Author response: Regulation of inflammation and protection against invasive pneumococcal infection by the long pentraxin PTX3

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Streptococcus pneumoniae is a major pathogen in children, elderly subjects, and immunodeficient patients. Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule (PRM) involved in resistance to selected microbial agents and in regulation of inflammation. The present study was designed to assess the role of PTX3 in invasive pneumococcal infection. In a murine model of invasive pneumococcal infection, PTX3 was strongly induced in non-hematopoietic (particularly, endothelial) cells. The IL-1β/MyD88 axis played a major role in regulation of the Ptx3 gene expression. Ptx3−/− mice presented more severe invasive pneumococcal infection. Although high concentrations of PTX3 had opsonic activity in vitro, no evidence of PTX3-enhanced phagocytosis was obtained in vivo. In contrast, Ptx3-deficient mice showed enhanced recruitment of neutrophils and inflammation. Using P-selectin-deficient mice, we found that protection against pneumococcus was dependent upon PTX3-mediated regulation of neutrophil inflammation. In humans, PTX3 gene polymorphisms were associated with invasive pneumococcal infections. Thus, this fluid-phase PRM plays an important role in tuning inflammation and resistance against invasive pneumococcal infection. Editor's evaluation This submission represents a holistic approach to how pentraxin 3 (PTX3) modulates susceptibility to experimental infection by Streptococcus pneumoniae. The authors have built robust findings on the importance of PTX3 for the survival of mice and they have extensively investigated all different aspects of the mechanism of PTX3 protection. One main strength of the manuscript is its usage of bone marrow chimeras in addition to total as well as tissue-specific mouse strains that support their claims. https://doi.org/10.7554/eLife.78601.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Streptococcus pneumoniae (or pneumococcus) is a Gram-positive extracellular pathogen which colonizes the respiratory mucosa of the upper respiratory tract and represents a major cause of bacterial pneumonia, meningitis, and sepsis in children, elders, and immunodeficient patients. Depending on the virulence factors expressed by the pathogen and on host factors, the disease can evolve to pneumococcal invasive infection, where pneumococcus invades the lower respiratory tract and translocates through the bloodstream into the systemic compartment (Weiser et al., 2018). As a first line of defense against respiratory pathogens, innate immune pattern recognition molecules (PRMs) recognize microbial components and modulate immune response to control infections. Among conserved fluid-phase PRMs, Pentraxin 3 (PTX3), is a member of the pentraxin family characterized by multifunctional properties, including regulation of innate immunity during infections (Garlanda et al., 2018). PTX3 is expressed by various hematopoietic and non-hematopoietic cells in response to microbial moieties and inflammatory cytokines (i.e IL-1β and TNF), and it has been associated with the control of various infections by promoting different anti-microbial mechanisms. Indeed, PTX3 participates directly to the elimination of selected microorganisms by promoting phagocytosis, activating the complement cascade and as a component of Neutrophil Extracellular Traps (NET) (Daigo et al., 2012; Jaillon et al., 2014; Jaillon et al., 2007; Moalli et al., 2010; Porte et al., 2019). Furthermore PTX3 modulates tissue remodeling (Doni et al., 2015) and inflammation by tuning complement activation and P-selectin-dependent transmigration (Deban et al., 2010; Lech et al., 2013), both involved in neutrophil recruitment and in the evolution of respiratory tract infections (Quinton and Mizgerd, 2015). In humans, PTX3 plasma levels increase in the context of inflammation and selected infectious diseases, including pneumococcal pathologies (i.e. community-acquired pneumonia, ventilator-associated pneumonia, pneumococcal exacerbated chronic obstructive pulmonary disease), correlating with the severity of the disease and predicting the risk of mortality (Bilgin et al., 2018; Kao et al., 2013; Mauri et al., 2014; Porte et al., 2019; Saleh et al., 2019; Shi et al., 2020; Siljan et al., 2019; Thulborn et al., 2017). In addition, single-nucleotide polymorphisms (SNPs) in the PTX3 gene have been associated with patient susceptibility to respiratory infections (Brunel et al., 2018; Chiarini et al., 2010; Cunha