IMMUNOLOGY

ORIGINAL ARTICLE

Interleukin-10 plays a key role in the modulation of neutrophils recruitment and lung inflammation during infection by Streptococcus pneumoniae Hernan F. Pe~ naloza,1 Pamela A. 1 Nieto, Natalia Mu~ noz-Durango,1 Francisco J. Salazar-Echegarai,1 Javiera Torres,2 Marıa J. Parga,3 Manuel Alvarez-Lobos,3 Claudia A. Riedel,4 Alexis M. Kalergis1,5,6 and Susan M. Bueno1,6 1

Millennium Institute on Immunology and Immunotherapy, Departamento de Genetica Molecular y Microbiologıa, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile, 2Departamento de Anatomıa Patologica, Facultad de Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile, 3Departamento de Gastroenterologıa, Facultad de Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile, 4Millennium Institute on Immunology and Immunotherapy, Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas y Facultad de Medicina, Universidad Andres Bello, Santiago, Chile, 5 Departamento de Inmunologıa Clınica y Reumatologıa, Facultad de Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile and 6INSERM U1064, Nantes, France

Summary Streptococcus pneumoniae is a major aetiological agent of pneumonia worldwide, as well as otitis media, sinusitis, meningitis and sepsis. Recent reports have suggested that inflammation of lungs due to S. pneumoniae infection promotes bacterial dissemination and severe disease. However, the contribution of anti-inflammatory molecules to the pathogenesis of S. pneumoniae remains unknown. To elucidate whether the production of the anti-inflammatory cytokine interleukin-10 (IL-10) is beneficial or detrimental for the host during pneumococcal pneumonia, we performed S. pneumoniae infections in mice lacking IL-10 (IL-10 / mice). The IL-10 / mice showed increased mortality, higher expression of proinflammatory cytokines, and an exacerbated recruitment of neutrophils into the lungs after S. pneumoniae infection. However, IL-10 / mice showed significantly lower bacterial loads in lungs, spleen, brain and blood, when compared with mice that produced this cytokine. Our results support the notion that production of IL-10 during S. pneumoniae infection modulates the expression of pro-inflammatory cytokines and the infiltration of neutrophils into the lungs. This feature of IL-10 is important to avoid excessive inflammation of tissues and to improve host survival, even though bacterial dissemination is less efficient in the absence of this cytokine. Keywords: bacterial infection; cytokines; lung inflammation; neutrophils; systemic bacterial infection.

doi:10.1111/imm.12486 Received 25 February 2015; revised 3 May 2015; accepted 27 May 2015. Correspondence: Dr Susan M. Bueno, Departamento de Genetica Molecular y Microbiologıa, Facultad de Ciencias Biol ogicas, Pontificia Universidad Cat olica de Chile. Avenida Libertador Bernardo O’Higgins N340, Santiago 8331010, Santiago, Chile. Email: [email protected] Senior author: Dr. Susan M Bueno

Introduction Streptococcus pneumoniae (S. pneumoniae) is an extracellular Gram-positive bacterium able to colonize and invade the respiratory tract.1,2 In 2009, it was estimated that approximately 800 000 children under 5 years old died due to S. pneumoniae infections and 90% of these 100

cases were documented in developing countries.3 Further, due to their impaired immune response and the incidence of chronic diseases, the elderly population is another highly susceptible group that suffer pneumococcal diseases. In fact, S. pneumoniae causes 50% of pneumonia cases in this population.4 In USA, pneumonia rates for individuals between 65 and 69 years old were ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

Role of IL-10 in S. pneumoniae infection 182/1000 person-years and 523/1000 person-years for individuals over 85 years of age.5 S. pneumoniae contains several virulence factors used to evade the immune response at different stages of infection.6–8 For instance, this bacterium is able to prevent phagocytosis and complement binding, induce necrosis of immune cells and cleavage of IgA antibodies.1,2 Current strategies used to prevent and treat pneumococcal infections are conjugate vaccines and antibiotics, respectively. Both approaches have shown only partial effectiveness at controlling the disease, especially in highly susceptible individuals.9,10 Based on the information provided above, S. pneumoniae is considered an important pathogen that causes a major burden to public health worldwide. The immune response against S. pneumoniae involves a broad range of cellular and soluble elements belonging to both the innate and adaptive immune system. S. pneumoniae is recognized in alveoli by alveolar macrophages, epithelial cells, dendritic cells and B cells,1,11 which secrete pro-inflammatory cytokines and chemokines that produce a burst of neutrophils and monocytes.1 A subsequent T helper type 17 (Th17) response developed by CD4+ and cd T cells enhances this neutrophil and monocyte recruitment into the lungs.12,13 However, in populations at higher risk, such as infants and the elderly, this response may not be enough to clear the bacterium because of its intrinsic virulence factors that promote the evasion of the immune response.9 Interleukin 10 (IL-10) is an anti-inflammatory cytokine that plays an important role in acute and persistent bacterial infections.14 The main sources of this cytokine are dendritic cells, macrophages, neutrophils, B and T cells.14–16 It reduces inflammation by inhibiting the action of natural killer cells, the differentiation of naive T cells into effector T cells, and the secretion of pro-inflammatory cytokines, such as tumour necrosis factor-a (TNF-a) by macrophages, dendritic cells and neutrophils.15,16 IL-10 also stimulates the proliferation of regulatory T cells, contributing to a balanced immune response consisting of pathogen clearance without excessive inflammation or damage to self tissues.16 The contribution of IL-10 to the immune response during respiratory infections has been evaluated on different models with varying results. For instance, during Klebsiella pneumoniae infection in mice, IL-10 neutralization led to improved bacterial clearance and higher survival rates,17 suggesting that the presence of IL-10 could be detrimental for host defense. This notion was further supported by the observation that IL-10 knockout mice (IL-10 / mice) intranasally infected with a non-lethal dose of Bortedella parapertussis showed higher lung inflammation due to increased neutrophil recruitment, with an improved bacterial clearance.18 However, other studies have shown that absence of IL-10 fails to enhance the host immune response against pathogenic bacteria. IL-10 / mice infected with a sublethal dose of Francisella tularensis LSV strain, showed higher mortality rates due to increased lung inflammation ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

and failure to clear the bacterium.19,20 Despite IL-10 is a key component of the immune response against many other pathogens,21,22 the effects of its deficiency vary from one microorganism to another. The capacity of IL-10 to modulate the host inflammatory immune response together with the fact that lung inflammation during S. pneumoniae infection is associated with increased bacterial dissemination and severe disease,23–25 would suggest that IL-10 is required to prevent bacterial dissemination during S. pneumoniae infection. Here we show that S. pneumoniae (strain D39) leads to a significant increase in the expression of IL-10 in the lungs of infected mice. Using IL-10 / mice, we observed that this cytokine is required to regulate the expression of soluble inflammatory mediators in lungs, such as IL-6, interferon-c (IFN-c), IL-1b and TNF-a. Although the absence of IL-10 improves bacterial clearance in lungs and other organs, this cytokine is required to prevent exacerbated neutrophil recruitment to the infected lungs. Our results support the notion that the immune system tightly regulates inflammation in the lung after bacterial infection to prevent tissue damage and organ failure, despite these regulatory mechanisms possibly favouring bacterial dissemination.

