Differential Outcome of TRIF-Mediated Signaling in TLR4 and TLR3 Induced DC Maturation

Differential outcome of TRIF-mediated signaling in TLR4 and TLR3 induced DC maturation

Wei 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 Board September 30, 2015 (received for review June 1, 2015) Recognition of pathogen-associated molecular patterns by Toll- activation of NF-κB and MAP kinases (2). The TRIF pathway like receptors (TLRs) on dendritic cells (DCs) leads to DC matura- of signaling, both downstream of TLR3 and TLR4, in addition to tion, a process involving up-regulation of MHC and costimulatory NF-κB, induces activation of IRF3, leading to production of IFN-β molecules and secretion of proinflammatory cytokines. All TLRs and -α4 (2). The type I IFNs induced by TLR3 and TLR4 activation except TLR3 achieve these outcomes by using the signaling play an important role in several facets of both innate and adaptive adaptor myeloid differentiation factor 88. TLR4 and TLR3 can both immunity (7). Because TLR3 recognizes viral RNA, type I IFN use the Toll–IL-1 receptor domain-containing adaptor inducing IFN-β production is important for induction of antiviral immunity. It has (TRIF)-dependent signaling pathway leading to IFN regulatory also been also demonstrated that type I IFN induction by the TLR3 factor 3 (IRF3) activation and induction of IFN-β and -α4. The TRIF ligand poly(I:C) is important for DC maturation and its subsequent signaling pathway, downstream of both of these TLRs, also leads ability to activate CD4 T cells (8). In contrast, the importance of to DC maturation, and it has been proposed that the type I IFNs act in type I IFN production for innate immunity by the TLR4 signaling cis to induce DC maturation and subsequent effects on adaptive pathway is not entirely clear. It has been proposed that the up- immunity. The present study was designed to understand the mo- regulation of costimulatory molecules on DCs by LPS is due to lecular mechanisms of TRIF-mediated DC maturation. We have dis- – – induction of type I IFNs by the TLR4 TRIF signaling axis (9). covered that TLR4 TRIF-induced DC maturation was independent of Recently, there has been considerable interest in designing both IRF3 and type I IFNs. In contrast, TLR3-mediated DC maturation INFLAMMATION adjuvants for human vaccines that target the TRIF pathway of IMMUNOLOGY AND was completely dependent on type I IFN feedback. We found that signaling downstream of TLR4 (10–13). It is clear that the TRIF differential activation of mitogen-activated protein kinases by the signaling pathway can induce DC maturation that is sufficient for TLR4– and TLR3–TRIF axes determined the type I IFN dependency induction of adaptive immunity without the overwhelming in- for DC maturation. In addition, we found that the adjuvanticity of LPS to induce T-cell activation is completely independent of type I flammatory response induced by the MyD88 signaling pathway IFNs. The important distinction between the TRIF-mediated signaling (14). Synthetic dsRNA, the ligand for TLR3, could also be an pathways of TLR4 and TLR3 discovered here could have a major important candidate to be considered for its adjuvant effect in vaccine impact in the design of future adjuvants that target this pathway. formulations. In this study, we examined the role of the TRIF signaling pathway downstream of TLR3 and TLR4 and discovered that MAP kinases | LPS | Poly I:C | MAVS | NF-κB activation TRIF signaling has differential outcomes downstream of these receptors. We find that the dsRNA analog poly(I:C) leads to effective oll-like receptors (TLRs) are a major family of pattern rec- Tognition receptors (PRRs) that recognize conserved microbial Significance products from a diverse class of pathogens (1). Upon recognition of cognate ligands, TLRs initiate a signaling cascade, resulting in Successful induction of protective immunity is critically dependent activation of several transcription factors including NF-κB, AP-1, on our ability to design vaccines that can induce dendritic cell (DC) and IFN regulatory factors (IRFs) (1). The