Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 16, 2015). doi:10.1152/ajplung.00141.2014

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Enhanced inflammation in aged mice following infection with Streptococcus

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pneumoniae is associated with decreased IL-10 and augmented chemokine

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production.

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Andrew E. Williams, Ricardo J. José, Jeremy S. Brown and Rachel C. Chambers.

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Centre for Inflammation and Tissue Repair, Rayne Institute, University College London

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(UCL), WC1E 6JF, United Kingdom.

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Running title: Ageing associated inflammation.

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Corresponding author.

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Rachel Chambers ([email protected])

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Centre for Inflammation and Tissue Repair, Rayne Institute, University College London

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(UCL), WC1E 6JF, United Kingdom.

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Phone: +44 (0)2076796978

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Fax: +44 (0)2076796973

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Author contribution.

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AEW designed and performed experiments, prepared and edited manuscript.

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RJJ performed experiments and edited manuscript.

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JSB edited manuscript and obtained funding.

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RCC prepared and edited manuscript and obtained funding.

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Copyright © 2015 by the American Physiological Society.

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Abstract.

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Streptococcus pneumoniae is the most common cause of severe pneumonia in the elderly.

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However, the impact of ageing on the innate inflammatory response to pneumococci is

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poorly defined. We compared the innate immune response in old versus young adult mice

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following infection with S. pneumoniae. The accumulation of neutrophils recovered from

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bronchoalveolar lavage fluid and lung homogenates was increased in aged compared to

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young adult mice, although bacterial outgrowth was similar in both age groups, as were

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markers of microvascular leak. Aged mice had similar levels of IL-1β, TNF, IFN-γ, IL-17 and

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G-CSF following S. pneumoniae infection, compared to young mice, but increased levels of

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CXCL9, CXCL12, CCL3, CCL4, CCL5, CCL11 and CCL17. Moreover, levels of IL-10 were

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significantly lower in aged animals. Neutralization of IL-10 in infected young mice was

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associated with increased neutrophil recruitment but no decrease in bacterial outgrowth.

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Furthermore, IL-10 neutralization resulted in increased levels of CCL3, CCL5 and CXCL10.

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We conclude that ageing is associated with enhanced inflammatory responses following S.

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pneumoniae infection as a result of a compromised immunomodulatory cytokine response.

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Keywords.

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Pneumonia, neutrophil, chemokine, inflammation, ageing.

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Introduction.

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The bacterium Streptococcus pneumoniae is the leading cause of community acquired

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pneumonia (CAP) and the most frequent cause of infectious disease-related admittance to

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the intensive care unit (32; 33). It has been estimated that S. pneumoniae affects 5-6 million

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people in the USA each year, resulting in 1 million hospitalizations. Furthermore, lower

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respiratory tract infections account for the highest number of infectious disease-related

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deaths worldwide, of which S. pneumoniae is the predominant etiological agent (4). Age is a

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major risk factor for S. pneumoniae infections, with the majority of cases occurring in the

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very young or in the elderly (51). Pneumonia often results in the development of septicemia,

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acute respiratory distress syndrome (ARDS), and respiratory failure, and these

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complications are also more common in the elderly (11; 22). However, why the frequency of

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pneumococcal infection is higher and the disease more severe in the elderly remains poorly

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defined.

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Even though current vaccine regimens have improved clinical outcome in children, age-

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related immunosenescence hinders vaccine efficacy and enhances disease susceptibility

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(33; 37). Immunosenescence is a well-recognized phenomenon in the elderly that has been

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directly related to an increased risk of infectious disease (1; 19). Age-related alterations in

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the adaptive immune system include an increased production of autoantibodies and

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decreased production of pneumococcal-specific antibodies (39; 45), decreased T cell

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proliferative responses (12; 31) and a diminished T cell repertoire (35). These factors are

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likely to enhance infectious disease susceptibility, exemplified by a higher incidence of

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infection to a broad spectrum of pathogens, such as influenza virus and S. pneumoniae (6;

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8; 19), by the reactivation of tuberculosis (23; 38), and a decrease in vaccine efficacy (39).

