PSGL-1 on Leukocytes is a Critical Component of the Host Immune Response against Invasive Pneumococcal Disease

Bacterial uptake by phagocytic cells is a vital event in the clearance of invading pathogens such as Streptococcus pneumoniae. A major role of the P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes against invasive pneumococcal disease is described in this study. Phagocytosis experiments using different serotypes demonstrated that PSGL-1 is involved in the recognition, uptake and killing of S. pneumoniae. Co-localization of several clinical isolates of S. pneumoniae with PSGL-1 was demonstrated, observing a rapid and active phagocytosis in the presence of PSGL-1. Furthermore, the pneumococcal capsular polysaccharide and the main autolysin of the bacterium ―the amidase LytA― were identified as bacterial ligands for PSGL-1. Experimental models of pneumococcal disease including invasive pneumonia and systemic infection showed that bacterial levels were markedly increased in the blood of PSGL-1 −/− mice. During pneumonia, PSGL-1 controls the severity of pneumococcal dissemination from the lung to the bloodstream. In systemic infection, a major role of PSGL-1 in host defense is to clear the bacteria in the systemic circulation controlling bacterial replication. These results confirmed the importance of this receptor in the recognition and clearance of S. pneumoniae during invasive pneumococcal disease. Histological and cellular analysis demonstrated that PSGL-1 −/− mice have increased levels of T cells migrating to the lung than the corresponding wild-type mice. In contrast, during systemic infection, PSGL-1 −/− mice had increased numbers of neutrophils and macrophages in blood, but were less effective controlling the infection process due to the lack of this functional receptor. Overall, this study demonstrates that PSGL-1 is a novel receptor for S. pneumoniae that contributes to protection against invasive pneumococcal disease.


Introduction
Streptococcus pneumoniae (pneumococcus) is one of the major causes of invasive disease accounting for more deaths than any other vaccine-preventable bacterial infection. This microorganism colonizes the human nasopharynx, being one of the leading causes of acute otitis media, community-acquired pneumonia and invasive pneumococcal disease (IPD) including sepsis and meningitis [1]. The World Health Organization estimates that nearly 14 million episodes of serious pneumococcal disease occur every year with a critical impact in childhood population as pneumonia kills more children than AIDS, malaria and measles combined [1,2].
Resolution of pneumococcal disease is regulated by the efficient recognition and clearance of the invading pathogen by professional phagocytes [3]. Leukocytes play an important role in inflammatory and immune responses against bacterial infection and bacterial clearance depends on the efficacy of different receptors on phagocytic cells to recognize, internalize and kill the pathogen [4][5][6][7]. Phagocytic receptors on the cell surface trigger phagocytosis following direct recognition of particulate targets. Interaction between selectins and selectin-ligand molecules is essential for the host-pathogen encounter due to its crucial role in leukocyte extravasation [8]. In this sense, expression of P-selectin and E-selectin by the endothelium provides protection against invading pathogens such as S. pneumoniae [9,10]. P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes mediates interactions with P-selectin and E-selectin expressed by endothelial cells [11]. PSGL-1 is a homodimeric mucin-like glycoprotein expressed on the surface of almost all circulating leukocytes with a great importance in leukocyte adhesion and transmigration as it is responsible for the initial steps of the extravasation cascade [8,12]. However, certain intracellular pathogens have developed sophisticated strategies exploiting specific receptors for their own benefit to enter eukaryotic cells and replicate intracellularly [13]. This is the case of the obliged intracellular pathogens Anaplasma phagocytophilum, Ehrlichia sp., and enterovirus 71 that get access inside the cell by binding PSGL-1, causing granulocytic anaplasmosis/ehrlichiosis and hand-foot-mouth disease respectively [14][15][16]. However, there is no experimental evidence indicating that PSGL-1 could act as a receptor on leukocytes participating in the recognition and clearance of extracellular invading pathogens such as S. pneumoniae. In this sense, the main goal of this study was to investigate the protective contribution of PSGL-1 in host defense against IPD.

