Novel Engagement of CD14 and Multiple Toll-Like Receptors by Group B Streptococci 1 Philipp Henneke,* †† Osamu Takeuchi, † Jos A. van Strijp, ‡ Hilde-Kari Guttormsen, § Jason A. Smith, ¶ Andra B. Schromm, ¶ Terje A. Espevik,  Shizuo Akira, † Victor Nizet,** Dennis L. Kasper, § and Douglas T. Golenbock 2 * Group B streptococcus (GBS) imposes a major health threat to newborn infants. Little is known about the molecular basis of GBS-induced sepsis. Both heat-inactivated whole GBS bacteria and a heat-labile soluble factor released by GBS during growth (GBS-F) induce nuclear translocation of NF-B, the secretion of TNF-, and the formation of NO in mouse macrophages. Macrophages from mice with a targeted disruption of MyD88 failed to secrete TNF- in response to both heat-inactivated whole bacteria and GBS-F, suggesting that Toll-like receptors (TLRs) are involved in different aspects of GBS recognition. Immune cell activation by whole bacteria differed profoundly from that by secreted GBS-F. Whole GBS activated macrophages independently of TLR2 and TLR6, whereas a response to the secreted GBS-F was not observed in macrophages from TLR2-deficient animals. In addition to TLR2, TLR6 and CD14 expression were essential for GBS-F responses, whereas TLR1 and TLR4 or MD-2 did not appear to be involved. Heat lability distinguished GBS-F from peptidoglycan and lipoproteins. GBS mutants deficient in capsular polysaccharide or -hemolysin had GBS-F activity comparable to that of wild-type streptococci. We suggest that CD14 and TLR2 and TLR6 function as coreceptors for secreted microbial products derived from GBS and that cell wall components of GBS are recognized by TLRs distinct from TLR1, 2, 4, or 6. The Journal of Immunology, 2001, 167: 7069 –7076. G roup B streptococcus (GBS) 3 inhabits a unique niche among infectious pathogens. GBS is a harmless com- mensal of the genitourinary tract in 20% of women but is the leading infectious threat to newborn infants in the western world (1, 2). Up to 70% of newborn infants from colonized women acquire GBS. About 1% of these infants develop the sepsis syn- drome and, depending on the gestational age, up to 42% of affected infants may die. Approximately one-half of infants that survive GBS meningitis suffer permanent neurological impairment (3). The expeditious elimination of microorganisms like GBS that invade from mucosal surfaces is a primary goal of the innate im- mune system. APCs recognize traces of microbial components and subsequently orchestrate the antibacterial defense. The crucial sen- sory function is assigned to pattern recognition receptors that dis- tinguish self from conserved microbial structures shared by differ- ent pathogens (4, 5). Mammalian Toll-like receptors (TLRs) lately have been identified as type I transmembrane signaling receptors with pattern recognition capabilities (6). Recent observations at- tributing the dependence of mammalian immune responses to LPS, the major cell wall component of Gram-negative bacteria, to the expression of functional TLR4 (7–10) serve as a paradigm for our understanding of the biology of TLRs. Previously enigmatic ob- servations that the inability to respond to LPS correlated to re- duced resistance to Gram-negative bacterial infections can now be understood in this context. Although Gram-positive and Gram-negative bacterial sepsis are due to distinct microorganisms, the similarities in the physiological consequences of invasive infection with the bacteria are extraor- dinary. There is extensive evidence that the immediate immune response to Gram-negative bacterial invasion is mediated by LPS, but no surface equivalent of LPS in Gram-positive bacteria has ever been identified. GBS subcellular cell wall components include its capsular polysaccharide, peptidoglycan, and lipoteichoic acid and have all been reported to have proinflammatory activities in vitro (11, 12). TLR2 has been identified as a receptor that is central to the innate immune response to several whole Gram-positive bacteria, as well as a receptor for peptidoglycan and lipoteichoic acid (13–16). Although it is an attractive hypothesis that capsular polysaccharide, peptidoglycan, and lipoteichoic acid are responsi- ble for the immune activity associated with GBS infection, none of these bacterial products is a potent inducer of cytokine production. Moreover, we failed to observe a role for TLR2 in the innate im- mune response to killed GBS, despite its content of peptidoglycan and lipoteichoic acid (17). This suggests that the ability of pepti- doglycan and lipoteichoic acid to activate cells via TLR2 and the ability of whole organisms to activate the innate immune system are not causally related. To further delineate the capabilities of different TLRs to dis- criminate microbial products, we hypothesized that GBS would be *Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605; † Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; ‡ Eijkman Winkler Institute of Medical and Clinical Microbiology, University of Utrecht, Utrecht, The Netherlands; § Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; ¶ Boston University School of Medicine, Boston, MA 02118;  Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway; **Depart- ment of Pediatrics, University of California at San Diego, La Jolla, CA 92093; and †† Department of Pediatrics, Free University, Berlin, Germany. Received for publication June 4, 2001. Accepted for publication October 3, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Deutsche Forschungsgemeinschaft (He 3127/1-1 to P.H. and Schr 621/1-1 to A.B.S.), by the National Institutes of Health (AI-23339 to D.L.K., RO1 GM54060, PO1 DK 50305, and AI32725 to D.T.G.), and by the Research Council of Norway and the Norwegian Cancer Society (to T.A.E.). 2 Address correspondence and reprint requests to Dr. Douglas T. Golenbock, Department of Medicine, University of Massachusetts Medical School, NRB Floor 3, 364 Plantation Street, Worcester, MA 01605. E-mail address: douglas.golenbock@umasssmed.edu 3 Abbreviations used in this paper: GBS, group B streptococcus; TLR, Toll-like re- ceptor; CHO, Chinese hamster ovary; huTLR2, human TLR2; GBS-F, soluble factor released by GBS; PEM, peritoneal exudate macrophage. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00