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Differential outcome of TRIF-mediated signaling inTLR4 and TLR3 induced DC maturationWei Hu1, Aakanksha Jain, Yajing Gao, Igor M. Dozmorov, Rajakumar Mandraju, Edward K. Wakeland,and Chandrashekhar Pasare2
Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9093
Edited by Akiko Iwasaki, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, and accepted by the Editorial BoardSeptember 30, 2015 (received for review June 1, 2015)
Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) on dendritic cells (DCs) leads to DC matura-tion, a process involving up-regulation of MHC and costimulatorymolecules and secretion of proinflammatory cytokines. All TLRsexcept TLR3 achieve these outcomes by using the signalingadaptor myeloid differentiation factor 88. TLR4 and TLR3 can bothuse the Toll–IL-1 receptor domain-containing adaptor inducing IFN-β(TRIF)-dependent signaling pathway leading to IFN regulatoryfactor 3 (IRF3) activation and induction of IFN-β and -α4. The TRIFsignaling pathway, downstream of both of these TLRs, also leadsto DCmaturation, and it has been proposed that the type I IFNs act incis to induce DC maturation and subsequent effects on adaptiveimmunity. The present study was designed to understand the mo-lecular mechanisms of TRIF-mediated DC maturation. We have dis-covered that TLR4–TRIF-induced DC maturation was independent ofboth IRF3 and type I IFNs. In contrast, TLR3-mediated DC maturationwas completely dependent on type I IFN feedback. We found thatdifferential activation of mitogen-activated protein kinases by theTLR4– and TLR3–TRIF axes determined the type I IFN dependencyfor DC maturation. In addition, we found that the adjuvanticity ofLPS to induce T-cell activation is completely independent of type IIFNs. The important distinction between the TRIF-mediated signalingpathways of TLR4 and TLR3 discovered here could have a majorimpact in the design of future adjuvants that target this pathway.
MAP kinases | LPS | Poly I:C | MAVS | NF-κB activation
Toll-like receptors (TLRs) are a major family of pattern rec-ognition receptors (PRRs) that recognize conserved microbial
products from a diverse class of pathogens (1). Upon recognitionof cognate ligands, TLRs initiate a signaling cascade, resulting inactivation of several transcription factors including NF-κB, AP-1,and IFN regulatory factors (IRFs) (1). The specificity of signaling isdictated both by the physical location of the receptor and by thesignaling adaptor use by each TLR (2). The outcome of TLRsignaling is robust activation of induced innate immunity in theform of enhanced phagocytosis (3) and increased reactive oxygenspecies production (4), as well as synthesis and secretion of severalproinflammatory cytokines and chemokines by cells of myeloidlineage (5). TLRs also regulate adaptive immunity by induction ofdendritic cell (DC) maturation. DC maturation is a process bywhich DCs up-regulate expression of MHC and costimulatorymolecules. Mature DCs migrate to the draining lymph nodes,interact with antigen-specific T cells, and induce their activationand differentiation. DC maturation is therefore an important con-trol point by which the innate immune system regulates the acti-vation of naïve T cells (6).All TLRs, with the exception of TLR3, use the adaptor mol-
ecule myeloid differentiation factor 88 (MyD88) for signal trans-duction (2). TLR3 recognizes double-stranded (ds) RNA in theendosomes and initiates signaling by using the adaptor Toll–IL-1receptor domain-containing adaptor inducing IFN-β (TRIF). TLR4recognizes LPS and uses both MyD88 and TRIF as signalingadaptors (2). The MyD88-dependent signaling pathway, down-stream of TLR4, uses the sorting adaptor TIRAP and induces
activation of NF-κB and MAP kinases (2). The TRIF pathwayof signaling, both downstream of TLR3 and TLR4, in addition toNF-κB, induces activation of IRF3, leading to production of IFN-βand -α4 (2). The type I IFNs induced by TLR3 and TLR4 activationplay an important role in several facets of both innate and adaptiveimmunity (7). Because TLR3 recognizes viral RNA, type I IFNproduction is important for induction of antiviral immunity. It hasalso been also demonstrated that type I IFN induction by the TLR3ligand poly(I:C) is important for DC maturation and its subsequentability to activate CD4 T cells (8). In contrast, the importance oftype I IFN production for innate immunity by the TLR4 signalingpathway is not entirely clear. It has been proposed that the up-regulation of costimulatory molecules on DCs by LPS is due toinduction of type I IFNs by the TLR4–TRIF signaling axis (9).Recently, there has been considerable interest in designing
