Neutrophils in hot pursuit of MRSA in the lymph nodes

Neutrophils traffic in the bloodstream as initial responders against invading pathogens. In particular, neutrophils are critically important in combatting Staphylococcus aureus, which is a Gram-positive extracellular bacterial pathogen that is the most common cause of skin infections and a prime cause of life-threatening infections such as pneumonia and bacteremia.

Over the past two decades, the public has become well aware of multidrug-resistant community-acquired methicillin-resistant S. aureus (MRSA), which has spread rapaciously through the population, creating a serious public health threat (1, 2). Thus, it is crucial to understand how neutrophils contain a local S. aureus infection to prevent systemic dissemination that often leads to death.

In PNAS, Bogoslowski et al. (3) describe a novel mechanism that prevents S. aureus dissemination from an initial site of S. aureus skin infection in the mouse footpad. Utilizing two-photon microscopy, Bogoslowski et al. report that S. aureus migrated from the skin via afferent lymphatics to popliteal lymph nodes where they encountered an accumulation of trafficking neutrophils, which deployed phagocytic and antimicrobial mechanisms to halt the spread of infection (Fig. 1A). A rapid neutrophilic response is critical to prevent bacterial dissemination beyond the local skin infection and lymph nodes, which filter draining extracellular fluids from the infected tissue. By imaging green fluorescent neutrophils, the authors visualized and enumerated neutrophils trafficking through the lymph node, essentially preventing S. aureus systemic dissemination. Recruitment of neutrophils into the lymph nodes involved (i) high endothelial venules (HEVs) (Fig. 1B), (ii) a chemotactic gradient of complement C5a, and (iii) L-selectin on neutrophils binding to peripheral node addressin (PNAd) on endothelial cells, which mediated neutrophil rolling, arrest, and migration into the lymph node (Fig. 1C). L-selectin was thought to be primarily involved in lymphocyte rolling, adhering, and entering HEVs in secondary lymphoid organs, but these new data suggest a new role for L-selectin on neutrophils. Finally, platelets helped neutrophil entry to lymph nodes by expressing P-selectin, which binds both P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils and PNAd on endothelial cells.

Fig. 1.

Neutrophil recruitment and clearance of S. aureus in draining lymph nodes. (A) S. aureus bacteria travel in afferent lymphatics from the initial footpad infection to the popliteal lymph node. (B) Neutrophils are recruited to lymph nodes through HEVs to promote bacterial clearance and prevent systemic bacterial dissemination. (C) Mechanisms in lymph node HEVs that facilitate neutrophil tethering, rolling, adhering, and migration into the lymph nodes.

Lymph Nodes—Sponges, Not Sieves

The mechanisms described by Bogoslowski et al. are distinct from those of prior reports. For example, in a Toxoplasma gondii infection model in mice, neutrophils were recruited to lymph nodes where they induced removal of subcapsular sinus macrophages (4). In a Mycobacterium bovis infection model, neutrophils captured these intracellular bacteria from ear skin and shuttled them through lymphatics to lymph nodes, but the neutrophils could not prevent bacteria from persisting there (5). Interestingly, in response to other extracellular bacteria (Pseudomonas aeruginosa and Salmonella typhimurium), a variety of immune cells [natural killer (NK) cells, NK T cells, γδ T cells, and innate-like CD8⁺ T cells] cooperated in the subcapsular sinus of lymph nodes through inflammasome-activated IL-18 to promote macrophage phagocytic bacterial clearance—but without a major role for neutrophils (6). Finally, in a model in which S. aureus was injected in close proximity to an inguinal lymph node, neutrophils were recruited from the blood to the lymph node where they inhibited antigen-specific IgG and IgM production (7). None of these prior studies reported how the neutrophils entered the lymph nodes or played a major role in pathogen clearance, highlighting a unique adaption in host prevention of S. aureus dissemination.

Given that subcapsular sinus macrophages promoted clearance of other extracellular bacteria (6), Bogoslowski et al. evaluated the role of these cells against S. aureus. By depleting lymph node macrophages (by injecting clodronate liposomes into the calf), a slight increase in dissemination of S. aureus to the spleen, liver, and lung was observed. Curiously, treatment of mice with the MECA-79 antibody (recognizing PNAd on HEV) to effectively block neutrophil recruitment to the lymph node did not prevent S. aureus clearance and dissemination. However, if the MECA-79 antibody was administered to mice first depleted of lymph node macrophages, there was a marked increase in bacterial dissemination to the liver and spleen, suggesting that both neutrophils and macrophages cooperate in the lymph node to prevent bacterial dissemination.

