Receptor 5/Toll-Like Receptor 4 Complexes Involves Signaling Via

Home , Cyclophilin, Flagellin, TIRAP, TLR5, Trif

Induction of Macrophage Nitric Oxide Production by Gram-Negative Flagellin Involves Signaling Via Heteromeric Toll-Like Receptor 5/Toll-Like Receptor 4 Complexes This information is current as of September 25, 2021. Steven B. Mizel, Anna N. Honko, Marlena A. Moors, Pameeka S. Smith and A. Phillip West J Immunol 2003; 170:6217-6223; ; doi: 10.4049/jimmunol.170.12.6217 http://www.jimmunol.org/content/170/12/6217 Downloaded from

References This article cites 50 articles, 26 of which you can access for free at:

http://www.jimmunol.org/content/170/12/6217.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Induction of Macrophage Nitric Oxide Production by Gram-Negative Flagellin Involves Signaling Via Heteromeric Toll-Like Receptor 5/Toll-Like Receptor 4 Complexes1

Steven B. Mizel,2 Anna N. Honko, Marlena A. Moors, Pameeka S. Smith, and A. Phillip West

The induction of cytokine synthesis by flagellin is mediated by a Toll-like receptor 5 (TLR5) signaling pathway. Although flagellin activation of the IL-1R-associated kinase and induction of TNF-␣ synthesis are dependent on TLR5 and not TLR4, we have found that flagellin stimulates NO in macrophages via a pathway that requires TLR5 and TLR4. Flagellin induced NO synthesis in HeNC2 cells, a murine macrophage cell line that expresses wild-type TLR4, but not in TLR4-mutant or -deficient GG2EE and 10ScNCr/23 cells. Flagellin stimulated an increase in inducible NO synthase (iNOS) mRNA and activation of the iNOS promoter. TLR5 forms heteromeric complexes with TLR4 as well as homomeric complexes. IFN-␥ permitted GG2EE and 10ScNCr/23 cells Downloaded from to produce NO in response to flagellin. Flagellin stimulated IFN-␤ synthesis and Stat1 activation. The effect of flagellin on iNOS gene expression was inhibited by a Stat1 mutant protein. Taken together, these results support the conclusions that flagellin induces distinct patterns of inflammatory mediators depending on the nature of the TLR5 signaling complex and that the induction of NO by flagellin involves signaling via TLR5/TLR4 complexes. The Journal of Immunology, 2003, 170: 6217Ð6223.

nnate immunity serves as an essential first-line defense a variety of TLR5-positive cell types, including monocytes, fibro- http://www.jimmunol.org/ against microbial pathogens and may also influence the na- blasts, and epithelial cells to produce cytokines such as TNF-␣, I ture of the subsequent adaptive immune response (1Ð3). The IL-1, and IL-8 (3, 10, 11, 13Ð19) and NO in monocytes (13). accumulated evidence indicates that host cells involved in the in- Although inducers such as LPS and flagellin have been linked nate immune response use the members of a relatively small fam- with individual TLRs, there is mounting evidence that different ily of structurally related membrane proteins termed Toll-like re- TLRs may interact to produce distinct signaling events. For ex- ceptors (TLR)3 to recognize and respond to products from diverse ample, the activation of NF-␬B-dependent gene expression in groups of bacterial, viral, and fungal pathogens. Signaling via CHO cells and the induction of TNF-␣ in murine macrophages in TLRs results in the production of an array of proinflammatory response to Gram-positive bacterial peptidoglycan are dependent

mediators, including cytokines (for example, IL-1, IL-8, and TNF- on the coexpression and physical interaction of TLR2 and TLR6 by guest on September 25, 2021 ␣), NO, leukotrienes, and platelet-activating factor. Presently, 10 (20). Although the expression of TLR2 is sufficient for a response human and nine murine TLRs have been identified that are char- to phenol-soluble modulin from Staphylococcus epidermidis, the acterized by the presence of a leucine-rich extracellular domain magnitude of the response is enhanced in the presence of TLR6 and cytoplasmic regions termed Toll/IL-1R homology (TIR) do- (21). In a prior study (13), we found that flagellin stimulated IRAK mains (see Ref. 4 for a review). Although TLRs do not, as a rule, activation and TNF-␣ production by GG2EE cells, a C3H/HeJ exhibit specificity for a single microbial product, they are individ- mouse-derived macrophage cell line (22) that expresses a mutant ually responsive to a limited group of molecules. For example, form of TLR4. These observations indicate that at least one form LPS (5Ð7), heat shock protein 60 (8), and Escherichia coli P fim- of flagellin signaling is not dependent on the expression of a wild- briae (9) signal via TLR4. type form of TLR4. However, as will be detailed in this report, the The results of several recent studies demonstrate that flagellin production of NO in response to flagellin is dependent on the co- from Gram-negative bacteria signals via TLR5 (10Ð12). As with expression and interaction of wild-type forms of TLR5 and TLR4. other TLRs, flagellin signaling via TLR5 results in activation of the IL-1R-associated kinase (IRAK) (12, 13). Flagellin stimulates Materials and Methods Cells and reagents Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27157 The C3H/HeN-derived macrophage cell line HeNC2 and the LPS-hyporesponsive C3H/HeJ-derived macrophage cell line GG2EE were maintained Received for publication February 7, 2003. Accepted for publication April 7, 2003. in RPMI 1640 containing 10% FBS and 20 ␮g/ml gentamicin. 10ScNCr/23 The costs of publication of this article were defrayed in part by the payment of page cells, a clonal derivative of primary C57BL/10ScNCr macrophages (23), charges. This article must therefore be hereby marked advertisement in accordance were provided by Dr. E. Lorenz. These cells do not express TLR4 mRNA with 18 U.S.C. Section 1734 solely to indicate this fact. or protein (5, 6). COS-1 cells and the murine macrophage cell line RAW 1 This work was supported by National Institutes of Health Grants AI38670 and 264.7 were maintained in DMEM with 10% FBS and gentamicin. Purified, AI51319 (to S. B. M.). endotoxin-free recombinant His-tagged Salmonella enteritidis flagellin was 2 Address correspondence and reprint requests to Dr. Steven B. Mizel, Department of prepared as previously described (16). As detailed in our prior studies Microbiology and Immunology, Wake Forest University School of Medicine, Medical (12Ð16), at the concentrations used in our studies the flagellin contained Center Boulevard, Winston-Salem, NC 27157. E-mail address: [email protected] insufficient residual LPS (usually Ͻ1 ng/ml) to produce a significant TLR4-dependent response (also see Fig. 1). Recombinant murine IFN-␥ 3 Abbreviations used in this paper: TLR, Toll-like receptor; HI, hemagglutinin; iNOS, inducible NO synthase; IRAK, IL-1R-associated kinase; IRF-3, IFN regulatory fac- was obtained from Life Technologies (Gaithersburg, MD). Anti-FLAG Ab tor-3; Mal, MyD88 adapter-like protein; TIR, Toll/IL-1 receptor homology; TIRAP, was obtained from Sigma-Aldrich (St. Louis, MO), high affinity anti- TIR domain-containing adaptor protein; TRIF, TIR domain-containing adaptor-in- hemagglutinin (anti-HA) Ab was obtained from Roche (Indianapolis, IN), ducing IFN-␤. anti-phospho-Stat1 Ab was purchased from Zymed (San Francisco, CA),