PSGL-1 is a functional receptor in neutrophils involved in the phagocytic process of S. pneumoniae
The plasma membrane of phagocytes expresses an array of receptors that interact with specific microbial ligands promoting the internalization and clearance of the potential pathogen. To evaluate the impact of PSGL-1 in pneumococcal phagocytosis, HL-60 cells differentiated to granulocytes were used as target cells because they express the same phagocytic receptors as peripheral blood neutrophils including PSGL-1 [14][15][16][17] (S1 Fig). To assess the role of PSGL-1 in the phagocytosis of S. pneumoniae, the receptor function was blocked using the specific monoclonal antibody KPL-1. This is an accepted method to assess the impact of PSGL-1 in microbial interaction [14][15][16]. To determine the generic role of PSGL-1 in host defense against this important pathogen, clinical isolates of S. pneumoniae belonging to different serotypes were assessed. Phagocytosis was significantly impaired when PSGL-1 was blocked, indicating that pneumococcal phagocytosis is more efficient when this receptor is fully active (Fig 1A and  1B). The contribution of FCγ-receptors was evaluated indicating that the effect of PSGL-1 in phagocytosis is independent of FCγ-receptors activity (S1 Fig). In addition, bacterial killing mediated by PSGL-1 was examined using three different clinical isolates. Our results showed that pneumococcal survival increased when PSGL-1 in phagocytic cells was blocked, demonstrating that this receptor is involved in the clearance of S. pneumoniae ( Fig 1C). Finally, phagocytosis experiments using neutrophils obtained from the spleen of wild-type and PSGL-1 -/mice by cell sorting, confirmed that PSGL-1 is involved in the phagocytosis of S. pneumoniae (Fig 1D and 1E).
To further analyze the kinetics of pneumococcal phagocytosis, cells with PSGL-1 -either active or antibody-blocked-were infected with the D39 (serotype 2) strain expressing the green fluorescent protein (GFP), and the phagocytosis process was monitored using live imaging confocal microscopy (Fig 2A and 2B, S1 and S2 Movies). When PSGL-1 was active, a rapid and active phagocytosis was observed, with the majority of the cells containing pneumococcal cells by the end of the process (Fig 2A and S1 Movie). However, when PSGL-1 receptor was blocked, the recognition and engulfment of S. pneumoniae was impaired, which confirmed the importance of PSGL-1 in pneumococcal phagocytosis ( Fig 2B and S2 Movie). To confirm the interaction of S. pneumoniae with PSGL-1, fluorescently-labeled pneumococcal isolates of serotypes 2, 3, 6B, 11A, 23F and 19A were used to observe co-localization with PSGL-1 (Fig 3 and  S2 Fig). Hence, our findings show that PSGL-1 is involved in the recognition and phagocytosis of a major human pathogen such as S. pneumoniae contributing therefore, to the variety of receptors on professional phagocytes that are needed to efficiently identify and destroy invading pathogens [4][5][6]18].
Autolysin LytA and pneumococcal capsular polysaccharide are bacterial ligands recognized by PSGL-1 Phagocytosis requires receptor-mediated recognition of microbial ligands that are usually expressed in the surface of invading pathogens. These ligands are frequently known as pathogen-associated molecular patterns (PAMPS) which are recognized by specific receptors of the innate immune system [19]. As LytA -the main cell wall hydrolase of S. pneumoniaeis located on the bacterial surface and it is essential to interact with critical components of the host immune response including neutrophils and macrophages [20,21], we explored the possible interaction of PSGL-1 with LytA. Pneumococcal recognition by PSGL-1 was hindered in the absence of LytA, suggesting that LytA might be a bacterial ligand for PSGL-1 (Fig 4A and  Error bars represent the SDs and asterisks indicate statistical significance after incubation with KPL-1 compared to the exposure to IgG isotype control or between OP using neutrophils from wild-type mice vs those from PSGL-1 -/mice. Error bars represent the SDs and asterisks indicate statistical significance after incubation with KPL-1 compared to the exposure to IgG isotype control. doi:10.1371/journal.ppat.1005500.g001 4B). Direct interaction between purified LytA and PSGL-1 molecules was observed confirming that this receptor recognizes LytA (Fig 4C). This interaction was dependent on the concentration of PSGL-1, suggesting that recognition of S. pneumoniae is enhanced when PSGL-1 levels are increased. Preincubation of HL-60 cells with purified LytA before infection reduced the phagocytosis in a similar way than KPL-1 antibody, supporting additional evidence that LytA interacts with PSGL-1 (S3 Fig). To confirm the interaction of LytA and PSGL-1 we included the non-capsulated strain (M11) and an isogenic lytA mutant strain (Fig 4D). Our results demonstrated that in the absence of LytA, the binding of S. pneumoniae to PSGL-1 is impaired. Although the capsular polysaccharide (CPS) is one of the major virulence factors of S. pneumoniae, resistance to phagocytosis can vary with the capsular type, which might explain differences of invasiveness among strains [22,23]. Capsule recognition by PSGL-1 was investigated using a non-capsulated strain (M11) and several isogenic transformants of M11 expressing different CPSs (Fig 4E). The absence of CPS caused impaired recognition by PSGL-1, in comparison to the corresponding encapsulated transformants, confirming that PSGL-1 recognizes the pneumococcal CPS ( Fig 4E). However, different levels of binding were observed depending on the CPS with the weakest recognition for the pneumococcal strain expressing serotype 19A ( Fig 4E). Experiments including purified CPS of type 3 and different concentrations of PSGL-1 were included to investigate the binding of PSGL-1 to the pneumococcal capsule ( Fig 4F). This CPS was assessed because is included in the current PCV-13 vaccine and clinical isolates of serotype 3 are a major cause of IPD [24]. Direct recognition of purified CPS by PSGL-1 was observed showing a concentration-dependent pattern, which confirms that PSGL-1 recognizes the capsule of S. pneumoniae ( Fig 4F).

