Phenotypically distinct neutrophils patrol uninfected human and mouse lymph nodes

Significance The classical view of neutrophils is as circulating phagocytes that are recruited to tissues following infection or injury. Here we show that neutrophils were present in mouse and human lymph nodes in the absence of perturbation. Lymph node neutrophils were phenotypically distinct, with increased expression of major histocompatibility complex II, and predominantly localized to the interfollicular zone, where CD4 T lymphocytes are activated. Neutrophils trafficked into lymph nodes via blood and lymphatic vessels, and were capable of rapidly carrying systemically acquired, IgG antibody-opsonized cargo to lymph nodes. These data support a novel role for neutrophils in homeostatic immune surveillance, sampling circulating antigens and delivering them to lymph nodes, with the potential to activate adaptive immunity.


Human LN analysis and neutrophil isolation
Whole human LNs were removed from deceased transplant organ donors. Human blood neutrophils were isolated as previously described (1) for ex vivo stimulation. All human blood and tissue samples were de-identified prior to use in the study. Ethical approval was obtained from East of England Research Ethics Committee (references 12/EE/0446, 15/EE/0152), and experiments were performed in accordance with the Declaration of Helsinki.

IC stimulation
IC was generated in vitro by incubating A647-conjugated whole OVA with rabbit anti-OVA (1:10 ratio) at 37°C for 1 hour. OVAIC (5 µg/ml OVA) was used to stimulate human blood neutrophils for 1 hour followed by flow cytometry, or murine BM neutrophils for 2 hours followed by reverse-transcriptase polymerase chain reaction (RT-PCR) using primers for H2-Aa, H2-Ab1, Cd80, Cd86 and Cd40. For co-culture experiments, OVA or OVAIC-stimulated neutrophils were incubated with OTII CD4 T cells (2:1) for 48 hours prior to staining for flow cytometry. For in vivo stimulation experiments, OVAIC was injected intravenously (5 µg OVA / mouse); PEIC was generated locally in vivo by intraperitoneal administration of 2 mg rabbit anti-PE followed by subcutaneous footpad injection of 15 µg PE.

Confocal microscopy
LNs were fixed in 1% paraformaldehyde, dehydrated in 30% sucrose, and frozen in optimal cutting temperature (OCT) compound. 30-50 µm sections were stained overnight at 4°C in a buffer containing 0.1 M TRIS, 1% bovine serum albumin, 1% mouse serum and 0.3% Triton X-100. Secondary antibodies were applied for 30 minutes before mounting with Vectashield medium. Images were acquired using a Leica TCS SP8 microscope and 40x oil immersion objective, with optical sections under 2 µm.

Intravital microscopy
Intravenous Qtracker655 (10-20 µl) and subcutaneous anti-LYVE-1-PE (5 µl to hock) were used to label blood and lymphatic vessels respectively. Popliteal LN was exposed and intravital imaging performed with a Chameleon Vision-S tuneable Ti:Sapphire multi-photon laser and Leica TCS SP8 microscope with the animal kept at 36°C throughout. Sequential excitation was used, with two-photon excitation at 920 nm for GFP, collagen second harmonics, PE and Qtracker, and single photon excitation at 633 nm for A647. For some experiments focal laser damage was achieved by two-photon excitation of a confined area at 860 nm and 90% power for 1 minute. Movies were acquired using a 25x water objective, with one Z stack every 40 seconds and 2 µm optical sections.

Image processing and data analysis
Intravital movies and confocal images were processed using Bitplane Imaris and ImageJ (NIH). Cells were tracked using the 'surfaces' function and dynamic parameters such as mean speeds generated. Time-lapse movies were recorded at 10 frames per second (fps), or at slower rates (as indicated) for clarity of presentation. Data were analyzed using GraphPad Prism, with statistical analysis performed using unpaired t-tests or one-way ANOVA and Holm-Sidak correction for between-group multiple comparisons. Data are shown as mean ± SEM unless otherwise indicated. p < 0.05 was considered statistically significant.