Significance
Staphylococcus aureus is a bacterium causing many community-acquired skin infections. However, as dissemination from skin infections is rare, we were interested in understanding what mechanisms the body has in place to prevent escape of bacteria from skin infections via lymphatics to the rest of the body. We found that after infection, neutrophils were almost immediately recruited to the lymph nodes via specialized blood vessels to cut bacteria off at the pass. Neutrophils worked together with resident macrophages to prevent S. aureus spread, as removing both these immune cells from the lymph nodes resulted in bacterial dissemination to peripheral organs. This study demonstrates the importance of the lymph node neutrophils in early infection to prevent systemic dissemination of bacteria.
Keywords: neutrophil, lymph node, L-selectin, Staphylococcus aureus, endothelium
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.
It is well appreciated that neutrophils, the most abundant immune cells in human blood, are the first and almost certainly the most critical responders in Staphylococcus aureus infections. The rapidity with which neutrophils are recruited serves to prevent bacterial dissemination by surrounding the infectious nidus and releasing powerful antimicrobial defenses (1). However, neutrophils need to be recruited from the bloodstream via a series of molecules, including selectins and integrins. Although these molecules are presynthesized and/or rapidly expressed in vasculature of the skin, it takes the neutrophils a few minutes to begin to accumulate at the infection site, giving the bacteria a head start to potentially disseminate to other tissues via either the lymphatics or the bloodstream. Therefore, neutrophils not only must enter the infection site, but also must chase after escaped bacteria. However, one could envisage a more efficient design than chasing after bacteria, either (i) having an immune barrier, such as the lymph node, in place and/or (ii) intercepting the bacteria by rapidly recruiting neutrophils into lymph nodes via the blood vessels, thereby “heading the bacteria off at the pass.”
While lymph nodes have received much attention as sites of the adaptive immune response, there is a growing body of evidence indicating that they also function as an important barrier to foreign particles and pathogens. Indeed, resident subcapsular sinus (SCS) macrophages are localized strategically so that they sample the incoming lymph and capture antigen, viruses (2–5), and even bacteria such as Salmonella typhimurium and Pseudomonas aeruginosa (6). Clearly, along with being considered the bastion of adaptive immunity, the lymph nodes also should be noted for their innate barrier function. However, in previously studied tissues, macrophages alone cannot contain infections and require other immune cells. Indeed, a tissue devoid of neutrophils despite having ample macrophages and other innate immune cells will become infected (7, 8). As such, it would seem reasonable that neutrophils also would be rapidly recruited to lymph nodes to further prevent bacterial dissemination and to sterilize the lymph nodes. Recruitment of neutrophils to lymph nodes has been reported in response to adjuvants (9, 10), parasites (11), and some (12), but not all, bacteria (6). Neutrophil recruitment to inguinal and cervical lymph nodes has also been reported following infection with heat-killed (13) and live S. aureus (14), respectively, the former via lymphatics and the latter via blood vessels when S. aureus was injected near lymph nodes.
Using two-photon intravital microscopy, we have identified numerous interesting mechanisms preventing dissemination of S. aureus in mice. We report that S. aureus disseminates from the footpad via the lymphatics and is stopped in the lymph node. We identify a small proportion of “temporarily resident” neutrophils already awaiting disseminating bacteria in the parenchyma of lymph nodes under basal conditions. A huge neutrophilic influx occurs very rapidly via complement activation, and the majority of these neutrophils use the lymph node high endothelial venules (HEVs) to ambush S. aureus. Importantly, neutrophil recruitment is entirely dependent on Peripheral Node Addressin (PNAd) and neutrophil L-selectin on neutrophils and P-selectin on platelets. Blocking the rapid recruitment of neutrophils led to a profound increase in bacterial dissemination to liver and other organs.
Results
Neutrophils Are Recruited to the Draining Lymph Node Following Peripheral Infection.
To model the breach of S. aureus across the skin barrier, S. aureus MW2, a clinically important strain for community-acquired methicillin-resistant S. aureus infection (15), was introduced either through a bacteria-coated needle puncture wound or a very small-volume injection into the hind footpad. Because only a small volume was injected, the response was similar to that from the needle prick, but the latter was 50% less robust (Fig. S1). In this study, we used both the injection model and puncture model of infection. We found that S. aureus was able to readily enter the popliteal lymph nodes (popLNs) but did not breach this barrier, as no bacteria were detected in the bloodstream or any other organ tested, including liver, kidney, spleen, and lungs, at multiple time points (Fig. 1A).