PSGL-1 on leukocytes controls the severity of pneumococcal infection
The development of IPD depends of the complex interplay of many factors including virulence determinants of the pathogen and the efficacy of the host immune response to clear the infection process. A failure to efficiently detect and destroy S. pneumoniae in the lower respiratory tract or the systemic circulation lead to severe pneumonia or disseminated infection which are associated to increased mortality rates [3]. Next, the protective role of PSGL-1 against IPD was investigated using pneumonia and sepsis models of infection ( Fig 5). In pneumonia, bacterial counts were slightly higher in the bronchoalveolar lavage fluid (BALF) and lung of For differences in survival between wild-type mice and PSGL1 −/− mice a long-rank test was used (P <0.01 for D39 strain and P <0.05 for TIGR4 strain). PSGL-1 −/− mice, and much higher in the blood of KO mice (Fig 5A). These bacterial levels were markedly elevated at 24 h in the blood of PSGL-1 −/− mice (with the progression of the infection), confirming that PSGL-1 contributes to control bacterial load by reducing the severity of pneumococcal dissemination (Fig 5A). In the sepsis model, PSGL-1 −/− mice had greater levels of bacteria in blood (Fig 5B). In addition, lethal infection developed faster in PSGL-1 −/− mice than in wild-type mice indicating that PSGL-1 plays a critical role in host defense against IPD by controlling bacterial infection in the systemic circulation (Fig 5B and5C). To confirm this hypothesis and exclude the contribution of cellular migration mediated by PSGL-1, mice were directly infected by the intravenous route ( Fig 5D). Mice lacking PSGL-1 had increased levels of bacteria in blood than wild-type mice both at 6 and 24 h confirming that a major function of PSGL-1 in host defense is to clear the bacteria in the bloodstream controlling the dissemination (Fig 5D). To extend the importance of PSGL-1 in the clearance of S. pneumoniae from the systemic infection, a sepsis model was repeated using a lethal dose of a different serotype such as TIGR4 strain. Our findings corroborated the results obtained with the D39 strain demonstrating that lack of PSGL-1 was associated with increased bacterial counts in blood and a more severe infection compared to wild-type mice (Fig 5E and 5F). Overall, our findings demonstrate that PSGL-1 plays an important role against IPD.

PSGL-1 contributes to cellular migration and the inflammatory response
The inflammatory response to infection with S. pneumoniae in PSGL-1 −/− and wild-type mice was characterized in BALF and serum. The pattern of the major cytokines associated to infection was similar in BALF (Fig 6A), although in serum of PSGL-1 −/− mice there were significant increased levels of IL-5, IL-6, and IFN-γ (P <0.05) which is compatible with the higher bacterial levels found in the blood of these mice (Fig 6B).
Neutrophils predominate within cellular infiltrates in pneumococcal pneumonia and the consequences of the neutrophil influx for the host can be advantageous or detrimental, depending on the degree of cellular influx and the ability of the pathogen to successfully avoid the immune response [3]. Leukocyte infiltration into lungs and circulating leukocytes in blood, were measured by flow cytometry, using wild-type and PSGL-1 −/− mice infected with S. pneumoniae D39 strain (Fig 7A and 7B). In the pneumonia model (intranasal inoculation), the number of neutrophils and macrophages were similar in the lungs of both types of mice whereas T cell counts were higher in PSGL-1 −/− mice (Fig 7A). In a systemic model of infection (intravenous inoculation), however, the number of T cells were similar, although the proportion of neutrophils and macrophages were significantly higher in PSGL-1 −/− mice (Fig 7B), which is compatible with the higher levels of bacteria in the blood of these mice ( Fig 5D). Overall, these results indicate that PSGL-1 −/− mice, despite having greater numbers of leukocytes in blood, had an impaired ability to clear the bacteria from the bloodstream confirming the importance of PSGL-1 in the recognition and killing of S. pneumoniae in the systemic circulation.
Immunohistochemical characterization of thin sections from lung tissues confirmed that mice deficient in PSGL-1 had greater infiltration of T cells and neutrophils compared to wildtype mice, which are consistent with the severity of the infection process developed in PSGL-1 −/− mice (Fig 7C).

