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Serum proinflammatory cytokines, receptor activator of nuclear factor kappa-Β ligand (RANKL), osteoprotegerin (OPG) and RANKL/OPG ratio in mild and severe COVID-19

BMC Infectious Diseases volume 24, Article number: 1047 (2024) Cite this article

Abstract

Introduction

Osteoporosis, a systemic skeletal disease, is characterized by a quantitative and qualitative, and progressive decrease in bone mass, which is related to inflammation. Since a cytokine storm is triggered in Coronavirus disease 2019 (COVID-19), this study aims to evaluate pro-inflammatory cytokines (TNF-α, IL-1β), Receptor activator of nuclear factor-κB ligand (RANKL)/serum osteoprotegerin (OPG) ratio, and their relationship in mild and severe COVID-19.

Methods

This study was performed on 48 adult patients (18 mild, 18 severe COVID-19, and 12 healthy subjects as a control group). Serum OPG, RANKL, TNF-α, IL-1β, 25-OH vitamin D, and ALKp were measured by ELISA and colorimetric assay.

Results

COVID-19 patients had a significant increase in RANKL, and RANKL/OPG in mild and severe form (p < 0.001) while OPG decreased significantly in severe form compared to healthy controls (p < 0.05). Inflammatory cytokines (TNF-α and IL-1β) increased in both groups of patients whereas Alkaline phosphatase (ALKp) increased only in severe patients (p < 0.001). Both groups had 25-OH vitamin D deficiency in comparison to healthy ones (p < 0.001). Pearson’s correlation coefficient was performed to determine the relationship between RANKL, OPG, ALKp, and 25-OH vitamin D with TNF-α and IL-1β in mild and severe COVID-19, which was statistically significant.

Conclusion

Serum RANKL/OPG ratio was elevated in COVID-19 individuals and is assumed to be a risk factor for BMD reduction and osteoporosis in these patients. Correlations between IL-1β, TNF-α, ALKp, 25-OH vitamin D, OPG, RANKL, and RANKL/OPG ratio offered the potential role of these proinflammatory markers in the mechanism of osteoporosis in COVID-19 patients.

Summary

Since cytokine storm, can stimulate osteoclastic activity, favoring bone resorption, in this study, we decided to evaluate its molecular mechanisms in COVID-19. We found an elevation of RANKL/OPG ratio with a positive correlation with inflammatory cytokines in COVID-19 which can be assumed as a risk factor for osteoporosis.

Peer Review reports

Introduction

SARS-CoV-2 is a viral pandemic of respiratory syndrome which was appeared in China since December 2019. It is a highly infectious virus with uncontrolled systemic inflammatory response, especially in severe COVID-19 patients. The physiopathology of SARS-CoV-2 has been unclear, however disproportionate reaction of the immune system has been considered as an important reason for disease outcome. It relaeases significant amounts of pro-inflammatory cytokines by immune and non-immune effector cells to develop inflammatory storms that lead to organ damage. Several pro-inflammatory cytokines such as interleukin (IL)-1β, Interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α) are the major cytokines produced in COVID-19 disease and can be considered as biomarkers of SARS-CoV-2 severity. Previously, it was indicated that severe COVID-19 patients show TNF-α rise in plasma [1]. Emerging data reported that there is a significant correlation between severe uncontrolled inflammation process and organ damage or mortality [2]. In fact, most of the people who are infected with the SARS-CoV-2 will manifest mild to moderate respiratory disease and recover without any special treatment [1]. It was declared that up to 17% of individuals infected with the virus, resulted in an acute respiratory distress syndrome (ARDS) that can cause various long-term ill-health consequences and organ damage [3]. Previous studies revealed that inflammatory storm-induced bone metabolism disturbances [4]. Salvio et al. suggest that various proinflammatory cytokines, particularly IL-1β and TNF-α, can stimulate osteoclastic activity, resulted in bone resorption, suggesting the measurement of bone metabolism markers in COVID-19 patients [5]. It is therefore critical to have a deep perception of the various molecules involved in bone damage, thus can be assumed as potential therapeutic targets in COVID-19. Tao et al. reported that immune imbalance may disturb bone metabolism resulting in bone destruction in immunization [6].Accordingly, immune cells including B and T cells, produce receptor activators of nuclear factor kappa-Β ligand (RANKL) and TNF-α thus, stimulating bone resorption [7]. Moreover, B cells are an important origin of osteoprotegerin (OPG) which can inhibit extensively the osteoclastogenesis process [8].

