Flagellin Induces Responses through a TLR5- and -Independent Pathway

This information is current as Américo Harry López-Yglesias, Xiaodan Zhao, Ellen K. of September 25, 2021. Quarles, Marvin A. Lai, Tim VandenBos, Roland K. Strong and Kelly D. Smith J Immunol 2014; 192:1587-1596; Prepublished online 17 January 2014;

doi: 10.4049/jimmunol.1301893 Downloaded from http://www.jimmunol.org/content/192/4/1587

Supplementary http://www.jimmunol.org/content/suppl/2014/01/20/jimmunol.130189 Material 3.DCSupplemental http://www.jimmunol.org/ References This article cites 71 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/192/4/1587.full#ref-list-1

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Flagellin Induces Antibody Responses through a TLR5- and Inflammasome-Independent Pathway

Ame´rico Harry Lo´pez-Yglesias,* Xiaodan Zhao,* Ellen K. Quarles,* Marvin A. Lai,* Tim VandenBos,† Roland K. Strong,† and Kelly D. Smith*

Flagellin is a potent immunogen that activates the innate via TLR5 and Naip5/6, and generates strong T and B responses. The adaptor MyD88 is critical for signaling by TLR5, as well as IL-1Rs and IL-18Rs, major downstream medi- ators of the Naip5/6 Nlrc4-inflammasome. In this study, we define roles of known flagellin receptors and MyD88 in Ab responses generated toward flagellin. We used mice genetically deficient in flagellin recognition pathways to characterize innate immune components that regulate isotype-specific Ab responses. Using purified flagellin from Salmonella, we dissected the contribution of innate flagellin recognition pathways to promote Ab responses toward flagellin and coadministered OVA in C57BL/6 mice. We demonstrate IgG2c responses toward flagellin were TLR5 and inflammasome dependent; IgG1 was the dominant isotype and Downloaded from partially TLR5 and inflammasome dependent. Our data indicate a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. IgA anti-FliC responses were TLR5 and MyD88 dependent and caspase-1 independent. Unlike C57BL/6 mice, flagellin-immunized A/J mice induced codominant IgG1 and IgG2a responses. Furthermore, MyD88-independent, flagellin-induced Ab responses were even more pronounced in A/J MyD882/2 mice, and IgA anti-FliC responses were suppressed by MyD88. Flagellin also worked as an adjuvant toward coadministered OVA, but it only promoted IgG1 anti-OVA responses. Our results demonstrate that a novel pathway for flagellin recognition contributes to Ab http://www.jimmunol.org/ production. Characterization of this pathway will be useful for understanding immunity to flagellin and the rationale design of flagellin-based vaccines. The Journal of Immunology, 2014, 192: 1587–1596.

nnate immunity is responsible for both sounding the alarm primary (8). FliC is a potent immunogen that is capable of of pathogen invasion and directing cellular and humoral inducing strong immune responses to itself (intrinsic adjuvancy) I immunity. The innate immune system recognizes pathogens and coadministered Ags (extrinsic adjuvancy) (9–17). The in- with germline-encoded pattern recognition receptors that respond trinsic and extrinsic adjuvancy of flagellin has been attributed to to conserved pathogen-associated molecular patterns (1–3). Two conserved structures in its D0 domain, recognized by Naip5 and by guest on September 25, 2021 key groups of pattern recognition receptors are membrane-bound Naip6 (Naip5/6), and its D1 domain, recognized by TLR5 (4, 6, TLRs and cytosolic NOD-like receptors (NLRs). TLRs recognize 18–24). Studies from several groups have established that recog- structurally diverse pathogen-associated molecular patterns, in- nition of FliC by the innate immune system leads to microbicidal cluding nucleic acids, glycolipids, lipoproteins, and . The activity, cytokine production, and dendritic cell (DC) activation only known protein ligand for human TLRs is bacterial flagellin, (25–27). Immunization of mice with FliC elicits robust ac- which is recognized by TLR5 (4). tivation and T cell–dependent Ab responses (14–16, 26, 28–33). Flagellin is exposed on the surface of flagellated and is Flagellin, the ligand for TLR5, has been shown to induce a a major antigenic target of the immune system in a wide variety of Th2-biased response (29, 30, 34), and it is currently being devel- hosts, ranging from plants and invertebrates to vertebrates (5–7). oped as a vaccine adjuvant (35, 36). Because flagellin is a protein, In Salmonella enterica serovar, Typhimurium (S. typhimurium) the molecule can be engineered for vaccine development to retain flagellin is encoded by the fliC and fljB, with fliC being the immunogenicity and display foreign epitopes of interest from pathogens such as influenza (hemagglutinin and matrix proteins),

