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Splenic Ly6Ghigh mature and Ly6Gint immature neutrophils contribute to eradication of S. pneumoniae

Splenic Ly6Ghigh mature and Ly6Gint immature neutrophils contribute to eradication of S. pneumoniae Ar ticle high int Splenic Ly6G mature and Ly6G immature neutrophils contribute to eradication of S. pneumoniae 1,3 1,3,4 1,3 1,2,3 Justin F. Deniset, Bas G. Surewaard, Woo-Y ong Lee, and Paul Kubes 1 2 3 Department of Physiology and Pharmacology, Department of Microbiology, Immunology, and Infectious Diseases, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada Department of Medical Microbiology, University Medical Centre, 3584 CX Utrecht, Netherlands t he spleen plays an integral protective role against encapsulated bacterial infections. our understanding of the associated mechanisms is limited to thymus-independent (tI) antibody production by the marginal zone (MZ) B cells, leaving the contri - bution of other splenic compartments such as the red pulp (rP) largely unexplored despite asplenic patients succumbing to the infection in the first 24 h, suggesting important antibody-independent mechanisms. In this study, using time-lapse intravital imaging of the spleen, we identify a tropism for Streptococcus pneumoniae in this organ mediated by tissue-resident MZ and hi intermediate rP macrophages and a protective role for two distinct splenic neutrophil populations (Ly6G and Ly6G ) residing in the splenic rP. Splenic mature neutrophils mediated pneumococcal clearance in the spleen by plucking bacteria off the surface of rP macrophages that caught the majority of bacteria in a complement-dependent manner. this neutrophil phagocytic capacity intermediate was further enhanced after tI antibody production. resident immature neutrophils (Ly6G ) in the spleen undergo emer- gency proliferation and mobilization from their splenic niche after pneumococcal stimulation to increase the effector mature neutrophil pool. We demonstrate that splenic neutrophils together with two macrophage populations and MZ B cells regulate systemic S. pneumoniae clearance through complementary mechanisms. IntroductIon The spleen is important for protection against encapsulated cells rapidly differentiate into plasmablasts and secrete patho- bacteria including Streptococcus pneumoniae, a major cause gen-binding serum IgM antibodies that are detectable in the of morbidity and mortality worldwide. Invasive pneumococ- serum only starting at 72–96 h after infection (Martin et al., cal disease, detected in 10–30% of pneumococcal pneumo- 2001; Belperron et al., 2005; Moens et al., 2007), meaning niae cases, has long been known to increase the mortality other cells must keep the pathogen in check until antibodies substantially beyond that seen with pneumonia alone (Chiou are made. Once made, these antibodies are thought to en- and Yu, 2006; Blasi et al., 2012). It is estimated to directly hance recognition and facilitate clearance of S. pneumoniae. cause >1.6 million deaths annually (World Health Organi- Indeed, splenectomized patients display impaired IgM anti- zation Geneva, 2007; O’Brien et al., 2009). Invasive disease body responses to polysaccharide antigen (Amlot and Hayes, is particularly serious in splenectomized or asplenic patients. 1985; Kruetzmann et al., 2003). As a result, pneumococcal These individuals have a 50-fold higher risk of developing research in the spleen has focused almost exclusively on the a fulminant septic infection to encapsulated bacteria, and a cells that regulate B cell–dependent antibody production. 50–70% mortality rate is associated with these cases (Hold- However, considering the kinetics of i.v. pneumococcal dis- sworth et al., 1991; Di Sabatino et al., 2011). Importantly, the ease in splenectomized patients, we hypothesize that other majority of deaths occur within the first 24 h (Gransden et al., very rapid innate immune mechanisms within the spleen 1985; Di Sabatino et al., 2011), highlighting the importance must not only contribute to early protection, but also stave of some very rapid as yet unknown innate immune mecha- off the infection until antibodies can be made. nisms within the spleen in providing protection during the Neutrophils are first responders in the innate immune acute stages of i.v. infection before antibody production. response to infection. Their multiple defense mechanisms, To date, the major protective mechanism of the spleen such as their phagocytic capacity, reactive oxygen species against S. pneumoniae is believed to be dependent on thy- production, and ability to degranulate and form neutrophil mus-independent (TI) antibody production by specialized B extracellular traps, contribute to the effective clearance of cells within the marginal zone (MZ). After stimulation, MZ B pathogens (Kolaczkowska and Kubes, 2013). Neutrophils typically access sites of infection via the circulation, following Correspondence to Paul Kubes: pkubes@ucalgary.ca © 2017 Deniset et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Abbreviations used: 3D, three dimensional; Cat, cathepsin; CLL, clodronate liposome; Alike–No Mirror Sites license for the first six months after the publication date (see http ://www .rupress .org IVM, intravital microscopy; MZ, marginal zone; RP, red pulp; SD-IVM, spinning-disk /terms /). After six months it is available under a Creative Commons License (Attribution–Noncommercial– IVM; TI, thymus independent. Share Alike 4.0 International license, as described at https ://creativecommons .org /licenses /by -nc -sa /4 .0 /). The Rockefeller University Press $30.00 J. Exp. Med. 2017 Vol. 214 No. 5 1333–1350 https://doi.org/10.1084/jem.20161621 The Journal of Experimental Medicine chemoattractant cues and responding to local inflammatory monas aeruginosa by the liver (Kolaczkowska et al., 2015 and mediators (Kolaczkowska and Kubes, 2013). In addition to unpublished data). In fact, removal of the spleen did not in- these circulating neutrophils, a large pool of neutrophils can crease blood levels or dissemination of S. aureus (unpublished be found in the bone marrow, and marginated pools of neu- data). The course of S. pneumoniae infection was tracked in trophils may also exist within peripheral organs. These sources the spleen and blood over a 5-d period (Fig. 1 b). Bacterial of neutrophils are thought to mobilize in response to stim- counts decreased in blood and spleen for the first 8  h after ulation (Athens et al., 1961a,b). There is some evidence to infection. This was followed by a rebound in levels at 24 and suggest that the spleen might function as a reservoir. Neu- 48 h before complete clearance by 5 d after infection. trophils, upon reinjection into humans and mice, appear to The spleen was integral for clearance, as a pneumococ- accumulate within the spleen (Peters et al., 1985; Ussov et al., cal challenge in splenectomized mice resulted in 100% mor- 1995; Suratt et al., 2001). Whether this is related to an artifact tality by 48  h, even at a fivefold decrease in infection dose of neutrophil isolation and reinjection or a bona fide physio- (Fig. 1 c). Much focus has been placed on the MZ B cells and logical event remains unclear. Recently, a B helper neutrophil MZ macrophages as contributors to antibody production and subset has been described to reside in the perifollicular zone pneumococcus clearance. Specific depletion of MZ B cells of the spleen in humans and mice to promote antibody pro- via an antibody-mediated protocol (Lu and Cyster, 2002) re- duction by MZ B cells (Puga et al., 2012; Magri et al., 2014; sulted in 40% mortality but only in the later phase (72 h) after Chorny et al., 2016). However, evidence on splenic neutro- S. pneumoniae infection (Fig. 1 d), coinciding with the MZ B phil turnover, behavior, and additional functions during basal cell–dependent production of IgM and IgG antibodies capa- and infectious conditions is lacking and would require live ble of binding S. pneumoniae (Fig. 1 e). Low-dose clodronate liposome (CLL) treatment, which depleted 80–90% of MZ cell imaging of these cells in the spleen, something that has to date not been done to our knowledge. macrophages but not RP macrophages (McGaha et al., 2011), In this study, we used a combination of spinning-disk resulted in a nearly identical temporal outcome (Fig.  1  d). intravital microscopy (IVM [SD-IVM]) and two-photon Combined MZ macrophage and MZ B cell depletion did IVM to evaluate the behavior of neutrophils in the spleen not further increase susceptibility to S. pneumoniae infection under steady state and after S. pneumoniae infection. Using (Fig. 1 d), suggesting a single collaborative protective mech- this platform, we found that much but not all of the S. pneu- anism of these two cell types via TI antibody production. moniae bypassed the MZ macrophages and were caught by Importantly, the combined depletion of these two cell popu- red pulp (RP) macrophages. We also identified two neu- lations only partially recapitulated the effect of a splenectomy trophil populations within the splenic RP: an immobilized, but with a delayed time frame, indicating that other earlier int ) population of neu- immature Ly6G-intermediate (Ly6G splenic innate immune mechanisms independent of these two hi trophils and a mature Ly6G-high (Ly6G ) population of cell types (and their ability to stimulate TI antibody produc- neutrophils that scan the tissue. Mature neutrophils mediated tion) are involved in splenic protection to pneumococcus. pneumococcal clearance by removing the bacteria from the surface of RP macrophages. During an emergency response S. pneumoniae localizes to both the MZ int to infection, the immobilized Ly6G immature neutrophils and the rP of the spleen increased their proliferative capacity and took on features of Dynamics of S. pneumoniae distribution in the spleen were the resident mature neutrophils. Circulating neutrophils re- determined using time-lapse two-photon IVM and SD-IVM cruited to the splenic MZ helped increase TI antibody pro- of the spleen. First, evaluation of S. pneumoniae movement in duction by MZ B cells, which further enhanced the ability the MZ revealed that, upon i.v. infection, the GFP-expressing bacteria flowed across the MZ, with some tethering inter- of mature splenic neutrophils to fully eradicate systemic pneumococcal infection. actions to MZ macrophages or other structures in this area (Fig. 2 a and Video 1). Quantification over the first 20 min reSuL tS after infection revealed that the vast majority of bacteria vi- the spleen is integral for protection against systemic sualized had a short transit time through this area with only S. pneumoniae infection a few bacteria (<20%) being detained permanently in the A pneumococcal bacteremia model was used to evaluate indi- MZ (>5 min in Fig. 2 b). Depletion of MZ macrophages by vidual components of immunity during S. pneumoniae infec- low-dose CLL treatment resulted in increased localization of tion. Intravenous infection with a very small dose (10 CFU) bacteria in the RP at 60 min after infection (Fig. 2 c). Visual- of S. pneumoniae resulted in a fivefold preferential sequestra- ization of the splenic RP revealed a progressive accumulation tion by the spleen compared with the lung and liver within of bacteria in this region over the first 20 min (Fig. 2, d and e; 60 min (Fig. 1 a). Bacterial counts in the brain, heart, and kid- and Video 2). RP macrophages are at least in part responsible neys were below detection limit (not depicted). This splenic for this retention, as their depletion through high-dose CLL tropism is unique to S. pneumoniae infection, as our previous treatment significantly impaired the number of bacteria in work has shown tremendous preferential sequestration of cir- the compartment (Fig. 2 e). This is despite increased bacteria culating Staphylococcus aureus, Escherichia coli, and Pseudo- migrating to the RP as a result of the concurrent depletion 1334 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. Figure 1. Splenic protection againstS. pneumoniae infection. (a) S. pneumoniae bacterial counts in blood, spleen, lung, and liver 1 h after i.v. infection. Black lines show the median. n = 6 pooled from two independent experiments. (b) Mean (± SD) bacterial counts in the blood (red circles) and spleen (blue squares) over 120 h. n = 5–7 pooled from two independent experiments. (c) Survival curve for S. pneumoniae i.v. infection at 10 -CFU dose in sham-operated (black line) or splenectomized (SPX; solid blue line) mice and at 2 × 10 –CFU dose in splenectomized mice (dotted red line). n = 5 from one experiment. (d) Representative flow cytometry plots of MZ B cell (MZB) depletion (dep), immunohistochemistry of MZ macrophage (MZM) depletion (green, RP macro- phage [RPM]; red, MZ macrophage) and survival curves to S. pneumonia infection at 10 -CFU dose in MZ macrophage–depleted (continuous green line), MZ B cell–depleted (continuous blue line), or MZ macrophage– and MZ B cell–depleted (dotted red line) animals. n = 6–10 from two independent experiments. Bar, 300 µm. Fo B, follicular B cell. Histograms and images are representative of two to three independent experiments. (e) Representative flow cytometry histograms and quantification of S. pneumoniae IgM (left) and IgG (right) serum antibodies after S. pneumoniae infection in WT (orange histogram; black lines) or MZ B cell–depleted S. pneumoniae (blue histogram; blue lines) mice. n = 3 for WT and n = 8 for MZ B cell depletion pooled from three independent experiments. Inf, infected. Data are represented as mean ± SEM, except in b. *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Kruskal-Wallis test (a), one-way ANO VA (b), log-rank Mantel-Cox test (c and d), and two-way ANO VA (e) statistical analyses were performed. of MZ macrophages. Localization of bacteria in both splenic tion to overall capture (20.1 ± 0.9%; Fig. 2 h). However, RP regions over time was also determined using fresh sections macrophages only accounted for 18.5 ± 2.1% of the total + + of spleen with labeled RP (F4/80) and MZ (CD209b ) (Fig.  2  h). Systematic assessment of the remaining popula- macrophages. This approach revealed a preferential localiza- tion of cells harboring S. pneumoniae using flow cytome- tion of S. pneumoniae in the MZ at 20 min after infection try revealed that the majority were neutrophils (42 ± 2.9%; (Fig. 2, f and g), whereas at 1 h after infection, bacteria load Fig.  2  h). The S. pneumoniae uptake appeared to be prefer- increased in the RP (Fig. 2 g). ential to splenic neutrophils, as the number of total S. pneu- Flow cytometry was used to identify the main cell types moniae-GFP–positive neutrophils was significantly higher in involved in S. pneumoniae capture in the spleen. Analysis of the spleen than the blood (Fig. 2 i). Furthermore, neutrophil splenic S. pneumoniae-GFP–positive cells from spleens har- numbers in the spleen did not increase within the first 30 min vested at 30 min after infection revealed that >60% of bac- after S. pneumoniae infection (Fig. 2 j), suggesting that a res- teria were in populations that resided within the RP of the ident neutrophil population within the spleen is responsible spleen, whereas MZ macrophages contributed a smaller por- for this bacterial uptake. JEM Vol. 214, No. 5 1335 Figure 2. Splenic localization ofS. pneumoniae. (a) Representative two-photon microscopy images of MZ macrophage interaction with S. pneumoniae during acute i.v. infection. Bar, 25 µm. Green, S. pneumoniae; red, MZ macrophages (MZM). White arrows indicate stationary bacteria. (b) Quantification of MZ dwell time of S. pneumoniae. n = 3 from three independent experiments. (c) Increased RP localization of S. pneumoniae 60 min after i.v. infection in MZ macrophage–depleted (low-dose CLLs; red bar) animals. n = 3–4 pooled from two independent experiments. Ctrl lipo, control liposome. (d) Representative spinning-disk confocal images of RP macrophage (RPM) interaction with S. pneumoniae during acute i.v. infection. Bars, 100 µm. Green, S. pneumoniae; purple, RP macrophages. (Inset) White arrows indicate stationary bacteria. Data are representative of n = 5 from three independent experiments. (e) S. pneu- moniae counts per field of view (FOV) in the RP from 0–20 min after i.v. infection in wild-type (black line), control liposome (blue line)–, low-dose CLL (red line)–, or high-dose CLL (gray line)–treated animals. There were four fields of view per animal. n = 5 for WT, n = 4 for control liposomes, n = 3 for high-dose CLL, and n = 5 for low-dose CLL pooled from four independent experiments. (f) Representative composite 10× stitched image of fresh spleen sections at 20 min after i.v. S. pneumoniae infection. Bar, 110 µm. Green, S. pneumoniae; red, MZ macrophages; purple, RP macrophages. (Inset) White arrows indicate bacteria. (g) Localization of S. pneumoniae in both the MZ (blue line) and RP (red line) regions at 20 and 60 min after i.v. infection. n = 3 pooled from + + − two independent experiments. (h) S. pneumoniae cell localization 30 minutes after infection in the spleen. MZ macrophages, CD11b CD209b F4/80 ; RP low − + + + int + − hi macrophages, CD11b CD209b F4/80 ; Neutrophils, CD11b Ly6G Ly6C ; monocytes, CD11b Ly6G Ly6C . n = 4 pooled from two independent experiments. 