Thus, increased inflammatory response due to COVID-19 may affect RANKL-OPG system as one of the molecular axis in bone remodeling and bone loss. We investigated RANKL, OPG, RANKL/OPG ratio, inflammatory cytokines (IL-1β, TNF-α), ALKp, 25-hydroxyvitamin D (25-OH vitamin D) as well as their relationship in patients with mild and severe COVID-19.

Materials and methods

Subjects

From October 2020 to December 2020, 48 samples were selected from COVID-19-infected patients (severe and mild phases) [9]. The available samples were randomly chosen for the sampling. These subjects were admitted to health centers (Universities) in Talegani Educational Hospital of Urmia University of Medical Sciences, Urmia, Iran; this study was a prospective case-control study. To assess and choose the appropriate sample size for this investigation, using the findings of the study by Y Liu et al. [10].

The study’s inclusion criteria for the severe phase group included pneumonia, acute respiratory symptoms, and a positive COVID-19 PCR test. Patients were selected based on the WHO classification and diagnostic tool of 2020 [11, 12] with confirmed COVID-19 by using reverse-transcriptase–polymerase-chain-reaction [RT-PCR]. Inclusion criteria for those in the moderate phase also included a positive PCR test for COVID-19 and the absence of acute respiratory symptoms. This study did not include people with chronic respiratory conditions, other underlying illnesses including hypertension, cancer, cardiovascular disease, or autoimmune inflammatory diseases like rheumatoid arthritis. Also, the participants did have not any endocrine or other disturbances that may affect bone tissue metabolism. The Urmia University of Medical Sciences Ethics Committee gave its approval to all experimental protocols (Ethical Code: IR.UMSU.REC.1400.119).

Demographic, clinical, and para-clinical information

Eventually, a questionnaire and a prepared checklist were used to gather the patients’ demographic, clinical, and paraclinical data. Moreover, the checklist contained the outcomes of laboratory and imaging findings as well as the contents of the medical file with a specialist’s opinion. Samples were taken from persons and patients with severe stages by healthcare staff relating to medical training facilities connected to Urmia University of Medical Sciences without respiratory symptoms. The molecular laboratory at the College received the samples from these facilities for molecular testing.

In this study, based on the fact that we wanted to measure the number of inflammatory genes and proteins, we first separated mononuclear cells (PBMCs) and blood serum according to the protocol based on the Ficoll method (Lymphodex, Inno-Train, Germany). Finally, the different phases of the samples, including the serum phase and the cell plate, were separated from each other. The values related to the protein level (OPG, RANKL, TNF-α, IL-1β) were evaluated by ELISA technique. ELISA technique was performed by the relevant protocol.

The blood samples for the measurement of OPG, RANKL, TNF-α, IL-1β, ALKp, 25-OH vitamin D were collected from patients on the first day of clinical admission. Blood samples were centrifuged then, plasma was frozen and stored at − 80 °C until assay. pro-inflammatory cytokines, OPG, RANKL, and 25-OH vitamin D levels were detected by High Sensitivity Especial Human ELISA kits: IL-1β (Quantikine R&D System, Inc., USA), TNF-α (eBioscience, Austria), OPG, and sRANKL (Biomedica, Austria), 25-OH vitamin D (Monobind Inc.USA). ALKp (pars Azmon) was measured by calorimetric assay.

Data analysis

The results were demonstrated as mean ± SD, and SPSS version 16.0 was used for data analyses. The parameters were checked for normality by one-sample Kolmogorov-Smirnov test. Data were analyzed by one-way analysis of variance (ANOVA) and Tukey’s test. Pearson’s correlation coefficient was used to test the relation between variables. The significant level was presented at p < 0.05.

Results

The mean age in the control group was 52.58 years, with no endocrine or other disturbances that may change bone tissue metabolism; they were all adult people from our hospital who volunteered to register in our study. The mean age in the mild disease group was 55.16 years, and in the severe disease group, was 57.19 years. In our study, 52.5% (19 subjects) were women, and 47.5% (17 subjects) were men. There was no significant difference regarding mean sex (p = 0.89) and age (p = 0.67). The mean BMI in the severe group was 28.92 kg/m2 and in the mild group was 26.68 kg/m2. There is no significant difference in mean BMI (p = 0.32) in the control, mild, and severe groups.