*Department of Pathology, University of Washington, Seattle, WA 98195; and Yersinia pestis, and (FlaA flagellin) (9, 37– †Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 41). Compared with the coadministration of flagellin with an Ag, 98109 flagellin fusion proteins elicit enhanced humoral responses and are Received for publication July 18, 2013. Accepted for publication December 11, 2013. therefore a more alluring alternative for vaccine design (35, 42– This work was supported by the National Institute of Allergy and Infectious Diseases 45). The greater efficacy of the flagellin–Ag fusions suggests that of the National Institutes of Health (Grant R01 AI084803 to K.D.S.) and Public Health Service National Research Service awards from the National Institutes of proximity of the Ag to the adjuvant allows for enhanced antige- Health (National Institute of Allergy and Infectious Diseases, University of Wash- nicity. ington Sexually Transmitted Diseases/AIDS Research Training Program Grant NLRs are cytosolic sensors that oligomerize after ligand rec- T32AI007140 to A.H.L.-Y.; and National Institute of General Medical Sciences, Molecular and Cellular Biology Training Program Grant T32 AI027757 to A.H.L.-Y.). ognition and form multiprotein complexes termed inflammasomes Address correspondence and reprint requests to Dr. Kelly D. Smith, Department (46, 47). A broad range of pathogen-derived and endogenous sig- of Pathology, University of Washington, 750 Republican Street, E681, Seattle, WA nals initiate inflammasome formation, and one of its triggers, alum, 98109. E-mail address: [email protected] has been used for decades as an adjuvant that elicits Th2 type The online version of this article contains supplemental material. responses toward coadministered Ags (48). The best studied NLR, Abbreviations used in this article: DC, dendritic cell; DKO, double knockout; NLR, Nlrp3, is required for alum-induced activation of the inflamma- NOD-like receptor; RT, room temperature; WT, wild-type. some (49, 50), but alum also uses an inflammasome-independent Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 pathway to induce Th2 immunity (51, 52). The Naip family of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301893 1588 FLAGELLIN INDUCES Ab RESPONSES THROUGH A NOVEL PATHWAY

