Toll-Like Receptor 4-Mediated Innate IL-10 Activates Antigen-Specific Regulatory T Cells and Confers Resistance to Bordetella pertussis by Inhibiting Inflammatory Pathology 1 Sarah C. Higgins, Ed C. Lavelle, Chantelle McCann, Brian Keogh, Edel McNeela, Patricia Byrne, Brian O’Gorman, Andrew Jarnicki, Peter McGuirk, and Kingston H. G. Mills 2 Signaling through Toll-like receptors (TLR) activates dendritic cell (DC) maturation and IL-12 production, which directs the induction of Th1 cells. We found that the production of IL-10, in addition to inflammatory cytokines and chemokines, was significantly reduced in DCs from TLR4-defective C3H/HeJ mice in response to Bordetella pertussis. TLR4 was also required for B. pertussis LPS-induced maturation of DCs, but other B. pertussis components stimulated DC maturation independently of TLR4. The course of B. pertussis infection was more severe in C3H/HeJ than in C3H/HeN mice. Surprisingly, Ab- and Ag-specific IFN- responses were enhanced at the peak of infection, whereas Ag-specific IL-10-producing T cells were significantly reduced in C3H/HeJ mice. This was associated with enhanced inflammatory cytokine production, cellular infiltration, and severe pathological changes in the lungs of TLR4-defective mice. Our findings suggest that TLR-4 signaling activates innate IL-10 production in response to B. pertussis, which both directly, and by promoting the induction of IL-10-secreting type 1 regulatory T cells, may inhibit Th1 responses and limit inflammatory pathology in the lungs during infection with B. pertussis. The Journal of Immu- nology, 2003, 171: 3119 –3127. P athogen recognition receptors, including the Toll-like re- ceptors (TLRs), 3 allow the innate immune system to detect conserved patterns of molecules on pathogens and to re- spond in the first line of defense against infection by producing inflammatory cytokines (1). Furthermore, the innate immune re- sponse to pathogens can shape the adaptive immune response, through the stimulation of dendritic cells (DCs), which act as APCs, but also direct the differentiation of naive T cells. A range of pathogen-derived molecules has been identified as ligands for TLRs. TLR4 was first implicated in LPS recognition (2), although more recent studies have shown that structurally distinct LPS from certain bacteria can signal through TLR2 (3, 4). The role of TLR4 in LPS signaling was identified using LPS-hyporesponsive C3H/ HeJ mice, which have a point mutation in the cytoplasmic region of TLR4 (2). Recognition of LPS is initialized by the cooperative interplay between the LPS-binding protein, CD14, and the TLR4- MD2 complex (5). Upon activation by LPS, TLR4 signals via a pathway involving the kinases of the IL-1R-associated kinase family, TNFR-associated factor-6 and NF-B (6). Binding of pathogens to TLRs on DCs results in their matura- tion, characterized by up-regulation of MHC class II, CD80, CD86, and CD40, but also activation of proinflammatory cytokine production, including TNF- and IL-12. The mature DCs migrate from the tissue to the lymph nodes, where they present Ag to naive T cells. Evidence is emerging that DCs activated by distinct pathogen- derived molecules can selectively promote the induction of distinct T cell subtypes. Many pathogen molecules, including Escherichia coli LPS (2), CpG motifs in bacterial DNA (7), flagellin (8), and viral dsRNA (9) that bind TLRs and stimulate IL-12 production by innate cells, such as DCs, direct the induction of Th1 cells. Other pathogen- derived molecules, such as yeast hyphae (10), helminth components (11), cholera toxin (12), and Porphyromonas gingivalis LPS (3), can activate DCs that drive the differentiation of naive T cells to a Th2 phenotype. Finally, we have recently reported that filamentous hem- agglutinin (FHA) from Bordetella pertussis can activate innate IL-10 production and stimulate DCs that selectively activate type 1 regula- tory T (Tr1) cells (13, 14). B. pertussis is a Gram-negative bacterium that causes whooping cough, a protracted respiratory disease in young children. Recov- ery from infection in both children and mice is associated with the development of B. pertussis-specific Th1 cells (15, 16). Adoptive transfer of Th1 cells from convalescent mice can confer protection (15), and IFN- receptor knockout mice develop lethal dissemi- nating infection (17). Athymic or SCID mice fail to clear the in- fection, but the bacteria do not disseminate from the lung (15, 18), suggesting that innate responses may prevent bacterial dissemina- tion before the development of adaptive immunity. However, in- duction of IgG and Th1 responses is suppressed during the acute stages of infection (19), and we have recently shown that Ag- specific Tr1 clones can be generated from the respiratory tract of B. pertussis-infected mice (14). The Tr1 cells were shown to sup- press B. pertussis-specific Th1 responses in vitro and in vivo. We concluded that induction of Tr1 cells may represent an evasion strategy by the pathogen to subvert protective Th1 responses, but also speculated that they may have a role in limiting immunopa- thology in the lungs (14). In the present study, we set out to examine the role of TLR4 in the pathogenesis of B. pertussis infection, specifically the role of Immune Regulation Research Group, Department of Biochemistry, Trinity College, Dublin 2, Ireland Received for publication March 3, 2003. Accepted for publication July 7, 2003. 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 by grants from Science Foundation Ireland and the Health Research Board of Ireland. 2 Address correspondence and reprint requests to Prof. Kingston Mills, Department of Biochemistry, Trinity College, Dublin 2, Ireland. E-mail address: kingston.mills@tcd.ie 3 Abbreviations used in this paper: TLR, Toll-like receptor; BAL, bronchoalveolar lavage; DC, dendritic cell; FHA, filamentous hemagglutinin; iDC, immature dendritic cell; MIP, macrophage-inflammatory protein; PT, pertussis toxin; Tr, regulatory T. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00