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. 2022 Feb 23;10(1):e0167121.
doi: 10.1128/spectrum.01671-21. Epub 2022 Feb 16.

Elevated Cytokine Levels in Plasma of Patients with SARS-CoV-2 Do Not Contribute to Pulmonary Microvascular Endothelial Permeability

Anita Kovacs-Kasa #  1 , Abdelrahman A Zaied #  1 , Silvia Leanhart  1 , Murat Koseoglu  1 , Supriya Sridhar  1 , Rudolf Lucas  1   2   3 , David J Fulton  1 , Jose A Vazquez  4 , Brian H Annex  1   3
Affiliations

Elevated Cytokine Levels in Plasma of Patients with SARS-CoV-2 Do Not Contribute to Pulmonary Microvascular Endothelial Permeability

Anita Kovacs-Kasa et al. Microbiol Spectr. .

Abstract

The vascular endothelial injury occurs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, but the mechanisms are poorly understood. We sought to determine the frequency and type of cytokine elevations and their relationship to endothelial injury induced by plasma from patients with SARS-CoV-2 versus controls. Plasma from eight consecutively enrolled patients hospitalized with acute SARS-CoV-2 infection was compared to controls. Endothelial cell (EC) barrier integrity was evaluated using ECIS (electric cell-substrate impedance sensing) on human lung microvascular EC. Plasma from all SARS-CoV-2 but none from controls decreased transendothelial resistance to a greater degree than that produced by tumor necrosis factor-alpha (TNF-α), the positive control for the assay. Thrombin, angiopoietin 2 (Ang2), and vascular endothelial growth factor (VEGF), complement factor C3a and C5a, and spike protein increased endothelial permeability, but to a lesser extent and a shorter duration when compared to SARS-CoV-2 plasma. Analysis of Ang2, VEGF, and 15 cytokines measured in plasma revealed striking patient-to-patient variability within the SARS-CoV-2 patients. Pretreatment with thrombin inhibitors, single, or combinations of neutralizing antibodies against cytokines, Ca3 and C5a receptor antagonists, or with ACE2 antibody failed to lessen the SARS-CoV-2 plasma-induced EC permeability. The EC barrier destructive effects of plasma from patients with SARS-CoV-2 were susceptible to heat inactivation. Plasma from patients hospitalized with acute SARS-CoV-2 infection uniformly disrupts lung microvascular integrity. No predicted single, or set of, cytokine(s) accounted for the enhanced vascular permeability, although the factor(s) were heat-labile. A still unidentified but potent circulating factor(s) appears to cause the EC disruption in SARS-CoV-2 infected patients. IMPORTANCE Lung vascular endothelial injury in SARS-CoV-2 patients is one of the most important causes of morbidity and mortality and has been linked to more severe complications including acute respiratory distress syndrome (ARDS) and subsequent death due to multiorgan failure. We have demonstrated that in eight consecutive patients with SARS-CoV-2, who were not selected for evidence of endothelial injury, the diluted plasma-induced intense lung microvascular damage, in vitro. Known endothelial barrier-disruptive agents and proposed mediators of increased endothelial permeability in SARS-CoV-2, induced changes in permeability that were smaller in magnitude and shorter in duration than plasma from patients with SARS-CoV-2. The effect on endothelial cell permeability of plasma from patients with SARS-CoV-2 was heat-labile. The main plasma factor that causes the increased endothelial permeability remains to be identified. Our study provides a possible approach for future studies to understand the underlying mechanisms leading to vascular injury in SARS-CoV-2 infections.