NLRs activates the inflammasome in an Nlrc4-dependent manner penicillin, 100 mg/ml streptomycin (Life Technologies) (19, 63, 64). All as- (21, 22). Murine Naip2 recognizes the rod proteins of some bac- says were performed in triplicate and each experiment was repeated at least terial type III secretory systems, whereas murine Naip5 and Naip6 twice. Bone marrow–derived macrophages were primed with 10 ng/ml ultrapure LPS (List Biologicals) for 3 h to induce pro–IL-1b expression recognize flagellin. Human NAIP recognizes the needle protein before protein transfection, using Profect-P1-lipid–based protein delivery of some bacterial type III secretion systems (21) and has been reagent (Targeting Systems) as previously described (19, 65); IL-1b se- reported to recognize flagellin (21, 53). Recognition of these cretion was determined by ELISA (DuoSet; R&D Systems). protein ligands by the Naip proteins induces oligomerization with Mice and immunizations Nlrc4, leading to recruitment and activation of caspase-1 (54). Active caspase-1 processes pro–IL-1b and pro–IL-18 into mature The University of Washington Institutional Animal Care and Use Com- mittees approved all animal protocols. Mice were bred and housed in forms for secretion, and initiates a form of cell-mediated death a specific pathogen-free facility at the University of Washington. A/J and termed pyroptosis (55). The Nlrc4 system has been recently found C57BL/6 animals were purchased from Jackson Laboratories and bred to contribute to flagellin-induced Ab production in mice, in a man- in-house. Naip52/2 (66), caspase-1 (Casp12/2) (67), MyD882/2 (68), and 2/2 ner that is redundant with TLR5 (16). In the absence of TLR5, TLR5 (69) mice were all generated on the C57BL/6 background and bred in-house. TLR52/2/Casp12/2 mice were generated and bred in our Nlrc4 is required for flagellin’s immunogenicity (16). The isotype 2/2 animal facility. A/J MyD88 mice were generated by backcrossing the specificity of Nlrc4 inflammasome-dependent Ab responses is MyD88 deletion onto the A/J background for nine generations and then unknown. crossing to generate homozygous MyD882/2 mice. Eight- to 14-wk-old TLR5 is expressed on the surface of epithelial cells, neutro- matched animals were used in all experiments. Retro-orbital bleeds were phils, monocytes, and DCs (4, 18, 56). Flagellin recognition by performed on all animals before immunization to obtain naive serum. Mice received two sequential i.p. immunizations with 30 mg FliC and 30 mg TLR5 induces its dimerization and signaling through adaptor OVA separated by 21 d. Blood was drawn 2 wk after each immunization. Downloaded from protein MyD88 (57, 58). Activation of DCs via TLR5 leads to the upregulation of MHC class II, CD80, and CD86, and the secretion Cytokine analysis of cytokines, such as IL-23, IL-6, and Cxcl1 (14, 26, 29). TLR5 Mouse sera were evaluated for cytokine responses after i.p. injections of also promotes flagellin uptake and presentation that is required for 30 mg FliC or PBS using commercially sourced ELISA kits according to efficient T cell activation (14, 33, 34, 45). Thus, TLR5 recognition manufacturer’s instructions (DuoSet). IL-18 cytokine analysis was deter- mined by ELISA, using anti-mouse IL-18 (clone 74; R&D Systems) as a of flagellin induces multiple pathways that are beneficial proper- http://www.jimmunol.org/ capture Ab and biotinylated anti-mouse IL-18 (clone 93-10C; R&D Sys- ties for adjuvants. In the absence of the Nlrc4 inflammasome, TLR5 tems) as a detection Ab. is required for flagellin’s immunogenicity (16). TLR5 and the major cytokine outputs of the Nlrc4 inflamma- Ab analysis some, IL-1b and IL-18, require MyD88 for signaling (59). Despite High binding capacity 96-well plates (COSTAR) were coated with 1 mg/ml this commonality, Ab responses toward flagellin are maintained in monomeric FliC or OVA diluted in PBS (OmniPur) and allowed to incu- MyD88-deficient mice (15). Thus, MyD88-independent pathways bate overnight at room temperature (RT). Plates were washed three times with PBS containing 0.05% Tween 20 and blocked for 1 h RT in PBS emanating from either TLR5, such as flagellin uptake (33, 60), or containing 1% BSA (Sigma). Plates were washed and serial dilutions of serum the Nlrc4 inflammasome (61) may also contribute to flagellin-specific were added to the wells and incubated for 1 h at RT. Plates were washed again Ab responses. In this article, we dissect the innate immune com- and HRP-conjugated secondary Abs (anti-IgG1, -IgG2a, –IgG2c-HRP by guest on September 25, 2021 ponents that are responsible for flagellin’s intrinsic and extrinsic [Jackson Immunoresearch], or –IgA-HRP [Biolegend]) were added and in- adjuvant properties, as well as the production of isotype-specific cubated for another hour at RT. Plates were developed with TMB substrate (Thermo Scientific), stopped with H2SO4, and absorbance was read at Ab responses to flagellin and a model coadministered Ag, OVA. 450 nm (Molecular Devices). Ab end-point titer was defined by the These studies define the innate immune components that are re- reciprocal of the maximal serum dilution that exceeded three times the quired to generate robust isotype-specific Ab responses toward SD above the mean background absorbance. FliC or coadministered Ags, and uncover a novel TLR5, inflam- Statistical analysis masome-, and MyD88-independent flagellin recognition pathway that contributes to flagellin’s immunogenicity. Significance was determined by one-way ANOVAwith Bonferroni multiple- comparison posttest or Mann–Whitney U test, using GraphPad Prism 5 software. Differences were noted as significant when p , 0.05. Materials and Methods Protein isolation and purification Results Wild-type (WT) flagellin monomers were isolated from S. typhimurium TLR5 and the Naip5 inflammasome control distinct early strain SL1344 (DflgM); purity was verified as previously described (18, cytokine responses in vivo 62). OVA was purchased from Sigma and ultrafiltered (Amicon) to reduce endotoxin. Removal of residual endotoxin from isolated flagellin mono- Innate recognition of FliC leads to cytokine and chemokine pro- mers and OVA (Sigma-Aldrich) was performed by using polymyxin B duction that contributes to host defense and adaptive immunity. columns (Thermo Scientific). Endotoxin levels were ,1pg/mgprotein,as To characterize similarities and differences in early phase of in- measured using the limulus colorimetric assay (Lonza). The purified fla- nate detection of flagellin, we defined the pathways necessary for gellin was characterized biochemically, and its biological activity for TLR5 triggering flagellin-dependent cytokine responses in vivo using and Naip5 was determined before mouse studies (Supplementary Fig. 1). WT, TLR52/2, Naip52/2, Casp12/2, MyD882/2, and TLR52/2/ NF-kB luciferase reporter assay Casp12/2 (double knockout [DKO]) mice. Mice were injected m CHO K1 cells were stably transfected with mouse TLR5 cDNA cloned into i.p. with 30 g FliC, isolated and purified from S. typhimurium the pEF6 V5/His TOPO vector (Invitrogen) or the empty vector, plus the (Supplementary Fig. 1), and sera were assessed at 2 and 4 h after ELAM-LUC plasmid; luciferase assays were performed as previously de- flagellin injection. WT mice produced IL-6, Cxcl1, IL-12/23p40, scribed (18, 62). and IL-18 (Fig. 1). At the 2-h time point, serum Cxcl1, IL-6, and Protein transfection IL-12/23p40 were TLR5 and MyD88 dependent (Fig. 1A, 1C, 1D). Serum IL-6 and IL-12/23p40 levels were also partially de- Bone marrow–derived macrophages were prepared from femurs of C57BL/6 mice, Naip5-deficient mice, caspase-1–deficient mice, or A/J mice, and pendent on Naip5 and Casp1 (Fig. 1C, 1D). In contrast, flagellin cultured in RPMI 1640 supplemented with 10% FBS (Atlas Biologicals), induction of IL-18 was TLR5 and MyD88 independent, but en- 10% L-cell supernatant (CSF1 source), 2 mM L-glutamine, and 100 U/ml tirely dependent on Naip5 and Casp1 (Fig. 1B). At 4 h postin- The Journal of Immunology 1589

