Amanda L. Blasius

Intracellular Toll-like Receptors

Foreign nucleic acids, the signature of invading viruses and certain bacteria, are sensed intracellularly. The nucleic acid-specific Toll-like receptors (TLRs) detect and signal within endolysosomal compartments, triggering the induction of cytokines essential for the innate immune response. These cytokines include proinflammatory molecules produced mainly by macrophages and conventional dendritic cells, as well as type I interferons, which are produced in great quantities by plasmacytoid dendritic cells. The cellular and molecular pathways by which nucleic acids and TLRs meet within the endosome assure host protection yet also place the host at risk for the development of autoimmunity. Here, we review the latest findings on the intracellular TLRs, with special emphasis on ligand uptake, receptor trafficking, signaling, and regulation.

Bone Marrow Stromal Cell Antigen 2 Is a Specific Marker of Type I IFN-Producing Cells in the Naive Mouse, but a Promiscuous Cell Surface Antigen following IFN Stimulation

Type I IFN-producing cells (IPC) are sentinels of viral infections. Identification and functional characterization of these cells have been difficult because of their small numbers in blood and tissues and their complex cell surface phenotype. To overcome this problem in mice, mAbs recognizing IPC-specific cell surface molecules have been generated. In this study, we report the identification of new Abs specific for mouse IPC, which recognize the bone marrow stromal cell Ag 2 (BST2). Interestingly, previously reported IPC-specific Abs 120G8 and plasmacytoid dendritic cell Ag-1 also recognize BST2. BST2 is predominantly specific for mouse IPC in naive mice, but is up-regulated on most cell types following stimulation with type I IFNs and IFN-γ. The activation-induced promiscuous expression of BST2 described in this study has important implications for the use of anti-BST2 Abs in identification and depletion of IPC. Finally, we show that BST2 resides within an intracellular compartment corresponding to the Golgi apparatus, and may be involved in trafficking secreted cytokines in IPC.

Development and function of murine B220+CD11c+NK1.1+ cells identify them as a subset of NK cells

Lymphoid organs contain a B220+CD11c+NK1.1+ cell population that was recently characterized as a novel dendritic cell (DC) subset that functionally overlaps with natural killer (NK) cells and plasmacytoid DCs (PDCs). Using Siglec-H and NK1.1 markers, we unambiguously dissected B220+CD11c+ cells and found that PDCs are the only professional interferon (IFN)-α–producing cells within this heterogeneous population. In contrast, B220+CD11c+NK1.1+ cells are a discrete NK cell subset capable of producing higher levels of IFN-γ than conventional NK cells. Unlike DCs, only a minute fraction of B220+CD11c+NK1.1+ cells in the spleen expressed major histocompatibility complex class II ex vivo or after stimulation with CpG. Consistent with being a NK cell subset, B220+CD11c+NK1.1+ cells depended primarily on interleukin 15 and common cytokine receptor γ chain signaling for their development. In terms of function, expression of distinctive cell surface receptors, and location in lymphoid organs, NK1.1+B220+CD11c+ appear to be the murine equivalent of human CD56bright NK cells.

Slc15a4, AP-3, and Hermansky-Pudlak syndrome proteins are required for Toll-like receptor signaling in plasmacytoid dendritic cells

Despite their low frequency, plasmacytoid dendritic cells (pDCs) produce most of the type I IFN that is detectable in the blood following viral infection. The endosomal Toll-like receptors (TLRs) TLR7 and TLR9 are required for pDCs, as well as other cell types, to sense viral nucleic acids, but the mechanism by which signaling through these shared receptors results in the prodigious production of type I IFN by pDCs is not understood. We designed a genetic screen to identify proteins required for the development and specialized function of pDCs. One phenovariant, which we named feeble, showed abrogation of both TLR-induced type I IFN and proinflammatory cytokine production by pDCs, while leaving TLR responses intact in other cells. The feeble phenotype was mapped to a mutation in Slc15a4, which encodes the peptide/histidine transporter 1 (PHT1) and has not previously been implicated in pDC function. The identification of the feeble mutation led to our subsequent observations that AP-3, as well as the BLOC-1 and BLOC-2 Hermansky-Pudlak syndrome proteins are essential for pDC signaling through TLR7 and TLR9. These proteins are not necessary for TLR7 or TLR9 signaling in conventional DCs and thus comprise a membrane trafficking pathway uniquely required for endosomal TLR signaling in pDCs.

Adhesive mechanisms governing interferon-producing cell recruitment into lymph nodes

Natural interferon-producing cells (IPCs) are found in peripheral lymph nodes (PLNs), where they support NK cell, T cell, and B cell responses to pathogens. However, their route of entry and the adhesive mechanisms used to gain access to PLNs remain poorly defined. We report that IPCs can enter PLNs via a hematogenous route, which involves a multistep adhesive process, and that transmigration is enhanced by inflammation. Results indicate that L-selectin on IPCs is required for efficient attachment and rolling on high endothelial venules in vivo in both nonstimulated and inflamed PLNs. IPCs, however, also possess functional ligands for E-selectin that contribute to this process only in the latter case. In conjunction with selectin-mediated adhesion, both β 1- and β 2-integrins participate in IPC attachment to the inflamed vessel wall, whereas chemotaxis relies in part on the chemokine receptor CCR5. Identification of the adhesive machinery required for IPC trafficking into PLNs may provide opportunities to regulate immune responses reliant on the activity of these cells.