porter (24) and Renilla luciferase control plasmid with or without TLR expression plasmids. The Renilla luciferase control plasmid provided a means to control for differences in transfection efﬁciency. The cells were transfected using the FuGene 6 reagent (Roche). After resting the cells overnight, the cultures were incubated in the presence or the absence of ﬂagellin or LPS for 6 h before harvesting the cells. Inducible NOS promoter-dependent and Renilla luciferase activities were measured using the Promega dual-luciferase reporter assay system according to the manufac- turer’s instructions.

Western blots To assess the association of TLR5 with TLR4, COS-1 cells were transiently transfected with TLR5-HA, TLR5-HA and TLR5-FLAG, or TLR5-HA and TLR4-FLAG expression plasmids using the Effectene transfection reagent (Qiagen, Valencia, CA) as previously described (12). After FIGURE 1. Activation of the iNOS promoter by flagellin is TLR5 de- resting the cells for 48 h, cell lysates were prepared and incubated with pendent. A, RAW264.7 cells were transiently transfected with the anti-FLAG Ab and recombinant protein G-agarose (Invitrogen, San Diego, p3XFlag-CMV-14 vector (0) or TLR5-Flag (TLR5), rested overnight, and CA)for2hat4¡C (12). The immunoprecipitates were washed, eluted in then incubated in the absence (0) or the presence of 10Ϫ9 M flagellin (F) sample buffer, and electrophoresed in a 7.5% SDS-PAGE, and the proteins or 100 ng/ml LPS (L) for 6 h. The cells were harvested and assessed for the were transferred to a polyvinylidene difluoride membrane. The membrane level of inducible luciferase activity. The values have been normalized was then probed for TLR5-HA using the anti-HA high affinity Ab. The blot using the constitutive Renilla luciferase activity in each sample. B, was stripped with Restore Western blot stripping buffer (Pierce, Rockford, Downloaded from RAW264.7 cells were transiently transfected with a TLR5 expression plas- IL) and reprobed for TLR5-FLAG and TLR4-FLAG with the anti-FLAG Ab. mid and the iNOS promoter and Renilla luciferase reporter constructs. After resting the cells overnight, the cultures were stimulated with increasing concentrations of flagellin for 6 h before analysis of luciferase activity. Results Flagellin activates the iNOS promoter in a TLR5-dependent and anti-Stat1 was obtained from Santa Cruz Biotechnology (Santa manner http://www.jimmunol.org/ Cruz, CA). The induction of IRAK activation (12, 13) and cytokine synthesis Plasmids by Gram-negative flagellin is dependent on signaling via TLR5 The p3XFLAG-CMV-14/TLR5 plasmid encoding a FLAG epitope-tagged (10Ð13), but not TLR4 (13). In addition to inducing cytokine syn- form of human TLR5 (12) was used as a template to generate a TLR5 PCR thesis, flagellin stimulates macrophages to produce NO (13). product for cloning into the pMH vector (Roche). The protein product Based on the results of a number of studies (24Ð29) demonstrating encoded by this construct contains a C-terminal HA tag. The human TLR4- that NO production in macrophages is dependent on the induction FLAG expression plasmid was provided by Tularik (South San Francisco CA). The inducible NO synthase (iNOS) promoter/luciferase reporter con- of iNOS gene transcription, we evaluated the TLR5 dependence of struct was provided by Dr. C. J. Lowenstein (The Johns Hopkins Univer- flagellin activation of the iNOS promoter. We took advantage of a sity School of Medicine, Baltimore, MD) (24). The Renilla luciferase con- preliminary observation (S.B. Mizel, unpublished observations) by guest on September 25, 2021 trol vector, pRL-TK, was purchased from Promega (Madison, WI). Stat1 that the murine macrophage cell line RAW264.7 does not express ¬ Y701F cDNA was provided by Dr. F. Oberg (University of Uppsala, Upp- TLR5 and therefore does not respond to flagellin. RAW 264.7 cells sala, Sweden). were transiently transfected with an iNOS promoter/luciferase re- Measurement of NO production porter plasmid and a Renilla luciferase control plasmid (to control The production of NO was determined by measuring nitrite formation as for transfection efficiency) with or without a TLR5 expression previously described (13). plasmid. The cells were rested overnight and then incubated for 6 h Analysis of iNOS and IFN-␤ mRNA levels in the presence or the absence of flagellin or LPS, and the level of iNOS promoter-dependent luciferase activity was determined for To assess the effect of flagellin on iNOS mRNA levels in HeNC2 and each culture (Fig. 1A). As expected (24), RAW 264.7 cells exhib- GG2EE cells, the cells (106 cells/culture) were stimulated for6hinthe absence or the presence of 2.8 ϫ 10Ϫ11 M flagellin or 10 ng/ml LPS, and ited a strong response to LPS, since these cells are TLR4 positive. then the cultures were harvested, and total RNA was isolated using the In contrast, flagellin had no significant effect on iNOS promoter- RNAqueous kit from Ambion (Houston, TX). RT-PCR was performed dependent gene expression in cells that had not received the TLR5 using the RETROscript first-strand synthesis kit (Ambion) and the follow- expression plasmid. However, flagellin did activate iNOS promot- Ј ing forward and reverse primers for murine iNOS: 5 -gaaaaccccttgtgctgt- er-dependent gene expression in cells that had been transfected tctc-3Ј (forward) and 5Ј-ccgatgtggccttgtggtgaag-3Ј (reverse). The following conditions were used for the PCR reaction: denaturing at 94¡C for 4 with a TLR5 expression plasmid. The effect of flagellin was con- min, 30 cycles of denaturing at 94¡C for 30 s, annealing 55¡C for 30 s, centration dependent; 50% of the maximal response was obtained extension at 72¡C for 50 s, and final extension at 72¡C for 5 min. Using with ϳ5 ϫ 10Ϫ11 M flagellin (Fig. 1B). Based on these results, it these primers, a product of 215 bp was generated that included sequences is evident that TLR5 is required for flagellin-mediated activation from two exons, thus reducing any problem associated with contamination