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

Abstract

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.

Fig. 1.

Identification of mutants with in vivo defects in pDC type I IFN production. Mice were injected with the TLR9 ligand CpG-A along with DOTAP and serum type I IFN levels measured six hours later. The screen was validated by testing mice with mutations in genes known to be essential for the type I IFN response to CpG-A. Tlr9^−/−, MyD88^poc/poc, Irak4^{otiose/otiose}, Unc93b1^3d/3d, Ifnar1^−/−, and Stat1^dom/dom mice all displayed little to no serum type I IFN (A). Screening ENU-mutagenized G3 mice revealed five unique pedigrees with defects in the CpG-induced type I IFN response. These mutations included alleles in Tlr9 (CpG6), Irf7 (inept), and Irf8 (gemini). Gemini and meager mice were assayed on a different day (B). All error bars represent SEM.

Fig. 2.

The feeble mutation causes defective pDC function. Injection of feeble homozygous mice with CpG-A (TLR9 stimulation) or resiquimod (TLR7 stimulation) does not induce serum type I IFN (A, Left), and highly enriched pDC cultures generated from homozygous feeble bone marrow lack type I IFN responses to the indicated TLR stimuli. Data are representative of three mice (A, Right). In vitro-derived feeble pDCs generate reduced levels of secreted proinflammatory cytokines in response to the indicated stimuli. Data are representative of three mice (B). Normal pDC numbers are present in the feeble spleen, and normal percentages of pDCs develop from feeble bone marrow in vitro (C). All error bars represent SEM.

Fig. 3.

The feeble mutation affects type I IFN mRNA expression and protein production. Lack of intracellular type I IFN in response to CpG stimulation in feeble pDCs even in the presence of exogenous recombinant IFN (rIFN-β). Populations were first gated on live cells expressing B220. FSC, forward scatter (A). Lack of IFN-α gene induction in CpG-stimulated feeble pDCs using two degenerate primer sets. Inept pDCs with a mutation in Irf7 were used as controls (B). Uptake of Alexa488 conjugated CpG is normal in feeble pDCs (C). Except for “Media,” all conditions included addition of CpG. Incubations were performed at 37 °C except for “Ice”. CpG on ice reflects background extracellular fluorescence occurring without active uptake. Treatment with DNase was to remove cell surface-bound DNA. Trypan blue was to quench extracellular fluorescence. Right hand graph depicts a merge of wild type and feeble data. All error bars represent SEM.

Fig. 4.

Mapping and identification of the feeble mutation. LOD score mapping the feeble mutation to chromosome 5 (A) and sequence showing the T-to-A transversion in the Slc15a4 gene (B). Slc15a4 mRNA is expressed in pDCs but not cDCs (C).

Fig. 5.

A membrane trafficking pathway is important for pDC function. Mice with defects in the Ap3b1 subunit of the AP-3 complex (pearl and bullet gray) and mutations in genes encoding BLOC-1 (salt and pepper) and BLOC-2 (toffee) components lack serum type I IFN in response to CpG injection compared with ENU-mutagenized G3 mice (A). Ap3b1^pearl/pearl mice display normal numbers of splenic pDCs and generate normal percentages of bone marrow-derived pDCs. C57BL/6J controls are the same as in Fig. 2 (B). Highly enriched in vitro-derived Ap3b1^pearl/pearl pDCs display defective type I IFN responses (C), but bone marrow-derived cDCs appear to be normal (D). Unc93b1^3d/3d pDCs were used as controls in C. The ARF1 inhibitor, Brefeldin A, prevents type I IFN gene production in wild type pDCs (E). All error bars represent SEM.