Smoking, Oxygen saturation, ESR and HCT factors showed dramatically a significant difference between the two groups of patients in the mild and severe phases (p < 0.05), as shown in Table 1 So that the ESR and HCT levels in the severe group increased markedly compared to the mild group (60.18 and 66.69 versus 46.75 and 36.34).

Table 1 Demographic details and median values of the studied parameters in control, mild and severe COVID-19
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For determination of bone resorption in COVID-19 patients, in the current study, we measured bone turnover markers. Our data showed that OPG decreased in mild (I.84 ± 0.65) ng/ml and severe (0.6 ± 0.05) ng/ml (p < 0.05) COVID-19. However, it is not significant in mild form. In addition, RANKL increased significantly (p < 0. 001) in mild (152.14 ± 9.81) pg/ml and severe (157.32 ± 9.74) pg/ml COVID-19 compared to healthy subjects. The mean value of the RANKL / OPG ratio was significantly higher in COVID-19 in mild (149.35 ± 32.1) (p < 0. 01) and severe (274.87 ± 21,94) (p < 0. 001) form compared to healthy control. Although in severely infected people with COVID-19 it is much higher than in mild ones (p < 0. 01) (Fig. 1).

Fig. 1
figure 1

OPG, RANKL and RANKL/OPG ratio in serum of each group. All data are expressed as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with healthy individuals. $P < 0.05, $$P < 0.01 compared with mild COVID-19. OPG: osteoprotegerin, RANKL: Receptor activator of nuclear factor-κB ligand

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TNF-α, IL-1β levels as proinflammatory cytokines, and accurate targets in the management of COVID-19, were assessed and compared among the groups. The results of this examination indicated that TNF-α and IL-1β levels were augmented significantly (p < 0. 001) in mild (25.1 ± 2.1 pg/ml, 13.6 ± 1.34 pg/ml) and severe (1.42 ± 3.8 pg/ml, 47.33 ± 4.7 pg/ml) COVID-19 patients respectively compared to the healthy controls (Fig. 2).

Fig. 2
figure 2

Assessment of proinflammatory cytokines (TNF-α, IL-1β) in the serum of different groups. All data are expressed as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, compared with healthy individuals. $$$P < 0.001 compared with mild COVID-19. TNF-α: tumor necrosis factor-alpha, IL-1β: interleukin-1β

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In patients infected with COVID-19, 25-OH vitamin D and ALKp were measured and compared among groups. According to our analysis, in comparison with the control group (31.2 ± 1.27) ng/ml, 25-OH vitamin D concentration was lower in both mild (23.22 ± 0.54) ng/ml and severe (24.55 ± 0.95) ng/ml groups (p < 0.001) (Fig. 3), while ALKp was higher in severe COVID-19 patients (35.94 ± 2.43) IU/L in comparison with mild (22 ± 1.74) IU/L and healthy (24.33 ± 1.44) IU/L individuals (p < 0.001) (Fig. 4).

Fig. 3
figure 3

25-hydroxyvitamin D in serum of each group. All data are expressed as the means ± SD. ***P < 0.001 compared with healthy individuals

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Fig. 4
figure 4

ALKp in serum of each group. All data are expressed as the means ± SD. ***P < 0.001 compared with healthy individuals. $$$P < 0.001 compared with mild COVID-19

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The results of the Pearson correlation are shown in Tables 2, 3 and 4. The correlation between OPG and TNF-α (R=-0.51, P-value = 0.016 ;R=-0.64, P-value = 0.002) and IL-1β (R=-0.44, P-value = 0.04; R=-0.61, P-value = 0.002) was reported in mild and severe COVID-19 respectively. The correlation of OPG with the inflammatory cytokines is inverse and with the increase of OPG, the amount of TNF-α and IL-1β decreased in mild and severe patients. In healthy ones, the correlation between OPG and TNF-α (R=-0.64, P-value = 0.002) was presented.

