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Commonly invasive serotypes of Streptococcus pneumoniae trigger a reduced innate immune response compared… Burgess, Thomas S.T.; Hirschfeld, Aaron F.; Tyrrell, Gregory J.; Bettinger, Julie A.; Turvey, Stuart E. 2007

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S HO R T COMMUN I C AT I O NCommonly invasiveserotypesofStreptococcuspneumoniae triggera reduced innate immune response comparedwith serotypes rarelyresponsible for invasive infectionThomas S.T. Burgess1, Aaron F. Hirschfeld1, Gregory J. Tyrrell2, Julie A. Bettinger1 & Stuart E. Turvey11Department of Pediatrics, Division of Infectious and Immunological Diseases, BC Children’s Hospital and Child & Family Research Institute, University ofBritish Columbia, Vancouver, BC, Canada; and 2Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, CanadaCorrespondence: Stuart E. Turvey,Department of Pediatrics, Division ofInfectious and Immunological Diseases, BCChildren’s Hospital and Child & FamilyResearch Institute, Rm 371, 950 West 28Avenue, Vancouver, BC, Canada, V5Z 4H4.Tel.:11 604 875 2345 ext 5094; fax:11 604875 2226; e-mail: sturvey@cw.bc.caReceived 24 August 2007; revised 20 November2007; accepted 17 December 2007.First published online 30 January 2008.DOI:10.1111/j.1574-695X.2008.00382.xEditor: Jennelle KydKeywordsStreptococcus pneumoniae ; pneumococcus;innate immunity; TNF-a.AbstractAlthough there are more than 90 serotypes of Streptococcus pneumoniae (orpneumococcus), it is not understood why a small number of serotypes accountfor most invasive infections. To investigate the human innate immune responsetriggered by different pneumococcal serotypes, monocyte-derived macrophageswere exposed to a group of commonly and rarely invasive pneumococcal clinicalisolates and tumor necrosis factor (TNF)-a production was measured. Commonlyinvasive pneumococcal serotypes triggered significantly less TNF-a productionthan serotypes rarely responsible for invasive infection (Po 0.004). These dataindicate that one factor influencing the invasive potential of a pneumococcalserotype is the magnitude of innate immune-mediated TNF-a productiontriggered by exposure to the organism and suggest that the integrated hostresponse generated against commonly invasive pneumococcal serotypes may beless effective than the response directed against rarely invasive serotypes.Streptococcus pneumoniae, also known as pneumococcus, isa pathogen of global importance causing invasive infec-tions such as sepsis, meningitis, and pneumonia. Morethan 90 immunologically distinct serotypes of pneumococ-cus have been described, each varying in the structureof their polysaccharide capsule. However, the prevalencewith which the serotypes are recovered from patientswith invasive disease varies considerably, presumably be-cause some serotypes have a much greater propensity tocause invasive disease than others. International pneumo-coccal surveillance has demonstrated that 70–88% of in-vasive pneumococcal disease in young children was causedby serotypes now included in the seven-valent protein-conjugated vaccine formulation (4, 6B, 9V, 14, 18C, 19F,and 23F) (Henriques et al., 2000; Hausdorff et al., 2005;Bettinger et al., 2007). This finding is consistent withthe hypothesis that, regardless of ethnic and socioeconomicdifferences, ‘commonly invasive’ serotypes as a grouphave a high level of intrinsic virulence relative to otherserotypes.Emerging evidence suggests that innate immune mechan-isms and particularly Toll-like receptor (TLR) signalling arecritical for defense against pneumococcus. Indeed, bothhumans with inherited immunodeficiencies affecting innateimmunity (Picard et al., 2003) and mice with engineereddefects affecting innate immune recognition (Echchannaouiet al., 2002; Malley et al., 2003; Albiger et al., 2005) showenhanced susceptibility to invasive pneumococcal infection.Given that innate immunity provides the sophisticated firstline of defense against infection and empowers the subse-quent adaptive immune response, it follows that the abilityof