by genomic DNA. Nonetheless, control reactions were performed with of NO production as well as cytokine synthesis. samples lacking reverse transcriptase. Cyclophilin primers were used to control for the quality of the RNA preparations and the efficiency of the Flagellin does not induce NO production in cells lacking RT-PCR reactions. The RT-PCR products were electrophoresed in 3% aga- functional TLR4 rose (NuSieve/agarose, 3/1; BioWhittaker, Walkersville, MD). IFN-␤ mRNA levels in HeNC2 cells were evaluated in a similar manner using the As noted previously, flagellin stimulation of IRAK activation and primers described by Toschchakov et al. (25). The relative levels of IFN-␤ cytokine production is dependent on TLR5, but not TLR4 (13). mRNA were determined by quantitating the intensity of the RT-PCR-gen- This conclusion is based on the observation that the TLR4 mutant erated bands and normalizing the values using internal TLR4 values for cell line, GG2EE, produces active IRAK and TNF-␣ in response to each sample. flagellin. In view of this finding, we assumed that the induction of Inducible NOS promoter-dependent reporter assays NO production by flagellin would also be TLR4 independent. To To assess the effect of flagellin on iNOS promoter activation, RAW 264.7 evaluate the validity of this assumption, we tested the ability of cells were transiently transfected with an iNOS promoter/luciferase re- flagellin to induce NO production in GG2EE cells. The GG2EE The Journal of Immunology 6219 cell line was derived from a C3H/HeJ mouse (22) and thus expresses a TLR4 containing a proline to histidine mutation at amino acid residue 714. The HeNC2 cell line was used as a positive control, since these cells produce NO in response to flagellin and express functional TLR4 (13). The GG2EE and HeNC2 cells were incubated for 48 h in the FIGURE 3. Flagellin does not elicit an increase in iNOS mRNA in presence of increasing concentrations of flagellin, and the culture GG2EE cells. HeNC2 and GG2EE cells were incubated in the absence (0) ϫ Ϫ11 medium was then analyzed for the level of nitrite using the Griess or the presence of 2.8 10 M flagellin (F) or 10 ng/ml LPS (L) for 6 h. Total RNA was isolated from each sample, and the presence or the absence reagent. In confirmation of our earlier studies (13), flagellin in- of iNOS mRNA was assessed using RT-PCR. Cyclophilin (CyP) levels duced NO production in HeNC2 cells in a concentration-depen- were measured to control for the quality of mRNA and the efficiency of the dent manner; 50% of the maximal response was achieved with individual RT-PCR reactions. ϳ2 ϫ 10Ϫ11 M flagellin (Fig. 2). In marked contrast to the results with HeNC2 cells, flagellin did not induce NO production in GG2EE cells, even at concentrations that were several 1000-fold it was also possible that the GG2EE cells possess a defect unre- higher than the concentration required for 50% of the maximal lated to TLR4 that prevents NO synthesis or flagellin signaling response in HeNC2 cells. These results are consistent with the idea leading to NO production. that flagellin stimulation of NO synthesis, unlike the activation of Previous studies on LPS hyporesponsive C3H/HeJ mice or mac- ␣ IRAK and the induction of TNF- , is dependent on functional rophages derived from these mice established that IFN-␥ permits Downloaded from TLR4 as well as TLR5. LPS responsiveness (30Ð33). In view of these findings, we ex- ␥ Effect of flagellin on iNOS mRNA levels in HeNC2 and plored the possibility that IFN- might permit GG2EE cells to GG2EE cells produce NO in response to flagellin. The cells were incubated for 48 h in the presence of increasing concentrations of flagellin. One Having found that flagellin does not induce NO production in set of cultures also received 100 U/ml recombinant murine IFN-␥. TLR4-mutant GG2EE cells, we determined whether the lack of As shown in Fig. 4, IFN-␥ restored the ability of flagellin to induce NO production was associated with an absence of iNOS mRNA. NO production in GG2EE cells. As with TNF-␣ production in http://www.jimmunol.org/ HeNC2 and GG2EE cells were incubated in the presence or the Ϫ GG2EE cells (13), 50% of the maximal NO response was achieved ϫ 11 Ϫ absence of 2.8 10 M flagellin or 10 ng/ml LPS for 6 h, and with 3Ð5 ϫ 10 11 M flagellin. To insure that this response was not then total RNA was isolated from the individual cultures and as- unique to GG2EE cells, we conducted the same experiment using sayed for iNOS mRNA using iNOS primers and RT-PCR. Cyclo- a cell line that does not express TLR4. Like the C57BL/10ScNCr philin primers were also used to control for sample variation in the mice from which they were derived, 10ScNCr/23 cells do not ex- quality of RNA or the RT-PCR reaction. As shown in Fig. 3, press TLR4 protein or mRNA. As with GG2EE cells, flagellin by flagellin and LPS increased HeNC2 cell iNOS mRNA levels over itself had a minimal effect on NO production in 10ScNCr/23 cells those observed with unstimulated cells. However, neither stimu- ␥

(Fig. 5). However, the addition of flagellin and IFN- resulted in by guest on September 25, 2021 lant triggered an increase in iNOS mRNA levels in GG2EE cells, a marked induction of NO synthesis. IFN-␥ by itself had no sig- a finding that is consistent with the lack of NO production in re- nificant effect on NO production. The results with GG2EE and sponse to flagellin (Fig. 2). 10ScNCr/23 cells are consistent with the idea that the lack of flagellin induction of NO in these cell lines is due to the absence IFN-␥ permits flagellin induction of NO production in of a TLR4-associated signal and not to some component unrelated TLR4-deficient cells to TLR signaling. IFN-␥ overcomes this condition by providing a Although the preceding results are consistent with a requirement compensatory second signal that is functionally equivalent to a for a wild-type form of TLR4 in flagellin-induced NO production, signal elicited in the presence of TLR5 and TLR4.