FIGURE 1. Flagellin-induced cytokines are differentially regulated by TLR5, Naip5, Casp1, and MyD88. WT (n = 7–12), TLR52/2 (n =4– 8), Naip52/2 (n = 3), Casp12/2 (n = 4–11), DKO (n = 3–4), MyD882/2 (n = 3–6), and mock (n = 8–10) mice were injected i.p. with FliC (30 mg) or PBS (mock). Serum was col- lected 2 h after injections, and cytokine levels were determined by ELISA. IL-12/23p40 (A), IL-18 (B), IL-6 (C), and Cxcl1 (D). All groups have a minimum n = 3. Statistical analysis was done using one-way ANOVA with Bonferroni multiple-comparisons posttest: **p , 0.01, ***p , 0.001. Downloaded from jection, the role of TLR5 and the Naip5 inflammasome in flagellin- with the same dose of flagellin 3 wk later. The immunized animals induced cytokine production was more complex (Fig. 1). IL-12/ were bled before immunization (naive serum) and at 2 wk after 23p40 and IL-18 continued to be dependent on the flagellin sen- primary and secondary immunizations. We tested the sera for sors TLR5 and Naip5 inflammasome, respectively (Fig. 1A, 1B). IgG1 and IgG2c Abs against FliC. After primary immunizations, 2/2 However, IL-6 and Cxcl1 were detected at 4 h in TLR5 ,but the IgG1 anti-FliC median titer was 3160 in WT mice and 316 in http://www.jimmunol.org/ not MyD882/2 or TLR5/Casp1 DKO mice, suggesting a delayed MyD882/2 mice (Fig. 2A). After the secondary immunization, cytokine cascade, where flagellin-induced IL-18 or other Casp1- IgG1 anti-FliC median titers increased .100-fold in both WT and dependent factors induce Cxcl1 and IL-6 in an MyD88-dependent MyD882/2 animals, and IgG1 titers remained significantly reduced manner (Fig. 1C, 1D). Thus, the innate immune receptors TLR5 in MyD882/2 compared with WT mice (Fig. 2A). and Naip5 function both independently and in concert to regulate In contrast with IgG1, FliC-immunized C57BL/6 mice generated early cytokine production induced by flagellin. Flagellin has low titers of IgG2c anti-FliC, which were best detected after the also been reported to induce low levels of TNF and IL-1b in mice secondary immunization (Fig. 2B). The IgG2c anti-FliC response (15, 16, 70). In our studies, we found no significant flagellin- was absolutely dependent on MyD88 (Fig. 2B). Our data demon- dependent induction of IL-1b or TNF at 2 and 4 h postinjection strate that IgG2c and a portion of IgG1 anti-flagellin responses are by guest on September 25, 2021 in any of the mice (data not shown). For TNF, it is likely that the MyD88 dependent. Our results also support an MyD88-independent 2-h time point missed the early and low-level induction, which pathway for IgG1 anti-flagellin Abs in C57BL/6 mice. typically peaks around 1–1.5 h postinjection (data not shown) (70, TLR5 and the inflammasome play largely redundant roles in 71). Other investigators have also had difficulty detecting IL-1b IgG1 anti-FliC responses in mouse serum (15, 70), suggesting that technical issues may have precluded our ability to detect the low levels reported by We next examined the individual components of innate flagellin Bedoui and colleagues (12). recognition for their contribution to the anti-FliC Ab responses. Compared with WT mice, TLR52/2, Naip52/2, and Casp12/2 IgG1 isotype-specific responses are MyD88 independent mice produced similar IgG1 titers toward flagellin (Fig. 3A–C). In Flagellin is a major antigenic target during bacterial infections contrast, the IgG2c anti-FliC responses were significantly reduced and when injected as a purified protein also induces Abs against in TLR52/2,Naip52/2, and Casp12/2 mice after secondary immu- itself and coadministered Ags. Because flagellin is being developed nizations (Fig. 3D–F). Our results suggest that each individual in- as a platform for recombinant vaccines, we dissected the innate nate recognition pathway for flagellin contributes to the generation immune pathways needed to generate isotype-specific Ab responses of IgG2c anti-FliC responses. In contrast, loss of TLR5 or the against flagellin or a coadministered Ag, OVA. C57BL/6 and individual inflammasome molecules, Naip5 or Casp1, did not af- MyD882/2 mice were injected i.p. with 30 mg FliC and boosted fect robust IgG1 anti-FliC responses.

FIGURE 2. IgG1 anti-FliC responses are partially and IgG2c anti-FliC are entirely MyD88 dependent. WT (n = 14) and MyD882/2 (n = 12) mice were im- munized with 30 mg FliC on days 1 and 21, and sera were collected on days 14 and 35. Naive, day 14, and day 35 sera were analyzed for IgG1 (A) and IgG2c (B) Ab responses against FliC by ELISA. Data are a com- bination of two independent experiments with n = 3–5 per experiment. Statistical analyses were done using Mann–Whitney analysis of individual groups: ***p , 0.001. 1590 FLAGELLIN INDUCES Ab RESPONSES THROUGH A NOVEL PATHWAY Downloaded from http://www.jimmunol.org/

FIGURE 3. IgG2c anti-FliC responses are partially TLR5 and inflammasome dependent. TLR52/2 (n = 19) (A, D), Naip52/2 (n =9)(B, E), and Casp12/2 (n = 14) (C, F) mice were immunized twice on days 1 and 21, and sera collected on days 14 and 35. Naive, day 14, and day 35 sera were analyzed for IgG1 (A–C) and IgG2c (D–F) isotype-specific Ab responses against FliC by ELISA. Data are a combination of two to three independent experiments with n = 3–7 per experiment. Statistical analyses were done using Mann–Whitney analysis of individual groups: *p , 0.05, **p , 0.01.

