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

1 Division of Infectious Diseases and Geographic Medicine, Dept. of Medicine, Stanford University School of Medicine, Beckman Center, 279 Campus Drive, Stanford, CA 94305 2 Department of Materials Science and Engineering, Stanford University, 476 Lomita Mall, Stanford, CA 94305 3 Department of Chemical Engineering, Stanford University, 443 Via Ortega, Stanford, CA 94305 4 Center for Excellence in Pulmonary Biology, Department of Pediatrics, Stanford University, 770 Welch Road, Stanford, CA 94305 5 Department of Pulmonology and Critical Care Medicine, Stanford University School of Medicine, Lane 235, 300 Pasteur Drive, Stanford, CA 94305 6 Department of Pathology, Stanford University School of Medicine, Lane 235, 300 Pasteur Drive, Stanford, CA 94305 7 Department of Emergency Medicine, Stanford University School of Medicine, Always Building, MC5119, 300 Pasteur Drive, Stanford, CA 94305

can result in acute respiratory distress syndrome (ARDS)(1), a condition marked by viscous respiratory secretions and respiratory distress (2). The compositional and rheological properties of these respiratory secretions impair their mucociliary clearance, resulting in a build-up of fluids in the lungs during ARDS (3). This greatly inhibits oxygen exchange, often necessitating endotracheal intubation and mechanical ventilation (4). Treatments that target these respiratory secretions are desperately needed to improve clinical outcomes for COVID-19 patients as well as for other patients suffering from severe cases of ARDS. It is therefore important to understand the composition of these secretions to better guide treatment development efforts.
Levels of hyaluronan (HA), a linear glycosaminoglycan, are elevated in respiratory secretions in other forms of respiratory inflammation (5)(6)(7)(8)(9) including ARDS (10,11). HA is produced at the cell surface by a variety of cell types (12) in response to viral DNA and other factors (13). HA is present in the body at molecular weights ranging from low kilodaltons to megadaltons (12,14) and is known to have major effects on the viscoelasticity of respiratory secretions and other materials (15,16). Additionally, HA plays important roles in innate immunity and antigenic responses in the lungs (17)(18)(19)(20).
DNA levels are also elevated in some forms of respiratory inflammation (21,22). This increase likely originates from dead cells, infiltrating neutrophils (23,24), and potentially microbial contaminants(25, 26). Relatively small increases in DNA concentrations can dramatically change the rheological properties of a solution, a phenomenon that has been leveraged both naturally in the production of bacterial biofilms (27) and synthetically in the development of DNA-based hydrogels (28). In the context of lung infections, extracellular DNA has been suggested to increase viscosity of mucosal fluid and provide colonization opportunities for bacterial infections (21).
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Solids and proteins are increased in COVID-19 respiratory secretions
We collected respiratory secretions from ventilated COVID-19 patients, ranging from 5 to 70 years of age (Table 1) via suction catheter, with only a single sample from each individual included in the dataset. Respiratory secretion samples were collected from patients with CF via spontaneous expectoration and from healthy volunteers via sputum induction.
We observed that samples from healthy patients were typically clear and colorless, whereas samples from patients with COVID-19 were nearly always colored and opaque, similar to samples from patients with CF ( Figure 1A), This suggested that the samples contain appreciable amounts of biopolymers and non-soluble debris.
The percent solids content of respiratory secretions, an index of hydration, impacts the difficulty with which respiratory secretions can be cleared and correlates with clinical outcomes in CF and other settings (32)(33)(34). We found that COVID-19 samples had significantly higher percent solids than healthy samples ( Figure 1B). We further observed that protein concentrations in COVID-19 samples were nearly 5.5 times greater than those seen in healthy samples ( Figure 1C, p = 0.003). COVID-19 and CF samples did not show statistically significant differences (p = 0.983). These data are consistent with infected and inflamed lungs being known to have protein deposits from increased mucin production (35), bacterial colonization (36), and infiltrating cells(24). However, there were no apparent changes in the density of the respiratory secretions ( Figure 1D), suggesting that the contribution to density of the suspended and dissolved solids was not different enough to deviate from the density of healthy aspirate samples. Additionally, the pH of all samples was observed to be between 7-8.
Of note, large variances in solids and protein were observed in the COVID-19 patient samples ( Figure 1B-C). Even though all the patient samples were collected from intubated patients with severe COVID-19 relatively early during mechanical ventilation, this variance may All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 19, 2021. ; https://doi.org/10.1101/2020.09.11.20191692 doi: medRxiv preprint reflect the differences in the individual response to the infection and the disease progression at the time of collection.