FIGURE 2. Flagellin does not induce NO production in GG2EE cells. FIGURE 4. IFN-␥ permits NO production in flagellin-stimulated HeNC2 (F) and GG2EE (Œ) cells were incubated with increasing concen- GG2EE cells. GG2EE cells were incubated with increasing concentrations trations of flagellin for 48 h. The culture medium from each sample was of flagellin (E)orflagellin and IFN-␥ (F) for 48 h before the measurement harvested and analyzed for the level of nitrite using Griess reagent. of nitrite in the culture medium. 6220 INTERACTION OF TLR5 AND TLR4 IN FLAGELLIN SIGNALING

FIGURE 6. TLR5 forms homomeric complexes as well as heteromeric complexes with TLR4. COS-1 cells were transiently transfected with TLR5-HA; TLR5-HA, TLR5, and TLR5-FLAG; or TLR5-HA and TLR4- FLAG. Lysates were prepared and incubated with anti-FLAG Ab and recombinant protein G to precipitate immune complexes. Following electrophoresis of the immune complexes, the proteins were transferred to polyvinylidene diﬂuoride membrane and probed with anti-HA Ab to detect FIGURE 5. IFN-␥ permits NO production in ﬂagellin-stimulated TLR5-HA (upper panel). The blot was then stripped and reprobed with anti-FLAG Ab to detect TLR5-FLAG or TLR4-FLAG (lower panel).

10ScNCr/23 cells. 10ScNCr/23 cells were incubated in the presence or Downloaded from the absence of flagellin (10Ϫ10 M) or flagellin and IFN-␥ (100 U/ml) for 48 h before measurement of nitrite in culture medium. translational initiation site. Since COS-1 cells were not exposed to flagellin, we also conclude that TLR5 self-association is not dependent on flagellin binding. Association of TLR5 and TLR4 Consistent with the postulated requirement for TLR4 and TLR5

Our results demonstrate that TLR5 by itself is not sufficient for collaboration in the NO response to flagellin (Figs. 2 and 3), we http://www.jimmunol.org/ flagellin stimulation of NO production. The responding cells must observed the formation of TLR5/TLR4 heteromeric complexes also express a functional form of TLR4. These results raised the (Fig. 6, lane 3). However, the level of these heteromeric com- possibility that TLR5 and TLR4 may interact to produce a signal- plexes consistently appeared to be less than the level of TLR5 ing complex with activity distinct from that of TLR5 alone. The homomeric complexes. This was the case even though the two hypothesis that TLR5 and TLR4 may form a heteromeric complex proteins appeared to be present at comparable levels. The data in was tested using coimmunoprecipitation analysis. For these exper- Fig. 6 not only demonstrate that TLR5 forms homomeric com- iments we used transfected COS-1 cells that express epitope- plexes, but also provide additional support for the involvement of tagged forms of TLR4 and TLR5. In a previous study (12) we TLR5/TLR4 heteromeric complexes in some forms of flagellin reported that transiently transfected COS-1 cells expressed high signaling. by guest on September 25, 2021 levels of ectopic full-length TLR5. Therefore, we used this cell ␤ line to examine the interaction between TLR5 and TLR4. In con- Flagellin stimulates IFN- production and Stat-1 activation in trast to COS-1 cells, transiently transfected monocyte and T cell HeNC2 cells lines such as RAW 264.7, Jurkat, and EL4 do not express high LPS signaling via TLR4 results in activation of IFN regulatory levels of ectopic TLR4 or TLR5, because the majority of the ex- factor-3 (IRF-3), induction of IFN-␤, and subsequent activation of pressed proteins are cleaved near the junction of the extracellular the transcription factor Stat1 that, in turn, binds to an IFN-␥-acti- and transmembrane domains (S. B. Mizel, unpublished observa- vated site in the iNOS gene (25, 27, 34Ð36). Like TLR4 agonists, tions). Furthermore, the transfection efficiency with GG2EE cells TLR3 agonists can also induce IRF-3 and IFN-␤ expression (34) is very low (regardless of the transfection reagent or protocol), and by a process that involves a protein termed TIR domain-containing thus these cells were not a useful alternative. adaptor-inducing IFN-␤ (TRIF) (37). In contrast to TLR4 and To determine whether TLR5 forms homomeric complexes, TLR3 agonists, TLR2 agonists do not induce IFN-␤ synthesis or COS-1 cells were transiently transfected with expression plasmids Stat1 activation (25). This finding indicates that the ability to trans- encoding TLR5-HA and TLR5-FLAG. The formation of hetero- duce signals culminating in IFN-␤ synthesis and Stat1 activation is meric complexes between TLR5 and TLR4 was evaluated using limited to a restricted subset of TLRs. To determine whether the TLR5-HA and TLR4-FLAG. Detergent cell lysates were prepared presence of TLR4 in the flagellin/TLR5 complex promotes IFN-␤ and incubated with anti-FLAG Ab to immunoprecipitate TLR5- synthesis and Stat1 activation, we tested the ability of flagellin to FLAG or TLR4-FLAG and any associated TLR5-HA. Following induce IFN-␤ mRNA and protein as well as Stat1 activation in electrophoresis and transfer to polyvinylidene difluoride mem- HeNC2 cells. The cells were incubated in the presence or the ab- branes, the blots were probed for the presence of TLR5-HA using sence of flagellin or LPS for 3 h before analysis of IFN-␤ mRNA an anti-HA Ab. Subsequently, the blots were stripped and reprobed content (Fig. 7). As a control we also evaluated IFN-␤ mRNA for TLR5-FLAG and TLR4-FLAG. As expected, TLR5-HA was expression in LPS-stimulated RAW264.7 cells. Identical amounts not found in anti-FLAG immunoprecipitates from cells transfected of RNA (based on OD260) were loaded in each lane. As an addi- with only TLR5-HA (Fig. 6, lane 1). However, TLR5-HA was tional control, we measured the relative level of TLR4 mRNA in present in immunoprecipitates from cells transfected with FLAG- each sample. Although other investigators have found that LPS can tagged TLR5 (lane 2), a finding that establishes the ability of reduce TLR4 expression to varying degrees (5, 38), we found the TLR5 to form homomeric complexes. For reasons that are not clones of RAW264.7 and HeNC2 cells we used did not exhibit a apparent at this time, FLAG-tagged TLR5, but not TLR5-HA, mi- significant reduction in TLR4 expression in response to flagellin or grated as two bands during electrophoresis. The presence of two LPS during the short time course of our experiments. LPS, as bands is not due to phosphorylation (S. B. Mizel, unpublished expected, induced an increase in the level of IFN-␤ mRNA (ϳ4.5- observations). However, it may be due to the use of an alternative fold) in HeNC2 and RAW cells. Although flagellin reproducibly The Journal of Immunology 6221