specificity of signaling is maturation. Here, we investigated the mechanisms by which Toll- dictated both by the physical location of the receptor and by the like receptor 4 (TLR4) and TLR3 induce DC maturation. We dis- signaling adaptor use by each TLR (2). The outcome of TLR covered that TLR4 that recognizes LPS from Gram-negative signaling is robust activation of induced innate immunity in the bacteria uses the signaling adaptor Toll–IL-1 receptor domain- β form of enhanced phagocytosis (3) and increased reactive oxygen containing adaptor inducing IFN- to induce robust activation κ species production (4), as well as synthesis and secretion of several of NF- B and MAP kinases that can directly lead to transcrip- proinflammatory cytokines and chemokines by cells of myeloid tion of genes necessary for DC maturation. However, TLR3 α β lineage (5). TLRs also regulate adaptive immunity by induction of that recognizes viral RNA depends on interferon / receptor dendritic cell (DC) maturation. DC maturation is a process by signaling to induce DC maturation. Discovery of these molecular which DCs up-regulate expression of MHC and costimulatory distinctions by which TLRs that recognize bacteria and viruses molecules. Mature DCs migrate to the draining lymph nodes, induce DC maturation will be beneficial to gaining critical insights interact with antigen-specific T cells, and induce their activation into induction of adaptive immunity and for successful design and differentiation. DC maturation is therefore an important con- of vaccines. trol point by which the innate immune system regulates the acti- Author contributions: W.H., E.K.W., and C.P. designed research; W.H., Y.G., and R.M. vation of naïve T cells (6). performed research; E.K.W. contributed new reagents/analytic tools; W.H., A.J., I.M.D., All TLRs, with the exception of TLR3, use the adaptor mol- R.M., and C.P. analyzed data; and W.H., A.J., Y.G., and C.P. wrote the paper. ecule myeloid differentiation factor 88 (MyD88) for signal trans- The authors declare no conflict of interest. duction (2). TLR3 recognizes double-stranded (ds) RNA in the This article is a PNAS Direct Submission. A.I. is a guest editor invited by the Editorial Board. – endosomes and initiates signaling by using the adaptor Toll IL-1 1Present address: Memorial Sloan Kettering Cancer Center, New York, NY 10065. β receptor domain-containing adaptor inducing IFN- (TRIF). TLR4 2To whom correspondence should be addressed. Email: chandrashekhar.pasare@ recognizes LPS and uses both MyD88 and TRIF as signaling utsouthwestern.edu. adaptors (2). The MyD88-dependent signaling pathway, down- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. stream of TLR4, uses the sorting adaptor TIRAP and induces 1073/pnas.1510760112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1510760112 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 DC maturation only when it also engages the cytosolic sensor and double-knockout (DKO) mice. We stimulated bone marrow- that DC maturation induced by dsRNA is completely dependent on derived DCs from these mice with LPS and analyzed DC matura- type I IFNs secreted by DCs. However, the TRIF-dependent TLR4- tion by measuring up-regulation of CD40, CD86, and MHC class II signaling-pathway-induced DC maturation is independent of type I on CD11c-positive DCs. We found that MyD88–IFNAR DKO DCs IFNs secreted by DCs. Furthermore, we find that this dependence on had comparable expression of DC maturation markers to MyD88 type I IFNs is dictated by differential activation of MAP kinases by the KO, suggesting that IFNAR signaling is dispensable for TLR4– TRIF signaling pathway downstream of TLR3 and TLR4. These data TRIF-driven DC maturation (Fig. 1A). We obtained similar results illustrate that the up-regulation of costimulatory molecules and the when we stimulated WT, IFNAR KO, and IRF3 KO DCs using adjuvanticity of LPS are direct outcome of TRIF-mediated signaling TLR4 ligand LPS (Fig. 1B). However, when DCs were stimulated and not due to indirect effects of autocrine type I IFN production. by using