Published online before print February 5, 2008
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B signaling during type-I IFN and TNF responses in human macrophagesInstitute of Veterinary Virology, University of Bern, Bern, Switzerland
1Correspondence: Institute of Veterinary Virology, University of Bern, Laenggassstrasse 122, Bern CH-3001, Switzerland. E-mail: reimer{at}ivv.unibe.ch
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B, were studied. We found that TNF production occurred rapidly after LPS stimulation. LPS induced a strong IFN-β mRNA response within a short time-frame, which subsided at 8 h. The IFN-stimulated genes (ISGs), ISG56 and IFN-inducible protein 10, were strongly induced by LPS. These responses were associated with NF-B and IRF3 activation, as shown by IRF3 dimerization and by nuclear translocation assays. poly(I:C), on the other hand, induced a strong and long-lasting (>12 h) IFN-β mRNA and protein response, particularly when transfected, whereas only a protracted TNF response was observed when poly(I:C) was transfected. However, these responses were induced in the absence of detectable IRF3 and NF-B signaling. Thus, in human macrophages, poly(I:C) treatment induces a distinct cytokine response when compared with murine macrophages. Additionally, a robust IFN-β response can be induced in the absence of detectable IRF3 activation.
Key Words: signal transduction • interferon • transcription factors • gene regulation • cytokines • innate immunity
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PAMPs frequently used to study the various pathways of innate immune activation are LPS and polyinosinic-polycytidylic acid [poly(I:C)]. LPS, a major component of the bacterial cell wall of gram-negative bacteria, is recognized by a receptor complex consisting of TLR-4/myeloid differentiation protein 2 and CD14 [4
5
6
7
]. poly(I:C) is a synthetic analog of dsRNA, a PAMP generated during the replication of RNA and DNA viruses [8
], and is recognized by distinct receptors depending on their localization. When added to the culture medium, poly(I:C) is mainly sensed by endosomally localized TLR-3 [9
, 10
], whereas transfected dsRNA is rather recognized via cytosolic melanoma differentiation-associated gene 5 (Mda-5) [11
, 12
]. LPS and dsRNA interacting with TLR-4, TLR-3, or Mda-5 activate different signaling pathways using various adaptor proteins leading to subsequent activation of TNFR-associated factor family member-associated NF-B activator-binding kinase 1 (TBK-1) and IFN regulatory factor 3 (IRF3) for the induction of type-I IFN and IFN-stimulated genes (ISGs), such as RANTES, ISG56, or IFN-inducible protein 10 (IP-10) [13
]. Furthermore, LPS as well as poly(I:C) induce the activation of NF-B [9
, 14
], followed by the release of TNF [15
, 16
].
This concept is based on findings made in mouse knockout models [17
]. This has revealed that the transcription factor IRF3 and its upstream kinase TBK-1 are essential components of the IFN-induction pathway, as cells deficient in IRF3 or TBK-1 only mount a reduced type-I IFN response after stimulation with various agents, including the TLR-4 agonist LPS and the dsRNA analog poly(I:C) [18
19
20
21
22
23
24
]. However, to understand disease processes in humans, it has to be secured that these findings can be extrapolated to relevant human host cells [25
26
27
]. Therefore, the activation of IRF3 and NF-B signaling in human macrophages was investigated. poly(I:C), added to the culture medium and transfected into the host cell, and LPS were used as stimuli to study the induction of IFN-β and TNF and activation of NF-B and IRF3 signaling.
We found a different signaling pattern in response to the TLR-4 agonist LPS and the TLR-3/Mda-5 agonist poly(I:C) in human macrophages. Despite the ability of both agents to activate a type-I IFN and TNF response, IRF3 and NF-B signaling was only activated after LPS stimulation. poly(I:C) neither induced detectable IRF3 dimerization nor nuclear translocation. Using an immunofluorescence staining for dsRNA, we show that these discrepancies cannot be the results of an insufficient uptake of poly(I:C) by human macrophages. Thus, LPS and poly(I:C) trigger a comparable cytokine response but induce a distinct activation pattern of the IRF3 and NF-B signaling pathways. Thus, although different PAMPs induce a similar cytokine response in human and mouse macrophages, the signaling pathways leading to their expression might be different. Most importantly, we show that poly(I:C), unlike LPS, can trigger a robust type-I IFN response without activating IRF3 signaling.