1336 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. int neutrophil populations reside in the splenic rP the Ly6G neutrophils are the resident population. These re- hi Time-lapse SD-IVM of the spleen was performed to study sults support the view that the mobile neutrophils are Ly6G int resident neutrophils in this organ. Under basal conditions, the and the immobilized resident neutrophils were the Ly6G spleen contained a large population of neutrophils that were population. This was not dissimilar to mature and immature localized in the RP compartment (Video 3). Based on their neutrophils, respectively, in bone marrow. int morphology and behavior, two neutrophil populations could To further characterize whether the Ly6G neutro- be identified: those that crawled around scanning the tissue phils expressed other markers of immaturity, we examined (mobile neutrophils) and those that formed large immobile the expression of c-KIT (CD117) as well as other maturity colonies (immobilized neutrophils) that could be as large as markers for neutrophils. Expression of CD117 was seen only int neutrophils (Fig. 4 c). CD117 staining was con- 30–50 cells (Fig.  3, a and b). Mobile neutrophils (Fig.  3  a, on Ly6G white arrowheads) migrated throughout the RP at varied ve- firmed on immobilized neutrophils by IVM (Fig. 4 d). A por- int locities (Fig. 3 c), and individual cells could be tracked for up tion of clustered Ly6G neutrophils also displayed increased to 1 h within the same field of view (Fig. 3, a–c). Neither of CD49d (consistent with immature neutrophils) and lower these populations was in the blood vessels. Immobilized neu- l-selectin expression (Fig. 4 c). No difference was noted in trophils (Fig. 3 a, green arrowheads) were stationary (Fig. 3 b any of the other markers between the two neutrophil pop- and Video  3), displayed a rounded morphology, and had a ulations (Fig.  4  c). Nuclear morphology assessment of im- perivascular localization (Fig. 3 a). Aside from these two pop- mobilized neutrophils in situ demonstrated the presence of ulations, neutrophils could also be seen rolling within blood banded (Fig. 4 e, white arrowheads) and segmented (Fig. 4 e, vessels, but they rarely stopped, and none emigrated out of the blue arrowheads) neutrophils. Collectively, these data further vasculature (Video 3). support the view that there are two phenotypically distinct T o evaluate the tur nover of these splenic populations with neutrophil populations in the RP, one immature expressing GFP blood neutrophils, parabiosis experiments pairing a LysM intermediate Ly6G levels and remaining immobilized in large mouse, in which neutrophils express GFP, with a nonfluores- colonies mostly as band cells and a second mature neutrophil cent wild-type mouse (C57) were undertaken. After 2 wk, population that is Ly6G high and that scans the splenic RP chimerism within the blood was 20–30%, as determined by under steady-state conditions. flow cytometry (Fig. 3 d), values consistent with that of pre- vious studies (Sawanobori et al., 2008; Guilliams et al., 2013). Mature splenic neutrophils mediate uptake of However, in the spleen, there was a dichotomy. IVM of the S. pneumoniae in the rP spleen in these same animals revealed that 35% of the mobile Next, we examined the behavior of the two populations of neutrophils in the splenic RP by SD-IVM. S. pneumoniae subset was derived from the parabiotic partner (Fig. 3 d and Video 4), indicating that, under steady state, this population uptake by neutrophils in the RP was predominantly limited was primarily replenished by neutrophils from the circula- to mature neutrophils, as the immobilized immature neutro- tion. Interestingly, only 10% of the immobilized clustered phils bound very little bacteria (Fig. 5, a and b; and Video 5). neutrophils appeared to derive from the parabiotic partner RP macrophages also bound the bacteria during this time (Fig. 3 d and Video 4), suggesting the majority of this popu- period. Visualization at a higher magnification revealed that lation was primarily resident. S. pneumoniae first bound to the surface of an RP macro- Intriguingly, analyzing all cells in the spleen, there were phage, and the mature neutrophil subsequently migrated clearly two populations of neutrophils: a large population of over and plucked the bacteria off the macrophage surface hi Ly6G neutrophils and a second smaller population of Ly- (Fig. 5 c and Video 6). At no point were mature neutrophils int 6G neutrophils. High concentrations of Ly6G (1A8) anti- S. pneumoniae freely flowing in the splenic RP. able to catch body were given to deplete neutrophils. IVM revealed that all Three-dimensional (3D) reconstruction of neutrophil-bound mobile neutrophils were depleted and only immobilized neu- bacteria further confirmed intracellular localization of the trophils remained (Fig. 4 a). Flow cytometric analysis of these pathogen. Increasing the transparency of Ly6G staining hi spleens revealed a significant decrease in Ly6G neutrophils, (Fig. 5 d, I–III) revealed GFP-expressing bacteria. Neutrophil int whereas Ly6G neutrophils remained unaffected (Fig.  4  a). phagocytosis of S. pneumoniae was complement dependent, When spleens from parabiosis animals were examined, the as capture was completely abrogated in C3-deficient mice hi percentage of Ly6G neutrophils of partner origin was akin (Fig. 5 e and Video 5). This was not because of the RP mac- int −/− to that of blood overall, whereas the percentage of Ly6G rophage not being able to catch the bacteria, as the C3 RP neutrophils was significantly reduced (Fig.  4  b), suggesting macrophages caught the S. pneumoniae, as well as wild-type (i) Flow cytometry quantification of total number of S. pneumoniae–GFP neutrophils in the blood (black bar) and spleen (teal bar). n = 4 pooled from two independent experiments. (j) Flow cytometry quantification of total neutrophil number in the spleen 30 min after saline (black bar) or S. pneumoniae (blue bar) injection. n = 3 for saline and n = 4 for S. pneumoniae pooled from two independent experiments. *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Student’s t test (c, i, and j) and two-way ANO VA (e and g) statistical analyses were performed. Data are represented as mean ± SEM. JEM Vol. 214, No. 5 1337 Figure 3. Steady-state neutrophil pop- ulations in the splenic rP. (a) Represen- tative time-lapse images of mobile (white arrowheads) and immobilized (encircled; green arrowheads) neutrophil populations in the splenic RP under basal conditions. Red, neutrophils; blue, vasculature. Bar, 50 µm. Data are representative of n = 5 from three independent experiments. (b) Cellular tracks representation of mobile and immobilized neutrophil populations from representative time-lapse images. (c) Quantification of cell velocity distribution of mobile (dark blue bars) and immobilized (light blue bar) neutrophils in the splenic RP under basal conditions. There were four fields of view (FOV) per animal. n = 5 from three independent experiments. (d) Representative flow cytometry plots (blood and spleen), IVM images (spleen), and quan- tification of neutrophil origin after parabiosis. SSC, side scatter. Bars, 25 µm. There were four fields of view per animal. n = 4 from three in- dependent experiments. *, P < 0.05. Student’s t test statistical analyses were performed. Data are represented as mean ± SEM. −/− RP macrophages (Fig. 5 e). Intriguingly, in C3 mice, where respiratory burst has long been regarded as an important in- neutrophils were not able to remove the bacteria from the tracellular killing mechanism for pathogens. To evaluate this surface of the macrophage, huge numbers of bacteria associ- capacity in mature splenic neutrophils,S . pneumoniae was la- ated with the RP macrophage and resulted in 100% mortality beled with an OxyBURST probe that fluoresces green in the within the first 18 h of infection (Fig. 5 f). This suggested the presence of oxidants. Visualization of OxyBURST -labeled macrophages were not able to directly kill the bacteria and bacteria upon neutrophil phagocytosis demonstrated a very required the immediate presence of the neutrophils. Even in rapid oxidative burst (Fig.  5, h and i). This rapid response the presence of complement, the macrophages were not able was NAD PH (nicotinamide adenine dinucleotide phosphate to kill bacteria, as depletion of mature neutrophils using a reduced) oxidase dependent, as the oxidant production was −/− Ly6G (1A8) antibody resulted in increases in splenic bacterial not seen in Cybb mice within this timeframe (Fig.  5  i). counts at 24 h after infection (Fig. 5 g). Neutrophil-mediated However, oxidation was not the predominant killing mech- 1338 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. Figure 4. characterization of mobile and immobilized splenic neutrophils. (a) Representative IVM images, flow cytometry plots, and quantification of splenic neutrophils 24 h after administration of isotype antibody (Ab) or Ly6G (1A8) antibody treatment. Green, neutrophils (neu). mono, monocyte. Bars, 50 µm. n = 5 for isotype and n = 6 for 1A8 antibody pooled from three independent experiments. (b) Representative flow cytometry plots and quantification JEM Vol. 214, No. 5 1339 anism, as Cybb-deficient animals were able to effectively that proliferation, maturation, and mobilization of the imma- int hi clear and control the pneumococcal infection (Fig. 5 j). Pre- ture Ly6G into the mature Ly6G neutrophil population vious in vitro work with human neutrophils has noted an occurred within the first 24 h after S. pneumoniae infection. important contribution of the serine proteases (e.g., cathep- sin G [CatG]) and neutrophil elastase in intracellular pneu- neutrophils from the blood stream are sequestered mococcal killing (Standish and Weiser, 2009). In our in vivo into the MZ by MZ macrophages and model, animals with a deficiency in CatC, a central regulator influence tI antibody production of serine proteases including CatG and neutrophil elastase, Under basal conditions, very few neutrophils could be found displayed nearly a log-fold increase in mean bacterial load in the MZ. However, at 24 and 48 h after infection, a very dramatic infiltration of neutrophils occurred into the MZ in the spleen, compared with wild-type animals, which was bordering on significant (P = 0.0628; Fig. 5 j). A significant (Fig. 7 a). Two-photon imaging of this area revealed that, after difference in bacterial loads was observed between CatC- and pneumococcal infection, neutrophils leaving the main circu- Cybb-deficient mice (Fig. 5 j). The increased clearance of S. lation in the marginal sinus were tethered right out of the pneumoniae observed in Cybb-deficient animals likely results mainstream of blood by the MZ macrophages, where they from increased proteolytic activity that has previously been remained rather than moving to the splenic RP (Fig.  7  b). reported in phagocytes from this mouse strain (Rybicka et Tracking individual neutrophils in the MZ under basal con- al., 2010, 2012). These data suggest an important role for the ditions revealed that the few cells that crawled within the local mature neutrophils in removing the bacteria from the area did so at speeds ranging from 2 to 10 µm/min (Fig. 7 c RP macrophage and helping in bacterial clearance. and Video  8). After the infection, the neutrophils that were recruited from the main stream of blood did not move like emergency neutrophil maturation occurs in the local neutrophils in the RP, but rather, many were not response to S. pneumoniae crawling at all at 24 and 48  h after S. pneumoniae seques- Neutrophil populations were further evaluated at later time tration in the MZ, and the remainder crawled at the lowest points after infection. Flow cytometric analysis revealed an in- detectable velocity (2–4 µm/s; Fig.  7  c). Indeed, the dwell hi crease in Ly6G neutrophils within the first 24 h after S. pneu- time (Fig. 7 d) and number of firm interactions of neutrophils int moniae infection (Fig.  6  a). Conversely, Ly6G neutrophil with MZ macrophages (Fig. 7 e) were both increased during numbers did not significantly change within this time period infections. Depletion of MZ macrophages using low-dose int (Fig. 6 a). Although 10% of Ly6G neutrophils were positive CLL treatment resulted in a significant decrease in MZ local- for the proliferative marker Ki67 at baseline, this value in- ization of neutrophils (Fig. 7 f). These data support a role for S. pneumoniae administration (Fig. 6 b) creased by 24 h after MZ macrophages in the retention of splenic neutrophils after int and only in the Ly6G neutrophil population, as none of the pneumococcal infection. Blocking antibodies against various hi Ly6G neutrophils were Ki67 positive after infection. integrins revealed that neutrophil retention in the MZ was int To see whether during infection the immature Ly6G partially β1 but not β2 integrin dependent (Fig.  7  g). This neutrophils could take on the more mature mobile pheno- decrease in retention could be recapitulated by VCAM-1 type, a photoactivatable (UBC-PaGFP) transgenic mouse blockade (Fig. 7 g), supporting an α4β1–VCAM-1–mediated system was used. This allowed the tracking of the cells over retention mechanism. It is worth mentioning that other as yet time. When immobilized immature neutrophils were photo- unidentified adhesion molecules make up a significant por- activated under control conditions, ∼10% of the population tion of this retention. mobilized over a 1-h period, so now, photoactivatable im- A consequence of this sequestration mechanism into mobilized GFP-positive neutrophils became GFP-positive the MZ of the spleen was that the neutrophils localized mature neutrophils (Fig.  6  c and Video  7), suggesting some not only with MZ macrophages, but also with MZ B cells immature neutrophils are always maturing in the spleen. After (Fig. 7 h). To see whether this impacted antibody output by 6-h pneumococcal stimulation, a significantly larger propor- these neutrophils, we attempted to deplete neutrophils with tion of immobilized progenitor neutrophils were able to start Ly6G (1A8) antibody. The results were somewhat ambiguous. mobilizing and scan the RP over 1  h after photoactivation Although there was a decreased density of neutrophils in the (Fig. 6 c and Video 7) and contribute to the pool of mature MZ and RP areas of the spleen after infection (not depicted) neutrophils within the spleen. Collectively, these data support when anti-Ly6G was used, the MZ B cell–dependent IgM hi int of Ly6G and Ly6G neutrophil origin in the blood, spleen, and bone marrow after parabiosis. n = 4 from two independent experiments. (a and b) Data are represented as mean ± SEM. *, P < 0.01; **, P < 0.01; ***, P < 0.001. Student’s t test (a) and one-way ANOVA (b) statistical analyses were performed. (c) Rep- hi int resentative flow cytometry histograms of cell-surface marker expression for both Ly6G (red) and Ly6G (blue) neutrophils under steady-state conditions from three independent experiments. (d) Representative immunohistochemistry CD117 staining of the immobilized neutrophil population in the splenic RP from four independent experiments. Bars, 25 µm. (e) Representative nuclear morphology of the immobilized neutrophil population including both banded (white arrowheads) and segmented (blue arrowheads) neutrophils from two independent experiments. Bars, 11.6 µm. 1340 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. Figure 5. Mature neutrophil phagocytosis of S. pneumoniae in the splenic rP. (a) Representative spinning-disk confocal image of neutrophils (red) and RP macrophages (purple) containing S. pneumoniae (green) at 30 min after infection. Data are representative of n = 6 from three independent experiments. Bar, 50 µm. White arrows, bacteria containing neutrophils; blue arrows, bacterial containing RP macrophages. (b) Proportion of splenic neu- trophil populations that bound S. pneumoniae at 30 min after infection. There were four fields of view per