LytA is relevant for the interaction with PSGL-1
To demonstrate that pneumococcal LytA is involved in the physiological effects mediated by PSGL-1, phagocytic assays were performed using HL-60 cells exposed or not to the KPL-1 antibody and a pneumococcal strain lacking LytA (Fig 8A and 8B). Hence, our findings confirmed that phagocytosis of a LytA deficient strain is not dependent on PSGL-1 on HL-60 cells (Fig 8A  and 8B). To confirm the relevance of LytA in the interaction with PSGL-1, pneumonia and sepsis models of infection were repeated in wild-type and PSGL-1 −/− mice using a D39 lytA deficient strain (Fig 8C and 8D). In contrast to mice infected with wild-type D39, there were no differences in lung or BALF CFU between PSGL-1 −/− and wild-type mice infected intranasally with the D39 lytA mutant strain. Similarly, in the sepsis model there were no differences in recovered D39 lytA deficient strain CFU between PSGL-1 −/− and wild-type mice. These results confirm that the interaction of LytA with PSGL-1 is important for innate immunity against S. pneumoniae. (Fig 8C and 8D).

Discussion
Neutrophils are key players in the innate and adaptive immune responses to microbial cells, since they are critical for rapid clearance of invading bacteria [25,26]. For this process, neutrophils must first detect the microorganisms using surface receptors that are essential to engulf and kill the pathogen [18,27]. PSGL-1 is a ligand of P-, E-and L-selectins, and is able to mediate the tethering and rolling of circulating leukocytes on the activated endothelium prior to their extravasation [8,11,12]. The role of P, E and L-selectin as well as certain integrins against pneumococcal infection has been previously characterized [9,10,28,29]. However, the direct role of PSGL-1 on leukocytes in host defense against S. pneumoniae including its contribution to the recognition and clearance of this microorganism is unknown. As this bacterium is highly variable with up to 96 serotypes described so far [30], we included different serotypes of S. pneumoniae to investigate the role of PSGL-1 in phagocytosis. Hence, our findings showed that PSGL-1 is involved in the recognition and phagocytosis of a major human pathogen such as S. pneumoniae contributing therefore to the variety of receptors on professional phagocytes that are needed to efficiently identify and destroy invading pathogens [4][5][6]18]. For the detection of the pathogen it is necessary the interaction of phagocytic receptors with bacterial ligands that are usually exposed on the surface of the microorganism [19]. Using LytA-deficient mutants, we have recently demonstrated that this surface-exposed protein is a major determinant in the virulence of S. pneumoniae by interacting with essential components of the immune system including phagocytes [20]. Our results show now that the LytA autolysin is recognized by PSGL-1 and this effect is dependent on the level of PSGL-1 suggesting that variations in the expression of PSGL-1 on leukocytes might contribute to the efficiency of this interaction. One of the major concerns in the outcome of the infection is that pneumococcal disease can be Role of PSGL-1 in Invasive Pneumococcal Disease produced by clinical isolates of a wide variety of polysaccharide capsules, the main virulence determinant of S. pneumoniae [22,23]. In this sense, using pneumococcal transformants expressing the same genetic background but different CPS, we have demonstrated that PSGL-1 recognizes the capsule of S. pneumoniae. This effect was variable depending on the CPS expressed, with the lowest binding related to the strain expressing serotype 19A. In this sense, incidence of IPD cases caused by this serotype has dramatically increased in the last few years and it has been linked to the emergence of vaccine escape variants that arise by switching the capsular locus from serotype 4 to 19A [31,32]. These results are important in terms of pathogenesis because differences in the recognition of pneumococcal CPS by receptors such as PSGL-1, might explain why certain serotypes of S. pneumoniae are more associated to IPD and dissemination worldwide than others [31][32][33].
