Peritoneal GATA6+ macrophages function as a portal for Staphylococcus aureus dissemination

Abstract

Essentially all Staphylococcus aureus (S. aureus) bacteria that gain access to the circulation are plucked out of the bloodstream by the intravascular macrophages of the liver - the Kupffer cells. It is also thought that these bacteria are disseminated via the bloodstream to other organs. Our data show that S. aureus inside Kupffer cells grew and escaped across the mesothelium into the peritoneal cavity and immediately infected GATA-binding factor 6-positive (GATA6+) peritoneal cavity macrophages. These macrophages provided a haven for S. aureus, thereby delaying the neutrophilic response in the peritoneum by 48 hours and allowing dissemination to various peritoneal and retroperitoneal organs including the kidneys. In mice deficient in GATA6+ peritoneal macrophages, neutrophils infiltrated more robustly and reduced S. aureus dissemination. Antibiotics administered i.v. did not prevent dissemination into the peritoneum or to the kidneys, whereas peritoneal administration of vancomycin (particularly liposomal vancomycin with optimized intracellular penetrance capacity) reduced kidney infection and mortality, even when administered 24 hours after infection. These data indicate that GATA6+ macrophages within the peritoneal cavity are a conduit of dissemination for i.v. S. aureus, and changing the route of antibiotic delivery could provide a more effective treatment for patients with peritonitis-associated bacterial sepsis.

Keywords: Infectious disease; Innate immunity.

Figure 1. S.aureus escapes from liver Kupffer cells and grows into the peritoneal cavity.

(A) SD-IVM images of GFP S. aureus (green) catching by Kupffer cells (F4/80; purple) 5 minutes (left; see also Supplemental Video 1) or 8 hours (right) after i.v. infection. Neutrophils are labelled in red (Ly6G). Scale bars: 50 μm. (B) Enlarged view of a Kupffer cell (F4/80; purple) 8 hours after infection with growing GFP S. aureus (green) inside. Scale bar: 10 μm. (C) Representative images of SD-IVM of liver at indicated time points after i.v. infection. Mesothelium in blue (podoplanin-EF660), GFP S. aureus in bright green, and hepatocytes in dark green autofluorescence. Scale bar: 90 μm (n = 3 per time point). (D and E) Kupffer cells were isolated from mice infected with GFP S. aureus for 30 minutes, cultured, and imaged over 11 hours ex vivo. (D) Representative images of time-lapse videos (Supplemental Video 2). Scale bar is 60 µm. (E) Analysis of time-lapse videos, 4 FOV per mouse; shown is the mean ± SEM (n = 6 from 4 independent experiments). (F) Mice were infected i.v. with 5 × 10⁷ CFU S. aureus. Blood was collected, peritoneal lavage was performed, and organs were harvested at the indicated time points from which CFU were determined. Shown is the geometric mean (n = 7–9 from 3 independent experiments), Kruskal-Wallis with Dunn’s post test; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the 0.5-hour time point for each organ.

Figure 2. S.aureus infects macrophages inside the peritoneal cavity.

(A–C) Flow cytometry analyses of peritoneal lavage at indicated time points after infection. The LPM and SPM gating strategy is shown in Supplemental Figure 3. (A) Total counts of free GFP S. aureus, mean ± SEM (n = 2–4 from 2 independent experiments). (B) S. aureus cell localization inside the peritoneal cavity, mean percentage (n = 6 from 3 independent experiments). (C) Quantitative analyses for total cell count of LPMs, SPMs, monocytes, and neutrophils over infection time. Boxes extend from the 25th to 75th percentiles, whiskers show minimum to maximum, and lines indicate the median. n = 3–8 from at least 2 independent experiments, Kruskal-Wallis with Dunn’s post test, *P < 0.05 and **P < 0.01. (D and E) Peritoneal cells were harvested 46 hours after GFP S. aureus bloodstream infection, and F4/80⁺ macrophages or Ly6G⁺ neutrophils were isolated, cultured, and imaged ex vivo. (D) Representative images of time-lapse videos are shown (Supplemental Video 3). (E) Analysis of time-lapse videos, 4 FOV per mouse (n = 4 from 2 independent experiments).