Keywords: ACE-2 receptor; SARS-CoV-2; SARS-CoV-2 plasma; antiinflammatory cytokines; barrier dysfunction; complements factors; cytokine; endothelial injury; endothelial permeability; heat inactivation; neutralizing antibodies; plasma; proinflammatory cytokines; spike protein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
SARS-CoV-2 plasma induces endothelial barrier disruption compared to control plasma. HLMVECs (human lung microvascular endothelial cells) were plated in an ECIS array. Transendothelial electrical resistance (TER) was measured in monolayers over time. The baseline was set, and cells were challenged with either normal or SARS-CoV-2 001–004 (A) patient plasma, or TNF-α (1 ng/mL) at the 0 time point, and TER was monitored for ∼20 h. Bar graphs represent resistance values for normal plasma, TNF-α, or SARS-CoV-2 plasma 001–008 at 2 h after plasma addition. (C) Commercially available pooled plasma or control patient plasma from our biobank was added to the lung EC monolayers, and TER was monitored for 15 h. Normal pooled/control plasma did not induce endothelial permeability increase. Results are expressed as mean ± SEM. Normal plasma and TNF-α are normalized averages from 8 separate experiments. SARS-CoV-2 plasma analyses were done at least 3 times, n = 4. The statistical significance was assessed by one‐way ANOVA followed by Bonferroni's multiple comparisons post hoc test using Graph Pad Prism. ****, p < 0.0001 normal plasma versus TNF-α and SARS-CoV-2 001–008; ####, p < 0.0001 TNF-α versus normal plasma and SARS-CoV-2 001–008; ###, P = 0.0001.
FIG 2
FIG 2
Thrombin inhibitors block thrombin but not SARS-CoV-2 plasma-induced EC permeability increase. HLMVECs (human lung microvascular endothelial cells) were plated in ECIS arrays. Transendothelial electrical resistance (TER) was measured in monolayers over time. The baseline was set, and dose-response analysis was performed with Thrombin. (A) Doses were ranging from 10 nM to 100 nM monitored for 15 h (the first 4 h are shown for better visibility). (B) Thrombin inhibitors, argatroban (1 uM), or antithrombin 3 (AT3, 25 nM) were used to treat the EC monolayers in the presence of SARS-CoV-2 005 patient plasma. (C) Direct thrombin inhibitors (argatroban, 1 uM) or antithrombin 3 (AT3, 25 nM) were used to block thrombin effect on EC permeability. At least 4 independent experiments, n = 4.
FIG 3
FIG 3
VEGF and Ang2 levels in patient plasma and their effect on EC permeability. (A) VEGF levels in 7 SARS-CoV-2 plasma (001–005, 007, 008) and in normal pooled plasma were determined by the Quantikine Human VEGF Immunoassay. Optical density (OD) values were measured at 450 nm. TER was measured in confluent HLMVEC after the baseline was set. Cells were treated either with normal pooled plasma, withSARS-CoV-2 001 plasma, or with 20, 50, or 100 ng/mL of VEGF. Changes in TER were monitored for 18 h. (B) Ang2 levels in 7 SARS-CoV-2 plasma (001–005, 007, 008) and in normal pooled plasma were analyzed by the Quantikine Human Ang2 Immunoassay. OD values were measured at 450 nm. TER was measured on HLMVEC after baseline was set. Cells were treated either with normal pooled plasma, with SARS-CoV-2 001 plasma, or with 1, 20, 50, or 100 ng/mL of Ang2. The dashed line on the left panels of the figure shows the level of the VEGF or Ang2 levels in the normal plasma samples. Changes in TER were monitored for 18 h.
FIG 4
FIG 4
Cytokine profiling of SARS-CoV-2 patient plasma. Inflammatory cytokine levels were analyzed in 8 SARS-CoV-2 patients’ plasma samples in duplicate; 25 μL plasma was analyzed in the assay, using the Milliplex High Sensitivity Human Cytokine Panel (MILLIPLEX MAP– Premixed 15 Plex, EMD Millipore). (A) TNF-α, (B) IFNγ, (C) IL-10, (D) IL-6, (E) IL-17A, (F) GM-CSF, (G) IL-21, (H) IL-12p70, (I) IL-8, (J) MIP1α, (K) IL-1β, (L) IL-13, (M) IL-7, (N) IL-4, (O) IL-2. Commercially available normal pooled plasma was used as the normal plasma group control, shown by mean and SEM. The dashed line shows the level of each cytokine in the normal plasma samples. n = 3.
FIG 5
FIG 5
