Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19

Abstract

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease including MIS-C and chilblain-like lesions (CLL), otherwise known as "COVID toes", remains unclear. Studying multinational cohorts, we found that, in CLL, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs post-disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased levels of NETs when compared to other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.

Summary: NET formation and degradation are dysregulated in pediatric and symptomatic adult patients with various complications of COVID-19, in association with disease severity. NET degradation impairments are multifactorial and associated with natural inhibitors of DNase 1, G-actin and anti-DNase1L3 and anti-NET antibodies. Infection with the Omicron variant is associated with decreased levels of NETs when compared to other SARS-CoV-2 strains.

Figure 1.. NETs are identified in MIS-C and CLL.

Levels of citrullinated histone H3 (citH3)- and elastase (Ela)-DNA complexes were quantified in serum or plasma MIS-C and CLL samples obtained from (A, B) Italy (MIS-C n=14, CLL n= 27, ctrl n= 21), (C)USA (CLL n= 5, ctrl n= 5) and (D)Chile (MIS-C n=27, ctrl n= 12) (D). Mann-Whitney and Kruskal-Wallis analysis were performed. (E) Detection of citrullinated histone H4 (citH4, red) and DNA (blue) was performed in skin tissue obtained from 3 CLL patients and one ctrl (F) Levels of citH3-DNA complexes were correlated with the absence or present of heart medication/pressors, pneumonia or shock in Chilean MIS-C patients. Results are the mean +/− SEM. Mann-Whitney analysis were performed *p< 0.05, **p<0.01.Ctrl: controls; OD: optical density; *p<0.05, ** p<0.01, ****p<0.001.

Figure 2.. Impaired NET degradation in MIS-C and CLL samples.

NET degradation capabilities were measured in serum or plasma from MIS-C samples obtained from (A) Italy (MIS-C n=12, control n= 12) and Chile (MIS-C n=27) and CLL obtained from (B) Italy (CLL n= 27, control n= 12) and US (n=5). Results are the mean +/− SEM. Kruskal-Wallis analysis was performed. (C) Pie charts representing the proportion of degrader (white) and non-degrader (black) per cohort. (D) Representative images of PMA-generated NETs incubated with serum or plasma from control or MIS-C, CLL patients. DNA is detected by Sytox green and scale bar is 100um. (E) NET degradation capabilities were measured in serum or plasma of MIS-C (n=7) and CLL (n= 4) samples in the presence or absence of recombinant DNase 1. Samples within dashed circle are those that did not respond to treatment with DNase1. Kruskal-Wallis analysis was performed. (F) Representative images of PMA generated NETs incubated with serum or plasma from MIS-C, CLL patients in the presence or absence of recombinant DNase 1. DNA is detected by Sytox green and scale bar is 100um; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 3.. Multiple factors impair NET degradation in MIS-C and CLL.

(A) Confocal images of immunofluorescence analysis of C1q (red) in PMA-generated NETs after incubation with serum or plasma from MIS-C or CLL patients. DNA is detected in blue and scale bar is 10um. (B) Levels of circulating G-actin measured in MIS-C (n=27) and CLL (n=26) samples. Results are the mean +/− SEM. Kruskal-Wallis analysis was performed *p< 0.05. (C) Confocal images of immunofluorescence analysis of immunoglobulin G (IgG) (red) in PMA generated NETs after incubation with serum or plasma from MIS-C or CLL patients. Scale bar is 10um. (D) Correlation analysis of levels of anti-NET antibodies and degradation capabilities, Pearson analysis was used, n=27.

Figure 4.. NET remnants are present in plasma and serum of COVID-19 patients from Italy.

Plasma levels of (A) citrullinated histone H3 (citH3) and (B) elastase (Ela)- DNA complexes were measured in COVID-19 samples obtained from Brescia, Italy (n= 99, control n= 7), Mann-Whitney was used. (C, D) Serum levels of citrullinated histone H3-DNA complexes were elevated in samples obtained up to 25 days (5d n= 118, 10d n= 117, 15d n= 77, 20d n=57, 25d n= 42) of hospitalization and sample collection. Kruskal-Wallis analysis was used. Levels of citH3-DNA complexes were measured in (E) symptomatic (n= 77) and asymptomatic COVID-19 (n= 12) samples. (F) Levels of citH3-DNA complexes at initial (n=20) and 3 months after diagnosis of infection with SARS-CoV-2. Results are the mean +/− SEM. Kruskal-Wallis analysis was used, *p< 0.05, **p<0.01, *** p<0.001, ****p<0.0001. OD: optical density; ctrl:control; d:days from diagnosis.

Figure 5.. NET remnants are present in plasma and serum of adult COVID-19 patients and correlate with severity and comorbidities.