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PAGE and Western blotting
Whole cell protein extracts were prepared using M-PER mammalian protein extraction reagent (Pierce Biotechnology, Rockford, IL, USA), supplemented with a set of protease inhibitors (Roche Applied Bioscience, Rotkreuz, Switzerland) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich). For SDS-PAGE, 40 µg protein per lane was electrophoresed on a 10% polyacrylamide gel. Native PAGE to detect IRF3 dimerization was done exactly as described previously [30
]. Proteins were transferred to a nitrocellulose membrane (Amersham, Piscataway, NJ, USA) and blocked using 5% milk in PBS containing 0.1% Tween-20. IFN-β was detected using mouse mAb MMHB-3 (PBL, Piscataway, NJ, USA) at a dilution of 1:250. GAPDH was detected using a mouse mAb from Abcam (Cambridge, UK). Mouse antibodies were detected using a peroxidase-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at a dilution of 1:7000. IRF3 was detected using a rabbit polyclonal antibody (Abcam) at a dilution of 1:1000. Rabbit antibodies were detected using a peroxidase-conjugated mouse anti-rabbit mAb (Sigma-Aldrich) at a dilution of 1:4000. ECL (Amersham) was used as a substrate and detected with a charged coupled device camera (Fujifilm LAS3000) and Aida image software (Raytest, Wetzikon, Switzerland).
Immunofluorescence staining
For immunfluorescence staining of IRF3 and dsRNA, macrophages were fixed with methanol and stored at –20°C until use. Samples were rehydrated with PBS containing 0.1% Tween-20. FcRs were blocked using human Ig (Berna Biotech, Bern, Switzerland) at a concentration of 10 mg/ml. IRF3 antibody (Abcam) was diluted 1:500 in PBS containing 10% FCS and 0.1% Tween-20 and was detected with an Alexa-Fluor 594-conjugated goat anti-rabbit F(ab)2 antibody (Invitrogen). dsRNA was detected using a mouse mAb (clone J2, Biocenter Ltd., Szeged, Hungary) at a dilution of 1:10,000 in PBS containing 10% FCS and 0.1% Tween-20. Mouse mAb was detected using an Alexa-Fluor 594-conjugated goat anti-mouse IgG2a antibody (Invitrogen). 4',6-Diamidino-2-phenylindole (DAPI) was used as nuclear counterstain. Samples were analyzed with a Nikon E80i epifluorescence microscope. Images were processed using OpenLab software (Improvision, Coventry, UK).
ELISA
TNF was measured in duplicates using an ELISA kit purchased from Biosource (Camarillo, CA, USA) according to the manufacturer’s instruction. 3,3',5,5'-Tetramethylbenzidine, purchased from Sigma-Alrich, was used as a substrate. ODs were determined at 650 nm using an automated ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). TNF was quantified using an international standard (NIBSC, Hertfordshire, UK). IFN-β ELISA was purchased from Biosource and performed according to the manufacturer’s instruction.