Blocking antibodies to elevated cytokines fail to prevent SARS-CoV-2 plasma mediated increases in EC permeability. Permeability profiling on HLMVEC pretreated for 2 h either with normal plasma, TNF-α (1 ng/mL) blocker, soluble TNF-receptor type I, IL-6 neutralizing antibody (nAb) (5 ng/mL), IL-10 nAb (25 ng/mL), IL-17 nAb (5 ng/mL), or IFNγ nAb (1 ng/mL), then cells were exposed to SARS-CoV-2 plasma (1:200 dilution in normal growth media). Arrows indicate the time point when blocking antibodies and SARS-CoV-2 plasma were added to the cells. TER was monitored for 18 h. Commercially available pooled normal human plasma was used as the normal plasma group control. Results are shown from 3 independent experiments, n = 3.
FIG 6
FIG 6
Multiple blocking antibody combinations fail to reduce endothelial injury after exposure to SARS-CoV-2 plasma. HLMVECs monolayers were incubated with combinations of the indicated blocking antibodies for 2 h prior to exposure to SARS-CoV-2 plasma, and TER was monitored for 18 h. For each cytokine target, patient plasma with the highest fold increase is shown: for IFNγ SARS-CoV-2 patient 006, for IL-10 SARS-CoV-2 patient 001, for IL-6 SARS-CoV-2 patient 002, for TNF-α SARS-CoV-2 patient 005, and for IL-17 SARS-CoV-2 patient 008. For each of this selected patient plasma, the three other indicated cytokines with the highest increase were selected as the second target. Arrows indicate the treatment time points. Bar graphs represent the selected time points for statistical analysis to compare each treatment group at 2 h after SARS-CoV-2 plasma treatment. Results are expressed as mean ± SEM of 3 independent experiments, n = 5. ****, P < 0.0001 normal plasma versus SARS-CoV-2 plasma and SARS-CoV-2 plasma/cytokine combinations. Ns, not significant between groups. The statistical significance was assessed by one‐way ANOVA followed by Tukey’s multiple comparisons post hoc test using Graph Pad Prism.
FIG 7
FIG 7
Destruction of the EC barrier by SARS-CoV-2 plasma does not involve ACE2. (A) Barrier function in HLMVEC monolayers was monitored using TER. Cells were exposed to an ACE2 blocking antibody for 2 h after which SARS-CoV-2 plasma from patients 001 and 002 were added (1:200 dilution, in normal growth media) and TER monitored for 18 h. Bar graphs represent the TER values for statistical analysis to compare each treatment group 2 h after SARS-CoV-2 plasma treatment. A one-way ANOVA test was performed to compare three groups, followed by Dunnett’s multiple-comparison test. ****, P < 0.0001 normal plasma versus SARS-CoV-2 001/ACE2 nAb, versus SARS-CoV-2 001, versus SARS-CoV-2 002. Ns, not significant SARS-CoV-2 001 versus SARS-CoV-2 001/ACE2 nAb and SARS-CoV-2 002 versus SARS-CoV-2 002/ACE2 nAb. (B) Endothelial monolayers were treated with 5 μg/mL recombinant SARS-CoV-2 spike protein, which increased permeability. ACE2 neutralizing antibody (10 μg/mL) blocked the effect of spike protein on EC permeability. Bar graphs represent the TER values for statistical analysis to compare each treatment group 2 h after SARS-CoV-2 plasma treatment. A one-way ANOVA test was performed to compare three groups, followed by Dunnett’s multiple-comparison test. ****, P < 0.0001 control versus spike protein and spike protein versus spike protein/ACE2 nAb. Data represent mean ± SEM, normal plasma. Results are shown from 3 independent experiments, n = 4.
FIG 8
FIG 8
Complement factors C3a and C5a are not responsible for SARS-CoV-2 patient plasma-induced permeability increase. HLMVEC monolayers on ECIS plates were treated either with plasma from the normal patient or plasma from SARS-CoV-2 patients 005 or 008, C3a (A) or C5a (B) with or without the C3a or C5a receptor antagonist (C3aRA, SB290157 100 nM or C5aRA W54011, 100 nM). TER was measured continuously for 20 h. Bar graphs represent the selected time points for statistical analysis to compare each treatment group at 2 h after SARS-CoV-2 plasma treatment. Results are expressed as mean ± SEM of 3 independent experiments, n = 3. ****, P < 0.0001 versus normal plasma; ***, P < 0.001 versus normal plasma; **, P < 0.01 C3a versus C3aRA; *, P < 0.01 C5a versus C5aRA; ns, SARS-CoV-2 versus SARS-CoV-2 C3aRA or C5Ara. The statistical significance was assessed by one‐way ANOVA followed by Tukey’s multiple comparisons post hoc test using Graph Pad Prism.
FIG 9
FIG 9
Heat inactivation of SARS-CoV-2 plasma abolishes its barrier disruptive effect. Plasma from SARS-CoV-2 patients (001–006) was heat-inactivated at 56°C for 15 min and applied to HLMVEC, and TER was measured for 18 h. Arrow indicates the time point when the plasma was added to the cells. Results are shown from 3 independent experiments, n = 4.
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