Connie H. Y. Wong, Craig N Jenne, Björn Petri, Navina L. Chrobok and Paul Kubes

Transcription

1 Nucleation of platelets with bloodborne pathogens on Kupffer cell precedes other innate immunity and contributes to bacterial clearance Connie H. Y. Wong, Craig N Jenne, Björn Petri, Navina L. Chrobok and Paul Kubes Supplemental information

2 Supplementary Figure 1. Nucleation of platelets on Kupffer cells post-infection is independent of the TLR pathways Quantification of the number of platelet aggregates per field of view (FOV) within liver sinusoids that were larger than 25 μm 2 following sham or following 10 mins of B. cereus infection in wild-type (WT) and Myd88 / mice. n 4 individual mice per group; error bars, SEM; NS denote not statistically significant by two way ANOVA.

3 Supplementary Figure 2. Nucleation of platelets on Kupffer cells post-infection precede the recruitment of neutrophils Quantification of the number of neutrophils per field of view (FOV) within liver sinusoids following sham or following 10 mins of B. cereus infection in wild-type (WT) and Myd88 / mice. n 4 individual mice per group; error bars, SEM.

4 Supplemental Figure 3. Host protection against intraperitoneal infection of B. cereus conferred by platelets Survival rate of intraperitoneal B. cereus-infected wild-type (WT), platelet-depleted (Platelet depl) and GpIbα / (GPIbα-def) mice. n 4 per group.

5 Supplementary Figure 4. Schematic diagram to illustrate the interactions between the different cell types and molecules Under basal conditions, platelets via GPIb formed transient touch-and-go interactions with vwf constitutively expressed on KCs. Bacteria, such as Bacillus cereus and human pathogen Methicillin-resistant Staphylococcus aureus, were rapidly caught by KCs and triggered a switch from touch-and-go to sustained platelet adhesion via GPIIb on the KC surface.

6 SUPPLEMENTARY VIDEO LEGENDS Supplementary Video 1 Platelet form "touch-and-go" interactions within liver sinusoids in vivo. Intravital visualization of circulating platelets within the liver microvasculature of a non-infected wild-type mouse using spinning-disk confocal fluorescence microscopy. Platelets were labeled with PE-conjugated anti-cd49b (red). Kupffer cells were labelled with Alexa Fluor 647-conjugated anti-f4/80. Elapsed time is shown at the top right. The time lapse was recorded at maximum speed and exported to video at 5 frames per second (fps). Objective: UPLANSAPO 20x/0.70. Scale bar, 50 μm. The video is representative of 2 independent experiments of minimum of 3 mice per experiment. Supplementary Video 2 Platelet interactions and aggregation within the liver of a B. cereus infected mouse. Intravital visualization of circulating platelets, adhering and forming firm aggregates within the liver microvasculature during the first 10 min of GFP-expressing B. cereus infection using spinning-disk confocal fluorescence microscopy. Platelets were labeled with PE-conjugated anti-cd49b (red). Elapsed time is shown at the top right. The time lapse was recorded at maximum speed and exported to video at 20 frames per second (fps). Objective: UPLANSAPO 10x/0.40. Scale bar, 100 μm. The video is representative of 2 independent experiments of minimum of 3 mice per experiment. Supplementary Video 3 Significant reduction of captured bacterium in KC-depleted animal. Intravital visualization of intravenous GFP-expressing B. cereus infection within the liver microvasculature of KC-depleted mouse using spinning-disk confocal fluorescence

7 microscopy. Platelets were labeled with PE-conjugated anti-cd49b (red). Elapsed time is shown at the top right. The time lapse was recorded at maximum speed and exported to video at 5 frames per second (fps). Objective: UPLANSAPO 10x/0.40. Scale bar, 100 μm. The video is representative of 2 independent experiments of minimum of 3 mice per experiment.