Material and methods Mice C57BL/6 wild-type (WT litter-mates) mice and B6.129P2Il10tm1Cgn/J (IL-10 / ) mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free animal facility at the Facultad de Ciencias Biol ogicas, Pontificia Universidad Cat olica de Chile. All experimental procedures performed in this study were revised and approved by the Bioethics and Biosafety Committee of the Facultad de Ciencias Biol ogicas, Pontificia Universidad Cat olica de Chile. All animal work was performed according to the Guide for Care and Use of Laboratory Animals (National Institute of Health, Bethesda, MD) and Institutional guidelines. Experiments using animals were overseen by a veterinarian at all times.

Pneumococcal infection S. pneumoniae D39 strain was grew on Todd Hewitt Yeast extract (THYE) medium until reach an optical density of 0.4. Then bacteria aliquots were frozen at -80C on THYE containing glicerol 10%. Finally each aliquot was thawed and diluted to reach the final concentration required. Six- to eight-week-old WT or IL-10 / mice were anaesthetized with a ketamine 16%/xylazine 4% solution and intranasally infected with 30 ll of THYE broth containing 3 9 105, 3 9 106 or 3 9 107 colony-forming units (CFUs) of S. pneumoniae D39. Weight was recorded daily, and animals that lost more than 25% of their original weight were 101

~aloza et al. H. F. Pen sacrificed. To evaluate cell infiltration in lungs, infected mice were sacrificed 24 and 48 hr post-infection (hpi) and bronchoalveolar lavage fluid (BALF) was obtained to measure total proteins by a Bradford protein assay. Bacterial loads were measured in lungs, brain, spleen and blood of infected animals by seeding serial dilutions of homogenized tissues on blood agar plates and incubating overnight at 37°C and 5% CO2. To corroborate that all mice were properly infected, a clinical scoring was performed as previously described.26

Flow cytometry Lungs were recovered 24 and 48 hpi, minced with sterile scissors and incubated in PBS/collagenase (1 mg/ml)/ DNAse I (50 lg/ml) for 1 hr at 37°C with agitation. Homogenized lungs were filtered using a 70-lM cell strainer. Cells were recovered by centrifugation, washed once with ACK lysis buffer and twice with PBS. Then, cells were resuspended in PBS/2% Fetal bovine serum (FBS) and stained with different combinations of the following antibodies: Anti-CD45 (PerCP: peridinin chlorophyll protein), Anti-CD3 (FITC: fluorescein isothiocyanate), Anti-CD8 (PE: phycoerythrin), Anti-CD4 (APC: allophycocyanin), Anti-CD11b (FITC: fluorescein isothiocyanate), Anti Ly6G (APC: allophycocyanin) and Anti-Ly6C (PeCy7: phycoerythrin Cy7). Before analysis, CountBright absolute counting beads (Life Technologies, Grand Island, NY) were added to quantify each cell population.

Histopathological analyses Lung lobes were removed from mice 24 and 48 hpi, fixed in 4% paraformaldehyde and embedded in paraffin. Then, paraffin blocks were sliced with a conventional microtome and stained with haematoxylin & eosin. Images were captured on an Olympus (Shinjuku, Tokyo, Japan) BX51 light microscope at 209 magnification. Blinded histopathological analyses were performed by a pathologist and the score criteria are listed in the Supplementary material (Table S1).

Quantitative RT-PCR RNA was extracted from lungs using the SV Total RNA Isolation System (Promega, Madison, WI), following the manufacturer’s instructions. Retrotranscription and amplification steps of the real-time PCR were performed using TaqMan RNA-to-Ct 1-step kit (Applied Biosystems, Foster City, CA, USA). Levels of mRNA were determined using TaqMan probes for the following cytokines: IFN-c, IL-6, IL-1b, IL-10 and TNF-a (Applied Biosystems). The b2-microglobulin gene was used as endogenous housekeeping control. Data were analysed with the DDCt comparative method of STEPONE software (Applied Biosystems), comparing all groups to the WT uninfected group. 102

ELISA Protein levels of IL-10, TNF-a, IL-6, IL-1b and IFN-c were measured at 24 and 48 hpi in BALF and lung tissue. Briefly, lung tissue was recovered and homogenized in PBS, treated with proteinase inhibitors (cOmplete ULTRA Tablets, Mini, EDTA-free; Roche, Basel, Switzerland) as suggested by the manufacturer. Each sample was diluted at a final concentration of 100 mg/ml of lung tissue, centrifuged at 9000 g/10 min at 4°C and supernatants were stored at 80°C until analyses. IL-10, TNF-a, IL-6, IL-1b, (OptEIA ELISA Sets; Becton Dickinson, Franklin Lakes, NJ) and IFN-c (ELISA Ready-Set-Go!; eBioscience, San Diego, CA) assays were performed according to the instructions provided by the manufacturer.

In vivo TNF-a neutralization The IL-10 / mice were divided into four groups: control treated group (Etanercept + THYE), infected treated group (Etanercept + S. pneumoniae), control untreated group (PBS + THYE) and infected untreated group (PBS + S. pneumoniae). One day before infection, 200 lg/500 ll of Etanercept was administered daily intraperitoneally, as previously described,27 while untreated mice received PBS. The efficiency of TNF-a neutralization was evaluated by ELISA in BALF at 48 hpi.

Statistical analyses One-way analysis of variance (ANOVA) test (or Kruskal– Wallis) followed by a Tukey multiple comparison test (Dunn test) were performed to analyse Il10 mRNA, IL-10 protein levels and TNF-a neutralization assays. A Holm–Sidak multiple comparison test was performed to evaluate the cell infiltration on TNF-a neutralization assays. A t-test and two-way ANOVA, followed by Tukey’s or Sidak multiple comparison tests, were performed to analyse mRNA determination. A t-test and two-way ANOVA followed by Tukey’s or Sidak multiple comparison tests were also performed to analyse the following parameters: protein quantification of TNF-a, IFN-c, IL-1b and IL-6; total protein quantification in BALF; histopathological score; cellular infiltrate and bacterial loads. Survival curves were compared using log-rank test. In all cases a P value < 005 was considered statistically significant. All comparisons were calculated using the GRAPHPAD PRISM 6.0c software for Macintosh (GraphPad software, San Diego, CA).

Results S. pneumoniae infection promotes increased IL-10 production in the lungs of mice Mice were intranasally infected with 3 9 107 CFU of S. pneumoniae D39, and 24 and 48 hpi the transcription ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

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Figure 1. S. pneumoniae infection induces IL-10 production in lungs at 48 hr post-infection (hpi) (a) Wild-type (WT) mice were intranasally infected with 3 9 107 CFU of S. pneumoniae D39, 24 and 48 hpi total lung RNA and proteins were obtained. Quantification of Il10 mRNA was performed by quantitative PCR, using Taqman probes. The b2-microglobulin gene was used as endogenous control. Results are relative to uninfected WT mice. *P < 005 by Kruskal–Wallis test with a posteriori Dunn multiple comparison test. (b) IL-10 in lungs was detected before infection and at 24 and 48 hpi in WT mice by the Mouse ELISA Duoset (R&D Systems, Minneapolis, MN). *P < 005 by one-way analysis of variance test with a posteriori Tukey multiple comparison test.

of Il10 gene in the lungs was determined by quantitative PCR. As shown in Fig. 1(a), Il10 mRNA was 4-fold higher at 24 hpi and 20-fold higher at 48 hpi in infected mice than in uninfected animals. Further, IL-10 protein levels were also measured in lungs at 24 and 48 hpi. In these experiments a significant increase of this cytokine at 48 hpi was detected (Fig. 1b). As expected, Il10 mRNA was not detected in lungs of IL-10 / mice (data not shown). Therefore, our results show that IL-10 is increased in the lungs of mice infected by S. pneumoniae.

