A lung-on-chip model reveals an essential role for alveolar epithelial cells in controlling bacterial growth during early tuberculosis

Context and background: Tuberculosis is a major healthcare burden, affecting a large proportion of the world (10 million individuals affected worldwide in 2018 (WHO)). A respiratory infection caused by Mycobacterium tuberculosis (Mtb), it begins with host-pathogen interactions at the air-liquid interface in the lung in early stages. Pulmonary surfactant, a mixture of lipids and proteins secreted by alveolar cells, has been traditionally known for its role in reducing surface tension and contributing to overall lung alveolar function [1]. Due to the fatality of surfactant deficiency and complexity of animal models, it has been difficult to probe the effects of surfactant on host-pathogen interactions. Organ-on-chip models combine the simplicity and feasibility of in vitro models with the ability to recapitulate physiologically relevant conditions like in in vivo models. This preprint looks at the role of surfactant released from alveolar epithelial cells using a model that recapitulates the air-liquid interface of lungs on a chip.

 

Experimental setup: The lung-on-chip model was made using polydimethylsiloxane (PDMS, a silicone-based organic polymer widely used for microfluidics) and had two chambers – air (alveolar) and liquid (vasculature) – separated by a porous membrane (see figure). The alveolar chamber contained alveolar epithelial cells (AECs), and the vascular chamber had endothelial cells. GFP-labelled macrophages were also added to the alveolar chamber, which could transverse the porous membrane to the endothelial side. Use of both AECs and macrophages allows one to study infection of both cell types. The chip was infected with fluorescently labelled Mycobacterium tuberculosis, WT, attenuated or avirulent mutants. Infection was visualized using time-lapse microscopy.

Important Results:

The lung-on-chip model recapitulates infection – This model makes use of two kinds of AECs – freshly isolated AECs which produce normal surfactant levels (NS), or in vitro passaged AECs (6-11 times) which have deficient surfactant levels (DS). These phenotypes were maintained in the chip, thus allowing  the study of endogenous pulmonary surfactant. Addition of Mtb to the chip lead to both cell types getting infected.

Surfactant reduces growth rates of Mtb – The intracellular growth rate of Mtb post infection was monitored over five days. Though highly variable, a pattern emerged with respect to the surfactant conditions. NS conditions reduced growth rates of Mtb, with a larger proportion of cells showing very slow growth (non-growing fraction) when compared to DS conditions. This was evident in both AECs and macrophages. Exogenous addition of a surfactant solution in the DS condition leads to an increase in the non-growing fraction of cells, restoring control of Mtb growth. This indicates that surfactant does play a role in host-pathogen interactions, by protecting the host cells.

An attenuated strain, deficient for ESX-1 type III secretion system, is unable to grow irrespective of surfactant conditions, indicating that ESX-1 is still required in DS conditions. Similarly, another attenuated strain, previously shown to be unable to grow in lungs of mice, showed a similar phenotype in the AECs and macrophages of the chip model. This underscores the importance and ability of the lung-on-chip model to reproduce similar phenotypes as animal models.

Surfactant plays a role in attenuation of growth by removing virulence-associated lipids of Mtb –

Surfactant solution was seen to coat the bacteria upon exposure (but this effect was heterogenous). Additionally, sulfoglycolipids and trehalose dimycolate were partially removed from the surface of Mtb cells. These lipids are known to be important for virulence in inducing granulomas in mice and inhibiting cytokine production and immune response [2,3].

 

What I found interesting about this preprint:

The use of organ-on-chip approach enabled the study of endogenously secreted surfactant from alveolar cells, while maintaining the physiology and microenvironment of the air-liquid interface of lungs. Organ-on-chip models are gaining popularity in the field of infection research due to the ability to dissect host-microbe interactions at higher resolutions compared to animal models. I think this study makes a strong case for the use of such models, reproducing phenotypes and features known from animal models.

This study also shows that surfactant does more than its widely known role of reducing surface tension in the lungs, as it also protects the host again a pulmonary infection-causing pathogen. It would be interesting to see if such a protective role is seen against other pulmonary pathogens as well.

 

Questions for the authors:

1. Where do you think the heterogeneity in coating of the Mtb surface with surfactant arises from? Does the correlate with any heterogeneity in the cell surface proteins of Mtb?
2. Did you look at bacteria that cross over from the porous membrane into the endothelial side?

 

References/Further Reading:

[1] Torrelles, J. B., & Schlesinger, L. S. (2017). Integrating lung physiology, immunology, and tuberculosis. Trends in microbiology, 25(8), 688-697.

[2] Hunter, R. L., Olsen, M., Jagannath, C., & Actor, J. K. (2006). Trehalose 6, 6′-dimycolate and lipid in the pathogenesis of caseating granulomas of tuberculosis in mice. The American journal of pathology, 168(4), 1249-1261.

[3] Blanc, L., Gilleron, M., Prandi, J., Song, O. R., Jang, M. S., Gicquel, B., … & Vercellone, A. (2017). Mycobacterium tuberculosis inhibits human innate immune responses via the production of TLR2 antagonist glycolipids. Proceedings of the National Academy of Sciences, 114(42), 11205-11210.

Tags: lung-on-chip, mycobacterium, tuberculosis

doi: https://doi.org/10.1242/prelights.23098

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