Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to Toxoplasma gondii profilin

Abstract

TLRs play a central role in the innate recognition of pathogens and the activation of dendritic cells (DCs). In this study, we establish that, in addition to TLR11, TLR12 recognizes the profilin protein of the protozoan parasite Toxoplasma gondii and regulates IL-12 production by DCs in response to the parasite. Similar to TLR11, TLR12 is an endolysosomal innate immune receptor that colocalizes and interacts with UNC93B1. Biochemical experiments revealed that TLR11 and TLR12 directly bind to T. gondii profilin and are capable of forming a heterodimer complex. We also establish that the transcription factor IFN regulatory factor 8, not NF-κB, plays a central role in the regulation of the TLR11- and TLR12-dependent IL-12 response of DCs. These results suggest a central role for IFN regulatory factor 8-expressing CD8(+) DCs in governing the TLR11- and TLR12-mediated host defense against T. gondii.

Figure 1. TLR12 localizes intracellularly and associates with TLR11 and UNC93B1

(A) HEK293 cells were plated on glass coverslips and co-transfected with TLR11-GFP and TLR12-mCherry. After 24 hours, the cells were imaged with a Leica SPE microscope equipped with a 63× objective. (B) HEK293 cells were plated on glass coverslips and co-transfected with TLR12-GFP and UNC93B1-mCherry. After 24 hours, the cells were imaged with a Leica SPE microscope equipped with a 63× objective. (C) HEK293 cells were co-transfected with (1) TLR11-GFP and myc-MyD88, (2) TLR11-GFP and myc-TLR11, (3) TLR11-GFP and myc-TLR12, (4) TLR12-GFP and myc-MyD88, (5) TLR12-GFP and myc-TLR11, (6) TLR12-GFP and myc-TLR12, (7) UNC93B1-GFP and myc-MyD88, (8) UNC93B1-GFP and myc-TLR11, (9) UNC93B1-GFP and myc-TLR12, (10) CD3z-GFP and myc-MyD88, (11) CD3z-GFP and myc-TLR11, or (12) CD3x-GFP and myc-TLR12. Thirty-six hours after transfection, the cell lysates were immunoprecipitated (IP) with anti-GFP antibody, and the immunoprecipitated proteins were analyzed by immunoblotting (IB) with anti-myc or anti-GFP antibodies to detect myc- or GFP-tagged proteins, respectively. A representative result from five experiments is shown.

Figure 2. T. gondii profilin directly interacts with TLR11 and TLR12

(A) ELISA plates were coated with T. gondii profilin (PFTg, 10µ/ml) or BSA (10 µg/ml) in 10 mM Tris (pH 6.0) and 150 mM NaCl. After blocking free binding sites with 5% fat-free milk, plates were incubated with 1 µg/ml of purified TLR11 (green), TLR12 (red), TLR13 (black) or BSA (blue). Binding of the TLR ectodomains was detected with a DED-specific monoclonal antibody. All ELISA steps were performed at pH 6.0. (B) ELISA plates were coated with T. gondii profilin (PFTg, 10µ/ml) or BSA (10 µg/ml) in 10 mM Tris (pH 8.0) and 150 mM NaCl. After blocking free binding sites with 5% fat-free milk, plates were incubated with 1 µg/ml of purified TLR11 (green), TLR12 (red), TLR13 (black) or BSA (blue). Binding of the TLR ectodomains was detected with a DED-specific monoclonal antibody. All ELISA steps were performed at pH 8.0. (C) The ELISA plates were coated with 10 µg/ml TLR11, TLR12, TLR13, or BSA in 10 mM Tris (pH 6.0) and 150 mM NaCl. After blocking and washing steps, recombinant profilin (10 µg/ml) was added to the wells. Profilin was detected with a T. gondii profilin-specific rabbit polyclonal antibody. All ELISA steps were performed at pH 6.0. A representative result from at least ten experiments is shown. (D) T. gondii profilin (2 µM) alone (blue) or pre-incubated with 0.2 µM purified TLR11 ectodomain (red). Biochemical fractionation of T. gondii profilin (PFTg) or PFTg+TLR11 (red) was performed by gel filtration chromatography, and each fraction was tested for T. gondii profilin by ELISA (left). In the right panel, the PFTg+TLR11 fractions (red) were additionally tested for TLR11 compared to TLR11 alone (green) and analyzed according to the same gel filtration chromatography procedure with a Superdex 200 10/300 GL chromatographic separation column. (E) PFTg alone (blue), PFTg+TLR12 (red), or TLR12 alone were analyzed by gel filtration chromatography as described in C. Each fraction was tested for T. gondii profilin (left) or TLR12 (right) by ELISA. The chromatograms are representative of three independent experiments. (F) PFTg alone (blue), PFTg+TLR13 (red), or TLR13 alone were analyzed by gel filtration chromatography as described in D and E. Each fraction was tested for T. gondii profilin (left) or TLR13 (right) by ELISA. The chromatograms are representative of three independent experiments.

