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Compartmentalization of nucleic acid sensing TLR9 has been implicated as a mechanism to prevent recognition of self nucleic acid structures. Furthermore, recognition of CpG DNA in different endosomal compartments leads to the production of the proinflammatory cytokine TNF-α, or type I IFN. We previously characterized a tyrosine-based motif at amino acid 888–891 in the cytoplasmic tail of TLR9 important for appropriate intracellular localization. Here we show that this motif is selectively required for the production of TNF, but not IFN. In response to CpG DNA stimulation, the proteolytically processed 80 kDa fragment is tyrosine phosphorylated. Although tyrosine 888 is not itself phosphorylated, the structure of this motif is necessary for both TLR9 phosphorylation and TNF-α production in response to CpG DNA. We conclude that bifurcation in TLR9 signaling is regulated by a critical tyrosine motif in the cytoplasmic tail.
Nucleic acid sensing TLRs are important for host defense against pathogens but are also potent immunomodulators. For example, the TLR9 ligand CpG DNA is an adjuvant for vaccines in non-human primates (1). CpG DNAs are also versatile, because depending on the sequence and chemical properties of the CpG DNA, TLR9 signaling can preferentially result in proinflammatory cytokine production and B cell proliferation (CpG DNA-B/D), or in type I IFN immune production (CpG DNA-A/K) (2, 3).
The outcome of response to the two different classes of CpG DNAs depends on the endosomal compartment where contact with TLR9 occurs (4). CpG DNA-A/D and CpG DNA B/K are endocytosed but then are preferentially retained in early endosomes (CpG DNA-A) to elicit IFN production, or lysosomes (CpG DNA-B) to elicit proinflammatory cytokines (4). TLR9 gains access to these DNAs by trafficking from the endoplasmic reticulum, through the Golgi (5–7). Recent data implicate adaptor protein 3 (AP3) in regulation of TLR9 trafficking from the Golgi to lysosome related organelles where IFN production occurs, but whether AP3 is selectively required for IFN production, or is also required for TNF production is controversial (8, 9). Regardless, distinct regulatory mechanisms selectively governing inflammatory cytokine production have not been identified.
We hypothesized that one of several discrete TLR9 localization motifs was phosphorylated and regulated signaling (YXXΦ, where X= any amino acid, Φ= a bulky hydrophobic amino acid) (10, 11, 12). We show that TLR9 with a single point mutation at tyrosine 888 (Y→A) selectively impairs TNF production and receptor phosphorylation. Mutation of tyrosine 888 to the structurally conserved phenylalanine (Y→F) retained TNF production, and phosphorylation. We conclude that while not directly phosphorylated, Y888 was structurally required for phosphorylation of TLR9 and thereby regulates TLR9-mediated signal bifurcation.
The following antibodies and reagents were used: Hemagglutinin (HA) (ABM and Roche), Rab5, phospho- and total-p38 (Cell Signaling Technologies), 4G10 (Millipore), CD107a (eBiosciences), secondary antibodies (Southern Biotech and Life Technologies), TNF-α ELISA kit (Biolegend), CpG DNA 2216 and 10104 (Eurofins MWG and Integrated DNA Technologies), LPS (Invivogen), DOTAP (Roche). QuikChange (Stratagene) was used for site directed mutagenesis of TLR9-HA. Primer sequences are available upon request.
TLR9−/− macrophages were cultured in DMEM with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM Hepes (complete DMEM), and 10 µg/ml ciprofloxacin. CpG DNA-DOTAP complexes were prepared as previously described (9). Retroviral supernatant with polybrene (8 µg/ml) was used in spin transductions. Cells were cultured at 37°C for 44–48 hours prior to stimulation. Immunoblotting was performed as previously described (7, 13).
RNA (QIAshredder, and RNeasy kit, Qiagen) was used to prepare cDNA (SuperScript® III First-Strand Synthesis,Invitrogen). PCR was performed with SYBR Green Supermix (Applied biosystems) on an ABI PRISM 7500 (Applied biosystems). Changes in gene expression were determined using the 2−ΔΔCT method.