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In contrast to the changes in adaptive immunity, age-related alterations in innate immunity

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are less well defined. Defects in innate immune cell function maybe associated with age,

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although innate leukocyte numbers in the elderly reflect those of young adults. 3

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Immunosenescence may affect specific functional aspects of innate immune cells (16), such

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as altered cytokine production and poor responses to inflammatory stimuli (3; 9; 19),

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including defective TLR2 activation (3). The innate immune system plays a pivotal role in

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controlling host-pathogen interactions and preventing invasive disease following S.

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pneumoniae colonization of the nasopharynx (27). In particular, alveolar macrophages and

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recruited neutrophils are thought to be important factors for the control of pneumococcal

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invasion (26).

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The present study aims to address whether the innate immune response of aged mice

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differs from that of young adult mice in response to S. pneumoniae infection. We compared

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the inflammatory response following infection with S. pneumoniae in young adult mice with

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aged mice, and characterized the neutrophil and macrophage response in bronchoalveolar

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lavage (BAL) fluid and whole lung homogenates. The levels of pro-inflammatory cytokines,

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the immunomodulatory cytokine IL-10 and several chemokines, were measured. Due to

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significant differences in IL-10 production between young and aged mice, the effect on

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pneumococci-induced inflammation, cytokine production and chemokine levels were

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assessed following neutralization of IL-10 in young mice. Taken together, these data

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demonstrate that the innate immune response to S. pneumoniae in aged mice is associated

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with heightened inflammation and diminished IL-10-mediated immunoregulation. These

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findings have important implications for our understanding of the mechanisms involved in the

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dysregulation of innate immune responses during ageing.

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Materials and Methods

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Bacterial strain and infection model

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The D39 strain (serotype 2) of S. pneumoniae was used for all experiments, a virulent strain

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which is fully infective in mouse lungs. S. pneumoniae was cultured on Columbia agar

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(Oxoid, UK) containing 5% defibrinated horse blood (TSC Biosciences, UK) at 37º C or in

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Todd-Hewitt medium (Oxoid, UK) containing 5% yeast extract (Oxoid, UK). Growth in liquid

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medium was assessed by measuring optical density (OD) at 580 nm with a

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spectrophotometer. Bacteria were grown to mid-log phase and numerated by performing

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serial dilutions and counting colony forming units (cfu) on Columbia agar.

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Young adult female Balb/c mice (6-7 weeks old) or aged Balb/c mice (24 months old) were

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housed in identical specified pathogen free conditions, in accordance with the Home Office,

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UK regulations. Mice were infected intra-nasally with 50 μl 5 × 106 cfu of S. pneumoniae,

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strain D39 (serotype 2), under general anesthesia (isofluorane). Mice were euthanized 2, 4,

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8, 24 or 48 h following infection, depending on the study, and blood, bronchoalveolar lavage

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fluid (BAL fluid) and whole lungs were removed post mortem.

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Cell isolation and analysis

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BAL fluid was obtained by making an excision in the trachea and inserting a plastic cannula.

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The lungs were inflated three times with 750 μl sterile saline and the fluid withdrawn. 100 μl

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BAL fluid was used for bacterial colony counts. The BAL fluid was centrifuged at 300 g for 5

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min in order to collect the cell pellet. The supernatant was removed and stored at -20º C until

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analysed. The cell pellet was re-suspended in 500 μl PBS (Gibco, UK) and total leukocytes

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counted using a hemocytometer. Cytospin preparations were performed on 200 μl of the cell

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suspension, centrifuged onto glass slides at 700 rpm for 7 min. Cytospins were then stained

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with a rapid Romanowski stain (Thermo-Fischer, UK). Differential cell counts were

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performed using optical microscopy.

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Whole lungs were homogenized into a single cell suspension. Lungs were passed through a

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40 μm nylon filter (BD Biosciences, USA) with 6 ml of sterile PBS (Gibco, UK). 100 μl of the

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neat cell suspension was used for bacterial colony counts. The cell suspension was

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centrifuged at 300 g for 5 min in order to obtain a cell pellet. The lung supernatant was

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removed and stored at -20º C until used. The cell pellet was re-suspended in 1 ml of sterile

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PBS and counted using a hemocytometer.