the innate immune system to recognize different pneu-mococcal serotypes may influence which serotypes are mostcommonly responsible for invasive infections. In this study,it was sought to investigate how commonly invasive sero-types of pneumococcus interact with human monocyte-derived macrophages to trigger an innate immune response,compared with pneumococcal serotypes rarely responsiblefor invasive infection. Specifically, macrophage productionof the inflammatory cytokine tumor necrosis factor (TNF)-aFEMS Immunol Med Microbiol 53 (2008) 136–139c 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reservedwas quantified following exposure to clinical isolates ofpneumococcus. Monocyte-derived macrophages were stu-died as these are key innate immune cells encountered bybacteria during the evolution of an invasive infection, andthe production of the proinflammatory cytokine TNF-a wasquantified as TNF-a production is known to influence theoutcome following pneumococcal infection (Takashimaet al., 1997; Wellmer et al., 2001).Based on Canadian epidemiological data (Fig. 1), alibrary of ‘commonly invasive’ serotypes (14, 6B, 19F, 18C,23F, 4, 9V) was acquired that collectively account for 80% ofinvasive pneumococcal infections in Canadian children, and‘rarely invasive’ serotypes (37, 45, 7C, 9L, 24F) that as agroup only caused 0.1% of the cases of invasive disease (Fig.1). These pneumococcal serotypes were all isolated from theblood of patients experiencing an invasive pneumococcalinfection. Formal growth curves were established for eachS. pneumoniae serotype. For macrophage stimulation, pneu-mococcal serotypes were grown in brain-heart infusion(BHI) medium to the mid-log phase (A600 nm = 0.3), har-vested by centrifugation, killed in 70% ethanol (v/v) for 1 hon ice, washed again, and resuspended in lipopolysacchar-ide-free phosphate-buffered saline. Inhibition of bacterialreplication was necessary to control for host responses thatcould be attributed to differential growth rates of livebacteria in vitro rather than growth-independent differencesdue to the serotype alone. Ethanol killing was selected topreserve pneumolysin, an ethanol-stable but heat-labileTLR4 ligand (Malley et al., 2003).Human monocyte-derived macrophages were preparedby differentiating THP-1 cells (ATCC #TIB-202) toward amacrophage phenotype in the presence of phorbol myristateacetate (PMA) (Tsuchiya et al., 1982). THP-1 cells weregrown in RPMI 1640 medium supplemented with 10% (v/v)fetal calf serum, 2mM L-glutamine and 1mM sodiumpyruvate (Gibco). Forty-eight hours before bacterial stimu-lation, the THP-1 cells were differentiated with PMA(0.1mM) and after 24 h the cells were washed and allowedto rest in fresh medium. THP-1 (2 105) cells were incubated(in triplicate) in a flat-bottom 96-well plate with ethanol-killed pneumococci at varying multiplicities-of-infection(MOIs). After 24h, the supernatant was harvested and theTNF-a concentration was quantified by an enzyme-linkedimmunosorbent assay (ELISA) (eBioscience). Stimulationwith each serotype was performed at least twice in triplicate.An MOI of 200 bacteria to 1 THP-1 cell (i.e. MOI= 200 : 1)was demonstrated to optimally trigger secretion of TNF-awhile avoiding cytotoxicity as demonstrated by the lactatedehydrogenase (LDH) cytotoxicity assay (data not shown).When the ability of different pneumococcal serotypes totrigger innate immune-mediated TNF-a secretion by mono-cyte-derived macrophages was compared, commonly inva-sive pneumococcal serotypes induced significantly lessproinflammatory cytokine production than serotypes rarelyresponsible for invasive infection (commonly invasiveserotypes: mean TNF-a concentration = 369 pgmL1,SEM= 38 pgmL1, n= 42; rarely invasive serotypes: meanTNF-a concentration = 712 pgmL1, SEM= 50 pgmL1,n= 30. Po 0.004 by Student’s t-test. Fig. 2).These data suggest that a difference in innate immune-mediated cytokine production is likely to be one factorinfluencing the capacity of pneumococcal serotypes to causeinvasive infection. TNF-a secretion as a consequence ofinnate immune