adjuvants for human vaccines that target the TRIF pathway ofsignaling downstream of TLR4 (10–13). It is clear that the TRIFsignaling pathway can induce DC maturation that is sufficient forinduction of adaptive immunity without the overwhelming in-flammatory response induced by the MyD88 signaling pathway(14). Synthetic dsRNA, the ligand for TLR3, could also be animportant candidate to be considered for its adjuvant effect in vaccineformulations. In this study, we examined the role of the TRIF sig-naling pathway downstream of TLR3 and TLR4 and discovered thatTRIF signaling has differential outcomes downstream of these re-ceptors. We find that the dsRNA analog poly(I:C) leads to effective
Significance
Successful induction of protective immunity is critically dependenton our ability to design vaccines that can induce dendritic cell (DC)maturation. Here, we investigated the mechanisms by which Toll-like receptor 4 (TLR4) and TLR3 induce DC maturation. We dis-covered that TLR4 that recognizes LPS from Gram-negativebacteria uses the signaling adaptor Toll–IL-1 receptor domain-containing adaptor inducing IFN-β to induce robust activationof NF-κB and MAP kinases that can directly lead to transcrip-tion of genes necessary for DC maturation. However, TLR3that recognizes viral RNA depends on interferon α/β receptorsignaling to induce DC maturation. Discovery of these moleculardistinctions by which TLRs that recognize bacteria and virusesinduce DC maturation will be beneficial to gaining critical insightsinto induction of adaptive immunity and for successful designof vaccines.
Author contributions: W.H., E.K.W., and C.P. designed research; W.H., Y.G., and R.M.performed research; E.K.W. contributed new reagents/analytic tools; W.H., A.J., I.M.D.,R.M., and C.P. analyzed data; and W.H., A.J., Y.G., and C.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. A.I. is a guest editor invited by the Editorial Board.1Present address: Memorial Sloan Kettering Cancer Center, New York, NY 10065.2To whom correspondence should be addressed. Email: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1510760112/-/DCSupplemental.
13994–13999 | PNAS | November 10, 2015 | vol. 112 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1510760112
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DC maturation only when it also engages the cytosolic sensor andthat DC maturation induced by dsRNA is completely dependent ontype I IFNs secreted by DCs. However, the TRIF-dependent TLR4-signaling-pathway-induced DC maturation is independent of type IIFNs secreted by DCs. Furthermore, we find that this dependence ontype I IFNs is dictated by differential activation ofMAP kinases by theTRIF signaling pathway downstream of TLR3 and TLR4. These dataillustrate that the up-regulation of costimulatory molecules and theadjuvanticity of LPS are direct outcome of TRIF-mediated signalingand not due to indirect effects of autocrine type I IFN production.
ResultsTRIF-Mediated DC Maturation by LPS and Poly(I:C) Use Type I IFN-Independent and -Dependent Pathways, Respectively. To understandthe importance of type I IFNs in TRIF-mediated DC maturation,we generated MyD88–interferon α/β receptor (IFNAR) and –IRF3
double-knockout (DKO) mice. We stimulated bone marrow-derived DCs from these mice with LPS and analyzed DC matura-tion by measuring up-regulation of CD40, CD86, and MHC class IIon CD11c-positive DCs. We found that MyD88–IFNARDKODCshad comparable expression of DC maturation markers to MyD88KO, suggesting that IFNAR signaling is dispensable for TLR4–TRIF-driven DC maturation (Fig. 1A). We obtained similar resultswhen we stimulated WT, IFNAR KO, and IRF3 KO DCs usingTLR4 ligand LPS (Fig. 1B). However, when DCs were stimulatedby using poly(I:C), a TLR3 ligand, IRF3 KO DCs were partiallycompromised in their ability to undergo maturation, but IFNARKO DCs did not undergo any maturation (Fig. 1B). This result wasalso evident when we measured DC maturation at different timepoints after stimulation with poly(I:C) (Fig. S1). When we tested theimportance of type I IFNs for DC maturation induced by othernucleic acid-sensing TLRs, we found that TLR9, which recognizes
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Fig. 1. TRIF-mediated DC maturation by LPS and poly(I:C) use IFN-independent and -dependent pathways, respectively. BMDCs of indicated genotypes werestimulated with LPS (A and B, 100 ng/mL), poly(I:C) (B, 20 μg/mL), or CpG (C, 1 μM) for 12 h and stained for surface expression of CD11c, CD86, CD40, and MHC-II. Histograms show CD11c+ cells expressing different maturation markers. Data are representative of two to five independent experiments.