FIGURE 8. Time course for the induction of IFN-␤ mRNA in flagellin- treated HeNC2 cells. The cells were incubated in the presence of flagellin or 0, 2, 4, and 6 h before the determination of IFN-␤ and TLR4 mRNA contents. induced an increase in IFN-␤ mRNA in HeNC2 cells, the effect was relatively modest (ϳ2-fold). Since the smaller effect of flagellin might be due to a difference in the kinetics of induction between flagellin and LPS, we measured the effect of flagellin on IFN-␤ mRNA levels over a 6-h period. As shown in Fig. 8, the level of FIGURE 10. Inhibition of flagellin and LPS signaling by a Stat1 mutant IFN-␤ was maximal after2h(ϳ2.5-fold increase) and declined to protein. RAW 264.7 cells were transiently transfected with TLR5 and an iNOS/luciferase reporter in the presence or the absence of Stat1 Y701F or background levels after 6 h. Downloaded from Given the small, but reproducible, effect of flagellin on IFN-␤ empty vector. The cells were rested overnight and then stimulated with Ϫ10 expression, we assessed the ability of flagellin to activate Stat1. To 10 M flagellin or 10 ng/ml LPS for 6 h before measuring inducible assess the effect of flagellin on Stat1 activation (a phosphorylation- luciferase activity. Samples were normalized using Renilla luciferase dependent process), HeNC2 cells were incubated in the presence activity. or the absence of flagellin for 4 h before Western blot analysis using an anti-phospho-Stat1-specific Ab. As a control, cells were ␥ ␥ http://www.jimmunol.org/ also incubated with recombinant IFN- . As expected, IFN- by activation of transcription (42). RAW264.7 cells were transiently itself induced substantial Stat1 activation (Fig. 9A). In contrast, transfected with TLR5, the iNOS promoter/luciferase reporter, and flagellin had little observable effect on Stat1 activation in HeNC2 the Renilla luciferase construct in the presence of an expression ␥ cells. Although not as potent as IFN- , LPS also stimulated sig- plasmid encoding the Stat1 Y701F or empty vector. The next day, nificant Stat1 phosphorylation in HeNC2 cells (Fig. 9B). Due to the cells were incubated in the presence or the absence of flagellin the lower level of Stat1 phosphorylation in the presence of LPS, it or LPS for 6 h and then assayed for inducible luciferase activity. was necessary to develop the Western blots for 5Ð10 min as op- As expected, expression of the Stat1 Y701F markedly reduced the ␥ posed to 30 s to 1 min for IFN- . Using the longer development response to LPS (Fig. 10). The response to flagellin was also re- time, we detected a very low level of Stat1-P in the flagellin- producibly reduced in cells expressing the Stat1 mutant protein. by guest on September 25, 2021 stimulated samples. Quantitative analysis revealed that the level of These results support the conclusion that TLR5/TLR4 signaling, Stat1-P in response to LPS was 20-fold greater than with flagellin. like TLR4/TLR4 signaling, involves the activation of Stat1. In view of the low level of Stat1 activation in response to flagellin, it was possible that flagellin induction of iNOS gene expres- Discussion sion may involve a non-Stat1-dependent pathway. To assess this The results presented in this study support the conclusion that the possibility, we tested the ability of a mutant form of Stat1 (Y701F) induction of NO synthesis by Gram-negative flagellin is dependent to block flagellin activation of the iNOS promoter. Previous stud- on signaling via TLR5/TLR4 complexes. Although TLR5-posi- ies (39Ð41) have demonstrated that this mutant Stat1 can act in a tive, TLR4-positive HeNC2 cells produce NO in response to ␥ dominant negative fashion to block IFN- and epidermal growth flagellin, TLR5-positive, TLR4-mutant (GG2EE), and deficient factor signaling, since the phosphorylation of Y701F is important (10ScNCr/23) cells are incapable of generating significant levels for dimerization, translocation to the nucleus, DNA binding, and of NO even at very high concentrations of flagellin (Figs. 2, 4, and 5). The absence of a functional TLR4 is associated with an inability of the cells to initiate iNOS gene expression in the presence of flagellin (Fig. 3). An interaction of TLR5 with TLR4 was demonstrated by the coimmunoprecipitation of these proteins from lysates of COS-1 cells (Fig. 4). However, it is formally possible that this interaction may be limited in nontransfected cells expressing both proteins. Although we have focused our analysis on the role of TLR4 in flagellin-induced NO production, it is likely that TLR5/ TLR4 complexes may be required for flagellin-induced expression of a number of other proinflammatory mediators. The observed formation of heteromeric TLR5-containing complexes and the resultant ability to initiate alternate signaling lead- FIGURE 9. Flagellin induces low level Stat1 phosphorylation in ing to iNOS gene expression are consistent with the results ob- HeNC2 cells. A, Cells were incubated in the presence or the absence of Ϫ tained with other TLRs. As noted earlier, the response to several flagellin (10 10 M) or flagellin and IFN-␥ (100 U/ml) for 4 h. Lysates were prepared for Western blot analysis. The blot was probed with a Stat1-P- stimuli that signal via TLR2 can be markedly enhanced by the pres- specific Ab, then stripped and reprobed with a Stat1-specific Ab. B, Cells ence of TLR1 or TLR6 (20). However, in some cases the formation were incubated for4hinthepresence or the absence of flagellin (10Ϫ10 M) of heteromeric TLR complexes may be inhibitory. For example, or LPS (10 ng/ml) for 4 h and analyzed for Stat1-P and total Stat1 TLR1 inhibits TLR4-dependent signaling in human microvascular en- expression. dothelial cells in response to LPS (43) as well as TLR2-dependent 6222 INTERACTION OF TLR5 AND TLR4 IN FLAGELLIN SIGNALING