activation is known to orchestrate a varietyof early and late protective host responses against infection,including triggering the initial acute phase response, em-powering the adaptive immune response through matura-tion of dendritic cells, and activating endothelial cells toenhance effector leukocyte recruitment (Locksley et al.,2001). Hence, these data suggest that the integrated hostresponse generated against commonly invasive pneumococ-cal serotypes may be less effective than the response directedagainst rarely invasive serotypes. The observation thatcommonly invasive serotypes of pneumococcus induce lessFig. 1. Distribution of invasive pneumococcalserotypes in Canadian children, 1991–2004.From January 1991 to September 2004, theImmunization Monitoring Program, Active(IMPACT) has collected data on a total of 3528cases of invasive pneumococcal infection inchildren 0–17 years of age across Canada. Thedistribution of invasive pneumococcal serotypesamong IMPACT cases is shown. Serotypesmarked as ‘commonly invasive’ and ‘rarelyinvasive’ were used in this study.FEMS Immunol Med Microbiol 53 (2008) 136–139 c 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved137Pneumococcus and innate immunityrobust TNF-a production is particularly intriguing, giventhat a wide spectrum of microorganisms have acquiredelegant mechanisms to interfere with host responsesmediated by TNF-a (Rahman & McFadden, 2006). In thisstudy, the authors elected to measure TNF-a secretionfollowing pneumococcal exposure, as production of TNF-ais known to influence the outcome in experimental modelsof pneumococcal infection (Takashima et al., 1997; Wellmeret al., 2001). Importantly, it has been demonstrated pre-viously that production of a variety of cytokines (specificallyIL-1b, IL-6, and IL-10) is well correlated with TNF-asecretion following innate immune activation of humanmononuclear cells (Hirschfeld et al., 2007).In this study, pneumococcal serotypes were classified aseither ‘commonly invasive’ or ‘rarely invasive’ using basicepidemiological surveillance data (Fig. 1). However, alter-native pneumococcal classification schemes have been pro-posed. For example, a published alternative is to dividepneumococcal serotypes into 3 major groups on the basis oftheir ability to cause invasive disease in humans (Sandgrenet al., 2004). The first group includes serotypes mostcommonly found in invasive disease and rarely found incarriers – such as serotypes 1, 4, and 7F – that have a highinvasive disease potential. The second group includes ser-otypes commonly found both in invasive disease and incarriers – such as serotypes 14 and 6B – that have anintermediate invasive disease potential. The third groupincludes serotypes primarily found in carriers – such asserotype 19F – that have a low invasive disease potential.Another possible classification scheme is by clonal proper-ties. The particular clonal type, in addition to capsularserotype, may be an important determinant of potential tocause disease. In one study, serotype 14 included clones thatwere only found among carriers, as well as clones causingonly invasive infection, suggesting that different clones havedifferent potential for disease (Sandgren et al., 2004).Ultimately, the ability of different pneumococcal sero-types to colonize and cause invasive disease is likely todepend on multiple host and pathogen factors. It is demon-strated that the capacity to trigger an innate immuneresponse is one factor that influences the potential of aspecific pneumococcal serotype to cause invasive infection(Fig. 2). Nevertheless, other factors certainly influence theevolution of invasive pneumococcal infection. For example,generation of specific antibody against the polysaccharidecapsule is important in determining the frequency ofinvasive infection as some of the most commonly invasivepneumococcal serotypes, such as 6B, 19F, and 23F, are slowto induce high titer antibody responses (Soininen et al.,2001). However, mechanisms in addition to antibody pro-duction must be at play, as serotype 14 is both commonlyinvasive and highly