Interferons (IFNs) are critical for protection from viral infection, but the pathways linking virus recognition to IFN induction remain poorly understood. Plasmacytoid dendritic cells produce vast amounts of IFN-alpha in response to the wild-type influenza virus. Here, we show that this requires endosomal recognition of influenza genomic RNA and signaling by means of Toll-like receptor 7 (TLR7) and MyD88. Single-stranded RNA (ssRNA) molecules of nonviral origin also induce TLR7-dependent production of inflammatory cytokines. These results identify ssRNA as a ligand for TLR7 and suggest that cells of the innate immune system sense endosomal ssRNA to detect infection by RNA viruses.

SummaryThe functions of Nr4a1-dependent Ly6Clow monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid “danger” signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6Clow monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6Clow monocyte development, crawling, or retention in Nr4a1−/−, Itgal−/−, and Tlr7host−/−BM+/+ and Cx3cr1−/− mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7host+/+BM−/− mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6Clow monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.

Cross-presentation of cell-associated antigens plays an important role in regulating CD8+ T cell responses to proteins that are not expressed by antigen-presenting cells (APCs). Dendritic cells are the principal cross-presenting APCs in vivo and much progress has been made in elucidating the pathways that allow dendritic cells to capture and process cellular material. However, little is known about the signals that determine whether such presentation ultimately results in a cytotoxic T cell (CTL) response (cross-priming) or in CD8+ T cell inactivation (cross-tolerance). Here we describe a mechanism that promotes cross-priming during viral infections. We show that murine CD8alpha+ dendritic cells are activated by double-stranded (ds)RNA present in virally infected cells but absent from uninfected cells. Dendritic cell activation requires phagocytosis of infected material, followed by signalling through the dsRNA receptor, toll-like receptor 3 (TLR3). Immunization with virus-infected cells or cells containing synthetic dsRNA leads to a striking increase in CTL cross-priming against cell-associated antigens, which is largely dependent on TLR3 expression by antigen-presenting cells. Thus, TLR3 may have evolved to permit cross-priming of CTLs against viruses that do not directly infect dendritic cells.

Toll-like receptors (TLR) recognize microbial and viral patterns and activate dendritic cells (DC). TLR distribution among human DC subsets is heterogeneous: plasmacytoid DC (PDC) express TLR1, 7 and 9, while other DC types do not express TLR9 but express other TLR. Here, we report that mRNA for most TLR is expressed at similar levels by murine splenic DC sub-types, including PDC, but that TLR3 is preferentially expressed by CD8 § + DC while TLR5 and TLR7 are selectively absent from the same subset. Consistent with the latter, TLR7 ligand activates CD8 § -DC and PDC, but not CD8 § + DC as measured by survival ex vivo, upregulation of surface markers and production of IL-12p40. These data suggest that the dichotomy in TLR expression between plasmacytoid and non-plasmacytoid DC is not conserved between species. However, lack of TLR7 expression could restrict the involvement of CD8 § + DC in recognition of certain mouse pathogens.

Type I interferons (IFN-I) are important cytokines linking innate and adaptive immunity. Plasmacytoid dendritic cells make high levels of IFN-I in response to viral infection and are thought to be the major source of the cytokines in vivo. Here, we show that conventional non-plasmacytoid dendritic cells taken from mice infected with a dendritic-cell-tropic strain of lymphocytic choriomeningitis virus make similarly high levels of IFN-I on subsequent culture. Similarly, non-plasmacytoid dendritic cells secrete high levels of IFN-I in response to double-stranded RNA (dsRNA), a major viral signature, when the latter is introduced into the cytoplasm to mimic direct viral infection. This response is partially dependent on the cytosolic dsRNA-binding enzyme protein kinase R and does not require signalling through toll-like receptor (TLR) 3, a surface receptor for dsRNA. Furthermore, we show that sequestration of dsRNA by viral NS1 (refs 6, 7) explains the inability of conventional dendritic cells to produce IFN-I on infection with influenza. Our results suggest that multiple dendritic cell types, not just plasmacytoid cells, can act as specialized interferon-producing cells in certain viral infections, and reveal the existence of a TLR-independent pathway for dendritic cell activation that can be the target of viral interference.

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8α+ and CD8α−CD4− DC to make IL-12 p70. In contrast, exposure of CD8α+, CD4+ and CD8α−CD4− DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

No abstract

Targeted gene delivery capitalizes on the presence of specific cell surface receptors for DNA uptake into cells by receptormediated endocytosis (1-3). Therefore, receptor binding ligands are coupled to polycationic compounds like polylysine (pL) 1 that bind and condense DNA. Following this concept, transferrin polylysine (TfpL)-based gene transfer systems were developed to target transferrin receptor for DNA delivery into cells (1, 4 -7). Binding of TfpL/DNA complexes to transferrin receptor causes internalization and DNA uptake into the endosomal compartment (8). To facilitate DNA release from this compartment, endosomolytic agents (such as inactivated adenoviruses) were included in transfection complexes and were demonstrated to effectively enhance gene transfer efficiency (8 -10). More recently, transferrin polyethylenimine (TfPEI) conjugates have been synthesized and used for DNA delivery, thereby combining the high intrinsic transfection efficacy of polyethylenimine (PEI) with receptor-targeted gene transfer (11,12). PEI possesses DNA binding and condensing activity together with a high pH buffering capacity that is believed to protect DNA from degradation and to enhance exit from the endosomal compartment. Accordingly, PEI is effective in gene delivery into a variety of cell types even without the addition of cell binding ligands or endosomolytic agents (13, 14). Here we investigated whether the mannose receptor that is abundantly expressed on dendritic cells (DC) represents a suitable entry site for targeted gene delivery into DC using mannosylated PEI (ManPEI).DC are professional antigen-presenting cells that occur in peripheral organs like skin, where these cells are exposed to antigens, which they capture and process (15-18). Upon inflammatory stimuli, DC migrate to lymphoid tissue and present processed antigens on major histocompatibility complex (MHC) class I and II molecules to T cells, to elicit an antigenspecific T cell response. Because of their central role in the initiation of primary immune responses, there is high interest in employing DC for immunotherapy of diseases, such as cancer (19 -21). Following such approaches, gene-modified DC offer several potential advantages over peptide/protein-pulsed DC. For example, gene-modified DC can be expected to induce T cell responses against multiple and/or undefined epitopes of tumor antigens, possibly in the context of both MHC class I and II, and with any MHC allele. Furthermore, the expression of chemokines and cytokines in DC simultaneously with tumorspecific and/or associated antigens would additionally allow modulation of the immune response. DC and T cell functions are effectively regulated by a variety of cytokines, and local cytokine production by DC might represent an important adjunct for T cell activation in medical therapy, for example in cancer patients who are often immunosuppressed. However, so far the generation of gene-modified immunocompetent DC has remained difficult mainly due to limitations in DNA delivery techniques (12,21,22)...