Plasma N-Cleaved Galectin-9 Is a Surrogate Marker for Determining the Severity of COVID-19 and Monitoring the Therapeutic Effects of Tocilizumab.

Galectin-9 (Gal-9) is known to contribute to antiviral responses in coronavirus disease 2019 (COVID-19). Increased circulating Gal-9 in COVID-19 is associated with COVID-19 severity. In a while, the linker-peptide of Gal-9 is susceptible to proteolysis that can cause the change or loss of Gal-9 activity. Here, we measured plasma levels of N-cleaved-Gal9, which is Gal9 carbohydrate-recognition domain at the N-terminus (NCRD) with attached truncated linker peptide that differs in length depending on the type of proteases, in COVID-19. We also investigated the time course of plasma N-cleaved-Gal9 levels in severe COVID-19 treated with tocilizumab (TCZ). As a result, we observed an increase in plasma N-cleaved-Gal9 levels in COVID-19 and its higher levels in COVID-19 with pneumonia compared to the mild cases (healthy: 326.1 pg/mL, mild: 698.0 pg/mL, and with pneumonia: 1570 pg/mL). N-cleaved-Gal9 levels were associated with lymphocyte counts, C-reactive protein (CRP), soluble interleukin-2 receptor (sIL-2R), D-dimer, and ferritin levels, and ratio of percutaneous oxygen saturation to fraction of inspiratory oxygen (S/F ratio) in COVID-19 with pneumonia and discriminated different severity groups with high accuracy (area under the curve (AUC): 0.9076). Both N-cleaved-Gal9 and sIL-2R levels were associated with plasma matrix metalloprotease (MMP)-9 levels in COVID-19 with pneumonia. Furthermore, a decrease in N-cleaved-Gal9 levels was associated with a decrease of sIL-2R levels during TCZ treatment. N-cleaved-Gal9 levels showed a moderate accuracy (AUC: 0.8438) for discriminating the period before TCZ from the recovery phase. These data illustrate that plasma N-cleaved-Gal9 is a potential surrogate marker for assessing COVID-19 severity and the therapeutic effects of TCZ.

Both Full-Length and Protease-Cleaved Products of Osteopontin Are Elevated in Infectious Diseases.

Circulating full-length osteopontin (FL-OPN) is elevated in plasma from patients with various infectious diseases, such as adult T-cell leukemia, Mycobacterium tuberculosis (TB), hepatitis virus infection, leptospirosis, acquired immune deficiency syndrome (AIDS), AIDS/TB, and coronavirus disease 2019 (COVID-19). Proteolysis of OPN by thrombin, matrix metalloproteases, caspase 8/3, cathepsin D, plasmin, and enterokinase generates various cleaved OPNs with a variety of bioactivities by binding to different target cells. Moreover, OPN is susceptible to gradual proteolysis. During inflammation, one of the cleaved fragments, N-terminal thrombin-cleaved OPN (trOPN or OPN-Arg168 [OPN-R]), induces dendritic cell (DC) adhesion. Further cleavage by carboxypeptidase B2 or carboxypeptidase N removes Arg168 from OPN-R to OPN-Leu167 (OPN-L). Consequently, OPN-L decreases DC adhesion. In particular, the differences in plasma level over time are observed between FL-OPN and its cleaved OPNs during inflammation. We found that the undefined OPN levels (mixture of FL-OPN and cleaved OPN) were elevated in plasma and reflected the pathology of TB and COVID-19 rather than FL-OPN. These infections are associated with elevated levels of various proteases. Inhibition of the cleavage or the activities of cleaved products may improve the outcome of the therapy. Research on the metabolism of OPN is expected to create new therapies against infectious diseases.

High Levels of the Cleaved Form of Galectin-9 and Osteopontin in the Plasma Are Associated with Inflammatory Markers That Reflect the Severity of COVID-19 Pneumonia.

Numbers of patients with coronavirus disease 2019 (COVID-19) have increased rapidly worldwide. Plasma levels of full-length galectin-9 (FL-Gal9) and osteopontin (FL-OPN) as well as their truncated forms (Tr-Gal9, Ud-OPN, respectively), are representative inflammatory biomarkers. Here, we measured FL-Gal9, FL-OPN, Tr-Gal9, and Ud-OPN in 94 plasma samples obtained from 23 COVID-19-infected patients with mild clinical symptoms (CV), 25 COVID-19 patients associated with pneumonia (CP), and 14 patients with bacterial infection (ID). The four proteins were significantly elevated in the CP group when compared with healthy individuals. ROC analysis between the CV and CP groups showed that C-reactive protein had the highest ability to differentiate, followed by Tr-Gal9 and ferritin. Spearman's correlation analysis showed that Tr-Gal9 and Ud-OPN but not FL-Gal9 and FL-OPN, had a significant association with laboratory markers for lung function, inflammation, coagulopathy, and kidney function in CP patients. CP patients treated with tocilizumab had reduced levels of FL-Gal9, Tr-Gal9, and Ud-OPN. It was suggested that OPN is cleaved by interleukin-6-dependent proteases. These findings suggest that the cleaved forms of OPN and galectin-9 can be used to monitor the severity of pathological inflammation and the therapeutic effects of tocilizumab in CP patients.