab,
characterized and then propagated, and titrated in African green monkey
kidney cell line Vero cells (2.85×106 TCID50 per ml).
The Vero E6 (ATCC, Rockville, MD) was cultured as monolayers in a 5%
CO2 atmosphere at 37°C in DMEM (Sigma-Aldrich,
Argentina) supplemented with 2mM L-glutamine, 10% FBS (Sigma-Aldrich,
Argentina), 100U/mL penicillin and 100μg/mL streptomycin.
In 24-well plates, 5×105 MDM/mL in RPMI (Gibco) were
cultured in RPMI complete medium with M-CSF (10ng/mL; StemCell
Technologies) for 3 days and separately infected with the two studied
SARS-CoV-2 variants using a MOI=0.1. When appropriate, MOI=0.01 and 1.0
were also used. The infection occurred in RPMI without FBS for 4 hours,
followed by 4-5 washes with PBS 1X (the last wash serving as T0), and a
complete RPMI medium with M-CSF (10ng/mL) was added. Finally, at 3 days
post-infection (dpi), α-MEM complete medium with M-CSF (10ng/mL) with or
without RANKL (30ng/mL) was added.
Detection and quantification of SARS-CoV-2 genomic RNA. RNA was
extracted from both, cells and culture supernatants using Chemagic™
Viral DNA/RNA kit special H96 on the automated Chemagic™ 360 instrument
(PerkinElmer). RNA was quantified using a NanoDrop™ (Thermo Scientific)
and its load was normalized before SARS-CoV-2 RNA detection was
performed using RT-qPCR (DisCoVery SARS-CoV-2 RT-PCR Detection Kit Rox)
amplifying ORF1ab and N viral genes following the manufacturer’s
instructions.
In culture supernatants, viral load was calculated by interpolation of
the corresponding Ct value with a standard curve, which had been built
with the Ct values obtained following PCR amplification of samples
containing serial dilutions of quantified SARS-CoV-2 positive RNA
control (GISAID EPI_ISL_420600). In cell lysates viral load was
normalized to human RNAse P gene and expressed as
2-Δct.
Titration of SARS-CoV-2 infectious particles. The release of
infectious viral particles into the supernatant was assessed using Vero
E6 cells. Briefly, Vero cells were seeded in 96-well plates, and the
culture supernatants to be tested were added for 1 h at 37°C. Afterward,
the supernatant was removed, and DMEM with 2% FBS was added. Plates
were then incubated for 3 days at 37°C. The infectious titer of each
supernatant was determined by TCID50 and expressed as plaque-forming
units (PFU) per mL.
Flow cytometry analysis . Cells detached using Accutase®
(StemCell Technologies) were stained for surface antigens for 30 minutes
at 4°C. To characterize the macrophage activation profile, anti-human
CD80, anti-human CD206, and anti-human CD163 were employed. The
osteoclast-specific CD51/61 marker was measured using an
anti-CD51/61-FITC antibody. The ACE2 expression was quantified using a
rabbit primary polyclonal antibody to human-ACE2. The determination of
intracellular SARS-CoV-2 nucleocapsid was performed on fixed and
permeabilized cells with Fixation/Permeabilization Kit (BD Biosciences)
according to the manufacturer’s using a rabbit primary antibody to
nucleocapsid. The Goat Anti-Rabbit IgG secondary polyclonal antibody was
used. For further information on antibodies used in flow cytometry see
Supplementary Table 1.
Cell death percentage was assessed using APC-conjugated annexin-V and
7-AAD staining with the apoptosis detection kit (BD Biosciences).
Freeze-thaw cycles served as a positive control for cell death.
Mitochondrial reactive oxygen species (mROS) were measured using 5 μM
MitoSOX™ (Thermo Fisher Scientific) staining for 30 min. Data were
acquired using Full Spectrum Flow Cytometry Cytek® Northern Lights 3000™
(Cytek Biosciences Inc.) and analyzed with FlowJo.v10.6.2 (Ashland).
Western Blot. Macrophages (5x105) were
sonicated in RIPA buffer (Santa Cruz Biotechnology, sc-29498) with
protease inhibitors cocktail (Roche). Protein concentration was
determined by the Bradford method (Bio-Rad dye reagent) as described by
the manufacturer with BSA as standard. Equal amounts of protein per lane
were run in an SDS-PAGE and then electrotransferred to PVDF membranes.