MyD88-independent IgG1 anti-FliC responses are also TLR5 alone failed to generate anti-OVA responses, whereas mice coin- and inflammasome independent jected with FliC and OVA generated anti-OVA Abs (Fig. 6A). We by guest on September 25, 2021 To determine whether TLR5 or the inflammasome contributes to also tested the isotype specificity of the FliC-dependent anti-OVA MyD88-independent IgG1 anti-FliC responses, we generated TLR5 Ab responses and found that in all animals tested, IgG1 was the and Casp1 DKO mice. TLR5/Casp1 DKO mice had significantly only isotype detected against OVA; no IgG2c or IgA was gener- ated against OVA (data not shown). The IgG1 anti-OVA responses reduced IgG1 anti-FliC titers but maintained moderate titers as 2/2 2/2 2/2 seen in MyD882/2 mice (Fig. 4A). As expected, the DKO animals were similar in WT, TLR5 , Naip5 , and Casp1 mice (Fig. 6B–D). The IgG1 anti-OVA titers were significantly reduced had significantly reduced IgG2c responses toward FliC (Fig. 4B). 2/2 These results strongly suggest that the MyD88-independent anti- in MyD88 mice and approached, but did not reach, statistical FliC IgG1 response is mediated by a novel pathway that is distinct significance in DKO animals (p = 0.07; Fig. 6E, 6F). Thus, the from the known TLR5 and inflammasome pathways for flagellin IgG1 and anti-OVA and IgG1 anti-FliC responses appear to be recognition. regulated by similar MyD88-dependent and -independent mech- anisms. Serum IgA anti-FliC responses are TLR5 and MyD88 dependent and Casp1 independent Defective flagellin-induced IL-18 in A/J mice We tested naive and boost sera of C57BL/6 mice and knockout A/J mice contain a hypofunctional Naip5 that is associated with mice, and assessed IgA anti-FliC titers after secondary immuni- susceptibility to Legionella pneumophila (21, 22, 47, 74). We zations of FliC because previous in vitro and in vivo studies have assessed the serum cytokines in response to FliC 2 h after i.p. m shown that monomeric flagellin is capable of inducing IgA re- injection with 30 g FliC. A/J and C57BL/6 mice produced sponses toward itself (72, 73). IgA anti-FliC responses were equivalent amounts of IL-6, IL-12/23p40, and Cxcl1 (Fig. 7A–C). detected at low levels after the secondary immunization (Fig. 5A). However, A/J mice failed to produce IL-18 (Fig. 7D). As with b Consistent with recent results of Cunningham and colleagues (72), C57BL/6 mice, A/J mice did not produce detectable TNF or IL-1 our results with TLR52/2, MyD882/2, and DKO mice show IgA (Fig. 7E, 7F). Thus, the flagellin-induced cytokine responses in A/J mice are consistent with intact TLR5 and impaired Naip5 anti-FliC responses are TLR5 and MyD88 dependent (Fig. 5B, 5E, A/J 5F). Furthermore, the IgA anti-FliC titers were detected in Naip52/2 recognition of flagellin due to the hypofunctional Naip5 allele. and Casp12/2 mice, suggesting that the inflammasome is not re- Augmented IgG1 and IgG2a anti-FliC responses in A/J mice. We quired for IgA anti-FliC responses (Fig. 5C, 5D). compared isotype-specific Ab responses against FliC in A/J and C57BL/6 mice and their respective MyD88 deficient strains. In Flagellin’s adjuvancy toward extrinsic Ags is partially contrast with C57BL/6 mice, A/J mice had more robust Ab re- dependent on TLR5 and the inflammasome sponses to flagellin after primary and secondary immunization, Next, we examined how flagellin regulates Ab responses toward with ∼10-fold increased IgG1 anti-FliC titers after primary and a coadministered (extrinsic) Ag, OVA. Mice immunized with OVA secondary, and .100-fold increased IgG2a/c anti-FliC titers after The Journal of Immunology 1591

FIGURE 4. IgG1 anti-FliC responses are partially TLR5 and caspase-1 independent. TLR52/2/Casp12/2 DKO (n =9) mice were immunized twice on days 1 and 21, and sera were collected on days 14 and 35. Naive, day 14, and day 35 sera were analyzed for IgG1 (A) and IgG2c (B) isotype-specific Ab responses against FliC by ELISA. Data are representative of two independent experiments with n = 4–5 per group. Statis- tical analyses were done using Mann–Whitney analysis of individual groups: *p , 0.05, **p , 0.01.