Quantitative RT-PCR (qRT-PCR)
RNA was extracted using a nucleic acid purification lysis solution (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instruction. RNA was isolated on RNA purification trays (Applied Biosystems) using an ABI Prism 6100 Prepstation (Applied Biosystems). Real-time qRT-PCR was performed on an Applied Biosystems 7300 or 7500 real-time PCR system. Primers were ordered from Microsynth (Balgach, Switzerland). Sequences were as follows: IFN-β probe: 5'-ACA AAG AAG CAG CAA TTT TCA GTG TCA GAA GCT; IFN-β forward: 5'-GAG CTA CAA CTT GCT TGG ATT CC; IFN-β reverse: 5'-CAA GCC TCC CAT TCA ATT GC; ISG56 probe: 5'-TTG CTA CAA GGC ACA AAT GAT CCA A; ISG56 forward: 5'-CTG TCT TAC TGC ATC ACC AGA TAG G; ISG56 reverse: 5'-CCC TCT AGG CTG CCC TTT TG; IP-10 probe: 5'-TGT CCA CGT GTT GAG ATC ATT GCT ACA ATG; IP-10 forward: 5'-TGA AAT TAT TCC TGC AAG CCA ATT; IP-10 reverse: 5'-CAG ACA TCT CTT CTC ACC CTT CTT T; GAPDH probe: 5'-CCC CCA TGT TCG TCA TGG GTG TG; GAPDH forward: 5'-CTC TGC CCC CTC TGC TGA T; GAPDH reverse: 5'-TGA TGA TCT TGA GGC TGT TGT CA. Data are expressed as mRNA expression levels to the levels of GAPDH mRNA (1/2
Ct).
Statistical analysis
Student’s t-test was used to detect statistical significances; *, P < 0.05; **, P < 0.01.
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10,000-fold increase in abundance of IFN-β mRNA, which was highest at 4 h p.i. At later time-points, such as 8 h and 12 h, mRNA levels were reduced again. IFN-β mRNA in mock-treated cells was only occasionally detected. Infection of macrophages with Sendai virus served as a positive control for IFN-β and ISG induction. Briefly, Sendai virus triggered IFN-β gene expression in a manner comparable with that obtained by transfected poly(I:C) (Fig. 1)
. We next assessed expression of the IFN-inducible genes ISG56 and IP-10. Transfection of poly(I:C) led to an increased expression of ISG56 and IP-10 when compared with the induction when poly(I:C) was only added to the cell culture medium (Fig. 1B
and 1C)
. The transfection reagent itself induced increased expression of ISG56 and IP-10, albeit to an at least 100-fold lesser extent when compared with transfected poly(I:C). Sendai virus induced expression of ISG56 and IP-10 mRNA, which was comparable with the induction triggered by transfected poly(I:C). When macrophages were treated with LPS, ISG56 and IP-10 were induced to a similar extent as with transfected poly(I:C) (Fig. 1B
and 1C)
. In contrast to the IFN-β mRNA expression induced by LPS, the high expression levels of IP-10 and ISG56 were maintained for 12 h of LPS stimulation. To assess whether the increase in IFN-β mRNA is reflected in the protein production, macrophages were stimulated for 12 h in the presence of Brefeldin to block cytokine release. Protein extracts were prepared and probed for the presence of IFN-β protein by immunoblotting (Fig. 2A
). IFN-β protein was observed after transfection of poly(I:C), whereas it remained undetectable when poly(I:C) was only added to the cell culture medium, even when increasing the concentration of poly(I:C) to 100 µg/ml (data not shown). LPS stimulation did not induce detectable amounts of IFN-β protein within 12 h of stimulation (data not shown). To confirm the results in a more quantitative manner, we performed an ELISA to detect IFN-β protein in the culture supernatant (Fig. 2B)
. Consistent with results obtained from qRT-PCR experiments (Fig. 1A)
and Western blotting (Fig. 2A)
, transfected poly(I:C) induced release of IFN-β protein to the culture supernatant that was readily detectable (Fig. 2B)
. LPS-triggered IFN-β protein was hardly detectable in the culture supernatant and only at the earliest time-point assessed (4 h).