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.
After footpad infection, S. aureus in the popLNs was imaged with two-photon intravital microscopy. To visualize neutrophil behavior, we used Lysozyme M (LysM) fluorescent EGFP reporter mice. Flow cytometry confirmed that >90% of the EGFP-positive cells in the lymph nodes were neutrophils (Fig. S2). Surprisingly, at steady state before any introduction of bacteria, there were already ∼1,000 neutrophils present per popLN, as seen by intravital imaging (Fig. 1B) and confirmed and counted by flow cytometry (Fig. 1 C and D). These cells were active in the lymph node, exhibiting random crawling (Movie S1). These tissue-resident neutrophils appeared to be constantly infiltrating into the lymph node, as depletion of neutrophils from the blood resulted in complete disappearance of the local lymph node population of neutrophils within 24 h (Fig. S3A), even though this route of administration did not deliver antibody to the lymph node parenchyma (Fig. S3B).
There was a spectacular increase in neutrophil number in the lymph node, from ∼1,000 neutrophils per lymph node to >250,000 neutrophils per lymph node within 5 to 6 h postinfection (hpi) with S. aureus, as assessed by flow cytometry (Fig. 1 C and D). At 4 hpi, the lymph nodes were fully seeded with neutrophils, which moved around the parenchyma, creating dynamic swarms (Movie S2). PBS injection in the same location and volume did not recruit neutrophils into the lymph node (Fig. S1).
Neutrophils Enter the Lymph Nodes via Blood Vessels.
To determine the route of entry of neutrophils into the lymph node, we used two-photon intravital microscopy of the LysM EGFP mice. Imaging of the afferent lymphatics was performed during the 4 h after infection along the length of the exposed lymphatic. Despite this extensive assessment of neutrophil trafficking through the lymphatics, only one in five lymphatic vessels contained any neutrophils following infection, and in the majority of mice, no neutrophils were seen in lymphatics. Fig. 2A presents a representative image of a lymphatic vessel at the peak time point of neutrophil recruitment into lymph nodes, with no visible neutrophils (quantified in Fig. 2B). Lymph was flowing in the lymphatics, as it was possible to see GFP-expressing S. aureus moving through the vessels (Fig. S4).
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.
Considering the large increase of neutrophils seen infiltrating the lymph node, it was unlikely the lymphatics were the main portal of entry. Imaging of the blood vessels of the lymph nodes at steady state showed few neutrophils rolling and adhering (Fig. 2C) but following infection, neutrophils exhibited crawling, adhesion, and transmigration in lymph node blood vessels (Fig. 2C). Fig. 2D quantifies the number of neutrophils that interacted with the endothelium over a 3-h period. Movie S3 shows neutrophils emigrating from blood vessels and swarming to a nearby nidus. Fluorescent-labeled antibody MECA-79, a specific antibody clone detecting HEVs, was injected i.v. at nonblocking concentrations, and intravital imaging revealed that most neutrophils used primarily MECA-79–positive vessels (Fig. 2E). Fig. 2E presents snapshots from time-lapse images of one of many neutrophils crossing through the HEVs into lymph node tissue.
Chemotactic Signals Are Required for Neutrophil Recruitment to the Lymph Node.
To understand the mechanisms leading to neutrophil recruitment via the HEVs, we first determined whether neutrophils were detecting secreted bacterial products released from live bacteria. However, heat-killed bacteria showed no differences in neutrophil recruitment compared with live S. aureus, as quantified by flow cytometry (Fig. 3A). Next we used the agr mutant, which, due to a defect in gene regulation does not produce many of the virulence factors of the S. aureus strain (16). However, similar numbers of neutrophils were detected in the lymph nodes regardless of whether the avirulent or wild-type S. aureus MW2 strain was used (Fig. 3A). Depletion of lymph node macrophages by clodronate-loaded liposomes (CLLs) also did not affect neutrophil recruitment (Fig. 3B). CLLs were effective in depleting the SCS macrophages (Fig. S5).
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.