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CpGDNA, activates IRF7, leading to IFN production, downstreamof MyD88 (15), induced DC maturation independent of IFNARsignaling in DCs (Fig. 1C). Similarly, TLR7, which recognizesssRNA, induced DC maturation independent of IFNAR signaling,suggesting that the dependency on type I IFN feedback for DCmaturation was restricted to dsRNA recognition. We also foundthat the kinetics and magnitude of DC maturation was similarwhen DCs were stimulated by poly(I:C) or when exogenous type IIFN was directly added to DC cultures (Fig. S2).It has been reported that both LPS- and dsRNA-induced DC
maturation is fully dependent on type I IFNs (8, 9). To validate ourfindings in vivo, we injected LPS or poly(I:C) subcutaneously intomice and measured DC maturation in the draining lymph nodes.Consistent with our in vitro data, we observed that LPS was able toinduce comparable DC migration (Fig. S3A), as well as maturation(Fig. S3B) in both WT and IFNARKOmice, whereas the ability ofpoly(I:C) to induce both DCmigration to the draining lymph nodesand DC maturation in vivo was completely dependent on IFNARsignaling (Fig. S3). These results provide clear evidence for thedifferential dependence of LPS and poly(I:C) on type I IFNs toinduce TRIF-dependent DC maturation in vivo.
Optimal DC Maturation Induced by dsRNA Requires both TRIF- andMAVS-Dependent Signaling.The poly(I:C) stimulation experimentsdescribed above are in agreement with previous findings thatIFNAR signaling is important for dsRNA-mediated DC matura-tion (8, 9, 16). Type I IFN synthesis and secretion can be inducedby dsRNA by activating either TLR3–TRIF signaling axis or RIG-I/MDA5–mitochondrial antiviral signaling protein (MAVS) sig-naling axis (17–21). To address the relative contribution of thesepathways in DC maturation, we stimulated either TRIF KO bonemarrow-derived DCs (BMDCs) or MyD88–MAVS DKO BMDCswith poly(I:C) and measured up-regulation of CD86, CD40, andMHC class II. Absence of either MAVS or TRIF reduced the abilityof the KO DCs to mature, suggesting that both TRIF and MAVScontributed to DC maturation (Fig. 2). It is also clear that theMAVS pathway had a larger contribution to the magnitude of DCmaturation compared with the TRIF pathway (Fig. 2). This resultcould in part be due to the ability of the RIG-I–MAVS pathway toinduce higher type I IFNs compared with TLR3–TRIF signaling axis(22), suggesting that type I IFN-positive feedback plays an importantrole in dsRNA-induced DC maturation.