IL-10 deficiency increases mortality due to S. pneumoniae infection To determine the role of IL-10 in the regulation of disease severity and survival during S. pneumoniae infection, IL-10 / and WT mice were intranasally infected with various sub-lethal doses. When mice were infected with 3 9 105 CFU, both groups showed 80% survival, with no changes in body weight and limited clinical scores (Fig. 2a). When mice were infected with 3 9 106 CFU of S. pneumoniae, both groups presented similar survival rates, a decrease of 5–10% of body weight and a slightly more severe clinical score (Fig. 2b). However, when mice were infected with 3 9 107 CFU, all IL-10 / mice died at day 5 post-infection, whereas 30% of WT mice survived until the end of the experiment. Both groups lost 10% of their body weight at day one post-infection; however, on the following days, IL-10 / mice continued losing weight, in contrast to surviving WT mice, which showed a progressive weight recovery. Furthermore, IL-10 / infected mice showed a more severe clinical score than WT infected mice (Fig. 2c). These data suggest that IL-10 plays a key role in the modulation of the immune response against S. pneumoniae at high bacterial infective doses, improving the host survival rate. ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

IL-10 modulates lung expression of pro-inflammatory cytokines triggered by S. pneumoniae infection Because the most significant survival difference between WT and IL-10 / mice was observed with an infective dose equal to 3 9 107 CFU, mice were challenged with this amount of bacteria to evaluate the effects of IL-10 in the production of local pro-inflammatory cytokines. As shown in Fig. 3, uninfected WT and IL-10 / mice showed similar basal mRNA and protein levels for most pro-inflammatory cytokines analysed, with the exception of IL-1b, which is elevated in the lungs of uninfected IL-10 / mice (Fig. 3d). These results indicate that the absence of IL-10 per se does not seem to generate a proinflammatory state in lungs of uninfected mice. Upon infection, a 30-fold increase of Tnf-a mRNA was observed for both WT and IL-10 / infected mice at 24 hpi. However, while infected IL-10 / mice showed a sustained increment of Tnf-a mRNA in lungs and TNF-a protein in BALF at 48 hpi, WT mice showed a significant decrease of mRNA in lungs and protein in BALF (Fig. 3a). No significant changes for TNF-a protein were observed in the lungs. Further, while S. pneumoniae infection promoted a 50-fold increase of Ifn-c mRNA at 24 hpi in the lungs of IL-10 / mice, only a 10-fold increase was observed in infected WT mice. These data correlate with the higher amount of IFN-c protein detected in the lungs at 24 hpi in IL-10 / mice (Fig. 3b). In BALF, the opposite pattern was observed for IFN-c protein production 24 hpi, in which WT mice showed significantly more IFN-c than IL-10 / mice. However, at 48 hpi an equivalent IFN-c concentration was observed for both WT and IL-10 / mice. As for the case of IL-6, both WT and IL-10 / mice showed a significant increase of mRNA and protein levels at 24 and 48 hpi. However, WT mice showed higher Il6 mRNA in lungs at 24 hpi, whereas at 48 hpi both groups 103

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Figure 2. Interleukin-10 knock out mice (IL-10 / ) are more susceptible to suffer a severe S. pneumoniae infection in a dose-dependent manner. Wild-type (WT) and IL-10 / mice were intranasally infected with (a) 3 9 105, (b) 3 9 106 and (c) 3 9 107 CFU of S. pneumoniae D39, and survival rate (left panels), body weight (middle panels) and clinical score (right panels) were recorded on a daily basis for 10 days. For dead mice, maximum score value was considered. *P < 005, log rank test.

showed similar values. The higher Il6 mRNA amount observed at 24 hpi in WT mice correlates with the higher IL-6 concentration observed in the lung tissue at 24 hpi. However, at 48 hpi the total amount of IL-6 was equivalent for both WT and IL-10 / mice (Fig. 3c). Finally, similar to IL-6, the amount of Il1b mRNA was higher in WT mice than IL-10 / mice at 24 hpi. However, protein concentrations were not significantly different between infected WT and IL-10 / mice. In addition, IL-10 / mice showed a higher basal production of IL-1b than did WT mice. These data suggest that lack of IL-10 during an infection with S. pneumoniae leads to an altered TNF-a, IFN-c and IL-6 transcription and translation pattern. Importantly, the significantly higher amount of TNF-a observed in BALF of IL-10 / mice at 48 hpi suggests that these animals develop a major pro-inflammatory state in the airways after infection.

IL-10 absence causes severe lung histopathology after S. pneumoniae infection To determine whether the pro-inflammatory environment observed in lungs of IL-10 / infected mice translates 104

into lung tissue damage and inflammation, we performed histopathological analyses of lung tissue at 24 and 48 hpi. At 24 hpi, WT and IL-10 / infected mice displayed similar lung damage, evidenced by the equivalent histopathology scores (Fig. 4a, b); however, at 48 hpi IL-10 / infected mice presented a sustained inflammation and loss of alveolar architecture in every lobe, whereas WT infected mice showed less inflammation, partial structure recovery and a reduced histopathology score (Fig. 4a, b). In both groups, there was no significant presence of cellular infiltrate in the alveoli, only in the parenchyma. The levels of total proteins in BALF, which directly reflect the severity of airway damage due to cytokine secretion and cell destruction, were 5-fold higher at 24 hpi in both WT and IL-10 / infected groups than in their respective controls. At 48 hpi the levels of total proteins in BALF of IL-10 / infected mice remained equivalent, whereas in WT infected mice they returned close to basal levels (Fig. 4c). These results are consistent with the amount of the pro-inflammatory cytokines TNF-a observed for IL-10 / mice (Fig. 3a), supporting the idea that infection of IL-10 / mice with S. pneumoniae results in sustained inflammation in lung tissue over time, ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

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Figure 3. Pro-inflammatory cytokine expression in lungs of wild-type (WT) and interleukin-10-knock out mice (IL-10 / ) after S. pneumoniae infection. WT and IL-10 / mice were intranasally infected with 3 9 107 CFU of S. pneumoniae D39. RNA from lungs and proteins from lungs and bronchoalveolar lavage fluid (BALF) were obtained at 24 hr and 48 hr. Transcripts and protein quantification of (a) Tumour Necrosis Factor-a (TNF-a), (b) Interferon-c (IFN-c), (c) IL-6 and (d) IL-1b mRNA was performed by quantitative PCR, using Taqman probes and ELISA kits, respectively. In transcripts analyses, the the b2-microglobulin gene was used as endogenous control. The results are shown as relative expression, compared with WT uninfected mice (DDCT). Data show mean + SEM. *P < 005, Student’s t-test between IL-10 / and WT mice at each time-point; two-way analysis of variance test with a posteriori Tukey multiple comparison test was performed to test the expression change across time; (a) P < 005 between uninfected and 24 hr post-infection (hpi); (c) P < 005 between 24 hpi and 48 hpi and; (b) P < 005 between uninfected and 48 hpi.

whereas WT mice show an important inflammatory response 24 hpi that decreases at 48 hpi.