To test whether endogenous molecules that stimulate GαI protein coupled receptor (GPCR) signaling were involved, we used pertussis toxin to inhibit these pathways and noted decreased neutrophil recruitment to the lymph nodes after infection with S. aureus (Fig. 3C). CCR7 is the key activating GPCR for lymphocyte recruitment into the lymph nodes (17). Therefore, we tested CCR7 knockout (KO) mice, and determined that neutrophil recruitment was not different in these mice (Fig. 3D). In contrast, neutrophil recruitment in complement-deficient (C3 KO) mice was reduced (Fig. S6), but not completely inhibited. However, we and others have detected C5a in C3 KO due to activation of complement via serine proteases (18). Indeed, blocking C5aR with a specific C5aR antibody completely prevented recruitment of neutrophils in response to S. aureus (Fig. 3E). This indicates that C5aR is absolutely necessary for directing neutrophils to the lymph nodes following infection.
Neutrophils Use L-Selectin and PSGL-1 at HEVs to Enter Lymph Nodes.
L-selectin binding to PNAd is required for B and T cells to enter lymph nodes at the HEVs (19), and neutrophils express high levels of L-selectin. To test whether neutrophils require L-selectin for entry into the lymph nodes, anti–L-selectin antibody was administered i.v. at 1 h before S. aureus infection. Anti–L-selectin treatment did not reduce the interactions of neutrophils with blood vessels (Fig. 4 A and B). Neutrophils predominately use PSGL-1 to enter into peripheral vascularized tissues (20). Intravital imaging of blood vessels within the infected lymph nodes showed essentially identical neutrophil–vasculature interactions following anti–PSGL-1 treatment (Fig. 4 A and B). However, when anti–L-selectin antibody and anti–PSGL-1 antibody were administered in tandem, neutrophil interactions with the vessel walls were significantly reduced (Fig. 4 A and B). We also examined the total number of neutrophils that infiltrated S. aureus-infected lymph nodes by flow cytometry, and found that L-selectin or PSGL-1 antibody alone reduced neutrophil recruitment by approximately 50%, whereas tandem administration of both antibodies reduced neutrophil recruitment into the lymph nodes by 95% (Fig. 4C).
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.
Neutrophil recruitment to the footpad was assessed to ensure specificity of the blocking effect to the lymph node vasculature, by measuring myeloperoxidase (MPO) levels, which are proportional to neutrophil numbers (21). MPO levels were reduced when PSGL-1 antibody was administered (Fig. S7A); however, the combination of anti–PSGL-1 and anti–L-selectin antibodies did not further reduce neutrophil recruitment into the footpad, suggesting that L-selectin has effects only in the lymph node HEVs, and not in the periphery (Fig. S7A). L-selectin antibody alone had no effect on neutrophil recruitment to infected footpads (Fig. S7A).
Platelets Contribute to PSGL-1–Dependent Neutrophil Recruitment to the Lymph Node via PNAds.
PNAds are ligands for L-selectin and are expressed almost exclusively on HEVs, whereas PSGL-1 typically binds P-selectin (22, 23). We blocked the L-selectin ligands with an anti-PNAd antibody, MECA-79, first alone and then together with an anti–PSGL-1 antibody. We expected to find complete elimination of recruitment with the tandem administration, but not with MECA-79 alone. Unexpectedly, blocking with MECA-79 both alone and in combination with anti–PSGL-1 before infection resulted in complete blockade of neutrophil recruitment (Fig. 4D). MECA-79 had no effect on neutrophils accumulating at the site of infection, suggesting that this is not just a nonspecific effect on all neutrophils (Fig. S7B).
According to the literature, PSGL-1 binds P-selectin, not PNAds (23). As expected, blocking of P-selectin alone had no effect on recruitment, whereas tandem inhibition of P-selectin together with L-selectin antibodies blocked all neutrophil recruitment into the lymph nodes (Fig. 4D). Clearly, neutrophil recruitment to the lymph nodes via the blood vessels is controlled by distinct pathways, including an L-selectin–PNAd interaction and a P-selectin–PSGL-1 interaction, but the latter is dependent on PNAd as well. While it is known that P-selectin binds PNAd (24, 25), there is no P-selectin on neutrophils, making this observation difficult to explain.