The repertoire of host receptors involved in the binding, uptake, signaling and response to invading pathogens is critical for the outcome of the infection. PSGL-1 is the main selectin receptor involved in neutrophil adhesion and migration [8]. Although the importance of leukocyte extravasation is relevant in pneumococcal infection [9,10,29], the contribution of PSGL-1 to the resolution of IPD was previously unknown. In this study we have used PSGL-1 −/− mice to investigate the in vivo role of this receptor in host defense against pneumococcal infection. Our findings confirm that PSGL-1 may act as a pathogen-recognition receptor of the immune system [34]. In this sense, PSGL-1 acts in host defense controlling bacterial proliferation, dissemination and tissue injury which are critical aspects of IPD. This is of great relevance from the respiratory perspective as invasive pneumonia caused by S. pneumoniae is one of the major causes of mortality in children and adults [1,3]. Lack of PSGL-1 has been linked to a greater susceptibility against the intracellular pathogens Salmonella typhimurium or Citrobacter rodentium, which is associated with dramatically increased levels of pro-inflammatory cytokines after intestinal infection [35,36]. In the case of S. pneumoniae, bacterial recognition by immune cells generates an array of cytokines which may play a significant role in host defense. Increase of IL-5, IL-6 and IFN-γ was observed in PSGL-1 -/mice sera. According to our findings, it has been described that increased serum levels of IL-5 and IL-6 were associated with reduced microbial clearance and higher mortality rates in sepsis [37,38]. In addition, the increased levels of IL-12 and IFN-γ in the lungs of wild-type mice might be protective as IFN-γ is involved in the IL-12 regulation of neutrophil-mediated host defense against pneumococcal pneumonia [39].
Bacterial infections of the lower respiratory tract are characterized by massive accumulation of neutrophils in the alveolar spaces [3,40]. Our results show that similar numbers of neutrophils and macrophages were observed in the lungs of PSGL-1 −/− mice. Our results might be unexpected as PSGL-1 is involved in cellular migration of these cells and therefore, increased numbers should be present in the lungs of wild-type mice [8]. Hence, our findings can be explained in the context of a bacterial infection as certain pathogens -including S. pneumoniaehave the ability to impair neutrophil migration to the site of infection by cleaving PSGL-1 [41,42]. Interestingly, this is in line with previous findings confirming that the absence of endothelial selectins E, L and P is not associated with impairment of leukocyte emigration to infection sites after pneumococcal disease [10,43]. In addition, PSGL-1 negatively regulates CD4 + T cell immune responses in vivo which can explain the increased levels of T cells observed in the lungs of PSGL-1 −/− infected mice [44].
Using an intravenous infection model when no collateral migration effects were expected, higher numbers of neutrophils and macrophages but not T cells, were observed in PSGL-1 −/− mice. This is consistent with previous observations showing that blood of PSGL-1 −/− mice contained similar numbers of lymphocytes although they had a significant increase in the proportion of leukocytes, with enhanced levels of granulocytes and monocytes in comparison to wildtype mice [45]. This is relevant from the phagocytosis perspective as PSGL-1 −/− mice, despite having increased numbers of these phagocytic cells in the bloodstream, were unable to control bacterial replication in the blood, leading to the rapid development of fatal infection. Overall, these results confirm that PSGL-1 on leukocytes plays a critical role in host defense against pneumococcal infection. As a consequence of pathogen-recognition by PSGL-1, S. pneumoniae is efficiently engulfed and destroyed, reducing bacterial replication and dissemination in the host, contributing to control the severity of IPD.

Ethics statement
All the experiments involving the use of animals have been performed following the guidelines of the Bioethical and Animal Welfare Committee of Instituto de Salud Carlos III (ISCIII) that reviewed and approved protocol CBA PA 52-2011-v2, to be performed at the National Centre for Microbiology of ISCIII. Animals were bred at Universidad Autónoma de Madrid animal facility following institutional guidelines for animal use and care. Infection experiments conformed to the Spanish government legislation (RD 1201/2005) and European Community regulations (86/609/EEC).

