Neutrophils recruited through high endothelial venules of the lymph nodes via PNAd intercept disseminating Staphylococcus aureus

Abstract

Staphylococcus aureus is a skin- and respiratory tract-colonizing bacterium and is the leading cause of community-acquired skin infections. Dissemination of these bacteria into systemic circulation causes bacteremia, which has a high mortality rate. Therefore, understanding the immunologic barriers that prevent dissemination is critical to developing novel treatments. In this study, we demonstrate that an S. aureus breach across skin leads to some migration of the pathogen to the draining lymph node, but no further. While subcapsular sinus (SCS) macrophage in lymph nodes were important in detaining S. aureus, a rapid complement-dependent neutrophil recruitment (independent of the SCS macrophage) via high endothelial venules (HEVs) resulted in high numbers of neutrophils that intercepted the bacteria in the lymph nodes. Peripheral Node Addressin together with its two ligands, L-selectin and platelet P-selectin, are critical for recruiting neutrophils via the HEVs. Almost no neutrophils entered the lymph nodes via lymphatics. Neutrophils actively phagocytosed S. aureus and helped sterilize the lymph nodes and prevent dissemination to blood and other organs.

Keywords: L-selectin; Staphylococcus aureus; endothelium; lymph node; neutrophil.

Fig. 1.

Neutrophils are recruited to the lymph nodes following S. aureus MW2 footpad infection. (A) CFU counts in popLN, spleen, liver, blood, kidney, and lung at 24, 48, and 72 hpi with 2.5 × 10⁷ S. aureus MW2. nd, not detected. Data shown as mean ± SD. n = 12 for 24 and 48 h; n = 6 for 72 h. (B) Representative image of intravital microscopy of popLN showing presence of LysM EGFP neutrophils (green) at steady state and accumulation at 4 hpi with S. aureus MW2. The dashed line indicates lymph nodes. (Scale bar: 30 μm.) (C) Representative flow cytometry plot of neutrophils in the popLN at steady state and at 4 hpi with S. aureus MW2 in the footpad. (D) Dynamics of neutrophil recruitment to the popLN following S. aureus MW2 infection as measured by flow cytometry. BL, baseline. Data are mean ± SD. n = 8 at 4 h; n = 4 for all other time points.

Fig. 2.

Neutrophils primarily use blood vessels to enter lymph nodes. (A) Representative images of afferent collecting lymphatic vessels of popLNs at steady state or at 3 hpi with S. aureus MW2 in an LysM EGFP reporter mouse. Green, neutrophils; magenta, secondary harmonic (SH) generation showing collagen structures; red, lymphatics labeled with an injection of Evans blue dye. Images are displayed as focus stacking of six 2-μm image slices. (Scale bar: 200 μm.) (B) Quantification of neutrophils seen passing through lymphatic vessels during 3 h of imaging. Data are mean ± SD. n = 3 for uninfected; n = 5 for infected. (C) PopLN blood vessels at steady state and at 3 hpi with S. aureus MW2 in an LysM EGFP reporter mouse. Green, neutrophils; red, i.v. TRITC-dextran. Images are displayed as focus stacking of six 2-μm image slices. (Scale bar: 52 μm.) (D) Quantification of neutrophils seen interacting with blood vessels over 3 h of imaging. Data are mean ± SD. n = 3. (E) Still image from a time-lapse video of LysM EGFP neutrophils (green) transmigrating at HEVs, labeled with MECA-79 in red, following S. aureus MW2 infection in the footpad. Images are displayed as focus stacking of six 2-μm image slices. (Scale bar: 32 μm.) The arrow indicates one of many examples of transmigration. Images are displayed as focus stacking of six 2-μm image slices. (Scale bar: 32 μm.) *P < 0.05. ns, not significant.

Fig. 3.

C5aR directs neutrophil recruitment to the lymph nodes independent of lymph node macrophages and virulence of S. aureus MW2. (A) Neutrophil recruitment to the popLN at 4 hpi with wild-type S. aureus MW2, heat-killed S. aureus MW2, and Δagr S. aureus MW2. Data are mean ± SD. n = 12 for WT S. aureus, n = 4 for mutant and heat-killed S. aureus. Data were pooled from two independent experiments. (B) Neutrophil recruitment to the popLNs at 4 hpi with S. aureus MW2 after treatment with CLLs to deplete lymph node macrophages. Data are mean ± SD. n = 8; data were pooled from two independent experiments. (C) Neutrophil recruitment to the popLNs at 4 hpi with S. aureus MW2 following pertussis toxin treatment (PTX). Data are mean ± SD. n = 3. (D) Neutrophil recruitment to the popLNs in a CCR7 KO mouse at 4 hpi with S. aureus MW2. Data are mean ± SD. n = 6; data were pooled from two independent experiments. (E) Neutrophil recruitment to the popLNs at 4 hpi with S. aureus MW2 following pretreatment with anti-C5aR antibody. Data are mean ± SD. n = 5. *P < 0.05; **P < 0.01. ns, not significant.

Fig. 4.

Neutrophils require L-selectin for recruitment to the lymph nodes following S. aureus MW2 infection. (A) Representative still images from a time-lapse intravital video of popLN showing LysM EGFP neutrophils (green) in i.v. dextran-labeled blood vessels (red) following blocking antibody pretreatment at 2–3 hpi. Images are displayed as focus stacking of six 2-μm slices. (Scale bar: 63 μm.) (B) Quantification of neutrophils in lymph node blood vessels following pretreatment with blocking antibodies from 2 hpi to 4 hpi. Data are mean ± SD number of neutrophils per total area of vessels (μm² × 10,000) to normalize data for differences in vessel size. n = 6. Each individual experiment was considered n = 1. (C) Number of neutrophils recruited to popLN at 4 hpi following pretreatment with anti–L-selectin antibody (n = 3), anti–PSGL-1 antibody (n = 3), or the two combined (n = 8) as determined by flow cytometry. Data are mean ± SD, pooled from two independent experiments. (D) Number of neutrophils in the popLNs at 4 hpi following pretreatment with MECA-79 (n = 5), combined MECA-79 and anti–PSGL-1 antibody (n = 3), anti–P-selectin antibody (n = 6), or combined anti–P-selectin and anti–L-selectin (n = 5) treatment as determined by flow cytometry. Data are mean ± SD, pooled from two independent experiments. (E) Number of neutrophils in the popLNs at 4 hpi following platelet depletion or combined platelet depletion and anti–L-selectin as determined by flow cytometry. Data are mean ± SD. n = 6; data pooled from two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Neutrophil recruitment to the lymph nodes is necessary to control S. aureus MW2 dissemination through lymphatics. (A) Bacteria load in peripheral organs and blood at 4 hpi without and with blockade of neutrophils by i.v. injection of MECA-79 antibody at 1 h before S. aureus MW2 infection in mice pretreated with CLL. nd, not detected. Data are shown as CFU/g of organ or CFU/mL of blood, mean ± SD. n = 5; data pooled from two independent experiments. (B) Image capture from intravital video of lymph node SCS showing neutrophil (red) containing phagocytosed bacteria (green). The image shows focus stacking of six 2-μm image slices. (Scale bar: 20 μm.) (C) Bacterial load in draining popLNs and subsequent draining inguinal LNs at 4 hpi with S. aureus MW2 with and without blockade of neutrophils by i.v. injection of MECA-79 antibody at 1 h before infection in mice pretreated with CLL. Data are mean ± SD CFU per lymph node. n = 5; data pooled from two independent experiments. *P < 0.05.