The only other source of P-selectin besides endothelium are platelets (26). Indeed, labeling platelets in vivo with CD49b revealed platelets interacting with the endothelium and neutrophils in the HEVs (Fig. S8A). We depleted platelets to check for an effect on neutrophil recruitment with and without L-selectin. Consistent with previous data, there was no change in neutrophil recruitment after platelet depletion alone, but tandem treatment with L-selectin–blocking antibody again dramatically inhibited neutrophil recruitment to the lymph nodes (Fig. 4E). Platelet depletion did not significantly affect neutrophil recruitment to the site of infection, suggesting that platelets only affected neutrophil recruitment into the lymph nodes (Fig. S8B).
Rapid Neutrophil Recruitment to the Lymph Nodes Is Necessary to Prevent Dissemination of Bacteria.
Wild-type mice have no dissemination of bacteria beyond the popLN into peripheral organs (Fig. 1A). Removing only the lymph node-resident macrophage with CLL injection into the calf as described previously (2) resulted in some dissemination of bacteria to peripheral organs, including the spleen, liver, and, to some extent, lung (Fig. 5A). In our previous work, blocking PNAd with MECA-79 completely blocked neutrophil recruitment to the lymph nodes without affecting neutrophil recruitment to the infection site. As such, we used this antibody to determine the importance of neutrophil recruitment into the lymph node. As MECA-79 treatment in WT mice resulted in CFU levels below the level of detection, MECA-79 treatment was used in CLL-treated mice. After treatment with MECA-79 in mice depleted of lymph node macrophages, there was a 10- to 100-fold significant increase in bacterial dissemination to the liver and spleen and a trend toward increased bacterial colonization in kidney and lung compared with untreated mice (Fig. 5A).
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.
Clearly, the neutrophils function as a very important barrier, intercepting bacteria before they can disseminate to other organs. Intravital microscopy showed neutrophils efficiently phagocytosing GFP-expressing S. aureus bacteria in the lymph nodes (Fig. 5B and Movie S4). In the popLN, there was a significant increase in bacterial load in the absence of neutrophils (Fig. 5C). Clearly, the neutrophils in lymph nodes have an antimicrobial function in the nodes themselves, after recruitment via the mechanism described in Fig. S9.
Discussion
Breach of the skin barrier by bacteria through cuts, abrasions, insect bites, and burns occurs daily, yet bacterial dissemination from the afflicted site to systemic circulation is rare in healthy individuals. This seems unusual, as bacteria always arrive at the skin site before neutrophils and should be able to escape to other sites. The concept that neutrophils would then chase after these pathogens seems to be a recipe for disaster. When we injected S. aureus into the footpad, bacteria always managed to reach the draining lymph node, but were never found in other organs. These data suggest that the bacteria must leave infected tissue via the lymphatics, not directly via blood vessels. This supports a previous study that found when bacteria breached the epithelial barrier in the gut, no bacteria could be detected in portal blood, but pathogens were found in the lymph nodes (27). There is growing evidence that the lymph nodes have a barrier function; the macrophages in the SCS can sample antigens, but can also capture viruses and bacteria (2, 4–6). However, in our preliminary experiments, depletion of these macrophages caused only small increases in dissemination, suggesting that the lymph nodes have multiple mechanisms that permit them to function as an effective barrier.
In this study, we found a small but persistent population of neutrophils present in the parenchyma of unperturbed lymph nodes detected by imaging and flow cytometry. In addition to waiting in ambush for pathogens to arrive in the lymph node, there is an intercept course that causes massive recruitment of neutrophils to the lymph nodes via the HEVs so that the pathogens can be captured. Indeed, together with the SCS macrophages, the neutrophil recruitment in the lymph nodes provides critical protection, ensuring that no bacteria reach the systemic circulation.
Rapid neutrophil recruitment to lymph nodes has previously been observed, although the mechanism was not the focus and thus was not delineated (11, 28). Herein, we clearly show that the majority of the neutrophils migrate via the HEVs, although on occasion a rare cell could be seen trafficking through the lymphatic vessels, as reported previously (12, 13).