IL-10 deficiency sustains infiltration of neutrophil in lungs upon S. pneumoniae infection To identify the cellular types infiltrating the lungs of IL-10 / mice after infection, we performed flow cytometry analyses of lung tissues at 24 and 48 hpi. Gating strategies for flow cytometry analyses of lungs and BALFs are shown in the Supplementary material (Fig. S1). We evaluated absolute numbers of neutrophils (CD45+, CD11b+, ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

Ly6Cmed, Ly6G+), inflammatory monocytes (CD45+, CD11b+, Ly6C+, Ly6Glow) and Ly6C myeloid cells (CD45+, CD11b+, Ly6C , Ly6G ), in lungs and BALFs (Fig. 5, see Supplementary material, Fig. S2). At 24 hpi, both infected groups showed a significant infiltration of neutrophils, a slight infiltration of inflammatory monocytes and reduced amounts of Ly6C myeloid cells in lungs (Fig. 5a–c). In BALF, an equivalent infiltration of neutrophils, inflammatory monocytes and Ly6C myeloid cells was observed in both IL-10 / and WT mice (see Supplementary material, Fig. S2). Although at 48 hpi both IL-10 / and WT infected mice showed a reduction 105

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in the number of neutrophils infiltrating the lungs, IL10 / mice showed a significantly higher amount of neutrophils in lungs than WT mice (Fig. 5a). The levels of inflammatory monocytes and Ly6C myeloid cells were equivalent in lungs as well as in BALFs of both groups at 48 hpi (Fig. 5a–b; see Supplementary material, Fig. S2). Given that T cells play an important role in the modulation of the immune response against S. pneumoniae,1,12,13,25,28 we quantified CD4+ and CD8+ T cells in lungs and BALF after infection. No significant differences were observed in the number of these cells at 24 and 48 hpi (see Supplementary material, Fig. S3a, b). Together, these results show that after a S. pneumoniae infection the production of IL-10 is crucial for the proper modulation of neutrophil recruitment to the lung parenchyma, but it does not have a detectable effect on CD4+ or CD8+ T cells recruitment (see Supplementary material, Fig. S3a, b). These results suggest that an excessive amount of neutrophils in lungs is the main feature of IL-10 / mice infected with S. pneumoniae, which could account for higher levels of TNF-a production in airways and higher mortality.

Bacterial loads are reduced in tissues derived from infected IL-10 / mice Our data suggest that high amounts of inflammatory cells in IL-10 / infected mice would be the main cause of death; but another factor that might contribute to death 106

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Figure 4. Lung damage after S. pneumoniae infection is more severe in interleukin-10knock out mice (IL-10 / ). Wild-type (WT) and IL-10 / mice were intranasally infected with 3 9 107 CFU of S. pneumoniae D39. At 24 and 48 hr post-infection (hpi) lungs and bronchoalveolar lavage fluid (BALF) were recovered. Lungs were removed, embedded in paraffin, 5-lM lung sections were stained with haematoxylin & eosin. (a) Representative lung tissue photographies observed in an optical microscope at 20 9 magnification. (b) Lung histopathological score (c) and total proteins in BALF were measured at 24 and 48 hpi, using the Bradford methodology. Data shown mean + SEM *P < 005, Student’s t-test between IL-10 / and WT mice at each timepoint; two-way analysis of variance tests with a posteriori Tukey multiple comparison test were performed to test the expression change across time; (a) P < 005 between uninfected and 24 hpi; (c) P < 005 between 24 hpi and 48 hpi and; (b) P < 005 between uninfected and 48 hpi.

is bacterial dissemination and sepsis development. To evaluate the dissemination of S. pneumoniae from lungs, bacterial loads in spleen, brain and blood were measured at 24 and 48 hpi. IL-10 / and WT infected mice presented equivalent levels of bacterial load in lung, brain and spleen at 24 hpi (Fig. 6a–d). However, bacterial loads increased from 24 to 48 hpi in WT mice, whereas they remained constant in IL-10 / infected mice. These results suggest that the exacerbated inflammatory response observed in IL-10 / mice allows more efficient bacterial clearance and limited bacterial dissemination, but leads to death due to inflammation. WT mice instead cannot control bacterial growth and dissemination, suggesting that WT mice die due to sepsis.

TNF-a neutralization does not prevent neutrophil recruitment to lungs and mortality of IL-10 / mice after S. pneumoniae infection As TNF-a was present in high levels in the BALF of IL10 / infected mice at 48 hpi (Fig. 3a), we evaluated whether TNF-a neutralization could reduce the severity of the disease caused by S. pneumoniae. Two hundred micrograms of Etanercept (a chimeric molecule that neutralizes TNF-a) were administered intraperitoneally daily, starting 1 day before infection. After infection, IL10 / treated mice showed 3-fold less TNF-a secretion in BALF (Fig. 7a). However, these mice did not show ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

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105

105 Neutrophils

+

Ly6C inflammatory monocytes

0

10

10

Neutrophils

101

Ly6C– myeloid cells

0

0

Ly6G

5

+

10

IL-10–/–

48 hpi

Neutrophils

Neutrophils 105

Ly6C inflammatory monocytes Ly6C– myeloid cells

101

0

100

100

101

102

103

104

105

100 101 102 103 104 105

Ly6C (b) 107 * 106 105

NS a a

c

104 103 102 Uninfected 24 hpi

48 hpi

(c) WT mice IL-10–/– mice

107 106

NS NS

NS

c

a

105 104 103 102

Uninfected 24 hpi

48 hpi

WT mice IL-10–/– mice

Lung CD45+CD11b+Ly6C–Ly6G– myeloid cells (cells/ml)

WT mice IL-10–/– mice

NS

Lung CD45+CD11b+Ly6C+Ly6Glow inf. monocytes (cells/ml)

Lung CD45+CD11b+Ly6CmedLy6G+ neutrophils (cells/ml)

(a)

107 106

NS NS NS

105 104 103 102

Uninfected 24 hpi

48 hpi

Figure 5. Interleukin-10 knock out mice (IL-10 / ) have higher numbers of neutrophils in lungs after S. pneumoniae infection. As a gate strategy, singlets were selected from total FSC/SSC events; then total CD45+ leucocytes were gated and Cd11b+ cells were selected. Finally, Ly6G and Ly6C antibodies were used to discriminate between neutrophils, Inflammatory monocytes and Ly6C- myeloid cells (see Supplementary material, Fig. S1). At 24 and 48 hr post-infection (hpi), absolute numbers of (a) neutrophils (CD45+ CD11b+ Ly6G+ Ly6Cmed), (b) inflammatory monocytes (CD45+ CD11b+ Ly6Glow Ly6C+) and (c) Ly6C- myeloid cells (CD45+ CD11b+ Ly6G Ly6C ) were measured by flow cytometry in lungs of wild-type (WT) and IL-10 / , using Countbright absolute counting beads (Life Technology, Grand Island, NY). Data shown are mean + SEM. *P < 005, Student’s t-test between IL-10 / and WT mice at each time-point; two-way analysis of variance test with a posteriori Tukey multiple comparison test were performed to test the expression change across time; (a) P < 005 between uninfected and 24 hpi; (c) P < 005 between 24 hpi and 48 hpi and; (b) P < 005 between uninfected and 48 hpi.

