Murine toll-like receptor 2 activation induces type I interferon responses from endolysosomal compartments

Abstract

Background: Toll-like receptors (TLRs) are among the first-line sentinels for immune detection and responsiveness to pathogens. The TLR2 subfamily of TLRs (TLR1, TLR2, TLR6) form heterodimers with each other and are thus able to recognize a broad range of components from several microbes such as yeast, Gram-positive bacteria and protozoa. Until now, TLR2 activation by bacterial ligands has long been associated with pro-inflammatory cytokines but not type I interferon responses.

Methodology/principal findings: Using a variety of transgenic mice, here we provide in vivo and in vitro data showing that TLR2 activation does in fact induce interferon-beta and that this occurs via MyD88-IRF1 and -IRF7 pathways. Interestingly, by microscopy we demonstrate that although a cell surface receptor, TLR2 dependent induction of type I interferons occurs in endolysosomal compartments where it is translocated to upon ligand engagement. Furthermore, we could show that blocking receptor internalization or endolysosomal acidification inhibits the ability of TLR2 to trigger the induction type I interferon but not pro-inflammatory responses.

Conclusion/significance: The results indicate that TLR2 activation induces pro-inflammatory and type I interferon responses from distinct subcellular sites: the plasma membrane and endolysosomal compartments respectively. Apart from identifying and characterizing a novel pathway for induction of type I interferons, the present study offers new insights into how TLR signaling discriminates and regulates the nature of responses to be elicited against extracellular and endocytosed microbes. These findings may also have clinical implication. Excessive production of pro-inflammatory cytokines and type I IFNs following activation of TLRs is a central pathologic event in several hyper-inflammatory conditions. The discovery that the induction of pro-inflammatory and type I IFN responses can be uncoupled through pharmacological manipulation of endolysosomal acidification suggests new avenues for potential therapeutic intervention against inflammations and sepsis.

Figure 1. TLR2 directly induces pro-inflammatory and type I IFN dependent responses via MyD88.

WT, TLR2^−/−, MyD88^−/−, IFN-β^−/− and IFNAR1^−/− macrophages were stimulated with 1.5 µg/ml of MALP-2 or PAM2CSK4 or PAM3CSK4 then analyzed 6 h later by real-time RT-PCR for mRNAs of TNF-α (A), IP-10 (B), Mx-2 (E) and IFN-β (C). In parallel, culture supernatants of cells stimulated for 24 h or 10 h were analyzed by ELISA for concentrations of IL-12p70 (D) or IFN-β (F) respectively. Data are representative of at least three independent experiments (mean ± s.e.m).

Figure 2. TLR2 activation induces IFN-β transcription independent of an auto/paracrine loop.

In A, BMDMs were stimulated with either MALP-2 (1.5 µg/ml) or Poly I:C (5 µg/ml) for the indicated time periods then analyzed by quantitative real time RT-PCR for IFN-β mRNA. B–D; TLR2 driven IFN-β induction is independent of an auto/paracrine mechanisms. BMDMs pre-treated for 1 h with or without 0.5 µg/ml CHX were stimulated for 6 h with either MALP-2 (1.5 µg/ml) or Poly I:C (5 µg/ml) then analyzed by real-time RT-PCR for IFN-β (B, please note logarithmic scale) and TNF-α (C). Culture supernatants of cells stimulated in parallel for 24 hours in the presence of cycloheximide were analyzed for nitric oxide (D). Data are representative of three independent experiments (mean ± s.e.m).

Figure 3. TLR2-driven IFN-β and type I IFN dependent responses in vivo.