Neutrophils were recruited to the lymph nodes directly from HEVs, which clearly required a chemoattractant that was produced very quickly to allow for such rapid neutrophil recruitment. Indeed, our data suggest a very important role for complement, specifically C5a, a potent end target chemoattractant. While one paper has suggested an important role for CCR7 in neutrophil recruitment to lymph nodes (10), our data and that of others have failed to indicate a role for this chemokine receptor (9, 13). In other infections, neutrophils were either not recruited to lymph nodes (S. typhimurium) or were observed to enter lymph nodes (P. aeruginosa) but in a process entirely dependent on SCS macrophage-producing cytokines (6). Here we observed no role for macrophages in neutrophil recruitment, but a much more rapid recruitment by C5a. Despite the fact that C5a appeared to be the dominant chemoattractant, we and others have predicted and seen a series of chemokine gradients directing neutrophils, and it is possible that while C5a initiates the recruitment, other molecules may contribute as well. For example, a molecule like LTB4, which is known to be produced by neutrophils as a relay chemoattractant to help induce neutrophil swarming (29), could also have a role in this system.
While in humans, L-selectin binds to E-selectin and can activate β-2 integrin (30, 31), in mice, investigators have struggled with why neutrophils express L-selectin, a well-known rolling molecule, when the only vascular ligand for L-selectin is in HEVs, where neutrophils were not thought to emigrate. Common extravasation molecules for neutrophils, lymphocytes, and other leukocytes include L-selectin (32). Our study shows that L-selectin on neutrophils, similar to lymphocytes (22), can bind PNAds as ligands to allow for tethering and rolling, which promote rapid adhesion and emigration during a bacterial infection.
In addition to L-selectin, the P-selectin ligand PSGL-1 was also necessary for neutrophil recruitment, while inhibition of PNAd eliminated both arms of this recruitment, establishing it as the sole ligand on HEVs and diminishing any role for P-selectin on HEV. Of course, platelets also expressed P-selectin, and when we labeled platelets, we could see platelets interacting with both neutrophils and HEVs, and platelet depletion reduced neutrophil recruitment. In contrast, a study by Gorlino et al. (9) revealed that neutrophils entered lymph nodes in immunized mice via both lymphatics and blood vessels, and that the latter was dependent on L-selectin and P-selectin. However, this group had not considered the possibility that platelets could be the source of P-selectin. Alternatively, our infection model for rapid neutrophil recruitment could differ from the immunization model, which may require endothelial P-selectin. Interestingly, Diacovo et al. (24) described a similar secondary mechanism for platelets for lymphocyte recruitment into lymph nodes during inflammation. This adds to the growing list of events to which platelets contribute in inflammation.
Approximately 30% of people are colonized with S. aureus (33), and even though this deadly pathogen can cause many skin infections, very few patients go on to develop bacteremia. Indeed, we show that in addition to resident macrophages and neutrophils, additional neutrophils are recruited rapidly via complement to induce a very important barrier function. In our working model, SCS macrophages partially detain the bacteria, while rapid recruitment of additional neutrophils via L-selectin and bridging platelets binding to PNAds on HEVs ensure that no dissemination occurs (Fig. S9).
Materials and Methods
All experimental animal protocols were approved by the University of Calgary Animal Care Committee and were in compliance with the Canadian Council for Animal Care Guidelines. To model a severe s.c. infection as a result of a puncture wound due to a contaminated object, we infected mice in footpads with MW2 S. aureus. Blocking treatments were administered before infection as described in SI Materials and Methods. Two-photon microscopy was used to assess the movement and location of neutrophils in reference to blood vessels. Mouse popLNs were imaged by anesthetizing the mouse and exposing the popLN in the right hind limb. Anesthetic and labeling antibodies were delivered by cannulation of the right jugular vein. For labeling of cells in the lymph nodes, labeled antibody was injected in the footpad for draining to the popLN. Additional information is provided in SI Materials and Methods.
Supplementary Material
Acknowledgments
We thank the members of the P.K. laboratory for training and assistance related to two-photon intravital microscopy. This work was supported by the Live Cell Imaging Facility, funded by the Snyder Institute at the University of Calgary. The P.K. laboratory is supported by a foundation grant from the Canadian Institute of Health Research. A.B. is a Beverley Phillips Scholarship Program trainee. This work was supported by NIH Grants R01-GM37734, R37-AI047822, and R01-CA0169354 (to E.C.B.).
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. V.N. is a guest editor invited by the Editorial Board.
See Commentary on page 2272.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1715756115/-/DCSupplemental.
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