Carboxypeptidase B blocks ex vivo activation of the anaphylatoxin-neutrophil extracellular trap axis in neutrophils from COVID-19 patients

The prospective study included 135 patients with confirmed diagnosis of COVID-19 who admitted to Beijing Ditan Hospital from January 20^th, 2020 to April 27^th, 2020. The following patients were excluded from the present study: 1. Age < 18; 2. Gestation; 3. Patients with any immunodeficiency such as neutrophilia, neutropenia, malignant tumor, using of immunosuppressants for more than 1 week; 4. The time from onset to admission is more than 2 weeks; 5. Dropout patients; 6. Patients or their guardians do not want to be included in the study. According to the inclusion/exclusion criteria, 148 patients were enrolled in the study cohort, and 13 of them quitted before the study was completed. According to the guidelines on the diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 7) [26] released by National Health Commission & National Administration of Traditional Chinese Medicine of China, the classification of COVID-19 are as follows: Mild: Clinical symptoms from mild fever, respiratory tract to pneumonia manifestation. Severe: Meeting any one of the following should be treated as severe cases, including respiratory distress, respiratory rate ≥ 30 breaths/min; oxygen saturation ≤ 93% at rest; and PaO₂/FiO₂ ≤ 300. In severe group, 31 patients were admitted to ICU, 15 cases received mechanical ventilation and 1 of them deceased. In severe group, 11 of 41 patients (26.8%) were first diagnosed as mild/moderate and then crossed over to severe COVID-19. In treating with coagulation disorders, 22 severe patients received enoxaparin sodium, 1 severe patient received low-molecular-weight heparin sodium and 1 severe patient received dabigatran treatment. The other 17 severe patients and all the mild patients did not receive anticoagulant therapy. Twenty-five healthy donors matched to the age and sex of mild COVID-19 patients were enrolled. Three volunteers donated their peripheral neutrophils for in vitro experiments.

The first sample of each patient was collected within 24 h after admission. Then, the blood was taken once a week until discharge from hospital. Blood samples were collected by venipuncture into ethylenediaminetetraacetic acid tubes. Plasma was separated from blood by centrifugation at 450 × g (break off) for 10 min at room temperature. Plasma samples were divided into small aliquots and stored at − 80 °C until the time of testing. The study was approved by Committee of Ethics at Beijing Ditan Hospital, Capital Medical University, Beijing, China. The approval number is JDLKZ(2020)D(036)-01.

Cell-free DNA in plasma was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. MPO-DNA complexes were quantified similarly to what has been previously described [27]. In brief, a capture antibody against MPO was coated on a 96-well flat-bottom plate at 1:200 (Abcam, Cambridge, MA, USA), and the amount of MPO-bound DNA was quantified using the Quant-iT PicoGreen dsDNA assay as described above.

Plasma levels of C5 (including the sum of C5 and C5a) and C3 (including the sum of C3, C3a and C3b) were detected using Human Complement C5 ELISA Kit and Human Complement C3 ELISA Kit (Abcam) according to the manufacturer’s instruction.

NETs were detected by immunofluorescence staining as previously reported [28]. Neutrophils were fixed, permeabilized and blocked after 3-h in vitro culture or stimulation. Cells were incubated with antibody against histone H3 citrulline R2 + R8 + R17 (H3Cit; Abcam) at 1:400 for 1 h at 37℃, followed by secondary antibody coupled with Alexa Fluor Dyes (Invitrogen) at 1:1000 for 1 h at room temperature. DNA was stained using 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, London, UK) at 1:2000 for 8 min at room temperature. Images were obtained with a confocal fluorescence microscope (Zeiss LSM 510 META; Carl Zeiss, Thornwood, NJ, USA). NETs were identified as structures positive for both histone H3Cit and DAPI staining.