primary and secondary immunizations (Fig. 8A, 8B). The IgG2a erate significant IgA anti-FliC after two immunizations (Fig. 9A). responses in A/J mice were partially MyD88 dependent, whereas Conversely, the A/J MyD882/2 mice produced a substantial IgA IgG1 anti-FliC titers were reduced in A/J MyD882/2 mice, trending anti-FliC titer after two immunizations (Fig. 9B). These results toward significance (p = 0.08; Fig. 8A, 8B). In contrast with illuminate the fine intricacies between different strains of mice, re- C57BL/6 mice, which have a strong IgG1 biased Ab response sulting in quantitative and qualitative differences in isotype-specific against flagellin, A/J mice had a balanced Ab response toward responses generated against FliC. Downloaded from flagellin, with equivalent IgG1 and IgG2a anti-FliC titers. Discussion Anti-OVA responses in A/J mice are partially dependent Dissecting the innate immune pathways that recognize flagellin’s on MyD88 structural properties and promote adaptive immune responses is 2/2 A/J and A/J MyD88 mice were immunized with FliC and OVA. vital for the rational design of flagellin-based vaccines. Flagellin- As with IgG2c responses in C57BL/6 mice, IgG2a anti-OVA Ab based fusion proteins are currently being developed as vaccines http://www.jimmunol.org/ responses were undetectable in A/J mice (data not shown). After for infectious diseases (35, 36) and assessed in phase I and II 2/2 primary immunizations in both A/J and A/J MyD88 animals, clinical trials (38–40). Flagellin is also being developed as a IgG1 anti-OVA Abs were at titers of #100 in all mice (Fig. 8C). In therapeutic agent to treat infectious diseases, toxic exposures, and A/J mice after secondary immunizations with FliC and OVA, IgG1 cancers (75–77). Several studies have shown that flagellin’s anti-OVA responses were robust, with titers reaching a median of adjuvancy is dependent on TLR5 recognition (35–37). More re- 5 2/2 2/2 10 (Fig. 8C). As for C57BL/6, MyD88 and A/J MyD88 cently, murine Naip5 and Naip6 have been demonstrated to rec- mice had reduced, but detectable, IgG1 anti-OVA titers compared ognize bacterial flagellin and activate the Nlrc4 inflammasome with that of their WT counterparts (Fig. 8C). (21, 22). Additional studies have indicated that inflammasome- by guest on September 25, 2021 MyD88 suppresses IgA anti-FliC responses in A/J mice. Sera from mediated detection of flagellin also contributes to its immunoge- A/J and A/J MyD882/2 were also assessed for IgA anti-FliC re- nicity (15). Vijay-Kumar et al. (16) demonstrated that TLR5 and sponses. In contrast with C57BL/6 mice, A/J mice did not gen- the Nlrc4 inflammasome play redundant roles in flagellin-induced

FIGURE 5. IgA anti-FliC responses are TR5 and MyD88 dependent. C57BL/6 (n = 17) (A), TLR52/2 (n = 14) (B), Naip52/2 (n =9)(C), Casp12/2 (n = 11) (D), TLR52/2/Casp12/2 DKO (n =9)(E), and MyD882/2 (n = 11) (F) mice were immunized on days 1 and 21, and sera were collected on days 0 (naive), 14, and 35. Naive and day 35 sera (boost) were analyzed for IgA-specific Ab responses against FliC by ELISA. Data are a combination of two to three independent experiments with n = 3–6 per experiment. Statistical analyses were done using Mann–Whitney analysis of individual groups: *p , 0.05, ***p , 0.001. 1592 FLAGELLIN INDUCES Ab RESPONSES THROUGH A NOVEL PATHWAY Downloaded from

FIGURE 6. Flagellin-induced IgG1 anti-OVA responses are partially MyD88 dependent. WT mice were immunized twice on days 1 and 21 with OVA alone (n = 5) or OVA plus FliC (n = 5), and sera were collected on days 14 and 35. (A) Naive, day 14, and day 35 sera were analyzed for IgG1-specific Ab http://www.jimmunol.org/ responses against OVA by ELISA. (B–F)WT,TLR52/2 (n =19)(B), Naip52/2 (n =9)(C), Casp12/2 (n =14)(D), MyD882/2 (n = 12) (E), and TLR52/2/Casp12/2 DKO (n =9)(F) mice were immunized twice on days 1 and 21 with OVA plus FliC, and sera were collected on days 14 and 35. Naive, day 14, and day 35 sera were analyzed for IgG1-specific Ab responses against OVA by ELISA. (B–F) Data are a combination of two to three independent experiments with n = 3–7 per ex- periment. Statistical analyses were done using Mann–Whitney analysis of individual groups: *p , 0.05, ***p , 0.001.

