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

  1. Vivek V Thacker
  2. Neeraj Dhar
  3. Kunal Sharma
  4. Riccardo Barrile
  5. Katia Karalis
  6. John D McKinney

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Abstract

We establish a murine lung-on-chip infection model and use time-lapse imaging to reveal the dynamics of host- Mycobacterium tuberculosis interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models and to probe the direct role of pulmonary surfactant in early infection. Surfactant deficiency results in rapid and uncontrolled bacterial growth in both macrophages and alveolar epithelial cells. In contrast, under normal surfactant levels, a significant fraction of intracellular bacteria are non-growing. The surfactant-deficient phenotype is rescued by exogenous addition of surfactant replacement formulations, which have no effect on bacterial viability in the absence of host cells. Surfactant partially removes virulence-associated lipids and proteins from the bacterial cell surface. Consistent with this mechanism, the attenuation of bacteria lacking the ESX-1 secretion system is independent of surfactant levels. These findings may partly explain why smokers and elderly persons with compromised surfactant function are at increased risk of developing active tuberculosis.

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  1. Version published to 10.7554/elife.59961 on eLife
  2. eLife

    ###Reviewer #3:

    This paper by Thaker et al describes the use of lung-on-a-chip microfluidic devices for early interactions during acute M. tuberculosis infection under conditions chosen to mimic the alveolar environment in vivo. The authors use time-lapse microscopy to study host-Mtb interactions in macrophages and alveolar epithelial cells, the role of the Mtb Type VII secretion system and the impact of surfactant on Mtb infection. This study suggests that organ-on-a chip systems might be able to reproduce host-microbe physiology during infection, which is difficult to reproduce ex vivo using single cells, air-liquid interface, organoids or organ explants. This is an exciting approach which has the potential to expand the ability to study host-pathogen interactions, but there are some limitations that dampen my enthusiasm.

    Major concerns:

    While I recognize that it is challenging to use live cell imaging with colocalization markers, much of the data of the paper, such as comparisons between AECs and macrophages, or mutant Mtb strain vs WT, or role of surfactant, rests on the ability to determine the precise localization of bacteria. However, neither AECs nor macrophages are specifically identified with high enough resolution to give confidence that the Mtb are associated with those cells specifically, and more importantly, that the bacteria are growing intracellularly rather than extracellularly. The authors show multiple bacterial microcolonies that grow in size over time, but whether these are inside or outside cells, and whether the cells are AECs or macrophages isn't overtly specified. Many of the images are of such low resolution that only tiny dots of bacteria are observed. To the author's credit, the quantitative and statistical analysis is very rigorous, however, better evidence for the issues raised above would increase confidence in the results. This point is highlighted in detail by by the following:

    Lines 60-63: "Inoculation of the LoC with between 200 and 800 Mtb bacilli led to infection of both macrophages (white boxes in Fig. 1M, P, zooms in Fig. 1O, R) and AECs (yellow boxes in Fig. 1M, P, zooms in Fig. 1N, Q) under both NS (Fig.1M-O) and DS (Fig. 1P-R) conditions." Identification of GFP-expressing macrophages can be assumed based on their expression of GFP (though the cells themselves aren't colocalized) on images but the same cannot be said of AECs. The yellow boxes could represent AECs or spaces on the chip with no cells at all. Furthermore, the 2D images showed in Figure 1 do not necessarily represent infected cells, and the possibility of visualization of Mtb outside the cells should be considered. Thus, higher resolution images, with clear colocalization and z-stacks, would increase the confidence in the results.

    The data arguing for attenuation of Esx-1 mutant Mtb in AECs and macrophages is not strong, and the authors do not actually make a direct statistical comparison between appropriate groups (i.e. AEC NS WT vs Esx-1, or Mac NS WT vs Esx-1). For example, it appears that the mean/median growth rate of WT Mtb in macs is ~0.25hr-1, which appears roughly the same for Esx-1 mutant Mtb in the same cells. There may be a difference under DS conditions, but since the comparisons aren't made directly it is impossible to know.