TLR9−/− macrophages were transduced prior to treatment with 3’ Cy3-10104 CpG DNA, 2216 CpG DNA in a complex with DOTAP or medium. Cells were stained for HA and CD107a or Rab5 followed by Alexa Fluor 488 and Alexa Fluor 647 antibodies. Coverslips were mounted with Prolong Gold Antifade Reagent with DAPI (Life Technologies) and visualized on a Lecia TCS SP5 confocal microscope using a 63× oil objective (Lecia Microsystems). Image analysis was performed with the co-localization tool. Figures were prepared in Photoshop (Adobe).
We previously identified a highly conserved four amino acid motif in the cytoplasmic tail of TLR9 that contributes to intracellular trafficking (11). To ask if this motif regulated production of the proinflammatory cytokine TNF, IFN, or both, we retrovirally transduced TLR9 deficient macrophages with wild-type TLR9 (WT), the tyrosine mutant (TLR9Y888A), or empty vector, and assayed for tumor necrosis factor alpha (TNF-α) production in response to CpG DNA. Vector transduced cells did not respond while cells expressing WT TLR9 produced TNF-α in response to CpG DNA-B (Fig. 1A). However, cells expressing TLR9Y888A produced no TNF-α in response to CpG DNA-B (Fig. 1A). LPS responses were similar in all cells (Fig. 1B).
We next asked if IFN production was also compromised in cells expressing mutant TLR9. WT TLR9, but not empty vector, reconstituted cells responded to CpG DNA-A in complex with DOTAP to induce interferon-beta (IFN-β) mRNA (Fig. 1C). CpG DNA-A alone, CpG DNA-B alone, or CpG DNA-B in complex with DOTAP did not induce IFN (Fig. 1C, and AC, CL unpublished observation). However, unlike for TNF production (Fig. 1A), TLR9Y888A supported near wild-type levels of IFN-β mRNA induction (Fig. 1C). Together these data showed that a specific tyrosine motif in the cytoplasmic tail of TLR9 was selectively required for production of TNF.
YXXΦ motifs can be phosphorylated and thereby regulate signaling (14); furthermore, TLR9 is phosphorylated in response to CpG DNA stimulation (12). Stimulation with CpG DNA-B resulted in phosphorylation of an 80 kDa form corresponding to the mature receptor (Fig. 2A) (13, 15–18). Full length TLR9 was not abundant in macrophages, so it is unclear if the phosphorylation event occurs exclusively on p80, or also on full length TLR9. Regardless, mutation of tyrosine 888 to alanine had no effect on proteolytic processing of TLR9 (Fig. 2A), but eliminated CpG DNA-dependent p80 phosphorylation (Fig. 2A) suggesting that TLR9 phosphorylation depended on tyrosine 888. Mutation of the critical tyrosine to F, a structurally conserved but non-phosphorylated amino acid, did not block phosphorylation. Therefore, phosphorylation depends on the structural motif, not on phosphorylation of Y888. Mutation of any one tyrosine in the cytoplasmic tail of TLR9 does not abrogate phosphorylation (CL unpublished observation). TLR9 phosphorylation is complex and likely involves more than one site. WT TLR9, but not the Y888A mutant supported downstream signaling as measured by time-dependent p38 phosphorylation in response to CpG DNA-B stimulation (Fig. 2B). Failure of TLR9Y888A to support TNF was not due to lack of binding to ligand, since TLR9Y888A in lysates interacted with CpG DNA-B as well, or better than, WT TLR9 (unpublished observation, AC and CL). Therefore, TLR9 phosphorylation occurred selectively on the p80 form of the receptor, depended on tyrosine 888, and correlated with TNF production.
