Increase in serum levels of phosphatidylserine-specific phospholipase A1 in COVID-19 patients.

The development of novel drugs to overcome the current global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important task. Although antiviral agents have been investigated, one of the important goals of treatment is also to control the biological response to the infection. A series of elegant basic studies have revealed that lysophosphatidylserine (LysoPS) might play important roles in inflammation through three kinds of G protein-coupled receptors [1]; LysoPS reportedly suppresses activation of T cells [2] and secretion of inflammatory cytokines from macrophages [3] and promotes phagocytosis of apoptotic cells, including apoptotic neutrophils, by macrophages [4]. Considering these proposed physiological effects, it appears that LysoPS might have important roles in the resolution of inflammation. Phosphatidylserine-specific phospholipase A1 (PS-PLA1) has been proposed to be involved in the production of LysoPS [5].

In the present study, we investigated the serum levels of PS-PLA1 in 58 healthy adult volunteers and 133 COVID-19 patients, consisting of 127 symptomatic patients and 6 asymptomatic patients. The 127 symptomatic COVID-19 patients were classified into three groups according to disease severity: severity level 1 (mild disease, did not require oxygen therapy), severity level 2 (moderate disease, required oxygen therapy but not mechanical ventilatory support), and severity level 3 (severe disease, required mechanical ventilatory support). The PS-PLA1 levels were determined by a two-site immunoenzymometric assay with the TOSOH AIA system (TOSOH, Tokyo, Japan) [6]. The method is described in detail in the Supplementary Materials and Methods section.

Compared to those in the healthy group, as shown in Fig. 1A, the serum PS-PLA1 levels were consistently and significantly higher during the clinical course, each day after the onset of symptoms, in the COVID-19 patients. From 7 COVID-19 patients, we collected serum samples 2–11 days prior to the onset of symptoms. The time course, shown in Fig. 1B, of the serum PS-PLA1 levels showed that the levels tended to increase on days 6–7 after the onset of symptoms (P = 0.07). The serum PS-PLA1 levels were also significantly higher from days 3–4 to days 11–12 than the levels measured after day 21 from symptom onset, suggesting that the serum PS-PLA1 levels did increase specifically in response to the infection in patients with symptomatic COVID-19. The time course of the serum PS-PLA1 levels in the asymptomatic subjects is shown in Supplementary Fig. 1. Comparison of the PS-PLA1 levels in the serum samples collected on days 1–2 to days 9–10 (n = 16) from the six asymptomatic patients with those in the serum samples from healthy subjects (n = 58) revealed that the serum PS-PLA1 levels were significantly higher in the asymptomatic COVID-19 patients than in the healthy control subjects (P < 0.01).

Fig. 1

Serum PS-PLA1 levels in subjects with COVID-19. The serum PS-PLA1 levels were measured in patients with COVID-19 (severity level 1, n = 41; severity level 2, n = 62; severity level 3, n = 24) and healthy subjects (n = 58). A Time course of serum PS-PLA1 levels in symptomatic COVID-19 patients and distribution of serum PS-PLA1 levels in healthy subjects. The differences in the levels between the healthy subjects and COVID-19 patients were assessed by the Mann–Whitney U test, *P < 0.01 vs. healthy subjects. Differences between the serum PS-PLA1 levels measured on specified days after the onset of COVID-19 symptoms and those measured after day 21 from symptom onset in individual subjects were assessed by the Wilcoxon signed-rank sum test, †P  0.01 vs. level measured after day 21. B Time course of the serum PS-PLA1 levels in the COVID-19 patients for whom samples collected before disease onset (Pre) were available (n = 7). C–F Time course of serum PS-PLA1 levels in patients with mild (C), moderate (D), and severe COVID-19 (E). *P < 0.01; †P < 0.05 vs. level measured after day 21 from symptom onset. F Differences in the serum PS-PLA1 levels on days 7–8, days 9–10, and days 11–12. The differences were assessed using an independent Kruskal–Wallis test, followed by the Games Howell test for post hoc analysis. *P < 0.01; †P < 0.05. The horizontal bars represent the means of independent samples

To date, elevated serum PS-PLA1 levels have been noted only in a limited number of pathological states, including cancers, SLE, and hyperthyroidism [7]. Among these, inflammation akin to that seen in SLE might possibly explain the increase in the serum PS-PLA1 levels in COVID-19 patients since several phenotypes of COVID-19 are characterized by the presence of anti-phospholipid antibody, antinuclear antibody, and systemic endotheliitis at rather high frequencies, with the possible involvement of NETosis [8].

In regard to the association of elevated PS-PLA1 levels with the severity and clinical parameters of COVID-19, in the subjects with mild COVID-19 (severity level 1), the serum PS-PLA1 levels were significantly higher than the levels measured after day 21 from symptom onset (Fig. 1C). In the patients with moderate COVID-19 (severity level 2), the serum PS-PLA1 levels were also higher than those measured after day 21 from symptom onset (Fig. 1D). In contrast, in the patients with severe COVID-19 (severity level 3), no significant elevation of the serum PS-PLA1 levels was observed (Fig. 1E). The PS-PLA1 levels were found to be higher in the patient group with severity level 2 than in the patient group with severity level 1 on days 7–8 and higher than those in the patient group with severity level 3 on days 7–8 and days 11–12 (Fig. 1F). As shown in Supplementary Fig. 2 and Supplementary Table 1, the serum PS-PLA1 levels showed significantly positive correlations with the serum CRP levels but a significantly negative correlation with the serum D-dimer levels on days 13–14. The serum PS-PLA1 levels showed significant negative correlations with the anti-SARS-CoV-2 IgM titers measured on days 11–12 and anti-SARS-CoV-2 IgG levels measured from days 11–12 to days 15–16.

Considering that LysoPS might have important roles in the resolution of inflammation [2-4], together with the result that the serum PS-PLA1 levels were lower in patients with severe COVID-19 than in those with moderate COVID-19 (Fig. 1C–F), we propose the hypothesis that failure of the serum PS-PLA1 levels to increase adequately to suppress an overreactive immune system could result in the development of severe COVID-19 as a result of a cytokine storm. The negative association with the serum D-dimer levels might be consistent with the serum PS-PLA1 levels being lower in patients with severe COVID-19 than in those with moderately severe disease, while the positive correlation with the serum CRP levels might be consistent with the elevated serum PS-PLA1 levels declining faster in patients with mild COVID-19 than in those with moderate COVID-19. PS-PLA1 is expressed in immune cells, such as dendritic cells, T cells, and macrophages, and in various tissues, including the lung and liver [7]. Considering these origins, a possible mechanism of modulation of the serum PS-PLA1 level in a bell-shaped manner depending on the severity of COVID-19 might be impaired upregulation of PS-PLA1 expression in immune cells in severe COVID-19 patients, resulting in inappropriate immune responses, and/or the severely injured lungs and/or liver in severe COVID-19 failing to maintain adequate serum PS-PLA1 levels. Interestingly, the serum PS-PLA1 levels were negatively correlated with the serum anti-SARS-CoV-2 antibody levels. Considering that LysoPS plays important roles in the biology of lymphocytes [2], PS-PLA1 might affect the generation of anti-SARS-CoV-2 antibodies through LysoPS.

In summary, COVID-19 patients showed elevated serum levels of PS-PLA1, an enzyme involved in the synthesis of LysoPS, in a bell-shaped manner depending on the severity of COVID-19. The alteration of the serum PS-PLA1 levels might represent compensatory biological responses directed at suppressing immunological overreaction of the body in COVID-19, which is an important risk factor for mortality from the disease.

Supplemental materials