The observed band gap of 3.03 eV is in excellent agreement with that (3.07 eV) calculated using density functional theory (DFT) at the B3LYP/6–311++G level. The Hirshfeld surface analysis indicates a high percentage of C⋯H/H⋯C (41.2%) contacts in the crystal.In the crystal, molecules are connected into chains along [100]  31H20O, was synthesized using a Claisen–Schmidt condensation. The enone group adopts an s-trans conformation and the anthracene ring systems are twisted at angles of 85.21 (19) and 83.98 (19)° from the enone plane. In the crystal, molecules are connected into chains along [100] via weak C—H⋯π interactions. The observed band gap of 3.03 eV is in excellent agreement with that (3.07 eV) calculated using density functional theory (DFT) at the B3LYP/6–311++G level. The Hirshfeld surface analysis indicates a high percentage of C⋯H/H⋯C (41.2%) contacts in the crystal.The title compound, C Anth A is formed by the aromatic rings labeled as Cg1(C1–C6), Cg2(C1/C6–C8/C13/C14) and Cg3(C8–C13). Anth B consists of Cg4 and Cg6(C26–C31).The molecular structure of (I)A: C20—C21, C22—C23, C27—C28 and C29—C30; Anth B: C2—C3, C4—C5, C9—C10 and C11—C12), which are much shorter. These observations are consistent with an electronic structure for the anthracene units where a central ring displaying aromatic delocalization is flanked by two isolated diene units  Å and DFT (see below) = 1.34 Å].The C—C distances in the central ring of the anthracene units show little variation compared to the other rings (Anth s-trans configuration with the O1—C15—C16—C17 torsion angle being −179.19 (19) and 179.64° in the experimental and calculated structures, respectively. Additionally, the enone moiety  forms dihedral angles of 85.21 (19) and 83.98 (19)° with the Anth A  and Anth B  groups, respectively. The large dihedral-angle deviation indicates that the possibility for electronic effects between the anthracene units through the enone moiety has decreased -1-(anthracen-9-yl)-3-(2-chloro-6-fluorophenyl)prop-2-en-1-one The enone moiety (O1/C15–C17) shows an A and Anth B), connecting the molecules into infinite zigzag chains propagating along the [100] direction.In the crystal of (I)Gaussian09 program package ] with the 6-311++G  basis set as implemented in A and enone moiety . The calculated λmax of 390 nm is shifted from the experimental value, which may be attributed to solvent effects, compared to the gas-phase calculation.The longest wavelength absorption maxima for (I)et al.  relates to the chemical activity of the molecule . The presence of this C—H⋯π interaction is also indicated through the combination of pale-orange and bright-red spots that are present on the Hirshfeld surfaces mapped over ed  and shape-index .Fig. 4de Fig. 5a and shex Fig. 5b.b) appear to be the major contributor to the Hirshfeld surface and are seen as one distinct spike with a minimum value for de + di that is less than the sum of the van der Waals radii (2.4 Å). The intermolecular C—H⋯π interactions are characterized by the short interatomic C⋯H/H⋯C (41.2%) contacts and their presence is indicated by the distribution of points around a pair of wings at de + di ∼2.6 Å .The two-dimensional fingerprint plots shown in Fig. 6 Å Fig. 6c.et al., 2016et al., 1975E)-1-(anthracen-9-yl)-3-[4-(propan-2-yl)phenyl]prop-2-en-1-one -1-(anthracen-9-yl)-3-(2-chloro-6-fluorophenyl) prop-2-en-1-one  and 9-anthracenecarboxaldehyde (0.5 mmol) was dissolved in methanol (20 ml). A catalytic amount of NaOH  was added to the solution dropwise with vigorous stirring. The reaction mixture was stirred for about 5-6 h at room temperature. After stirring, the contents of the flask were poured into ice-cold water (50 ml). The resultant crude products were filtered, washed successively with distilled water and recrystallized from acetone solution as yellow blocks. The single crystal  used for data collection was obtained by the slow-evaporation technique using acetone as the solvent.Uiso(H)=1.2Ueq(C).Crystal data collection and structure refinement details are summarized in Table 210.1107/S2056989018003791/hb7739sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018003791/hb7739Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018003791/hb7739sup3.pdfSupplemetary figures and table. DOI: Click here for additional data file.10.1107/S2056989018003791/hb7739Isup4.cmlSupporting information file. DOI: 1817217CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