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Bacterial culture

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Bacteria recovered from BAL fluid, whole lung homogenates and blood were counted by

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performing serial dilutions and cultured on Columbia agar containing 5% defibrinated horse

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serum as described. Cultures were incubated at 37º C for 18 h. Colony forming units (cfu)

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were counted and numbers transformed into log10 values.

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Flow cytometric analysis of lung cells.

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Leukocyte recruitment into lung tissue was analyzed by flow cytometry. 100 μl containing 2 ×

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105 cells from homogenized lung tissue were used for each stain. Cells were centrifuged at

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400 g for 2 min and re-suspended in staining medium containing PBS, 4% bovine serum

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albumin (Sigma, UK) and 0.1% sodium azide (Sigma, UK). The following antibodies specific

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to mouse surface markers were used: Ly6G-PE (clone 1A8, BD Biosciences, USA), CD11b-

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APC (BD Biosciences, USA) and CD11c-PerCP (BD Biosciences). Isotype controls (BD

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Biosciences, USA) and fluorescent minus one (FMO) control stains were performed. Flow

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cytometry analysis was performed on a FACSVerse (BD Biosciences, USA) and analysed

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using FlowJo software (Tree Star, USA). The total leukocyte population was gated based on

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FSc and SSc and neutrophils were analyzed based on their specific expression of Ly6G and

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CD11b (Ly6GhighCD11bhiCD11clo population). Differential cell counts were calculated based

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on the percentage of neutrophils within the total leukocyte population.

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Protein, cytokine and chemokine analysis 6

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BAL fluid was used to analyse endothelial-epithelial barrier permeability and surrogate

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markers of coagulopathy. Endothelial-epithelial barrier permeability was measured by

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analyzing the amount of serum albumin recovered in BAL fluid with an ELISA (Bethyl

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Laboratories, USA). Coagulopathy was assessed by measuring thrombin-anti-thrombin

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(TAT) complexes (EnymeResearch, USA) or activated protein C (APC) (Antibodies Online,

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USA) recovered from BAL fluid by ELISA. Neutrophil elaste (ELA2) was measured by ELISA

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(Caltag-MedSystems) and the ELA2/neutrophil ratio for BAL fluid calculated. The level of

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cytokines and chemokines were analyzed in supernatant derived from whole lung

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homogenates. The pro-inflammatory cytokines IL-1β, TNF, IFN-γ, IL-17 and G-CSF, and the

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immunomodulatory cytokine IL-10 were all measured by ELISA (Peprotech). Chemokines

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were measured using a multi-array ELISA platform (Qiagen, USA).

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IL-10 neutralisation in vivo.

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Mice were intra-nasally infected with 50 μl 5 × 106 cfu of S. pneumoniae strain D39

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(serotype 2). In addition, the inoculum contained 10 μg of functional grade anti-IL-10

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antibody per mouse (eBiosciences, UK) or poly-Ig control (Peprotech, UK). This antibody

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has been shown to inhibit 50% the biological activity of IL-10 (at a concentration of 4 ng/ml of

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antibody in the presence of 1 ng/ml antigen), as reported by the manufacturers. Animals

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were euthanized 2, 4, 8, 24 or 48 h following challenge, as described, and BAL fluid, lung

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tissue and blood removed post mortem.

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Statistical analaysis.

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Data were analyzed using one-way analysis of variance (ANOVA) with Neuman-Keuls post-

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test to compare all groups, or with 2-way ANOVA across time courses, or with a Student’s t-

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test when comparing two groups. A p value of less than 0.05 was considered significant.

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Results

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Enhanced inflammation in aged mice following S. pneumoniae infection.

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In order to assess whether there are age-related differences in the innate immune response

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to S. pneumoniae, young adult mice (6-7 weeks) or aged mice (24 months) mice were

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challenged with 5 × 106 cfu of the D39 S. pneumoniae strain and euthanized 24 h later.