TLR3–TRIF Signaling Axis Fails to Directly Induce Genes Associatedwith DC Maturation. To gain deeper insights into the mechanismsof DC maturation downstream of the TRIF signaling pathway, we
decided to examine the global gene transcription of early genes,induced by TLR4 and TLR3 signaling restricted to TRIF. Toeliminate the contribution of MAVS- and MyD88-dependent sig-naling, we performed the RNA sequencing in MyD88–MAVSDKO BMDCs after 3 h of stimulation with poly(I:C) and LPS,respectively. This approach allowed us to directly compare out-come of TRIF signaling downstream of TLR3 and TLR4 (Fig. 3A).Although we found that LPS was able to induce robust transcrip-tion of close to 800 genes (Fig. 3B), including genes associated withDC maturation (Fig. 3 A and A′), poly(I:C) could induce only asubset of those genes (Fig. 3 B and C). Both the TLR4– andTLR3–TRIF signaling axes were able to induce genes associatedwith IRF3 activation (Fig. 3D). TLR3–TRIF signaling was alsocapable of inducing several IFN-stimulated genes (ISGs) (Fig. 3E),comparable to TLR4–TRIF signaling. Although activation of IRFand genes associated with type I IFN receptor signaling weresimilar in LPS and poly(I:C) stimulation (Fig. 3 E and F), pathwayanalysis revealed that TRIF signaling downstream of TLR3 wasdefective in inducing genes associated with NF-κB, TNFR2,p38MAP kinase signaling, and CD40 signaling (Fig. 3F). Absenceof TNF-α induction and genes associated with TNFR2 signaling(Fig. 3G) by the TLR3–TRIF signaling axis is particularly impor-tant because it has been shown that TNF-α induced by TLR4–TRIF signaling axis is important for stable NF-κB activation (23).We therefore tested whether TNF-α induced by TLR4–TRIF sig-naling is important to induce DC maturation and found that it wasdispensable for LPS-induced drive DC maturation via the TRIFpathway (Fig. S4).
Differential Activation of MAP Kinases, JNK and P38, by the TRIFSignaling Pathway Downstream of TLR4 and TLR3. Our DC matu-ration and gene expression data above prompted us to examineearly upstream signaling events after TRIF signaling downstreamof TLR3 and TLR4. Although NF-κB and ERK activation bypoly(I:C) was delayed compared with LPS, in MyD88–MAVSDKO macrophages, there was a striking deficiency in activationof MAP kinases JNK and P38 (Fig. 4A). These results suggestthat strong NF-κB and MAP kinases downstream of the TLR4–TRIF axis can directly induce transcription of genes necessaryfor DC maturation, whereas dsRNA recognition pathways de-pend on type I IFN–IFNAR signaling axis to achieve DC mat-uration (Fig. 4B). Based on the above results, we predicted that,in the absence of p38 and JNK activation, TRIF-mediated sig-naling downstream of TLR4 would no longer be able to induceDC maturation. To test this hypothesis directly, we performedDC maturation experiments in the presence of MAP kinase
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Fig. 2. Optimal DC maturation induced by dsRNA requires both TRIF- and MAVS-dependent signaling. WT, Trif−/− (A), or Myd88−/−Mavs−/− (B) BMDCs werestimulated with poly(I:C) (20 μg/mL) for 12 h and stained for surface expression of CD11c, CD86, CD40, and MHC-II. Histograms show maturation markers onCD11c+ population. Data are representative of two independent experiments.
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inhibitors. Consistent with the above experiments, we saw thatLPS-mediated maturation of MyD88-deficient DCs was abro-gated by both p38 and JNK inhibitors, but not by a MEKK1/2inhibitor (Fig. S5) that functions to inhibit MEKK1/2, which hasbeen shown to control ERK activation (24). Although therecould be potential off-target effects of these inhibitors, thesedata are consistent with differential ability of TLR4 and TLR3 toinduce MAP kinase activation (Fig. 4).