During acute infections and chronic illnesses, the pro-inflammatory cytokine interleukin-1β (IL-1β) acts within the brain to elicit metabolic derangements and sickness behaviors. It is unknown which cells in the brain are the proximal targets for IL-1β with respect to the generation of these illness responses. We performed a series of in vitro experiments to (1) investigate which brain cell populations exhibit inflammatory responses to IL-1β and (2) examine the interactions between different IL-1β-responsive cell types in various co-culture combinations.t tests or by ANOVA followed by Bonferroni-corrected t tests.We treated primary cultures of murine brain microvessel endothelial cells (BMEC), astrocytes, and microglia with PBS or IL-1β, and then performed qPCR to measure inflammatory gene expression or immunocytochemistry to evaluate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. To evaluate whether astrocytes and/or BMEC propagate inflammatory signals to microglia, we exposed microglia to astrocyte-conditioned media and co-cultured endothelial cells and glia in transwells. Treatment groups were compared by Student’s IL-1β increased inflammatory gene expression and NF-κB activation in primary murine-mixed glia, enriched astrocyte, and BMEC cultures. Although IL-1β elicited minimal changes in inflammatory gene expression and did not induce the nuclear translocation of NF-κB in isolated microglia, these cells were more robustly activated by IL-1β when co-cultured with astrocytes and/or BMEC. We observed a polarized endothelial response to IL-1β, because the application of IL-1β to the abluminal endothelial surface produced a more complex microglial inflammatory response than that which occurred following luminal IL-1β exposure.Inflammatory signals are detected, amplified, and propagated through the CNS via a sequential and reverberating signaling cascade involving communication between brain endothelial cells and glia. We propose that the brain’s innate immune response differs depending upon which side of the blood-brain barrier the inflammatory stimulus arises, thus allowing the brain to respond differently to central vs. peripheral inflammatory insults.The online version of this article (doi:10.1186/s12974-017-0908-4) contains supplementary material, which is available to authorized users. Infectious illnesses and chronic diseases elicit a constellation of metabolic and behavioral responses, including anorexia, weight loss, fever, lethargy, and disrupted sleep , 2. ThesSystemic inflammation is a common feature of the disparate infections and illnesses that cause sickness behaviors and cachexia . ActivatIl1r1 mRNA is primarily expressed by blood vessels, meninges, choroid plexus, and ependymal cells lining the cerebroventricles, but has also been reported in glia and discrete neuronal populations  for 45 min at 37 °C in a shaking water bath. Cells were resuspended in endothelial culture medium containing 20% BSA (pH 7.4) and were spun at 1000×g for 20 min at 4 °C. After aspirating the myelin-rich layer and supernatant, the cells were mixed with collagenase/dispase  and DNase I (10 μg/mL) in ggDMEM and were incubated in a shaking 37 °C water bath for 30 min. Cells were then layered on a 33% Percoll  gradient and spun at 1000×g for 20 min at 4 °C. The microvessel layer was removed, mixed with ggDMEM, and spun for 8 min at 700×g. At the end of the