HA is increased in COVID-19 respiratory secretions and lung sections
We next evaluated HA content in the respiratory secretions. We observed a statistically significant, 10-fold increase in HA concentration in COVID-19 patient samples compared to samples from healthy subjects (Figure 2A, p = <0.0001). The average concentration of HA found in samples from COVID-19 subjects was comparable to that observed in samples from CF subjects (p = 0.333), a disease state associated with greatly increased sputum HA(37).
Similar to our findings with percent solids and protein, we observed larger variance in the amounts of HA in COVID-19 and CF patient samples than compared to samples from healthy donors.
Given that the molecular weight of HA is known to influence both the immunogenic as well as the rheological properties of the resulting solution(38-40), we measured the molecular weight of the HA found in the different samples ( Figure 2B; Supplemental Figure 1). We found that while all samples of respiratory secretions had HA of molecular weight less than 500 kDa, HA size in samples from patients with COVID-19 donors skewed smaller than that seen in samples from donors with CF and healthy controls. Given that low molecular weight HA polymers promote inflammation in some systems(38, 39) this is consistent with the highly inflammatory nature of respiratory disease in COVID-19 infection.

HA is increased in COVID-19 cadaveric lung sections
We next examined cadaveric lung sections from patients with COVID-19, patients with CF, and patients with healthy lungs (i.e., without a diagnosed pulmonary disease) for HA deposits by staining with HA binding protein (HABP). We observed very little HABP staining in sections treated with hyaluronidase (HAdase) ( Figure 2C-E), suggesting very low non-specific All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 19, 2021. ; https://doi.org/10.1101/2020.09.11.20191692 doi: medRxiv preprint background staining. However, we observed a substantial increase in HA staining in lung sections from both COVID-19 and CF donors compared to healthy samples when no prior HAdase treatment was used ( Figure 2F-H). Higher magnification images demonstrated the accumulation of HA within alveolar spaces ( Figure 2I-K). These data, together with the aforementioned respiratory secretion studies, indicated that the respiratory secretions of patients with severe COVID-19 have elevated levels of HA.

DNA is increased in COVID-19 respiratory secretions
We next examined the double-stranded DNA (dsDNA) content in these respiratory secretion samples. We observed that the respiratory secretions collected from patients with COVID-19 had a statistically significant increase in dsDNA content compared to healthy subjects ( Figure 3A

