Biochemical, Biophysical, and Immunological Characterization of Respiratory Secretions in Severe SARS-CoV-2 (COVID-19) Infections

Abstract

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We find the percent solids and protein content are greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibit heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observe increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factorâ€"stimulated gene-6 (TSG6) staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicate that increases in HA and DNA in COVID-19 respiratory secretion samples correlate with enhanced inflammatory burden and suggest that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Figure 1.. Respiratory secretions from patients with COVID-19 are high in solids and protein compared to healthy subjects.

(A) Representative images of respiratory secretions collected from ventilated patients with COVID-19 are viscous and tenacious. Similar to CF samples, COVID-19 samples are often colored and opaque, whereas healthy samples are clear and colorless. (B) Quantification of solids found in COVID-19 (n=18), CF (n=4), and healthy (n=15) respiratory secretions. (C) Quantification of protein concentration in COVID-19 (n=16), Cystic Fibrosis (n=4), and healthy (n=15) respiratory secretions. One-way ANOVA with Tukey multiple comparisons tests. *p<0.05, **p<0.01.

Figure 2.. COVID-19 human lung sections have high levels of HA.

(A) Quantification of HA in respiratory secretion samples. COVID-19 (n=8), CF (n=4), Healthy (n=7) respiratory secretion samples. One-way ANOVA with Tukey multiple comparisons tests; **p<0.01, ****p<0.0001. (B) Representative chromatogram of HA molecular weight (MW). Solid traces are the averages of COVID-19, CF, and healthy respiratory secretion samples. The dotted trace is the chromatogram of standard loaded with HA of known MWs, as indicated on graph. The dashed trace is representative of a commercially available 100 kDa MW HA. The bar graph represents the % low MW HA in respiratory secretion samples (Healthy (n=6), COVID-19 (n=8)). Unpaired t test with Welch’s correction; **p<0.005. (C-H) Representative histological cadaveric lung sections from donors with COVID-19, donors with CF, and healthy donors, both with (C-E) and without (F-H) HAdase treatment. Nuclei are stained in blue, and HA binding proteins (HABP) are stained in brown. (I-K) Enlarged sections from panels F-H, respectively. Scale bars C-H 800 μm, I-K 400 μm.

Figure 3.. HA and hyaladherins are increased in blood vessels of COVID-19 lung sections.

Representative histological cadaveric lung sections from donors with COVID-19 ARDS, donors with non-COVID-19 ARDS, donors with CF, and healthy donors stained with (A-D) HABP, (E-H) versican, (I-L) TSG6 (40X magnification). Nuclei are stained in blue, and HABP, versican, or TSG6 are stained in brown. Scale bar A-L 400 μm. Tissues were examined using an Amscope T720Q microscope and images (40X) were acquired using Amscope digital camera (MU1403) and imaging software. (M) % HABP+, (N) % versican+, and (O) % TSG6+ area in lung sections from COVID-19 ARDS (n=5), non-COVID-19 ARDS (n=4), CF (n=3), and healthy donors (n=5). One-way ANOVA with Dunnett’s multiple comparisons tests; **p<0.01, **p<0.001, ****p<0.0001.

Figure 4.. Increased levels of dsDNA in COVID-19 respiratory secretions.

(A) Quantification of dsDNA in respiratory secretion samples. COVID-19 (n=17), CF (n=4), Healthy (n=15) respiratory secretion samples. One-way ANOVA with Tukey multiple comparisons tests. *p<0.05. (B) Representative chromatogram of dsDNA molecular weight. Solid traces are the averages of COVID-19, CF, and healthy respiratory aspirate samples. The dashed line trace is the chromatogram of a DNA standard ladder with the dsDNA base pair-lengths labeled above the respective peaks.

Figure 5.. Enzymatic treatments impact the rheology of COVID-19 respiratory secretions in proportion to their pre-treatment moduli.

(A) The difference in modulus of COVID-19 lung secretions (n=15) upon control saline dilution and enzymatic DNase treatment (ΔG_Saline-ΔG_DNase) versus the initial, pre-treatment modulus (blue). Healthy controls shown for comparison (n=6) (81). (B) The difference in modulus of COVID-19 lung secretions (n=15) upon control saline dilution and enzymatic HAdase treatment (ΔG_Saline-ΔG_HAdase) versus the initial, pre-treatment modulus (green). Healthy controls shown for comparison (n=6) (magenta). The Bayesian Information Criterion (BIC) was used as the statistical metric to compare the impact of enzymatic treatment. (BIC = 35.75 for DNase, and BIC = 36.92 for HAdase).

Figure 6.. Immunological characterization of respiratory secretions from COVID-19 ARDS patients.

(A) Heat map of mean fluorescence intensity (MFI) data, log2 transformed, and normalized to average of the healthy controls per cytokine. Data are ordered by KNN clustering of the cytokines (y-axis). Cytokines, chemokines, adhesion molecules, and growth factors were measured in the respiratory secretion samples of healthy controls (n = 6) and in COVID-19 ARDS patients (n=8) using a bead-based multiplexed immunoassay system, Luminex-EMD Millipore Human 80 Plex assays. Upregulated cytokines are shown in orange and downregulated in blue. (B) Bar graphs of raw MFI values for representative cytokines (IFNa2, PDGFAA, IL13, IL10, IL6, and MIP1B/CCL4) in healthy control and COVID-19 ARDS group (not normalized). Mann Whitney test; *p<0.05, **p<0.005, ****p<0.0005.