Adjuvanticity of LPS Is Not Dependent on IFNAR Signaling. There isvery good evidence that poly(I:C)-mediated activation T- andB-cell responses in vivo is completely dependent on type I IFNsand IFNAR signaling (8, 25), and our data support these findingsbecause DC maturation is completely abrogated in the absenceof IFNAR signaling. However, because our data demonstratethat LPS can induce DC maturation in the absence of IFNARsignaling, we decided to test the role of IFNAR signaling in LPS-mediated T-cell activation. We primed OT-II T cells in vitrousing WT and IFNAR KO DCs and observed that LPS inducedcomparable IL-2 secretion by activated T cells, irrespective ofthe source of the DCs (Fig. 5A). Consistent with previous studies(8), poly(I:C)-stimulated IFNAR KO DCs were unable to primeT cells (Fig. 5A). We also found that IFNAR signaling in DCswas necessary for poly(I:C), but dispensable for LPS to instructTh1 commitment in vitro (Fig. 5A). It has been demonstratedthat IFNAR signaling is important for T-cell activation in vivo,when poly(I:C) was used as the adjuvant (8). However, we weremore interested in understanding whether IFNAR signaling isimportant for the adjuvant effects of LPS in vivo and examinedCD4 T-cell priming in IFNAR-deficient mice. We found that
TLR4 activation in vivo resulted in comparable CD4 T-cellpriming in both WT and IFNAR-deficient mice (Fig. 5B). Ad-ditionally, the primed CD4 T cells were also able to differentiateand commit to a Th1 lineage in the absence of IFNAR signaling.These results clearly establish that the ability of TLR4 to driveDC maturation in vivo (Fig. S3A), and subsequent activation ofthe adaptive CD4 T-cell responses is completely independent oftype I IFNs.
DiscussionDC maturation is a critical first step in priming and differentia-tion of antigen-specific naïve T cells (26). DC maturation can beinduced by a variety of ligands that activate different classes ofPRRs (27). Two TLR ligands in particular have been of great in-terest because of their clinical use. Poly(I:C), a mimic of dsRNAthat activates TLR3, has been used for treatment of cancer due itits ability to induce type I IFNs (28–31). In addition, derivativesof LPS that specifically activate the TRIF pathway of signalingdownstream of TLR4 are being considered as vaccine adjuvants(10, 13). Earlier studies have found that the autocrine IFNARsignaling is important for DC maturation by poly(I:C) to induceCD4 T-cell priming and Th1 differentiation (8). It has also beenproposed that the DC maturation induced by LPS via TLR4 isalso dependent on IFNAR signaling (9). In this study, we carefullyexamined the need of IFNAR signaling for DC maturationdownstream of TLR4– and TLR3–TRIF signaling axes anddiscovered that, although dsRNA-mediated DC maturation isdependent on IFNAR signaling, TLR4–TRIF-mediated DCmaturation is completely independent of both type I IFN pro-duction and IFNAR signaling.
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Fig. 3. TRIF signaling induces robust transcription of genes associated with DC maturation downstream of TLR4, but not TLR3. BMDCs (CD11c+) fromMyd88−/−Mavs−/− mice were stimulated with LPS (100 ng/mL) or poly(I:C) (20 μg/mL) for 3 h, and RNA was prepared for RNA sequencing analysis. (A) Heat maprepresents the gene expression values of LPS or poly(I:C)-treated vs. untreated DCs. (A′) Heat map of selected genes critical for antigen presentation. (B) Venndiagram represents number of genes up-regulated more than twofold, uniquely or in both LPS (red) and poly(I:C) (green) compared with unstimulated DCs.(C) Total number of genes up-regulated (black) or down-regulated (gray), more than twofold, upon stimulation compared with unstimulated control.(D) Activation z-score for IRF3 calculated based on the IPA Upstream Regulator analysis. Overlap –logP values are shown above the bars. IRF3 in both con-ditions is predicted to be activated. (E) Fold induction of ISGs upon stimulation with LPS and poly(I:C). (F) Pathway analysis was performed on the genesinduced >1.5-fold (P < 0.05). Each bar represents the percentage of core genes of the pathway induced upon stimulation based on ingenuity pathway analysissoftware. (G) Expression of core genes of TNFR2 signaling pathway upon stimulation with LPS and poly(I:C).