et al., 2015; Cunha et al., 2014; He et al., 2018; Olesen et al., 2007; Wójtowicz et al., 2015). The involvement of PTX3 in the control of selected respiratory pathogens and in the modulation of infection prompted us to investigate the role of this molecule in the control of pneumococcal infections. In a murine model of invasive pneumococcal infection, we observed that PTX3 genetic deficiency was associated with higher disease severity and higher respiratory tract inflammation. PTX3, mainly produced by stromal non-hematopoietic cells during pneumococcal infection, modulated neutrophil recruitment by dampening P-selectin-dependent neutrophil migration. Hence, PTX3 plays a non-redundant role in the control of S. pneumoniae infection, modulating neutrophil-associated respiratory tissue damage and pneumococcal systemic dissemination. Results PTX3 expression during pneumococcal invasive infection In order to define the relevance of PTX3 in pneumococcal respiratory disease, we first investigated whether the protein is induced during infection. Thus, we used a murine model of pneumococcal invasive infection induced by S. pneumoniae serotype 3. Mice were challenged intranasally with 5 × 104 CFU and sacrificed at different time points. As already described, S. pneumoniae serotype 3 causes bacterial colonization of the respiratory tract, then disseminates through the blood circulation and infects other organs like the spleen, resulting in death within 3–4 days (Figure 1—figure supplement 1A ,B; de Porto et al., 2019). When compared to uninfected mice, infected animals also developed organ dysfunction, as demonstrated by increased circulating levels of creatinine and enzymatic activity of alanine transaminase and creatine phosphokinase 36 hr post-infection (Figure 1—figure supplement 1C). As early as 6 hr post-infection, we detected a local expression of PTX3 in the alveolar compartment near the pulmonary veins (Figure 1A, B). At 12 hr post-infection, we were able to detect PTX3-specific staining in endothelial cells in the area where we can appreciate inflammatory cells infiltration. This association was confirmed 24 hr post-infection, when a strong PTX3 staining was present near the recruitment site of inflammatory cells forming inflammatory foci (Figure 1A). The kinetic of PTX3 production was confirmed by the quantification of PTX3+ area (Figure 1B) and by analysis of mRNA in the lung (Figure 1—figure supplement 1D). Interestingly, local and systemic production of PTX3 was strongly induced by the infection during the disseminating phase (Figure 1C). During this invasive infection we observed that Ptx3 was upregulated mainly in the lung, aorta, and heart, while other organs like brain, kidneys, and liver did not show higher Ptx3 expression compared to the uninfected mice (Figure 1—figure supplement 1E). Figure 1 with 1 supplement see all Download asset Open asset Invasive pneumococcal infection induces PTX3 expression. Wild-type (WT) mice were infected intranasally with 5 × 104 CFU of S. pneumoniae serotype 3 and sacrificed at the indicated time points for tissue collection. (A, B) Immunohistochemical analysis and quantification of PTX3 expression in lung sections (magnification 20x) from uninfected mice and mice sacrificed 6, 12, and 24 hr post-infection (n = 3–6). (A) One representative image of at least three biological replicates for each condition is reported. Inflammatory cell infiltrates are indicated by arrows. (B) Sections were scanned and analyzed to determine the percentage of PTX3+ area at the indicated time points. (C) PTX3 protein levels determined by ELISA in serum and lung homogenates collected at the indicated time points (n = 4–10). Results are reported as mean ± standard error of the mean (SEM). Statistical significance was determined using the Mann–Whitney test comparing results to uninfected mice (φ or *p < 0.05 and **p < 0.01). Figure 1—source data 1 Individual data values for the graph in Figure 1B. https://cdn.elifesciences.org/articles/78601/elife-78601-fig1-data1-v2.xlsx Download elife-78601-fig1-data1-v2.xlsx Figure 1—source data 2 Individual data values for the graph in Figure 1C. https://cdn.elifesciences.org/articles/78601/elife-78601-fig1-data2-v2.xlsx Download elife-78601-fig1-data2-v2.xlsx Induction of PTX3 by IL-1β during S. pneumoniae infection PTX3 has been described to be induced by primary inflammatory cytokines in particular IL-1β (Garlanda et al., 2018; Porte et al., 2019). In this pneumococcal invasive infection model we observed a rapid induction of IL-1β (Figure 2A), and a strong