Wnt5a and Wnt11 as acute respiratory distress syndrome biomarkers for SARS-CoV-2 patients

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).

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FIGURE 1

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.