activity must be more limited, relative to their activity in TLR4/ TLR4 signaling. This conclusion is based on our observations that the levels of NO, IFN-␤ mRNA, and Stat1 activation in response to flagellin are lower than those observed with LPS. It may be that FIGURE 7. Flagellin induces low level IFN-␤ mRNA expression in the affinity of TIRAP/Mal or TRIF for TLR5/TLR4 complexes is HeNC2 cells. HeNC2 cells were incubated in the presence or the absence of flagellin or LPS for 3 h, and the presence of IFN-␤ mRNA was assessed markedly reduced relative to that for TLR4/TLR4 complexes. Al- by RT-PCR. RAW cells were incubated in the presence or the absence of ternatively, a molecule other than TIRAP/Mal or TRIF may be LPS for 3 h. The relative levels of TLR4 mRNA were also assessed and recruited to TLR5/TLR4 complexes that subserves the same func- used to normalize the results with the individual samples. tion, but does so in a more limited manner. These possibilities are currently under study. signaling in HEK 293 cells stimulated with S. epidermidis phenol- References soluble modulin (21). These types of observations are consistent with 1. Kaisho, T., and S. Akira. 2002. Toll-like receptors as adjuvant receptors. Bio- the idea that changes in the composition of TLR signaling complexes chim. Biophys. Acta 1589:1. may result in the recruitment of different cofactors and thus the ini- 2. Re, F., and J. L. Strominger. 2001. Toll-like receptor 2 (TLR2) and TLR4 dif- ferentially activate human dendritic cells. J. Biol. Chem. 276:37692. tiation of distinct second messenger pathways. 3. Sierro, F., B. Dubois, A. Coste, D. Kaiserlian, J.-P. Kraehenbuhl, and J.-C. Sirard. As noted earlier, signaling via TLR4 is associated with the in- 2002. Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated duction of IFN-␤, an event followed by IFN-␤-mediated activation migration of dendritic cells. Proc. Natl. Acad. Sci. USA 98:13722. 4. Aderem, A., and R. J. Ulevitch. 2000. Toll-like receptors in the induction of the of Stat-1 (25, 27, 34Ð36). The addition of IFN-␥ to TLR4 mutant innate immune response. Nature 406:782. Downloaded from or deficient GG2EE and 10ScNCr/23 cells circumvents the need 5. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. V. Huffel, X. Du, D. Birdwell, E. Alejos, M. Silva, C. Galanos, et al. 1998. Defective LPS signaling in C3H/HeJ for TLR4 in flagellin signaling leading to NO production, because and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085. IFN-␥, like IFN-␤, can promote Stat-1 activation and thus com- 6. Qureshi, S. T., L. Lariviere, G. Leveque, S. Clermont, K. J. Moore, P. Gros, and plements the signals provided by TLR5/TLR5 complexes. Thus, Malo. 1999. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J. Exp. Med. 189:615. our findings are consistent with the idea that the role of TLR4 in 7. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda, the TLR5/TLR4 complex is to provide the necessary signaling that and S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are http://www.jimmunol.org/ culminates in IFN-␤ expression. hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162:3749. There are a number of possible mechanisms by which TLR4 8. Ohashi, K., V. Burkart, S. Flohe, and H. Kolb. 2000. Heat shock protein 60 is a may contribute to flagellin signaling that culminates in the expres- putative endogenous ligand of the Toll-like receptor-4 complex. J. Immunol. sion of iNOS and NO production. TLR4 may participate in the 164:558. 9. Frendeus, B., C. Wachtler, M. Hedlund, H. Fischer, P. Samuelsson, M. Svensson, binding of flagellin to TLR5/TLR4 complexes. In view of the in- and C. Svanborg. 2001. Escherichia coli P fimbriae utilize the Toll-like receptor ability of neutralizing anti-TLR4 Ab to block flagellin induction of 4 pathway for cell activation. Mol. Microbiol. 40:37. NO production in HeNC2 cells (13) and the lack of flagellin re- 10. Hayashi, F., K. D. Smith, A. Ozinsky, T. R. Hawn, E. C. Yi, D. R. Goodlett, J. K. Eng, S. Akira, D. M. Underhill, and A. Aderem. 2001. The innate immune sponsiveness in TLR5-negative RAW 264.7 cells, this possibility response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410: is unlikely. Alternatively, the major contribution of TLR4 in 1099. by guest on September 25, 2021 11. Gewirtz, A. T., T. A. Navas, S. Lyons, P. J. Godowski, and J. L. Madara. 2001. flagellin signaling may be to contribute to the formation of a dock- Bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial ing site for one or more factors that initiate a signaling pathway proinflammatory gene expression. J. Immunol. 167:1882. distinct from that used by TLR5 homomeric complexes. An in- 12. Mizel, S. B., and J. A. Snipes. 2002. Gram-negative flagellin-induced self-toler- ance is associated with a block in interleukin-1 receptor-associated kinase release creasing body of evidence is consistent with a model for LPS/ from Toll-like receptor 5. J. Biol. Chem. 277:22414. TLR4 activation of NO production that involves an autocrine loop 13. Moors, M. A., L. Li, and S. B. Mizel. 2001. Activation of interleukin-1 receptor- that begins with LPS-induced IRF-3 activation and IFN-␤ synthe- associated kinase by Gram-negative flagellin. Infect. Immun. 69:4424. ␤ 14. Ciacci-Woolwine, F., I. C. Blomfield, S. H. Richardson, and S. B. Mizel. 1998. sis and results in the subsequent IFN- -mediated activation of Salmonella flagellin induces tumor necrosis factor ␣ in a human promonocytic Stat1␣, a transcription factor that promotes iNOS gene transcrip- cell line. Infect. Immun. 66:1127. tion (24Ð27, 29, 35). Using this model, one would predict that the 15. Ciacci-Woolwine, F., P. F. McDermott, Mizel, and SB. 1999. Induction of cytokine synthesis by flagella from Gram-negative bacteria may be dependent on inability of a given TLR/agonist complex to promote IFN-␤ ex- the activation or differentiation state of human monocytes. Infect. Immun. 67: pression would prevent that TLR/agonist complex from triggering 5176. the induction of iNOS gene expression. The observations that 16. McDermott, P. F., F. Ciacci-Woolwine, J. A. Snipes, and S. B. Mizel. 2000. High-affinity interaction between Gram-negative flagellin and a cell surface TLR2 agonists do not induce NO production (44) and that TLR2 polypeptide results in human monocyte activation. Infect. Immun. 68:5525. agonists are very poor inducers of IFN-␤ expression and Stat1␣ 17. Eaves-Pyles, T., K. Murthy, L. Liaudet, L. Virag, G. Ross, F. G. Soriano, C. Szabo, and A. L. Salzman. 2001. Flagellin, a novel mediator of Salmonella- activation (25) provide important evidence in support of this induced epithelial activation and systemic inflammation: I␬B␣ degradation, in- model. Our observations indicate that TLR5 by itself cannot me- duction of nitric oxide synthase, induction of proinflammatory mediators, and diate a signaling pathway that is required for IFN-␤ expression and cardiovascular dysfunction. J. Immunol. 166:1248. 18. Gewirtz, A. T., Jr., P. O. Simon, C. K. Schmitt, L. J. Taylor, C. H. Hagedorn, ultimately iNOS expression. Since TLR5 and TLR4 signaling are A. D. O’Brien, A. S. Neish, and J. L. Madara. 2001. Salmonella typhimurium individually associated with the activation of IRAK, it is likely that translocates flagellin across intestinal epithelia, inducing a proinflammatory re- the contribution of TLR4 must involve a mechanism unrelated to sponse. J. Clin. Invest. 107:99. 19. Eaves-Pyles, T., H. R. Wong, K. Odoms, and R. B. Pyles. 2002. Salmonella the binding of MyD88/IRAK complexes. The results of several flagellin-dependent proinflammatory responses are localized to the conserved recent studies indicate that TLR4 signaling may be MyD88 de- amino and carboxyl regions of the protein. J. Immunol. 167:7009. pendent or independent (25, 45Ð48). Although LPS induction of 20. Ozinsky, A., D. M. Underhill, J. D. Fontenot, A. M. Hajjar, K. D. Smith, C. B. Wilson, L. Schroeder, and A. Aderem. 2000. The repertoire for pattern macrophage IL-6 and TNF-␣ synthesis is MyD88 dependent (45), recognition of pathogens by the innate immune system is defined by cooperation the expression of IFN-␤ is MyD88 independent and involves TIR between Toll-like receptors. Proc. Natl. Acad. Sci. USA 97:13766. 21. Hajjar, A. M., D. S. O’Mahony, A. Ozinsky, D. M. Underhill, A. Aderem, domain-containing adaptor protein (TIRAP)/MyD88 adapter-like S. J. Klebanoff, and C. B. Wilson. 2001. Functional interactions between Toll-like (Mal) (25, 49) or TRIF (37). The action of TIRAP/Mal appears to receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. be selective, as evidenced by the lack of involvement of this pro- J. Immunol. 166:15. 22. Blasi, E., D. Radzioch, S. K. Durum, and L. Varesio. 1987. A murine macrophage tein in TLR9 (48), TLR2 (25), or TLR5 (50) signaling. If TIRAP/ cell line, immortalized by v-raf and v-myc oncogenes, exhibits normal macro- Mal or TRIF are involved in TLR5/TLR4 signaling, then their phage functions. Eur. J. Immunol. 17:1491. The Journal of Immunology 6223