correlation between the levels of IL-1β expressed in the respiratory tract with the levels of lung PTX3 (Figure 2B). A similar correlation was observed with TNF and IL-6 levels measured in homogenates from infected lungs (Figure 2—figure supplement 1A, B), whereas CXCR1 levels were not correlated with the level of lung PTX3 (Figure 2—figure supplement 1C). Moreover, Il1r−/− mice infected by S. pneumoniae showed lower PTX3 levels, locally and systemically (i.e. in the lung and the serum, respectively) (Figure 2C, D). S. pneumoniae infected Myd88−/− mice were not able to produce PTX3 in the lung and presented the same impairment of PTX3 production as Il1r−/− mice (Figure 2C, D). These data suggest that, similar to what occurs in other models, PTX3 production during pneumococcal infection requires IL-1β sensing or contribution of MyD88-dependent pathways (Doni et al., 2015; Jaillon et al., 2014; Salio et al., 2008). Figure 2 with 1 supplement see all Download asset Open asset Role of IL-1β in induction of PTX3 during S. pneumoniae infection. WT mice were infected intranasally with 5 × 104 CFU of S. pneumoniae serotype 3 and sacrificed at the indicated time points for tissue collection. (A) IL-1β protein levels in lung homogenates collected at the indicated time points determined by ELISA (n = 3–4). (B) Correlation between PTX3 and IL-1β protein levels in lung homogenates of all infected mice sacrificed from 2 to 48 hr post-infection (data pooled from five independent experiments, n = 60); Pearson correlation coefficient is reported. PTX3 protein levels determined by ELISA in lung homogenates (C) and serum (D) collected 36 hr post-infection in WT, Il1r−/− and Myd88−/− mice (n = 7–8). Results are reported as mean ± SEM. Statistical significance was determined using the Mann–Whitney test comparing results to uninfected mice (A, B) or the non-parametric Kruskal–Wallis test with post hoc corrected Dunn’s test comparing means in Il1r-/- and Myd88-/- mice to WT infected mice (C, D) (*p < 0.05, ***p < 0.001, and ****p < 0.0001). Figure 2—source data 1 Individual data values for the graph in Figure 2A. https://cdn.elifesciences.org/articles/78601/elife-78601-fig2-data1-v2.xlsx Download elife-78601-fig2-data1-v2.xlsx Figure 2—source data 2 Individual data values for the graph in Figure 2B. https://cdn.elifesciences.org/articles/78601/elife-78601-fig2-data2-v2.xlsx Download elife-78601-fig2-data2-v2.xlsx Figure 2—source data 3 Individual data values for the graph in Figure 2C. https://cdn.elifesciences.org/articles/78601/elife-78601-fig2-data3-v2.xlsx Download elife-78601-fig2-data3-v2.xlsx Figure 2—source data 4 Individual data values for the graph in Figure 2D. https://cdn.elifesciences.org/articles/78601/elife-78601-fig2-data4-v2.xlsx Download elife-78601-fig2-data4-v2.xlsx Non-hematopoietic cells are a major source of PTX3 during pneumococcal infection It has been previously reported that neutrophils contain preformed PTX3, representing an important source of the protein, rapidly released in response to pro-inflammatory cytokines or microbial recognition (Jaillon et al., 2007). In agreement, we observed that human neutrophils can release PTX3 upon stimulation with S. pneumoniae (Figure 3—figure supplement 1A). To investigate the involvement of neutrophils in the production of PTX3 in our model, we used mice lacking granulocyte colony-stimulating factor receptor (Csf3r−/−). These mice are characterized by chronic neutropenia, granulocyte, and macrophage progenitor cell deficiency and impaired neutrophil mobilization (Liu et al., 1996; Ponzetta et al., 2019). Following pneumococcal infection, Csf3r−/− mice presented lower levels of myeloperoxidase (MPO), a marker of neutrophil recruitment, in lung homogenates at 36 hr post-infection (Figure 3—figure supplement 1B). By contrast, even though these mice presented lower amount of neutrophils recruited in response to the infection, they expressed the same pulmonary levels of PTX3 as WT mice (Figure 3—figure supplement 1B). These results suggest that neutrophils are not the main source of PTX3 in our murine model of pneumococcal invasive infection. Since PTX3 can be produced by hematopoietic and non-hematopoietic cells, bone marrow chimeras were used to evaluate the cellular compartment responsible for PTX3 production. During pneumococcal infection, we did not observe any difference in the levels of PTX3 in the respiratory tract and in the serum of WT mice receiving bone marrow from Ptx3−/− or WT animals, while no PTX3 was measured in Ptx3−/− mice receiving WT or Ptx3−/− bone marrow (Figure 