Membranes were blocked with 5% non-fat milk for 1 hour and incubated
overnight with a specific antibody against ACE2 (PA5-20040, Thermo
Fisher). Anti-Na+/K+ ATPase (Thermo Fisher, MA532184) was used as a
loading control. Peroxidase conjugated anti-rabbit (NA934, GE Healthcare
Life Sciences) was used as the secondary antibody. Bands were visualized
with enhanced chemiluminescence reagent (ECL, Millipore) and a Chemidoc
Station (Bio-Rad) and quantified using Image Lab software (Bio-Rad).
Assessment of osteoclast differentiation. Cells were fixed with
PFA 4% and stained for evaluating the generation of mature
multinucleated osteoclasts using Tartrate Resistant Acid Phosphatase
(TRAP) (Sigma-Aldrich, USA) according to the manufacturer’s protocol.
Briefly, at the end of the experimental timeline cells were washed twice
with 1XPBS and fixed with a fixative solution comprised of citrate,
acetone, and 4% formaldehyde for 10 min at 37°C. After washing twice
with 1XPBS, fixed cells were stained for TRAP at 37°C in the dark for 1
hour. Multinucleated TRAP-positive cells with ≥ 3 nuclei were considered
mature osteoclasts. TRAP-positive multinucleated cells were further
counted and imaged using an inverted microscope 200x (ECLIPSE, TS100,
Nikon).
Measurement of TNF-α, IL-6, and IL-1β concentration. TNF-α,
IL-6, and IL-1β were measured by sandwich ELISA in culture supernatants
using paired cytokine-specific monoclonal antibodies, according to the
manufacturer’s instructions (BD Pharmingen).
Quantitative real-time PCR . Quantitative RQ-PCR was utilized to
detect the gene expression of M1 and M2-related cytokines as well as key
regulators of osteoclast differentiation. Total RNA was isolated using a
GenElute™ Total RNA isolation kit from Sigma-Aldrich, and RNA
quantification was performed on a nanodrop spectrophotometer. cDNA was
synthesized using the ImPromII Reverse Transcription System (Promega).
Real-time PCR was conducted on a StepOne PCR system (Applied Biosystems)
with SYBR green for PCR product detection. Cycling conditions and
primers’ sequences for various target genes, including GAPDH, IL-10,
TGF-β, TNF-α, IL-1β, NFATc1, DC-STAMP, MMP-9, and RANK. For primer
sequences and cycling conditions see Supplementary Table 2.
The fold-change (relative expression) in gene expression was calculated
using the relative quantification method
(2−ΔΔCt)(19). Relative
expression levels were normalized against GAPDH. Intra-experiment CT
value differences between samples were less than 0.5.
Assessment of bone resorption . To assess bone resorption
activity, macrophages were seeded on bovine cortical bone slices
(Boneslices, Inc.) and differentiated into osteoclasts. Following
complete cell removal by several washes with water, bone slices were
stained with toluidin blue (Sigma-Aldrich) to detect resorption pits
under a light microscope (ECLIPSE, TS100, Nikon). The surface of bone
degradation areas was quantified manually with ImageJ software (version
1.47).
Statistical Analysis .
The figure legends provide the exact values of n (donors). For datasets
with a normal distribution (confirmed by the Kolmogorov-Smirnov test),
two-tailed paired or unpaired t-tests were employed. Alternatively,
two-tailed Mann-Whitney (unpaired test) or Wilcoxon matched-paired
signed rank tests were applied, using GraphPad Prism 7.0 (GraphPad
Software Inc.). A significance level of p < 0.05 was
considered (*p ≤0.05; **p ≤0.01; ***p ≤0.001, ****p ≤0.0001).
Results
SARS-CoV-2 causes
an abortive infection in osteoclast precursors during their
differentiation.
The experimental timeline, outlined in Figure 1A, involved separately
exposing monocyte-derived macrophages (MDM) to each of the two studied
SARS-CoV-2 variants (ancestral and Omicron). Remarkably, comparable
viral infection kinetics were observed even during MDM differentiation
into osteoclasts, as illustrated in Figure 1B. The assessment, conducted
at 3, 6, 9, and 12 days post-infection (dpi), included measuring viral
load in cell supernatants (via RT-qPCR) and intracytoplasmic viral load
in cell lysates. In addition, infection efficiency was evaluated through
flow cytometry, measuring the relative abundance of intracellular
nucleocapsid protein (N)-expressing cells. The viral load in MDM culture
supernatants for each SARS-CoV-2 variant, measured using both N-gene and
ORF-1a-gene, exhibited an initial increase from inoculation to 3 dpi,
followed by a progressive decline at 6, 9, and up to 12 dpi (Figure 1B).