Ab responses, with neither being necessary and either being suf- Naip5 inflammasome alone were sufficient to reduce IgG1 anti- ficient for flagellin-induced Ab responses. This same group de- FliC Abs, deletion of both TLR5 and Casp1 or elimination of termined that MyD88 is not required for flagellin-induced IgG Ab MyD88 significantly reduced IgG1 responses toward flagellin. responses, suggesting that an MyD88-independent pathway ema- However, IgG1 anti-FliC responses were still detected in MyD88 by guest on September 25, 2021 nates from either TLR5 or the Nlrc4 inflammasome, and con- and TLR5/Casp1 DKO mice, suggesting flagellin is recognized by tributes to anti-flagellin IgG production. a third uncharacterized pathway. This conclusion is supported by In our study, we dissected the role of flagellin detection by the our studies in A/J MyD882/2 mice, which are naturally impaired innate immune system in generating isotype-specific Ab responses in Naip5 (74). The A/J MyD882/2 mice produced reduced, but to flagellin itself (intrinsic adjuvancy) and coadministered OVA moderate, IgG1 and IgG2a anti-FliC responses. The combined (extrinsic adjuvancy). Although neither deletion of TLR5 nor the data from immunizations performed in C57BL/6 and A/J mice

FIGURE 7. Defective flagellin-induced IL-18 in A/J mice. C57BL/6 (n = 5) and A/J (n = 5) mice were injected i.p. with FliC (30 mg). Serum was collected 2 h after injections, and cytokine levels were determined by ELISA: IL-6 (A), IL-12/23p40 (B), Cxcl1 (C), IL-18 (D), IL-1b (E), and TNF (F). Statistical analysis was done using one-way ANOVA with Bonferroni multiple-comparisons posttest: *p , 0.05. The Journal of Immunology 1593

FIGURE 8. Augmented IgG1 and IgG2a anti-FliC responses in A/J mice are partially MyD88 dependent. C57BL/6 (n = 9) and A/J mice (n = 7) and their MyD88- deficient counterparts (n = 7–9) were immunized twice on days 1 and 21 with FliC plus OVA, and sera collected on days 14 and 35 were analyzed for IgG1 (A) and IgG2a or IgG2c (B) responses against FliC by ELISA. (C) Naive, day 14, and day 35 sera were assessed for IgG1 anti-OVA responses by ELISA. Data are a combi- nation of two independent experiments with n = 3–5 per experiment. Statistical analyses were done using Mann– Whitney analysis of individual groups: *p , 0.05, **p , 0.01. Downloaded from http://www.jimmunol.org/

support our working hypothesis that a novel third pathway contrib- Although this MyD88-independent pathway did not contribute utes to Ab responses toward flagellin, and that this pathway is in- to IgG2c responses in C57BL/6 mice, this may be because of dependent of MyD88 and known innate detection pathways for the low magnitude of the IgG2c responses toward flagellin ob- flagellin recognition, TLR5, and Naip5. Uncloaking this third path- served in C57BL/6 mice. Thus, it will be important to further way would help to understand the complexities of innate immune dissect the components of the immune systems in C57BL/6 and

recognition of flagellated pathogens and will also be critical for the A/J mice that are responsible for MyD88-independent responses by guest on September 25, 2021 rationale design of flagellin-based vaccines. It will be critical to as- against flagellin. Similarly, the structural components of bacte- sess whether this third pathway is conserved in humans and how this rial flagellin that dictate MyD88-independent Ab production are pathway may influence the generation of robust cellular and humoral uncharacterized. Understanding this third pathway of flagellin is responses, and the establishment of long-term immunity. another piece to the puzzle that can be used to enhance flagellin- Our data also demonstrate that, in C57BL/6 mice, TLR5 and based vaccine design. the Naip5/Nlrc4/caspase-1 inflammasome work in parallel to drive Our results from C57BL/6 mice demonstrate that IgA anti-FliC IgG2c anti-FliC responses in an MyD88-dependent manner. The responses are TLR5 and MyD88 dependent, consistent with a re- MyD88 dependency of the IgG2a/c Ab response is even more ap- cently published report (72). We add to this body of work by parent in A/J mice, which have quantitatively greater Ab responses demonstrating that the inflammasome does not impact IgA anti- and markedly enhanced IgG2a response that is equivalent to the FliC titers, and thus does not compensate for TLR5 deficiency. IgG1 response. The studies in A/J mice demonstrate that the Our results from the C57BL/6 mice are congruent with the data IgG2a primary response has greater dependence on MyD88 than from Cunningham and colleagues (72), which indicate that lamina the IgG1 response, as seen in C57BL/6 mice. However, unlike propria TLR5+ CD103+ DCs prime Foxp3+ regulatory T cells to C57BL/6, A/J mice also had an MyD88-independent compo- induce flagellin-specific IgA in the mesenteric lymph node. Con- nent for the IgG2a response. This suggests that underlying ge- versely, our data from A/J and A/J MyD882/2 mice indicate that netic differences between C57BL/6 and A/J mice contribute to IgA anti-FliC responses may be regulated differently in differ- isotype specificity and overall quantity of anti-flagellin immune ent strains of mice. In A/J mice, MyD88-dependent signals sup- responses. pressed IgA production. The mechanism for suppression of IgA