We next asked if Y888 was directly phosphorylated by mutation of the critical tyrosine to phenylalanine, which is structurally related to tyrosine, but can not be phosphorylated due to the lack of the hydroxyl group (19). When expressed in TLR9−/− macrophages, TLR9Y888F was proteolytically cleaved (Fig. 2A), and supported downstream signaling as indicated by CpG DNA-induced p38 phosphorylation (Fig. 2B), and TNF-α production in response to CpG DNA-B (Fig. 1A). LPS responses were similar regardless of the transduction conditions (Fig. 1B). Therefore, while tyrosine 888 was not directly phosphorylated, it was structurally required for TLR9 to support production of TNF.
We next asked if the mechanism by which the Y888 motif selectively regulated cytokine production was through access to endolysosomes. CpG DNA is endocytosed to early endosomes within 5 minutes and traffics to late endosomes/endolysosomes at 1 hour (4, 7). The endolysosomal compartment is where proinflammatory cytokine production initiates and is identified by the presence of lysosomal associated membrane protein (LAMP) -1. WT and mutant TLR9 were similarly localized in untreated cells (AC, WR, CL, unpublished observation). However, after a one-hour incubation with 3’-Cy3-labeled CpG DNA, WT TLR9 and TLR9Y888F, but not TLR9Y888A colocalized with LAMP-1 positive compartments as determined by four-color confocal microscopy (Figure 3). Pearson’s coefficients calculated from line scan analyses from multiple sections of multiple cells (not shown) demonstrated colocalization of WT and the F mutant with CpG DNA (WT: 0.4949, TLR9Y888F: 0.6192), and LAMP-1 (WT: 0.7344, TLR9Y888F: 0.7015) (Fig. 3). Pearson’s colocalization coefficients were significantly lower for TLR9Y888A with CpG DNA (0.1141) and LAMP-1 (0.2186). Together these data show that TLR9Y888A fails to induce production of TNF because it fails to reach LAMP-1 positive endolysosomes.
Since TLR9Y888A supported IFN production, we asked if TLR9Y888A localized with early endosome markers following stimulation with CpG DNA-A-DOTAP complexes. Within 10 minutes of exposure to CpG DNA-A-DOTAP complexes, both WT and TLR9Y888A colocalized with Rab5 (Figure 4). Together with the observation that TLR9Y888A did not colocalize with LAMP-1 one hour after CpG DNA-B incubation, we conclude that this structural motif is required to enable trafficking of TLR9 from early endosomes to late endosomes where proinflammatory signaling is initiated.
TLR9 signals to induce proinflammatory cytokines and IFN from different endosomal compartments. The mechanisms regulating this signal bifurcation are not fully understood although they likely require AP3 (8, 9). Here we showed that a single tyrosine mutation in the cytoplasmic tail of TLR9 selectively impairs TNF production. Tyrosine phosphorylation of mature TLR9 correlated with proinflammatory cytokine production, but not IFN production, and depended on Y888. Our observation that mutation of tyrosine 888 to alanine inhibits TLR9 phosphorylation, yet selectively impairs only TNF production, seemingly contradicts observations by Sanjuan et al (12). They show that Src kinase inhibitors, such as PP2, reduce both TNF and IFN production, as well as inhibit tyrosine phosphorylation of TLR9. However, PP2 inhibition of IFN and TNF responses does not necessarily mean that blocking tyrosine phosphorylation of TLR9 itself inhibits both responses. AP3 has recently been implicated in regulation of TLR9 access to the IFN- and proinflammatory cytokine-inducing compartments (8, 9). Our motif likely does bind AP3 since IFN production was normal for TLR9Y888A. Thus, our data support a model where TLR9 traffics to early endosomes and induces IFN production then is sorted to endolysosomal compartments and induces proinflammatory cytokine production. Together these motifs, and the regulatory proteins that bind them, would be targets to therapeutically manipulate cellular response to CpG DNA.
AC, MH, WAR and CJ performed experiments; AC and CL designed the research and wrote the manuscript. The macrophage cell line derived from TLR9 Knockout Mice, NR-9569, was from the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH.
1This work was supported by RO1AI076588 and RO1AI076588-S1 (CAL).