  3. eLife

    ###Reviewer #2:

    The manuscript by Thacker et al, entitled "A lung-on-chip model reveals an essential role for alveolar epithelial cells in controlling bacterial growth during early tuberculosis" is an interesting study describing a new in vitro model to determine the early events of Mycobacterium tuberculosis infection. This model is important and novel; however, this study is descriptive and some of the findings (e.g., attenuated growth of M. tuberculosis after exposure to surfactant in macrophages and alveolar epithelial cells, as well as changes on the M. tuberculosis cell wall after exposure to surfactant, or that exposure to surfactant does not alter the extracellular viability of M. tuberculosis) have been reported by others using other in vitro models. The use of the ESX-1 attenuated mutant is not clear in this study, as well as the concept that exposure to surfactant may change the attenuation of this strain. The composition of mouse surfactant and human surfactant is also quite different, thus extrapolating results need to be done with caution.

    Major concerns:

    1. Results provided in Figures 1, 2 and Fig. 3 supplement 1 are confusing, and readers need to guess what they are looking at, especially in Figure 1 M-R. As this is an important model , it will be appropriate to have detailed and better images showing well-defined cells, and quantify their findings in Tables (e.g. number of alveolar epithelial cells type I and II, number of macrophages, numbers of endothelial cells, bacteria per cell, etc.). In Fig. 3 supplement 1 one needs to guess what is intracellular or extracellular within the studied system.

    2. The definition of Normal surfactant (NS) vs. Deficient surfactant (DS) is confusing as used. Alveolar epithelial cells type II (AT-IIs) become type I (AT-I) over time in in vitro cultures (in 5 to 7 days) and thus, these stop secreting surfactant. Authors found that after 6-11 passages AT-IIs stopped producing surfactant but also lost their cellular characteristics as well as the expected characteristics of AT-Is. This needs to be further studied in detail to ensure that this cell is not an artifact produced by multi-passaging in vitro. Authors need to use several AT-IIs and AT-Is markers to be certain that the DS cell monolayers indeed still are ATs. Surfactant protein C, although used as a marker for AT-IIs, is a soluble protein that has been shown to interact with many cells within a cellular system. A correlation between SPTPC and AQP5 expression over time is also necessary as points out the differentiation of AT-IIs to AT-Is, a key feature of the role of AT-IIs as progenitors of AT-Is.

    3. Authors did not consider that M. tuberculosis can form micro-colonies on the cell surface of alveolar epithelial cells and thus, the intracellular growth that they are reporting could be extracellular growth. Did the authors after infection treat the system with an antibiotic to kill extracellular M. tuberculosis bacilli attached to the alveolar epithelial cell surface? In addition, the concept of M. tuberculosis micro-colonies growing inside cells need to be better explained. Are these bacterial clumps? How the authors discern that the ones that are not growing vs. the ones that are dead?

    4. If I understand the described method well, the staining of Curosurf (poractant alfa) is not as such. Authors used a commercial labeled phosphatidylcholine (PC) added into the Curosurf. This labeled PC may or may not interact with Curosurf components, but what is obvious is that it makes micelles. What it is quantified is the interaction of the labeled PC with M. tuberculosis. Moreover, the artificial addition of this phospholipid (at 10%) is changing the original composition of Curosurf, and this may have physiological implications. Authors need to confirm if the PC added was indeed DPPC. Authors also need to come up with a better way to demonstrate that Curosurf components are opsonizing M. tuberculosis bacilli. In addition, why authors used 1% Curosurf for their experiments. Is there a dose titration effect? Why authors did not use Survanta or Infasurf or mouse surfactant?

    5. The in vivo simulation of infection using grow rates randomly chosen from the kernel density estimations for the respective populations. In this graph, it is very important to discern the bacteria with high growth rates from the bacteria with low growth and intermediate growth rates (at the 99 percentile, 75 percentile, at the 50 percentile, at the 25 percentile and at the 1 percentile) and assess how these are projected to behave in vivo. As presented it is not very informative about the impact of NS ATs vs. DS ATs on M. tuberculosis infectivity in this model system.