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Figure 1. Expression of IFN-β, ISG56, and IP-10 mRNA. Human macrophages were stimulated with 100 ng/ml LPS or 1 µg/ml or 10 µg/ml poly(I:C) (pIC), which was added to the cell culture medium or transfected using lipofectin (L.). Sendai virus was used as a positive control, and macrophages were infected at a multiplicity of infection (MOI) of 3. At 4 h, 8 h, and 12 h, cells were lysed, and RNA was isolated and subjected to real-time RT-PCR. Relative amounts of (A) IFN-β mRNA, (B) ISG56 mRNA, and (C) IP-10 mRNA are expressed as mRNA expression levels to GAPDH mRNA (1/2Ct). IFN-β mRNA remained undetected in mock-treated cells except in one blood donor at 8 h after treatment. Data of four different blood donors are pooled, and means and SEM are shown.
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Figure 2. Expression of IFN-β protein. Macrophages were stimulated with 1 µg/ml or 10 µg/ml poly(I:C), which was added to the culture medium or transfected using lipofectin (Lipof.). (A) Cytokine release was blocked using Brefeldin at a concentration of 1 µg/ml. At 12 h poststimulation, whole cell protein extracts were prepared and separated by SDS-PAGE. Western blotting was performed to detect IFN-β protein levels. GAPDH served as a control for equal loading. Data from one blood donor out of two are shown. (B) Culture supernatant was collected at time-points between 4 and 24 h after stimulation, and ELISA was performed to assess IFN-β released into the culture supernatant. The amount of IFN-β detected is expressed as U/ml.
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Figure 3. Release of TNF. Macrophages were treated with poly(I:C), which was added to the cell culture medium or transfected using lipofectin or stimulated with 100 ng/ml LPS. Between 4 and 24 h, TNF release was assessed using an ELISA kit. OD was determined at 650 nm. Data are expressed as IU/ml. The number of blood donors tested is indicated in each graph; bars indicate mean; error bars SEM. Statistical significances were assessed to mock-treated cells (n.s., not significant).
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B signaling is activated immediately after LPS but not after poly(I:C) treatmentB signaling was assessed, as NF-B is important for TNF release and contributes to IFN-β expression [16
, 19
]. Therefore, macrophages were treated with poly(I:C), which was added to the cell culture medium, transfected using lipofectin, or stimulated with LPS, which induced the phosphorylation of the NF-B subunit p65 at serine 536 and the degradation of IB
within 15 min of treatment (Fig. 4
). IB was expressed at higher levels after LPS stimulation when compared with mock-treated cells at 4 h, pointing to transcriptional activity of NF-B (Fig. 4)
. These responses were not observed after treatment of macrophages with poly(I:C): Neither the phosphorylation of NF-B p65 nor the degradation of IB was detected within 4 h of poly(I:C) treatment.
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Figure 4. Activation of NF-B signaling. Macrophages were treated with 1 µg/ml and 10 µg/ml poly(I:C), which was added to the culture medium or transfected using lipofectin. Alternatively, macrophages were treated with 100 ng/ml LPS. At the indicated time-points, whole cell protein extracts were prepared and subjected to SDS-PAGE. Western blotting was performed to detect the expression of IB and the phosphorylation of NF-B p65 (P-p65) at serine 536 (S536). Tubulin served as a control for equal loading. Representative data of two blood donors out of three tested are shown.
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B signaling is not a result of a failure of macrophages to immediately take up poly(I:C).
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Figure 5. IRF3 dimer formation. Whole cell protein extracts were prepared between 1 and 8 h after stimulation of macrophages with (A) 100 ng/ml LPS or (B) 1 µg/ml and 10 µg/ml poly(I:C), which was added to the cell culture medium or transfected using lipofectin. A sample of VSV (MOI of 3 for 4 h)-infected macrophages served as a positive control to validate the method. One of two blood donors with similar results is shown.
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Figure 6. Nuclear translocation of IRF3. Macrophages were stimulated with 100 ng/ml LPS or 1 µg/ml and 10 µg/ml poly(I:C), which was added to the culture medium or transfected using lipofectin. Cells were methanol-fixed at the indicated time-points and stained for IRF3 (labeled gray and red, respectively) to detect nuclear translocations. DAPI (blue) served as a nuclear counterstain. Data from two blood donors are shown.