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Both LPS and poly(I:C) have the ability to induce maturationof MyD88-deficient DCs (21, 32). Although the MyD88-dependentsignaling pathway downstream of TLR4 is important for inductionof most proinflammatory cytokines such as IL-6, IL-12, TNF-α, etc.,the MyD88-independent pathway or the TRIF-dependent pathwayof signaling is responsible for activation of IRF3 and subsequenttranscription of IFN-β and -α4 (21). Evidence for utilization ofIFN-α/β for DC maturation and subsequent induction of adap-tive immunity by dsRNA has been presented before (16–18).Our studies establish that dependency on type I IFNAR signalingto induce DC maturation is restricted only to dsRNA.The obvious question is how and why the TRIF signaling path-
way downstream of TLR4 and TLR3 are different. It has becomeapparent that both TLR4 and TLR3 engage TRIF from anendosomal compartment (33, 34). Direct comparison of signalinginduced by LPS and poly(I:C) is not very informative because ofparticipation of MyD88-mediated and RIG-I–mediated signalingpathways, respectively. The MyD88–MAVS DKO mouse allowedus to compare gene expression, as well as signaling outcomes,downstream of TRIF in response to LPS and poly(I:C). Pathwayanalysis of RNA sequencing data from LPS- or poly(I:C)-stimu-lated MyD88–MAVS DKO BMDCs provides enormous distinc-tions between the two groups. TLR3–TRIF signaling axis, althoughcapable of inducing ISGs, is unable to activate genes that are de-pendent on NF-κB and MAP kinases. Strikingly, TRIF also fails torobustly activate MAP kinases JNK and P38, downstream of TLR3
compared with their activation downstream of TLR4. Inhibition ofp38 and JNK, but not ERK, led to reduction in the ability of LPSto induce TRIF-dependent DC maturation, suggesting that acti-vation of these MAP kinases is critical for TLR4–TRIF signaling toinduce DC maturation. Failure of poly(I:C) to robustly induceMAP kinase activation is reflected by its inability to induce directDC maturation. One possible explanation for lack of robust MAPkinase activation downstream of TLR3 could be differential use ofTRIF-related adaptor molecule (TRAM) for signaling. It would bethen possible to predict that altering the C-terminal domain ofTLR3 to engage TRAM would make it behave similar to TLR4and induce robust NF-κB and MAP kinase signaling and typeI IFN-independent DC maturation.We finally evaluated the ability of LPS to induce T-cell acti-
vation and differentiation in vivo in the absence of type I IFNsignaling. This investigation is important because induction ofCD4 Th1 immunity when poly(I:C) is used as an adjuvant in vivois critically dependent on type I IFNs (8). Another study dem-onstrated that LPS-induced DC maturation and its adjuvanticitywere also dependent on type I IFNAR-signaling–induced DCmaturation (9). We found no defect in the ability of LPS toinduce DC migration and maturation in IFNAR KO mice.Consistently, LPS was able to induce robust activation anddifferentiation of antigen-specific Th1 cells in both WT andIFNAR-deficient mice. These results are clearly different fromthe earlier study, in which LPS-induced DC maturation wascompletely abrogated in IFNAR-deficient mice (9). Our ex-periments clearly demonstrate that earlier conclusions on therole of type I IFNs in regulating DC maturation and adaptive
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Fig. 4. TRIF signaling pathway downstream of TLR4 induces strongerp38 and JNK activation compared with TLR3. (A) WT or Myd88−/−Mavs−/−
BMDMs were stimulated with LPS or poly(I:C) for indicated times. Phos-phorylation of IκBα, ERK, JNK, and p38 were analyzed by Western blot. Dataare representative of three independent experiments. (B) Schematic repre-sentation of signaling molecules involved in DC maturation downstream ofTLR4, TLR3, and RIG-I/MDA5. Although TLR4–TRIF signaling can directly in-duce DC maturation, TLR3–TRIF cooperates with MDA5/RIG-I–MAVS signal-ing pathway to induce DC maturation through type I IFN feedback. Eventhough TLR4-TRIF axis induces type I IFNs (not drawn in the schematic), DCmaturation after LPS stimulation is independent of IFN–IFNAR signaling.
A
B
Fig. 5. The ability of LPS to induce T-cell priming is independent of IFNARsignaling. (A) Purified OT-II T cells were cultured with WT or Ifnar−/− BMDCsin the presence of OVA and LPS (100 ng/mL) or poly(I:C) (20 μg/mL) for 3 d.IL-2 or IFN-γ in the culture supernatants were measured by ELISA. (B) WT orIfnar−/− mice were immunized in the foot pad (fp) with OVA (25 μg per fp)and LPS (2.5 μg per fp) emulsified in IFA. Draining lymph nodes were har-vested on day 7 after immunization, and purified CD4 T cells were restimulatedin the presence of Tlr2−/−Tlr4−/− B cells as APCs and titrating doses of OVA for72 h. Proliferation of CD4 T cells was measured by 3H-thymidine incorporation.IFN-γ concentrations in the culture supernatants were determined by ELISA.Data are representative of three independent experiments.