Table 2 Correlation between Tumour necrosis factor alpha (TNF-α), Interleukin-1β (IL-1β), osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand, (RANKL), ALKp (alkaline phosphatase), 25-hydroxyvitamin D, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) in control group
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Table 3 Correlation between Tumour necrosis factor alpha (TNF-α), Interleukin-1β (IL-1β), osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand, (RANKL), ALKp (alkaline phosphatase), 25-hydroxyvitamin D, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) in mild COVID-19
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Table 4 Correlation between Tumour necrosis factor alpha (TNF-α), Interleukin-1β (IL-1β), osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand, (RANKL), ALKp (alkaline phosphatase), 25-hydroxyvitamin D, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) in severe COVID-19
Full size table

Also, the correlation between RANKL and TNF-α (R = 0.47, P-value = 0.009 ;R = 0.62, P-value < 0.001) and IL-1β (R = 0.37, P-value = 0.032; R = 0.56, P-value = 0.001) was reported in mild and severe COVID-19 respectively. The correlation of RANKL with TNF-α and IL-1β is direct and with the increase of RANKL, the amount of both cytokines also increased. In healthy ones, there is a positive correlation between RANKL, TNF-α (R = 0.36, P-value = 0.01), and IL-1β (R = 0.31, P-value = 0.04).

In addition, ALKp had a significant direct correlation with TNF-α (R = 0.52, P-value = 0.002; R = 0.76, P-value < 0.001) and IL-1β (R = 0.61, P-value = 0.001; R = 0.62, P-value < 0.001) in mild and severe patients respectively. The correlation between ALKp and IL-1β (R = 0.52, P-value = 0.02) was demonstrated in healthy ones.

It was also found that 25-OH vitamin D had a significant correlation with TNF-α (R=-0.31, P-value = 0.048; R=-0.39, P-value = 0.029), IL-1β (R=-0.38, P-value = 0.028; R=-0.41, P-value = 0.02) and RANKL (R=-0.51, P-value = 0.003; R=-0.69, P-value < 0.001) in mild and severe patients respectively. In healthy ones the correlation between 25-OH vitamin D and RANKL (R=-0.47, P-value = 0.03) was reported. None of these factors (OPG, RANKL, ALKp, 25-OH vitamin D) had a significant correlation with ESR and CRP.

Discussion

It has been well known that inflammation has an outstanding role in the pathogenesis of COVID-19 infection with a strong relation to several organ disorders [6]. Some evidence has potentially indicated that increasing inflammatory cytokines can stimulate osteoclastic activity, favoring bone resorption [5, 6]. However, little is known about bone disorders in COVID-19 as a cytokine storm event. Accumulating evidence proposed that low bone mass is an outstanding risk factor for vertebral fractures as a hallmark of osteoporosis in patients infected with COVID-19 in the rehabilitation stage [13,14,15,16]. Recently, It was displayed that thoracic vertebral fractures happened in about 36% of COVID-19 patients and cause noninvasive mechanical ventilation [17]. Some studies reported that SARS-CoV-2 infection can cause bone loss in ACE2-transgenic mouse invitro, which may exhibit disturbances in bone formation or resorption markers in COVID-19 [18, 19].

Elucidating the molecular mechanisms underlying bone lesions in COVID-19 we investigated the bone mineral markers in COVID-19 patients.

To our knowledge, our research is the first to indicate the influence of COVID-19 on serum RANKL and OPG levels and also on the RANKL/OPG ratio in adults, then compare the results to those obtained from healthy individuals. We found significantly higher bone metabolic-related markers including RANKL and RANKL/OPG ratio levels as well as lower OPG levels in mild and severe patients infected with COVID-19 compared with controls.