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Figure 7. Immunofluorescence staining for poly(I:C). Macrophages were stimulated with 1 µg/ml and 10 µg/ml poly(I:C), which was added to the cell culture medium or transfected using lipofectin. At 1 h after treatment, macrophages were fixed with methanol and stained for dsRNA (red). Image overlays with DAPI (blue) are shown to reveal that every macrophage contains poly(I:C) when transfected. Data from two blood donors are shown.
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B, LPS induced an activation of both pathways: IRF3 dimerization was detected in native PAGE, and nuclear translocation was observed in immunofluorescence stainings. NF-B signaling was detected at the levels of IB degradation, NF-B p65 phosphorylation and subsequent enhanced expression of IB, revealing transcriptional activity of NF-B. When extrapolating from studies conducted in the mouse, a comparable response would have to be expected following poly(I:C) stimulation. As poly(I:C) can be recognized predominantly by TLR-3 when just added to the culture medium or predominantly via Mda-5 when transfected [11
], both administration routes to assess a poly(I:C)-induced cytokine response were used. When transfecting poly(I:C), a considerable and sustained type-I IFN response at the mRNA level as well as at the level of IFN-β protein was observed (Figs. 1
and 2)
. When poly(I:C) was added to the culture medium, only a minimal induction of IFN-β mRNA was observed, and IFN-β protein was not detected. Lipofectin itself induced IFN-β gene expression and induction of IFN-inducible genes. We also observed induction of Mx protein after lipofectin treatment of THP-1 cells, a human monocytoid cell line (data not shown), confirming the observation made by others that liposomal transfection reagents may induce a type-I IFN response [31
]. This induction is unlikely to be mediated by endotoxin, as TNF release was not triggered by lipofectin but by LPS. When poly(I:C) was transfected, TNF release was only induced at later time-points (Fig. 3)
, despite that transfection was immediately efficient, as shown by the type-I IFN response (Fig. 1)
and the immunostaining for dsRNA (Fig. 7)
. For unknown reasons, we observed the release of TNF after transfection of poly(I:C) in the absence of detectable signs of an activation of the NF-B pathway (Figs. 3
and 4)
. This discrepancy may be a result of a varying sensitivity of the methods used; alternatively, TNF may be induced in an indirect manner after poly(I:C) transfection.
When investigating the activation of the IRF3 and NF-B signaling pathways, neither addition of poly(I:C) to the culture medium nor transfection using lipofectin induced detectable IRF3 dimer formations or nuclear translocations (Figs. 5
and 6)
. This is a striking result, as transfection strongly affected IFN-β, ISG56, and IP-10 expression (Fig. 1)
, and these genes are thought to depend on IRF3 or type-I IFN [13
, 32
]. Furthermore, when compared with LPS, induction of IFN-β and ISG by transfected poly(I:C) was similar. Additionally, IFN-β protein was readily detected after poly(I:C) transfection but remained undetectable following LPS treatment (Fig. 2
, and data not shown). However, a LPS-triggered response was accompanied by IRF3 dimer formation and nuclear translocation, and a poly(I:C)-triggered response was not. Thus, the absence of detectable IRF3 signaling after poly(I:C) transfection is unlikely a result of an insufficient induction of IFN-β gene expression. As IRF3 is thought to be a key signal transducer for a type-I IFN response, this result suggests that at least in human macrophages, additional factors might determine quantity and kinetics of IFN-β and ISG expression. Finally, to rule out the possibility that not every macrophage takes up poly(I:C), we performed immunofluorescence staining with an antibody against poly(I:C) (Fig. 7)
. These stainings revealed that when poly(I:C) was only added to the culture medium, it was hardly detected in human macrophages, whereas transfection of poly(I:C) revealed a specific staining pattern. Thus, the failure of macrophages to translocate IRF3 to the nucleus after poly(I:C) transfection is unlikely to be a result of defective poly(I:C) uptake. Additionally, the delayed TNF response cannot be attributed to a slow uptake of poly(I:C).