animal. n = 6 pooled from three independent experiments. (c) Time-lapse spinning-disk confocal images of S. pneumoniae (green) cell adhesion (white arrow) to RP macrophage (purple) and subsequent JEM Vol. 214, No. 5 1341 and IgG antibody responses occurred in a dichotomous fash- into splenectomized mice. The data reveal that benefit of ion (Fig.  7  i). A portion of the animals displayed low anti- the antibodies required splenic neutrophils, as injection of body production similar to MZ B cell depletion (Fig.  1  e), these antibodies into splenectomized mice provided no sur- whereas normal responses were detected in other animals vival benefit (Fig. 8 d). Collectively, these data highlight the (Fig. 7 i). It became very clear that although neutrophil de- important phagocytic role of mature splenic neutrophils for pletion efficiency was ∼90% at 24  h, at 72  h it was highly antibody-mediated clearance of S. pneumoniae. variable in the spleen ranging from as little as 30 to as high as 80%. This likely accounts for much of the antibody vari- dIScuSSIon ability. Consistent with these data, a similar dichotomy was This study used imaging to track the dynamic progression of S. pneumoniae infection in spleen and has revealed previously also observed in both blood and spleen bacterial counts at 72 h after infection (Fig. 7 j). Clearly, the more effective the unknown mechanisms that increase our understanding of neutrophil depletion was, the fewer antibodies were produced spleen-specific protective mechanisms against this pathogen. and the more bacteria were noted. Correlational analysis of This includes the collaboration of local neutrophils and mac- these mixed responses revealed that splenic bacterial titers in rophages as early innate immune components in the splenic neutrophil-depleted animals negatively correlated with both RP helping to dampen bacterial proliferation. Indeed, we vi- IgM (r = −0.6950; P = 0.0402) and IgG (r = −0.813 and sualized the macrophage of the splenic RP being able to cap- P = 0.0092) serum antibody levels (not depicted). These data ture and present bacteria to local mature neutrophils. Based suggest that retained neutrophils in the MZ participate in on our parabiosis experiments, these local mature neutrophils the TI antibody response after S. pneumoniae infection, but are recruited constantly from the mainstream of blood under one has to be cautious when trying to deplete neutrophils basal conditions. Furthermore, we identify the presence of a in the spleen long term. second local population of splenic neutrophils that appear to be an immature population that, based on photoactivation Mature splenic neutrophils mediate tI antibody– experiments, can rapidly mature and mobilize during emer- dependent pneumococcal clearance gency situations, and this turns out to be key for helping to To assess whether MZ B cell–dependent TI antibod- control the infection until antibodies are made. We also deter- ies enhanced mature neutrophil function, serum transfer mine the requirement for the TI antibody response by MZ B experiments were performed (Fig.  8  a). Transfer of pneu- cells aiding neutrophil phagocytosis for final clearance. mococcus-immunized serum (72 h) resulted in an increased Tissue-resident macrophages play important homeo- phagocytosis of S. pneumoniae by mature neutrophils at 30 static roles including immune surveillance for pathogens. In min after infection, compared with both control serum (72 h) the context of encapsulated bacterial infection, MZ macro- and pneumococcus-immunized serum from MZ B cell–de- phages have been suggested to be important with their ability pleted animals (TI antibody deficient, 72  h; Fig.  8  b). This to bind the capsule of S. pneumoniae via the c-type lectin antibody-dependent mechanism was limited to neutrophils, SIGN-R1 expressed on its surface (Kang et al., 2004). Our as no differences in RP macrophage uptake of bacteria were data support this work but also provide new information on noted between serum transfers (Fig.  8  b). This enhanced a second population of splenic macrophages, the RP mac- neutrophil phagocytic capacity resulted in rapid removal of rophages, which appear to contribute in a major way to ini- pneumococcus from the bloodstream (Fig. 8 c). Next, to de- tial binding and tropism of S. pneumoniae to the spleen. This termine whether these TI antibodies could enhance killing in macrophage function appears to be limited to the spleen, as the absence of the splenic environment, they were injected Kupffer cells in the liver were less effective at catching encap- phagocytosis by neutrophils (red). Data are representative of n = 4 from three independent experiments. Bar, 10 µm. (d) 3D reconstruction of intracellular S. pneumoniae (green) within neutrophil (red) at 30–60 min after infection. Transparency of neutrophils is increased from left to right. Bar, 23.3 µm. Data are representative of n = 3 from two independent experiments. White arrows indicate intracellular bacteria. (e) IVM and flow cytometric quantification of S. pneumoniae phagocytosis by neutrophils (Neu) and RP macrophages (RPM) from 30–60 min after i.v. infection in wild-type (black bars) and C3-deficient (blue bars) animals. n = 3–6 pooled from three and two independent experiments for IVM and flow cytometry, respectively. FOV, field of view; S. p., S. pneu- moniae. (f) Survival curve for S. pneumoniae i.v. infection at 10 -CFU dose in wild-type (black line) or C3 KO (blue line) mice. n = 5 from one experiment. (g) S. pneumoniae bacterial counts in the spleen at 1 and 24 h after i.v. infection in isotype antibody (black circles)– and Ly6G (1A8) antibody (1A8 Ab; blue triangles)–treated animals. n = 4–6 pooled from two independent experiments. Red lines show the median. (h) Time-lapse spinning-disk confocal images of OxyBUR ST probe activation (green) after S. pneumoniae (red) phagocytosis by mature splenic neutrophil (blue). Data are representative of n = 6 from two independent experiments. Bar, 5 µm. White arrows indicate OxyBURST activation on the surface of intracellular bacteria. (i) Quantification of intracellular −/− OxyBUR ST probe activation within neutrophils in wild-type (blue line) and Cybb (gray line) mice. n = 3–6 pooled from two independent experiments. −/− AU, arbitrary units. (j) S. pneumoniae bacterial counts in the spleen at 24  h after i.v. infection in wild-type (black circles), Cybb (gray squares), and −/− CatC (blue triangles) animals. n = 4–6 pooled from two independent experiments. Black lines show the median. *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Student’s t test (b, e, and i), log-rank Mantel-Cox test (f), and Mann-Whitney test (g and j) statistical analyses were performed. Data are represented as mean ± SEM. 1342 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. Figure 6. Splenic immature neutrophil proliferation and mobilization after pneu- mococcal stimulation. (a) Representative flow cytometry plots (baseline and 24  h) and hi int quantification of Ly6G (blue line) and Ly6G (teal line) neutrophil populations at baseline and after S. pneumoniae infection. n = 4–6 pooled from four independent experiments. **, P < 0.01 versus 0  h (baseline). (b) Represen- tative flow cytometry histograms and quanti- hi fication of Ki67 staining in both Ly6G (blue int line) and Ly6G (teal line) neutrophil popula- tions at baseline and after S. pneumoniae in- fection. Positive gates were established by use of an AF488-conjugated isotype control within each experiment. n = 3–4 pooled from two in- dependent experiments. ***, P < 0.001 versus 0 and 12 h. FSC, forward scatter. (c) Time-lapse (0–60 min) spinning-disk confocal images of photoactivated neutrophil cluster mobilization (top left, white box) in the splenic RP at 6  h after saline (gray bar) or S. pneumoniae (blue bar) administration. Green, photoactivated (PA) cells; red, neutrophils; blue, vasculature; multicolored, tracks. Bar, 50 µm. n = 4 pooled from four independent experiments. *, P < 0.05. One-way ANOVA (a and b) and Student’ s t test (c) statistical analyses were performed. Data are represented as mean ± SEM. sulated S. pneumoniae in the sinusoidal circulation (unpub- had a larger contribution to initial binding and subsequent lished data). Although both splenic macrophage populations clearance of S. pneumoniae mediated by mature neutrophils. sequestered pneumococcus early, they contributed to S. pneu- RP macrophage binding of S. pneumoniae in our model was moniae infection defense by distinct mechanisms. MZ mac- not dependent on complement and was not enhanced with rophages contributed to TI antibody production by MZ B passive immunization, suggesting an alternative mechanism. cells, consistent with another study (Koppel et al., 2008). This In addition to resident macrophages, the spleen also is likely by facilitating the direct interaction of MZ B cells serves as an important reservoir for other myeloid cells. with the pathogen on the surface of the macrophages and in- Swirski et al. (2009) described a pool of monocytes that directly enhancing MZ B cell activation through recruitment reside within the RP under steady-state conditions and that of neutrophils at the MZ. Interestingly, MZ macrophages dis- can be mobilized to the sites of injury. We demonstrate that played only a small amount of catching of S. pneumoniae mature neutrophils are also present within the RP compart- as observed by intravital imaging. In fact, RP macrophages ment under basal conditions and carry out an important fil- JEM Vol. 214, No. 5 1343 Figure 7. neutrophil sequestration by MZ macrophages contributes to tI antibody response after pneumococcal infection. (a) Representative confocal images and quantification of neutrophil (green) colocalization with MZ macrophages (MZM; red) and RP macrophages (RPM; purple) after saline (black bars) or S. pneumoniae (S. p.; 24 h, blue bars; 48 h, red bars) i.v. injection. Bars, 50 µm. Eight fields of view were averaged per animal. n = 4–6 pooled from four independent experiments. (b) Time-lapse two-photon microscopy images of neutrophil behavior in MZ 24 h after S. pneumoniae injection. Green, neutrophils; red, MZM; multicolored, tracks. Bars, 25 µm. (c–e) Tracking quantification of neutrophil cell velocity (c), dwell time (d), and MZ macrophage interaction time (e) at 24 h (blue bars) and 48 h (red bars) after S. pneumoniae i.v. infection. Two fields of view were averaged per animal. n = 3–4 pooled from four independent experiments. (f) Quantification of neutrophil colocalization with MZ B cells and RP macrophages at 24 h after S. pneumoniae i.v. in- fection in control (Ctrl) liposomes and low-dose CLL–treated animals. n = 3–5 pooled from three independent experiments. (g) Quantification of neutrophil 1344 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. tering function within the organ by mediating phagocytosis ter inhibition of antibody production and increased bacterial of bacteria. This protective mechanism is not restricted to load, consistent with a potential role for regulation of anti- encapsulated bacteria, as phagocytosis of S. aureus and Liste- body production by neutrophils within the spleen. ria monocytogenes has also been observed with our system Although most tissues rely on circulating neutrophils (unpublished data), supporting an immune surveillance role for recruitment, the spleen appeared to compartmentalize for this pool of neutrophils. The spleen also has the capacity its recruitment; in the MZ, there was classical recruitment to increase both neutrophil and monocyte numbers by facil- of neutrophils from the vasculature, whereas the RP had its itating extramedullary hematopoiesis during inflammation own population of immature and mature neutrophils that (Swirski et al., 2009; Robbins et al., 2012). This is thought to were key for local eradication of S. pneumoniae. The obvious advantage of this type of system is that, at the first sign of occur by mobilization of hematopoietic stem and progen- itor cells from the bone marrow to the spleen, where they infection, neutrophils are already in the RP ready to help kill seed preestablished niches and undergo expansion (Swirski bacteria caught by the macrophage. Without the neutrophils et al., 2009). Intriguingly, the recently described hemato- there, the bacteria were able to increase their numbers dra- poietic niche in the splenic RP (Dutta et al., 2015; Inra et matically within the spleen, killing the host. Although at this al., 2015) shares common features with the local environ- point we can only conclude that it is the immediate presence int ment surrounding our described Ly6G immature neutro- of neutrophils in spleen that helped to eradicate bacteria, an phils, including the perivascular location and the presence additional possibility is that the circulating neutrophils that of VCAM-1–positive macrophages around the colonies enter the RP further mature to perform a necessary function (unpublished data). It is likely that our immature neutro- that circulating neutrophils are incapable of doing. In fact, phils represent the consequence of continuous extramedul- under steady-state conditions, we do note a more activated lary hematopoiesis during the steady state, albeit at a very phenotype for mature splenic neutrophils compared with slow rate. Upon inflammatory stimulation, these immature circulating neutrophils (unpublished data). Many of these neutrophils can mobilize as observed and contribute to the features are reminiscent of B helper neutrophils, which are mature neutrophil population. proposed to polarize locally in response to bacterial factors Neutrophils in the spleen have previously been shown and GM-CSF, which is released locally by innate lymphoid to contribute to MZ B cell activation and subsequent pro- cells (Magri et al., 2014; Chorny et al., 2016). It will also be duction of anti-capsular polysaccharide antibodies (Puga et interesting to know whether during systemic infections such al., 2012; Magri et al., 2014; Chorny et al., 2016). Pentraxin as sepsis, where most neutrophils are recruited to lungs and 3 has recently been identified as a natural adjuvant produced other infectious sites and in some instances patients become and released by these B helper neutrophils to help mediate neutropenic, this local pool helps to maintain neutrophil pres- this response (Chorny et al., 2016). Although, some have not ence in the spleen for critical innate immune purposes. been able to confirm these observations in humans (Nagelk- erke et al., 2014), our data does support a role for newly re- MaterIaLS and MethodS cruited splenic neutrophils in promoting anti-pneumococcal animals TI antibody production during systemic mouse pneumococ- 8–12-wk-old male and female mice were used for experi- cal infection. Although it is very easy to deplete circulating ments. C57BL/6, Pep BoyJ(B6), LysM-eGFP/eGFP, UBC- −/− neutrophils to determine their function, the resident mature PaGFP, and Cybb-deficient (Cybb ) mice were obtained −/− population in the spleen was extremely difficult to eradicate from The Jackson Laboratory, and CatC-deficient (CatC) during infection, and the immature population was resistant mice were a gift from GlaxoSmithKline. All mice were housed under a specific pathogen–free, double-barrier unit to 1A8 depletion. In fact, similar depletion inefficiency of splenic neutrophils has previously been noted in a chronic at the University of Calgary. Mice were fed autoclaved ro- inflammatory setting of tumor-bearing mice (Moses et al., dent feed and water ad libitum. All protocols used were in 2016). It was unclear whether this resident population could accordance with the guidelines drafted by the University of impact on B cell antibody production. Nevertheless, in those Calgary Animal Care Committee and the Canadian Council mice where we obtained good depletion, we also got bet- on the Use of Laboratory Animals. colocalization with MZ B cells (left) and RP macrophages (right) at 24 h after S. pneumoniae i.v. infection in animals treated with isotope control, anti-CD18, anti-CD29, and anti–VCAM-1 blocking antibodies. Eight fields of view were averaged per animal. n = 3–5 pooled from three independent experiments. (h) Representative image of neutrophil localization within the MZ B cells (MZB) 24 h after i.v. S. pneumoniae infection. Green, MZ B cells; red, neutrophils; purple, RP macrophages. Data are representative of n = 4 from two independent experiments. Bar, 25 µm. (i) Quantification of S. pneumoniae–specific serum IgM and IgG antibody levels in isotype antibody–treated and Ly6G (1A8) antibody (1A8 Ab)–treated animals at 72 h after infection. Red