isolation, pellets were resuspended in endothelial culture medium [ggDMEM + endothelial cell growth supplement  + 20% fetal bovine serum ) + heparin ] supplemented with puromycin . Seventy-two h after seeding fragments into collagen-coated flasks, cells were washed with PBS and switched to puromycin-free culture medium. Medium was changed every 2–3 days until the cells reached confluence. Cells were then trypsinized and re-plated into 6-well plates for RNA analysis or 24-well plates containing poly-d-lysine/laminin-coated coverslips  for immunocytochemistry (ICC).Primary brain microvessel endothelial cell (BMEC) cultures were generated as previously described, with a few modifications . For eac3, 6 g/L d-glucose, 300 mg/L BSA, 1.44 g/L MgSO4*7H2O, and 10 mM HEPES in Hank’s Balanced Salt Solution) and digested with papain (Worthington) for 5 min. The enzymatic digestion was terminated by the addition of culture medium [DMEM  + 10% FBS + 1% penicillin/streptomycin (Life Technologies)]. Cell suspensions were filtered and spun at 150×g for 10 min. Cells were seeded into T-75 flasks and placed in a 37 °C incubator with 5% CO2/95% O2. Culture medium was changed 24 h later, and then every 3–5 days thereafter. Between days in vitro (DIV)10–DIV20, mixed glia cultures were dissociated with 0.05% trypsin-EDTA and seeded into 6-well plates , 24-well plates containing poly-d-lysine/laminin-coated coverslips for ICC, or the upper inserts of transwells for luciferase assays.The brains were harvested from neonatal mouse pups on P1-P5. Cortices were dissected, separated from the meninges, rinsed in dissecting buffer (350 mg/L NaHCO6 cells/well) or 24-well (1.5 × 105 cells/well) plates. Seventy-two h later, 1 mM l-LME was added to each well to selectively deplete microglia [l-LME treatment was repeated again 3 days later on DIV6. On DIV9, astrocytes were treated with 0.05% trypsin-EDTA, spun at 150×g for 10 min, and then seeded into 6-well plates for RNA analysis.Neonatal mouse cortices were processed as described above, and cells were seeded into 6-well  after coating the dishes with 0.01% poly-l-lysine (Sigma). The HIV-1-based lentiviral vector stocks were produced by co-transfecting the helper constructs pLP1, pLP2, and pLP/VSVG [Human embryonic kidney cells (HEK293T) were seeded at a density of 1.5 × 10pLP/VSVG  and the pLP/VSVG . SIM-A9 pLP/VSVG . Cells wd-lysine/laminin-coated coverslips. PBS or IL-1β (50 ng/mL) was added to both the upper and lower chambers for 30 min before proceeding with immunostaining.Primary BMEC were isolated as described above, with a few deviations. After spinning the endothelial fragments in the Percoll gradient, cells were resuspended in culture medium containing endothelial cell growth supplement (60 μg/mL), 20% plasma-derived platelet-free serum , heparin (100 μg/mL), Glutamax , and puromycin (4 μg/mL). Endothelial fragments were seeded onto collagen-coated transwell inserts . After 72 h, BMEC were switched to puromycin-free culture medium and were co-cultured with WT primary mixed glia in the lower wells. BMEC were confluent after 4 days in co-culture with mixed glia, at which time the transwell inserts were moved to wells containing SIM-A9 or NF-kB Luc SIM-A9 cells. We used SIM-A9 cells as surrogates for primary microglia because we had difficulty isolating the large numbers of primary microglia that would be necessary for these experiments. BMEC were treated with PBS or IL-1β (50 ng/mL) added to either the luminal surface (upper chamber) or abluminal surface (lower chamber) for 6 or 8 h. Cells were lysed and then assayed for luciferase activity or frozen at −80 °C for gene expression analysis. Transwell inserts were pooled (two inserts pooled together) for measuring BMEC gene expression. In a separate experiment, once the BMEC reached confluence, the transwell inserts were moved to wells that contained CX3CR1-EYFP-Cre primary mixed glia on poly-l-glutamine