High modulus secretions are more susceptible to enzymatic treatment
We evaluated the rheological properties of COVID-19 respiratory secretions and the contribution of HA and dsDNA to the physical flow properties of the secretions. These flow properties are of crucial importance for the removal of the secretions from the lungs. Dynamic light scattering microrheology, a non-invasive rheology technique, was used to evaluate the rheological properties of the sample due to the small sample volume required and the ability of the technique to not alter the sample properties during measurement (41,42). The samples were All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 19, 2021. ; https://doi.org/10.1101/2020.09.11.20191692 doi: medRxiv preprint measured both before and following enzymatic treatment (microrheology protocol further described in Methods and Supplemental Figures 3-4). Specifically, we examined the impact of enzymatic treatment with HAdase (to degrade HA) or deoxyribonuclease(43) (DNase; to degrade dsDNA) on the flow properties of respiratory secretions, as we hypothesized that enzymatic degradation of these biopolymers would lower the modulus (i.e. the resistance to flow). As a non-enzymatic treatment control, samples were diluted with an equivalent volume of saline. When compared to dilution, the average modulus relative to pre-treatment was lower following enzymatic treatment with either HAdase or DNase, although the comparison was not statistically significant (Supplemental Figure 4). However, we suspected the lack of significance was largely impacted by the wide variance of pre-treatment moduli of the secretions.
To account for this large variance in pre-treatment samples, we then evaluated the impact of enzymatic treatment as a function of the measured pre-treatment modulus of the respiratory secretions ( Figure 4). As expected, samples that had a higher pre-treatment modulus (i.e. thicker samples that were more resistant to flow) had a larger response to enzymatic treatment by either DNase or HAdase compared to a control saline dilution. We found a statistically significant linear relationship between the pre-treatment modulus of the secretions and the difference between the change of modulus with dilution and change of modulus with enzymatic treatment (ΔG Saline -Δ G Enzyme ). If the enzyme had no effect compared to the dilution control, then (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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Discussion
Here, COVID-19 respiratory secretions were observed to be similar to the notoriously thick and tenacious sputum produced by patients with CF. COVID-19 respiratory secretions have significantly elevated levels of solids, with HA and DNA contributing to the elevated viscosity. Low-molecular weight HA is greatly increased in the respiratory secretion samples from intubated patients with COVID-19. Consistent with this, HA is abundant in histologic sections from cadaveric lung tissue obtained from an individual with COVID-19-associated ARDS. Together these data support the hypothesis that low-molecular weight HA is elevated in the respiratory secretions of patients with COVID-19-associated ARDS and may be a contributing factor in the inflammatory state in the lungs. Respiratory secretions with higher modulus are expected to be more challenging to clear from the airway and hence hinder oxygen exchange in the lungs (4,26,45). Thus, thinning of the fluid to improve lung clearance is a common goal across a range of diseases with respiratory inflammation (46)(47)(48)(49). As we observed in our study, treatment of respiratory secretions with an enzyme to digest the biopolymers (and hence decrease the polymer entanglements) will decrease the flow resistance of thick samples with an initial high modulus. The impact of the DNase is established clinically, as it has been used in treating CF lung disease(50, 51); All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 19, 2021. ; https://doi.org/10.1101/2020.09.11.20191692 doi: medRxiv preprint however, the use of HAdase for improving the flow properties of respiratory secretions is a relatively new approach and requires further investigation. More research is needed to identify ideal treatment conditions such as dosages and dosage regimens. Further, targeting the production of the HA during infection may be more successful than relying on a post-production degradation approach. Treatment with a pharmaceutical HA-inhibitor such as 4methylumbiliferone(52) may be a viable approach to limit the deposition of HA during infection.
These studies have several limitations. Most notable is the small numbers of cases and samples of secretions involved. These findings need to be confirmed in larger, multi-center studies involving individuals with diverse backgrounds and case presentations. The underlying mechanisms that lead to increased HA would also benefit from further research to identify the causative cell types and signalling pathways. In addition, data in SARS-CoV-2 animal models would enable improved understanding of the contribution of HA to pathogenesis in this disease.
Finally, to safely acquire the rheology data, the COVID-19 samples were heat treated to render the samples non-infectious. In control CF samples, this same heat treatment was found to decrease the modulus (Supplemental Figure 4A), presumably due to the denaturation of biopolymers in the sample. Since we observed that higher modulus samples had larger responses to enzymatic treatment, the true effect of enzymatic treatment on COVID-19 lung secretion is likely larger than that reported here using heat-treated samples. Based on the promising data presented here, future studies should further evaluate a range of enzymatic treatment dosages and durations to assess the rheological effects on non-heat-treated COVID-19 lung secretions.
In summary, these data indicate elevated dsDNA and HA levels in COVID-19 respiratory secretions. These studies may have important implications for the development of much needed therapeutics for patients with COVID-19. Developing treatments that render the respiratory secretions of lungs less viscous, and thus easier to clear via natural mucociliary clearance, could be pivotal to improving clinical outcomes in severe COVID-19 and ARDS. All rights reserved. No reuse allowed without permission.
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Methods
Histologic staining of lung tissues for HA: Human lung tissue was obtained from a deidentified autopsy specimen provided through the Stanford Pathology Department in the form of formalin-fixed, paraffin-embedded histologic specimen. Histological staining for HA was performed as described previously (53). In brief, 5-µm thick sections were cut on a Leica RM bilateral infiltrates on chest X-ray). For controls, sputum was collected from asymptomatic adult donors. Healthy control subjects were asymptomatic, aged 24-50 years. Sputum samples from CF patients were collected during routine care. All samples were frozen at -80°C immediately All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Gel electrophoresis to characterize DNA molecular weight: Respiratory secretion samples were mixed with loading solution and separated on a 1% agarose gel with 0.5 µg/mL ethidium bromide at 120 V. Samples were separated on the same gel with a 2-Log DNA Ladder (New England Biolabs). Gels were imaged on a BioRad ChemiDoc MP imaging system.