A–B; Five mice per group WT, TLR2^−/− and IFNAR^−/− were injected with control medium or PAM3CSK4 (100 µg/mouse) then sacrificed at different time points between 0–18 hours and their sera (A) or peritoneal exudates (B) analyzed by ELISA for IFN-β and IP-10 respectively Data representative of two independent experiment show ± s.e.m. C; Three mice per group of TLR2^+/+IFN-β^+/Δ-luc (TLR2^+/+) and TLR2^−/−IFN-β^+/Δ-luc (TLR2^−/−) mice were injected with MALP-2 (100 µg/mouse) and expression of luciferase was monitored for 24 h by luminescence imaging. C shows two representative mice out of a group of four mice before (0 hours) and after (3 hours) MALP-2 administration. Fold increase and kinetics of luciferase induction of corresponding mice are depicted in Fig. S2A. D–I; TLR2 mediated IFN-β induction in macrophages. Macrophages matured from bone marrows of TLR2^+/+IFN-β^+/Δ-luc or TLR2^−/−IFN-β^+/Δ-luc mice were stimulated with indicated concentrations of MALP-2 (D, G) or PAM2CSK4 (E, H) or PAM3CSK4 (F, I) for 4 hours and analyzed for luciferase expression. D–F; show representative luminescence images of macrophages while G–I indicate the fold increase in luciferase in corresponding cells. Luciferase expression is represented by a colour shift from blue to red. Similar results were obtained from three independent experiments (mean ± s.e.m). Note that the background response in reporter mice, mainly in the liver region, might be due to the liver's role in trapping and detoxifying noxious substances from the system. Such substances and the metabolic products thereof could account for the higher MALP-2 associated background in TLR2−/− mice.

Figure 4. TLR2 activation induces type I IFN-β via IRF1 and IRF7.

A–C, WT, TLR2 ^−/−, IRF1 ^−/−, IRF3 ^−/−, and IRF7 ^−/− BMDMs were stimulated with ligands for TLR2/6 (1.5 µg/ml MALP-2 or PAM2CSK4) or TLR2/1 (1.5 µg/ml PAM3CSK4). After 10 hours, supernatants were analyzed for IFN-β by ELISA. Similar data represent an average of two independent experiments (mean ± s.e.m, *: p<0.05).

Figure 5. Ligand induced internalization and trafficking of TLR2 into endolysosomal compartments.

RAW 264.7 macrophages transfected with hTLR2-GFP were labelled with lysotracker then left untreated (A) or stimulated with 1 µg/ml of MALP-2 and imaged by confocal microscopy after 3 h (B). Panel C shows cells first labelled with lysotracker then incubated for 1 hour with 50 µM Bafilomycin A before stimulation with MALP-2. The co-localization fluorographs displayed in the right panels show the intensities and scatter pattern of all pixels within the merged images. Pixels with mostly one fluorescent component are placed along the axes 1 and 2 while the pixels with equal fluorescence intensity from both components (due to co-localization) are placed along the diagonal. Axis 1 and 2 show the green (TLR2-GFP) and red (Lysotracker) fluorescence intensities on an arbitrary scale of 0 to 250.

Figure 6. Blockers of receptor internalization and endosome maturation pathways abrogate TLR2 driven type I IFN responses.

BMDM, untreated or pre-treated with either 25 µM Cytochalasin D (A–C) or 50 µM Bafilomycin A (D–F) for 1 hour, were stimulated with 1.5 µg/ml of the indicated TLR2 ligands. After 6 h cell pellets were analyzed by quantitative real-time RT-PCR for IFN-β (A and D). Supernatants of cells stimulated in parallel for 24 hours were also analyzed for Nitric Oxide (Nitrite) by Griess reaction (B and E) or for TNF-α. Data represent similar results from three independent experiments (mean ± s.e.m, *: p<0.05). G; Model for TLR2 signaling. Upon ligand engagement at the plasma membrane TLR2 activates a MyD88-NF-κB pathway leading to the induction of pro-inflammatory cytokines like TNF-α and IL-12. The initial trigger at the plasma membrane also induces endocytic internalization and transportation of the TLR2 complex into endolysosomal compartments from where it activates MyD88-IRF1/IRF7 pathways of IFN-β induction. Once produced, IFN-β in turn activates STAT1 which alone or in synergy with other transcription factors like NF-κB activates the transcription of IFN-inducible genes; Mx-2, IP-10, iNOS, IL-6.