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immune responses need to be revisited to highlight the differencesbetween LPS and dsRNA mediated signaling pathways.Together, our data demonstrate that TRIF signaling pathway
has differential outcomes downstream of TLR4 and TLR3 andthat there is a restricted role for type I IFNs in regulating DCactivation and T-cell differentiation in vivo. As we move forwardwith designing adjuvants for human use, it will be important tounderstand the ability of PRR ligands to directly activate DCsthemselves or indirectly through induction of cytokines such astype I IFNs as that might affect the specificity of the response.This study provides important insights that would need to beconsidered in future design and use of vaccine adjuvants thattarget TRIF pathway of signaling.
Materials and MethodsMice. Myd88−/−, Myd88−/−Ifnar−/−, Myd88−/−Irf3−/−, Myd88−/−MAVS−/−, Trif−/−,Ifnar−/−, Irf3−/−, OT-II, and Tlr2−/−Tlr4−/− mice were bred and maintained at theanimal facility of University of Texas (UT) Southwestern Medical Center. ControlC57BL/6 mice were obtained from the UT Southwestern mouse breeding corefacility. All mouse experiments were performed as per protocols approved bythe Institutional Animal Care and Use Committee at UT SouthwesternMedical Center.
DC–T-Cell Cocultures. Purified OT-II T cells (4 × 105 per well) and BMDCs (8 ×104 per well) were cultured in 48-well plates with 3 μg/mL ovalbumin (OVA)for 3 d. Concentrations of IL-2 and IFN-γ in the supernatant were measuredby using paired antibody ELISAs from BD Biosciences.
T-Cell Proliferation Assay. Purified CD4 T cells (2 × 105) from draining thelymph nodes of immunized mice were cultured in flat-bottom 96-well plates
with Tlr2−/−Tlr4−/− B cells (3 × 105) and titrating doses of antigen for 72–84 h.Tlr2−/−Tlr4−/− B cells were used to rule out any possibility of B-cell proliferationinduced by potential contamination of LPS in OVA. Proliferation of T cells wasdetermined by incorporation of 3H-thymidine for the last 12–16 h of the culture.
Western Blotting. BMDMs were plated in six-well plates (1 × 106 per well) andstimulated with LPS (100 ng/mL) or poly(I:C) (20 μg/mL). Cells were lysed in20 mM Tris·HCl (pH 7.6) containing 1% Triton X-100, 30 mM NaCl, 2 mMEDTA, 1 mM Na3VO4, 20 mM glycerol 2-phosphate, and Complete ProteaseInhibitor Mixture (Roche). Lysates were resolved on 10% (wt/vol) SDS/PAGE, transferred to PVDF membrane, and blotted with the relevantantibodies. Stained membranes were developed by using Super SignalWest Pico Chemiluminescent Substrate (Thermo Scientific) and exposedto film (Kodak).
RNA Sequencing and Analysis. BMDCs were stimulated with LPS or poly(I:C) for3 h. RNA was extracted by using the miRNeasy kit (Qiagen). Methods for datanormalization and analysis are based on the use of “internal standards” (35–37),which was slightly modified to the needs of RNA sequencing data analysis.Functional analysis of identified genes was performed with Ingenuity PathwayAnalysis (IPA; Ingenuity Systems). The two-step normalization procedure and theAssociative analysis functions were implemented inMatLab (Mathworks) and areavailable from authors upon request. Functional analysis of identified genes wasperformed with IPA (Ingenuity Systems).
ACKNOWLEDGMENTS. We thank Zhijian (James) Chen from University ofTexas Southwestern Medical Center for generous sharing of MyD88 mito-chondrial antiviral signaling protein (MAVS) double-knockout (DKO) mice.This work was supported by National Institutes of Health Grants AI082265,AI115420, and AI113125 (to C.P.).
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