Apparent Yield Stress of Sputum as a Relevant Biomarker in Cystic Fibrosis

Abstract

The mucus obstructing the airways of Cystic Fibrosis (CF) patients is a yield stress fluid. Linear and non-linear rheological analyses of CF sputa can provide relevant biophysical markers, which could be used for the management of this disease. Sputa were collected from CF patients either without any induction or following an aerosol treatment with the recombinant human DNAse (rhDNAse, Pulmozyme^®). Several sample preparations were considered and multiple measurements were performed in order to assess both the repeatability and the robustness of the rheological measurements. The linear and non-linear rheological properties of all CF sputa were characterized. While no correlation between oscillatory shear linear viscoelastic properties and clinical data was observed, the steady shear flow data showed that the apparent yield stress of sputum from CF patients previously treated with rhDNAse was approximately one decade lower than that of non-treated CF patients. Similar results were obtained with sputa from non-induced CF patients subjected ex vivo to a Pulmozyme^® aerosol treatment. The results demonstrate that the apparent yield stress of patient sputa is a relevant predictive/prognostic biomarker in CF patients and could help in the development of new mucolytic agents.

Keywords: Cystic Fibrosis; airway mucus; biomarker; rheology.

Figure 1

Repeatability and effect of centrifugation: storage modulus (G′) and loss modulus (G″) for 6 samples taken from the same sputum (obtained from patient p15); sample 6 is the pellet obtained after centrifugation at 2000× g, for 10 min.

Figure 2

(A) Graphical comparison of rheological data obtained in oscillatory and steady shear flow of mucus from spontaneous or induced CF expectoration collected for this study. Storage modulus (G′) and loss modulus (G″) data were measured at a strain amplitude of 1% and at an angular frequency of 0.1 rad.s⁻¹ and tanδ = G″/G′. (B) Graphical comparison of apparent yield stress (τ_γ) and Newtonian viscosity (η) of untreated and rhDNAse-treated mucus. Statistical analysis with the non-parametric Mann–Whitney test was performed to compare τ_γ and η values in patients from the two groups; the symbol “*” denotes a p value < 5%.

Figure 3

Flow curves, Newtonian viscosity (η₀) and apparent yield stress (τ_γ) of sputum collected from p18 (collected without rhDNAse induction) and treated or not with either rhDNAse or N-acetyl-cysteine (NAC). The apparent yield stress is defined as the stress at the flow curve near discontinuity.

Figure 4

Schematic representation of CF sputum composed of two distinct networks and bacterial biofilm. Mucin and DNA/actin networks are interconnected. Untreated CF sputum exhibits high yield stress (τ_γ 1). When CF sputum is treated with rhDNAse, the mucin network is preserved but DNA/actin network is broken, leading to the decrease in the apparent yield stress to the same level as that of non-CF sputum (τ_γ 2). On the other hand, when CF sputum is exposed to NAC (N-acetyl-cysteine), some of the disulfide bridges of the mucin network are broken, but the DNA/actin network is largely preserved, resulting in a slightly lower decrease in the yield stress (τ_γ 3).