23. Lorenz, E., D. D. Patel, T. Hartung, and D. A. Schwartz. 2002. Toll-like receptor 36. Hoshino, K., T. Kaisho, T. Iwabe, O. Takeuchi, and S. Akira. 2002. Differential 4 (TLR4)-deﬁcient murine macrophage cell line as an in vitro assay system to involvement of IFN-␤ in Toll-like receptor-stimulated dendritic cell activation. show TLR4-independent signaling of Bacteroides fragilis lipopolysaccharide. In- Int. Immunol. 14:1225. fect. Immun. 70:4892. 37. Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Takeuchi, K. Takeda, and 24. Lowenstein, C. J., E. W. Alley, P. Raval, A. M. Snowman, S. H. Snyder, S. Akira. 2002. A novel Toll/IL-1 receptor domain-containing adapter that pref- S. W. Russell, and W. J. Murphy. 1993. Macrophage nitric oxide synthase gene: erentially activates the IFN-␤ promoter in the Toll-like receptor signaling. J. Im- two upstream regions mediate induction by interferon ␥ and lipopolysaccharide. munol. 169:6668. Proc. Natl. Acad. Sci. USA 90:9730Ð9734. 38. Medvedev, A. E., K. M. Kopydlowski, and S. N. Vogel. 2000. Inhibition of 25. Toschchakov, V., B. W. Jones, P. Y. Perera, K. Thomas, M. J. Cody, S. Zhang, lipopolysaccharide-induced signal transduction in endotoxin-tolerized mouse B. R. G. Williams, J. Major, T. A. Hamilton, M. J. Fenton, et al. 2002. TLR4, but macrophages: dysregulation of cytokine, chemokine, and Toll-like receptor 2 and not TLR2, mediates IFN-␤-induced STAT1␣␤-dependent gene expression in 4 gene expression. J. Immunol. 164:5564. macrophages. Nat. Immunol. 3:392. 39. Nakajima, K., Y. Yamanaka, K. Nakae, H. Kojima, M. Ichiba, N. Kiuchi, 26. Gao, J., D. C. Morrison, T. J. Parmely, S. W. Russell, and W. J. Murphy. 1997. T. Kitaoka, T. Fukada, M. Hibi, and T. Hirano. 1996. A central role for Stat3 in An interferon-␥-activated site (GAS) is necessary for full expression of the mouse IL-6-induced regulation of growth and differentiation in M1 leukemia cells. iNOS gene in response to interferon-␥ and lipopolysaccharide. J. Biol. Chem. EMBO J. 15:3651. 272:1226. 40. Bromberg, J. F., Z. Fan, C. Brown, J. Mendelsohn, and J. E. Darnell, Jr. 1998. 27. Gao, J. J., M. B. Filla, M. J. Fultz, S. N. Vogel, S. W. Russell, and W. J. Murphy. Epidermal growth factor-induced growth inhibition requires Stat1 activation. Cell 1998. Autocrine/paracrine IFN-␣␤ mediates the lipopolysaccharide-induced ac- Growth Differ. 9:505. tivation of transcription factor Stat1␣ in mouse macrophages: pivotal role of 41. Walter, M. J., D. C. Look, R. M. Tidwell, W. T. Roswit, and M. J. Holtzman. ␥ Stat1␣ in induction of the inducible nitric oxide synthase gene. J. Immunol. 1997. Targeted inhibition of interferon- -dependent intercellular adhesion mol- 161:4803. ecule-1 (ICAM-1) expression using dominant-negative Stat1. J. Biol. Chem. 272: 28. Chan, E. D., and D. W. H. Riches. 1998. Potential role of the JNK/SAPK signal 28582. transduction pathway in the induction of iNOS by TNF-␣. Biochem. Biophys. 42. Levy, D. E., and J. E. Darnell, Jr. 2002. Stats: transcriptional control and bio- Res. Commun. 253:790. logical impact. Mol. Cell. Biol. 3:651. 43. Spitzer, J. H., A. Visintin, A. Mazzoni, M. N. Kennedy, and D. M. Segal. 2002.