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Infection with S. pneumoniae resulted in an acute inflammatory response in both young and

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aged animals. The total number of cells recovered from the BAL fluid (Figure 1A) and from

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whole lung homogenates (Figure 1B) of aged mice were higher than those recovered from

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young mice. Differential cell counts revealed that the number of neutrophils isolated from the

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BAL fluid and lung homogenates were significantly higher in aged mice compared to young

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mice following infection (Figure 1C and D), indicating that aged mice mount a heightened

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innate inflammatory response to S. pneumoniae. Furthermore, although macrophage

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numbers were slightly increased in aged mice following infection, macrophage numbers at

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baseline were similar between young and aged mice in both BAL fluid and lung

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homogenates (Figure 1E and F).

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We next determined the effect of age on the ability of the host to control bacterial outgrowth.

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The bacterial cfu recovered from the BAL fluid of aged and young mice were comparable

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(Figure 1G), as was the bacterial burden based on cfu recovered from whole lung

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homogenates (Figure 1H). In addition, neither young mice nor aged mice developed

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septicemia after 24 h, as no bacteria were recovered from blood (data not shown).

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Considering aged mice had significantly increased neutrophil numbers in the BAL fluid

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compared to young mice, but no corresponding decrease in bacterial cfu, we next assessed

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neutrophil function by measuring neutrophil elastase production in BAL fluid of infected mice.

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Aged mice had a decrease in the ELA2/neutrophil ratio compared to young mice (fugure 1I),

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indicating reduced capacity to produce ELA2 in aged neutrophils.

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Aged mice do not exhibit increased barrier permeability.

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We next aimed to investigate whether aged mice display evidence of increased endothelial-

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epithelial barrier permeability or coagulopathy. Serum albumin levels, recovered from BAL

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fluid, increased following S. pneumoniae infection in both young and aged mice, although

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there was no difference between the two age groups (Figure 2A), indicating that increased

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neutrophil influx does not result in increased barrier permeability. In order to assess potential

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differences in coagulation, APC and TAT were measured in BAL fluid. There was no

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difference in APC levels in young compared to old mice (Figure 2B). However, aged mice

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had a small but significant increase in BAL fluid TAT levels (Figure 2C), indicating that aged

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animals exhibit an enhanced pro-coagulant state.

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Decreased IL-10 production in aged mice.

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In order to determine whether the heightened inflammatory response in aged mice was

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associated with an increase in pulmonary cytokine levels, we measured several pro-

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inflammatory cytokines and the immunomodulatory cytokine IL-10 in lung tissue. Although

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the levels of IL-1β, TNF and G-CSF increased following infection with S. pneumoniae in both

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young and aged mice, no differences between the two age groups were detected (Figure

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3A-C). The levels of IFN-γ and IL-17 did not significantly increase following infection,

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although baseline levels were higher in aged mice compared to young (Figure 3D and E). In

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contrast, baseline levels of IL-10 were higher in young compared to aged mice (Figure 3F),

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while young mice exhibited increased IL-10 levels following infection with S. pneumoniae.

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Taken together, these data suggest that the heightened inflammatory response in aged mice

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is associated with reduced IL-10 levels rather than an increased pro-inflammatory cytokine

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response.

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Elevated chemokine production in aged mice.

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We next measured the levels of several chemokines in lung tissue. Aged animals exhibited

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enhanced production of a number of chemokines following infection with S. pneumoniae 9

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compared to young animals (Figure 4). Baseline levels of these chemokines were low or

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undetectable in PBS instilled mice regardless of age. Increased production of CCL3 (MIP-

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1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL11 (Eotaxin), CCL17 (TARC), CXCL9 (MIG) and

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CXCL12 (SDF-1) were detected in aged mice compared to young mice (Figure 4),

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suggesting that the increased neutrophil margination into the lungs of aged mice may be

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associated with increased levels of several chemokines. Of note, although the known

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neutrophil chemoattractants, CXCL1 (KC) and CCL2 (MCP-1), increased following infection,

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the levels were similar in aged versus young mice (Figure 4A and I).

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IL-10 modulates the inflammatory response to S. pneumoniae.