Contrarily, intracytoplasmic RNA in cell lysates, measured using both
N-gene and ORF-1a-gene, exhibited a progressive decrease from
inoculation to 12 dpi (Figure 1C). The undetectable levels (%) of the
SARS-CoV-2 capsid protein (N) were found for both variants at 3 dpi by
flow cytometry (Figure 1D).
Furthermore, an assay to detect infective viral particles in
supernatants for the SARS-CoV-2 ancestral strain revealed undetectable
de novo production of viral particles from the infectious inoculum to 72
hpi (Figure 1E).
In summary, after exposure to SARS-CoV-2 (Wh and BA.5 strains), abortive
infections were observed in MDM both before and during their
differentiation into osteoclasts, highlighting the intricate dynamics of
viral replication and host response throughout the experimental
timeline.
Cell viability and redox balance are preserved when
macrophages are abortively infected by SARS-CoV-2
Even when the infection is abortive it might trigger macrophage
activation and several biochemical and morphological changes leading to
programmed cell death (20). Thus, to
ensure that cellular-related parameters are comparable between
virus-infected and control groups, the level of programmed cell death
was measured.
As shown in Figures 2A and 2B, cell viability (%) remained preserved
among macrophages after SARS-CoV-2 exposure from 3 dpi and throughout
osteoclastogenesis until 12 dpi.
Reactive oxygen species (ROS) production increases during M-CSF-induced
macrophage differentiation from monocytes and they are mainly produced
by mitochondria (mROS)(21). It may
influence osteoclast differentiation. As depicted in Figures 2C and 2D,
cellular mROS levels (%) were assessed post SARS-CoV-2 infection before
RANKL treatment. A slight increase in mROS production was observed
immediately after virus-cell contact, but no significant differences
were evident during the follow-up at 4, 24, and 72 hpi.
Taken together, both cell viability and mROS levels were maintained
under the experimental conditions before and after the SARS-CoV-2
challenge.
SARS-CoV-2 induces upregulation of ACE2 expression in
macrophages
Given that ACE2 serves as the primary receptor for SARS-CoV-2, the level
of its expression on macrophages delineates susceptibility to infection
and replication. Moreover, the potential viral-induced impact on its
expression was also assessed. This investigation involved a
comprehensive analysis employing two distinct technical approaches.
First, the expression level of ACE2 in MDMs using flow cytometry after
3-days of infection was examined. As shown in Figures 3A and 3B, the
analysis revealed that ACE2 was expressed in low frequency (4.7±1.6 %)
in control cells. However, among SARS-CoV-2–infected MDMs from the same
donors, a significantly increased level of ACE2 expression was measured
(Wh: 10.5±4.1%; p<0.05). Second, ACE2 protein expression
measured by western blotting from MDM homogenates revealed that these
osteoclast precursors infected with SARS-CoV-2 had a significant
increase (3.5-fold-change; p<0.01) in receptor expression
compared with control-MDM (Figures 3C, 3D).
These results demonstrate that MDM express the ACE2 receptor at a low
level, which is upregulated after the SARS-CoV-2 challenge.
OC formation is enhanced by ancestral and Omicron
SARS-CoV-2 strains in a dose-dependent manner
To determine the influence of SARS-CoV2 in osteoclastogenesis, human MDM
(from 4-5 donors) were exposed to two viral variants, (Wh, BA.5). At
12-dpi, osteoclast formation in culture was quantified and its relative
abundance (%±SD) was calculated after manually counting TRAP-positive,
multinucleated (≥3 nuclei) cells visualized under the microscope (x200).