3A, B). These results suggest that PTX3 is mainly produced by the non-hematopoietic compartment after pneumococcal infection. Endothelial cells were described as an important source of PTX3 (Garlanda et al., 2018), thus we evaluated their contribution to PTX3 production during pneumococcal infection. To this aim we crossed conditional Ptx3-deficient mice (Ptx3Lox/Lox) with Cdh5Cre/+ mice to generate animals with the deletion of PTX3 in endothelial cells. When Ptx3Lox/LoxCdh5Cre/+ mice were infected with S. pneumoniae, they presented approximately 50% reduction of PTX3 levels compared to PTX3-competent mice (Figure 3C, D). In vitro experiments confirmed the ability of both murine and human endothelial cells to produce PTX3 after stimulation with S. pneumoniae (Figure 3—figure supplement 1C). Thus, in our setting, non-hematopoietic cells, mainly endothelial cells, are a major source of PTX3. Figure 3 with 1 supplement see all Download asset Open asset Non-hematopoietic cells are a major source of PTX3 during pneumococcal infection. Mice were infected intranasally with 5 × 104 CFU of S. pneumoniae serotype 3 and sacrificed 36 hr post-infection for tissue collection. (A, B) PTX3 protein levels determined by ELISA in lung homogenates (n = 12–14, A) and serum (n = 6, B) from chimeric mice. Two independent experiments were performed with similar results. (C, D) PTX3 protein levels determined by ELISA in lung homogenates (C) and serum (D) collected from Ptx3Lox/LoxCdh5+/+, Ptx3Lox/LoxCdh5Cre/+ (n = 10–13). Results are reported as mean; PTX3 detection limit is 2 ng/ml in lung homogenates (A) and 0.25 ng/ml in serum (B) and is represented by a dotted line. Statistical significance was determined using the non-parametric Kruskal–Wallis test with post hoc corrected Dunn’s test comparing means to the WT recipient mice reconstituted with WT bone marrow (A, B) or the Mann–Whitney test (C, D) (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns: not significant). Figure 3—source data 1 Individual data values for the graph in Figure 3A. https://cdn.elifesciences.org/articles/78601/elife-78601-fig3-data1-v2.xlsx Download elife-78601-fig3-data1-v2.xlsx Figure 3—source data 2 Individual data values for the graph in Figure 3B. https://cdn.elifesciences.org/articles/78601/elife-78601-fig3-data2-v2.xlsx Download elife-78601-fig3-data2-v2.xlsx Figure 3—source data 3 Individual data values for the graph in Figure 3. https://cdn.elifesciences.org/articles/78601/elife-78601-fig3-data3-v2.xlsx Download elife-78601-fig3-data3-v2.xlsx Figure 3—source data 4 Individual data values for the graph in Figure 3D. https://cdn.elifesciences.org/articles/78601/elife-78601-fig3-data4-v2.xlsx Download elife-78601-fig3-data4-v2.xlsx Non-redundant role of PTX3 in resistance to pneumococcal infection Next, we evaluated the role of PTX3 in resistance against pneumococcus. When Ptx3−/− mice were infected with S. pneumoniae (5 × 104 CFU), a significant increase of the bacterial load in the lung was observed during the invasive phase of infection (i.e. 36 hr post-infection), compared to WT mice (Figure 4A). Defective local control of bacterial growth was associated to an increase of bacterial load in the spleen (Figure 4B). Interestingly there was no difference at earlier time points (i.e. 18 hr post-infection, Figure 4—figure supplement 1A), suggesting that PTX3 exerted a role in the control of pneumococcal infection mainly during the invasive phase. Using a bacterial dose (5 × 103 CFU) inducing around 30% mortality in WT animals, Ptx3−/− mice showed a significant higher mortality (83.3%; p < 0.001) (Figure 4C). The phenotype described so far is not restricted to serotype 3 pneumococcus. In fact, when mice were infected with S. pneumoniae serotype 1, we observed a strong PTX3 production during the invasive phase of the infection (Figure 4—figure supplement 1B) and a correlation with IL-1β levels (Figure 4—figure supplement 1C). Ptx3−/− mice infected by serotype 1 presented a higher sensitivity to the infection compared to WT animals, with a higher number of bacteria at the local site of infection and also in the spleen 24 hr post-infection (Figure 4—figure supplement 1D, E). Thus, in the applied model of S. pneumoniae infection, the protection conferred by PTX3 is not limited to serotype 3, and embraces other bacterial serotypes of clinical relevance, including serotype 1. Figure 4 with 1 supplement see all Download asset Open asset Defective resistance of Ptx3-deficient mice to invasive pneumococcal infection. WT and Ptx3-/- mice were infected intranasally with different