Blood samples from healthy donors were collected into ethylenediaminetetraacetic acid tubes as described above for plasma separation. The anticoagulated blood was then fractionated by density-gradient centrifugation using Percoll (Stemcell Technologies, Vancouver, Canada). Neutrophils were further purified by dextran sedimentation of the red blood cell layer before lysing residual red blood cells with sodium chloride. Neutrophil preparations were at least 95% pure as confirmed by nuclear morphology.

Purified neutrophils were resuspended in RPMI-1640 medium supplemented with heat-inactivated 5% fetal bovine serum and 2 mM L-glutamine. Neutrophils were seeded into 96-well plate (5 × 10⁴/well) for supernatant detection and 24-well plate (2 × 10⁵/well) with polylysine-coated coverslips for NET immunofluorescence staining. Cells were rested for 1 h at 37 °C and 5% CO₂ followed by replacement with plasma from HCs or COVID-19 patients at a final concentration of 5% in RPMI-1640 medium for 3 h according to previous report [20]. Recombinant CPB (YaxinBio, Shanghai, China) was used at 100 μg/ml to digest C3a and C5a in the plasma at 37 °C for 30 min prior to neutrophil stimulation. One hour prior to neutrophil stimulation, 100 μg/ml anti-human C3a (Merck, Darmstadt, Germany) or 10 μg/ml anti-human C5a antibody (R&D Systems, Minneapolis, MN, USA) were added into the culture system for neutralizing C3a or C5a in the plasma. Three hours later, the supernatant was collected to quantify MPO-DNA content. The results were calculated by deducting the background levels of MPO-DNA in the plasma.

Neutrophils from healthy donors were cultured with RPMI-1640 medium supplied with 5% plasma from patients with COVID-19 as described above. The peptidylarginine deiminase inhibitor Cl-amidine was added at 200 μM for blocking NET formation. Three hours later, the supernatant was collected carefully and used as NET-conditioned medium. HUVEC cells (3 × 10³/well) were seeded into 96-well plate and cultured for 24 h. Cells culture media were replaced with 100 μl NET-conditioned media or new cell culture media as control for 24 h. Cell viability assay was performed using a cell counting kit 8 (CCK‐8) (Dojindo, Kumamoto, Japan) as per the manufacturer's protocol. Absorbance was detected at 590 nm using a microplate reader. The experiments were performed in sextuplicate.

All statistical analyses were performed with the SPSS 25.0 statistical package (IBM, Armonk, NY, USA). Values are presented as the mean ± standard deviation for data that were normally distributed or median and interquartile range for data that were not normally distributed for continuous variables and number (%) for categorical variables. The Kolmogorov–Smirnov test was used to inspect the normality and homogeneity of variance of all the data. For two-group comparison, P values were derived from the one-way Student t test to determine differences between groups with normally distributed data and Mann–Whitney nonparametric test with other data. For multi-group comparison, P values were derived from one-way ANOVA (continuous variables) or Chi-square test (categorical variables). For all comparisons, P < 0.05 was considered statistically significant.

We recruited 135 confirmed COVID-19 patients admitted to Beijing Ditan Hospital. Among them, 94 (69.6%) were mild cases and 41 (30.4%) were severe cases according to the guidelines on the diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 7) [26] released by National Health Commission & National Administration of Traditional Chinese Medicine of China. Twenty-five age and gender-matched healthy donors (HDs) were enrolled as controls. Demographics and relevant clinical characteristics are reported in Table 1. The study was approved by the Committee of Ethics at Beijing Ditan Hospital, Capital Medical University, Beijing, China.

We collected plasma once a week from hospitalized patients and evaluated the levels of complements and NETs by ELISA. In comparison with HDs, COVID-19 patients had significantly higher levels of NETs in the first week of admission quantified by measuring cell-free DNA (cfDNA) and myeloperoxidase (MPO)-DNA (all P < 0.001; Fig. 1a). Linear regression analysis showed that severe patients had higher levels of NETs than mild cases dynamically within 60 days of hospitalization (Fig. 1b). Both cfDNA and MPO-DNA levels in severe cases showed an upward trend with the disease progression (Fig. 1b).