    6. Similar alterations on the M. tuberculosis cell wall and release of cell wall components to the milieu when exposed to physiological concentrations of human lung surfactant have been already described. The same is applicable to the slower replication rate in ATs (an intracellular killing in macrophages) after M. tuberculosis exposure to human lung surfactant. Although two different systems, authors need to contrast their findings with these reported ones in their discussion. In addition, it is not clear how many times this was performed. Statistics are mentioned on the figure legends, but there are no stats in the figure.

  4. eLife

    ###Reviewer #1:

    1. What quality control is done for each experiment to determine the ratio of type I and type II AECs in each chip set up for each experiment? This is of particular importance because the authors do not show any images where they stain for both type I and type II AECs in the same chip. Do the authors have images stained for both type of cells to illustrate the composition of each chip? After figure 1, what staining is done to confirm the DS cells decrease proSPC expression for each experiment?

    2. The authors focus on the difference in surfactant gene expression in the newly isolated AECs (NS) versus in vitro passaged AECs (DS), but they also observe that aqp5 is downregulated. In fact, the data supports that the cells are just de-differentiating during passage in culture, which will have multiple effects on the cells, not just surfactant production. This should be commented on and discussed. After loss of those markers, how do the authors confirm they still have type I and type II AECs in their cultures? Is there microscopy data with other markers that are retained in the AECs? The add back experiments with Curosurf support that surfactant can contribute to bacterial control, but this imparts only a partial complementation and the evidence for de-differentiation implies other pathways at play.

    3. One of the biggest concerns is that the authors never stain for type I or type II AECs after infection and make the conclusion that the bacteria are within type II cells based on the absence of macrophage staining. However, the bacteria may not even be in a cell, or the AECs could be dying during infection. On a related note, there is no data presented that shows that type I cells are not infected in the lung on chip system with Mtb.

    4. The authors state that their data with the Esx1 mutant "demonstrates that ESX-1 secretion is necessary for rapid intracellular growth in the absence of surfactant, consistent with the hypothesis that surfactant may attenuate Mtb growth by depleting ESX-1 components on the bacterial cell surface". This seems like quite a jump in interpretation of the data since the Esx1 mutant is likely attenuated for many reasons, and this attenuation is dominant to any effect that surfactant is having. The authors also show that PDIM levels are not different in the presence or absence of surfactant, and this is an Esx1 dependent lipid.

    5. What is the purpose for including the icl1/icl2 mutant? This experiment is not included in the data quantification.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    This manuscript is in revision at eLife.

    ###Summary:

    This paper by Thacker et al. describes the use of lung-on-a-chip microfluidic devices to study early interactions during M. tuberculosis infection under conditions meant to mimic the alveolar environment in vivo. The authors use time-lapse microscopy to study host cell-Mtb interactions in macrophages and alveolar epithelial cells and the impact of surfactant on Mtb infection. This study suggests that organ-on-a-chip systems might be able to reproduce elements of host-microbe physiology during infection, which is difficult to reproduce ex vivo using single cells, air-liquid interface, organoids or organ explants.

    This is an exciting approach which has the potential to expand the ability to study host-pathogen interactions. However, the reviewers all agree that the manuscript requires a major revision and additional data. Specifically, the manuscript requires improvement in the cell identification/classification, co-localization of Mtb with epithelial cells and macrophages, and distinction between intracellular and extracellular growth in order for the authors to provide convincing data to support their interpretations and conclusions.

    While the reviewers recognize that it is challenging to use live cell imaging in this system, much of the data of the paper, such as comparisons between infection of AECs and macrophages, rests on the ability to determine the precise localization of bacteria. However, neither AECs nor macrophages are specifically identified with high enough resolution to give confidence that the Mtb are associated with those cells specifically, and more importantly, that the bacteria are growing intracellularly rather than extracellularly. Many of the images are of such low resolution that only tiny dots of bacteria are observed.

    In addition, the findings of attenuated growth of Mtb after exposure to surfactant in macrophages and alveolar epithelial cells, changes in the Mtb cell wall after exposure to surfactant, and the finding that exposure to surfactant does not alter the extracellular viability of M. tuberculosis have been reported by others using other in vitro models and should be discussed in manuscript.

  7. Version published to 10.1101/2020.02.03.931170v2 on bioRxiv
  8. Version published to 10.1101/2020.02.03.931170v1 on bioRxiv