doses of S. pneumoniae serotype 3 and sacrificed at the indicated time points for tissue collection. Bacterial load in lung (A) and spleen (B) was analyzed at 36 hr post-infection with 5 x 104 CFU of bacteria (data pooled from two independent experiments, n = 16–21). (C) Survival of WT and Ptx3−/− mice (data pooled from two independent experiments, n = 18) was monitored every 6 hr after infection with 5 × 103 CFU. (D) Bacterial load was analyzed in lungs collected 36 hr post-infection from WT, Ptx3−/− and Ptx3−/− mice treated intraperitoneally with recombinant PTX3 (10 µg/100 µl) before the infection and 24 hr post-infection (n = 18–23). (E) Bacterial load in lungs collected 36 hr post-infection from WT mice treated intranasally before the infection (prophylaxis, data pooled from two independent experiments, n = 22–26) or 12 hr post-infection (treatment, data pooled from three independent experiments, n = 37–40) with 1 µg/30 µl of recombinant PTX3 or phosphate-buffered saline (PBS). Results are reported as median CFU. Detection limit in the spleen is 5 CFU (dotted line in panel B). Statistical significance was determined using the Mann–Whitney test (A, B, E), the non-parametric Kruskal–Wallis test with post hoc corrected Dunn’s test comparing means to the WT mice (D) and log-rank (Mantel–Cox) test for survival (C) (*p < 0.05, **p < 0.01, and ***p < 0.001). Figure 4—source data 1 Individual data values for the graph in Figure 4A. https://cdn.elifesciences.org/articles/78601/elife-78601-fig4-data1-v2.xlsx Download elife-78601-fig4-data1-v2.xlsx Figure 4—source data 2 Individual data values for the graph in Figure 4B. https://cdn.elifesciences.org/articles/78601/elife-78601-fig4-data2-v2.xlsx Download elife-78601-fig4-data2-v2.xlsx Figure 4—source data 3 Individual data values for the survival graph in Figure 4C. https://cdn.elifesciences.org/articles/78601/elife-78601-fig4-data3-v2.xlsx Download elife-78601-fig4-data3-v2.xlsx Figure 4—source data 4 Individual data values for the graph in Figure 4D. https://cdn.elifesciences.org/articles/78601/elife-78601-fig4-data4-v2.xlsx Download elife-78601-fig4-data4-v2.xlsx Figure 4—source data 5 Individual data values for the graph in Figure 4E. https://cdn.elifesciences.org/articles/78601/elife-78601-fig4-data5-v2.xlsx Download elife-78601-fig4-data5-v2.xlsx Systemic administration of recombinant PTX3 to Ptx3−/− mice rescues the phenotype. As reported in Figure 4D, PTX3 administration in Ptx3−/− mice reduced lung colonization to the same level observed in WT mice. We then evaluated the antibacterial activity of PTX3 on S. pneumoniae serotype 3. WT animals were treated locally with 1 µg of recombinant protein before infection or 12 hr post-infection. Under both conditions we observed a significant reduction (44% and 57%, respectively; p < 0.01) of the pulmonary bacterial load compared with the CFU found in mice treated with vehicle alone (Figure 4E). Lack of effective opsonic activity of PTX3 In an effort to explore the mechanism responsible for PTX3-mediated resistance, we first assessed the effect of the recombinant protein on the in vitro growth of S. pneumoniae. The incubation of S. pneumoniae with 25–250 µg/ml of recombinant PTX3 did not have any effect on the growth rate of the bacteria (Figure 5—figure supplement 1). PTX3 has the capability to act as an opsonin binding selected pathogens and increasing their removal by phagocytosis (Garlanda et al., 2002; Jaillon et al., 2014; Moalli et al., 2010). To assess whether the control of the pneumococcal infection by PTX3 was due to opsonic activity, we first analyzed PTX3 binding to S. pneumoniae. By using a flow cytometry assay, we analyzed PTX3 binding to S. pneumoniae serotype 3 mimicking the bacteria/PTX3 ratio found in the infected lung (106 CFU/100 ng PTX3). Under these conditions, we did not observe any interaction of PTX3 with bacteria and, even with an amount of PTX3 5- to 10-fold higher than the one produced in the entire lung, less than 1% of the bacteria were bound (Figure 5A). At 500 µg/ml of PTX3 (5000-fold higher than in the lung homogenates) we observed binding to only 36.4% of bacteria (Figure 5A). Figure 5 with 2 supplements see all Download asset Open asset Role of phagocytosis in PTX3-mediated resistance to S. pneumoniae. (A) Binding of biotinylated recombinant PTX3 at the indicated concentration with 106 CFU of S. pneumoniae serotype 3 was analyzed by flow cytometry after incubation with Streptavidin-Alexa Fluor 647. (B) S. pneumoniae serotype 1 expressing GFP (S pneumoniae-GFP; 106 CFU) was pre-opsonized with