dotted lines indicate antibody levels in MZ B cell–depleted animals. n = 5–9 total pooled from three independent experiments. (j) S. pneumoniae bacterial counts in the blood and spleen at 72 h after i.v. infection in isotype antibody–treated and Ly6G (1A8) antibody–treated animals. Black lines show the median, and red dotted lines show the detection limit. n = 5–9 total pooled from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One-way ANO VA (a and e), two-way ANO VA (c and d), Student’s t test (f, g, and i), and Mann-Whitney test (j) statistical analyses were performed. JEM Vol. 214, No. 5 1345 Figure 8. Passive immunization facili- tates neutrophil-mediated capture and clearance of S. pneumoniae in the spleen. (a) Schematic of serum transfer protocol. (b) Representative spinning disk confocal im- ages and quantification of neutrophils (Neu; red) and RP macrophages (RPM; purple) con- taining S. pneumoniae (green) at 30–60 min after infection in animals given control serum (blue bars), immunized serum (solid red bars), and immunized serum from MZ B cell–de- pleted (MZB dep) animals (checkered red bars). Bars, 30 µm. White arrows indicate neutrophil containing bacteria, and blue arrows indicate RP macrophage containing bacteria. Eight fields of view (FOV) were averaged per an- imal. n = 3–4 pooled from two independent experiments. Data are represented as mean ± SEM. (c) S. pneumoniae bacterial counts in the blood and spleen at 1  h after i.v. infection in wild-type mice receiving either control serum (blue circles) or immunized serum (red squares). n = 5–6 pooled from two independent experi- ments. (d) Survival curve to S. pneumoniae in- fection at 10 -CFU dose in sham-operated and splenectomized (Spx) animals receiving control (CT) serum or immunized serum. n = 4–5 from one experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One-way ANO VA (b), Mann-Whitney test (c), and Log-rank Mantel-Cox test (d) sta- tistical analyses were performed. IVM together with the appropriate band-pass filters (Semrock). A Multichannel spinning-disk confocal and two-photon con- back-thinned electron-multiplying charge-coupled device focal microscopes were used to image mouse spleens. Mice 512 × 512–pixel camera (Hamamatsu Photonics) was used were anaesthetized (10 mg/kg xylazine hydrochloride and for fluorescence detection. Volocity software (PerkinElmer) 200 mg/kg ketamine hydrochloride). The right jugular vein was used to drive the confocal microscope and for 3D ren- was cannulated to administer fluorescent dyes and additional dering, acquisition, and analysis of images. Bacteria and neu- anesthetic. After skin incision, the mouse was placed in a left trophil behavior was evaluated using Volocity software. Both lateral position, and the spleen was exteriorized onto a glass populations were identified and tracked using the Find Ob- coverslip on the inverted microscope stage. The stage was kept ject and Track functions, respectively, within the measurement at 37°C to maintain the mouse body temperature. modality. For each animal, tracking data were averaged for Image acquisition of the splenic RP was performed multiple fields of view and considered an n = 1. using an inverted microscope (IX81; Olympus), equipped Image acquisition of the splenic MZ was performed with a focus drive (Olympus) and a motorized stage (Ap- using an inverted multiphoton microscope (TCS SP8; Leica plied Scientific Instrumentation) and fitted with a motor- Biosystems) or upright multiphoton microscope (TCS SP8; ized objective turret equipped with 4×/0.16 UPLANS APO, Leica Biosystems). Typically, MZ macrophages were stained 10×/0.40 UPLANS APO, and 20×/0.70 UPL ANS APO ob- by i.v. injection of 1 µg anti-CD209b fluorescent conjugated jective lenses and coupled to a confocal light path (WaveFx; mAbs. i.v. injection of 2 µg anti-MAR CO (macrophage re- Quorum Technologies) based on a modified CSU-10 head ceptor with collagenous structure) fluorescent conjugated (Yokogawa Electric Corporation). Cells of interest were vi- mAbs was also used to label MZ macrophages in preliminary experiments to confirm that 1 µg anti-CD209b did not affect sualized using fluorescently labeled antibodies, fluorescent reporter mice, and fluorescent reporter bacteria. Typically, RP S. pneumoniae binding. The dynamic behaviors of immune macrophages and neutrophils were stained by i.v. injection of cells and bacteria were visualized simultaneously with a single- 2.5 µg anti–F4-80 and 1 µg anti-Ly6G fluorescent conjugated pulsed laser (Ti-Sapphire; Spectra-Physics or Coherent) at mAbs, respectively. Laser excitation wavelengths of 491, 561, 950 nm or 1,040 nm in combination with appropriate band- 642, and 730 nm (Cobolt) were used in a rapid succession pass emission filters (Semrock). The fluorescence was detected 1346 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. by HyD hybrid or high-sensitivity photomultiplier non- spleen were removed after thoracotomy, weighed, and ho- descanned detectors. Leica Biosystems software was used to mogenized. For determination of CFUs, 50 µl of blood and drive the confocal microscope and for 3D rendering and ac- 100 µl of tissue homogenate were serially diluted, plated onto quisition of images. Files were converted from LIF format into blood agar plates, and incubated at 37°C with 5% CO for multiple TIFFs using Volocity and subsequently imported into 18 h, and bacterial colonies were counted. ImageJ (National Institutes of Health) for analysis. The plugin StackReg was used for movement correction purposes. Both Serum antibody measurement bacteria and neutrophil tracking were completed using the Blood was collected by cardiac puncture at multiple time Manual Tracking plugin. For each animal, tracking data were points after either saline or S. pneumoniae injection and spun n = 1. For down at 400 g for 10 min. Then, serum was collected, and 25 µl averaged for multiple images and considered an photoactivation experiments, multiphoton excitation at 800 was diluted with an equal volume of FACS wash buffer (1× nm was used to activate neutrophil populations of interest, PBS, 2% FBS, and 0.2% EDTA) and subsequently incubated and images were acquired using a spinning-disk microscope with 10 CFU of freshly cultured S. pneumoniae for 30 min as described in the previous paragraph. on ice. After a wash step, 1 µg of APC-conjugated anti–mouse IgG or IgM antibody was added in a resuspension volume Parabiosis of 50  µl and incubated for an additional 30 min. Antibody Age-matched female mice were housed together for 2 wk concentration used was determined by a preliminary serial before surgery. Parabiotic pairs of mice were generated sur- dilution experiment to ensure optimal signal saturation. Sam- gically as previously described (Ajami et al., 2007). In brief, ples were subsequently fixed with 0.5% paraformaldehyde in a skin incision running from elbow to knee along the flank flow cytometry wash buffer and run using a flow cytometer was generated on opposite sides of the mice to be paired. The (LSR II; BD). Samples were first gated on GFP bacteria, and mice were first joined with a suture through the shoulder and then, the median fluorescence intensities were determined thigh muscles, and next, the inside faces of the skin flaps were for APC-labeled anti-IgG or IgM. Values were normalized to juxtaposed and sutured. Complete blood sharing was moni- control animal (saline injected) levels within each experiment tored between 12 and 17 d by tail vein blood sampling. 24 h and then averaged among multiple experiments. after chimerism detection in the blood, pairs were separated and prepared for subsequent intravital imaging of the spleen Serum transfer experiments and/or flow cytometry of the spleen, blood, and bone marrow. Serum was collected as described in the previous paragraph at 72 h after injection (saline or S. pneumoniae). After confir- Bacterial growth and infection mation of antibody production, serum from multiple animals The S. pneumoniae D39-GFP strain was grown on blood (same treatment) was combined and heat inactivated at 56°C agar plates overnight at 37°C with 95% O /5% CO . Single for 20 min. 100 µl of the control, immunized, or immunized 2 2 colonies were picked from plates and grown in brain-heart (MZ B cell depletion) heat-inactivated serum was adminis- infusion broth at 37°C with CO until an OD 600 of 0.5 tered i.v. 20–30 min before infection with S. pneumoniae. was reached. Bacteria were resuspended in saline and diluted to achieve appropriate dose with a 100-µl volume. For long- Flow cytometry term infection and acute bacteria infection, doses of 2 × 10 Mice were anesthetized, and the spleen was removed and 4 6 CFU or 10 CFU and 5 × 10 CFU were administered i.v., placed in PBS on ice. Then, blood was collected in a hep- respectively. Generation of reporter bacteria for oxidation was arinized syringe by cardiac puncture. The spleen was passed performed as previously described (Surewaard et al., 2016). through a 70-µm filter. For S. pneumoniae–GFP detection In brief, fresh streptococcal cultures were washed twice in experiments, cells were immediately stained for 20 min with saline, resuspended at 5 × 10 CFU in 500 µl in carbonate, fluorescently labeled antibodies and subsequently incubated pH 8.3, buffered saline, and labeled for 30 min with 20 µg in 1× Fix/Lyse buffer (eBioscience). For the remainder of −1 ml AF647 N-hydroxysuccinimide ester (Thermo Fisher experiments, residual red blood cells were lysed using ACK −1 Scientific) and 60 µg ml OxyBUR ST Green H2DCF DA lysing buffer (Invitrogen). The cells were blocked using an- SE (DMSO stock; Thermo Fisher Scientific) under vigorous ti-CD16/32 antibody (2.4G2 clone; Bio X Cell) for 20–30 agitation. Activation of OxyBURST w as accomplished by min. Then, cells were stained for 30 min with specified mark- adding 250 µl of 1.5 M hydroxylamine, pH 8.5, and incubat- ers including FITC-labeled B220 (RA3-6B2; BD), CD18 ing for 30 min on ice. Reporter bacteria were washed twice (C71/16; BD), CD62P (RB40.3; BD), CD45.2 (104; BD), 6 7 with PBS and injected i.v. into mice at 5 × 10 –10 CFU. CD43 (eBioR2/60; eBioscience), PE-labeled anti–mouse Ly6G (1A8; eBioscience), F4/80 (BM8; eBioscience), CD Bacteriological analysis 21/35 (4E3; eBioscience), CD117 (2B8; eBioscience), Anesthetized mice were washed with 70% ethanol under CD49d (R1-2; eBioscience), CxCR4 (2B11; eBioscience), aseptic conditions. Blood was collected in a heparinized sy- CD62L (MEL-14; eBioscience), CD169 (3D6.112; BioLeg- ringe by cardiac puncture. The lungs, liver, heart, kidneys, and end), APC-labeled anti–mouse CD209b (22D1; eBioscience), JEM Vol. 214, No. 5 1347 CD23 (B3B4; eBioscience), CD45.1 (A20; eBioscience), unless otherwise specified. Data, with the exception of bac- PerCP Cy5.5-labeled Ly6C (HK 1.4; eBioscience), PE-Cy7– terial CFUs, were compared either by unpaired two-tailed labeled CD11b (M1/70; eBioscience), Pacific blue–labeled Student’s t test or one-way or two-way ANO VA followed Ly6G (1A8; eBioscience), AF647-labeled CD54 (YN1/1.7.4; by Bonferroni posthoc test for multiple comparisons adjust- BioLegend), efluor 660–labeled CD11c(N418; eBioscience), ment. Bacterial CFU data were compared by Mann-Whitney Brilliant violet 510–labeled CD45 (30-F11; BioLegend), and test or Kruskal-Wallis test followed by Dunn’s multiple com- APC-Cy7–labeled CD45 (30-F11; eBioscience). Appropriate parisons test. Survival curves were compared using log-rank isotype control antibodies were used to confirm positive sig- (Mantel-Cox) test. Statistical significance was set at P < 0.05. nals. Nonviable cells were identified using propidium iodide The applied statistical analyses and the numbers of indepen- n) are reported in the figure legends. or viability dye efluor 780 (eBioscience). Samples were run dent replicates ( using a flow cytometer (FAC SCanto; BD) and analyzed using FlowJo software (Tree Star). Neutrophils were identified as online supplemental material + hi int hi hi CD11b Ly6G Ly6C or LysM GFP SSC (for depletion Video 1 displays the movement of S. pneumoniae in the splenic experiments). Proinflammatory monocytes were identified MZ after infection. Video 2 shows S. pneumoniae accumula- + − hi as CD11b Ly6G Ly6C . RP macrophages were identi- tion in the splenic RP after infection. Video 3 demonstrates in +/lo − hi fied as CD11b CD209b F4/80 . MZ macrophages were vivo behavior of splenic neutrophil populations under steady- + hi − identified as CD11b CD209b F4/80 . Metallophilic mac- state conditions. Video  4 shows chimerism of both mature + + −/lo rophages were identified as CD11b CD169 F4/80 . Fol- and immature splenic neutrophils after parabiosis. Video  5 + hi int licular B cells were identified as B220 CD23 CD21/35 , displays in vivo capture of S. pneumoniae by splenic neu- + lo int/hi MZ B cells as B220 CD23 CD21/35 , and B1 B cells as trophils in the presence or absence of complement. Video 6 + lo lo + B220 CD23 CD21/35 CD43 . shows neutrophil-mediated removal and phagocytosis of S. pneumoniae after initial binding to RP macrophages. Video 7 depletion and blocking antibody protocols shows mobilization of photoactivated immature neutrophils Depletion protocols for various cell types have been used as after S. pneumoniae infection. Video  8 displays the in vivo previously described (Lu and Cyster, 2002; McGaha et al., 2011; behavior of circulating neutrophils in the splenic MZ. Kolaczkowska et al., 2015). In brief, transient MZ B cell de- pletion was achieved by intraperitoneal injection of 100 µg of acknoWLedGMentS both anti-CD11a (M17/4; Bio X Cell) and anti-CD49d (PS/2; Bio X Cell) 96 h before S. pneumoniae infection. Depletion of We thank Trecia Nussbaumer and Dr. Robin Yates for the breeding of mice. We thank Dr. Craig Jenne and Dr. Bryan Yipp for use of their multiphoton microscopes. We MZ macrophages was completed by i.v. delivery of a low-dose thank Dr. Pina Colarusso at the Snyder Live Cell Imaging Facility for technical support clodronate treatment (100 µg) 96 h before infection. Neutro- for photoactivation experiments and Dr. Karen Poon (Snyder Institute Molecular phils were depleted using an anti-Ly6G (1A8; Bio X Cell) Core) for assistance with flow cytometry. antibody. For acute infection experiments, a single 500-µg dose P. Kubes is supported by a foundation grant from the Canadian Institutes of Health Research and a Heart and Stroke Foundation of Canada grant. J.F. Deniset is was given intraperitoneally 24 h before infection. For long- financially supported by a postgraduate fellowship from Alberta Innovates–Health term infection, supplemental 200-µg doses were given daily. Solutions. B.G. Surewaard is partially funded by Marie Curie Actions (FP7-PEOPLE- Isotype antibodies or control liposomes were given as con- 2013-IOF; grant no. 627575) and a postgraduate fellowship from Alberta Innovates– trol injections. The specificity of all three depletion protocols Health Solutions. The authors declare no competing financial interests. was verified using both flow cytometry and in some instances Author contributions: J.F. Deniset and B.G. Surewaard conceived the study, immunohistochemistry. LysM-GFP reporter mice for our de- performed experiments, analyzed data, and wrote the manuscript. W.-Y. Lee contrib- pletion studies were used to evaluate neutrophil depletion. uted to parabiosis experiments. P. Kubes wrote the manuscript and directed the study. hi int hi int Ly6G neutrophils were identified as Ly6C GFP , Ly6G int int hi hi neutrophils as Ly6C GFP , and Ly6C monocytes as Ly6C Submitted: 27 September 2016 int GFP . These gates were established in experiments performed Revised: 27 January 2017 with control and isotype antibody–treated LysM-GFP mice Accepted: 3 March 2017 with the inclusion of the Ly6G antibody (1A8 clone). For blocking antibody experiments, 100 µg of pu- rified anti-CD18 (clone M18/2; BioLegend), anti-CD29 (clone HMβ1-1; BioLegend), or anti-CD106 (clone 429; reFerenceS eBioscience) was added i.v. 10 min before S. pneumoniae Ajami, B., J.L. Bennett, C. Krieger, W. Tetzlaff, and F.M. Rossi. 2007. Local self-renewal can sustain CNS microglia maintenance and function infection. Appropriate isotype control antibodies were used throughout adult life. Nat. Neurosci. 10:1538–1543. http ://dx .doi .org for control experiments. /10 .1038 /nn2014 Amlot, P.L., and A.E. 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Splenic Ly6Ghigh mature and Ly6Gint immature neutrophils contribute to eradication of S. pneumoniae

The Journal of Experimental Medicine , Volume 214 (5) – May 1, 2017

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© 2017 Deniset et al.