Serum levels of (A) citrullinated histone H3 (citH3) were elevated in critical patients (mild n= 20, moderate n=18, severe n= 41, critical n=156). Kruskal-Wallis analysis was used. (B) COVID-19 patients in intensive care unit (ICU) displayed elevated levels of citH3-DNA complexes (non-ICU n= 175, ICU n= 73) (C) Patient with pneumonia displayed elevated levels of citH3-DNA complexes (absent n= 30, pneumonia n= 203), Mann-Whitney was used. (D) Plasma levels of elastase-DNA complexes (Ela-DNA) were elevated COVID-19 samples with comorbidities such as cardiovascular disease (CVD) (absent n= 56, CVD n= 27), (E) chronic kidney disease (CKD) (absent n= 62, CKD n= 21), and (F)congestive heart failure (CHF) (absent n= 73, CHF n= 10). Results are the mean +/− SEM. Mann-Whitney was used. *p<0.05, ***p<0.001, ****p<0.0001. OD: optical density.

Figure 6.. G-actin associates to decreased degradation of NETs in adult Italian COVID-19 subjects.

(A) NET degradation capabilities were measured in COVID-19 serum samples obtained from Brescia Italy (COVID-19 n=153, asymptomatic n= 26, control n=12), pie chart depicting the proportion of degrader (white) and non-degrader (black). Results are the mean +/− SEM. Kruskal-Wallis analysis was performed. (B) Representative images of PMA generated NETs incubated with serum from control or COVID-19 patients. DNA is detected by Sytox green and scale bar is 100um. (C) NET degradation capabilities were assessed in twenty patients at initial infection and 3 months (3 mo) after. Pie charts depicting the proportion of degrader (white) and non-degrader (black) at initial and 3 months after infection with SARS-CoV-2. Kruskal-Wallis analysis was performed. (D) Confocal images of immunofluorescence analysis of C1q deposition (red) in PMA-generated NETs after incubation with serum from symptomatic and asymptomatic COVID-19 patients. DNA is detected in blue and scale bar is 10um. Pearson correlation analysis of (E) levels of anti-NET, (F) anti-DNase1, (G) anti-DNase1L3 Abs and (H) G-actin measured in serum from COVID-19 patients with NET degradation capabilities (DNA). (I) Levels of serum G-actin in COVID-19 patients (n=20) at initial and 3 months after diagnosis of infection with SARS-CoV-2. Kruskal-Wallis analysis was performed. (J) Pearson correlation analysis of G-actin measured in serum from COVID-19 patients at initial and 3 months after infection with SARS-CoV-2 with NET degradation capabilities (DNA). *p< 0.05, **p<0.01, ****p<0.0001; ctrl:contrl; mo:months; RFU: relative fluorescence units.

Figure 7.. Impairment in NET degradation correlates with comorbidities, while NETs are detected in pulmonary and extrapulmonary tissues in COVID-19.

(A) Decreased serum NET degradation capabilities in severe COVID-19 patients (mild n= 17, moderate n= 8, severe n= 13, critical n=89). Kruskal-Wallis analysis was used. COVID-19 patients with (B) pneumonia (absent n=17, pneumonia n=109), (C) neurologic manifestations (absent n=103, neurologic n=17), and (D) malignancy (absent n=110, malignancy n=17) displayed decreased capabilities of NET degradation. Results are the mean +/− SEM. Mann-Whitney was used. (E) Representative confocal images of citrullinated histone H4 (citH4, red) and DNA (blue) detected in lung, heart, kidney, and spleen tissues obtained from post-mortem COVID-19 patient. (F) Summary of tissue NET detection in each patient (n=13). (G) Pie charts depicting global NET detection per tissue analyzed. Red indicates positive to citH4 (NETs), black indicates no presence of citH4 signal (no NETs); *p<0.05, ***p<0.001.

Figure 8.. NET remnants are lower in adult unvaccinated patients infected with the Omicron variant.

Plasma levels of (A) citrullinated histone H3 (citH3) and (B) elastase-DNA complexes (ela-dsDNA) were measured in COVID-19 patients infected with SARS-CoV-2 Alpha or Omicron variants (ctrl n= 14, Alpha n=14, Omicron= 21). Kruskal-Wallis analysis was used. (C) Male patients infected with the alpha variant of SARS-CoV-2 displayed elevated levels of citH3-DNA complexes (Alpha, n=12, Omicron, n=13). (D) Patients with critical severity of disease have lower levels of NETs when infected with Omicron infected patients displayed decreased levels of plasma citH3-DNA complexes (Alpha n= 14, Omicron n= 16). (E) COVID-19 patients in the intensive care unit (ICU) displayed decreased elevated levels of citH3-DNA complexes when infected with Omicron variant (Alpha, n=11, Omicron n= 3) (F) COVID-19 patients with concomitant hypertension displayed decreased elevated levels of citH3-DNA complexes when infected with Omicron variant (Alpha n= 7, Omicron n= 6), Mann-Whitney was used. Results are the mean +/− SEM. Mann-Whitney was used. **p<0.01, ***p<0.001, ****p<0.0001. OD: optical density