poly(I:C), a TLR3 ligand, IRF3 KO DCs were partially compromised in their ability to undergo maturation, but IFNAR Results KO DCs did not undergo any maturation (Fig. 1B). This result was TRIF-Mediated DC Maturation by LPS and Poly(I:C) Use Type I IFN- also evident when we measured DC maturation at different time Independent and -Dependent Pathways, Respectively. To understand points after stimulation with poly(I:C) (Fig. S1). When we tested the the importance of type I IFNs in TRIF-mediated DC maturation, importance of type I IFNs for DC maturation induced by other we generated MyD88–interferon α/β receptor (IFNAR) and –IRF3 nucleic 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 were stimulated with LPS (A and B, 100 ng/mL), poly(I:C) (B,20μg/mL), or CpG (C,1μM) 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.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1510760112 Hu et al. Downloaded by guest on October 2, 2021 CpG DNA, activates IRF7, leading to IFN production, downstream decided to examine the global gene transcription of early genes, of MyD88 (15), induced DC maturation independent of IFNAR induced by TLR4 and TLR3 signaling restricted to TRIF. To signaling in DCs (Fig. 1C). Similarly, TLR7, which recognizes eliminate the contribution of MAVS- and MyD88-dependent sig- ssRNA, induced DC maturation independent of IFNAR signaling, naling, we performed the RNA sequencing in MyD88–MAVS suggesting that the dependency on type I IFN feedback for DC DKO BMDCs after 3 h of stimulation with poly(I:C) and LPS, maturation was restricted to dsRNA recognition. We also found respectively. This approach allowed us to directly compare out- that the kinetics and magnitude of DC maturation was similar come of TRIF signaling downstream of TLR3 and TLR4 (Fig. 3A). when DCs were stimulated by poly(I:C) or when exogenous type I Although we found that LPS was able to induce robust transcrip- IFN was directly added to DC cultures (Fig. S2). tion of close to 800 genes (Fig. 3B), including genes associated with It has been reported that both LPS- and dsRNA-induced DC DC maturation (Fig. 3 A and A′), poly(I:C) could induce only a maturation is fully dependent on type I IFNs (8, 9). To validate our subset of those genes (Fig. 3 B and C). Both the TLR4– and findings in vivo, we injected LPS or poly(I:C) subcutaneously into TLR3–TRIF signaling axes were able to induce genes associated mice and measured DC maturation in the draining lymph nodes. with IRF3 activation (Fig. 3D). TLR3–TRIF signaling was also Consistent with our in vitro data, we observed that LPS was able to capable of inducing several IFN-stimulated genes (ISGs) (Fig. 3E), induce comparable DC migration (Fig. S3A), as well as maturation comparable to TLR4–TRIF signaling. Although activation of IRF (Fig. S3B) in both WT and IFNAR KO mice, whereas the ability of and genes associated with type I IFN receptor signaling were poly(I:C) to induce both DC migration to the draining lymph nodes similar in LPS and poly(I:C) stimulation (Fig. 3 E and F), pathway and DC maturation in vivo was completely dependent on IFNAR analysis revealed that TRIF signaling downstream of TLR3 was signaling (Fig. S3). These results provide clear evidence for the defective in inducing genes associated with NF-κB, TNFR2, differential dependence of LPS and poly(I:C) on type I IFNs to p38MAP kinase signaling, and CD40 signaling (Fig. 3F). Absence induce TRIF-dependent DC maturation in vivo. of TNF-α induction and genes associated with TNFR2 signaling (Fig. 3G)bytheTLR3–TRIF signaling axis is particularly impor- Optimal DC Maturation Induced by dsRNA Requires both TRIF- and tant because it has been shown that TNF-α induced by TLR4– MAVS-Dependent Signaling. The poly(I:C) stimulation experiments TRIFsignalingaxisisimportantforstableNF-κB activation (23). described above are in agreement with previous findings that We therefore tested whether TNF-α induced by TLR4–TRIF sig- IFNAR signaling is important for dsRNA-mediated DC matura- naling is important to induce DC maturation and found that it was tion (8, 9, 16). Type I IFN synthesis and secretion can be induced dispensable for LPS-induced drive DC maturation via the TRIF

