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. 2012 Feb 1;188(3):1019-26.
doi: 10.4049/jimmunol.1102181. Epub 2012 Jan 6.

TLR2 Signaling Depletes IRAK1 and Inhibits Induction of Type I IFN by TLR7/9

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Free PMC article

TLR2 Signaling Depletes IRAK1 and Inhibits Induction of Type I IFN by TLR7/9

Yi C Liu et al. J Immunol. .
Free PMC article

Abstract

Pathogens may signal through multiple TLRs with synergistic or antagonistic effects on the induction of cytokines, including type I IFN (IFN-I). IFN-I is typically induced by TLR9, but not TLR2. Moreover, we previously reported that TLR2 signaling by Mycobacterium tuberculosis or other TLR2 agonists inhibited TLR9 induction of IFN-I and IFN-I-dependent MHC-I Ag cross processing. The current studies revealed that lipopeptide-induced TLR2 signaling inhibited induction of first-wave IFN-α and IFN-β mRNA by TLR9, whereas induction of second-wave IFN-I mRNA was not inhibited. TLR2 also inhibited induction of IFN-I by TLR7, another MyD88-dependent IFN-I-inducing receptor, but did not inhibit IFN-I induction by TLR3 or TLR4 (both Toll/IL-1R domain-containing adapter-inducing IFN-β dependent, MyD88 independent). The inhibitory effect of TLR2 was not dependent on new protein synthesis or intercellular signaling. IL-1R-associated kinase 1 (IRAK1) was depleted rapidly (within 10 min) by TLR2 agonist, but not until later (e.g., 2 h) by TLR9 agonist. Because IRAK1 is required for TLR7/9-induced IFN-I production, we propose that TLR2 signaling induces rapid depletion of IRAK1, which impairs IFN-I induction by TLR7/9. This novel mechanism, whereby TLR2 inhibits IFN-I induction by TLR7/9, may shape immune responses to microbes that express ligands for both TLR2 and TLR7/TLR9, or responses to bacteria/virus coinfection.