Microrheology measurements:
Dynamic light scattering (DLS) microrheology data was collected as previously described 41,42 with minor modifications as described below. Due to the presence of naturally occurring particulates within all samples, no additional beads were required to induce light scattering. Light scattering was collected from a Malvern Nano Zetasizer Nano ZS with a 633 nm laser operated in 173º backscatter mode. The raw intensity autocorrelation function of a respiratory secretion sample was measured at a specified measurement position for 30 minutes at 37 o C. Following initial microrheology measurements, the same respiratory secretion sample was then treated with either 1) benzonase nuclease (250 U/mL) for 1 hour at 37ºC, 2) hyaluronidase (50 mg/mL, Sigma Aldrich) for 2 hour at 37ºC, or 3) 1x phosphate buffered saline for 1 hour at 37ºC as a dilution control. After the allotted reaction time, the DLS measured the raw intensity autocorrelation function of the sample at the same settings as before. To safely determine the effect of heat on the rheological behavior of respiratory secretions, we measured the intensity autocorrelation function of CF sputum, which is similar to COVID-19 respiratory secretions in both composition and rheological behavior, before and after the same heat treatment that all COVID-19 respiratory secretion samples were subjected to prior to handling. The heat treatment significantly decreased the resistance to flow (i.e. the elastic modulus) of the CF sputum (Supplemental Figure 4A). All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 19, 2021. ; https://doi.org/10.1101/2020.09.11.20191692 doi: medRxiv preprint using the custom analysis package found at dlsur.readthedocs.io. The size of the particulates was assumed to be 500 nm in diameter for all samples. While this assumption affects the absolute value of the modulus derived from the scattering autocorrelation function, it has the same proportional effect across all samples. Thus, the trends observed in the microrheology data, along with the conclusions drawn from those trends, are unaffected by this assumption. All rheological measurements in this study obtained the complex modulus over a wide range of frequencies (from about 10 1 to 10 6 Hz), but only the modulus value at one frequency was used when comparing the modulus across samples and conditions. This is a common approach when comparing rheological results of lung secretions (21). To determine this frequency, the complex moduli of the pre-and post-treatment were compared. In the spectrum with the higher complex modulus (typically the pre-treatment), a single frequency was determined by selecting either a) the middle of the "plateau" region of the elastic modulus (Supplemental Figure 3A)  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 19, 2021. ; https://doi.org/10.1101/2020.09.11.20191692 doi: medRxiv preprint Statistics: Data are expressed as mean +/-SEM of n independent measurements. Significance of the difference between the means of two or three groups of data was evaluated using a oneway ANOVA followed by Tukey's post hoc test. A p value less than <0.05 was considered statistically significant. The linear model fit used to compare enzyme effects on rheological properties of different samples was evaluated using the Bayesian Information Criterion. The Bayesian Information Criterion for a linear fit and a random model fit were compared, and a Bayesian Information Criterion above 10 was considered to be very strong evidence against the random model. Data Availability: All data generated and analyzed during the current study are available from the corresponding author upon reasonable request.
Study Approval: All secretion samples were obtained under the auspices of research protocols approved the Stanford Institutional Review Board (IRB) (Stanford IRB approval #28205, #53685, #55650, #37232, and #43805). Samples were collected after written informed consent from patients or their surrogates prior to inclusion in the study. All rights reserved. No reuse allowed without permission.
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A B
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Antibiotics at time of collection 6 (43%) 0 For adult patients, values listed as N (%) or Median (IQR). In pediatric patients, values listed as N (%) or given for both patients.
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