Protection from septic peritonitis by rapid neutrophil recruitment through omental high endothelial venules

Abstract

Acute peritonitis is a frequent medical condition that can trigger severe sepsis as a life-threatening complication. Neutrophils are first-responders in infection but recruitment mechanisms to the abdominal cavity remain poorly defined. Here, we demonstrate that high endothelial venules (HEVs) of the greater omentum constitute a main entry pathway in TNFα-, Escherichia coli (E. coli)- and caecal ligation and puncture-induced models of inflammation. Neutrophil transmigration across HEVs is faster than across conventional postcapillary venules and requires a unique set of adhesion receptors including peripheral node addressin, E-, L-selectin and Mac-1 but not P-selectin or LFA-1. Omental milky spots readily concentrate intra-abdominal E. coli where macrophages and recruited neutrophils collaborate in phagocytosis and killing. Inhibition of the omental neutrophil response exacerbates septic progression of peritonitis. This data identifies HEVs as a clinically relevant vascular recruitment site for neutrophils in acute peritonitis that is indispensable for host defence against early systemic bacterial spread and sepsis.

Figure 1. HEVs of the greater omentum enable rapid neutrophil exit in TNFα-peritonitis.

(a) Confocal micrographs of the naive omentum show vascularization and milky spots (left panel). PNAd⁺PECAM-1⁺-HEVs are distinct vascular compartments within the milky spot (right panel). Scale bars, 120 μm (left) and 40 μm (right). (b) Non-treated (NT), omentectomized (OE) or sham-operated mice were compared by analysing the peritoneal lavage after 60 min i.p. treatment with TNFα. Cell number as mean±s.d. n=5 per condition of three independent experiments. Student's t-test **P<0.01, ***P<0.001. (c) Confocal micrograph of an inflamed omentum of a LysM-eGFP reporter mouse after 60 min i.p. TNFα treatment. Panlaminin is used as a vessel marker. Note the luminal accumulation of GFP^bright neutrophils in the PNAd⁻-postcapillary venule (arrow). Scale bar, 40 μm. Representative of five independent experiments. (d) Transmission electron micrographs of naive and TNFα-inflamed milky spot HEVs. Scale bar, 5 μm. Representative of >10 milky spots in two independent experiments. (e) Representative snapshots from a 4D intravital spinning disc microscopy video with PNAd- and panlaminin staining using a LysM-eGFP mouse (video 3). The arrow indicates extravasating GFP^bright neutrophils. Scale bar, 20 μm. (f) Video recordings of PNAd⁺ HEVs and PNAd⁻ PCV were used for LysM-eGFP neutrophil tracking 60 min after TNFα application. Colour-coding of tracks indicates time (20 min). HEV stained by MECA-79 in white. Arrows indicate active neutrophil transmigration sites. Representative of three independent experiments. Scale bar, 20 μm. (g) Analysis of an intravital recording of a milky spot HEV and a conventional PCV in the same field of view over a time course of 100 min in a LysM-eGFP mouse. TNFα was topically applied at 0 min and the integral of the pixel intensity as a measure of leucocyte accumulation in the region of interest was plotted over time. Data representative of four milky spots in two independent experiments. (h) Intravital microscopy of the inflamed exteriorized omentum in LysM-eGFP mice with PNAd staining. The dashed line marks the omental border. Scale bar, 100 μm. Representative of two independent experiments.

Figure 2. Unique adhesion receptor requirements of HEV-mediated neutrophil recruitment.

(a) Flow cytometry gating strategy and analysis of the peritoneal lavage after 30 or 60 min of TNFα-induced peritonitis with and without (NT, non-treated) PNAd inhibition by high-dose i.v. MECA-79 mAb injection. Data representative of two independent experiments. (b) The effect of different blocking antibodies on neutrophil accumulation in the abdominal lavage was evaluated after 1 h TNFα treatmensing flow cytometry as in (a). n=6 of least three independent experiments. Data as mean total cell number±s.d. Statistical significance is indicated relative to NT with TNFα. Student's t-test **P<0.01, ***P<0.001. (c) To investigate the luminal neutrophil behaviour, E-selectin, L-selectin or Mac-1 was blocked by i.v. injection of monoclonal antibodies and intravital microscopy of inflamed omental milky spots was performed in LysM-eGFP mice at high magnification. Time is colour-coded and visualizes cell motion within 3–5 min duration. Dashed lines indicate vessel borders. Scale bar, 20 μm. Quantification of intraluminal neutrophil behaviour in HEVs shown as pie charts for each condition. Cell numbers for rolling/crawling/arrest/time without any neutrophil–endothelium interaction in seconds: NT n=25/33/34/0, α-PNAd=1/1/0/210, α-E-selectin n=9/34/0/0 and α-Mac-1 n=34/2/2/0.

Figure 3. Bacterial peritonitis triggers HEV-mediated neutrophil recruitment required to prevent septic peritonitis.

(a) Polymicrobial sepsis was induced by caecal ligation and puncture and the greater omentum was imaged by confocal microscopy 4 h after surgery. Sham control did not show major signs of inflammation (Supplementary Fig. 4a). Representative of three independent experiments. Scale bar, 30 μm. (b) Fluorescently labelled E. coli were injected i.p. and confocal micrographs of the omentum were taken after 2 h. The left panel shows a low magnification image of two milky spots. The right panel shows LysM-eGFP neutrophils adjacent to HEVs and labelled E. coli. Scale bars, 100 μm (left) and 10 μm (right). (c) Flow cytometry data of the peritoneal lavage showing colocalization of fluorescently labelled E. coli and CD11b⁺F4/80⁺ macrophages or CD11b⁺Ly6G⁺ neutrophils 2 h after infection in untreated (NT, non-treated), L-selectin- or PNAd-blocked mice. Similar data has been obtained for omental extracts (Supplementary Fig. 4c). Data is representative of two independent experiments. Gating strategy on CD45⁺CD11b⁺ cells in Supplementary Fig. 4b. (d) Clinical evaluation of mice i.p. injected with live E. coli using a murine sepsis score with a maximum of 28 points. PBS-treated (NT, n=6), blocking anti-L-selectin antibody (n=4) or blocking anti-PNAd antibody treated (n=5) mice were compared over a 2-h time course in 2–3 independent experiments. Data shows mean±s.d. Significance shown compared with untreated condition. Two-way analysis of variance with Bonferroni posthoc correction. **P<0.01, ***P<0.001. (e) The abdominal lavage, the greater omentum and blood was collected after 2 h E. coli peritonitis for the determination of colony forming units (CFUs). Data shows mean±s.d. (f) Correlation of omental E. coli bacterial load and the maximal clinical sepsis score obtained during 2 h after infection. Each data point represents one mouse of the untreated or anti-L-selectin treated groups at the 120 min time point.