– INFLAMMATION

by dsRNA by activating either TLR3 TRIF signaling axis or RIG-I/ pathway (Fig. S4). IMMUNOLOGY AND MDA5–mitochondrial antiviral signaling protein (MAVS) signaling axis (17–21). To address the relative contribution of these Differential Activation of MAP Kinases, JNK and P38, by the TRIF pathways in DC maturation, we stimulated either TRIF KO bone Signaling Pathway Downstream of TLR4 and TLR3. Our DC matu- marrow-derived DCs (BMDCs) or MyD88–MAVS DKO BMDCs ration and gene expression data above prompted us to examine with poly(I:C) and measured up-regulation of CD86, CD40, and early upstream signaling events after TRIF signaling downstream MHC class II. Absence of either MAVS or TRIF reduced the ability of TLR3 and TLR4. Although NF-κB and ERK activation by of the KO DCs to mature, suggesting that both TRIF and MAVS poly(I:C) was delayed compared with LPS, in MyD88–MAVS contributed to DC maturation (Fig. 2). It is also clear that the DKO macrophages, there was a striking deficiency in activation MAVS pathway had a larger contribution to the magnitude of DC of MAP kinases JNK and P38 (Fig. 4A). These results suggest maturation compared with the TRIF pathway (Fig. 2). This result that strong NF-κB and MAP kinases downstream of the TLR4– couldinpartbeduetotheabilityoftheRIG-I–MAVS pathway to TRIF axis can directly induce transcription of genes necessary induce higher type I IFNs compared with TLR3–TRIF signaling axis for DC maturation, whereas dsRNA recognition pathways de- (22), suggesting that type I IFN-positive feedback plays an important pend on type I IFN–IFNAR signaling axis to achieve DC mat- role in dsRNA-induced DC maturation. uration (Fig. 4B). Based on the above results, we predicted that, in the absence of p38 and JNK activation, TRIF-mediated sig- TLR3–TRIF Signaling Axis Fails to Directly Induce Genes Associated naling downstream of TLR4 would no longer be able to induce with DC Maturation. To gain deeper insights into the mechanisms DC maturation. To test this hypothesis directly, we performed of DC maturation downstream of the TRIF signaling pathway, we DC 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 were stimulated 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 on CD11c+ population. Data are representative of two independent experiments.

Hu et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 A A’ B Control LPS Poly I:C LPS Poly I:C Ciita Cd40 Cd86 Cd80 H2-Q6 H2-M2 H2-Q7 396 374 3 H2-K2 H2-Q5 H2-Q4 H2-T22

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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+) from Myd88−/−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 map represents 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) Venn diagram 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 genes induced >1.5-fold (P < 0.05). Each bar represents the percentage of core genes of the pathway induced upon stimulation based on ingenuity pathway analysis software. (G) Expression of core genes of TNFR2 signaling pathway upon stimulation with LPS and poly(I:C).

inhibitors. Consistent with the above experiments, we saw that TLR4 activation in vivo resulted in comparable CD4 T-cell LPS-mediated maturation of MyD88-deficient DCs was abro- priming in both WT and IFNAR-deficient mice (Fig. 5B). Ad- gated by both p38 and JNK inhibitors, but not by a MEKK1/2 ditionally, the primed CD4 T cells were also able to differentiate inhibitor (Fig. S5) that functions to inhibit MEKK1/2, which has and commit to a Th1 lineage in the absence of IFNAR signaling. been shown to control ERK activation (24). Although there These results clearly establish that the ability of TLR4 to drive could be potential off-target effects of these inhibitors, these DC maturation in vivo (Fig. S3A), and subsequent activation of data are consistent with differential ability of TLR4 and TLR3 to the adaptive CD4 T-cell responses is completely independent of induce MAP kinase activation (Fig. 4). type I IFNs.

