Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF- β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

Highly antibiotic resistant Staphylococcus aureus (S. aureus) are an important human pathogen and major cause of hospital acquired infections. An early host defense mechanism against bacterial infection is neutrophil recruitment, which helps eliminate the bacteria at the site of invasion. However, unless quickly neutralized, pathogens such as S. aureus can gain access to nearby lymph nodes via draining lymphatics. Lymph nodes protect the host by mobilizing additional resources that limit further pathogen dissemination. These include recruitment of neutrophils to the lymph node to directly target pathogens and the initiation of adaptive immune mechanisms, such as the humoral immune response, which transforms B lymphocytes capable of making pathogen specific antibodies into antibody producing plasma cells. Using a mouse model that allows direct visualization of lymphocytes, neutrophils, and fluorescently-labeled S. aureus in lymph nodes, we document the rapid appearance of bacteria in the lymph node following local S. aureus infection. We characterize the dynamic influx of neutrophils that occurs as a consequence and reveal direct B cell-neutrophil interactions within the lymph node parenchyma. We find that while lymph node neutrophils rapidly engage bacteria, they limit the subsequent humoral immune response likely by producing Transforming Growth Factor-β1, a factor known to limit B cell responses. These finding have important implication for our understanding of B cell responses against potent pathogens such as S. aureus and for the design of effective vaccines.

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We analyzed the mobilization of neutrophils to the inguinal LN (iLN) challenged with heat-inactivated or live S. aureus using intravital two-photon laser scanning microscopy (TP-LSM). Our in vivo data indicate that the migration areas of mobilized neutrophils and activated B cells in the iLN often overlapped, while neutrophils and B cells established multiple intercellular interactions enriched with F-actin. The early humoral response to S. aureus in the iLN was significantly boosted after neutrophil depletion in vivo, and BLIMP1 + GC B cell numbers were elevated. Shown in vitro, activated neutrophils secreted TGF-β1, which potently suppressed IgM production by iLN B cells. To specify the origin of neutrophils recruited to the iLN, we performed intravital microscopy of mouse calvarium and demonstrated neutrophil egress from the BM prior to their mobilization to the iLN. Our results suggest that the mobilization of bone marrow neutrophils to LNs following immunization or infection acts to limit the early humoral response.

Staphylococcus aureus (S. aureus) is a potent human pathogen and the most common cause of skin and soft tissue infections in the USA. The host mobilizes both innate and adaptive immune responses to counter the infection. While neutrophils provide an initial line of defense arriving rapidly at the invasion site, the importance of humoral immunity in pathogen clearance is unresolved [ 27 , 28 ]. Some studies dispute its importance emphasizing the role of cellular immunity and in particular the importance of T h 1 and T h 17 cells [ 29 ]. Supporting this view B cell deficiency does not worsen the level of S. aureus bacteremia [ 30 ]. Yet multiple bacterial virulence factors specifically target humoral immunity [ 31 ]. For example, the humoral immune response is suppressed by S. aureus superantigens, which activate antimicrobial B cell populations triggering activation-induced cell death [ 32 ] and S. aureus protective antigens suppress B cell response [ 33 ]. LAC is a clone of methicillin-resistant S. aureus (MRSA) strain USA300 (known as Los-Angeles County clone) that compromises severely both innate and adaptive immunity of the host [ 34 ]. Detailed understanding the mechanisms of neutrophil and B cell responses to LAC is an urgent need in order to develop an effective anti-Staphylococcal vaccine strategy [ 35 ]. In this study we asked how the massive neutrophil recruitment that occurs during local S. aureus infection might impact the humoral immune response in the draining LN.

The formation of a productive humoral response in LNs depends upon proper B cell trafficking and highly orchestrated intercellular interactions. After B cells exit high endothelial venules (HEVs), they migrate through the medullary region (MR) and interfollicular zones (IFZ) to populate follicular areas near the subcapsular sinus (SCS) [ 20 ]. Follicular B cells exposed to cognate antigen migrate to the follicle border to acquire T cell help, and either proceed to the IFZ to differentiate into early antibody secreting cells or re-enter follicles to form germinal centers (GCs). GC B cells clonally expand and differentiate into plasma cells (PCs) or memory B cells [ 21 ]. Terminal B cell differentiation is accompanied by increasing expression of the transcription factor BLIMP-1 [ 22 ], and often takes place within the IFZ, and along the medullary cords. PCs predominately reside in the MR, or leave the LN to localize in splenic red-pulp or in specialized BM niches [ 23 ]. B cell proliferation and maturation can be boosted by cytokines like BAFF, APRIL and IL-6 released by innate cells [ 24 ], or inhibited in T cell contact-depended manner [ 25 ] or by cytokines like TGF- β 1 [ 26 ]. Sites or niches where recruited neutrophils reside in LN and their regulatory effects on LN B cells are largely unknown.

