Fig 1.
Fixation of murine gastric mucosal tissue for intravital microscopy imaging.
(A) Cartoon representing the closed (or intact) stomach. The dashed line delineates the area of cutting to open the stomach. (B) Open stomach showing the inner gastric mucosa where the yellow square represents the imaging window. (C) After sedation, the mouse is placed in the right lateral decubitus position. The stomach is exposed by making an incision of approximately 150 mm across the lateral side. (D) Steps demonstrating the gastric mucosa preparation and its fixation by the suction ring device with an imaging window of 8 mm, which was designed, and 3D printed. (D-I) After opening the skin, the stomach is opened with a cauterizing scalpel and the gastric mucosa is exposed. (D-II) Any remaining gastric contents are carefully removed with a moist cotton swab and irrigation with isotonic solution. Rinsing with isotonic solution, taking care not to injure the gastric mucosa. (D-III) The vacuum chamber can now be placed on to the lining of the stomach to permit multiphoton imaging of the gastric mucosa. (D-IV) 3D-printed suction ring with an imaging window of 8 mm. (E) Overview of the gastric mucosa preparation for multiphoton imaging.
Fig 2.
VivoFollow the real time drift correction software.
(A) Time-lapse sequence imaging of LysM-GFP mouse gastric mucosa showing drift along the X and Y axis. The white dotted lines are for reference; the red dotted lines show the drift, and the red solid lines indicate the magnitude of the shift. The yellow arrow indicates the followed immobilized reference structure showing the imaging drift in (A) and the stable corrected time series in (B). (B) Effects of the live drift correction stabilization. There is no major drift from the yellow arrow and white dotted lines serving as references (see also S1 Video). Numbers indicate minutes. Scale bars are 50 μm. (C) VivoFollow cartoon showing the path of image acquisition and correction in x, y and z, from microscope to GPU-equipped computer.
Fig 3.
Neutrophil recruitment after H. pylori infection monitored by multiphoton microscopy in vivo.
Mice were infected with H. pylori for 24 hours or 6 weeks. (A) Multiphoton 4D time-lapse images of uninfected CX3CR1-GFP+ mice, which were injected with anti-Ly6G-PE antibodies to visualize neutrophils in red. (B) LysM-eGFP mice show neutrophil recruitment (green) after H. pylori 24h weeks of infection (red), second harmonic generation (SHG) showing the vessels walls (blue) or 6 weeks after infection in (C). (D) Histogram showing neutrophil velocity at 24 hours and 6 weeks post-infection. Neutrophils show significantly reduced mobility in the gastric mucosa in vivo after the first 24 hours of H. pylori infection compared to control. After 6 weeks of H. pylori infection, neutrophil velocity is significantly increased compared to control 24-hours post infection. Immunostaining image of 6 weeks infected gastric mucosal section showing neutrophils stained with anti-Ly6G-488 (green) and H. pylori-mRFP (red), DAPI (blue). Immunohistology of non-infected gastric mucosa (E), scale bar is 20 μm. (F) 24h after infection, showing H. pylori (magenta) localized inside the crypts and neutrophils (yellow). Scale bar is 10 μm. (G) 6 weeks after infection, showing H. pylori (red) located inside the crypts and neutrophils (green), DAPI (nucleus), scale is 20 μm. The arrows indicate the localization of the H. pylori inside the crypts. (H) Quantification of neutrophil influx after 24 hours of H. pylori infection and decrease after 6 weeks of infection. n = 4 mice. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, or not significant (ns). One way ANOVA t-test. Mean ± S.D.
Fig 4.
Interaction of macrophages with H. pylori monitored by multiphoton microscopy in vivo.
(A) Multiphoton imaging of CX3CR1-GFP mouse showing macrophages (green) promoting cytoplasmic extensions in closer contact with H. pylori, as indicated by the arrows in the upper panel. In the lower panel a 3D image reconstruction of the macrophages (green) and bacteria (red) in a closer proximity. Scale bar is 5 μm. (B-E) Multiphoton imaging of CX3CR1-GFP mouse showing H. pylori (red) localized inside macrophages (green) in vivo after 24 hours (B-C) or 6 weeks of infection (D-E), as indicated by the arrows. (C and D) the images represent the zoomed-out macrophages with H. pylori phagocytized. (F) Histogram showing the internalization of H. pylori by macrophages after 24 hours or 6 weeks of infection. (G-I) Immunohistology showing resident macrophages peripherally localized in crypts. Scale bars are 20 μm. (J) Quantification of macrophages in gastric mucosa after 24 hours and 6 weeks of H. pylori infection. n = 4 mice, ns (non-significant difference), ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. One way ANOVA t test. Mean ± S.D.
Fig 5.
Macrophage depletion and neutrophils response under PLX 5622 treatment.
(A-B) Multiphoton imaging of CX3CR1-GFP mouse gastric mucosa showing macrophages (green) and neutrophils, labelled by the injection of anti-Ly6G-PE antibodies (red), treated with PLX 5622 chow (B) and control chow treated (A), SHG (blue), scale bar is 20 μm. (C) Quantification of the CX3CR1-GFP positive cells after 7 days of PLX 5622 treatment. (D) Quantification of neutrophils in gastric mucosa after PLX treatment versus untreated (control). n = 8 mice, ns (non-significant difference), ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. One way ANOVA t-test. Mean ± S.D.
Fig 6.
Continuous macrophage depletion is associated with reduced H. pylori infection.
(A) Schematic representation of H. pylori infection (2–3 weeks) and PLX 5622 food treatment (3–4 weeks) to reduce macrophage replication. (B) Quantification of CX3CR1+ cells in the gastric mucosa after PLX treatment versus untreated (control) for 2 or 3 weeks of infection. n = 4 mice for each treatment. (C) Determination of colony forming units (CFU) of H. pylori infected CX3CR1+ mice after PLX treatment (PLX 7 days) or untreated (control). ns (non-significant difference), ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. One way ANOVA t-test. Mean ± S.D. Mouse cartoon by lemmling, sourced from Open Clip Art, licensed under CC0 1.0.
Fig 7.
H. pylori survival in infected macrophages.
(A) Bone marrow-derived mouse macrophages were incubated with H. pylori PMSS1-RFP, P12-GFP or P12ΔvacAΔcagA. After 6h or 24h infected macrophages were lysed, and H. pylori were re-isolated for colony forming unit (CFU) assay. (B) CFU quantification indicating the bacterial survival after 6h of internationalization by macrophages. (C) Infected macrophages were fixed and stained with anti-F4/80-Alexa-488 (green) or Alexa-594 (red) for macrophages, and DAPI for nucleus (blue). Images show H. pylori internalized by macrophages. Scale bar is 10 μm. (D) Quantification of macrophages infected with H. pylori strains PMSS1-RFP (red) or P12-GFP (green) for 6h or 24h. n = 5–6, ****p<0.0001, ***p<0.001, **p<0.01,*p<0.05, or not significant (ns). Two-way ANOVA t-test. Mean ± S.D.
Fig 8.
H. pylori internalization by macrophages.
3D rendering image sequences showing the localization of PMMS1-RFP (A) or P12-GFP (B) after 6h of infection inside macrophages. The images series are displayed from different angles and were extracted from S9 and S10 Videos. Scale bar is 10μm. DAPI is labelling the nucleus. Anti-F4/80 antibodies were used to label macrophages.