Dual Role of TLR2 and Myeloid Differentiation Factor 88 in a Mouse Model of Invasive Group B Streptococcal Disease 1 Giuseppe Mancuso,* Angelina Midiri,* Concetta Beninati,* Carmelo Biondo,* Roberta Galbo,* Shizuo Akira, † Philipp Henneke, ‡§ Douglas Golenbock, ‡ and Giuseppe Teti 2 * Toll-like receptors (TLRs) are involved in pathogen recognition by the innate immune system. Different TLRs and the adaptor molecule myeloid differentiation factor 88 (MyD88) were previously shown to mediate in vitro cell activation induced by group B streptococcus (GBS). The present study examined the potential in vivo roles of TLR2 and MyD88 during infection with GBS. When pups were infected locally with a low bacterial dose, none of the TLR2- or MyD88-deficient mice, but all of the wild-type ones, were able to prevent systemic spread of GBS from the initial focus. Bacterial burden was higher in MyD88- than in TLR2-deficient mice, indicating a more profound defect of host defense in the former animals. In contrast, a high bacterial dose induced high level bacteremia in both mutant and wild-type mice. Under these conditions, however, TLR2 or MyD88 deficiency significantly protected mice from lethality, concomitantly with decreased circulating levels of TNF- and IL-6. Administration of anti-TNF- Abs to wild-type mice could mimic the effects of TLR2 or MyD88 deficiency and was detrimental in the low dose model, but protective in the high dose model. In conclusion, these data highlight a dual role of TLR2 and MyD88 in the host defense against GBS sepsis and strongly suggest TNF- as the molecular mediator of bacterial clearance and septic shock. The Journal of Immunology, 2004, 172: 6324 – 6329. G roup B streptococci (GBS) 3 are a frequent cause of sys- temic infections in neonates and adults with underlying chronic conditions (1, 2). The incidence of lethality and permanent neurological damage is high despite the availability of modern therapy. Several observations suggest that an exaggerated production of proinflammatory cytokines is responsible for many of the manifestations of GBS sepsis. In neonates with early onset GBS sepsis, plasma levels and gene expression of proinflammatory cytokines are extremely elevated (3, 4). Experimental rodent mod- els of GBS disease indicate that TNF- has a crucial role in me- diating lethality, as evidenced by the protective effects of prophy- lactic administration of anti-TNF- Abs (5, 6). Finally, GBS most potently activate phagocytes, as evidenced by maximal cytokine levels induced in vitro using human monocytes or mouse macro- phages (7, 8). Toll-like receptors (TLRs) belong to a family of evolutionarily conserved type I transmembrane proteins capable of detecting the presence of microbial products or damaged host tissues (9). A leucine-rich domain, which is presumably responsible for ligand recognition, is present in the extracellular portion of TLRs, whereas the cytoplasmic portion contains a region that is highly homologous to the intracellular signaling domain of the IL-1R (the so-called TIR domain). Activation of TLRs initiates a cascade of intracellular events resulting in cytokine production and the ex- pression of costimulatory molecules (10). There is strong evidence that TLRs play a crucial role in innate immunity responses, in- cluding inflammation, activation of antimicrobial mechanisms, and initiation of adaptive immune responses (9 –11). Different TLRs are engaged by different microbial stimuli (9– 11). The LPS component of the Gram-negative cell wall activates TLR4 in conjunction with the secreted molecule MD2. Bacterial flagellin and bacterial DNA activate TLR5 and TLR9, respec- tively. TLR2 can recognize, in conjunction with TLR6 or TLR1, a wide array of microbial products, including peptidoglycan, li- poproteins, and glycolipids from bacteria, as well as yeast cell walls and mycobacterial products (8 –11). This latter observation suggested that TLRs have the ability to establish a combinatorial repertoire to discriminate among the large number of different mi- crobial products, although to date such a combinatorial repertoire has only been found for TLR2 and its partners. The signaling pathways activated by TLRs have been well char- acterized and include at least five different TIR domain-containing adapter molecules, protein kinases, and transcription factors. My- eloid differentiation factor 88 (MyD88) is the best studied of the adaptor molecules and has an important role in transducing acti- vation signals from TLRs and the IL-1R (12, 13). MyD88-deficient mice are more susceptible to infection by different pathogens (14– 18), indicating a crucial role of MyD88 in antimicrobial defenses. Surprisingly, MyD88-deficient mice have also been reported to be more resistant to the effects of septic polymicrobial peritonitis, implying a pathophysiological role of MyD88 in sepsis (19). These apparently conflicting observations in the MyD88 knockouts raise the possibility that the responses to septic insults mediated by MyD88 depend upon the precise conditions of infection and may differ substantially based on inoculum, the location of the infec- tion, and other variables that are, as yet, poorly understood. *Department of Pathology and Experimental Microbiology, University of Messina Medical School, Messina, Italy; † Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; ‡ Department of Medicine, Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605; and § Children’s Hospital, Freiburg University, Freiburg, Germany Received for publication August 21, 2003. Accepted for publication March 16, 2004. 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 the European Commission (HOSPATH Contract QLK2-CT-2000-00336), National Institutes of Health Grants RO1AI52455 and RO1GM54060 (to D.T.G.), and Deutsche Forschungssgemeinschaft Grant HE 3127/ 2-1 (to P.H.). 2 Address correspondence and reprint requests to Dr. Giuseppe Teti, Policlinico Uni- versitario, Via C. Valeria 1, I-98125 Messina, Italy. E-mail address: teti@eniware.it 3 Abbreviations used in this paper: GBS, group B streptococci; MyD88, myeloid differentiation factor 88; TIR domain, intracellular domain of the TLR/IL-1R; TLR, Toll-like receptor; WT, wild type. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00