Figure 2. Surfactant deficiency results in uncontrolled intracellular growth of Mtb.

Snapshots from live-cell imaging at 1.5–2.0 hr intervals between 3 and 5 days post-infection (dpi) in normal surfactant (NS) (A-D) and deficient surfactant (DS) (E-H) lung-on-chip (LoCs). Macrophages are false-colored magenta; Mtb is false-colored green. The calibration bar (inset in A, D, E, H) indicates the absolute intensities in the Mtb channel, the scales were chosen to achieve a similar saturation level in the images across surfactant conditions. Representative examples of infected AT (yellow boxes) and macrophage (white boxes) are highlighted, and zooms (B, C, F, G) reveal growth in both cell types over this period. Scale bar, 10 µm. (I, J) Plots of the logarithm of total fluorescence intensity over time confirm exponential Mtb growth for representative infections in ATs (I, K) and macrophages (J, L) under NS and DS conditions, respectively. In each case, an intracellular microcolony with growth rate close to the population maximum (red), median (yellow), and minimum (blue) is shown. The growth rate is the gradient of the linear fit. (M) Scatter plots of Mtb growth rates in ATs (n=122 for NS, n=219 for DS) and macrophages (n=185 for NS, n=122 for DS). Growth is significantly slower in macrophages than ATs in NS conditions (p = 7.8E-8) but not in DS conditions (p = 0.64), and is more heterogenous in both conditions compared to single-cell Mtb growth rate data from axenic microfluidic cultures. The green- and red-shaded regions indicate the growing bacteria and the non-growing fraction (NGF), respectively. (N) Uncontrolled growth in DS conditions can be rescued by exogenous administration of Curosurf. Scatter plots represent Mtb growth rates in macrophages in a DS LoC treated with Curosurf (‘Chiptreat’) or infected with Mtb preincubated with Curosurf (‘Mtbtreat’). Data from DS and NS LoC infections (no Curosurf) are included for comparison. Growth attenuation for both treatments is significant relative to DS conditions as reflected by the average growth rate and the size of the NGF (n = 122 for DS and n = 121 for Chiptreat; p = 2.5E-12 and n = 63 for Mtbtreat; p = 2.2E-12 and n = 122 for DS). p-Values were calculated using the Kruskal-Wallis one-way ANOVA test.

Figure 2—figure supplement 1. Characterization of intracellular Mtb growth in a lung-on-chip (LoC) treated with surfactant (chiptreat) at 4 days post-infection.

(A) A maximum intensity projection of a Z stack and (B) a 3D projection of a 155 × 155 μm² field of view on the epithelial face of the LoC. Three areas marked in (A) are expanded in (C-H). In each case, 3D view is shown (C, E, G), followed by an individual slice from the stack (D, F, H) together with YZ and XZ orthogonal views for the entire stack at the positions indicated by the horizontal and vertical lines. Examples are chosen to highlight slow intracellular Mtb growth (C, D), rapid intracellular Mtb growth (E, F) and growth within macrophages (G, H). In all panels, cell nuclei are shown in indigo, fluorescent macrophages are shown in azure, fluorescent Mtb are shown in spring green, and actin is shown in amber LUTs, respectively.

Figure 2—figure supplement 2. Comparison of Mtb growth rates on-chip vs. single-cell growth rates in axenic conditions.

Scatter plots of bacterial intracellular growth rates for infected ATs (n = 122 for NS, n = 219 for DS) or macrophages (n = 185 for NS, n = 122 for DS) in normal surfactant (NS) conditions (A) or deficient surfactant (DS) conditions (B). These measurements reveal greater cell-to-cell heterogeneity in both conditions (NS and DS) when compared to single-cell Mtb growth rates from axenic cultures grown in microfluidic devices. p-Values for comparisons of distributions were calculated using the Kruskal-Wallis one-way ANOVA test.

Figure 2—figure supplement 3. Characterization of the growth characteristics of WT and the Δicl1Δicl2 Mtb strain in ALI media.

Growth curves for WT Mtb and the Δicl1Δicl2 strain grown to exponential phase in 7H9 media and then sub-cultured in the ALI media used in the lung-on-chip experiments.

Figure 2—figure supplement 4. Curosurf does not affect Mtb viability or growth in vitro.

Figure 2—figure supplement 5. Heterogenous host-Mtb interactions are not restricted to a particular spatial niche on-chip.

3D scatter plots of growth rates of individual microcolonies of intracellular bacteria vs. their spatial coordinates on a representative LoC for normal surfactant (A) and deficient surfactant (B) conditions for infected ATs (cubes) and infected macrophages (spheres). Growing microcolonies are colored green and non-growing fraction (NGF) microcolonies are colored red, as in Figure 2. In both examples shown, heterogenous growth is observed throughout the chip, with no discernable spatial pattern. The X-axis refers to position along the length of the chip, with the approximate position of the origin (0 mm) set roughly in the middle of the chip, as indicated in the schematic.

Figure 2—video 1. Live-cell imaging over 3-5 days post-infection at the ALI for an LoC infected with WT Mtb in normal surfactant conditions and corresponding to snapshots in Figure 2A-D.

Download video file ^{(5.4MB, mp4)}

Imaging frequency is 1.5 hr. Macrophages are false-colored magenta, Mtb is false-colored green.

Figure 2—video 2. Live-cell imaging over 3-5 days post-infection at the ALI for an LoC infected with WT Mtb in deficient surfactant conditions and corresponding to snapshots in Figure 2E-H.

Download video file ^{(3.9MB, mp4)}

Imaging frequency is 2 hr. Macrophages are false-colored magenta, Mtb is false-colored green.