Fig 1.
SEM micrographs of uncoated and biofilm-coated microbeads before and after microinjection, and after sonication.
SEM micrographs of uncoated microbeads at X800 (A) and X5000 (B) magnification, biofilm-coated microbeads before passing through the 34G needle used for microinjections at X2000 (C) and X4000 (D) magnification, biofilm-coated microbeads after passing through the 34G needle used for microinjections at X2000 (E) and X3700 (F) magnification, and sonicated biofilm-coated microbeads at X2000 (G) and X8000 (H) magnification. Filled red arrowheads in C and E indicate microbeads and red arrows in C and E indicate biofilm extracellular matrix. Scale bar: 20 μm (A), 10 μm (C, E, G), 5 μm (B, D, F), 2 μm (H).
Fig 2.
Confocal images of a biofilm-coated microbead and representative flow cytometry plots verifying coating of microbeads.
Confocal images (A) of biofilm-coated microbeads, after passing through the micro-injection needle, treated with Alexa Fluor 488 WGA at X63 magnification. Images show microbeads and maximum intensity projections of mCherry (magenta) and Alexa Fluor 488 WGA (green) fluorescence. The merged image shows maximum intensity projections for both fluorescence channels. Scale bar: 5 μm. Representative FACS plots showing the gates used to determine uncoated microbeads (B), planktonic bacteria and microbeads (C) and biofilm-coated microbead (D) populations. The subsequent counts for each population are also presented according to their fluorescence intensities.
Fig 3.
Intravital confocal imaging of CDy11 labelled biofilm-coated microbeads.
Intravital confocal imaging after microinjection of only the CDy11 fluorescent probe (A), of biofilm-coated microbeads after in vitro CDy11 labelling (B) or of biofilm-coated microbeads after in vivo CDy11 labelling (C) in the ear pinna of WT C57BL/6 mice. (A to C) Images show maximum intensity projections of GFP (green) and CDy11 (red) fluorescence. The merged image shows maximum intensity projections for both fluorescence channels. Scale bar: 100 μm.
Fig 4.
Global inflammatory response after microinjection of different inocula into the transgenic mice ear.
Reconstituted confocal images of LyM-EGFP transgenic mice ear pinna tissue after microinjection of either PBS (control) (A), planktonic bacteria and microbeads (B) or biofilm-coated microbeads (C) at early (6 hours post-infection or 6 hpi) and late time points (after 26 hpi). Images show maximum intensity projections of EGFP (green) fluorescence that correspond to phagocytic cells (neutrophils and macrophages). The yellow line indicates the Region of Interest (ROI) where the “Sum of EGFP fluorescence intensities” was measured. One representative experiment is shown for each group of mice from four independent experiments. Scale bar: 2 mm. (D) Ratio of the sum of EGFP fluorescence intensities to ROI area. Data are expressed as median and interquartile ranges for four mice per group. p≤0.05 was considered statistically significant (symbols: ****p≤0.0001; ***p≤0.001; **p≤0.01; *≤0.05; ns = non-significant).
Fig 5.
Live confocal imaging of innate immune responses after microinjection of the different inocula.
Live confocal imaging after microinjection of planktonic bacteria and microbeads (A, B) in the ear pinna of LysM-EGFP transgenic mice at early time points (4–6 hpi). Innate immune cell recruitment towards injection sites was observed between 4.35 (A) to 5.05 hpi (B). Live confocal imaging after microinjection of biofilm-coated microbeads (C, D, E, F) in the ear pinna of LysM-EGFP transgenic mice at early time points (4–6 hpi). Innate immune cell recruitment towards injection sites was observed between 4.15 (C) to 4.45 hpi (D). Images show maximum intensity projections of EGFP (green) and mCherry (magenta) fluorescence. A progressive recruitment of EGFP+ innate immune cells was observed (white empty circles) at the injection sites with cell-bacteria contact areas (filled white arrowheads). *: autofluorescent hair (also in magenta). Filled white arrowheads indicate cell-bacteria contact. Live confocal imaging of two additional experiments at early time points after microinjection of biofilm-coated microbeads, at 4.15 hpi (E) and 4.00 hpi (F). Scale bar: 100 μm.
Fig 6.
Analysis of average speed and straightness of innate immune cells in the mouse ear pinna.
Average speed (A and C) and straightness (B and D) of EGFP+ cells recruited to injection sites at early time points after microinjection of PBS (control), planktonic bacteria and microbeads, or biofilm-coated microbeads. Data are expressed as median and interquartile ranges pooled from three different mice in three independent experiments for each group. Average speed (A) and straightness (B) of all cells (in contact with visible bacteria or not) in infected and control mice. Number of cells (N) analysed for each group at early time points: Control: N = 190 cells; Planktonic form: N = 721 cells; Biofilm form: N = 771. Average speed (C) and straightness (D) of cells either in contact (bacteria contact) or not (no bacteria contact) with planktonic or biofilm bacteria at early time points. Number of cells (N) analysed at early time points that were in contact or not in contact with bacteria, respectively: Planktonic form: N = 238 and 386 cells; Biofilm form: N = 433 and 276 cells. p≤0.05 was considered statistically significant (symbols: ****p≤0.0001; ***p≤0.001; **p≤0.01; *≤0.05; ns = non-significant).