Cutting Edge: A TLR9 Cytoplasmic Tyrosine Motif Is Selectively Required for Proinflammatory Cytokine Production

A cytoplasmic tyrosine motif is required for TNF production

We previously identified a highly conserved 4-aa motif in the cytoplasmic tail of TLR9 that contributes to intracellular trafficking (11). To determine whether this motif regulated production of the proinflammatory cytokine TNF, IFN, or both, we retrovirally transduced TLR9-deficient macrophages with wild-type (WT) TLR9, the tyrosine mutant (TLR9^Y888A), or empty vector, and assayed for TNF-α production in response to CpG DNA. Vector-transduced cells did not respond, whereas cells expressing WT TLR9 produced TNF-α in response to CpG DNA-B (Fig. 1A). However, cells expressing TLR9^Y888A produced no TNF-α in response to CpG DNA-B (Fig. 1A). LPS responses were similar in all cells (Fig. 1B).

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FIGURE 1.

A tyrosine motif in TLR9 cytoplasmic domain is required for proinflammatory cytokine response. A, TLR9^−/− macrophages were retrovirally transduced with empty vector (Vector), WT TLR9, TLR9^Y888A (Y888A), or TLR9^Y888F (Y888F). Cells were stimulated with media or CpG DNA-B (10 μg/ml) for 8 h. Secreted TNF-α concentration was determined by ELISA. Error bars indicate SD (n = 4). B, As in A, except cells were stimulated with LPS (100 ng/ml). C, Vector, WT, or Y888A cells were stimulated with CpG DNA-A (10 μg/ml) or CpG DNA-B (10 μg/ml) in complex with DOTAP, and IFN-β mRNA relative expression level (REL%) was determined by real-time PCR. Results are representative of three experiments.

We next asked whether 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 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 CpG DNA-A alone, CpG DNA-B alone, or CpG DNA-B in complex with DOTAP did not induce IFN (Fig. 1C; A.C. and C.A.L., unpublished observations). However, unlike for TNF production (Fig. 1A), TLR9^Y888A supported near WT 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.

TLR9 receptor phosphorylation correlates with TNF production and depends on Y888

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 whether the phosphorylation event occurs exclusively on p80 or also on full-length TLR9. Regardless, mutation of Y888 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 Y888. Mutation of the critical tyrosine to phenylalanine, a structurally conserved but nonphosphorylated 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 (C.A.L., unpublished observations). 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 TLR9^Y888A to support TNF was not due to lack of binding to ligand, because TLR9^Y888A in lysates interacted with CpG DNA-B as well as, or better than, WT TLR9 (A.C. and C.A.L., unpublished observations). Therefore, TLR9 phosphorylation occurred selectively on the p80 form of the receptor, depended on Y888, and correlated with TNF production.

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FIGURE 2.

WT TLR9 and TLR9^Y888F, but not TLR9^Y888A, are tyrosine phosphorylated in response to CpG DNA and support downstream signaling. TLR9^−/− macrophages were retrovirally transduced with empty vector (V), TLR9-HA (WT), TLR9^Y888F-HA (Y888F), or TLR9^Y888A-HA (Y888A). Cells were stimulated with CpG DNA-B (10 μg/ml) for the indicated times. A, HA immunoprecipitates were immunoblotted for phosphotyrosine or HA. Arrow indicates full-length TLR9; asterisks indicate p80. B, Whole-cell lysates were immunoblotted for phospho-p38 and total p38. Representative of two experiments.

The structure of Y888 is critical for TNF production

We next asked whether Y888 was directly phosphorylated by mutation of the critical tyrosine to phenylalanine, which is structurally related to tyrosine, but cannot be phosphorylated because of the lack of the hydroxyl group (19). When expressed in TLR9^−/− macrophages, TLR9^Y888F 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, although Y888 was not directly phosphorylated, it was structurally required for TLR9 to support production of TNF.

Y888 is required for TLR9 trafficking to lysosomal-associated membrane protein 1-positive endolysosomes

We next asked whether 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 min and traffics to late endosomes/endolysosomes at 1 h (4, 7). The endolysosomal compartment is where proinflammatory cytokine production initiates and is identified by the presence of lysosomal-associated membrane protein 1 (LAMP-1). WT and mutant TLR9 were similarly localized in untreated cells (A.C., W.R., and C.A.L., unpublished observations). However, after a 1-h incubation with 3′-Cy3–labeled CpG DNA, WT TLR9 and TLR9^Y888F, but not TLR9^Y888A, colocalized with LAMP-1⁺ compartments as determined by four-color confocal microscopy (Fig. 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, TLR9^Y888F: 0.6192) and LAMP-1 (WT: 0.7344, TLR9^Y888F: 0.7015; Fig. 3). Pearson’s colocalization coefficients were significantly lower for TLR9^Y888A with CpG DNA (0.1141) and LAMP-1 (0.2186). Together, these data show that TLR9^Y888A fails to induce production of TNF because it fails to reach LAMP-1⁺ endolysosomes.

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FIGURE 3.

Amino acid structure at Y888 is important for trafficking of TLR9 to the lysosomal compartment after CpG stimulation in macrophages. Cells were transduced as in Figs. 1 and 2, and stimulated with CpG DNA 10104-Cy3 for 1 h. Overlay of TLR9 (HA, green), CpG DNA (magenta), lysosomes (CD107a, red), and nuclei (DAPI, blue) is shown to the left. Scale bars, 5 μm. Representative of 3 experiments, 10 fields per experiment.

Because TLR9^Y888A supported IFN production, we asked whether TLR9^Y888A localized with early endosome markers after stimulation with CpG DNA-A–DOTAP complexes. Within 10 min of exposure to CpG DNA-A–DOTAP complexes, both WT and TLR9^Y888A colocalized with Rab5 (Fig. 4). Together with the observation that TLR9^Y888A did not colocalize with LAMP-1 1 h 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.

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FIGURE 4.

CpG DNA-A induces trafficking of WT and TLR9^Y888A to early endosomes. Cells were transduced as in Figs. 1 and 2, and stimulated with CpG DNA 2216 in complex with DOTAP for 10 min. Overlay of TLR9 (HA, green), early endosomes (Rab5, red), and nuclei (DAPI, blue) is shown to the left. Scale bars, 5 μm. Representative of 3 experiments, 10 fields per experiment.

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). In this study, 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 Y888 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 because IFN production was normal for TLR9^Y888A. 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.