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We next aimed to determine the relationship between IL-10 and the modulation of the

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inflammatory response following S. pneumoniae infection. Young adult mice were infected

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with 5 × 106 cfu of S. pneumoniae with or without 10 μg of a neutralizing anti-IL-10 antibody

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given within the intra-nasal challenge volume. Compared to IgG control antibody treated

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mice, those that received anti-IL-10 had an increase in total cell (Figure 5A and B) and

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neutrophil numbers (Figure 5C and D) recovered from the BAL fluid and lung tissue,

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although macrophage numbers were similar (Figure 5E and F). These data indicate that IL-

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10 modulates the magnitude of the neutrophil influx into the lungs of S. pneumoniae

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challenged mice.

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In order to assess whether the anti-IL-10 neutralizing antibody was inducing inflammation

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itself, mice were treated with anti-IL-10 antibody or control antibody via the intransal route in

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the asbcence of concurrent bacterial infection. No differences in the total cell or neutrophil

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numbers (figure 5G) were observed 2, 4 or 8 h after treatment. Furthermore, no differences

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in IL-1β production between control antibody and anti-IL-10 antibody were detected (figure

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5H) indicating an absence of inflammation.

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No decrease in bacterial outgrowth in the absence of IL-10. 10

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We next measured S. pneumoniae recovered from BAL fluid and lung homogenates

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following treatment with anti-IL-10 antibody. No difference in bacterial counts was observed

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in recovered BAL fluid (Figure 6A) or lungs (Figure 6B) at 4, 24 or 48 h compared to control

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mice following infection with S. pneumoniae, suggesting that the control of bacterial

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outgrowth is independent of IL-10. Bacteremia was not detected at any of the time points

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(data not showm), measured by cfu counts from the blood. Furthermore, neutralization of IL-

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10 at the point of infection with S. pneumoniae resulted in elevated neutrophil and

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macrophage numbers in the BALF up to 48 h post infection compared to control antibody

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treated mice (Figure 6 C and D), despite a significant decrease in bacterial numbers within

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the BAL fluid and lung at this time point.

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In addition, IL-10 treatment did not alter barrier permeability as determined by serum

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albumin levels in recovered BAL fluid (Figure7A) 24 hours after infection with S.

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pneumoniae. No differences in the levels of IL-1β or TNF were detected following anti-IL-10

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treatment compared to IgG controls (Figure 7B and C), suggesting that IL-10 is not involved

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in modulating these cytokines, consistent with the data obtained for young versus aged

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mice.

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IL-10 regulates CCL3, CCL5 and CXCL10 production.

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In order to determine whether IL-10 has a modulatory effect on chemokine production, the

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levels of several chemokines were assessed following infection. Of all the chemokines

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tested, treatment with anti-IL-10 neutralizing antibody only led to a significant increase in the

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pulmonary levels of CCL3, CCL5 and CXCL10 (Figure 8). Increased expression of CCL3

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and CCL5 following anti-IL-10 treatment is consistent with the enhanced chemokine

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response in aged mice compared to young mice (Figure 4). These data indicate that IL-10

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modulates the expression of a subset of chemokines associated with the innate immune

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response following infection with S. pneumoniae.

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Discussion.

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S. pneumoniae is the leading cause of community acquired pneumonia, accounting for more

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than 1 million hospitalizations in the US and 1 million childhood deaths worldwide each year

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(2). Although vaccination has been successful at reducing disease burden in infants,

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immunocompromised patients are still at risk of developing invasive disease (20; 40). In

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particular, the elderly population (>65 years of age) represents a substantial and growing

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group of individuals who are at particular risk of developing life-threatening pneumococcal

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pneumonia (6; 29). As global demographics shift towards an expanding elderly population,

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understanding host-pathogen interactions in ageing individuals is therefore paramount.