immunogenic in terms of antibodyinduction (Soininen et al., 2001).A potential weakness of this study is that the authorsquantified the innate immune response generated by onlyone cell type – human monocyte-derived macrophages –following exposure to S. pneumoniae. In the process ofcausing invasive infection, pneumococci must interact witha variety of host cells and the most immunologically activeof these are respiratory epithelial cells, monocytes, macro-phages, and dendritic cells. Therefore, it would be valuableto expand the authors’ studies to include analysis of innateimmune responses generated by human respiratory epithe-lial cells and human dendritic cells following exposure todifferent pneumococcal serotypes.Despite vaccination strategies, pneumococcus remainsthe leading cause of invasive bacterial infections in childrenand the elderly. Moreover, rising rates of antibiotic non-susceptibility around the world make this infection increas-ingly more difficult to treat with currently availableantimicrobial agents. New treatment and prevention strate-gies are likely to arise through an improved understandingof the basic immunological mechanisms involved in invasivepneumococcal disease, and the present data indicate thatone factor influencing the invasive potential of a pneumo-coccal serotype is the magnitude of innate immune-mediated TNF-a production triggered by exposure to theorganism.AcknowledgementsThe authors acknowledge the Canadian Paediatric Society’sImmunization Monitoring Program, Active (IMPACT) net-work for providing epidemiological data describing theFig. 2. Commonly invasive pneumococcal serotypes induce significantlyless pro-inflammatory TNF-a production than serotypes rarely responsi-ble for invasive infection. Human monocytic cells (THP-1) werestimulated with a variety of pneumococcal serotypes at an MOI of200 : 1. Clinical pneumococcal isolates were classified as eithercommonly or rarely invasive based on previous epidemiological studies.After 24 h of exposure to the pneumococci, culture supernatants wereharvested and TNF-a production was quantified by ELISA. All bacterialstimulations were performed in triplicate and repeated twice. Valuesrepresent mean TNF-a production from all experiments. Groups werecompared by Student’s t-test and Po 0.004.FEMS Immunol Med Microbiol 53 (2008) 136–139c 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved138 T.S.T. Burgess et al.etiology of invasive pneumococcal infections in Canadianchildren. This work was supported by the SickKids Founda-tion/IHDCYH-CIHR National Grants Program and a NewInvestigator in Infection and Immunity award from theCanadian Institutes of Health Research. SET was supportedby a Career Development Award from the Canadian ChildHealth Clinician Scientist Program (CCHCSP)-a CIHRStrategic Training Program.ReferencesAlbiger B, Sandgren A, Katsuragi H, Meyer-Hoffert U, Beiter K,Wartha F, Hornef M, Normark S & Normark BH (2005)Myeloid differentiation factor 88-dependent signallingcontrols bacterial growth during colonization and systemicpneumococcal disease in mice. Cell Microbiol 7: 1603–1615.Bettinger JA, Scheifele DW, Halperin SA, Kellner JD & Tyrrell G(2007) Invasive pneumococcal infections in Canadianchildren, 1998–2003: implications for new vaccinationprograms. Can J Public Health 98: 111–115.Echchannaoui H, Frei K, Schnell C, Leib SL, Zimmerli W &Landmann R (2002) Toll-like receptor 2-deficient mice arehighly susceptible to Streptococcus pneumoniae meningitisbecause of reduced bacterial clearing and enhancedinflammation. 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Cancer Res 42:1530–1536.Wellmer A, Gerber J, Ragheb J, Zysk G, Kunst T, Smirnov A,Bruck W & Nau R (2001) Effect of deficiency of tumornecrosis factor alpha or both of its receptors on Streptococcuspneumoniae central nervous system infection and peritonitis.Infect Immun 69: 6881–6886.FEMS Immunol Med Microbiol 53 (2008) 136–139 c 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved139Pneumococcus and innate immunity


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