The Toll-like receptor (TLR) family has important roles in microbial recognition and dendritic cell activation. TLRs 7 and 9 can recognize nucleic acids and trigger signalling cascades that activate plasmacytoid dendritic cells to produce interferon-alpha (IFN-alpha) (refs 7, 8). TLR7/9-mediated dendritic cell activation is critical for antiviral immunity but also contributes to the pathogenesis of systemic lupus erythematosus, a disease in which serum IFN-alpha levels are elevated owing to plasmacytoid dendritic cell activation. TLR7/9-induced IFN-alpha induction depends on a molecular complex that contains a TLR adaptor, MyD88, and IFN regulatory factor 7 (IRF-7) (refs 10-14), but the underlying molecular mechanisms are as yet unknown. Here we show that IkappaB kinase-alpha (IKK-alpha) is critically involved in TLR7/9-induced IFN-alpha production. TLR7/9-induced IFN-alpha production was severely impaired in IKK-alpha-deficient plasmacytoid dendritic cells, whereas inflammatory cytokine induction was decreased but still occurred. Kinase-deficient IKK-alpha inhibited the ability of MyD88 to activate the Ifna promoter in synergy with IRF-7. Furthermore, IKK-alpha associated with and phosphorylated IRF-7. Our results identify a role for IKK-alpha in TLR7/9 signalling, and highlight IKK-alpha as a potential target for manipulating TLR-induced IFN-alpha production.

Plasmacytoid dendritic cells (pDCs), originating from hematopoietic progenitor cells in the BM, are a unique dendritic cell subset that can produce large amounts of type I IFNs by signaling through the nucleic acid-sensing TLR7 and TLR9 (TLR7/9). The molecular mechanisms for pDC function and development remain largely unknown. In the present study, we focused on an Ets family transcription factor, Spi-B, that is highly expressed in pDCs. Spi-B could transactivate the type I IFN promoters in synergy with IFN regulatory factor 7 (IRF-7), which is an essential transcription factor for TLR7/9-induced type I IFN production in pDCs. Spi-B-deficient pDCs and mice showed defects in TLR7/9-induced type I IFN production. Furthermore, in Spi-Bdeficient mice, BM pDCs were decreased and showed attenuated expression of a set of pDC-specific genes whereas peripheral pDCs were increased; this un-

Material

Double-stranded RNA (dsRNA) is produced upon viral infection and can activate innate immunity. Polyinosinic:polycytidylic acids [poly(I:C)] is a synthetic mimetic of dsRNA and functions through an endosomal receptor, Toll-like receptor (TLR) 3 or cytosolic receptors. Another type of dsRNA, polyadenylic:polyuridylic acids [poly(A:U)], can also act as an immune adjuvant, but it remains unclear how it exhibits its adjuvant effects. Here, we have characterized the adjuvant effects of poly(A:U). Poly(A:U) could induce both IFN-alpha and IL-12p40 from murine bone marrow dendritic cells (DCs). Poly(A:U)-induced IFN-alpha production depended on a DC subset, plasmacytoid dendritic cell (pDC), and required TLR7. IL-12p40 was also produced by poly(A:U)-stimulated pDC in a TLR7-dependent manner. In addition to pDC, conventional dendritic cell (cDC) also produced IL-12p40 in response to poly(A:U). This IL-12p40 induction resulted from two cDC subsets, CD24(high) cDC and CD11b(high) cDC in a TLR3- and TLR7-dependent manner, respectively. In vivo injection of poly(A:U) with antigen led to clonal expansion of and IFN-gamma production from antigen-specific CD8(+) T cells. Consistent with the in vitro findings, TLR3 and TLR7 were required for the clonal T-cell expansion. Notably, TLR3, rather than TLR7, was critical for generating IFN-gamma-producing CD8(+) T cells. CD8(+) T-cell responses induced by poly(A:U) were independent of type I IFN signaling. Our results demonstrate that poly(A:U) functions as an in vivo immunoadjuvant mainly through TLR3 and TLR7.

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