Figure 2. Neutrophil diapedesis and NET formation on-chip.

(A1–A5) Schematic of the UPEC infection, introduction of neutrophils and diapedesis of neutrophils across the epithelial-endothelial barrier to sites of infection. Flow in the epithelial and endothelial channels is indicated by arrows; the flow rate was increased upon introduction of neutrophils in the vascular channel to increase attachment. Increased flow rate in the epithelial and endothelial channel is indicated by black arrows. (B1–B5) Snapshots from time-lapse imaging highlighting each stage in the infection cycle shown in (A1–A5). Bladder epithelial cells (magenta) and neutrophils (amber) were identified with membrane (Cell Mask Orange) and cytoplasmic (Cell Tracker Deep Red) dyes, respectively. UPEC identified via constitutive expression of YFP are shown in green. Neutrophil swarms could either control bacterial growth (yellow dashed square, compare B4 vs. B5) or did not manage to restrict bacterial growth (white dashed square, compare B4 vs. B5). (C) Bar charts for relative frequency of neutrophil diapedesis (black) in n=three uninfected control bladder-chips and n=four infected bladder-chips. Data obtained from n=95 and n=116 fields of view, each of which was 206 x 206 μm². (D) Quantification of the number of neutrophils detected on the epithelial layer, in control and infected bladder-chips. The red bar represents the median value, p<1E-15. In many instances in the uninfected control bladder-chips, no neutrophil diapedesis is detected. Data obtained from n=95 and n=130 fields of view, each of which was 206 x 206 μm². (E) A plot of the maximum neutrophil swarm volume on the epithelial layer normalized to the total volume for n=67 and n=118 fields of view on n=three uninfected control and n=three infected bladder-chips. The red bar represents the median value. p<1E-15. (F) A plot of neutrophil swarm volume on the epithelial layer over time for n=51 and n=40 fields of view for n=two technical replicates indicated by squares and circles. For each time profile, the volume is normalized to the maximum volume attained over the timeseries and t=0 refers to the timepoint at which neutrophils are introduced into the vascular channel (G) Plot of the time to reach the maximum swarm volume in n=154 fields of view across n=four infected bladder-chips. (H1–H5, I1–I5) NET formation by neutrophils on the epithelial layer. The neutrophils are identified by a cytoplasmic dye (CellTracker Deep Red, false colored in amber) (H2, I2) and immunostaining with an anti-myeloperoxidase antibody (H3, zooms in H5) or an anti-neutrophil elastase antibody (I3, zooms in I5). Merged images in each case are shown in H4 and I4. UPEC identified via YFP expression is shown in spring green. Nuclear labelling with DAPI is shown in azure. (H5) NETs, identified via anti-myeloperoxidase staining or anti-elastase staining are indicated with white arrows in (H5) and (I5), respectively. (J1–J4) Scanning electron micrographs of the epithelial layer of an infected bladder-chip 2 hr after the introduction of neutrophils in the endothelial channel. (J1) Neutrophils (amber arrowheads) are visible above a layer of epithelial cells. Thick bundles consisting of many thinner NET structures between neutrophils are indicated by white arrowheads, and examples of individual UPEC bacteria within the NETs are indicated by green arrowheads. A heavily infected epithelial cell with UPEC visible on the epithelial cell (purple arrowhead), and appendages between epithelial cells (cyan arrowheads) are also visible. (J2) Micrographs showing thick bundles consisting of many thinner NET structures (white arrowheads) that extend between cells and trap many individual bacteria. Thinner NET fibers (yellow arrowheads) are also visible. (J3) Zooms of the regions in J2 identified by a yellow dashed square. Two bacteria held by a thick bundle composed of many thinner NET fibers are shown. (J4) Micrograph highlighting multiple bacteria trapped between NET bundles. p-Values in D and E were calculated using a Mann-Whitney test. Scale bar = 50 μm in B1–B5, H1-H5, I1-I5.

Figure 2—figure supplement 1. Quantification of UPEC attachment to bladder epithelial cells on-chip under flow.

Ratio of number of attached UPEC to the average number of epithelial cells in n=25 (magenta line), n=38 (orange line), and n=34 (blue line) fields of view, each 206 x 206 μm² across on the epithelial layer of n=three infected bladder-chips. In each case, the protocol results in less than 1 focus of infection per epithelial cell at the end of the 90 min infection period. The dotted lines and the shaded regions represent the standard deviation.

Figure 2—figure supplement 2. Timeseries highlighting neutrophil diapedesis and swarm formation.

Additional images that highlight the diapedesis of neutrophils across the epithelial-endothelial barrier and the formation of neutrophil swarms. Bladder epithelial cells (magenta) and neutrophils (amber) were identified with membrane (Cell Mask Orange) and cytoplasmic (Cell Tracker Deep Red) dyes, respectively. UPEC identified via constitutive expression of YFP are shown in green. In all images, scale bar = 50 μm.

Figure 2—figure supplement 3. Neutrophils isolated from human blood are CD15+.

Images of neutrophils isolated via negative depletion from human blood and labelled with a cytoplasmic dye (Cell Tracker Deep Red) (A) and immunostained with an anti-CD15 antibody (B). (C) Merged image for both channels confirms that all neutrophils are CD15+.

Figure 2—figure supplement 4. Neutrophil attachment to endothelial cells is enhanced upon bacterial infection.