Phagocytosis by HL-60 cells and neutrophils isolated from mice
HL-60 cells (CCL-240; ATCC) differentiated to granulocytes were used and the general conditions of the assay were based on those described previously [17,48]. Briefly, S. pneumoniae strains were fluorescently labeled by incubation with FAM-SE (Molecular Probes) in 0.1 M sodium bicarbonate buffer for 1 h at 37°C, washed five times with Hanks balanced salt solution (HBSS) and stored at −70°C in 10% glycerol as aliquots for further assays. HL-60 cells were harvested by centrifugation and washed twice with HBSS and once with HBSS in the presence of calcium and magnesium ions. Infection assays were performed in the absence of serum to avoid complement-dependent phagocytosis and 10 5 HL-60 cells were infected with 2×10 6 colony forming units (CFU) of viable FAM-SE labeled bacteria. To block PSGL-1, HL-60 cells were incubated for 1 h at room temperature with 25 μg/ml of the KPL-1 antibody (mouse antihuman PSGL-1; MBL) or IgG isotype control (mouse anti-human IgG; Novus Biologicals) as previously described [15,16]. A similar approach was performed using purified LytA. A minimum of 6,000 cells were analyzed using a FACS Calibur flow cytometer (BD Biosciencies). Using cytochalasin D, an inhibitor of actin polymerization, we have previously shown that the majority of the effect on the association of fluorescent S. pneumoniae with HL-60 cells is due to phagocytosis rather than to adhesion of bacteria to the cell surface [49]. Results were expressed as a fluorescence index defined as the proportion of positive cells for fluorescent bacteria multiplied by the geometric mean of fluorescence intensity which correlates with the amount of bacteria phagocytosed per cell [48].
Opsonophagocytosis killing assays were performed in the absence of serum using 10 5 HL-60 cells with the PSGL-1 receptor either active or blocked as mentioned above and 2.5 × 10 4 CFU/ml of S. pneumoniae as previously described [50]. Serial dilutions of culture supernatants were plated on blood agar plates for bacterial counts determination and results were expressed as bacterial survival after 45 min incubation of the pneumococcal strains with HL-60 cells expressing (or not) PSGL-1.
Phagocytosis assays were repeated using neutrophils purified from the spleen of wild-type and PSGL-1 −/− mice by FACS using a FACSAria I (BD Biosciences) dispositive with DIVA version 6.1 software as previously described [51]. Briefly, single-cell suspensions were prepared in staining buffer (2% fetal calf serum in PBS), and non-specific binding was blocked with Fc block (BD Biosciences). Staining was performed using standard protocols with the following antibodies diluted 1/200 in staining buffer including propidium iodide (rat anti-mouse CD11b-allophycocyanin (APC) and rat anti-mouse GR-1-phycoerythrin; Biolegend).

Confocal microscopy
S. pneumoniae strains labeled with FAM-SE were used for immunofluorescence microscopy. HL-60 cells previously infected as described above were seeded on 12-mm circular coverslips for immunofluorescence staining. As HL-60 cells are in suspension, cells were cytofuged at 70 × g for 2 min using a Cytospin centrifuge (Thermo Electron, Pittsburgh, PA), as described elsewhere [16]. For the detection of PSGL-1 in HL-60 cells differentiated to granulocytes, cells were fixed with 3% paraformaldehyde (PFA) for 10 min at room temperature and after two washes with PBS, coverlips were kept in a solution 1 M NH 4 Cl-PBS solution. Coverslips containing the infected cells were washed twice in PBS containing 0.1% saponin and once in PBS and incubated for 30 min with the primary antibody. Staining was performed in PBS containing 10% horse serum, 0.1% saponin and the primary antibody using a mouse anti-human PSGL-1 monoclonal antibody (KPL-1; MBL) diluted 1/300. Cellular DNA was stained with Hoechst (Invitrogen) diluted 1/2500. After 30 min incubation with the primary antibody at room temperature, coverlips were washed twice with PBS-saponin 0.1%, and once with PBS pH 7.0 before incubation during 30 min at room temperature with a dilution 1/200 of the secondary antibody (goat anti-mouse Texas Red; Serotec). Finally, coverslips were washed twice in PBS containing 0.1% saponin, once in PBS, and once in H 2 O, mounted with Aqua Poly/ Mount (Polysciences), and analyzed with a Leica spectral SP5 confocal microscope using the Leica software (LAS-AF).