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In the present study we specifically focused on the acute phase of S. pneumoniae infection

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and compared the innate immune response in aged versus young mice. Aged mice exhibited

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an enhanced inflammatory response to the pneumococcus, as demonstrated by higher

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numbers of total leukocytes, neutrophils and macrophages recovered from the BAL fluid and

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lung tissue following challenge. The enhanced neutrophil response may be of particular

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relevance to the pathogenesis of disease due to the pivotal role that these cells play in host

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defense (30). The increased macrophage response in aged mice further suggests a

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dysregulated inate immune response in these animals. Moreover, alveolar macrophages

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often marginate and adhere to the epithelium during acute inflammatory reactions (25), as

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seen in young mice in this study, a process that may be defective in aged mice. Indeed,

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macrophages from aged animals have previously been shown to be less responsive to

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inflammatory signals (24). Alternatively, the increased number of macrophages in aged mice

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may be due to the underlying pro-inflammatory state, which may result in increases in both

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resident and recruited macrophage popualtions. However, it is accepted that there are some

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limitations in using aged mice to model responses in elderly humans, as not all age-related

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changes can be modeled accurately. For example, aged mice are not exposed to a life time

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of environmental insults and do not possess many of the comorbidities associated with aged

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humans. Despite these caveats this model does represent a useful means of studying acute 12

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inflammation in the lung in order to understand how the ageing process affects innate

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immune responses to bacterial pathogens.

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Taking into account the enhanced recruitment of neutrophils in aged mice, we next assessed

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how this would affect bacterial clearance. Despite higher neutrophil numbers, pneumococcal

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clearance from the lungs was not enhanced in aged compared to young mice. This may be

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explained by the finding that the relationship between neutrophil recruitment and pathogen

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clearance is not linear. An increase in neutrophil numbers therefore does not necessarily

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mean that the pneumococcus will be cleared more effectively. This may also reflect

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discrepencies between neutrophil accumulation and normal function, whereby a decline in

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bacterial killing may further compromise host defence (18). Neutrophil function has

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previously been demonstrated to be compromised in aged mice, so that bacteria are cleared

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less efficiently. Indeed, neutrophils from aged mice have a reduced phagocytic killing

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potential (46) and are less capable of generating reactive oxygen species (53). Neutrophils

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from aged humans also have reduced phagocytic activity (13), which may be related to lower

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CD16 expression (5), or defective Fcγ-receptor-mediated uptake of opsonized microbes

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(15). In addition, elderly neutrophils are less effective at phagocytosing and killing

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pneumococci opsonized by immunoglobulin (46). In the present study we further

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demonstrate that ELA2 production per neutrophil is compromised in aged mice, which could

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explain the unaffected bacterial counts, despite increased cell numbers, in our model. It may

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be the case that aged individuals recruit more neutrophils into sites of infection in order to

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compensate for a reduced antimicrobial capacity.

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Due to the potential for enhanced tissue damage as a result of excessive neutrophilia, we

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next assessed endothelial-epithelial barrier permeability in young and aged mice. Although

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aged mice recruited more neutrophils, endothelial-epithelial barrier permeability, as

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measured by serum albumin levels in the BAL fluid, was not correspondingly increased.

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There is some debate as to whether neutrophil migration directly contributes to barrier

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permeability, while it has been suggested that the activation of neutrophils is required for 13

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bystander tissue damage to occur (10; 47; 54). Although barrier integrity was compromised

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following S. pneumoniae challenge, this was not directly associated with the observed

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neutrophil influx. This apparent paradoxical observation may be accounted for by the poor

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functionality of neutrophils derived from aged compared to young animals (46; 53). Indeed,

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the release of proteinases, such as matrix metalloproteinases, ELA2 and other granule-

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derived enzymes, have been shown to contribute, and even be required for, the tissue

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damage associated with neutrophil recruitment into the lung (55-57). We further found that

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BAL fluid TAT complex levels were elevated in aged compared to young mice, suggesting

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that ageing is associated with an enhanced pulmonary pro-coagulant state in these animals.

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Coagulation markers in the circulation have been shown to increase in patients hospitalized

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with CAP, although no correlations were made with clinical outcome (28; 34).

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In order to account for the heightened recruitment of leukocytes into the lungs following S.

