Respiratory distress when a lung surfactant loses one of its two hydrophobic tails

Lung homeostasis results from a complex interplay of anatomical and interfacial mechanics in which biomolecular amphiphiles—species with polar and nonpolar segments—play an essential role (1–4). Amphiphiles are surface active molecules (surfactants), which adsorb onto air/water interfaces to form monolayers with their polar parts remaining in contact with water and their hydrophobic parts removed to the air phase. Adsorption lowers the surface tension of the air/water interface. The job of lung (pulmonary) surfactants is to adsorb to the air/water interfaces of the liquid film lining the air sacs of the lung (alveoli) and maintain a low tension so that the sacs can inflate and deflate during respiration. A recent PNAS study by Ciutara et al. (5) examines how inflammation due to infection can change the lung surfactant composition preventing the alveoli from properly expanding and contracting, and leading to acute respiratory distress syndrome (ARDS) (6), a common complication of COVID-19 (7). Surprisingly, the study shows how the change in composition principally affects the compliance (elasticity) of the air/water surface, and it is this alteration, rather than a change in tension-lowering ability, that interferes with alveoli function and can lead to respiratory distress.

In normal respiration (8), air from the trachea travels through bifurcating and narrowing bronchi into the smallest passageways (the bronchioles) which terminate in the alveoli, air sacs enclosed by a continuous cell epithelium (9) surrounded by capillary beds. The inside of the sacs (atrium) is covered by a liquid film (alveolar fluid). When inspiring, the cavity enclosing the lung expands so that the pressure within the lung cavity $p_L$ is less than the atmospheric pressure $p_{\mathit{atm}}$ so that the alveoli can inflate. However, the curvature of the alveoli creates a Laplace overpressure in the alveoli atrium ($p_{\mathit{alv}}$) relative to $p_L$, $p_{\mathit{alv}}-p_L=2\gamma /R$ where $R$ is the radius of the sac and $\gamma$ is the tension of the air/water interface lining the sac. Lung surfactant, secreted by the epithelium cells and released into the alveolar fluid (3), aims to reduce this tension by adsorption so that the $p_{\mathit{alv}}<p_{\mathit{atm}}$ and the alveoli can inflate. During expiration, the lung cavity reduces in volume, increasing the pressure in the cavity and causing air to flow out of the air sac as the sac contracts. In expiration, pulmonary surfactant aims to maintain a low tension to decrease the work required for breathing (10).

Lung surfactant is a proteolipidic mixture of biomolecules in which the most abundant are the phospholipids (PLs), Nature’s familiar design for amphiphilicity, with a glycerol backbone linked to two long nonpolar hydrocarbon acyl chains (tails) and a phosphatidyl polar head group. Of the phospholipids, DPPC (dipalmitoylphosphatidylcholine) is the largest component. Phospholipids have a very low water solubility because of their long acyl chains, and when dispersed in an aqueous phase, are driven by the hydrophobic effect to self-assemble into aggregated structures which screen the hydrocarbon chains from water. As the tails occupy a large molecular volume relative to the polar headgroup, lipids spontaneously form bilayers, contiguous assemblies of lipids in two layers with the hydrocarbon tails in the sheet interior and the polar groups on the outside of which vesicles are the most elementary. The hydrophobic effect also drives adsorption of PLs to an air/water interface, where PLs lower the tension γ —their major task in respiration. As phospholipids in aqueous solution (and in the alveolar fluid in particular) are retained in vesicles (or other bilayer structures), current models envision adsorption proceeding through intermediates which “dock” underneath the surface to form a bridge which unloads lipid onto the surface (3, 11–13). During exhalation, the alveolar air sacs contract, leading to a “squeezing out” of lipid material into lamellar or vesicular structures which retain proximity (or even attachment) to the interface so that on inhalation and expansion of the air sacs, they can re-adsorb and re-spread over the surface (14, 15) to complete the cyclic process.

A recent PNAS study by Ciutara et al. examines how inflammation due to infection can change the lung surfactant composition preventing the alveoli from properly expanding and contracting, and leading to acute respiratory distress syndrome (ARDS), a common complication of COVID-19.

Ciutara et al. (5) focus on the role of the elasticity of the lung surfactant monolayer on respiration. Since the adsorbed lipids are insoluble in water, upon compression (expansion) of the interface area $A$, the tension decreases (increases), creating an elastic effect with surface dilatational elasticity ${\displaystyle E^{\ast }=A\frac{d\gamma }{\mathit{dA}}}$. Ciutara et al. (5) draw attention to the fact that in the lung, alveoli sacs of unequal radius can potentially disproportionate as the smaller sacs have a higher Laplace pressure causing air to flow to the larger sacs. For constant tension, the pressure increases with a decrease in the radius (${\displaystyle \frac{\partial \mathrm{\Delta }P}{\partial R}=-\frac{2\gamma }{R^2}<0}$) and the smaller sacs can potentially deflate (a phenomenon known as the (the Laplace instability). When lung surfactant covers the surface, as the area $A$ decreases the tension decreases, offsetting the Laplace instability. Here, ${\displaystyle \frac{\partial \mathrm{\Delta }P}{\partial R}=2\frac{2E^{\ast }-\gamma }{R^2}}$. If $2E^{\ast }-\gamma >0$ — i.e., when the elasticity is large enough—the pressure in the smaller bubbles does not increase as the radius decreases preventing instability and allowing the pressure in the large and small sacs to balance.