Mature neutrophils express Ly6G hi , CXCR2, and CXCR4; and reside in the bone marrow (BM) niche retained by high concentration of SDF-1α [ 8 ], and in the red pulp of the spleen [ 9 ]. During inflammation neutrophils are mobilized to the blood and migrate toward the source of CXC chemokines and other mediators released by affected cells or pathogens [ 10 ] to liquidate the source of danger [ 11 ]. Concurrently, they infiltrate adjacent lymphoid tissues to perform other highly specialized tasks, often linking innate and adaptive immunity [ 12 ]. In challenged LNs, neutrophils support cell-mediated responses during the differentiation of T h 1 and T h 17 cells, and development of efficient T h 2 mediated response [ 13 , 14 ]. However, suppressive effect of neutrophils on T cell mediated response have also been shown [ 15 , 16 ]. Neutrophils augment antibody production by facilitating marginal zone B cell responses in spleen [ 17 ], and can favor the transition from autoimmunity to lymphoma [ 18 ]. Conversely, depletion of neutrophils in mice immunized with protein antigens in adjuvants leads to elevated levels of serum antibodies [ 19 ].

Lymph nodes (LNs) are secondary lymphoid organs where pathogenic antigens are captured and processed, and antigen-specific (adaptive) responses are generated. T and B cells arrive to the LNs with the blood flow or via the afferent lymphatics, and occupy highly specialized compartments (niches) to differentiate into effector cells [ 1 , 2 ]. At the same time, LN residing innate cells shape these adaptive response directly by capturing antigens and either eliminating or presenting them, and indirectly by creating cytokine-rich surroundings [ 3 ]. Among the latter, neutrophils are the most dynamic cells mobilized to the LNs following infection or immunization [ 4 , 5 ]. While activated neutrophils are known for their capability to either support lymphocyte proliferation and activation [ 6 ] or suppress adaptive cell function [ 7 ], the physiological roles of their influx to the LNs following vaccination or during the course of an infection remain only partially understood.