better survival than IL-10 / Etanercept untreated mice after infection (Fig. 7b). Further, both IL-10 / Etanercept treated and untreated mice showed similar amounts of neutrophils in lungs after infection (Fig. 7c). However, only IL-10 / Etanercept treated mice presented a high infiltration of inflammatory monocytes in lungs, which resulted in an infiltration ratio of neutrophils/ monocytes close to 2, whereas IL-10 / untreated mice showed ratio close to 4 (Fig. 7e). As we expected, no differences were observed in neutrophils and inflammatory monocytes recruited to BALF (Fig. 7d), or CD4+ and CD8+ T cells recruited to lungs (not shown). These observations support the notion that the unbalanced recruitment/migration of neutrophils to lungs of IL-10 / mice is the main cause of death after S. pneumoniae infection. Further, these data indicate that TNF-a ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

secretion does not contribute significantly to neutrophil recruitment to or migration from the lungs (Fig. 7c). Nevertheless, these data suggest that high amounts of TNF-a in airways prevent the recruitment of inflammatory monocytes (Fig. 5a), whereas reduced amounts of TNF-a improve the recruitment of these cells to the lungs (Fig. 7c). However, in the experimental setting tested here, this effect of Etanercept did not improve survival of IL-10 / mice after S. pneumoniae infection.

Discussion Interleukin-10 is an anti-inflammatory cytokine that modulates the pro-inflammatory response against pathogenic microorganisms, ensuring a proper microbial clearance with a limited tissue immunopathology.14–16 In some cases, 107

~aloza et al. H. F. Pen WT mice 106

NS

(b) *

106

a CFU/mg brain

CFU/mg lungs

(a)

IL-10–/– mice

104

102

100

NS 102

a

100 24 hpi

48 hpi *

(c) 10

* 104

24 hpi (d)

6

48 hpi *

108

10

CFU/ml blood

CFU/mg spleen

* 4

NS 10

2

106

a

104 102 100

100 24 hpi

48 hpi

24 hpi

lack of IL-10 promotes a better microbial clearance and better survival rates,29 although in others the lack of IL-10 leads to higher mortality due to uncontrolled inflammation with no effect in bacterial clearance.20 Here we demonstrate that pneumococcal pneumonia infection induces the production of IL-10 in lungs, which is necessary to control inflammation, neutrophil infiltration and migration, tissue damage and host survival when mice are infected with a high dose of bacteria (3 9 107 CFU). We also demonstrate that, despite the fact that absence of IL-10 improves bacterial clearance and reduces bacterial dissemination, IL-10 / mice showed an altered proinflammatory environment after infection by S. pneumoniae, characterized by significantly higher transcription and translation of TNF-a in the airway and IFN-c in lung tissue. This observation is related to increased neutrophil accumulation in lungs at 48 hpi, severe lung immunopathology and in higher mortality rate, which were not prevented when TNF-a was neutralized. Mechanistically, TNFa neutralization did not affect neutrophil recruitment or migration, supporting the notion that the inability to control the influx of neutrophils to the lungs, as well as inflammation in the absence of IL-10, is the main cause of death in these mice. When pro-inflammatory cytokine production was analysed, we found that IL-10 / infected mice developed a different cytokine pattern compared with WT mice, characterized by higher transcription levels of Tnf-a and Ifn-c in lungs, a higher concentration of IFNc in lungs, a high concentration of TNF-a in BALF and lower amounts of IL-6 and IL-1b in lung tissue. In mice, IL-6 and IL-1b are necessary for the proper development of Th17 immune responses. The Th17 response 108

48 hpi

Figure 6. Interleukin-10 knock out mice (IL10–/–) show reduced S. pneumoniae dissemination after infection. Bacterial loads of (a) lungs, (b) brain, (c) spleen and (d) blood were quantified at 24 and 48 hr post-infection (hpi) by seeding organs onto blood agar, as described in the Materials and methods. *P < 005, Student’s t test between IL-10 / and wild-type (WT) mice at each time-point; two-way analysis of variance test with a posteriori Sidak’s multiple comparison test were performed to test the expression change across time, (a) P < 005 between 24 hpi and 48 hpi.

is induced by IL-6, IL-1b and TNF-a, which are secreted at early times after infection by alveolar macrophages and epithelial cells.1 These signals promote the subsequent activity of cd T cells and CD4+ T cells, which contribute to the Th17 response.1,11,12 In contrast, IFN-c is thought to inhibit Th17 immune responses.30 As the effect of the presence or absence of IL-10 in the proinflammatory pattern was observed at 48 hpi, without significant changes in the frequency of T cells, it is likely that IL-10 is required for the proper secretion of cytokines by innate and epithelial cells, which will in turn promote a Th17 response profile.30 Our data show that the lack of IL-10 during S. pneumoniae infection, leads to higher levels of IFN-c and TNF-a, which might inhibit the development of Th17 immune responses and lead to an exacerbated innate inflammation. Another important difference in the inflammatory response of IL-10 / mice after S. pneumoniae infection is the sustained production of TNF-a in airways. Mostly macrophages and T cells produce this cytokine, but neutrophils and B cells are also able produce TNF-a.31 TNF receptors, present in several cell types, recognize this cytokine and promote different effects such as cell death/survival, cell differentiation, proliferation/migration, increased tissue permeability and leucocyte adhesion.31 An important feature of TNF-a is the capacity to induce apoptosis under certain conditions,32 as for example when the nuclear factor-jB pathway is blocked.33 IL-10 can inhibit nuclear factor-jB (NF-kB) by the induction of p50/p50 homodimerization and its nuclear translocation;34 therefore in the presence of IL-10, TNF-a will induce cell apoptosis. When IL-10 is absent, TNF-a will not induce apoptosis, instead it will promote the expresª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

Role of IL-10 in S. pneumoniae infection 1500

(b) 100

*

IL-10–/– + VEH (n = 8) IL-10–/– + ETA (n = 8)

80 Survival (%)

1000

500

60 40 20

0 Inf

Uninf

Vehicle

103

2

2

10

+



Ly6C myeloid cells

10

4

10

Neutrophils

ETA

10

10 10

Ly6G

+

Ly6C inflammatory monocytes – Ly6C myeloid cells

100 10

1

10

2

103 10

10 10

4

100 0 10 101 102 103 104 104

Neutrophils

3

+

Ly6C inflammatory monocytes – Ly6C myeloid cells

Neutrophils

103

102

2

10 +

10

+

Ly6C inflammatory Ly6C inflammatory 101 monocytes monocytes – – Ly6C myeloid cells Ly6C myeloid cells

101 0 0

10

1

10

2

10

3

10

4

10

100 100 101 102 103 104

Ly6C

106 5

10

+

104 3

10

Uninf

Inf

Uninf

Neutrophils/inf. monocytes ratio

(e)

Inf

Etanercept

Vehicle

107

*

6

10

5

10

104

108

NS

106 104 102

+

NS

107

Lung + + + low CD45 CD11b Ly6C Ly6G inf. monocytes (cells/ml)