As shown in Figures 4A and 4B, MDM exposure to Wh or BA.5 virus strain
triggered significantly higher MDM fusion into osteoclasts compared to
non-infected cells (Ctrl:5.5±1; Wh:9.6±1.2; BA.5:10.1±2%). Moreover,
for both viral variants, SARS-CoV2-enhanced osteoclastogenesis depicts
an inoculum dose response. As shown in Figures 4C and 4D, when MDM were
exposed to a ten-fold increased viral inoculum (MOI 0.010.11.0), the
abundance of OC (%) was significantly higher for both, ancestral
(6.3±2.39.0±1.210.0±1.4) and Omicron (7.2±1.19.7±1.010.5±0.5) strain.
These findings show that osteoclast formation is significantly boosted
in a dose-dependent manner when its precursors are abortively infected
by SARS-CoV-2.
Osteoclasts CD51/61 expression and resorptive ability is
up-modulated by SARS-CoV-2
The integrin complex CD51/CD61, also known as αVβ3, is expressed at high
levels on osteoclasts. The SARS-CoV2 influence on the CD51/61 expression
was evaluated. Besides their morphology and TRAP expression, this marker
is expressed in differentiated osteoclasts as a receptor for
vitronectin. Figures 5A and 5B show that, after 9-days of
osteoclastogenesis with M-CSF+RANKL (12 dpi), a significantly higher
level of CD51/61 expression (%) was measured when OC precursors were
exposed to SARS-CoV2 (Ctrl:20.7±9.2; Wh:30.7±14.0).
To assess the impact of SARS-CoV-2 on osteoclastogenesis and subsequent
resorptive ability, infected osteoclast precursors were cultured on bone
slices and differentiated into mature osteoclasts by RANKL for 9 days.
Figures 5C and 5D illustrate the defined resorption pits, showing
significantly greater osteoclast activity in SARS-CoV-2-exposed cultures
(p<0.05). The resorbed bone area (%) was markedly increased
compared to control (Ctrl: 10.7±1.1; Wh:22.2±2.1; BA.5: 20.4±2.4). These
findings highlight the SARS-CoV-2-mediated upregulation of CD51/61
expression among osteoclasts, which would facilitate interactions with
the extracellular matrix, cell adhesion, and bone resorption. In line,
both SARS-CoV-2 strains can increase osteoclast traits, such as
multinuclearity and bone resorption competence.
RANK, NFATc1, DC-STAMP, and MMP9 mRNA levels are
differently regulated during osteoclastogenesis in response to
infection
The SARS-CoV-2 influence on different osteoclastogenesis-related genes
was evaluated. The RANKL and its receptor RANK play critical roles in
controlling the development, activation, and survival of osteoclasts. As
shown in Figure 6A, at 3dpi the RANK mRNA level in infected macrophages
was higher than non-infected control (7.5-fold-change). According to the
timeline, at 3 dpi the macrophages are exposed to RANKL activating other
signals involved in differentiation and fusion. As the RANK/RANKL
interaction triggers NFATc1 transcription, a master regulator of
osteoclast differentiation, its mRNA level post-RANKL stimulus at 6 dpi
was measured. As presented in Figure 6B, the mRNA levels in infected
cultures were significantly elevated compared to those Ctrl
(7.4-fold-change). Additionally, mRNA levels of DC-STAMP, a surface
receptor necessary for osteoclast fusion, and MMP9, a collagenase
abundantly expressed in osteoclasts, were examined at 12 dpi. As
illustrated in Figures 6C and 6D, both DC-STAMP and MMP9 mRNA were
notably increased in cells exposed to SARS-CoV-2 (1.9 and
2.5-fold-change respectively).
Thus, the mechanism of the SARS-CoV-2-enhanced osteoclast
differentiation includes the RANK/RANKL pathway, resulting in increased
expression of NFATc1, DC-STAMP, and MMP9.
SARS-CoV-2 drives early pro-inflammatory M1 macrophage
polarization shifting towards an M2-like profile during infection.