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10.1084/jem.20161621
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Abstract

Ar ticle high int Splenic Ly6G mature and Ly6G immature neutrophils contribute to eradication of S. pneumoniae 1,3 1,3,4 1,3 1,2,3 Justin F. Deniset, Bas G. Surewaard, Woo-Y ong Lee, and Paul Kubes 1 2 3 Department of Physiology and Pharmacology, Department of Microbiology, Immunology, and Infectious Diseases, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada Department of Medical Microbiology, University Medical Centre, 3584 CX Utrecht, Netherlands t he spleen plays an integral protective role against encapsulated bacterial infections. our understanding of the associated mechanisms is limited to thymus-independent (tI) antibody production by the marginal zone (MZ) B cells, leaving the contri - bution of other splenic compartments such as the red pulp (rP) largely unexplored despite asplenic patients succumbing to the infection in the first 24 h, suggesting important antibody-independent mechanisms. In this study, using time-lapse intravital imaging of the spleen, we identify a tropism for Streptococcus pneumoniae in this organ mediated by tissue-resident MZ and hi intermediate rP macrophages and a protective role for two distinct splenic neutrophil populations (Ly6G and Ly6G ) residing in the splenic rP. Splenic mature neutrophils mediated pneumococcal clearance in the spleen by plucking bacteria off the surface of rP macrophages that caught the majority of bacteria in a complement-dependent manner. this neutrophil phagocytic capacity intermediate was further enhanced after tI antibody production. resident immature neutrophils (Ly6G ) in the spleen undergo emer- gency proliferation and mobilization from their splenic niche after pneumococcal stimulation to increase the effector mature neutrophil pool. We demonstrate that splenic neutrophils together with two macrophage populations and MZ B cells regulate systemic S. pneumoniae clearance through complementary mechanisms. IntroductIon The spleen is important for protection against encapsulated cells rapidly differentiate into plasmablasts and secrete patho- bacteria including Streptococcus pneumoniae, a major cause gen-binding serum IgM antibodies that are detectable in the of morbidity and mortality worldwide. Invasive pneumococ- serum only starting at 72–96 h after infection (Martin et al., cal disease, detected in 10–30% of pneumococcal pneumo- 2001; Belperron et al., 2005; Moens et al., 2007), meaning niae cases, has long been known to increase the mortality other cells must keep the pathogen in check until antibodies substantially beyond that seen with pneumonia alone (Chiou are made. Once made, these antibodies are thought to en- and Yu, 2006; Blasi et al., 2012). It is estimated to directly hance recognition and facilitate clearance of S. pneumoniae. cause >1.6 million deaths annually (World Health Organi- Indeed, splenectomized patients display impaired IgM anti- zation Geneva, 2007; O’Brien et al., 2009). Invasive disease body responses to polysaccharide antigen (Amlot and Hayes, is particularly serious in splenectomized or asplenic patients. 1985; Kruetzmann et al., 2003). As a result, pneumococcal These individuals have a 50-fold higher risk of developing research in the spleen has focused almost exclusively on the a fulminant septic infection to encapsulated bacteria, and a cells that regulate B cell–dependent antibody production. 50–70% mortality rate is associated with these cases (Hold- However, considering the kinetics of i.v. pneumococcal dis- sworth et al., 1991; Di Sabatino et al., 2011). Importantly, the ease in splenectomized patients, we hypothesize that other majority of deaths occur within the first 24 h (Gransden et al., very rapid innate immune mechanisms within the spleen 1985; Di Sabatino et al., 2011), highlighting the importance must not only contribute to early protection, but also stave of some very rapid as yet unknown innate immune mecha- off the infection until antibodies can be made. nisms within the spleen in providing protection during the Neutrophils are first responders in the innate immune acute stages of i.v. infection before antibody production. response to infection. Their multiple defense mechanisms, To date, the major protective mechanism of the spleen such as their phagocytic capacity, reactive oxygen species against S. pneumoniae is believed to be dependent on thy- production, and ability to degranulate and form neutrophil mus-independent (TI) antibody production by specialized B extracellular traps, contribute to the effective clearance of cells within the marginal zone (MZ). After stimulation, MZ B pathogens (Kolaczkowska and Kubes, 2013). Neutrophils typically access sites of infection via the circulation, following Correspondence to Paul Kubes: pkubes@ucalgary.ca © 2017 Deniset et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Abbreviations used: 3D, three dimensional; Cat, cathepsin; CLL, clodronate liposome; Alike–No Mirror Sites license for the first six months after the publication date (see http ://www .rupress .org IVM, intravital microscopy; MZ, marginal zone; RP, red pulp; SD-IVM, spinning-disk /terms /). After six months it is available under a Creative Commons License (Attribution–Noncommercial– IVM; TI, thymus independent. Share Alike 4.0 International license, as described at https ://creativecommons .org /licenses /by -nc -sa /4 .0 /). The Rockefeller University Press $30.00 J. Exp. Med. 2017 Vol. 214 No. 5 1333–1350 https://doi.org/10.1084/jem.20161621 The Journal of Experimental Medicine chemoattractant cues and responding to local inflammatory monas aeruginosa by the liver (Kolaczkowska et al., 2015 and mediators (Kolaczkowska and Kubes, 2013). In addition to unpublished data). In fact, removal of the spleen did not in- these circulating neutrophils, a large pool of neutrophils can crease blood levels or dissemination of S. aureus (unpublished be found in the bone marrow, and marginated pools of neu- data). The course of S. pneumoniae infection was tracked in trophils may also exist within peripheral organs. These sources the spleen and blood over a 5-d period (Fig. 1 b). Bacterial of neutrophils are thought to mobilize in response to stim- counts decreased in blood and spleen for the first 8  h after ulation (Athens et al., 1961a,b). There is some evidence to infection. This was followed by a rebound in levels at 24 and suggest that the spleen might function as a reservoir. Neu- 48 h before complete clearance by 5 d after infection. trophils, upon reinjection into humans and mice, appear to The spleen was integral for clearance, as a pneumococ- accumulate within the spleen (Peters et al., 1985; Ussov et al., cal challenge in splenectomized mice resulted in 100% mor- 1995; Suratt et al., 2001). Whether this is related to an artifact tality by 48  h, even at a fivefold decrease in infection dose of neutrophil isolation and reinjection or a bona fide physio- (Fig. 1 c). Much focus has been placed on the MZ B cells and logical event remains unclear. Recently, a B helper neutrophil MZ macrophages as contributors to antibody production and subset has been described to reside in the perifollicular zone pneumococcus clearance. Specific depletion of MZ B cells of the spleen in humans and mice to promote antibody pro- via an antibody-mediated protocol (Lu and Cyster, 2002) re- duction by MZ B cells (Puga et al., 2012; Magri et al., 2014; sulted in 40% mortality but only in the later phase (72 h) after Chorny et al., 2016). However, evidence on splenic neutro- S. pneumoniae infection (Fig. 1 d), coinciding with the MZ B phil turnover, behavior, and additional functions during basal cell–dependent production of IgM and IgG antibodies capa- and infectious conditions is lacking and would require live ble of binding S. pneumoniae (Fig. 1 e). Low-dose clodronate liposome (CLL) treatment, which depleted 80–90% of MZ cell imaging of these cells in the spleen, something that has to date not been done to our knowledge. macrophages but not RP macrophages (McGaha et al., 2011), In this study, we used a combination of spinning-disk resulted in a nearly identical temporal outcome (Fig.  1  d). intravital microscopy (IVM [SD-IVM]) and two-photon Combined MZ macrophage and MZ B cell depletion did IVM to evaluate the behavior of neutrophils in the spleen not further increase susceptibility to S. pneumoniae infection under steady state and after S. pneumoniae infection. Using (Fig. 1 d), suggesting a single collaborative protective mech- this platform, we found that much but not all of the S. pneu- anism of these two cell types via TI antibody production. moniae bypassed the MZ macrophages and were caught by Importantly, the combined depletion of these two cell popu- red pulp (RP) macrophages. We also identified two neu- lations only partially recapitulated the effect of a splenectomy trophil populations within the splenic RP: an immobilized, but with a delayed time frame, indicating that other earlier int ) population of neu- immature Ly6G-intermediate (Ly6G splenic innate immune mechanisms independent of these two hi trophils and a mature Ly6G-high (Ly6G ) population of cell types (and their ability to stimulate TI antibody produc- neutrophils that scan the tissue. Mature neutrophils mediated tion) are involved in splenic protection to pneumococcus. pneumococcal clearance by removing the bacteria from the surface of RP macrophages. During an emergency response S. pneumoniae localizes to both the MZ int to infection, the immobilized Ly6G immature neutrophils and the rP of the spleen increased their proliferative capacity and took on features of Dynamics of S. pneumoniae distribution in the spleen were the resident mature neutrophils. Circulating neutrophils re- determined using time-lapse two-photon IVM and SD-IVM cruited to the splenic MZ helped increase TI antibody pro- of the spleen. First, evaluation of S. pneumoniae movement in duction by MZ B cells, which further enhanced the ability the MZ revealed that, upon i.v. infection, the GFP-expressing bacteria flowed across the MZ, with some tethering inter- of mature splenic neutrophils to fully eradicate systemic pneumococcal infection. actions to MZ macrophages or other structures in this area (Fig. 2 a and Video 1). Quantification over the first 20 min reSuL tS after infection revealed that the vast majority of bacteria vi- the spleen is integral for protection against systemic sualized had a short transit time through this area with only S. pneumoniae infection a few bacteria (<20%) being detained permanently in the A pneumococcal bacteremia model was used to evaluate indi- MZ (>5 min in Fig. 2 b). Depletion of MZ macrophages by vidual components of immunity during S. pneumoniae infec- low-dose CLL treatment resulted in increased localization of tion. Intravenous infection with a very small dose (10 CFU) bacteria in the RP at 60 min after infection (Fig. 2 c). Visual- of S. pneumoniae resulted in a fivefold preferential sequestra- ization of the splenic RP revealed a progressive accumulation tion by the spleen compared with the lung and liver within of bacteria in this region over the first 20 min (Fig. 2, d and e; 60 min (Fig. 1 a). Bacterial counts in the brain, heart, and kid- and Video 2). RP macrophages are at least in part responsible neys were below detection limit (not depicted). This splenic for this retention, as their depletion through high-dose CLL tropism is unique to S. pneumoniae infection, as our previous treatment significantly impaired the number of bacteria in work has shown tremendous preferential sequestration of cir- the compartment (Fig. 2 e). This is despite increased bacteria culating Staphylococcus aureus, Escherichia coli, and Pseudo- migrating to the RP as a result of the concurrent depletion 1334 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. Figure 1. Splenic protection againstS. pneumoniae infection. (a) S. pneumoniae bacterial counts in blood, spleen, lung, and liver 1 h after i.v. infection. Black lines show the median. n = 6 pooled from two independent experiments. (b) Mean (± SD) bacterial counts in the blood (red circles) and spleen (blue squares) over 120 h. n = 5–7 pooled from two independent experiments. (c) Survival curve for S. pneumoniae i.v. infection at 10 -CFU dose in sham-operated (black line) or splenectomized (SPX; solid blue line) mice and at 2 × 10 –CFU dose in splenectomized mice (dotted red line). n = 5 from one experiment. (d) Representative flow cytometry plots of MZ B cell (MZB) depletion (dep), immunohistochemistry of MZ macrophage (MZM) depletion (green, RP macro- phage [RPM]; red, MZ macrophage) and survival curves to S. pneumonia infection at 10 -CFU dose in MZ macrophage–depleted (continuous green line), MZ B cell–depleted (continuous blue line), or MZ macrophage– and MZ B cell–depleted (dotted red line) animals. n = 6–10 from two independent experiments. Bar, 300 µm. Fo B, follicular B cell. Histograms and images are representative of two to three independent experiments. (e) Representative flow cytometry histograms and quantification of S. pneumoniae IgM (left) and IgG (right) serum antibodies after S. pneumoniae infection in WT (orange histogram; black lines) or MZ B cell–depleted S. pneumoniae (blue histogram; blue lines) mice. n = 3 for WT and n = 8 for MZ B cell depletion pooled from three independent experiments. Inf, infected. Data are represented as mean ± SEM, except in b. *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Kruskal-Wallis test (a), one-way ANO VA (b), log-rank Mantel-Cox test (c and d), and two-way ANO VA (e) statistical analyses were performed. of MZ macrophages. Localization of bacteria in both splenic tion to overall capture (20.1 ± 0.9%; Fig. 2 h). However, RP regions over time was also determined using fresh sections macrophages only accounted for 18.5 ± 2.1% of the total + + of spleen with labeled RP (F4/80) and MZ (CD209b ) (Fig.  2  h). Systematic assessment of the remaining popula- macrophages. This approach revealed a preferential localiza- tion of cells harboring S. pneumoniae using flow cytome- tion of S. pneumoniae in the MZ at 20 min after infection try revealed that the majority were neutrophils (42 ± 2.9%; (Fig. 2, f and g), whereas at 1 h after infection, bacteria load Fig.  2  h). The S. pneumoniae uptake appeared to be prefer- increased in the RP (Fig. 2 g). ential to splenic neutrophils, as the number of total S. pneu- Flow cytometry was used to identify the main cell types moniae-GFP–positive neutrophils was significantly higher in involved in S. pneumoniae capture in the spleen. Analysis of the spleen than the blood (Fig. 2 i). Furthermore, neutrophil splenic S. pneumoniae-GFP–positive cells from spleens har- numbers in the spleen did not increase within the first 30 min vested at 30 min after infection revealed that >60% of bac- after S. pneumoniae infection (Fig. 2 j), suggesting that a res- teria were in populations that resided within the RP of the ident neutrophil population within the spleen is responsible spleen, whereas MZ macrophages contributed a smaller por- for this bacterial uptake. JEM Vol. 214, No. 5 1335 Figure 2. Splenic localization ofS. pneumoniae. (a) Representative two-photon microscopy images of MZ macrophage interaction with S. pneumoniae during acute i.v. infection. Bar, 25 µm. Green, S. pneumoniae; red, MZ macrophages (MZM). White arrows indicate stationary bacteria. (b) Quantification of MZ dwell time of S. pneumoniae. n = 3 from three independent experiments. (c) Increased RP localization of S. pneumoniae 60 min after i.v. infection in MZ macrophage–depleted (low-dose CLLs; red bar) animals. n = 3–4 pooled from two independent experiments. Ctrl lipo, control liposome. (d) Representative spinning-disk confocal images of RP macrophage (RPM) interaction with S. pneumoniae during acute i.v. infection. Bars, 100 µm. Green, S. pneumoniae; purple, RP macrophages. (Inset) White arrows indicate stationary bacteria. Data are representative of n = 5 from three independent experiments. (e) S. pneu- moniae counts per field of view (FOV) in the RP from 0–20 min after i.v. infection in wild-type (black line), control liposome (blue line)–, low-dose CLL (red line)–, or high-dose CLL (gray line)–treated animals. There were four fields of view per animal. n = 5 for WT, n = 4 for control liposomes, n = 3 for high-dose CLL, and n = 5 for low-dose CLL pooled from four independent experiments. (f) Representative composite 10× stitched image of fresh spleen sections at 20 min after i.v. S. pneumoniae infection. Bar, 110 µm. Green, S. pneumoniae; red, MZ macrophages; purple, RP macrophages. (Inset) White arrows indicate bacteria. (g) Localization of S. pneumoniae in both the MZ (blue line) and RP (red line) regions at 20 and 60 min after i.v. infection. n = 3 pooled from + + − two independent experiments. (h) S. pneumoniae cell localization 30 minutes after infection in the spleen. MZ macrophages, CD11b CD209b F4/80 ; RP low − + + + int + − hi macrophages, CD11b CD209b F4/80 ; Neutrophils, CD11b Ly6G Ly6C ; monocytes, CD11b Ly6G Ly6C . n = 4 pooled from two independent experiments. 