FIGURE 9. A/J MyD882/2 mice generate robust IgA anti- FliC responses. A/J mice (n = 8) and their MyD88-deficient counterparts (n = 7) were immunized twice on days 1 and 21 with FliC plus OVA, and sera were collected on days 0 (naive), 14, and 35. Naive and day 35 sera were analyzed for IgA- specific Ab responses against FliC by ELISA (A, B). Data are a combination of two independent experiments with n = 3–4 per experiment. Statistical analyses were done using Mann– Whitney analysis of individual groups: **p , 0.01. 1594 FLAGELLIN INDUCES Ab RESPONSES THROUGH A NOVEL PATHWAY anti-FliC responses in A/J mice is currently unknown and requires mers (87–89). Our results from TLR5/Casp1 DKO mice indicate further investigation. that the third pathway does not use either TLR5 or the inflamma- It is well publicized that flagellin, when used as an adjuvant, some. Thus, the possibility that TLR5 heterodimerizes with another imparts an IgG1 isotype-specific response toward coadministered TLR, such as the proposed TLR4/5 heterodimers, is an unlikely Ags, presumably because of TLR5 recognition (9, 15, 26, 30). Our explanation for the third pathway (27, 90). Gangliosides have been data support the conclusion that TLR5 and the Naip5/Nlrc4 in- implicated as receptors for bacterial flagellin (91, 92), and it is flammasome play overlapping roles for IgG1 responses toward possible that flagellin binding to glycolipids may induce receptor- OVAwhen coadministered with FliC (extrinsic adjuvancy). Because independent signaling in DCs, as has been proposed for mono- the anti-OVA IgG1 is reduced, but detectable, in both MyD882/2 sodium urate crystals (93). It is also formally possible that TLR11 and DKO, our data also indicate that the MyD88-independent could function through an MyD88-independent mechanism, or that pathway contributes to the adjuvancy of FliC toward OVA. There- Naip5 or Naip6 could signal independently of Casp1. The existence fore, our immunization studies support the existence of a third of both of these novel pathways would provide a complex expla- pathway for flagellin recognition that also contributes to flagellin’s nation for our results. Although we cannot exclude this possibility adjuvancy. of multiple novel signaling pathways emerging from the known Interestingly, humans contain one full-length Naip homolog, flagellin receptors, we favor the most parsimonious explanation for NAIP (78, 79). Human NAIP recognizes needle proteins from our data, which is a third pathway for flagellin recognition that bacterial type three secretory systems (21); however, there are also functions independently of MyD88, TLR5, and the inflammasome. reports indicating that NAIP recognizes flagellin (53, 80) and that Our results demonstrate that flagellin’s adjuvancy toward in- copy number variation for the human NAIP gene may affect ligand trinsic and extrinsic Ags can be attributed to at least three path- Downloaded from detection (79, 81). Although flagellin recognition by human NAIP ways in our animal model: TLR5, the Naip5/Nlrc4/Casp1 inflam- has been implicated with cellular assays (53), it still remains un- masome, and a novel MyD88-independent pathway. Currently, it substantiated by biochemical or genetic analysis (21, 53, 82). Further is unclear what constitutes the third novel pathway for flagellin- studies are required to determine the ligand specificity of human dependent Ab responses and what structures on flagellin are re- NAIP and the contribution of NAIP to flagellin-induced immune quired for this activity. In addition, it is unknown whether the third

responses in humans. pathway is evolutionarily conserved and contributes to flagellin- http://www.jimmunol.org/ In addition, it has been reported that TLR11 can recognize induced immunity in humans. These questions beg further inves- flagellin in mice of the C57BL/6 background (83). It is possible tigation to enhance our understanding of the biological properties that TLR11 contributed to Ab responses toward FliC. However, of flagellin and how flagellin may be used as an adjuvant, a vac- like all other TLRs, except TLR3 (84, 85), TLR11 is MyD88 cine platform, and a therapeutic agent. dependent (86). Because FliC-immunized MyD882/2 and TLR5/ Casp1 DKO mice have similar phenotypes, TLR11 does not ap- Acknowledgments pear to contribute significantly to flagellin-dependent Ab responses. We thank Lynn Hajjar for helpful comments. We thank Tom Hawn and his In any case, the translational implications of TLR11 recognition of laboratory for technical assistance. We thank Shizuo Akira, Richard Flavell, flagellin for vaccine development in humans are of little relevance and Russel Vance for providing knockout mice. by guest on September 25, 2021 because human TLR11 is a nonfunctional pseudogene (7). Our data indicate that flagellin is a potent adjuvant for IgG1 and Disclosures IgG2a/c isotype-specific responses toward itself. In addition, our The authors have no financial conflicts of interest. data indicate that TLR5 and MyD88 contribute to the IgG2a/c immune responses in both C57BL/6 and A/J mice. Our studies revealed that the IgG2a/c versus IgG1 bias of the anti-flagellin References humoral response was strongly influenced by mouse genetic back- 1. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197–216. ground. Because C57BL/6 mice have a weaker and IgG1-biased 2. Pedra, J. H., S. L. Cassel, and F. S. Sutterwala. 2009. 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