To analyze the impact of SARS-CoV-2 on macrophage activation during
osteoclast differentiation, a three-day exposure to the virus was
conducted before RANKL treatment. As shown in Figure 7A, at 1 and 3 dpi,
non-infected macrophages predominantly appeared as small, roundish
cells. SARS-CoV-2-infected macrophages exhibited mainly amoeboid
morphology, while at 3 dpi, roundish and amoeboid macrophages, as well
as large bipolar spindeloid macrophages, were observed, resembling M1
and M2-like phenotypes, respectively. Phenotypically these cells
revealed early increases in CD80 (M1 marker) at 1 dpi and comparable
CD206 levels (M2 marker) in infected macrophages compared to
non-infected controls but lower CD163 levels (M1 to M2 shifting marker)
were observed. Then, among SARS-CoV-2-infected cells, CD80 decreased,
CD206 and CD163 increased at 2 dpi, and later (3 dpi), CD80 levels
normalized, while CD206 and CD163 peaked in virus-infected cultures
(Figure 7B, 7C). Pro-inflammatory cytokines, significantly increased in
SARS-CoV-2 infected macrophages compared to undetectable or very low
levels in controls, were observed with TNF-α peaking at 1 dpi, IL-1β at
2 dpi, and constant IL-6 levels (Figure 7D). Figure 7E presents results
obtained by RT-qPCR at 3 dpi, indicating the upregulation of M2-related
cytokine mRNA (TGF-β and IL-10: 6.7 and 4.2-fold change, respectively)
and TNF-α (2.5-fold change), but not IL-1β (1.1-fold change).
Collectively, the abortive infection by SARS-CoV-2 initiates an early
M1-like activation profile, transitioning to an M2-like state, as
indicated by altered cell surface markers and cytokine production
patterns.
SARS-CoV-2 variants promote RANKL-independent osteoclast
formation
To ascertain whether the early pro-inflammatory environment induced by
SARS-CoV-2 could prompt the differentiation of macrophages into
osteoclasts, MDM were exposed to two viral variants (Wh, BA.5) and
maintained in a media with MCSF alone in the absence of RANKL. At 12
dpi, osteoclast formation in culture was quantified by manually counting
TRAP-positive, multinucleated (≥3 nuclei) cells visualized under the
microscope (x200; mean number ± SD). As depicted in Figures 8A and 8B,
exposure of MDM to the ancestral or BA.5 virus strains resulted in a
significantly higher fusion of MDM into osteoclasts compared to
non-infected cells (Ctrl:26.5±3.3; Wh:51.0±5.1; BA.5:52.3±7.5).
These findings demonstrate that SARS-CoV-2-enhanced osteoclastogenesis
involves not only RANKL-dependent differentiation but also
RANKL-independent mechanisms.
Discussion
In this investigation, despite causing an abortive infection in
precursor macrophages of osteoclasts, the SARS-CoV-2’s ability to
accelerate osteoclastogenesis and bone resorption is highlighted.
Limited studies on the direct impact of SARS-CoV-2 on osteoclast
susceptibility and differentiation, primarily conducted in rodent
models, have provided insights(16,
17, 22,
23). Studying SARS-CoV-2’s impact on the
skeletal system is challenging due to delayed onset, but lasting effects
include bone metabolism disorders observed in COVID-19
patients(24,
25). Osteoclasts, originating from the
monocyte/macrophage lineage (pre-OC), are exclusive bone-resorbing cells
crucial for bone development and remodeling, actively contributing to
musculoskeletal tissue damage. While consensus suggests that human
monocyte-derived macrophages (MDMs) exposed to SARS-CoV-2 do not release
infectious virions(20,
26-30), conflicting studies propose MDMs’
potential support for initial infection stages, including viral entry,
RNA replication, and protein
synthesis(20,
26, 27,
30). Conversely, other research indicates
MDM resistance to SARS-CoV-2 entry. Interestingly, despite their ability
to support de novo synthesis of viral RNA and proteins, respiratory
viruses like influenza A infect macrophages
abortively(29).
In this study, utilizing both ancestral and Omicron strains, abortive
infection of SARS-CoV-2 among human primary monocyte-derived macrophages
(MDM), acting as pre-osteoclasts (pre-OCs) is demonstrated. This finding
remains consistent despite a notable increase in ACE2 expression among
pre-OC following exposure to SARS-CoV-2. A transient synthesis of viral
RNA without de novo protein synthesis or virion release was observed.
Early after the virus challenge, a pro-inflammatory cytokine profile,
including TNF-α, IL-1β, and IL-6, was detected, gradually declining.