1336 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. int neutrophil populations reside in the splenic rP the Ly6G neutrophils are the resident population. These re- hi Time-lapse SD-IVM of the spleen was performed to study sults support the view that the mobile neutrophils are Ly6G int resident neutrophils in this organ. Under basal conditions, the and the immobilized resident neutrophils were the Ly6G spleen contained a large population of neutrophils that were population. This was not dissimilar to mature and immature localized in the RP compartment (Video 3). Based on their neutrophils, respectively, in bone marrow. int morphology and behavior, two neutrophil populations could To further characterize whether the Ly6G neutro- be identified: those that crawled around scanning the tissue phils expressed other markers of immaturity, we examined (mobile neutrophils) and those that formed large immobile the expression of c-KIT (CD117) as well as other maturity colonies (immobilized neutrophils) that could be as large as markers for neutrophils. Expression of CD117 was seen only int neutrophils (Fig. 4 c). CD117 staining was con- 30–50 cells (Fig.  3, a and b). Mobile neutrophils (Fig.  3  a, on Ly6G white arrowheads) migrated throughout the RP at varied ve- firmed on immobilized neutrophils by IVM (Fig. 4 d). A por- int locities (Fig. 3 c), and individual cells could be tracked for up tion of clustered Ly6G neutrophils also displayed increased to 1 h within the same field of view (Fig. 3, a–c). Neither of CD49d (consistent with immature neutrophils) and lower these populations was in the blood vessels. Immobilized neu- l-selectin expression (Fig. 4 c). No difference was noted in trophils (Fig. 3 a, green arrowheads) were stationary (Fig. 3 b any of the other markers between the two neutrophil pop- and Video  3), displayed a rounded morphology, and had a ulations (Fig.  4  c). Nuclear morphology assessment of im- perivascular localization (Fig. 3 a). Aside from these two pop- mobilized neutrophils in situ demonstrated the presence of ulations, neutrophils could also be seen rolling within blood banded (Fig. 4 e, white arrowheads) and segmented (Fig. 4 e, vessels, but they rarely stopped, and none emigrated out of the blue arrowheads) neutrophils. Collectively, these data further vasculature (Video 3). support the view that there are two phenotypically distinct T o evaluate the tur nover of these splenic populations with neutrophil populations in the RP, one immature expressing GFP blood neutrophils, parabiosis experiments pairing a LysM intermediate Ly6G levels and remaining immobilized in large mouse, in which neutrophils express GFP, with a nonfluores- colonies mostly as band cells and a second mature neutrophil cent wild-type mouse (C57) were undertaken. After 2 wk, population that is Ly6G high and that scans the splenic RP chimerism within the blood was 20–30%, as determined by under steady-state conditions. flow cytometry (Fig. 3 d), values consistent with that of pre- vious studies (Sawanobori et al., 2008; Guilliams et al., 2013). Mature splenic neutrophils mediate uptake of However, in the spleen, there was a dichotomy. IVM of the S. pneumoniae in the rP spleen in these same animals revealed that 35% of the mobile Next, we examined the behavior of the two populations of neutrophils in the splenic RP by SD-IVM. S. pneumoniae subset was derived from the parabiotic partner (Fig. 3 d and Video 4), indicating that, under steady state, this population uptake by neutrophils in the RP was predominantly limited was primarily replenished by neutrophils from the circula- to mature neutrophils, as the immobilized immature neutro- tion. Interestingly, only 10% of the immobilized clustered phils bound very little bacteria (Fig. 5, a and b; and Video 5). neutrophils appeared to derive from the parabiotic partner RP macrophages also bound the bacteria during this time (Fig. 3 d and Video 4), suggesting the majority of this popu- period. Visualization at a higher magnification revealed that lation was primarily resident. S. pneumoniae first bound to the surface of an RP macro- Intriguingly, analyzing all cells in the spleen, there were phage, and the mature neutrophil subsequently migrated clearly two populations of neutrophils: a large population of over and plucked the bacteria off the macrophage surface hi Ly6G neutrophils and a second smaller population of Ly- (Fig. 5 c and Video 6). At no point were mature neutrophils int 6G neutrophils. High concentrations of Ly6G (1A8) anti- S. pneumoniae freely flowing in the splenic RP. able to catch body were given to deplete neutrophils. IVM revealed that all Three-dimensional (3D) reconstruction of neutrophil-bound mobile neutrophils were depleted and only immobilized neu- bacteria further confirmed intracellular localization of the trophils remained (Fig. 4 a). Flow cytometric analysis of these pathogen. Increasing the transparency of Ly6G staining hi spleens revealed a significant decrease in Ly6G neutrophils, (Fig. 5 d, I–III) revealed GFP-expressing bacteria. Neutrophil int whereas Ly6G neutrophils remained unaffected (Fig.  4  a). phagocytosis of S. pneumoniae was complement dependent, When spleens from parabiosis animals were examined, the as capture was completely abrogated in C3-deficient mice hi percentage of Ly6G neutrophils of partner origin was akin (Fig. 5 e and Video 5). This was not because of the RP mac- int −/− to that of blood overall, whereas the percentage of Ly6G rophage not being able to catch the bacteria, as the C3 RP neutrophils was significantly reduced (Fig.  4  b), suggesting macrophages caught the S. pneumoniae, as well as wild-type (i) Flow cytometry quantification of total number of S. pneumoniae–GFP neutrophils in the blood (black bar) and spleen (teal bar). n = 4 pooled from two independent experiments. (j) Flow cytometry quantification of total neutrophil number in the spleen 30 min after saline (black bar) or S. pneumoniae (blue bar) injection. n = 3 for saline and n = 4 for S. pneumoniae pooled from two independent experiments. *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Student’s t test (c, i, and j) and two-way ANO VA (e and g) statistical analyses were performed. Data are represented as mean ± SEM. JEM Vol. 214, No. 5 1337 Figure 3. Steady-state neutrophil pop- ulations in the splenic rP. (a) Represen- tative time-lapse images of mobile (white arrowheads) and immobilized (encircled; green arrowheads) neutrophil populations in the splenic RP under basal conditions. Red, neutrophils; blue, vasculature. Bar, 50 µm. Data are representative of n = 5 from three independent experiments. (b) Cellular tracks representation of mobile and immobilized neutrophil populations from representative time-lapse images. (c) Quantification of cell velocity distribution of mobile (dark blue bars) and immobilized (light blue bar) neutrophils in the splenic RP under basal conditions. There were four fields of view (FOV) per animal. n = 5 from three independent experiments. (d) Representative flow cytometry plots (blood and spleen), IVM images (spleen), and quan- tification of neutrophil origin after parabiosis. SSC, side scatter. Bars, 25 µm. There were four fields of view per animal. n = 4 from three in- dependent experiments. *, P < 0.05. Student’s t test statistical analyses were performed. Data are represented as mean ± SEM. −/− RP macrophages (Fig. 5 e). Intriguingly, in C3 mice, where respiratory burst has long been regarded as an important in- neutrophils were not able to remove the bacteria from the tracellular killing mechanism for pathogens. To evaluate this surface of the macrophage, huge numbers of bacteria associ- capacity in mature splenic neutrophils,S . pneumoniae was la- ated with the RP macrophage and resulted in 100% mortality beled with an OxyBURST probe that fluoresces green in the within the first 18 h of infection (Fig. 5 f). This suggested the presence of oxidants. Visualization of OxyBURST -labeled macrophages were not able to directly kill the bacteria and bacteria upon neutrophil phagocytosis demonstrated a very required the immediate presence of the neutrophils. Even in rapid oxidative burst (Fig.  5, h and i). This rapid response the presence of complement, the macrophages were not able was NAD PH (nicotinamide adenine dinucleotide phosphate to kill bacteria, as depletion of mature neutrophils using a reduced) oxidase dependent, as the oxidant production was −/− Ly6G (1A8) antibody resulted in increases in splenic bacterial not seen in Cybb mice within this timeframe (Fig.  5  i). counts at 24 h after infection (Fig. 5 g). Neutrophil-mediated However, oxidation was not the predominant killing mech- 1338 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. Figure 4. characterization of mobile and immobilized splenic neutrophils. (a) Representative IVM images, flow cytometry plots, and quantification of splenic neutrophils 24 h after administration of isotype antibody (Ab) or Ly6G (1A8) antibody treatment. Green, neutrophils (neu). mono, monocyte. Bars, 50 µm. n = 5 for isotype and n = 6 for 1A8 antibody pooled from three independent experiments. (b) Representative flow cytometry plots and quantification JEM Vol. 214, No. 5 1339 anism, as Cybb-deficient animals were able to effectively that proliferation, maturation, and mobilization of the imma- int hi clear and control the pneumococcal infection (Fig. 5 j). Pre- ture Ly6G into the mature Ly6G neutrophil population vious in vitro work with human neutrophils has noted an occurred within the first 24 h after S. pneumoniae infection. important contribution of the serine proteases (e.g., cathep- sin G [CatG]) and neutrophil elastase in intracellular pneu- neutrophils from the blood stream are sequestered mococcal killing (Standish and Weiser, 2009). In our in vivo into the MZ by MZ macrophages and model, animals with a deficiency in CatC, a central regulator influence tI antibody production of serine proteases including CatG and neutrophil elastase, Under basal conditions, very few neutrophils could be found displayed nearly a log-fold increase in mean bacterial load in the MZ. However, at 24 and 48 h after infection, a very dramatic infiltration of neutrophils occurred into the MZ in the spleen, compared with wild-type animals, which was bordering on significant (P = 0.0628; Fig. 5 j). A significant (Fig. 7 a). Two-photon imaging of this area revealed that, after difference in bacterial loads was observed between CatC- and pneumococcal infection, neutrophils leaving the main circu- Cybb-deficient mice (Fig. 5 j). The increased clearance of S. lation in the marginal sinus were tethered right out of the pneumoniae observed in Cybb-deficient animals likely results mainstream of blood by the MZ macrophages, where they from increased proteolytic activity that has previously been remained rather than moving to the splenic RP (Fig.  7  b). reported in phagocytes from this mouse strain (Rybicka et Tracking individual neutrophils in the MZ under basal con- al., 2010, 2012). These data suggest an important role for the ditions revealed that the few cells that crawled within the local mature neutrophils in removing the bacteria from the area did so at speeds ranging from 2 to 10 µm/min (Fig. 7 c RP macrophage and helping in bacterial clearance. and Video  8). After the infection, the neutrophils that were recruited from the main stream of blood did not move like emergency neutrophil maturation occurs in the local neutrophils in the RP, but rather, many were not response to S. pneumoniae crawling at all at 24 and 48  h after S. pneumoniae seques- Neutrophil populations were further evaluated at later time tration in the MZ, and the remainder crawled at the lowest points after infection. Flow cytometric analysis revealed an in- detectable velocity (2–4 µm/s; Fig.  7  c). Indeed, the dwell hi crease in Ly6G neutrophils within the first 24 h after S. pneu- time (Fig. 7 d) and number of firm interactions of neutrophils int moniae infection (Fig.  6  a). Conversely, Ly6G neutrophil with MZ macrophages (Fig. 7 e) were both increased during numbers did not significantly change within this time period infections. Depletion of MZ macrophages using low-dose int (Fig. 6 a). Although 10% of Ly6G neutrophils were positive CLL treatment resulted in a significant decrease in MZ local- for the proliferative marker Ki67 at baseline, this value in- ization of neutrophils (Fig. 7 f). These data support a role for S. pneumoniae administration (Fig. 6 b) creased by 24 h after MZ macrophages in the retention of splenic neutrophils after int and only in the Ly6G neutrophil population, as none of the pneumococcal infection. Blocking antibodies against various hi Ly6G neutrophils were Ki67 positive after infection. integrins revealed that neutrophil retention in the MZ was int To see whether during infection the immature Ly6G partially β1 but not β2 integrin dependent (Fig.  7  g). This neutrophils could take on the more mature mobile pheno- decrease in retention could be recapitulated by VCAM-1 type, a photoactivatable (UBC-PaGFP) transgenic mouse blockade (Fig. 7 g), supporting an α4β1–VCAM-1–mediated system was used. This allowed the tracking of the cells over retention mechanism. It is worth mentioning that other as yet time. When immobilized immature neutrophils were photo- unidentified adhesion molecules make up a significant por- activated under control conditions, ∼10% of the population tion of this retention. mobilized over a 1-h period, so now, photoactivatable im- A consequence of this sequestration mechanism into mobilized GFP-positive neutrophils became GFP-positive the MZ of the spleen was that the neutrophils localized mature neutrophils (Fig.  6  c and Video  7), suggesting some not only with MZ macrophages, but also with MZ B cells immature neutrophils are always maturing in the spleen. After (Fig. 7 h). To see whether this impacted antibody output by 6-h pneumococcal stimulation, a significantly larger propor- these neutrophils, we attempted to deplete neutrophils with tion of immobilized progenitor neutrophils were able to start Ly6G (1A8) antibody. The results were somewhat ambiguous. mobilizing and scan the RP over 1  h after photoactivation Although there was a decreased density of neutrophils in the (Fig. 6 c and Video 7) and contribute to the pool of mature MZ and RP areas of the spleen after infection (not depicted) neutrophils within the spleen. Collectively, these data support when anti-Ly6G was used, the MZ B cell–dependent IgM hi int of Ly6G and Ly6G neutrophil origin in the blood, spleen, and bone marrow after parabiosis. n = 4 from two independent experiments. (a and b) Data are represented as mean ± SEM. *, P < 0.01; **, P < 0.01; ***, P < 0.001. Student’s t test (a) and one-way ANOVA (b) statistical analyses were performed. (c) Rep- hi int resentative flow cytometry histograms of cell-surface marker expression for both Ly6G (red) and Ly6G (blue) neutrophils under steady-state conditions from three independent experiments. (d) Representative immunohistochemistry CD117 staining of the immobilized neutrophil population in the splenic RP from four independent experiments. Bars, 25 µm. (e) Representative nuclear morphology of the immobilized neutrophil population including both banded (white arrowheads) and segmented (blue arrowheads) neutrophils from two independent experiments. Bars, 11.6 µm. 1340 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. Figure 5. Mature neutrophil phagocytosis of S. pneumoniae in the splenic rP. (a) Representative spinning-disk confocal image of neutrophils (red) and RP macrophages (purple) containing S. pneumoniae (green) at 30 min after infection. Data are representative of n = 6 from three independent experiments. Bar, 50 µm. White arrows, bacteria containing neutrophils; blue arrows, bacterial containing RP macrophages. (b) Proportion of splenic neu- trophil populations that bound S. pneumoniae at 30 min after infection. There were four fields of view per