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pneumoniae challenge in aged mice, we next analyzed the levels of several pro-

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inflammatory cytokines. We found that IL-1β, TNF, G-CSF, IFN-γ and IL-17 levels were

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similar in young and old mice. However, IL-10 levels were significantly lower in aged mice

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compared to young mice at baseline levels and in response to S. pneumoniae infection. IL-

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10 is an immunosuppressive cytokine capable of modulating both innate and adaptive

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immune responses. Initially recognized as a regulatory cytokine produced by T cells, it is

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now known to be expressed by a variety of cells, including macrophages, neutrophils and

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epithelial cells, and has diverse modulatory effects on the immunopathology induced by

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infectious microorganisms (42). However, ageing is associated with chronic inflammation,

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termed inflammaging (14), which may modulate the phenotype of both immune and non-

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immune cells. However, the cell-specific changes in propensity for IL-10 production remain

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poorly defined and findings have often been paradoxical. For example, the T cell system in

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aged humans is biased toward an inflammatory Th17 phenotype under basal condition but

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shifts towards a regulatory T cell phenotype upon stimulation, indicating an imbalance exists

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between pro- and anti-inflammatory immune responses (43). There may also be tissue14

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specific differences in the aging immune system, as exemplified by decreased production of

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TGF-β and IL-10 in the gut mucosa (41). The reduced levels of IL-10 in aged mice may

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therefore suggest a lack of immunoregulation in the lungs following bacterial infection,

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thereby contributing to enhanced recruitment of neutrophils and inflammatory disease.

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In order to further evaluate the contribution of IL-10 on the acute phase of S. pneumoniae

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infection, we inhibited IL-10 with a specific neutralizing antibody. This treatment caused a

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similar result in young mice to that observed in aged mice, with increased neutrophil

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recruitment to BAL and lungs, similar levels of pro-inflammatory cytokines, and an increase

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in some chemokines (CCL3 and CCL5). These data indicate that IL-10 plays an important

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functional role in regulating the recruitment of neutrophils into infected lungs. This

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heightened recruitment occured despite a significant reduction in bacterial burden by 48 h

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post infection, further suggesting a lack of resolution of inflammation in these aged animals.

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However, bacterial lung burden was not altered after anti-IL-10 treatment, despite the

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corresponding increase in neutrophils, comparable to the findings in aged mice following S.

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pneumoniae infection.This suggests that bacterial clearance may be independent of IL-10,

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whereas the extent of neutrophilic inflammation is dependent on IL-10. Previous reports

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have been somewhat conflicting regarding the role of IL-10 in host defense against S.

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pneumoniae. Some report that IL-10 actually impairs host defense against S. pneumoniae

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(50; 52), while others report that IL-10 is protective in models of septic peritonitis (49).

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Further studies have shown that the absence of IL-10 results in immunopathology

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independent of pathogen load (17; 21), an observation which the current study supports.

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These findings and the data reported herein indicate that the balance between host defense

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and the regulation of excessive inflammation is highly complex.

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Chemokines play an instrumental role in directing leukocyte trafficking into sites of

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inflammation. CXCL1 (KC) is considered to be the archetypal neutrophil chemoattractant,

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which is the murine functional homologue of human CXCL8 (IL-8). Even though aged mice 15

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recruited higher numbers of neutrophils than young mice, there were no differences in the

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levels of CXCL1. This suggests that excessive neutrophil influx in aged mice may be

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dependent on other chemokines. Of the chemokines tested, CCL3, CCL4, CCL5, CCL11,

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CXCL9 and CXCL12 were all elevated in the lungs of aged mice, suggesting they might be

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involved in mediating the heightened inflammatory response of aged mice. Furthermore,

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inhibiting IL-10 resulted in an increase in CCL3 and CCL5, two of the chemokines that were

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increased in aged mice following pneumococcal infection. This would suggest that IL-10

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modulates the expression of these chemokines following challenge with S. pneumoniae and

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that these chemokines may be important in mediating the enhanced inflammatory response

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of aged mice. IL-10 has previously been shown to modulate neutrophil and eosinophil

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recruitment in a mouse model of ovalbumin (OVA) sensitization (58). In a mouse model of

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LPS-induced inflammation, IL-10 was shown to regulate the expression of CCL3 and CXCL2

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and decrease neutrophil accumulation into airspaces (44). This likely reflects the dynamic

415

relationship between various pro- and anti-inflammatory mediators during an innate immune

416

response to a bacterial infection. The way in which the immune system responds to a

417

microorganism is therefore a critical factor that determines the severity of disease (36).

