Abstract
Wnt5a/Wnt11 as ARDS biomarker for SARS-CoV-2 patients
The coronavirus disease-19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has spread globally, thereby resulting in declaration of pandemic emergency [1]. COVID-19 patients suffer from various infectious symptoms, including pneumonia, acute respiratory distress syndrome (ARDS) and sepsis. Some known-antiviral drugs, including remdesivir, have been proposed as effective agents for the treatment of SARS-CoV-2 infection [2, 3]. Along with the development of potential therapeutics, there is also urgency to mitigate the transmission and economic crisis of SARS-CoV-2 via identification of biomarkers that can rapidly indicate the severity of the disease in infected patients. Wnt ligands are secreted glycoproteins and their downstream signalling plays a pivotal role in embryonic development and tissue homeostasis. With remarkable progress in the immunology field, Wnt signalling has gained much attention as a critical regulator in various inflammatory diseases. A large body of evidence has suggested that Wnt ligands were secreted by immune cells, such as PBMCs, and non-immune cells, including stroma cell, to regulate inflammatory response and immune cell modulation [4–7]. In addition to their roles in inflammation, recent studies have reported that these Wnt ligands play key roles in tissue damage and repair [6]. Interestingly, previous studies have reported significant alterations in Wnt5a and Wnt11 expression compared to other Wnt ligands by analysing sera of patients with severe sepsis or sepsis mouse model [4, 8]. Wnt5a signalling has been known to activate in sepsis or ARDS and play a pivotal role in lung inflammation and fibrosis [5, 9], whereas Wnt11 protein has been reported to suppress induction of inflammatory cytokines by regulating NF-κB activity [10, 11]. Previous reports have demonstrated that Wnt5a and Wnt11 have opposite functions to one another in response to inflammation [12, 13]; hence it is thought that Wnt5a has pro-inflammatory effect and Wnt11 may be anti-inflammatory effect. Therefore, we focused on Wnt5a and Wnt11 to explore their potential relevance to COVID-19-related diseases. In this study, we report Wnt5a and Wnt11 as reliable biomarkers for monitoring of pathological progression in SARS-CoV-2 patients.
Materials and Methods
Whole blood was collected from admitted SARS-CoV-2 patients at Yeungnam University Medical Center when these patients were diagnosed with the SARS-CoV-2 infection at the public health centre in Daegu. None of the patients had taken any medications nor used any mechanical devices upon admission to the hospital. The study protocol (YUH 2020-03-057, 2020-05-031-001) was approved by the Institutional Review Board of Yeungnam University Hospital at Daegu in Korea.
The concentrations of Wnt5a, Wnt11, or cytokines in SARS-CoV-2 patients' plasma was quantified according to the manufacturer's instructions using a commercially available ELISA kit. Human recombinant WNT11 protein (H00007481-P01, Abnova), anti-Wnt5a antibody (MAB645, R&D system), anti-Wnt11 antibody (ab31962, Abcam), Human Protein Wnt-5a ELISA Kit (MBS2886311, MyBioSource), and Human Protein Wnt-11 ELISA Kit (MBS281148, MyBioSource) were used.
Heparinised blood samples were used fresh within 4 h, and peripheral blood mononuclear cells (PBMCs) were separated from blood using Ficoll–Hypaque or NycoPrep. Following this, more refined PBMCs were obtained via MACSprep™ PBMC Isolation Kit. To verify the effect of Wnt5a neutralising antibodies or recombinant human Wnt11 on the suppression of cytokine secretion and NF-κB activation, PBMCs isolated from SARS-CoV-2 patients were incubated with the Wnt5a antibody (20 μg·mL−1) or recombinant human Wnt11 (10 ng·mL−1) for 6 h. The supernatant was used for analysis of cytokines ELISA, and lysate was used for NF-κB activity analysis by an ELISA-based NF-κB family transcription factor assay kit (43 296; Active Motif). All experiments were performed independently at least three times. Statistically significant differences were determined using unpaired t-test. Prism software was used for statistical analyses.