Figure 3. TLR12 participates in the regulation of IL-12 responses triggered by T. gondii profilin

(A) Flt3L-derived DCs were transduced with lentiviral constructs expressing miRNA against MyD88 (blue) or TLR12 (red). After 48 hours, the DCs were stimulated with 1 µg/ml T. gondii profilin, and IL-12/23p40 production was measured by ELISA twenty hours after stimulation. (B) Flt3L-derived DCs were transfected with control or TLR12-targeting siRNA constructs and stimulated with T. gondii profilin. IL-12/23p40 production was measured by ELISA twenty hours after stimulation. The data shown are representative of four experiments.

Figure 4. TLR12 and TLR11 did not induce detectable NF-κB activation

(A) HEK 293 cells were transfected with a plasmid encoding TLR11, TLR12, or TLR11 and TLR12, in addition to a 5×-NF-κB-luciferase reporter. After overnight stimulation with 1 µg/ml of T. gondii profilin (green) or media alone (grey), the cells were lysed and assayed for luciferase activity. The error bars represent the standard deviation. (B) Purified CD8+ DCs were stimulated with 1 µM CpG or 1 µg/ml PFTg, and NF-kBp65 was analyzed six hours after stimulation. At least 100 CD8+ DCs were analyzed in each of the three experiments. (C) Splenic DCs were isolated from WT (black), NF-κB1−/− (red), or NF-κB2−/− (blue) mice and stimulated with 1 µg/ml PFTg or 1 µM CpG. IL-12/23p40 and TNF productions were measured by ELISA twenty hours after stimulation. The data shown are representative of two experiments. (D) Splenic DCs were isolated from WT mice and stimulated with 1 µg/ml PFTg (red) or 1 µM CpG (blue) in the presence of increasing concentrations of a pan-NF-kB inhibitor BAY-11-708 (0 uM, 1 uM, and 5 uM). IL-12/23p40 and TNF productions were measured by ELISA twenty hours after stimulation. The data shown are representative of three experiments.

Figure 5. IRF8 regulates the TLR12- and TLR11-dependent IL-12 responses of DCs triggered by T. gondii profilin

(A) Splenic CD11c+ DCs were prepared from WT, IRF8−/−, and IRF4−/− mice and stimulated with 1 µg/ml of T. gondii profilin (PFTg) or 1 µM CpG for 20 hours. IL-12/23p40 (left) and TNF (right) production were measured by ELISA. (B) Flt3L-derived DCs were generated from bone marrow cells prepared from WT, IRF8−/−, TLR11−/−, and MyD88−/− mice and were retrovirally transduced with the IRF8 (left) or control (right) vectors. Both IRF8-rescued and control DCs were stimulated with 1 µg/ml PFTG or 1 µM CpG for twenty hours. IL-12/23p40 productions were analyzed by ELISA. The data shown are representative of four experiments. (C) WT or IRF8−/− bone-marrow macrophages or (D) sort-purified CD11c+CD8−CD11b+ DC were incubated with PBS or rIFN-γ (10ng/ml) overnight and then were stimulated with 1 µg/ml PFTg or 1 µM CpG. IL-12/23p40 production was analyzed by ELISA twenty hours later. The data shown are representative of four experiments. (E) WT and IRF8−/− mice were injected with T. gondii profilin (10 µg/mouse) and IL-12/23p40 production by splenic DCs was analyzed by intracellular staining four hours later by intracellular staining for IL-12/23p40 and IRF8. (F) WT mice were injected with PBS or rIFN-γ (1µg/mouse) overnight. The next day mice were injected with T. gondii profilin (10 µg/mouse) and IL-12/23p40 production by splenic CD8+ and CD8− DCs was analyzed by intracellular staining. The data shown are representative of two experiments.