418 419

Although the current study suggests that aged mice have a heightened innate immune

420

response to bacterial infection, several questions regarding the role of IL-10 in this process,

421

and the functional capacity of aged neutrophils, remain. For example, restoring the levels of

422

IL-10 in aged animals to similar levels in young adult mice would provide important

423

information regarding the role of this cytokine in regulating inflammation during the ageing

424

process. Extending these studies to strains of mice that are more susceptible to S.

425

pneumoniae infection, such as C57BL/6 or CD1, may also help to delineate how IL-10

426

regulates the host response to this pathogen. Further studies on the microbicidal capacity of

427

aged neutrophils would also be useful to determine whether these cells have reduced

428

functionality in aged mice. In addition, studies designed to address the effect of IL-10 on

16

429

neutrophil recruitment and on the regulation of the pulmonary chemokine network, would be

430

informative and should form the basis of future research activity in this area.

431 432

To summarize, we provide evidence that aged mice respond to S. pneumoniae infection with

433

a heightened pulmonary innate immune response compared to young adult mice,

434

represented by increased neutrophil recruitment, even though this did not seem to affect

435

lung protective immunity. We further show that IL-10 production in response to S.

436

pneumoniae is reduced in aged mice and is associated with increased chemokine

437

production. These data support the notion that loss of IL-10 production with age, during an

438

innate immune response within the lung, is associated with enhanced inflammatory

439

responses to this common pathogen. This study also supports the immune dysregulation

440

theory of ageing, whereby normal regulatory components of the immune system, in this case

441

IL-10, are lost, resulting in an underlying pro-inflammatory state (7). This study also supports

442

the concept of inflammaging (14), whereby age-related diseases are often associated with

443

chronic inflammation. A similar mechanism might underlie the greater degree of morbidity

444

and mortality reported in elderly patients during S. pneumoniae pneumonia compared to

445

younger subjects (6; 48); although we accept there are physiological differences between

446

aged mice and elderly humans. As global demographics shift towards an aged population,

447

understanding the mechanisms underlying the fine control of innate immune responses to S.

448

pneumoniae during ageing may reveal new molecular targets that could improve outcome in

449

elderly individuals.

450 451

17

452 453

Funding statement.

454

This work was supported by funding from the Rosetrees Trust (Grant No: A420 to R.C.C.

455

and A.E.W.) and the NIHR UCLH Biomedical Research Centre (Grant No: BRC/76HI/RC to

456

R.C.C and A.E.W.), The Wellcome Trust (Grant No: 097216/Z/11/Z to R.J.J and R.C.C.);

457

and the NIHR UCLH Biomedical Research Centre (to J.S.B.).

458 459

Conflict of interest statement.

460

The authors can confirm that we have no conflicts of interest.

461 462 463

18

464

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26

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Figure Legends.

618

Figure 1. Differential inflammatory response between young and aged mice. Young

619

adult Balb/c mice (6-7 weeks of age, n=4-6 per group) or aged Balb/c mice (24 months of

620

age, n=3-5 per group) were intra-nasally challenged with 5 × 106 cfu S. pneumoniae strain

621

D39 (serotype 2) and euthanized 24 h later. Total cells were recovered from BAL fluid (A)

622

and lung tissue (B). Differential cell counts were performed on BAL fluid cytospin

623

preparations or flow cytometry analysis was performed on whole lung cell homogenates.

624

Analysis determined total neutrophil (C and D) and macrophages numbers (E and F) in BAL

625

fluid and lung tissue, respectively. Bacteria recovered from BAL fluid (G) and whole lung

626

homogenates (H) were cultured on Columbia agar containing 5% defibrinated horse serum.

627

Cultures were incubated at 37º C for 18 h. Cfu were counted and numbers transformed into

628

log10 values. Neutrophil elsatse-2 (ELA2) was measured in the BAL fluid of infected mice

629

and the ELA2/neutrophil number ratio calculated (I). Statistical analysis was performed using

630

an ANOVA with Neuman-Keuls post-test to compare all groups (**p