Results
In order to establish reliable diagnostic biomarkers, we have conducted a prior study by exploring clinical manifestations and various risk factors on severe SARS-CoV-2 patients admitted to Yeungnam university medical center in Korea [14, 15]. We conducted research to discover new biomarkers in blood based on patient information such as age, BMI, and comorbidities (fig. 1A). The SARS-CoV-2 patient blood plasma samples were divided according to the severity of the disease; normal individuals (control group, tested for SARS-CoV-2 infection but negative), SARS-CoV-2 patients, SARS-CoV-2 patients with acute respiratory distress syndrome (SARS-CoV-2 ARDS), and discharged individuals after hospitalisation by SARS-CoV-2 infection. ELISA analysis showed marginal difference in the Wnt5a secretion level between the SARS-CoV2 infection and the control group. Irrespectively, the Wnt5a protein level was dramatically increased in the blood of SARS-CoV-2 ARDS (fig. 1Ba). Interestingly, the Wnt5a protein level was rescued in discharged individuals (fig. 1Ba). This was consistent in the survived patients, where the Wnt5a level remained low in the plasma but significant high level of Wnt5a was still observed in the dead patients, thus demonstrating correlation of the Wnt5 level with the severity of the disease (fig. 1Bb). On the contrary, Wnt11 protein level was robustly induced in the plasma of SARS-CoV2 patients and discharged individuals, but remained at normal levels in SARS-CoV-2 ARDS, where the Wnt5a level was detected at its highest (fig. 1Bc and Bd).
Wnt5a/Wnt11: a promising diagnostic marker for SARS-CoV-2 ARDS. Plasma was secured from 20 healthy volunteers, 80 SARS-CoV-2 patients, 25 patients who progressed to ARDS (SARS-CoV-2 ARDS) and 25 discharged patients (No SARS-CoV-2 detected). A) Baseline characteristics and clinical outcomes of COVID-19 patients admitted to Yeungnam University Hospital. Ba–Bd) Analysis of Wnt5a and Wnt11 concentrations in SARS-CoV-2 patients. Be and Bf) The mRNA expression of Wnt5a and Wnt11 in PBMCs was quantified by qRT-PCR. Ca) Wnt5a or Wnt11 protein was visualised in PBMCs isolated from normal and SARS-CoV-2 ARDS patients by immunofluorescence staining (x 200). Cb and Cc) Wnt5a or Wnt11 secreted from SARS-CoV-2, SARS-CoV-2 ARDS, and discharged patients were detected by ELISA. SARS-CoV-2 Sepsis patient plasma was incubated with Wnt5a antibody (20 μg·mL−1) or recombinant human Wnt11 (10 ng·mL−1) for 6 h (each group n=18). Da) Binding activity of NF-κB (p65) in PBMC and (Db–Dg) plasma cytokines levels in SARS-CoV-2 ARDS patients PBMCs treated by anti-Wnt5a antibody- or recombinant human Wnt11. Data are reported as mean±sem with significance set at p<0.05; ***p<0.001 versus Normal or Survival; ###p<0.001 versus SARS-CoV-2. p<0.05 ***p<0.001 versus Normal; *p<0.05 versus SARS-CoV-2; ##p<0.01 versus SARS-CoV-2 ARDS; **p<0.01 versus PBS treat.
