Biochemical, biophysical, and immunological characterization of respiratory secretions in severe SARS-CoV-2 infections

Abstract

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19, 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 found the percentages of solids and protein content were greatly elevated in COVID-19 compared with heathy control samples and closely resembled levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) were major components of respiratory secretions in COVID-19 and were likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors, with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observed increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor-stimulated gene-6 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 indicated that increases in HA and DNA in COVID-19 respiratory secretion samples correlated with enhanced inflammatory burden and suggested that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Keywords: COVID-19; Innate immunity; Pulmonology; Respiration.

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

(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), CF (n = 4), and healthy (n = 15) respiratory secretions. One-way ANOVA with Tukey’s 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), and healthy (n = 7) respiratory secretion samples. One-way ANOVA with Tukey’s 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. (C) The bar graph represents the percentage low–molecular weight HA in respiratory secretion samples: healthy (n = 6) and COVID-19 (n = 8). We chose 250 kDa as the cutoff to define low–molecular weight HA. Unpaired t test with Welch’s correction; **P < 0.005. (D–I) Representative histologic cadaveric lung sections from donors with COVID-19, donors with CF, and healthy donors, both with (D–F) and without (G–I) HAdase treatment. Nuclei are stained in blue, and HA binding proteins (HABPs) are stained in brown. (J–L) Enlarged sections from G–I, respectively. Scale bars: 800 μm (D–I), 400 μm (J–L).

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

Representative histologic 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, and (I–L) TSG-6 (original magnification, 40×). Nuclei are stained in blue, and HABP, versican, or TSG-6 are stained in brown. Scale bar: 400 μm (A–L). Tissues were examined using an Amscope T720Q microscope, and images (original magnification, 40×) were acquired using Amscope digital camera (MU1403) and imaging software. (M) Percentage HABP⁺, (N) versican⁺, and (O) TSG-6⁺ 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), and healthy (n = 15) respiratory secretion samples. One-way ANOVA with Tukey’s 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 bp lengths labeled above the respective peaks.

Figure 5. Enzymatic treatments affect the rheology of COVID-19 respiratory secretions in proportion to their pretreatment moduli.

(A) The difference in modulus of COVID-19 lung secretions (n = 15) upon control saline dilution and enzymatic DNase treatment (ΔG_Saline – G_DNase) versus the initial, pretreatment modulus (blue). Healthy controls shown for comparison (n = 6). (B) The difference in modulus of COVID-19 lung secretions (n = 15) upon control saline dilution and enzymatic HAdase treatment (ΔG_Saline – G_HAdase) versus the initial, pretreatment 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) Heatmap of mean fluorescence intensity (MFI) data, log₂ transformed and normalized to average of the healthy controls per cytokine. Data are ordered by k-nearest neighbors (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 (IFN-α2, PDGFAA, IL-13, IL-10, IL-6, and MIP-1β/CCL4) in healthy control and COVID-19 ARDS groups (not normalized). Mann-Whitney test; *P < 0.05, **P < 0.005, ***P < 0.0005.