PSGL-1 binding to S. pneumoniae
Binding of PSGL-1 to S. pneumoniae, purified LytA or CPS was analyzed by ELISA as previously described [20]. Briefly, whole cell ELISA was performed by coating 96-well plates with 200 μl of exponentially growing bacteria and resuspended in PBS to an OD 550 of 1.0. Plates were air dried at room temperature and blocked with 200 μl of PBS-0.5% BSA-NaN 3 for 1 h before 50 μl of different concentrations of PSGL-1 (R&D systems, USA) were added to each well. After overnight incubation at 4°C, plates were washed 5 times with PBS-Tween 0.1% and incubated overnight at 4°C with 50 μl of mouse anti-human PSGL-1 (KPL-1; MBL) diluted 1/ 4000. After 5 washes with PBS-Tween 0.1%, plates were incubated with goat anti-mouse IgG HRP (Southern Biotech) for 30 min at room temperature and developed with o-phenylenediamine (Sigma-Aldrich). Plates were measured at OD 492 using a microtiter plate reader (Anthos 2020). Direct binding of PSGL-1 to purified LytA protein or type 3 CPS (ATCC 169-X, Merck Sharp & Dohme) was performed as described above except that the 96-well plates were coated with 50 μg of purified LytA protein or CPS per well. Purified LytA protein was obtained by overexpression in Escherichia coli [52].

Experimental models of infection
Wild-type C57BL/6 mice and PSGL-1 −/− mice were bred in a conventional animal facility at the School of Medicine, Universidad Autónoma de Madrid (UAM). PSGL-1 −/− mice were kindly provided by Dr. D Vestweber and Dr. MK Wild (Max Plank Institute for Molecular Biomedicine, Münster, Germany). Wild-type C57BL/6 mice obtained from the Jackson Laboratory and PSGL-1 −/− mice were backcrossed and the wild-type and PSGL-1 −/− littermates obtained from crosses of the resulting heterozygous mice were used to breed our wild-type and PSGL-1 −/− colonies used in this study. Animal procedures were approved by the Animal Care and Use Committee of ISCIII. All mice used were 8-16 weeks old, and within each experiment, groups of mice were matched for age and sex. Studies investigating pneumococcal sepsis or pneumonia were performed using groups of at least 5 mice and infected as previously described [48]. Briefly, for sepsis, mice were challenged with 5 × 10 6 CFU/ml for D39 strain or 3 × 10 4 CFU/ml for TIGR4 strain (in a volume of 200 µl) by the intraperitoneal route, whereas for pneumonia, mice under anesthesia with isofluorane were inoculated intranasally with 50 µl containing 10 7 CFU/mouse of D39 strain. For intravenous inoculation, mice were infected with 2 × 10 7 CFU/mouse of D39 strain through the tail vein. At 6 h and 24 h after challenge, a lethal dose of pentobarbital was administered and bacterial counts were determined from samples recovered from BALF, lung and blood. Experiments were repeated twice using 5 mice in each group and results were expressed as Log 10 CFU/ml of bacteria recovered from the different sites. Cytokines were measured from BALF and blood of wild-type mice and PSGL-1 −/− mice infected with D39 strain by the intranasal route as explained above. Cytokines levels (IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, GM-CSF, TNF-α and IFN-γ) were determined by using a Luminex magnetic bead array assay (Bio-Rad) according to manufacturer protocols.

Lung sectioning and histology
Mice were euthanized with pentobarbital and lungs were inflated and fixed with 4% PFA in PBS. Lungs were paraffin-embedded and 5-μm sections were obtained. Infiltrates of granulocytes and T cells were measured by staining with anti-Ly-6G/6C (antigen retrieval pH 6.5, 1/ 400, Abcam ab2557) and anti-CD3 (antigen retrieval pH 6.0, 1/200, Santa Cruz Biotechnology sc-1127) antibodies respectively. Immunohistochemistry was performed with the Dako LSAB + System-HRP following manufacturer's instructions.

Statistical analysis
Data are representative of results obtained from repeated independent experiments, and each data point represents the mean and standard deviations (SD) for 3 to 5 replicates. Statistical analysis was performed by using two-tailed Student's t test (for two groups), whereas analysis of variance (ANOVA) followed by a Dunnett's post hoc test was chosen for multiple comparisons. Survival was analyzed by the log-rank test. GraphPad InStat version 5.0 (GraphPad Software, San Diego, CA) was used for statistical analysis. Differences were considered statistically significant with P <0.05 ( Ã ) and highly significant with P <0.01 ( ÃÃ ) and P <0.001 ( ÃÃÃ ).