Results

Depletion of neutrophils boosts antibody production by LN B cells, while activated neutrophils suppress antibody production via TGF-β1 production The large influx of neutrophils and their observed interactions with B cells following local injection of S. aureus suggested that these interactions might influence the subsequent humoral response. To test this possibility we depleted neutrophils in vivo and measured antibody production by iLN B cells in mice immunized with S. aureus bioparticles or infected with LAC-GFP. The mice received an intraperitoneal injection of Ly6G-specific antibodies (1A8) or isotype control antibodies at day -1, 0 and 1 of immunization/infection with S. aureus. 24 h after first 1A8 injection, neutrophils were mobilized to the blood and LNs in S. aureus immunized isotype control-injected, but not 1A8-injected mice (S5A–S5C Fig). At day 5, the iLNs in neutrophil-depleted mice were larger, and more heavily vascularized than in isotype control mice (Fig 5A). Analysis of the kinetics of lymphocyte recruitment to S. aureus bioparticle-immunized iLN revealed an increase in B220+ cell population and decrease in CD4+ and CD8+ populations in neutrophil-depleted mice (Fig 5B). B220+ cell numbers increased in neutrophil-depleted mice correlating with the total iLN cell numbers (S5D Fig). We harvested the iLN B cells at days 5–6 post S. aureus injection, cultured them for 3 days and measured the levels of IgG and IgM in the supernatants. We compared amounts of antibodies produced by B cells derived from a single iLN (S5D Fig). In the LNs from mice injected with S. aureus bioparticles, neutrophil depletion caused a 12-fold increase in total IgG and 30-fold increase in total IgM production (Figs 5C and S5E). When quantified as amount of antibodies per B cell number, antibody production was also increased in B cell cultures derived from neutrophil-depleted mice (S5F Fig). Total IgG levels were elevated in the serum of neutrophil-depleted mice starting at day 14 after immunization with S. aureus bioparticles (Fig 5D). In the LNs harvested from LAC-GFP infected mice, neutrophil depletion resulted in over a 100-fold increases in both IgG and IgM production by LN B cells (Fig 5E). Thus, the fold increase in antibody production after neutrophil depletion was higher in LAC-GFP infected mice than in the S. aureus bioparticle immunized mice (Fig 5F). Using LAC or LAC spa lysates as antigens, we found that LAC-specific IgG and IgM responses were elevated in neutrophil-depleted mice (Fig 5G). At day 5 after infection, LAC was found in the LNs of neutrophil depleted mice but not of isotype control-injected mice (S3G Fig). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Neutrophils limit the humoral response following local immunization or infection. C57BL/6 mice were given PBS as a baseline control, immunized with S. aureus bioparticles, or infected with LAC-GFP. Isotype control or 1A8 antibody was injected intraperitoneally at 100 μg/mouse on day -1, 0 and 1 of immunization/infection. (A) Images of iLNs in isotype control (left) and neutrophil depleted (right) mice immunized with S. aureus bioparticles are shown at day 5 after immunization. Indicated are LN edges (black arrows), blood vessels (blue arrows). Scale bars: 5 mm. (B) Flow cytometry analysis of B and T cell populations in the iLN of isotype control or neutrophil-depleted mice 3 days after immunization. N = 4 iLNs; 3 repeats. Means ± SEM. (C) ELISA of total IgG and IgM produced by iLN B cells isolated from PBS control, immunized isotype control and immunized neutrophil-depleted (1A8) mice. B cells were isolated from the iLNs at day 6 after immunization and cultured for 3 days. N = 4 iLNs. Data are shown as fold change. 3 repeats. Means ± SEM. (D) ELISA of total IgG in the serum of immunized isotype control and immunized neutrophil-depleted mice measured at days 0, 7, 14, 21 and 28 after immunization. N = 4 mice; 2 repeats; means ± SEM. (E-G) Mice were depleted of neutrophils as above and infected with LAC-GFP. (E) ELISA of total IgG and IgM produced by iLN B cells. ILNs were harvested at day 5 after infection, B cells were isolated and cultured for 3 days. N = 5–7 iLNs. Data are shown as antibody concentration in B cell supernatants. Means ± SD. (F) Comparison of fold changes in IgG and IgM production in neutrophil-depleted and isotype iLNs, calculated for S. aureus immunized versus LAC-GFP infected mice. Data shown as fold increases (Means ± SD). (G) ELISA of LAC-specific (upper panel) and LAC spa-specific IgG and IgM. N = 5–7 iLNs. Means ± SD. (H) ELISA of IgM produced in vitro by LPS or S. aureus activated LN B cells in presence of neutrophils, activated correspondingly. No LPS in culture used as a baseline control. (I) ELISA of IgM produced by LPS activated iLN B cells in presence of LPS-activated neutrophils supernatants from LPS-activated (SN act) and non-activated neutrophils (SN NA), neutralizing anti-TGF-β1 antibody, and TGF-β1. (J) ELISA results measuring TGF-β1 produced by activated neutrophils, non-activated neutrophils or LPS-activated B cells in 24 h cultures. (H-J) Final concentration of LPS in all B cell cultures: 2 μg/mL. 3 to 5 repeats for each of in vitro experiments. Means ± SEM. https://doi.org/10.1371/journal.ppat.1004827.g005 To determine if neutrophil depletion also augmented LN B cells responses to protein antigens we isolated LN B cells 7 days after immunization and measured their secretion of IgG and IgM. In case of SRBCs we measured total IgG and IgM production and for the NP-KLH immunized mice we measured NP-KLH specific IgG and IgM produced by LN B cells. In both instances neutrophil depletion resulted in a higher production of antibody (S5H and S5I Fig). To provide insight into the mechanism by which activated neutrophils suppress LN B cell antibody production we established an in vitro system. We isolated B cells from the iLNs of naïve mice and activated them with either LPS or S. aureus in the presence of absence of neutrophils. In the co-culture we chose a ratio of 10 B cells to 1 neutrophil as that is the approximate ratio of B cells to neutrophils in the immunized iLN. We relied on the ability of LPS or S. aureus to activate both B cell antibody production and to stimulate neutrophils. We found that both inductive signals increased IgM production in the B cell cultures. When neutrophils were present we observed a potent suppression of IgM production (Figs 5H and S5J, left). At the same time, we did not observe such a pronounced reduction of IgA levels in LN B cell cultures in presence of S. aureus bioparticle-activated neutrophils (S5J Fig, right). Seeded at the same cell density, by day 5 B cell numbers in B cell/neutrophil co-cultures were 1.5-fold lower than in pure B cell cultures (S5K Fig). Thus, in presence of activated neutrophils, IgM production by total LN B cell cultures was 5-fold suppressed and IgA production 2-fold suppressed (S5L Fig, left). When normalized for B cell number (production by 1 x 106 B cells), IgM production was still 4-fold decreased, and IgA production only 35% decreased (S5L Fig, right). Next, we tried to identify the inhibitor present in the activated neutrophil cultures. As TGF-β1 is known as a potent inhibitor of B cell antibody production [26, 40], we added a neutralizing TGF-β1 antibody to B cell-neutrophil co-culture. The suppressive effect of neutrophils was nearly completely reversed (Fig 5I, left). In addition, supernatant from LPS-activated neutrophils (SN act), but not from non-stimulated cells (SN non-act), also suppressed IgM production, and this effect was reversed by adding a neutralizing TGF-β1 antibody (Fig 5I, middle and right). We also verified that LPS-activated neutrophils secrete TGF-β1, much more than non-activated neutrophils or LPS-activated B cells (Fig 5J). These data indicate that neutrophils mobilized to antigen stimulated LNs can suppress B cell antibody production and suggest that this may occur via neutrophil TGF-β1 production. An increase in humoral immune response in neutrophil-depleted mice infected with live S. aureus is more pronounced than in those immunized with S. aureus bioparticles, SRBC or NP-KLH.