Ciutara et al. (5) note that the inflammatory immune response resulting from infection drives an elevation in phospholipase A₂, a lipase which hydrolyzes double chain phospholipids into single chain lysolipids and a fatty acid. This lipase transports through the capillary/endothelium barrier of the alveoli sacs (whose permeability is also increased by inflammation) into the fluid lining as evidenced by the fact that the bronchial fluid of ARDS patients contains higher than usual levels (see for example Notter et al. (16)). In the alveolar fluid, this lipase hydrolyzes one tail of the phospholipids to form a lysolipid and a fatty acid. Thus for DPPC, the lipase hydrolyzes this lipid to lysopalmitoylphosphatidylcholine (LysoPC). The lysolipids, with one tail, are much more soluble in water than the phospholipids and resemble the commercial one tail surfactants used for example in detergency (17). At a critical concentration these single tail surfactants also form aggregates, but in the form of spheroids (micelles) with the chains (of smaller volume and therefore more easily accommodated in a spherical volume) surrounded by the polar groups. (For LysoPC, the critical micelle concentration (CMC) is ≈6 μM.)

To understand the effect of LysoPC in the alveolar fluid on the interfacial properties of the lung surfactant monolayer, Ciutara et al. (5) examine monolayers of DPPC as the pulmonary mimic. The key experiment (Fig. 1) is the following: DPPC is spread on an aqueous subphase in a Langmuir trough, and the monolayer is compressed by surface barriers to a tension of ≈40 mN/m (measured by a Wilhelmy plate in the monolayer), after which the area is held constant. LysoPC is then injected in the subphase to concentrations (20 and 40 μM) which are above the CMC. The surface tension is observed to decrease over time indicating penetration of the LysoPC into the spread monolayer. While the insertion of a soluble amphiphile from an underlying subphase onto an existing monolayer of an insoluble amphiphile has been observed in many interfacial systems (18), what Ciutara et al. (5) find next is surprising. For both LysoPC concentrations above the CMC, after a tension reduction to a common value of ≈25 mN/m, the interface area is oscillated at low amplitude by movement of the barriers at frequencies corresponding to normal respiration (0.1 Hz). The oscillations in tension and area are measured from which the mean tension and elasticity $E^{\ast }$ are computed. Ciutara et al. (5) find that the mean tension after oscillation increases to the values corresponding to an interfacial layer of only LysoPC adsorbed from a LysoPC subphase at concentrations above the CMC (a tension of ≈ 37 mN/m). More importantly, the elasticity has decreased to 25 mN/m, the value for monolayers of pure LysoPC adsorbed from subphases above the CMC. For DPPC compressed to 25 mN/m, the elasticity is ≈250 mN, hence there is a reduction in $E^{\ast }$ of the monolayer upon oscillation of an order of magnitude. These results indicate that oscillation has driven an exchange of the DPPC in the monolayer for LysoPC. Ciutara et al. (5) only conjecture on the mechanism of this exchange: The oscillation can squeeze out lipid which then can become incorporated in micelles of LysoPC in the subphase or, alternatively, aggregates of LysoPC and lipid can form directly at the interface in a manner similar to how surfactants “solubilize” cell membranes by forming mixed micelles with the lipid. Confocal microscopy sectioning is used to create 3D images near the interface which demonstrate the shedding of resolution-limited fluorescent spots—possibly mixed micelles near the interface.

Fig. 1.

A model for lung surfactant exchange of an insoluble with a soluble amphiphile due to interfacial oscillation: (A) DPPC is spread on the air/water surface of a Langmuir trough and LysoPC is injected underneath at concentrations above the CMC, (B) LysoPC penetrates the DPPC monolayer and (C) barriers are oscillated causing DPPC to be squeezed-out into mixed micelles of LysoPC and DPPC on compression and LysoPC populates the surface on expansion.

The fact that $E^{\ast }$ for LysoPC is much smaller than the modulus for DPPC is a result of its higher solubility which allows LysoPC to exchange quickly with the bulk on expansion and contraction of the interface so that the change in tension is small relative to the insoluble phospholipids. This is expected, but the exposure of the DPPC monolayer to LysoPC in a subphase—combined with oscillation of the interface—has had the effect of reducing the elastic modulus by an order of magnitude by exchanging amphiphiles. The implications for ARDS are clear: the elevation in phospholipase A₂ due to inflammation can elevate the concentration of the soluble amphiphile LysoPC in the alveolar fluid. Upon contraction and expansion of the alveoli during normal respiration, LysoPC replaces the lung surfactant (here DPPC) forming an interfacial layer of markedly lower elasticity which makes the alveoli susceptible to the Laplace instability ($2E^{\ast }-\gamma <0$) and collapse of the smaller air sacs leading to respiratory distress.

Much work still needs to be done: in terms of the mechanism described by Ciutara et al. (5), studies aimed at demonstrating that LysoPC in the subphase can displace monolayers of lung surfactant should study mimics that more closely resemble the composition of human pulmonary lung surfactant (i.e., containing the proteins and the other unsaturated phospholipids). And the mechanism behind the removal of DPPC on oscillation needs to be more carefully investigated. Research indicates that in the normal lung function, lung surfactant components are “squeezed-out” on area compression into aggregates which remain proximal to the interface and then re-adsorb and re-spread on areal expansion. Here the excluded DPPC never returns to the interface, indicating either permanent removal into micelles of LysoPC or simply a rapid adsorption of the more soluble LysoPC or both. And of course, other questions remain, including the role of the endothelium and its compliance (19) and the fact that the alveoli sacs are not independent spheres but share planar surfaces forming polyhedra (20) which may require a rethinking of the Laplace instability. But this careful study will make us all breathe a little easier.