Fluorescent (A) and bright-field (B) imaging of the endothelial layer of an uninfected bladder-chip. The few neutrophils (identified by CellTracker Deep Red, amber) attached to the endothelial layer are indicated by white arrowheads. Fluorescent (C) and bright-field (D) imaging of the endothelial layer of an infected bladder-chip, 1.5 hr after infection of the epithelial layer. Neutrophils attached to the endothelial layer are marked by white arrowheads and examples of diapedesis, although the PDMS pores to the epithelial layer are marked with magenta arrowheads. In all panels, neutrophils (amber) were introduced into the vascular channel of the bladder-chip under a flow rate of 3 ml/hr corresponding to a shear stress η=one dyne/cm².

Figure 2—figure supplement 5. Neutrophil diapedesis is stimulated by a pro-inflammatory cytokine gradient across the epithelial-endothelial barrier.

(A) Representative images of the epithelial layer of an uninfected control bladder-chip, 2 hr after the introduction of neutrophils in the endothelial channel. No neutrophil diapedesis is observed. (B) Representative image of the epithelial layer of an uninfected control bladder-chip exposed to a cocktail of pro-inflammatory cytokines (Interleukin-1α, Interleukin-1β, Interleukin-6 and Interleukin-8, each at 100 ng/ml) added to the diluted urine on the epithelial side and maintained under flow for 2 hr. Epithelial cells (magenta, identified via CellMask Orange) and neutrophils (amber, identified via CellTracker Deep Red) are shown. (C) Representative image of the epithelial layer of an infected bladder-chip 2 hr after the introduction of neutrophils in the endothelial channel. UPEC identified via constitutive expression of YFP are shown in green. (D) Scatterplot of maximum number of neutrophils detected in 206 x 206 μm² fields of view under control (n = 26), cytokine stimulation (n=26) and infection (n=130). p-Values were calculated using Kruskal-Wallis ANOVA Test. Red lines represent median values. Scale bars, 50 μm.

Figure 2—figure supplement 6. NETs formation on the epithelial and endothelial layers of an infected bladder-chip.

(A1–A3) Additional example of NETs formation by neutrophils on the epithelial layer (epi) of an infected bladder-chip. Neutrophils are identified via immunostaining with an anti-myeloperoxidase antibody (A1–A2). UPEC identified via YFP expression are shown in spring green (A1). Nuclear labeling with DAPI is shown in azure (A2). A merged image is shown in A3. (B1–B3) An example of NETs formation by neutrophils on the endothelial layer (endo) of an infected bladder-chip. UPEC identified via YFP expression are shown in spring green and nuclear labeling with DAPI is shown in azure (B1). Neutrophils are identified via immunostaining with an anti-neutrophil elastase antibody (B2). A zoomed image corresponding to the white dashed box in B2 is shown in B3. In all images, scale bar = 50 μm.

Figure 2—figure supplement 7. SEM characterization of uninfected and infected bladder-chips.

(A) SEM image of the confluent epithelial layer of an uninfected bladder-chip. Appendages between epithelial cells are indicated by cyan arrowheads. (B) Example from an infected bladder-chip without the addition of neutrophils. Long filaments characteristic of NET formation is not observed. Individual UPEC on the surface of the epithelial cells are indicated by green arrowheads. Appendage between epithelial cells is indicated by a cyan arrowhead (C) Additional example of formation of NETs around a large swarm of neutrophils on the epithelial layer of an infected bladder-chip. NETs are indicated by white arrowheads, and large clusters of neutrophils are indicated by dashed amber circles.

Figure 2—figure supplement 8. Neutrophils do not form NETs in response to shear stress in the bladder-chip.

Neutrophils infused through the vascular channel of an infected bladder-chip were collected and characterized via immunofluorescence for myeloperoxidase (A-C) and neutrophil elastase expression (D-F) to identify the formation of NETs, indicated by dotted white circles. All neutrophils are labeled by the cytoplasmic CellTracker dye (shown in amber in B, E). Both myeloperoxidase expression (marked with dotted white circles in C) and elastase expression (marked with dotted white circles in F) coincide with infected neutrophils (A, D). UPEC are identified via YFP expression and colored spring green, nuclear labeling is indicated in azure. Scale bars, 50 μm in all panels.

Figure 2—video 1. Description: Infection of bladder-chip with UPEC, diapedesis of neutrophils across the epithelial-endothelial barrier and formation of swarms (Figure 2B1–B5).

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Stage 1: Prior to infection, the uninfected cells of the epithelial layer (magenta) are imaged (0–120 mins). Stage 2: UPEC (green) are introduced into the epithelial channel via flow and infection proceeds under flow for 120–210 min. Stage 3: Neutrophils (amber) are introduced into the vascular channel of the infected bladder-chip via flow (210 min onwards). Stage 4: Neutrophils undergo diapedesis within 15–30 min and are visible on the epithelial side (210–240 min). UPEC can be seen internalized by neutrophils. Stage 5: Neutrophils aggregate and form a neutrophil swarm (240 min onwards) on the epithelial side.

Figure 2—video 2. Description: Neutrophil diapedesis and swarm formation on the epithelial side of UPEC infection (Figure 2—figure supplement 2A1-A5).

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Stage 1: Prior to infection, the uninfected cells of the epithelial layer (magenta) are imaged (0–120 mins). Stage 2: UPEC (green) are introduced into the epithelial channel via flow and infection proceeds under flow for 120–210 min. Stage 3: Neutrophils (amber) are introduced into the vascular channel of the infected bladder-chip via flow (210 min onwards). Stage 4: Neutrophils undergo diapedesis within 15 min and are visible on the epithelial side (210–240 min). UPEC can be seen internalized by neutrophils. Stage 5: Neutrophils aggregate and form a neutrophil swarm (240 min onwards) on the epithelial side.