Adjuvanticity of LPS Is Not Dependent on IFNAR Signaling. There is Discussion very good evidence that poly(I:C)-mediated activation T- and DC maturation is a critical first step in priming and differentia- B-cell responses in vivo is completely dependent on type I IFNs tion of antigen-specific naïve T cells (26). DC maturation can be and IFNAR signaling (8, 25), and our data support these findings induced by a variety of ligands that activate different classes of because DC maturation is completely abrogated in the absence PRRs (27). Two TLR ligands in particular have been of great in- of IFNAR signaling. However, because our data demonstrate terest because of their clinical use. Poly(I:C), a mimic of dsRNA that LPS can induce DC maturation in the absence of IFNAR that activates TLR3, has been used for treatment of cancer due it signaling, we decided to test the role of IFNAR signaling in LPS- its ability to induce type I IFNs (28–31). In addition, derivatives mediated T-cell activation. We primed OT-II T cells in vitro of LPS that specifically activate the TRIF pathway of signaling using WT and IFNAR KO DCs and observed that LPS induced downstream of TLR4 are being considered as vaccine adjuvants comparable IL-2 secretion by activated T cells, irrespective of (10, 13). Earlier studies have found that the autocrine IFNAR the source of the DCs (Fig. 5A). Consistent with previous studies signaling is important for DC maturation by poly(I:C) to induce (8), poly(I:C)-stimulated IFNAR KO DCs were unable to prime CD4 T-cell priming and Th1 differentiation (8). It has also been T cells (Fig. 5A). We also found that IFNAR signaling in DCs proposed that the DC maturation induced by LPS via TLR4 is was necessary for poly(I:C), but dispensable for LPS to instruct also dependent on IFNAR signaling (9). In this study, we carefully Th1 commitment in vitro (Fig. 5A). It has been demonstrated examined the need of IFNAR signaling for DC maturation that IFNAR signaling is important for T-cell activation in vivo, downstream of TLR4– and TLR3–TRIF signaling axes and when poly(I:C) was used as the adjuvant (8). However, we were discovered that, although dsRNA-mediated DC maturation is more interested in understanding whether IFNAR signaling is dependent on IFNAR signaling, TLR4–TRIF-mediated DC important for the adjuvant effects of LPS in vivo and examined maturation is completely independent of both type I IFN pro- CD4 T-cell priming in IFNAR-deficient mice. We found that duction and IFNAR signaling.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1510760112 Hu et al. Downloaded by guest on October 2, 2021 WT Myd88-/-Mavs-/- compared with their activation downstream of TLR4. Inhibition of A p38 and JNK, but not ERK, led to reduction in the ability of LPS LPS Poly I:C LPS Poly I:C to induce TRIF-dependent DC maturation, suggesting that acti- Time (min) 0 15 30 60 90 120180 15 30 60 90 120180 0 15 30 60 90 120180 15 30 60 90 120180 vation of these MAP kinases is critical for TLR4–TRIF signaling to p-IB induce DC maturation. Failure of poly(I:C) to robustly induce MAP kinase activation is reflected by its inability to induce direct p-ERK DC maturation. One possible explanation for lack of robust MAP kinase activation downstream of TLR3 could be differential use of p-JNK TRIF-related adaptor molecule (TRAM) for signaling. It would be then possible to predict that altering the C-terminal domain of p-p38 TLR3 to engage TRAM would make it behave similar to TLR4 -tubulin and induce robust NF-κB and MAP kinase signaling and type I IFN-independent DC maturation. We finally evaluated the ability of LPS to induce T-cell acti- B TLR4 IFNAR vation and differentiation in vivo in the absence of type I IFN