29. Jacobs, A. T., and L. J. Ignarro. 2001. Lipopolysaccharide-induced expression of Downloaded from Toll-like receptor 1 inhibits Toll-like receptor 4 signaling in endothelial cells. interferon-␤ mediates the timing of inducible nitric-oxide synthase induction in Eur. J. Immunol. 32:1182. RAW 264.7 macrophages. J. Biol. Chem. 276:47950. 44. Jones, B. W., T. K. Means, K. A. Heldwein, M. A. Keen, P. J. Hill, J. T. Belisle, 30. Beutler, B., V. Tkacenko, I. Milsark, N. Krochin, and A. Cerami. 1986. Effect of ␥ and M. J. Fenton. 2001. Different Toll-like receptor agonists induce distinct mac- interferon on cachectin expression by mononuclear phagocytes: reversal of the rophage responses. J. Leukocyte Biol. 69:1036. lpsd (endotoxin resistance) phenotype. J. Exp. Med. 164:1791. 45. Kawai, T., O. Adachi, T. Ogawa, K. Takeda, and S. Akira. 1999. Unresponsive- 31. Akagawa, K. S., K. Kamoshita, S. Onodera, and T. Tokunaga. 1987. Restoration ness of MyD88-deﬁcient mice to endotoxin. Immunity 11:115. of lipopolysaccharide-mediated cytotoxic macrophage induction in C3H/HeJ 46. Schnare, M., A. C. Holt, K. Takeda, S. Akira, and R. Medzhitov. 2000. Recog- mice by interferon-␥ or a calcium ionophore. Jpn. J. Cancer Res. 78:279.

nition of CpG DNA is mediated by signal pathways dependent on the adaptor http://www.jimmunol.org/ 32. Hogan, M. M., and S. N. Vogel. 1987. Lipid A-associated proteins provide an protein MyD88. Curr. Biol. 10:1139. ␥ alternative “second signal” in the activation of recombinant interferon- -primed, 47. Kaisho, T., O. Takeuchi, T. Kawai, K. Hoshino, and S. Akira. 2001. Endotoxin- C3H/HeJ macrophages to a fully tumoricidal state. J. Immunol. 139:3697. induced maturation of MyD88-deﬁcient dendritic cells. J. Immunol. 166:5688. 33. Chapes, S. K., J. W. Killion, and D. C. Morrison. 1988. Tumor cell killing and 48. Horng, T., G. M. Barton, and R. Medzhitov. 2001. TIRAP: an adapter molecule cytostasis by C3H/HeJ macrophages activated in vitro by lipid A-associated pro- in the Toll signaling pathway. Nature Immunol. 2:835. tein and interferon ␥. J. Leukocyte Biol. 43:232. 49. Fitzgerald, K. A., E. M. Palsson-McDermott, A. G. Bowie, C. A. Jefferies, 34. Doyle, S., S. Vaidya, R. O’Connell, H. Dadgostar, P. Dempsey, T. Wu, G. Rao, A. S. Mansell, G. Brady, E. Brint, A. Dunne, P. Gray, M. T. Harte, D. McMurray, M. Haberland, R. Modlin, and G. Cheng. 2002. IRF3 mediates a TLR3/TLR4- D. E. Smith, J. E. Sims, T. A. Bird, and L. A. O’Neill. 2001. Mal (MyD88- speciﬁc antiviral gene program. Immunity 17:251. adapter-like) is required for Toll-like receptor-4 signal transduction. Nature 413: 35. Weinstein, S. L., A. J. Finn, S. H. Dave, F. Meng, C. A. Lowell, J. S. Sanghera, 78. and A. L. DeFranco. 2000. Phosphatidylinositol 3-kinase and mTOR mediate 50. Horng, T., G. M. Barton, R. Flavell, and R. Medzhitov. 2002. The adaptor mol-

lipopolysaccharide-stimulated nitric oxide production in macrophages via inter- ecule TIRAP provides signaling speciﬁcity for Toll-like receptors. Nature by guest on September 25, 2021 feron-␤. J. Leukocyte Biol. 67:405. 420:329.