Figure 3. A lack of peritoneal macrophages results in more neutrophil recruitment and less dissemination of S. aureus to the kidneys.

GATA6-KO^mye mice or littermate controls were infected i.v. (A–H) or i.p. (I and J) for indicated time points with 5 × 10⁷ S. aureus Newman strain. (A–E and J) Organs were collected and the CFU determined; the geometric mean is shown. n = 5–10 from at least 2 independent experiments, Mann-Whitney test, *P < 0.05. (F) S. aureus cell localization inside the peritoneal cavity determined by flow cytometry, mean percentage. n = 5 (WT) or 6 (GATA6-KO^mye). (G and I) Neutrophil numbers in the peritoneal cavity assessed by flow cytometry. n = 5–6, mean ± SEM, unpaired t test, **P < 0.01. (H) Body weight loss, n = 6–9 from 3 independent experiments, mean ± SEM, unpaired t test, *P < 0.05.

Figure 4. S.aureus infects the kidney initially on the capsule and then infiltrates into the interstitium.

(A) Representative 2-photon stitched image from kidney around 150 μm deep where approximately 20 glomeruli can be seen (left). Vasculature including glomeruli appear in grey (AF680-albumin), tubules appear in dark yellow autofluorescence, and S. aureus is in bright green GFP (not visible). The right panel shows 3 enlarged glomeruli. (B) Two-photon IVM images from a glomerulus (Supplemental Video 5) during the first 10 minutes of i.v. infection with 5 × 10⁷ S. aureus Newman. Vasculature (glomerulus) is shown in blue (Qtracker 655), tubules are autofluorescent in all channels and appear in brown around the glomerulus, neutrophils are labelled in red (Ly6G-PE), and GFP S. aureus appears in yellow inside a neutrophil (white arrow). (C) Representative stitched images from kidney surfaces at indicated time points after infection. Scale bar: 500 μm. S. aureus (bright green) is circled in white and an enlarged view of bacteria on the surface at 48 hours is shown. (D) 3D representation FOV of a kidney 48 hours after infection with GFP S. aureus (bright green) on the capsule (purple collagen/second harmonic generation [SHG]) and the vasculature in grey (AF680-albumin). (E) Representative FOV of a kidney 72 hours after infection, view from the bottom of the Z-stack. Tubules appear autofluorescent in brown, GFP S. aureus is bright green and indicated with white arrows. (F) Analysis of location of bacteria as determined using 2-photon IVM of the kidney. Six FOV per mouse were analyzed (n = 3–4 per time point).

Figure 5. Intraperitoneal liposomal vancomycin (vancosomes) targets tissue resident macrophages to eradicate infections and sustain survival.

Mice were infected i.v. with 5 × 10⁷ S. aureus Newman and treated 2 (24 hours plus 48 hours, A–C) or 3 times (24 hours plus 48 hours plus 72 hours, D) with either vancomycin i.v (red), vancosomes i.p. (blue), a combination of vancomycin i.v. plus i.p. (orange), or vancomycin i.v. plus vancosomes i.p. (green). (A) Seventy-two hours after infection, organs were harvested, peritoneal lavage was performed, and CFU were determined (n = 6–11 from 3 independent experiments). Data are presented as the geometric mean; Kruskal-Wallis with Dunn’s post test; *P < 0.05 and **P < 0.01. (B) Representative 2-photon IVM images of kidneys 72 hours after infection with or without treatment 2 times. Dead tubular cells are stained with Sytox Orange (red), tubules appear in dark green autofluorescence. Scale bar: 75 μm. (C) Analysis of B. Number of Sytox Orange⁺ (dead) cells from 8 FOV per mouse, data represent the mean ± SEM (n = 3, 1-way ANOVA with Bonferroni’s multiple comparisons test). *P < 0.05, ***P < 0.001, and ****P < 0.0001. (D) Survival curve of S. aureus-infected and treated mice (n = 4 mice per group from 2 independent experiments). Log-rank test compared with vancomycin i.v., *P < 0.05 and **P < 0.01.