To assess the regulation of Wnt5a and Wnt11 expression by SARS-CoV-2, peripheral blood mononuclear cells (PBMCs) were isolated from SARS-CoV-2, SARS-CoV-2 ARDS, and discharged patients. Based on transcriptional analysis by real-time qPCR (RT-qPCR), it was observed that the secretion in each plasma sample is associated with differential WNT5a and WNT11 mRNA expression level in the PBMCs (fig. 1Be and Bf). Likewise, Wnt5 level was significantly higher and Wnt11 level was significantly lower in the SARS-CoV-2 ARDS patients. Similar restoration in the Wnt5 level and high level of Wnt11 were observed in discharged patients (fig. 1Be and Bf). These results suggest that increased level of Wnt5a is associated with the severity of the disease in SARS-CoV-2 ARDS patients, while low level of Wnt11 is related with insufficient capability to suppress and alleviate the inflammatory cytokine-induced SARS-CoV-2. To further confirm our findings and expand to clinical significance, PBMCs isolated from normal, SARS-CoV-2, SARS-CoV-2 ARDS, and discharged patients were cultured for immunocytochemical analysis. The patient PBMCs were immunostained with specific antibodies and a high level of Wnt5a expression was observed from the SARS-CoV-2 ARDS patients, while less was detectable for Wnt11 (fig. 1Ca). Indeed, PBMCs isolated from SARS-CoV-2 ARDS showed increase in Wnt5a protein secretion than other groups (fig. 1Cb). Conversely, Wnt11 protein secretion was remained at minimum level in PBMCs isolated from SARS-CoV-2 ARDS, and dramatic increase in Wnt11 protein secretion was observed in PBMCs isolated from discharged individuals (fig. 1Cc). The effects of Wnt5a and Wnt11 on anti-inflammatory responses were further investigated. Patient PBMCs were treated with anti-Wnt5a neutralising antibody or recombinant Wnt11 (rWnt11). NF-κB activation analysis demonstrated that the treatment with anti-Wnt5a antibody does not significantly reduce the anti-inflammatory response in the PBMCs of SARS-CoV-2 ARDS (fig. 1Da). Moreover, secretion of various cytokines, including IL-6, IL-1β, IL-4, IFN-γ and TNF-α, was not significantly altered upon anti-Wnt5a antibody treatment (fig. 1Db–Dg). However, treatment of rWnt11 to the PBMCs of SARS-CoV-2 ARDS showed dramatic inhibitory effect on NF-κB activation as well as on cytokine production (fig. 1Db–Dg). These results suggest that Wnt11 protein has great efficacy in reducing inflammatory responses caused by SARS-CoV-2 infection, but Wnt5a is unlikely to be the potential therapeutic target. Previously, Wnt5a expression was up-regulated by TGF-β, which induce pulmonary fibrosis [9], and recent study has reported a significant increase of TGF-β in COVID-19 patent sera [16]. Thus, it is possible that elevation of Wnt5a in patient sera with severe COVID-19 may be due to TGF-mediated lung injury progression, whereby Wnt5a inhibition may not be effective in recovering of inflammatory responses.
In summary, we investigated a single-centre and observational study in South Korea to identify biomarkers that could be used to monitor the progression and severity of the disease in the SARS-CoV-2 patients. By analysing plasma and PBMCs from patients with different pathological severities, our findings reveal that Wnt5a and Wnt11 show opposite expression pattern in SARS-CoV-2 ARDS patients. Based on our results, the measurement of Wnt5a level in SARS-CoV-2 ARDS patients may be a good indicator for poor prognosis, whereas Wnt11 levels may be a good indicator for ability to survive the disease. Given that Wnt11, not Wnt5a, efficiently inhibits inflammatory responses and cytokines production, it could be exploited as a therapeutic target for the treatment of SARS-CoV-2 ARDS patients.
Acknowledgements
We thank the patients who participated in this study.
Footnotes
Author contributions: E.Y.C., H.H.P., W.K., J.-S.B, and W.L. designed and directed the study. H.K., H.N.K, and W.L. carried out ELISA, western blot, immunocytochemistry and cytokine assays. E.Y.C. collected blood samples from patients. E.Y.C., J.-S.B., I.Y.K, S.Y.J and W.L. directed the data analysis. H.H.P., W.K., and W.L. wrote the manuscript. All authors reviewed the manuscript and consented to the description of author contribution.
Support statement: This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07050422 and NRF-2018R1C1B6002749). This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020R1A2C1004131). This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI15C0001). National Research Foundation; DOI: http://dx.doi.org/10.13039/501100001321; Grant: NRF-2018R1C1B6002749, NRF-2018R1D1A1B07050422, NRF-2020R1A2C1004131; Korea Health Industry Development Institute; DOI: http://dx.doi.org/10.13039/501100003710; Grant: HI15C0001.
Conflict of interest: Dr. Choi has nothing to disclose.
Conflict of interest: Dr. Park has nothing to disclose.
Conflict of interest: Dr. Kim has nothing to disclose.
Conflict of interest: Dr. Kim has nothing to disclose.
Conflict of interest: Dr. Kim has nothing to disclose.
Conflict of interest: Dr. Jeon has nothing to disclose.
Conflict of interest: Dr. Kim has nothing to disclose.
Conflict of interest: Dr. Bae has nothing to disclose.
Conflict of interest: Dr. Lee has nothing to disclose.
- Received May 2, 2020.
- Accepted August 21, 2020.
- Copyright ©ERS 2020
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