Future Challenges and Questions Remaining

Several significant issues associated with the host response to S. aureus emerge. First, the role neutrophils play in lymph nodes as a fail-stop mechanism to prevent S. aureus dissemination should be interpreted in the context of the animal model used. A prior skin infection model in which wild-type mice were inoculated in the back skin with a different CA-MRSA strain [USA300 LAC (8) isolated from a skin infection outbreak in the Los Angeles County Jail in 2002] reported S. aureus dissemination to the kidneys, liver, and spleen (9). However, Chan et al. (9) did not determine if the route of entry from the skin to the bloodstream was via lymph nodes.

This is in contrast to findings in the footpad infection model in which dissemination did not occur in wild-type mice despite using a similar inoculum (10⁷ CFU range). One important difference may be attributed to differential virulence factors produced by the distinct S. aureus strains (10). The MW2 CA-MRSA strain used by Bogoslowski et al. (3) was not isolated from a skin infection outbreak, but rather from a fatal septicemia and septic arthritis infection in a 16-mo-old girl in North Dakota (9). In addition, the different vasculature and lymphatic drainage anatomy of the inoculation sites on the footpad or back skin might have played a role in determining whether invasive bacterial dissemination occurred via lymph nodes or the bloodstream.

Second, in translating these results to humans, it is important to take into account that many virulence factors of S. aureus exhibit strong activity against human but not mouse neutrophils, as well as diverse effects on other leukocytes. For example, Panton–Valentine leukocidin has potent cytolytic activity against human but not mouse neutrophils (11). Other leukocidins and virulence factors (i.e., certain superantigens) have activity in humans but not mice (12, 13). Thus, in humans, the effectiveness of the neutrophilic response in the lymph nodes might not be as robust as observed in mouse models.

Remarkably, none of the recruitment processes described by Bogoslowski et al. (i.e., C5a, PNAd-expressing HEV, L-selectin on neutrophils, and P-selectin on platelets) contributed to neutrophil recruitment to the site of S. aureus infection in the skin. Typically, neutrophil recruitment involves E-selectin recognition of PSGL-1 that mediates rolling and outside-in signaling that converts to β₂-integrin–dependent arrest (14). This suggests that alternate mechanisms are involved for neutrophil recruitment to defend against S. aureus skin infections. In our prior work, we found inflammasome-activated IL-1β production, which signaled via IL- 1R/MyD88 signaling recruited neutrophils to a S. aureus skin infection in mice (15–18). In addition, IL-1β triggered IL-17–producing γδ T cells, which together with IL-1β, promoted production of neutrophil-attracting chemokines (e.g., KC and MIP2) and granulopoiesis factors (e.g., G-CSF) that likely facilitated neutrophil recruitment (18–23). We also identified a role for LTB₄ and formylated peptides of S. aureus in contributing to effective neutrophil recruitment to a S. aureus skin infection (17, 24), which might have provided chemotactic gradients for neutrophils in skin vasculature similar to the role of C5a in HEVs of lymph nodes. Nonetheless, the specific addressins, integrins, and other neutrophil–endothelial cell interactions that enables tethering and arrest of surveilling neutrophils at the site of a S. aureus skin infection have yet to be fully defined.

Finally, two recent studies have shed light on the role of lymphatics and platelets in combatting S. aureus infections. Jones et al. (25) found that a localized S. aureus skin infection in mice reduced both lymphatic vessel contractility and lymph flow as a result of S. aureus exotoxins that induced death and disorganization of lymphatic muscle cells. These data indicated that S. aureus possesses evasion mechanisms to prevent lymphatic vessel function and subsequent bacterial clearance in lymph nodes. Gaertner et al. (26) reported that, in response to S. aureus bacteremia in mice, platelets collected and bundled fibrin-trapped S. aureus bacteria. In this context, platelets facilitated neutrophil clearance of the bacteria by promoting phagocytosis and formation of neutrophil extracellular traps. Thus, platelets not only are involved in neutrophil recruitment to lymph nodes but can also participate in bacterial clearance by neutrophils.

Future work will address these challenges and questions, but it is now clear that neutrophils are playing a critical host defense role in the lymph nodes.