Previous studies have reported that NETs were associated with a variety of pathological changes such as immune status, coagulation disorder, and multiple organ dysfunction [27]. In COVID-19 patients, NET levels were positively correlated with the neutrophil count, fibrin(-ogen) degradation products (FDP), lactate dehydrogenase, and creatine kinase (Table 2). In addition, greater levels of cfDNA were detected in patients with mechanical ventilation (without vs with: 570.4 vs 897 ng/mL, P = 0.001; Fig. 1c) or those with sequential organ failure assessment (SOFA) scores ≥ 2 (SOFA < 2 vs SOFA ≥ 2: 554.9 vs 897 ng/mL, P = 0.001; Fig. 1d). These results indicated that the NET levels were closely related to the disease severity of COVID-19.

We measured the levels of complement C3 and C5 in the plasma of patients and HDs. Within the first week of admission, both of C3 and C5 were dramatically increased in mild patients with COVID-19 compared with HDs (Median with interquartile range, C3: 0.75 [0.65, 0.79] mg/mL vs 22.61 [14.86, 44.22] mg/mL, P < 0.001; C5: 41.66 [30.19, 47.7] μg/mL vs 274.8 [209.3, 344.7] μg/mL, P < 0.001; Fig. 2a). The levels of C3 and C5 were further raised to 56.23 [31.91, 76.04] mg/mL and 326.1 [245, 388.8] μg/mL in the severe COVID-19 patients (Fig. 2a). Consistent with the longitudinal changes of NETs, linear regression analysis showed that severe patients had higher levels of C3 and C5 than mild cases as the disease progresses (Fig. 2b). There was an upward trend of C3 in patients with disease progression (Fig. 2b). Furthermore, C3 levels were positively correlated with the neutrophil count, FDP, direct bilirubin, and total bilirubin in COVID-19 patients (Table 2).

To determine the regulation of complement anaphylatoxin C3a and C5a on NET formation, we isolated peripheral neutrophils from HCs (Additional file 1: Figure S1, and Additional file 2: Figure S2a), then cultured these cells in the presence of the plasma from severe patients with COVID-19 or the plasma from HDs. The COVID-19 plasma was capable of activating neutrophils to form NETs (Fig. 3a and Additional file 2: Figure S2b) and to release MPO-DNA in the media (Fig. 3b), which were significantly restrained by anti-C3a and anti-C5a neutralizing antibodies. Moreover, we added recombinant CPB, a stable homolog of enzyme CPB2 which is capable of inactivating anaphylatoxin C3a and C5a by specifically degrading their arginine residues [29]. As shown in Fig. 3a, CPB significantly reduced NET release induced by the plasma from COVID-19 patients. Consistently, we treated neutrophils with recombinant C3a and C5a for 3 h and found that both C3a and C5a could significantly promoted NET release (Additional file 3: Figure S3). These data indicated that the over-production of NETs in the plasma of COVID-19 patients at least in part was induced by anaphylatoxin C3a and C5a, and the induction could be blocked by recombinant CPB.

To further investigate the pathological roles of the anaphylatoxin-NET axis, we stimulated neutrophils with COVID-19 plasma to prepare a NET-conditioned medium and then added the conditioned medium to the culture system of vascular endothelial HUVEC cells. Compare with the plasma from HDs, the plasma from mild and severe COVID-19 patients reduced cell viability of HUVEC cells (Fig. 3c). However, the cytotoxicity of COVID-19 plasma was significantly relieved after in vitro digestion of plasma by CPB for 30 min (Fig. 3c). Similarly, the supplement of anti-C3a antibody plus anti-C5a antibody, or the NET inhibitor Cl-amidine to the cultured neutrophils to block NET production, could also reduce the cytotoxicity to HUVEC cells (Fig. 3c). These results suggested that recombinant CPB could protect vascular endothelial cells from damage by reducing C3a- and C5a-induced NET production in COVID-19 patients.