TRAM signaling. This investigation is important because induction of RIG-I/MDA5 TRIF CD4 Th1 immunity when poly(I:C) is used as an adjuvant in vivo is critically dependent on type I IFNs (8). Another study dem- TLR3 onstrated that LPS-induced DC maturation and its adjuvanticity were also dependent on type I IFNAR-signaling–induced DC P TRIF JNK MAVS maturation (9). We found no defect in the ability of LPS to ERK P NF-B p-38 P induce DC migration and maturation in IFNAR KO mice. TBK1 JNK Consistently, LPS was able to induce robust activation and IKK P differentiation of antigen-specific Th1 cells in both WT and ERK P NF-B DC MaturaƟon DC MaturaƟon P IFNAR-deficient mice. These results are clearly different from p-38 IRF3 the earlier study, in which LPS-induced DC maturation was completely abrogated in IFNAR-deficient mice (9). Our ex- IFN-/ INFLAMMATION periments clearly demonstrate that earlier conclusions on the IMMUNOLOGY AND roleoftypeIIFNsinregulating DC maturation and adaptive Fig. 4. TRIF signaling pathway downstream of TLR4 induces stronger p38 and JNK activation compared with TLR3. (A)WTorMyd88−/−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. Data A are representative of three independent experiments. (B) Schematic repre- sentation of signaling molecules involved in DC maturation downstream of TLR4, TLR3, and RIG-I/MDA5. Although TLR4–TRIF signaling can directly induce DC maturation, TLR3–TRIF cooperates with MDA5/RIG-I–MAVS signaling pathway to induce DC maturation through type I IFN feedback. Even though TLR4-TRIF axis induces type I IFNs (not drawn in the schematic), DC maturation after LPS stimulation is independent of IFN–IFNAR signaling.

Both LPS and poly(I:C) have the ability to induce maturation of MyD88-deficient DCs (21, 32). Although the MyD88-dependent signaling pathway downstream of TLR4 is important for induction of most proinflammatory cytokines such as IL-6, IL-12, TNF-α,etc., the MyD88-independent pathway or the TRIF-dependent pathway of signaling is responsible for activation of IRF3 and subsequent transcription of IFN-β and -α4 (21). Evidence for utilization of B IFN-α/β for DC maturation and subsequent induction of adaptive immunity by dsRNA has been presented before (16–18). Our studies establish that dependency on type I IFNAR signaling to induce DC maturation is restricted only to dsRNA. The obvious question is how and why the TRIF signaling pathway downstream of TLR4 and TLR3 are different. It has become apparent that both TLR4 and TLR3 engage TRIF from an endosomal compartment (33, 34). Direct comparison of signaling induced by LPS and poly(I:C) is not very informative because of – participation of MyD88-mediated and RIG-I mediated signaling Fig. 5. The ability of LPS to induce T-cell priming is independent of IFNAR – − − pathways, respectively. The MyD88 MAVS DKO mouse allowed signaling. (A) Purified OT-II T cells were cultured with WT or Ifnar / BMDCs us to compare gene expression, as well as signaling outcomes, in the presence of OVA and LPS (100 ng/mL) or poly(I:C) (20 μg/mL) for 3 d. downstream of TRIF in response to LPS and poly(I:C). Pathway IL-2 or IFN-γ in the culture supernatants were measured by ELISA. (B)WTor − − analysis of RNA sequencing data from LPS- or poly(I:C)-stimu- Ifnar / mice were immunized in the foot pad (fp) with OVA (25 μgperfp) μ lated MyD88–MAVS DKO BMDCs provides enormous distinc- and LPS (2.5 g per fp) emulsified in IFA. Draining lymph nodes were har- vested on day 7 after immunization, and purified CD4 T cells were restimulated tions between the two groups. TLR3–TRIF signaling axis, although −/− −/− in the presence of Tlr2 Tlr4 B cells as APCs and titrating doses of OVA for capable of inducing ISGs, is unable to activate genes that are de- 72 h. Proliferation of CD4 T cells was measured by 3H-thymidine incorporation. pendent on NF-κB and MAP kinases. Strikingly, TRIF also fails to IFN-γ concentrations in the culture supernatants were determined by ELISA. robustly activate MAP kinases JNK and P38, downstream of TLR3 Data are representative of three independent experiments.