These data show critical differences in the initiation of cytokine responses in human macrophages when compared with murine macrophages or cell lines. First, neither TLR-3- nor Mda-5-mediated recognition of poly(I:C) leads to an immediate activation of NF-B signaling and only a delayed TNF response. Second, despite robust and sustained expression of IFN-β, ISG56, and IP-10 mRNA after poly(I:C) transfection, no sign of an activation of IRF3 signaling was detected. This is in contrast to a LPS stimulation, which led to a similar type-I IFN response but to readily detectable IRF3 dimerization and nuclear translocation. This suggests that IRF3 is differentially involved in the induction of a type-I IFN response in human macrophages when compared with other cell types or cells of other species and also points to additional factors that might function as on-off switches for IFN responses in this particular cell type. A study by Romieu-Mourez et al. [33
] revealed that a constitutively active form of IRF3 induces cell death rather than type-I IFN in human macrophages, whereas a constitutively active form of IRF7 induced a type-I IFN response. We suggest that a particular signaling pathway in specialized cells can have different biological outcomes. In a recently published study, Lundberg et al. [34
] describe an observation comparable with ours: Human myeloid cells such as macrophages and dendritic cells (DC) failed to activate IRF3 after poly(I:C) treatment, whereas endothelial cells and cells of synovial fluid did so. Yet, IFN-β is induced in all cell types. This study and our results show that the function of a signaling pathway in one model cannot necessarily be extrapolated to other biological systems, and extrapolation of an observation made in one model should only be applied with extreme caution to other models and species.
Honda et al. [35
] suggest IRF7 to be a master regulator of type-I IFN-mediated responses. Thus, it would be intriguing to investigate whether IFN-induction mechanisms not relying on IRF3 may exist in cells other than plasmacytoid DC. Different cells may use different signaling pathways to induce expression of IFN-β. Using human monocytoid cell lines, Lu and Pitha [36
] suggested that IRF7 is required for monocyte-to-macrophage transition. However, we [28
] only observed IRF7 protein during the course of a type-I IFN response but never in unstimulated or unprimed cells, suggesting that human macrophages do not constitutively express IRF7 protein. Therefore, it seems unlikely that IRF7 acts in place of IRF3 in human macrophages [28
]. Another study investigated cytokine responses of human fetal astrocytes [37
]. It was observed that stimulation with IL-1β induces activation of IRF3 and expression of a set of ISGs. Thus, a specialization or "fine tuning" of IRF3 signaling in different cell types may exist, and IRF3 might not only function as an on-off switch for type-I IFN as other models may suggest [23
, 24
, 38
]. Furthermore, IRF1 has been suggested to be a transcription factor contributing to type-I IFN induction [39
], and recently, Schmitz et al. [40
] implicated IRF1 in mediating CpG-triggered induction of IFN-β in myeloid DC and macrophages. This may suggest that the trigger applied determines the signaling pathway activated for the induction of IFN-β. We also tested for an activation of IRF1 signaling following poly(I:C) stimulation but only observed a nuclear translocation of IRF1 in response to IFN-
. poly(I:C) did not induce a detectable activation of IRF1 signaling (data not shown).
As a result of the fast kinetics of LPS-triggered cytokine induction, we may have missed the time-point of maximal response. Nevertheless, this does not invalidate the major finding of this study.
In summary, we show that poly(I:C) and LPS induce an overlapping cytokine response, albeit at different kinetics. Remarkably, transfected poly(I:C) led to the induction of IFN-β and ISGs in the absence of detectable IRF3 activation in human monocyte-derived macrophages.
Received June 19, 2007; revised November 16, 2007; accepted November 30, 2007.
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