Plasmacytoid dendritic cells: in search of their niche in immune responses.

Plasmacytoid dendritic cells (pDCs) have been associated with several names and functions over time, reflecting the limited availability of specific markers and their ability to produce large amounts of type I interferons, present antigens, as well as prime disparate T-cell helper responses. Yet, there is increasing evidence that pDCs are a distinct cell type in the innate immune system. This review high-lights aspects in which pDCs are unique in comparison to other antigen-presenting cells in regulating innate and adaptive immune responses.

Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1, MyD88, and a microbial trigger.

A recessive phenotype called spin (spontaneous inflammation) was induced by N-ethyl-N-nitrosourea (ENU) mutagenesis in C57BL/6J mice. Homozygotes display chronic inflammatory lesions affecting the feet, salivary glands and lungs, and antichromatin antibodies. They are immunocompetent and show enhanced resistance to infection by Listeria monocytogenes. TLR-induced TNF and IL-1 production are normal in macrophages derived from spin mice. The autoinflammatory phenotype of spin mice is fully suppressed by compound homozygosity for Myd88poc, Irak4otiose, and Il1r1-null mutations, but not Ticam1Lps2, Stat1m1Btlr, or Tnf-null mutations. Both autoimmune and autoinflammatory phenotypes are suppressed when spin homozygotes are derived into a germ-free environment. The spin phenotype was ascribed to a viable hypomorphic allele of Ptpn6, which encodes the tyrosine phosphatase SHP1, mutated in mice with the classical motheaten alleles me and me-v. Inflammation and autoimmunity caused by SHP1 deficiency are thus conditional. The SHP1-deficient phenotype is driven by microbes, which activate TLR signaling pathways to elicit IL-1 production. IL-1 signaling via MyD88 elicits inflammatory disease.

Inflammatory arthritis can be reined in by CpG-induced DC-NK cell cross talk

Unmethylated CpG-oligodeoxynucleotides (ODNs) are generally thought of as potent adjuvants with considerable therapeutic potential to enhance immune responses against microbes and tumors. Surprisingly, certain so-called stimulatory CpG-ODNs strongly inhibited the effector phase of inflammatory arthritis in the K/BxN serum transfer system, either preventively or therapeutically. Also unexpected was that the inhibitory influence did not depend on the adaptive immune system cells mobilized in an immunostimulatory context. Instead, they relied on cells of the innate immune system, specifically on cross talk between CD8α+ dendritic cells and natural killer cells, resulting in suppression of neutrophil recruitment to the joint, orchestrated through interleukin-12 and interferon-γ. These findings highlight potential applications of CpG-ODNs and downstream molecules as antiinflammatory agents.

Essential requirement for IRF8 and SLC15A4 implicates plasmacytoid dendritic cells in the pathogenesis of lupus

In vitro evidence suggests that plasmacytoid dendritic cells (pDCs) are intimately involved in the pathogenesis of lupus. However, it remains to be determined whether these cells are required in vivo for disease development, and whether their contribution is restricted to hyperproduction of type I IFNs. To address these issues, we created lupus-predisposed mice lacking the IFN regulatory factor 8 (IRF8) or carrying a mutation that impairs the peptide/histidine transporter solute carrier family 15, member 4 (SLC15A4). IRF8-deficient NZB mice, lacking pDCs, showed almost complete absence of anti-nuclear, anti-chromatin, and anti-erythrocyte autoantibodies, along with reduced kidney disease. These effects were observed despite normal B-cell responses to Toll-like receptor (TLR) 7 and TLR9 stimuli and intact humoral responses to conventional T-dependent and -independent antigens. Moreover, Slc15a4 mutant C57BL/6-Faslpr mice, in which pDCs are present but unable to produce type I IFNs in response to endosomal TLR ligands, also showed an absence of autoantibodies, reduced lymphadenopathy and splenomegaly, and extended survival. Taken together, our results demonstrate that pDCs and the production of type I IFNs by these cells are critical contributors to the pathogenesis of lupus-like autoimmunity in these models. Thus, IRF8 and SLC15A4 may provide important targets for therapeutic intervention in human lupus.

Mice with mutations of Dock7 have generalized hypopigmentation and white-spotting but show normal neurological function

The classical recessive coat color mutation misty (m) arose spontaneously on the DBA/J background and causes generalized hypopigmentation and localized white-spotting in mice, with a lack of pigment on the belly, tail tip, and paws. Here we describe moonlight (mnlt), a second hypopigmentation and white-spotting mutation identified on the C57BL/6J background, which yields a phenotypic copy of m/m coat color traits. We demonstrate that the 2 mutations are allelic. m/m and mnlt/mnlt phenotypes both result from mutations that truncate the dedicator of cytokinesis 7 protein (DOCK7), a widely expressed Rho family guanine nucleotide exchange factor. Although Dock7 is transcribed at high levels in the developing brain and has been implicated in both axon development and myelination by in vitro studies, we find no requirement for DOCK7 in neurobehavioral function in vivo. However, DOCK7 has non-redundant role(s) related to the distribution and function of dermal and follicular melanocytes.