Hu et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 − − − − immune responses need to be revisited to highlight the differences with Tlr2 / Tlr4 / B cells (3 × 105) and titrating doses of antigen for 72–84 h. − − − − between LPS and dsRNA mediated signaling pathways. Tlr2 / Tlr4 / B cells were used to rule out any possibility of B-cell proliferation Together, our data demonstrate that TRIF signaling pathway induced by potential contamination of LPS in OVA. Proliferation of T cells was has differential outcomes downstream of TLR4 and TLR3 and determined by incorporation of 3H-thymidine for the last 12–16 h of the culture. that there is a restricted role for type I IFNs in regulating DC activation and T-cell differentiation in vivo. As we move forward Western Blotting. BMDMs were plated in six-well plates (1 × 106 per well) and with designing adjuvants for human use, it will be important to stimulated with LPS (100 ng/mL) or poly(I:C) (20 μg/mL). Cells were lysed in understand the ability of PRR ligands to directly activate DCs 20 mM Tris·HCl (pH 7.6) containing 1% Triton X-100, 30 mM NaCl, 2 mM themselves or indirectly through induction of cytokines such as EDTA, 1 mM Na3VO4, 20 mM glycerol 2-phosphate, and Complete Protease type I IFNs as that might affect the specificity of the response. Inhibitor Mixture (Roche). Lysates were resolved on 10% (wt/vol) SDS/ This study provides important insights that would need to be PAGE, transferred to PVDF membrane,andblottedwiththerelevant considered in future design and use of vaccine adjuvants that antibodies. Stained membranes were developed by using Super Signal target TRIF pathway of signaling. West Pico Chemiluminescent Substrate (Thermo Scientific) and exposed to film (Kodak). Materials and Methods −/− −/− −/− −/− −/− −/− −/− −/− RNA Sequencing and Analysis. BMDCs were stimulated with LPS or poly(I:C) for Mice. Myd88 , Myd88 Ifnar , Myd88 Irf3 , Myd88 MAVS , Trif , Ifnar−/−, Irf3−/−,OT-II,andTlr2−/−Tlr4−/− mice were bred and maintained at the 3 h. RNA was extracted by using the miRNeasy kit (Qiagen). Methods for data animal facility of University of Texas (UT) Southwestern Medical Center. Control normalization and analysis are based on the use of “internal standards” (35–37), C57BL/6 mice were obtained from the UT Southwestern mouse breeding core which was slightly modified to the needs of RNA sequencing data analysis. facility. All mouse experiments were performed as per protocols approved by Functional analysis of identified genes was performed with Ingenuity Pathway the Institutional Animal Care and Use Committee at UT Southwestern Analysis (IPA; Ingenuity Systems). The two-step normalization procedure and the Medical Center. Associative analysis functions were implemented in MatLab (Mathworks) and are available from authors upon request. Functional analysis of identified genes was DC–T-Cell Cocultures. Purified OT-II T cells (4 × 105 per well) and BMDCs (8 × performed with IPA (Ingenuity Systems). 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 measured ACKNOWLEDGMENTS. We thank Zhijian (James) Chen from University of by using paired antibody ELISAs from BD Biosciences. Texas Southwestern Medical Center for generous sharing of MyD88 mitochondrial antiviral signaling protein (MAVS) double-knockout (DKO) mice. T-Cell Proliferation Assay. Purified CD4 T cells (2 × 105) from draining the This work was supported by National Institutes of Health Grants AI082265, lymph nodes of immunized mice were cultured in flat-bottom 96-well plates AI115420, and AI113125 (to C.P.).

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