Subsequently, SARS-CoV-2-exposed pre-OC exhibited increased
transcriptional levels of anti-inflammatory cytokine genes (IL-10,
TGF-β), elevated CD163 expression as a marker of inflammation
resolution(31), and maintained cell
viability. Morphological changes in pre-OC were noted, initially
resembling an M1 profile, transitioning to an elongated cell reminiscent
of the M2 macrophage profile(32). These
morphological alterations, influenced by biophysical cues in the
microenvironment, indicate the reversible repolarization capacity of
macrophages(33). This observation aligns
with previous reports of reversible repolarization in the context ofCryptococcus neoformansinfection(34). The SARS-CoV-2-induced
M1-to-M2 switch resulted in increased osteoclast differentiation,
suggesting a higher bone-resorptive
capacity(35). This M2-osteoclastogenic
potential, associated with the silencing of factors enhancing M1
polarization, such as IRF5, highlights the intricate mechanisms by which
SARS-CoV-2 modulates macrophage phenotypes and accelerates
osteoclastogenesis(36).
Upon initiation of in vitro RANKL-induced osteoclastogenesis, a
direct impact of SARS-CoV-2 abortive infection on pre-osteoclasts
(pre-OCs) was observed, leading to a concurrent increase in
osteoclastogenesis in a dose-dependent manner. Additionally, SARS-CoV-2
abortive infection prompted the formation of larger osteoclasts,
significantly enhancing the expression of osteoclast-specific markers,
including tartrate-resistant acid phosphatase and CD51/61, while also
upregulating osteoclast bone resorption activity. Such findings further
revealed elevated mRNA levels of inducible genes associated with
osteoclast differentiation, such as RANK, NFATc1 (master regulator
controlling genes like TRAP and MMP-9), and DC-STAMP (master fusogenic
protein). The heightened mROS levels in SARS-CoV-2-exposed pre-OC
correlated with an upregulated TNFα-mediated inflammatory response,
characteristic of the initial M1 profile and crucial for M2 macrophage
differentiation(37,
38). Remarkably, unlike other target
cells such as epithelial, hepatocytes, and cholangiocytes, the redox
balance and viability of abortively infected osteoclasts (OC) remained
intact. This suggests the potential for these infected cells to persist
for an extended period within bones.
These findings unveil an intriguing aspect: priming with transforming
growth factor β (TGF-β) enables TNF-α to induce osteoclastogenesis
effectively, bypassing the canonical RANKL pathway. This discovery holds
significance as inflammatory cytokines, including TNF-α, typically have
limited capacity to directly stimulate osteoclast differentiation. The
production of TGF-β induced by SARS-CoV-2 transforms TNF-α’s
pro-inflammatory action on macrophages into a highly efficient
osteoclastogenic function, creating a chromatin environment favorable
for the expression of osteoclastic
genes(39). Consequently, even in the
absence of productive infection in osteoclast precursor cells,
SARS-CoV-2 accelerates osteoclastogenesis through both RANKL-dependent
and independent mechanisms.
In symptomatic COVID and even in patients who survive its initial stage
but with persistent symptoms, often referred to as ”long COVID” or
”post-acute sequelae of SARS-CoV-2 infection” (PASC), arthralgia or
rheumatic complaints are frequently reported
(8, 40-42),
but fewer efforts have been put forward to explore the underlying
factors behind SARS-CoV-2-induced arthralgia.
This study has limitations. Firstly, the experiments primarily utilized
peripheral blood mononuclear cells (PBMCs) from multiple donors,
potentially introducing subtle differences between donors. However, the
consistent and robust observations across multiple assays validate the
stimulatory effect of SARS-CoV-2 infection. Secondly, the current model
relies on an isolated culture system, valuable for screening direct
effects on the osteoclast phenotype. Nevertheless, these findings
necessitate further validation in in vivo models. Thirdly, the observed
phenotypic effects lack complete support from gene expression profiles,
partly due to the chosen time point, inherent cell population
heterogeneity, and concurrent expression of key markers. Future studies
will focus on investigating these markers and their interactions at the
protein level.
In summary, this study sheds light on the role of osteoclastogenesis in
SARS-CoV-2 pathogenesis using a primary in vitro human model. This model
enhances the understanding of the factors contributing to bone loss
following SARS-CoV-2 infection. These findings reveal disruptions in
osteoclast differentiation and function due to SARS-CoV-2 infection. An
alternative mechanism driving bone pathology, which results in
accelerated bone resorption, is proposed. This model presents potential
targets for therapeutic and preventive interventions against SARS-CoV-2,
especially concerning long-term complications associated with COVID-19.