Ly6C Lung CD45 CD11b+Ly6CmedLy6G+ neutrophils (cells/ml)

0 2 3 100 101 10 10 104

100 2 3 100 101 10 10 104

4

102 +

Ly6C inflammatory monocytes – 101 Ly6C myeloid cells

10

+

0

10 10

Ly6C inflammatory Ly6C inflammatory 1 monocytes monocytes 10 – – Ly6C myeloid cells Ly6C myeloid cells

Neutrophils

103

2

1

Neutrophils

+

1

10

48 hpi

3

102

2

8

4

10

Neutrophils

10

103

103

6

Uninfected

100 4 3 1 2 100 10 10 10 10

100 4 3 1 2 100 10 10 10 10 4

4

4

10

ETA

10

Ly6C inflammatory monocytes 101

1

(d)

Neutrophils

BALF CD45 CD11b+Ly6CmedLy6G+ neutrophils (cells/ml)

PBS

104

Neutrophils

103 10

2

Days

48 hpi

Uninfected 104

0

Etanercept

Ly6G

(c)

0

Inf

PBS

Uninf

3

10

Inf

Uninf Vehicle

Uninf

Inf

100 Uninf Vehicle

Inf

Uninf

Inf

Etanercept

BALF + + + low CD45 CD11b Ly6C Ly6G inf. monocytes (cells/ml)

pg/ml of TNF-α BALF

(a)

108

NS

106 10

4

102 100 Uninf Vehicle

Inf

Uninf

Inf

Etanercept

Etanercept

*

8 6 4 2 0 Uninf Vehicle

Inf

Uninf

Inf

Etanercept

Figure 7. Etanercept neutralizes tumour necrosis factor-a (TNF-a) in vivo but does not improve the survival rate of interleukin-10 knock out mice (IL-10 / ) after an infection with S. pneumoniae. (a) IL-10 / mice were treated intraperitoneally daily with 200 lg/500 ll of Etanercept from 1 day before the infection. At day 0, mice were intranasally infected with 3 9 107 CFU of S. pneumoniae D39, at 48 hr post-infection (hpi) TNF-a neutralization was measured in bronchoalveolar lavage fluid (BALF) by ELISA, *P < 005 by one-way analysis of variance test with Tukey’s multiple comparisons test. (b) Survival rate was followed by 10 days. *P < 005, log rank test. Neutrophils (CD45 + CD11b + Ly6G+ Ly6Cmed) and inflammatory monocytes (CD45 + CD11b + Ly6Glow Ly6C+) absolute numbers in (c) lungs and (d) BALF were measured at 48 hpi. (e) Neutrophils/Ly6C+ monocytes ratio in lungs at 48 hpi. *P < 005, one-way analysis of variance test and with Holm–Sidak multiple comparisons test.

sion of genes encoding pro-inflammatory cytokines, increasing immunopathology.32 Our data show that WT mice produce higher amounts of IL-10 after 48 hr of infection with S. pneumoniae, which correlate with reduced amounts of neutrophils infiltrating lungs (Fig. 5a,b) and a reduced histopathology ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

score at that time after infection (Fig. 4b). Probably, because IL-10 could promote TNF-a-mediated cell apoptosis at 48 hpi, IL-10 / mice would be unable to clear excessive cellular infiltrate by this mechanism at this time post infection. As a result, IL-10 / mice would be rendered deficient at removing inflammatory cells from 109

Role of IL-10 in S. pneumoniae infection mouse mortality after a challenge with Pseudomonas aeruginosa.48 Therefore, the levels of IL-10 must be tightly regulated during an infection in order to ensure a controlled recruitment of inflammatory cells and an adequate clearance of the pathogen. In summary, our study describes how the absence of IL-10 in the early stages of pneumococcal pneumonia renders the host more susceptible to death, due to excessive neutrophil recruitment into the lung and production of pro-inflammatory cytokines, which lead to excessive inflammation. Therefore, we conclude that IL10 plays a key role in the regulation of the development of an immune response against S. pneumoniae. This knowledge will be useful to explore new therapeutic strategies and ultimately prevent severe infections caused by this pathogen.

Acknowledgements This study was supported by the following grants: FONDO NACIONAL DE CIENCIA Y TECNOLOGIA DE CHILE (FONDECYT numbers 1140010, 1110604, 1100971, 1131012, 1150862 and 1110397), Millennium Institute on Immunology and Immunotherapy P09/016-F and Grant ‘Nouvelles Equipes-nouvelles thematiques’ from the La Region Pays De La Loire. HFP, PAN, NMD and MJP are supported by the Comisi on Nacional de Investigaci on Cientıfica y Tecnol ogica (CONICYT). AMK is a Chaire De  La Region Pays De La Loire, Chercheur Etranger D’excellence, France.

Disclosures The authors have not financial conflict of interest.

References 1 Nieto PA, Riquelme SA, Riedel CA, Kalergis AM, Bueno SM. Gene elements that regulate Streptococcus pneumoniae virulence and immunity evasion. Curr Gene Ther 2013; 13:51–64. 2 Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008; 6:288–301. 3 O’Brien KL, Wolfson LJ, Watt JP, et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet 2009; 374: 893–902. 4 Drijkoningen JJ, Rohde GG. Pneumococcal infection in adults: burden of disease. Clin Microbiol Infect 2014; 20(Suppl. 5):45–51. 5 Jackson ML, Neuzil KM, Thompson WW, Shay DK, Yu O, Hanson CA, Jackson LA. The burden of community-acquired pneumonia in seniors: results of a populationbased study. Clin Infect Dis 2004; 39:1642–50. 6 Beiter K, Wartha F, Albiger B, Normark S, Zychlinsky A, Henriques-Normark B. An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr Biol 2006; 16:401–7. 7 Hyams C, Camberlein E, Cohen JM, Bax K, Brown JS. The Streptococcus pneumoniae capsule inhibits complement activity and neutrophil phagocytosis by multiple mechanisms. Infect Immun 2010; 78:704–15. 8 Janoff EN, Rubins JB, Fasching C, Charboneau D, Rahkola JT, Plaut AG, Weisser JN. Pneumococcal IgA1 protease subverts specific protection by human IgA1. Mucosal Immunol 2014; 7:249–56.

ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

9 Feikin DR, Kagucia EW, Loo JD, et al. Serotype- specific changes in invasive pneumococcal disease after pneumococcal conjugate vaccine introduction: a pooled analysis of multiple surveillance sites. PLoS Med 2013; 10:e1001517. 10 Fitzwater SP, Chandran A, Santosham M, Johnson HL. The worldwide impact of the seven-valent pneumococcal conjugate vaccine. Pediatr Infect Dis J 2012; 31: 501–8. 11 van der Poll T, Opal SM. Pathogenesis, treatment, and prevention of pneumococcal pneumonia. Lancet 2009; 374:1543–56. 12 Kirby AC, Newton DJ, Carding SR, Kaye PM. Pulmonary dendritic cells and alveolar macrophages are regulated by gammadelta T cells during the resolution of S. pneumoniae-induced inflammation. J Pathol 2007; 212:29–37. 13 Kadioglu A, Coward W, Colston MJ, Hewitt CR, Andrew PW. CD4-T-lymphocyte interactions with pneumolysin and pneumococci suggest a crucial protective role in the host response to pneumococcal infection. Infect Immun 2004; 72:2689–97. 14 Duell BL, Tan CK, Carey AJ, Wu F, Cripps AW, Ulett GC. Recent insights into microbial triggers of interleukin-10 production in the host and the impact on infectious disease pathogenesis. FEMS Immunol Med Microbiol 2012; 64:295–313. 15 Mege JL, Meghari S, Honstettre A, Capo C, Raoult D. The two faces of interleukin 10 in human infectious diseases. Lancet Infect Dis 2006; 6:557–69. 16 Cyktor JC, Turner J. Interleukin-10 and immunity against prokaryotic and eukaryotic intracellular pathogens. Infect Immun 2011; 79:2964–73. 17 Greenberger MJ, Strieter RM, Kunkel SL, Danforth JM, Goodman RE, Standiford TJ. Neutralization of IL-10 increases survival in a murine model of Klebsiella pneumoniae. J Immunol 1995; 155:722–9. 18 Wolfe DN, Karanikas AT, Hester SE, Kennett MJ, Harvill ET. IL-10 induction by Bordetella parapertussis limits a protective IFN-c response. J Immunol 2010; 184:1392–400. 19 Metzger DW, Salmon SL, Kirimanjeswara G. Differing effects of interleukin-10 on cutaneous and pulmonary Francisella tularensis live vaccine strain infection. Infect Immun 2013; 81:2022–7. 20 Slight SR, Monin L, Gopal R et al. IL-10 restrains IL- 17 to limit lung pathology characteristics following pulmonary infection with Francisella tularensis live vaccine strain. Am J Pathol 2013; 183:1397–404. 21 Jeong ES, Won YS, Kim HC, Cho MH, Choi YK. Role of IL-10 deficiency in pneumonia induced by Corynebacterium kutscheri in mice. J Microbiol Biotechnol 2009; 19:424– 30. 22 van der Poll T, Marchant A, Keogh CV, Goldman M, Lowry SF. Interleukin-10 impairs host defense in murine pneumococcal pneumonia. J Infect Dis 1996; 174:994–1000. 23 Rijneveld AW, de Vos AF, Florquin S, Verbeek JS, van der Poll T. CD11b limits bacterial outgrowth and dissemination during murine pneumococcal pneumonia. J Infect Dis 2005; 191:1755–60. 24 van Zoelen MA, Schouten M, de Vos AF, Florquin S, Meijers JC, Nawroth PP, Bierhaus A, van der Poll T. The receptor for advanced glycation end products impairs host defense in pneumococcal pneumonia. J Immunol 2009; 182:4349–56. 25 Weber SE, Tian H, Pirofski LA. CD8+ cells enhance resistance to pulmonary serotype 3 Streptococcus pneumoniae infection in mice. J Immunol 2011; 186:432–42. 26 Mook-Kanamori B, Geldhoff M, Troost D, van der Poll T, van de Beek D. Characterization of a pneumococcal meningitis mouse model. BMC Infect Dis 2012; 12:71. 27 Christadoss P, Goluszko E. Treatment of experimental autoimmune myasthenia gravis with recombinant human tumor necrosis factor receptor Fc protein. J Neuroimmunol 2002; 122:186–90. 28 Mertens J, Fabri M, Zingarelli A, et al. Streptococcus pneumoniae serotype 1 capsular polysaccharide induces CD8CD28 regulatory T lymphocytes by TCR crosslinking. PLoS Pathog 2009; 5:e1000596. 29 Jeong ES, Lee KS, Heo SH, Seo JH, Choi YK. Modulation of immune response by interleukin-10 in systemic Corynebacterium kutscheri infection in mice. J Microbiol 2012; 50:301–10. 30 Mills KH. Induction, function and regulation of IL-17-producing T cells. Eur J Immunol 2008; 38:2636–49. 31 Bradley JR. TNF-mediated inflammatory disease. J Pathol 2008; 214:149–60. 32 Flusberg DA, Sorger PK. Surviving apoptosis: life-death signaling in single cells. Trends Cell Biol 2015; doi: 10.1016/j.tcb.2015.03.003. 33 Walczak H. Death receptor-ligand systems in cancer, cell death, and inflammation. Cold Spring Harb Perspect Biol 2013; 5:a008698. 34 Driessler F, Venstrom K, Sabat R, Asadullah K, Schottelius AJ. Molecular mechanisms of interleukin-10-mediated inhibition of NF-jB activity: a role for p50. Clin Exp Immunol 2004; 135:64–73. 35 Williams AE, Jose RJ, Brown JS, Chambers RC. Enhanced inflammation in aged mice following infection with Streptococcus pneumoniae is associated with decreased IL-10 and augmented chemokine production. Am J Physiol Lung Cell Mol Physiol 2015; 308: L539–49.

111

Role of IL-10 in S. pneumoniae infection mouse mortality after a challenge with Pseudomonas aeruginosa.48 Therefore, the levels of IL-10 must be tightly regulated during an infection in order to ensure a controlled recruitment of inflammatory cells and an adequate clearance of the pathogen. In summary, our study describes how the absence of IL-10 in the early stages of pneumococcal pneumonia renders the host more susceptible to death, due to excessive neutrophil recruitment into the lung and production of pro-inflammatory cytokines, which lead to excessive inflammation. Therefore, we conclude that IL10 plays a key role in the regulation of the development of an immune response against S. pneumoniae. This knowledge will be useful to explore new therapeutic strategies and ultimately prevent severe infections caused by this pathogen.

Acknowledgements This study was supported by the following grants: FONDO NACIONAL DE CIENCIA Y TECNOLOGIA DE CHILE (FONDECYT numbers 1140010, 1110604, 1100971, 1131012, 1150862 and 1110397), Millennium Institute on Immunology and Immunotherapy P09/016-F and Grant ‘Nouvelles Equipes-nouvelles thematiques’ from the La Region Pays De La Loire. HFP, PAN, NMD and MJP are supported by the Comisi on Nacional de Investigaci on Cientıfica y Tecnol ogica (CONICYT). AMK is a Chaire De  La Region Pays De La Loire, Chercheur Etranger D’excellence, France.

Disclosures The authors have not financial conflict of interest.

References 1 Nieto PA, Riquelme SA, Riedel CA, Kalergis AM, Bueno SM. Gene elements that regulate Streptococcus pneumoniae virulence and immunity evasion. Curr Gene Ther 2013; 13:51–64. 2 Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008; 6:288–301. 3 O’Brien KL, Wolfson LJ, Watt JP, et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet 2009; 374: 893–902. 4 Drijkoningen JJ, Rohde GG. Pneumococcal infection in adults: burden of disease. Clin Microbiol Infect 2014; 20(Suppl. 5):45–51. 5 Jackson ML, Neuzil KM, Thompson WW, Shay DK, Yu O, Hanson CA, Jackson LA. The burden of community-acquired pneumonia in seniors: results of a populationbased study. Clin Infect Dis 2004; 39:1642–50. 6 Beiter K, Wartha F, Albiger B, Normark S, Zychlinsky A, Henriques-Normark B. An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr Biol 2006; 16:401–7. 7 Hyams C, Camberlein E, Cohen JM, Bax K, Brown JS. The Streptococcus pneumoniae capsule inhibits complement activity and neutrophil phagocytosis by multiple mechanisms. Infect Immun 2010; 78:704–15. 8 Janoff EN, Rubins JB, Fasching C, Charboneau D, Rahkola JT, Plaut AG, Weisser JN. Pneumococcal IgA1 protease subverts specific protection by human IgA1. Mucosal Immunol 2014; 7:249–56.

ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

9 Feikin DR, Kagucia EW, Loo JD, et al. Serotype- specific changes in invasive pneumococcal disease after pneumococcal conjugate vaccine introduction: a pooled analysis of multiple surveillance sites. PLoS Med 2013; 10:e1001517. 10 Fitzwater SP, Chandran A, Santosham M, Johnson HL. The worldwide impact of the seven-valent pneumococcal conjugate vaccine. Pediatr Infect Dis J 2012; 31: 501–8. 11 van der Poll T, Opal SM. Pathogenesis, treatment, and prevention of pneumococcal pneumonia. Lancet 2009; 374:1543–56. 12 Kirby AC, Newton DJ, Carding SR, Kaye PM. Pulmonary dendritic cells and alveolar macrophages are regulated by gammadelta T cells during the resolution of S. pneumoniae-induced inflammation. J Pathol 2007; 212:29–37. 13 Kadioglu A, Coward W, Colston MJ, Hewitt CR, Andrew PW. CD4-T-lymphocyte interactions with pneumolysin and pneumococci suggest a crucial protective role in the host response to pneumococcal infection. Infect Immun 2004; 72:2689–97. 14 Duell BL, Tan CK, Carey AJ, Wu F, Cripps AW, Ulett GC. Recent insights into microbial triggers of interleukin-10 production in the host and the impact on infectious disease pathogenesis. FEMS Immunol Med Microbiol 2012; 64:295–313. 15 Mege JL, Meghari S, Honstettre A, Capo C, Raoult D. The two faces of interleukin 10 in human infectious diseases. Lancet Infect Dis 2006; 6:557–69. 16 Cyktor JC, Turner J. Interleukin-10 and immunity against prokaryotic and eukaryotic intracellular pathogens. Infect Immun 2011; 79:2964–73. 17 Greenberger MJ, Strieter RM, Kunkel SL, Danforth JM, Goodman RE, Standiford TJ. Neutralization of IL-10 increases survival in a murine model of Klebsiella pneumoniae. J Immunol 1995; 155:722–9. 18 Wolfe DN, Karanikas AT, Hester SE, Kennett MJ, Harvill ET. IL-10 induction by Bordetella parapertussis limits a protective IFN-c response. J Immunol 2010; 184:1392–400. 19 Metzger DW, Salmon SL, Kirimanjeswara G. Differing effects of interleukin-10 on cutaneous and pulmonary Francisella tularensis live vaccine strain infection. Infect Immun 2013; 81:2022–7. 20 Slight SR, Monin L, Gopal R et al. IL-10 restrains IL- 17 to limit lung pathology characteristics following pulmonary infection with Francisella tularensis live vaccine strain. Am J Pathol 2013; 183:1397–404. 21 Jeong ES, Won YS, Kim HC, Cho MH, Choi YK. Role of IL-10 deficiency in pneumonia induced by Corynebacterium kutscheri in mice. J Microbiol Biotechnol 2009; 19:424– 30. 22 van der Poll T, Marchant A, Keogh CV, Goldman M, Lowry SF. Interleukin-10 impairs host defense in murine pneumococcal pneumonia. J Infect Dis 1996; 174:994–1000. 23 Rijneveld AW, de Vos AF, Florquin S, Verbeek JS, van der Poll T. CD11b limits bacterial outgrowth and dissemination during murine pneumococcal pneumonia. J Infect Dis 2005; 191:1755–60. 24 van Zoelen MA, Schouten M, de Vos AF, Florquin S, Meijers JC, Nawroth PP, Bierhaus A, van der Poll T. The receptor for advanced glycation end products impairs host defense in pneumococcal pneumonia. J Immunol 2009; 182:4349–56. 25 Weber SE, Tian H, Pirofski LA. CD8+ cells enhance resistance to pulmonary serotype 3 Streptococcus pneumoniae infection in mice. J Immunol 2011; 186:432–42. 26 Mook-Kanamori B, Geldhoff M, Troost D, van der Poll T, van de Beek D. Characterization of a pneumococcal meningitis mouse model. BMC Infect Dis 2012; 12:71. 27 Christadoss P, Goluszko E. Treatment of experimental autoimmune myasthenia gravis with recombinant human tumor necrosis factor receptor Fc protein. J Neuroimmunol 2002; 122:186–90. 28 Mertens J, Fabri M, Zingarelli A, et al. Streptococcus pneumoniae serotype 1 capsular polysaccharide induces CD8CD28 regulatory T lymphocytes by TCR crosslinking. PLoS Pathog 2009; 5:e1000596. 29 Jeong ES, Lee KS, Heo SH, Seo JH, Choi YK. Modulation of immune response by interleukin-10 in systemic Corynebacterium kutscheri infection in mice. J Microbiol 2012; 50:301–10. 30 Mills KH. Induction, function and regulation of IL-17-producing T cells. Eur J Immunol 2008; 38:2636–49. 31 Bradley JR. TNF-mediated inflammatory disease. J Pathol 2008; 214:149–60. 32 Flusberg DA, Sorger PK. Surviving apoptosis: life-death signaling in single cells. Trends Cell Biol 2015; doi: 10.1016/j.tcb.2015.03.003. 33 Walczak H. Death receptor-ligand systems in cancer, cell death, and inflammation. Cold Spring Harb Perspect Biol 2013; 5:a008698. 34 Driessler F, Venstrom K, Sabat R, Asadullah K, Schottelius AJ. Molecular mechanisms of interleukin-10-mediated inhibition of NF-jB activity: a role for p50. Clin Exp Immunol 2004; 135:64–73. 35 Williams AE, Jose RJ, Brown JS, Chambers RC. Enhanced inflammation in aged mice following infection with Streptococcus pneumoniae is associated with decreased IL-10 and augmented chemokine production. Am J Physiol Lung Cell Mol Physiol 2015; 308: L539–49.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Flow cytometry gating strategy used to identify neutrophils, inflammatory monocytes, Ly6C- myeloid cells and T cells in lungs and bronchoalveolar lavage fluid of mice infected with Streptococcus pneumoniae. Figure S2. Interleukin-10-deficient (IL-10–/–) and wildtype (WT) mice have similar amounts of neutrophils, inflammatory monocytes and myeloid Ly6C- cells in bronchoalveolar lavage fluid after Streptococcus pneumoniae infection. Figure S3. Interleukin-10-deficient (IL-10–/–) and wildtype (WT) infected mice have similar infiltration pattern of T cells in lungs and bronchoalveolar lavage fluid after Streptococcus pneumoniae infection. Table S1. Histopathological score of lung tissue.

ª 2015 John Wiley & Sons Ltd, Immunology, 146, 100–112

Interleukin-10 plays a key role in the modulation of neutrophils recruitment and lung inflammation during infection by Streptococcus pneumoniae.

Streptococcus pneumoniae is a major aetiological agent of pneumonia worldwide, as well as otitis media, sinusitis, meningitis and sepsis. Recent repor...
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