OPG acts as a specific decoy receptor for RANKL to prevent RANK-RANKL reaction contributing to osteoclastogenesis and also bone resorption [20]. Based on the mentioned points, serum measurement of RANKL/OPG ratio showed an elevation surge in COVID-19 and thus can be considered as a risk factor for reduced bone density in these patients. Our findings are in line with Qiao et al. research, which described the effect of SARS-CoV-2 infection on the bone turnover markers in an animal model for COVID-19. Accordingly, they found that remarkable bone resorption was mediated by an imbalance in bone metabolism mediators including OPG and RANKL at the acute inflammatory phase after SARS-CoV-2 infection invivo, and in vitro [19]. Consistent with our work, bone remodeling markers including OPG and RANKL were changed in bone lesions thus contributing to the development of osteoporosis process in girls with anorexia nervosa [21], advanced ages of b-Thalassemia patients [22], , Duchenne muscular dystrophy patients [20], post-transplantation period of hematopoietic stem cells [23], and Systemic lupus erythematosus [24]. Queiroz et al. indicated that Coronavirus induces an osteoporotic phenotype in mice dependent on TNF-α and on macrophage/osteoclast infection [25]. In another study, it was shown that intermediate COVID-19 infection interferes with bone metabolism including lower levels of the bone resorption marker C-terminal telopeptide of type 1 collagen and the bone formation marker osteocalcin and higher levels of serum Dickkopf-1 and sclerostin [26].

These obtained findings could be elucidated by disturbances in osteoblastic activity as manifested by RANKL and OPG changes [20]. Hence, the RANKL/OPG ratio elevation in part explains the decreased the bone density observed in patients with COVID-19. However, we did not measure bone mineral density (BMD) of infected patients in this study, but according to Tsourdi et al., mice infected with SARS-CoV2 even in the absence of symptoms show two weeks after infection a decrease in trabecular bone mass as a result of an increase in osteoclast counts [27].

It was estimated that pro-inflammatory cytokines originating from the respiratory system as the main mediators for pathological bone resorption can stimulate generally pro-osteoclastogenesis activity [19].

Several evidence suggests that highly activated neutrophils, monocytes, and macrophages may lead to promote adaptive immune responses and possible cytokine storm [28]. These alterations have been related to increased levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α), which play a critical role in the pathogenesis of various disease [29]. IL-1β production occurs after the virus binds to cell surface receptors resulting in inflammasome activation [28]. TNF-α as a major regulator of inflammatory responses contributes to the development of inflammation and is elevated in both the blood and other tissues of COVID-19 patients [30]. Numerous clinical studies have demonstrated a remarkable rise of several cytokines/chemokines (TNF-α, IL-17, IL-6, IL-8, and IL-1β) contributing to cytokine release syndrome (CRS ) onset in COVID-19 which may lead to organ failure [31,32,33,34,35]. TNF-α can stimulate the production of the other cytokines for example IL-1β and IL-6 [36]. Many researchers have reported that severe COVID-19 patients exhibit high levels of plasma TNF-α [32,33,34]. Recently, it was displayed that IL-1β is highly produced as a series of immune responses in both forms of COVID-19 subjects [33]. The IL-1β can enhance the release of several hematopoietic factors, especially IL-6, similar to those of TNF-α [37]. In agreement with these studies, our results exhibited a significant increase in TNF-α and IL-1β levels in mild and severe COVID-19 patients. In addition in the present study, the positive correlation between RANKL and inflammatory cytokines (TNF-α and IL-1β) and also the negative correlation between OPG and aforementioned cytokines seems to suggest the relationship between proinflammatory cytokines and bone turnover markers in mild and severe patients infected with COVID-19. Previously, it was reported that pro-inflammatory cytokines predominantly IL-1β, IL-6, and TNF-α influence the bone mass directly and/or indirectly, by the RANK/OPG system and activate bone resorption cascade both in vitro and in vivo [38,39,40,41,42]. These studies are in line with our research. So, it seems reasonable to assume that increasing of these cytokine levels in patients with COVID-19 might have a noticeable role in the development of osteoporosis [43].

TNF-α and IL-1β stimulate ALKp activity and mineralization [44]. Lencel et al. reported that PPARγ inhibition may contribute to ALKp expression and mineralization induced by TNF-α surge [45].

The ALKp enzyme as a marker of bone metabolism, is the most important index of osteoblast differentiation [46]. It is found in several tissues, especially in the liver, kidney and bone. Elevated serum ALKp levels indicate bone disorders, including osteomalacia, and osteoporosis [47].

A remarkable positive association was reported between high levels of alkaline phosphatase and the risk of bone fracture [48, 49]. Our data demonstrated that ALKp increased in severe COVID-19 patients which was highlighted the bone fracture possibility as a chronic complication of COVID-19 infection. High levels of ALKp in severe COVID-19 casesare primarily related to significant disruptions in bone remodeling processes and increasing osteoblastic activity driven by the severity of the disease. Inflammatory state, hypoxia and vitamin D deficiency, can exacerbate bone remodeling issues, leading to increased ALKp levels as the body attempts to compensate for impaired bone metabolism in severe cases than mild [26, 50].