Figures

Figure 1
Figure 1. Mtb and LpqH-lipopeptide inhibit TLR9-induced IFN-I in a TLR2-dependent manner
A, B, Flt3L-derived DCs from wild-type C57BL/6 mice or TLR2−/− mice were cultured for 24 h with CpG-A ODN (300 nM) with or without Mtb H37Ra (MOI=1) (A) or LpqH-lipopeptide (400 nM) (B). C–F, Flt3L-derived DCs were cultured for 24 h with or without CpG-A ODN (300 nM) and various doses of LpqH-lipopeptide. Supernatants were assessed by ELISA for IFN-α (A, C) IFN-β (B, D), IL-12p40 (E) or IL-10 (F). Data represent means and standard deviations of triplicate wells and are representative of 3 or more independent experiments. Student’s t-test was performed to compare results with CpG-A ODN only and CpG-A ODN with Mtb or LpqH-lipopeptide (** = p < 0.01, *** = p < 0.001; n.d., not detected; n.s., not significant).
Figure 2
Figure 2. LpqH-lipopeptide inhibits TLR9-induced IFN-I mRNA expression
A–C, Flt3L-derived DCs (A, B) or GM-CSF DCs (C) were cultured for 3 h with medium, LpqH-lipopeptide (400 nM), CpG-A ODN (300 nM) or both agonists. Expression of mRNA for IFN-α and IFN-β was determined by qRT-PCR with normalization to GAPDH and is expressed as fold-change relative to expression with medium alone. Data represent means and standard deviations for triplicate samples and are representative of three or more independent experiments. D–G, FLt3L-derived DCs were cultured for various times (0, 2, 4, 7.5, 16, 24 h) with CpG-A ODN (300 nM) or CpG-A ODN + LpqH-lipopeptide (400 nM). Expression of mRNA for IFN-α and IFN-β was determined as above. Results are expressed as fold-change relative to expression in the non-treated (0 h) condition on a logarithmic scale (D, E) or as percent of the response to CpG-A ODN alone at each time point (F, G). Data represent means and standard deviations of triplicate samples and are representative of two independent experiments. Student’s t-test was performed to compare results with CpG-A ODN alone to results with CpG-A ODN plus LpqH-lipopeptide (* = p < 0.05, ** = p < 0.01, *** = p < 0.001; #, results are plotted but are too low to be visualized on this graph).
Figure 3
Figure 3. LpqH-lipopeptide inhibits induction of first wave IFN-I but not second wave IFN-I
A, Flt3L-derived DCs were left untreated or pretreated with LpqH-lipopeptide (400 nM) for 3 h before a 6-hour incubation with or without IFN-β (200 pg/ml) in the continued presence of LpqH-lipopeptide. B, DCs were simultaneously treated with medium, LpqH-lipopeptide, IFN-β or both agonists for 8 h. C–F, Flt3L-derived DCs from wild-type or IFN-IR−/− mice were cultured with medium, LpqH-lipopeptide (400 nM), CpG-A ODN (300 nM) or both agonists for 3 h (C, D) or 24 h (E, F). A–D, Expression of mRNA for IFN-β was determined by qRT-PCR with normalization to GAPDH and is expressed as fold-change relative to expression with medium alone. E, F, Supernatants were assessed by ELISA for IFN-β̃ Data in all panels represent means and standard deviations of triplicate samples and are representative of three or more independent experiments. Student’s t-test was performed to compare results with CpG-A ODN alone to results with CpG-A ODN plus LpqH-lipopeptide (*** = p < 0.001; #, results are plotted but are too low to be visualized on this graph; n.d., not detected).
Figure 4
Figure 4. TLR2-mediated inhibition of IFN-I production occurs by an intracellular mechanism
Incubations included Flt3L-derived DCs (3–5×106/well) from wild-type, TLR2−/− or TLR9−/− mice, or a 1:1 mixture of TLR2−/− and TLR9−/− DCs (total of 3–5×106 cells). DCs were cultured for 3 h in 12-well plates with medium, LpqH-lipopeptide (400 nM), CpG-A ODN (300 nM), or both agonists. Expression of mRNA for IFN-β was determined by qRT-PCR with normalization to GAPDH and is expressed as fold-change relative to expression with medium alone. Data represent means and standard deviations of triplicate samples and are representative of three or more independent experiments. Student’s t-test was performed to compare results with CpG-A ODN alone to results with CpG-A ODN plus LpqH-lipopeptide (*** = p < 0.001; #, results are plotted but are too low to be visualized on this graph).
Figure 5
Figure 5. TLR2-mediated inhibition is not dependent on new protein synthesis
Flt3L-derived DCs (3–5×106) were incubated without (A) or with (B) cycloheximide (5 μg/ml) for 1 h before the addition of medium, LpqH-lipopeptide (400 nM) or CpG-B ODN (3 μM) with or without CpG -A ODN (300 nM) for 3 h in the continued presence or absence of cycloheximide. Expression of mRNA for IFN-β was determined by qRT-PCR with normalization to GAPDH and is expressed as percent of control expression observed with CpG-A ODN alone within each cycloheximide treatment group. Data represent means and standard deviations of triplicate samples and are representative of three independent experiments. Student’s t-test was performed to compare results with CpG-A ODN only to results with CpG-A ODN plus LpqH-lipopeptide or results with CpG-A ODN plus CpG-B ODN (** = p < 0.01, *** = p < 0.001).
Figure 6
Figure 6. LpqH-lipopeptide inhibits MyD88-dependent but not TRIF-dependent induction of IFN-I expression
A, B, Flt3L-derived DCs were cultured for 3 h with medium, poly(I:C) (10 μg/ml), LPS (50 ng/ml), CpG-A ODN (300 nM) or ssRNA (1 μg/ml) with or without LpqH-lipopeptide (400 nM). Expression of mRNA for IFN-β was determined by qRT-PCR with normalization to GAPDH and is expressed as fold-change relative to expression with medium alone. C, Flt3L-derived DCs were cultured for 24 h with medium, LpqH-lipopeptide, ssRNA or both agonists. Supernatants were assessed by ELISA for IFN-β̃ Data represent means and standard deviations for triplicate samples and are representative of three or more independent experiments. Student’s t-test was performed to compare conditions as indicated (** = p < 0.01, *** = p < 0.001; #, results are plotted but are too low to be visualized on this graph; n.d., not detected).
Figure 7
Figure 7. LpqH-lipopeptide induces rapid IRAK1 depletion in DCs
A, Flt3L-derived DCs from wild-type C57BL/6 or IRAK1−/− mice were cultured for 3 h with medium, LpqH-lipopeptide (400 nM), CpG-A ODN (300 nM) or both agonists. Expression of mRNA for IFN-β was determined by qRT-PCR with normalization to GAPDH and is expressed as fold-change relative to expression with medium alone with wild type DCs B, Flt3L-derived DCs were left untreated or treated for various periods with CpG-A ODN (300 nM) or LpqH-lipopeptide (400 nM). C, Flt3L-derived DCs were cultured for 30 min or 120 min with medium, LpqH-lipopeptide (400 nM), CpG-A ODN (300 nM) or both. Cell lysates were prepared and analyzed by Western blot for IRAK1 and β-actin (loading control). Data in panel A represent the means and standard deviations of triplicate wells. Data are representative of 3 or more independent experiments. Student’s t-test was performed to compare conditions as indicated (*** = p < 0.001; #, results are plotted but are too low to be visualized on this graph).

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