animal. n = 6 pooled from three independent experiments. (c) Time-lapse spinning-disk confocal images of S. pneumoniae (green) cell adhesion (white arrow) to RP macrophage (purple) and subsequent JEM Vol. 214, No. 5 1341 and IgG antibody responses occurred in a dichotomous fash- into splenectomized mice. The data reveal that benefit of ion (Fig.  7  i). A portion of the animals displayed low anti- the antibodies required splenic neutrophils, as injection of body production similar to MZ B cell depletion (Fig.  1  e), these antibodies into splenectomized mice provided no sur- whereas normal responses were detected in other animals vival benefit (Fig. 8 d). Collectively, these data highlight the (Fig. 7 i). It became very clear that although neutrophil de- important phagocytic role of mature splenic neutrophils for pletion efficiency was ∼90% at 24  h, at 72  h it was highly antibody-mediated clearance of S. pneumoniae. variable in the spleen ranging from as little as 30 to as high as 80%. This likely accounts for much of the antibody vari- dIScuSSIon ability. Consistent with these data, a similar dichotomy was This study used imaging to track the dynamic progression of S. pneumoniae infection in spleen and has revealed previously also observed in both blood and spleen bacterial counts at 72 h after infection (Fig. 7 j). Clearly, the more effective the unknown mechanisms that increase our understanding of neutrophil depletion was, the fewer antibodies were produced spleen-specific protective mechanisms against this pathogen. and the more bacteria were noted. Correlational analysis of This includes the collaboration of local neutrophils and mac- these mixed responses revealed that splenic bacterial titers in rophages as early innate immune components in the splenic neutrophil-depleted animals negatively correlated with both RP helping to dampen bacterial proliferation. Indeed, we vi- IgM (r = −0.6950; P = 0.0402) and IgG (r = −0.813 and sualized the macrophage of the splenic RP being able to cap- P = 0.0092) serum antibody levels (not depicted). These data ture and present bacteria to local mature neutrophils. Based suggest that retained neutrophils in the MZ participate in on our parabiosis experiments, these local mature neutrophils the TI antibody response after S. pneumoniae infection, but are recruited constantly from the mainstream of blood under one has to be cautious when trying to deplete neutrophils basal conditions. Furthermore, we identify the presence of a in the spleen long term. second local population of splenic neutrophils that appear to be an immature population that, based on photoactivation Mature splenic neutrophils mediate tI antibody– experiments, can rapidly mature and mobilize during emer- dependent pneumococcal clearance gency situations, and this turns out to be key for helping to To assess whether MZ B cell–dependent TI antibod- control the infection until antibodies are made. We also deter- ies enhanced mature neutrophil function, serum transfer mine the requirement for the TI antibody response by MZ B experiments were performed (Fig.  8  a). Transfer of pneu- cells aiding neutrophil phagocytosis for final clearance. mococcus-immunized serum (72 h) resulted in an increased Tissue-resident macrophages play important homeo- phagocytosis of S. pneumoniae by mature neutrophils at 30 static roles including immune surveillance for pathogens. In min after infection, compared with both control serum (72 h) the context of encapsulated bacterial infection, MZ macro- and pneumococcus-immunized serum from MZ B cell–de- phages have been suggested to be important with their ability pleted animals (TI antibody deficient, 72  h; Fig.  8  b). This to bind the capsule of S. pneumoniae via the c-type lectin antibody-dependent mechanism was limited to neutrophils, SIGN-R1 expressed on its surface (Kang et al., 2004). Our as no differences in RP macrophage uptake of bacteria were data support this work but also provide new information on noted between serum transfers (Fig.  8  b). This enhanced a second population of splenic macrophages, the RP mac- neutrophil phagocytic capacity resulted in rapid removal of rophages, which appear to contribute in a major way to ini- pneumococcus from the bloodstream (Fig. 8 c). Next, to de- tial binding and tropism of S. pneumoniae to the spleen. This termine whether these TI antibodies could enhance killing in macrophage function appears to be limited to the spleen, as the absence of the splenic environment, they were injected Kupffer cells in the liver were less effective at catching encap- phagocytosis by neutrophils (red). Data are representative of n = 4 from three independent experiments. Bar, 10 µm. (d) 3D reconstruction of intracellular S. pneumoniae (green) within neutrophil (red) at 30–60 min after infection. Transparency of neutrophils is increased from left to right. Bar, 23.3 µm. Data are representative of n = 3 from two independent experiments. White arrows indicate intracellular bacteria. (e) IVM and flow cytometric quantification of S. pneumoniae phagocytosis by neutrophils (Neu) and RP macrophages (RPM) from 30–60 min after i.v. infection in wild-type (black bars) and C3-deficient (blue bars) animals. n = 3–6 pooled from three and two independent experiments for IVM and flow cytometry, respectively. FOV, field of view; S. p., S. pneu- moniae. (f) Survival curve for S. pneumoniae i.v. infection at 10 -CFU dose in wild-type (black line) or C3 KO (blue line) mice. n = 5 from one experiment. (g) S. pneumoniae bacterial counts in the spleen at 1 and 24 h after i.v. infection in isotype antibody (black circles)– and Ly6G (1A8) antibody (1A8 Ab; blue triangles)–treated animals. n = 4–6 pooled from two independent experiments. Red lines show the median. (h) Time-lapse spinning-disk confocal images of OxyBUR ST probe activation (green) after S. pneumoniae (red) phagocytosis by mature splenic neutrophil (blue). Data are representative of n = 6 from two independent experiments. Bar, 5 µm. White arrows indicate OxyBURST activation on the surface of intracellular bacteria. (i) Quantification of intracellular −/− OxyBUR ST probe activation within neutrophils in wild-type (blue line) and Cybb (gray line) mice. n = 3–6 pooled from two independent experiments. −/− AU, arbitrary units. (j) S. pneumoniae bacterial counts in the spleen at 24  h after i.v. infection in wild-type (black circles), Cybb (gray squares), and −/− CatC (blue triangles) animals. n = 4–6 pooled from two independent experiments. Black lines show the median. *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Student’s t test (b, e, and i), log-rank Mantel-Cox test (f), and Mann-Whitney test (g and j) statistical analyses were performed. Data are represented as mean ± SEM. 1342 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. Figure 6. Splenic immature neutrophil proliferation and mobilization after pneu- mococcal stimulation. (a) Representative flow cytometry plots (baseline and 24  h) and hi int quantification of Ly6G (blue line) and Ly6G (teal line) neutrophil populations at baseline and after S. pneumoniae infection. n = 4–6 pooled from four independent experiments. **, P < 0.01 versus 0  h (baseline). (b) Represen- tative flow cytometry histograms and quanti- hi fication of Ki67 staining in both Ly6G (blue int line) and Ly6G (teal line) neutrophil popula- tions at baseline and after S. pneumoniae in- fection. Positive gates were established by use of an AF488-conjugated isotype control within each experiment. n = 3–4 pooled from two in- dependent experiments. ***, P < 0.001 versus 0 and 12 h. FSC, forward scatter. (c) Time-lapse (0–60 min) spinning-disk confocal images of photoactivated neutrophil cluster mobilization (top left, white box) in the splenic RP at 6  h after saline (gray bar) or S. pneumoniae (blue bar) administration. Green, photoactivated (PA) cells; red, neutrophils; blue, vasculature; multicolored, tracks. Bar, 50 µm. n = 4 pooled from four independent experiments. *, P < 0.05. One-way ANOVA (a and b) and Student’ s t test (c) statistical analyses were performed. Data are represented as mean ± SEM. sulated S. pneumoniae in the sinusoidal circulation (unpub- had a larger contribution to initial binding and subsequent lished data). Although both splenic macrophage populations clearance of S. pneumoniae mediated by mature neutrophils. sequestered pneumococcus early, they contributed to S. pneu- RP macrophage binding of S. pneumoniae in our model was moniae infection defense by distinct mechanisms. MZ mac- not dependent on complement and was not enhanced with rophages contributed to TI antibody production by MZ B passive immunization, suggesting an alternative mechanism. cells, consistent with another study (Koppel et al., 2008). This In addition to resident macrophages, the spleen also is likely by facilitating the direct interaction of MZ B cells serves as an important reservoir for other myeloid cells. with the pathogen on the surface of the macrophages and in- Swirski et al. (2009) described a pool of monocytes that directly enhancing MZ B cell activation through recruitment reside within the RP under steady-state conditions and that of neutrophils at the MZ. Interestingly, MZ macrophages dis- can be mobilized to the sites of injury. We demonstrate that played only a small amount of catching of S. pneumoniae mature neutrophils are also present within the RP compart- as observed by intravital imaging. In fact, RP macrophages ment under basal conditions and carry out an important fil- JEM Vol. 214, No. 5 1343 Figure 7. neutrophil sequestration by MZ macrophages contributes to tI antibody response after pneumococcal infection. (a) Representative confocal images and quantification of neutrophil (green) colocalization with MZ macrophages (MZM; red) and RP macrophages (RPM; purple) after saline (black bars) or S. pneumoniae (S. p.; 24 h, blue bars; 48 h, red bars) i.v. injection. Bars, 50 µm. Eight fields of view were averaged per animal. n = 4–6 pooled from four independent experiments. (b) Time-lapse two-photon microscopy images of neutrophil behavior in MZ 24 h after S. pneumoniae injection. Green, neutrophils; red, MZM; multicolored, tracks. Bars, 25 µm. (c–e) Tracking quantification of neutrophil cell velocity (c), dwell time (d), and MZ macrophage interaction time (e) at 24 h (blue bars) and 48 h (red bars) after S. pneumoniae i.v. infection. Two fields of view were averaged per animal. n = 3–4 pooled from four independent experiments. (f) Quantification of neutrophil colocalization with MZ B cells and RP macrophages at 24 h after S. pneumoniae i.v. in- fection in control (Ctrl) liposomes and low-dose CLL–treated animals. n = 3–5 pooled from three independent experiments. (g) Quantification of neutrophil 1344 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. tering function within the organ by mediating phagocytosis ter inhibition of antibody production and increased bacterial of bacteria. This protective mechanism is not restricted to load, consistent with a potential role for regulation of anti- encapsulated bacteria, as phagocytosis of S. aureus and Liste- body production by neutrophils within the spleen. ria monocytogenes has also been observed with our system Although most tissues rely on circulating neutrophils (unpublished data), supporting an immune surveillance role for recruitment, the spleen appeared to compartmentalize for this pool of neutrophils. The spleen also has the capacity its recruitment; in the MZ, there was classical recruitment to increase both neutrophil and monocyte numbers by facil- of neutrophils from the vasculature, whereas the RP had its itating extramedullary hematopoiesis during inflammation own population of immature and mature neutrophils that (Swirski et al., 2009; Robbins et al., 2012). This is thought to were key for local eradication of S. pneumoniae. The obvious advantage of this type of system is that, at the first sign of occur by mobilization of hematopoietic stem and progen- itor cells from the bone marrow to the spleen, where they infection, neutrophils are already in the RP ready to help kill seed preestablished niches and undergo expansion (Swirski bacteria caught by the macrophage. Without the neutrophils et al., 2009). Intriguingly, the recently described hemato- there, the bacteria were able to increase their numbers dra- poietic niche in the splenic RP (Dutta et al., 2015; Inra et matically within the spleen, killing the host. Although at this al., 2015) shares common features with the local environ- point we can only conclude that it is the immediate presence int ment surrounding our described Ly6G immature neutro- of neutrophils in spleen that helped to eradicate bacteria, an phils, including the perivascular location and the presence additional possibility is that the circulating neutrophils that of VCAM-1–positive macrophages around the colonies enter the RP further mature to perform a necessary function (unpublished data). It is likely that our immature neutro- that circulating neutrophils are incapable of doing. In fact, phils represent the consequence of continuous extramedul- under steady-state conditions, we do note a more activated lary hematopoiesis during the steady state, albeit at a very phenotype for mature splenic neutrophils compared with slow rate. Upon inflammatory stimulation, these immature circulating neutrophils (unpublished data). Many of these neutrophils can mobilize as observed and contribute to the features are reminiscent of B helper neutrophils, which are mature neutrophil population. proposed to polarize locally in response to bacterial factors Neutrophils in the spleen have previously been shown and GM-CSF, which is released locally by innate lymphoid to contribute to MZ B cell activation and subsequent pro- cells (Magri et al., 2014; Chorny et al., 2016). It will also be duction of anti-capsular polysaccharide antibodies (Puga et interesting to know whether during systemic infections such al., 2012; Magri et al., 2014; Chorny et al., 2016). Pentraxin as sepsis, where most neutrophils are recruited to lungs and 3 has recently been identified as a natural adjuvant produced other infectious sites and in some instances patients become and released by these B helper neutrophils to help mediate neutropenic, this local pool helps to maintain neutrophil pres- this response (Chorny et al., 2016). Although, some have not ence in the spleen for critical innate immune purposes. been able to confirm these observations in humans (Nagelk- erke et al., 2014), our data does support a role for newly re- MaterIaLS and MethodS cruited splenic neutrophils in promoting anti-pneumococcal animals TI antibody production during systemic mouse pneumococ- 8–12-wk-old male and female mice were used for experi- cal infection. Although it is very easy to deplete circulating ments. C57BL/6, Pep BoyJ(B6), LysM-eGFP/eGFP, UBC- −/− neutrophils to determine their function, the resident mature PaGFP, and Cybb-deficient (Cybb ) mice were obtained −/− population in the spleen was extremely difficult to eradicate from The Jackson Laboratory, and CatC-deficient (CatC) during infection, and the immature population was resistant mice were a gift from GlaxoSmithKline. All mice were housed under a specific pathogen–free, double-barrier unit to 1A8 depletion. In fact, similar depletion inefficiency of splenic neutrophils has previously been noted in a chronic at the University of Calgary. Mice were fed autoclaved ro- inflammatory setting of tumor-bearing mice (Moses et al., dent feed and water ad libitum. All protocols used were in 2016). It was unclear whether this resident population could accordance with the guidelines drafted by the University of impact on B cell antibody production. Nevertheless, in those Calgary Animal Care Committee and the Canadian Council mice where we obtained good depletion, we also got bet- on the Use of Laboratory Animals. colocalization with MZ B cells (left) and RP macrophages (right) at 24 h after S. pneumoniae i.v. infection in animals treated with isotope control, anti-CD18, anti-CD29, and anti–VCAM-1 blocking antibodies. Eight fields of view were averaged per animal. n = 3–5 pooled from three independent experiments. (h) Representative image of neutrophil localization within the MZ B cells (MZB) 24 h after i.v. S. pneumoniae infection. Green, MZ B cells; red, neutrophils; purple, RP macrophages. Data are representative of n = 4 from two independent experiments. Bar, 25 µm. (i) Quantification of S. pneumoniae–specific serum IgM and IgG antibody levels in isotype antibody–treated and Ly6G (1A8) antibody (1A8 Ab)–treated animals at 72 h after infection. Red dotted lines indicate antibody levels in MZ B cell–depleted animals. n = 5–9 total pooled from three independent experiments. (j) S. pneumoniae bacterial counts in the blood and spleen at 72 h after i.v. infection in isotype antibody–treated and Ly6G (1A8) antibody–treated animals. Black lines show the median, and red dotted lines show the detection limit. n = 5–9 total pooled from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One-way ANO VA (a and e), two-way ANO VA (c and d), Student’s t test (f, g, and i), and Mann-Whitney test (j) statistical analyses were performed. JEM Vol. 214, No. 5 1345 Figure 8. Passive immunization facili- tates neutrophil-mediated capture and clearance of S. pneumoniae in the spleen. (a) Schematic of serum transfer protocol. (b) Representative spinning disk confocal im- ages and quantification of neutrophils (Neu; red) and RP macrophages (RPM; purple) con- taining S. pneumoniae (green) at 30–60 min after infection in animals given control serum (blue bars), immunized serum (solid red bars), and immunized serum from MZ B cell–de- pleted (MZB dep) animals (checkered red bars). Bars, 30 µm. White arrows indicate neutrophil containing bacteria, and blue arrows indicate RP macrophage containing bacteria. Eight fields of view (FOV) were averaged per an- imal. n = 3–4 pooled from two independent experiments. Data are represented as mean ± SEM. (c) S. pneumoniae bacterial counts in the blood and spleen at 1  h after i.v. infection in wild-type mice receiving either control serum (blue circles) or immunized serum (red squares). n = 5–6 pooled from two independent experi- ments. (d) Survival curve to S. pneumoniae in- fection at 10 -CFU dose in sham-operated and splenectomized (Spx) animals receiving control (CT) serum or immunized serum. n = 4–5 from one experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One-way ANO VA (b), Mann-Whitney test (c), and Log-rank Mantel-Cox test (d) sta- tistical analyses were performed. IVM together with the appropriate band-pass filters (Semrock). A Multichannel spinning-disk confocal and two-photon con- back-thinned electron-multiplying charge-coupled device focal microscopes were used to image mouse spleens. Mice 512 × 512–pixel camera (Hamamatsu Photonics) was used were anaesthetized (10 mg/kg xylazine hydrochloride and for fluorescence detection. Volocity software (PerkinElmer) 200 mg/kg ketamine hydrochloride). The right jugular vein was used to drive the confocal microscope and for 3D ren- was cannulated to administer fluorescent dyes and additional dering, acquisition, and analysis of images. Bacteria and neu- anesthetic. After skin incision, the mouse was placed in a left trophil behavior was evaluated using Volocity software. Both lateral position, and the spleen was exteriorized onto a glass populations were identified and tracked using the Find Ob- coverslip on the inverted microscope stage. The stage was kept ject and Track functions, respectively, within the measurement at 37°C to maintain the mouse body temperature. modality. For each animal, tracking data were averaged for Image acquisition of the splenic RP was performed multiple fields of view and considered an n = 1. using an inverted microscope (IX81; Olympus), equipped Image acquisition of the splenic MZ was performed with a focus drive (Olympus) and a motorized stage (Ap- using an inverted multiphoton microscope (TCS SP8; Leica plied Scientific Instrumentation) and fitted with a motor- Biosystems) or upright multiphoton microscope (TCS SP8; ized objective turret equipped with 4×/0.16 UPLANS APO, Leica Biosystems). Typically, MZ macrophages were stained 10×/0.40 UPLANS APO, and 20×/0.70 UPL ANS APO ob- by i.v. injection of 1 µg anti-CD209b fluorescent conjugated jective lenses and coupled to a confocal light path (WaveFx; mAbs. i.v. injection of 2 µg anti-MAR CO (macrophage re- Quorum Technologies) based on a modified CSU-10 head ceptor with collagenous structure) fluorescent conjugated (Yokogawa Electric Corporation). Cells of interest were vi- mAbs was also used to label MZ macrophages in preliminary experiments to confirm that 1 µg anti-CD209b did not affect sualized using fluorescently labeled antibodies, fluorescent reporter mice, and fluorescent reporter bacteria. Typically, RP S. pneumoniae binding. The dynamic behaviors of immune macrophages and neutrophils were stained by i.v. injection of cells and bacteria were visualized simultaneously with a single- 2.5 µg anti–F4-80 and 1 µg anti-Ly6G fluorescent conjugated pulsed laser (Ti-Sapphire; Spectra-Physics or Coherent) at mAbs, respectively. Laser excitation wavelengths of 491, 561, 950 nm or 1,040 nm in combination with appropriate band- 642, and 730 nm (Cobolt) were used in a rapid succession pass emission filters (Semrock). The fluorescence was detected 1346 Splenic neutrophils eradicate S. pneumoniae | Deniset et al. by HyD hybrid or high-sensitivity photomultiplier non- spleen were removed after thoracotomy, weighed, and ho- descanned detectors. Leica Biosystems software was used to mogenized. For determination of CFUs, 50 µl of blood and drive the confocal microscope and for 3D rendering and ac- 100 µl of tissue homogenate were serially diluted, plated onto quisition of images. Files were converted from LIF format into blood agar plates, and incubated at 37°C with 5% CO for multiple TIFFs using Volocity and subsequently imported into 18 h, and bacterial colonies were counted. ImageJ (National Institutes of Health) for analysis. The plugin StackReg was used for movement correction purposes. Both Serum antibody measurement bacteria and neutrophil tracking were completed using the Blood was collected by cardiac puncture at multiple time Manual Tracking plugin. For each animal, tracking data were points after either saline or S. pneumoniae injection and spun n = 1. For down at 400 g for 10 min. Then, serum was collected, and 25 µl averaged for multiple images and considered an photoactivation experiments, multiphoton excitation at 800 was diluted with an equal volume of FACS wash buffer (1× nm was used to activate neutrophil populations of interest, PBS, 2% FBS, and 0.2% EDTA) and subsequently incubated and images were acquired using a spinning-disk microscope with 10 CFU of freshly cultured S. pneumoniae for 30 min as described in the previous paragraph. on ice. After a wash step, 1 µg of APC-conjugated anti–mouse IgG or IgM antibody was added in a resuspension volume Parabiosis of 50  µl and incubated for an additional 30 min. Antibody Age-matched female mice were housed together for 2 wk concentration used was determined by a preliminary serial before surgery. Parabiotic pairs of mice were generated sur- dilution experiment to ensure optimal signal saturation. Sam- gically as previously described (Ajami et al., 2007). In brief, ples were subsequently fixed with 0.5% paraformaldehyde in a skin incision running from elbow to knee along the flank flow cytometry wash buffer and run using a flow cytometer was generated on opposite sides of the mice to be paired. The (LSR II; BD). Samples were first gated on GFP bacteria, and mice were first joined with a suture through the shoulder and then, the median fluorescence intensities were determined thigh muscles, and next, the inside faces of the skin flaps were for APC-labeled anti-IgG or IgM. Values were normalized to juxtaposed and sutured. Complete blood sharing was moni- control animal (saline injected) levels within each experiment tored between 12 and 17 d by tail vein blood sampling. 24 h and then averaged among multiple experiments. after chimerism detection in the blood, pairs were separated and prepared for subsequent intravital imaging of the spleen Serum transfer experiments and/or flow cytometry of the spleen, blood, and bone marrow. Serum was collected as described in the previous paragraph at 72 h after injection (saline or S. pneumoniae). After confir- Bacterial growth and infection mation of antibody production, serum from multiple animals The S. pneumoniae D39-GFP strain was grown on blood (same treatment) was combined and heat inactivated at 56°C agar plates overnight at 37°C with 95% O /5% CO . Single for 20 min. 100 µl of the control, immunized, or immunized 2 2 colonies were picked from plates and grown in brain-heart (MZ B cell depletion) heat-inactivated serum was adminis- infusion broth at 37°C with CO until an OD 600 of 0.5 tered i.v. 20–30 min before infection with S. pneumoniae. was reached. Bacteria were resuspended in saline and diluted to achieve appropriate dose with a 100-µl volume. For long- Flow cytometry term infection and acute bacteria infection, doses of 2 × 10 Mice were anesthetized, and the spleen was removed and 4 6 CFU or 10 CFU and 5 × 10 CFU were administered i.v., placed in PBS on ice. Then, blood was collected in a hep- respectively. Generation of reporter bacteria for oxidation was arinized syringe by cardiac puncture. The spleen was passed performed as previously described (Surewaard et al., 2016). through a 70-µm filter. For S. pneumoniae–GFP detection In brief, fresh streptococcal cultures were washed twice in experiments, cells were immediately stained for 20 min with saline, resuspended at 5 × 10 CFU in 500 µl in carbonate, fluorescently labeled antibodies and subsequently incubated pH 8.3, buffered saline, and labeled for 30 min with 20 µg in 1× Fix/Lyse buffer (eBioscience). For the remainder of −1 ml AF647 N-hydroxysuccinimide ester (Thermo Fisher experiments, residual red blood cells were lysed using ACK −1 Scientific) and 60 µg ml OxyBUR ST Green H2DCF DA lysing buffer (Invitrogen). The cells were blocked using an- SE (DMSO stock; Thermo Fisher Scientific) under vigorous ti-CD16/32 antibody (2.4G2 clone; Bio X Cell) for 20–30 agitation. Activation of OxyBURST w as accomplished by min. Then, cells were stained for 30 min with specified mark- adding 250 µl of 1.5 M hydroxylamine, pH 8.5, and incubat- ers including FITC-labeled B220 (RA3-6B2; BD), CD18 ing for 30 min on ice. Reporter bacteria were washed twice (C71/16; BD), CD62P (RB40.3; BD), CD45.2 (104; BD), 6 7 with PBS and injected i.v. into mice at 5 × 10 –10 CFU. CD43 (eBioR2/60; eBioscience), PE-labeled anti–mouse Ly6G (1A8; eBioscience), F4/80 (BM8; eBioscience), CD Bacteriological analysis 21/35 (4E3; eBioscience), CD117 (2B8; eBioscience), Anesthetized mice were washed with 70% ethanol under CD49d (R1-2; eBioscience), CxCR4 (2B11; eBioscience), aseptic conditions. Blood was collected in a heparinized sy- CD62L (MEL-14; eBioscience), CD169 (3D6.112; BioLeg- ringe by cardiac puncture. The lungs, liver, heart, kidneys, and end), APC-labeled anti–mouse CD209b (22D1; eBioscience), JEM Vol. 214, No. 5 1347 CD23 (B3B4; eBioscience), CD45.1 (A20; eBioscience), unless otherwise specified. Data, with the exception of bac- PerCP Cy5.5-labeled Ly6C (HK 1.4; eBioscience), PE-Cy7– terial CFUs, were compared either by unpaired two-tailed labeled CD11b (M1/70; eBioscience), Pacific blue–labeled Student’s t test or one-way or two-way ANO VA followed Ly6G (1A8; eBioscience), AF647-labeled CD54 (YN1/1.7.4; by Bonferroni posthoc test for multiple comparisons adjust- BioLegend), efluor 660–labeled CD11c(N418; eBioscience), ment. Bacterial CFU data were compared by Mann-Whitney Brilliant violet 510–labeled CD45 (30-F11; BioLegend), and test or Kruskal-Wallis test followed by Dunn’s multiple com- APC-Cy7–labeled CD45 (30-F11; eBioscience). Appropriate parisons test. Survival curves were compared using log-rank isotype control antibodies were used to confirm positive sig- (Mantel-Cox) test. Statistical significance was set at P < 0.05. nals. Nonviable cells were identified using propidium iodide The applied statistical analyses and the numbers of indepen- n) are reported in the figure legends. or viability dye efluor 780 (eBioscience). Samples were run dent replicates ( using a flow cytometer (FAC SCanto; BD) and analyzed using FlowJo software (Tree Star). Neutrophils were identified as online supplemental material + hi int hi hi CD11b Ly6G Ly6C or LysM GFP SSC (for depletion Video 1 displays the movement of S. pneumoniae in the splenic experiments). Proinflammatory monocytes were identified MZ after infection. Video 2 shows S. pneumoniae accumula- + − hi as CD11b Ly6G Ly6C . RP macrophages were identi- tion in the splenic RP after infection. Video 3 demonstrates in +/lo − hi fied as CD11b CD209b F4/80 . MZ macrophages were vivo behavior of splenic neutrophil populations under steady- + hi − identified as CD11b CD209b F4/80 . Metallophilic mac- state conditions. Video  4 shows chimerism of both mature + + −/lo rophages were identified as CD11b CD169 F4/80 . Fol- and immature splenic neutrophils after parabiosis. Video  5 + hi int licular B cells were identified as B220 CD23 CD21/35 , displays in vivo capture of S. pneumoniae by splenic neu- + lo int/hi MZ B cells as B220 CD23 CD21/35 , and B1 B cells as trophils in the presence or absence of complement. Video 6 + lo lo + B220 CD23 CD21/35 CD43 . shows neutrophil-mediated removal and phagocytosis of S. pneumoniae after initial binding to RP macrophages. Video 7 depletion and blocking antibody protocols shows mobilization of photoactivated immature neutrophils Depletion protocols for various cell types have been used as after S. pneumoniae infection. Video  8 displays the in vivo previously described (Lu and Cyster, 2002; McGaha et al., 2011; behavior of circulating neutrophils in the splenic MZ. Kolaczkowska et al., 2015). In brief, transient MZ B cell de- pletion was achieved by intraperitoneal injection of 100 µg of acknoWLedGMentS both anti-CD11a (M17/4; Bio X Cell) and anti-CD49d (PS/2; Bio X Cell) 96 h before S. pneumoniae infection. Depletion of We thank Trecia Nussbaumer and Dr. Robin Yates for the breeding of mice. We thank Dr. Craig Jenne and Dr. Bryan Yipp for use of their multiphoton microscopes. We MZ macrophages was completed by i.v. delivery of a low-dose thank Dr. Pina Colarusso at the Snyder Live Cell Imaging Facility for technical support clodronate treatment (100 µg) 96 h before infection. Neutro- for photoactivation experiments and Dr. Karen Poon (Snyder Institute Molecular phils were depleted using an anti-Ly6G (1A8; Bio X Cell) Core) for assistance with flow cytometry. antibody. For acute infection experiments, a single 500-µg dose P. Kubes is supported by a foundation grant from the Canadian Institutes of Health Research and a Heart and Stroke Foundation of Canada grant. J.F. Deniset is was given intraperitoneally 24 h before infection. For long- financially supported by a postgraduate fellowship from Alberta Innovates–Health term infection, supplemental 200-µg doses were given daily. Solutions. B.G. Surewaard is partially funded by Marie Curie Actions (FP7-PEOPLE- Isotype antibodies or control liposomes were given as con- 2013-IOF; grant no. 627575) and a postgraduate fellowship from Alberta Innovates– trol injections. The specificity of all three depletion protocols Health Solutions. The authors declare no competing financial interests. was verified using both flow cytometry and in some instances Author contributions: J.F. Deniset and B.G. Surewaard conceived the study, immunohistochemistry. LysM-GFP reporter mice for our de- performed experiments, analyzed data, and wrote the manuscript. W.-Y. Lee contrib- pletion studies were used to evaluate neutrophil depletion. uted to parabiosis experiments. P. Kubes wrote the manuscript and directed the study. hi int hi int Ly6G neutrophils were identified as Ly6C GFP , Ly6G int int hi hi neutrophils as Ly6C GFP , and Ly6C monocytes as Ly6C Submitted: 27 September 2016 int GFP . These gates were established in experiments performed Revised: 27 January 2017 with control and isotype antibody–treated LysM-GFP mice Accepted: 3 March 2017 with the inclusion of the Ly6G antibody (1A8 clone). For blocking antibody experiments, 100 µg of pu- rified anti-CD18 (clone M18/2; BioLegend), anti-CD29 (clone HMβ1-1; BioLegend), or anti-CD106 (clone 429; reFerenceS eBioscience) was added i.v. 10 min before S. pneumoniae Ajami, B., J.L. Bennett, C. Krieger, W. Tetzlaff, and F.M. Rossi. 2007. Local self-renewal can sustain CNS microglia maintenance and function infection. Appropriate isotype control antibodies were used throughout adult life. Nat. Neurosci. 10:1538–1543. http ://dx .doi .org for control experiments. /10 .1038 /nn2014 Amlot, P.L., and A.E. 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The Journal of Experimental MedicinePubmed Central

Published: May 1, 2017

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