Furthermore, there is a positive correlation between ALKp and inflammatory cytokines (TNF-α, IL-1β) in mild and severe COVID-19 patients, suggesting the relationship between proinflammatory cytokines and the risk of bone fracture.

Further investigation is needed to describe the other bone turnover markers to provide further evidence to validate and confirm the present research. The small number of studied patients and also follow-up to monitor their BMD is another limitation of this study. Statistical significance does not directly translate to clinical relevance because of small sample size. Small sample sizes can lead to inflated effect sizes and may not adequately represent the population, making it crucial to interpret findings cautiously. Therefore, a larger number of patients are recommended to in future studies to enhance the statistical power, improve the reliability of findings, and better understand the relationship between inflammation and bone disorders to provide better-targeted therapy in patients infected with COVID-19.

Conclusion

Our study is the first to define the involvement of RANKL and OPG and their correlation with inflammatory cytokines in mild and severe COVID-19 patients and their possible roles as biomarkers to monitor their bone density. So, therapeutic interventions against the onset and progression of pathological bone loss in patients who have recovered from SARS-CoV-2 disease are recommended.

Data availability

Data willbe available on a reseanable request.

Abbreviations

RANKL:

Receptor activator of nuclear factor-κB ligand

OPG:

Osteoprotegerin

TNF-α:

Tumor necrosis factor-alpha

IL-1β:

Interleukin − 1β

25-OH:

vitamin D 25-hydroxyvitamin D

IL-6:

Interleukin 6

ARDS:

Respiratory distress syndrome

RT-PCR:

Rreverse-transcriptase–polymerase-chain-reaction

BMD:

Bone mineral density

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Acknowledgements

We are thankful to Nephrology and Kidney Transplant Research Center, Clinical Research Institute, Urmia University of Medical Sciences, Urmia, Iran.

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Nothing.

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Authors and Affiliations

  1. Department of Orthopedics, School of Medicine, Urmia University of Medical Sciences, Urmia, Iran

    Siamak Kazemi-Sufi

  2. Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences, Urmia, Iran

    Shahriar Alipour

  3. Department of Clinical Biochemistry and Applied Cell Sciences, Urmia University of Medical Sciences, Urmia, Iran

    Shahriar Alipour

  4. Department of Internal Medicine, School of Medicine, Urmia University of Medical Sciences, Urmia, Iran

    Masome Rabieepour

  5. Neurophysiology Research Center, Cellular and Molecular Medicine Research Institute, Urmia University of Medical Sciences, Urmia, Iran

    Shiva Roshan-Milani

  6. Nephrology and Kidney Transplant Research Center, Clinical Research Institute, Urmia University of Medical Sciences, Urmia, Iran

    Shiva Roshan-Milani & Roya Naderi

  7. Department of Physiology, School of Medicine, Urmia University of Medical Sciences, Urmia, Iran

    Roya Naderi

Authors
  1. Siamak Kazemi-Sufi
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  2. Shahriar Alipour
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  3. Masome Rabieepour
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  4. Shiva Roshan-Milani
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  5. Roya Naderi
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Contributions

SKS: Data curation, Methodology, SA: Data curation, Formal analysis, Methodology, MR: Data curation, Methodology. SRM: Conceptualization, Methodology. RN: Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

Corresponding author

Correspondence to Roya Naderi.

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Ethics approval and consent to participate

The Urmia University of Medical Sciences Ethics Committee gave its approval to all experimental protocols (Ethical Code: IR.UMSU.REC.1400.119). Informed consent was obtained from all subjects.

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Competing interests

The authors declare no competing interests.

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The authors declare no conflict of interests.

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Kazemi-Sufi, S., Alipour, S., Rabieepour, M. et al. Serum proinflammatory cytokines, receptor activator of nuclear factor kappa-Β ligand (RANKL), osteoprotegerin (OPG) and RANKL/OPG ratio in mild and severe COVID-19. BMC Infect Dis 24, 1047 (2024). https://doi.org/10.1186/s12879-024-09941-6

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Keywords

BMC Infectious Diseases

ISSN: 1471-2334

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