the indicated concentration of recombinant PTX3 and incubated 30 min with 105 purified human neutrophils from six independent donors. GFP-positive neutrophils were analyzed by flow cytometry. Results are expressed as mean of five technical replicates for each time and (C) Bacterial load in lungs collected at indicated time points from WT mice infected intranasally with S. pneumoniae serotype 3 pre-opsonized with µg/ml of recombinant PTX3 or (data pooled from two independent experiments, n = (D) Neutrophil phagocytosis of S. was analyzed by flow cytometry. and lungs from WT and Ptx3−/− mice were collected 24 hr after infection with a of S. pneumoniae (data pooled from two independent experiments, n = (E) performed with neutrophils purified from WT and Ptx3−/− mice assessed after 1 and 3 hr incubation at a S. median values (C) or mean values E). Statistical significance was determined using the analysis of with test the non-parametric Kruskal–Wallis test with post hoc corrected Dunn’s test comparing means to the WT mice of each time (C, and the Mann–Whitney test (D) < 0.01). Figure data 1 Individual data values for the graph in Figure Download Figure data 2 Individual data values for the graph in Figure Download Figure data 3 Individual data values for the graph in Figure Download Figure data 4 Individual data values for the graph in Figure Download We then assessed phagocytosis in vitro and in using S. pneumoniae serotype 1 In a first of experiments, human neutrophils were incubated with S. We confirmed that PTX3 opsonic increasing the phagocytosis of pneumococcus by only at high that is higher than µg/ml (Figure We then to an in Since the of as as 1 µg of PTX3 was to an antibacterial effect when locally before the infection (Figure we incubated 5 × 104 CFU of S. pneumoniae serotype 3 (i.e. the used for a infection in our with µg/ml of recombinant PTX3. Mice infected with S. pneumoniae serotype 3 showed the same local bacterial at hr after infection as mice infected with pneumococcus incubated with (Figure We then evaluated the ability of neutrophils recruited in during the infection comparing WT and Ptx3-deficient mice. Interestingly, we did not observe any difference in the percentage of neutrophils S. in the or in the lung (Figure we assessed the ability of neutrophils collected from WT and Ptx3-deficient mice. We did not observe any difference in the percentage of S. pneumoniae serotype 3 by purified murine neutrophils after 1 hr of incubation ± and ± p = or 3 hr of incubation when all pneumococcus were ± and ± p = (Figure These results suggest that the role of PTX3 in resistance to invasive pneumococcus infection is not for by its opsonic of inflammation by PTX3 In pneumococcal invasive disease induced by S. pneumoniae serotype 3, infection was characterized by a (Figure supplement 1A). The main inflammatory cell recruitment was observed during the invasive phase of the infection from 24 hr after when the pulmonary was increased (Figure supplement 1B). We analyzed more neutrophil recruitment in the lung and in the of infected mice and we observed two of neutrophil recruitment, characterized by an increased (i.e. compared to uninfected number of neutrophils both in the and in the lung was observed during the first 6 hr of infection. In the hr of infection we observed an important recruitment of neutrophils in the lung (i.e. compared to uninfected that into the alveolar to compared to uninfected (Figure supplement D). These two of recruitment have been described to et al., 2015). Indeed, the first phase is important for the early control of the infection, the number of In the phase has been associated with the of the inflammatory to tissue damage that growth and of the bacteria et al., 2015). the expression of PTX3 during the first (Figure 1A), we investigated the phase of neutrophil recruitment, comparing Ptx3-deficient and WT mice 18 hr after infection. At this time Ptx3 deficiency was not associated with a higher respiratory bacterial load (Figure 4—figure supplement 1A). Interestingly, the inflammatory was increased in Ptx3−/− mice, as by an increased of foci in the lung induced by a higher inflammatory cell recruitment (Figure Moreover, at the time of the of respiratory we observed that Ptx3−/− mice had a and more severe of inflammatory foci compared to the WT (Figure these mice presented also an increased damage on higher and (Figure supplement 1E). cytometry analysis that the higher inflammation in Ptx3-deficient mice was due to a significant increase of neutrophil recruitment in the and the lung (Figure Moreover, we