and 50 μg/mL gentamicin (phenol red-free ggDMEM). l-NAME (1 mM) or phenol red-free ggDMEM was added to both the upper and lower chambers for 1 h. Then, PBS or IL-1β (50 ng/mL) was added to the upper chambers  for an additional 8 h. SIM-A9 cells were lysed and frozen at −80 °C for gene expression analysis. Supernatants from the upper chambers were analyzed for nitrate/nitrite levels using a nitrate/nitrite colorimetric assay kit  following the manufacturer’s instructions.BMEC and SIM-A9 cells were seeded into transwells as described above. Cells were switched to phenol red-free DMEM supplemented with 2 mM WT astrocyte-enriched cultures were treated with PBS or IL-1β (50 ng/mL) for 24 h, after which the astrocyte-conditioned media was removed and added to wells containing WT primary microglia or SIM-A9 cells for an additional 4 h. Gene expression was measured in the astrocytes, primary microglia, and SIM-A9 cells.−ΔΔCt method. Statistical analyses were performed on the ΔCt values for each gene.All gene expression studies were conducted 24–48 h after cells were seeded into 6- or 12-well plates. Cells were washed with PBS and then switched to serum-free media for 30 min. Cells were then treated with vehicle (PBS or PBS + 0.1% BSA), IL-1β (50 ng/mL), TNF-α (50 ng/mL), or LPS (10 ng/mL) for 4 h prior to lysis and stored at −80 °C. SIM-A9 cells were treated with PBS or IL-1β (50 ng/mL) for 1, 2, 4, 8, or 24 h. Total cellular RNA was extracted using RNeasy kits (Qiagen). cDNA was generated using Taqman reverse transcription reagents as previously described . Real-tiNF-kB Luc SIM-A9 cells were seeded into empty 12-well or 48-well plates, 12-well plates containing WT primary mixed glia, or in the lower chambers of transwell plates with WT primary mixed glia in the upper chambers. Forty-eight h later, cells were washed in PBS and incubated in serum-free media for 30 min prior to treatment. Cells were treated with vehicle (PBS or PBS + 0.1% BSA), IL-1β (50 or 100 ng/mL), or LPS (10 or 100 ng/mL) for 6 h and then lysed in Glo Lysis buffer . Luciferase activity in the lysates was measured using the Bright-Glo Luciferase Assay System (Promega) and a BioTek Gen5 microplate reader . Luminescence was normalized to total protein content, which was measured using a Pierce BCA protein assay kit  according to the manufacturer’s instructions.n = 4 each for SIM-A9 + PBS, SIM-A9 + IL-1β, empty lower chambers + PBS, and empty lower chambers + IL-1β). After 8 h, the supernatant was removed from both the upper and lower chambers and frozen at −80 °C. Supernatant IL-1β was measured by ELISA  according to the manufacturer’s instructions. Sensitivity of the assay was 8 pg/mL.WT BMEC were seeded into the upper inserts of 16 transwells. In eight of the transwells, SIM-A9 cells were seeded into the lower chambers. The other eight transwells contained empty lower chambers. PBS or IL-1β (50 ng/mL) was added to the upper chambers (d-lysine/laminin-coated coverslips. Cells were washed in PBS and incubated in serum-free media for 30 min. Cells were then treated with vehicle (PBS or PBS + 0.1% BSA), IL-1β (50 ng/mL), or LPS (10 ng/mL) for 30 min. Cells were then fixed in 4% paraformaldehyde for 30 min and blocked for 30 min in 0.3% Triton-X 100 + 1% BSA in PBS. Cells were incubated with primary antibodies diluted in PBS + 0.3% Triton-X 100 + 5% normal serum at 4 °C overnight. Primary antibodies were used at the following concentrations: rabbit anti-p65 NF-κB , mouse anti-GFAP , rat anti-PE-CAM , and chicken anti-GFP . The next day, cells were incubated in Alexa Fluor secondary antibody  for 2 h at room temperature. The secondary antibodies were donkey anti-rabbit 555, goat anti-mouse 633, donkey anti-rat 488, and goat anti-chicken 488 (Life Technologies). Cells were stained with DAPI and mounted onto gelatin-coated slides using Aqua-Poly/Mount . Cells were imaged with a Nikon Ti Eclipse inverted microscope and NIS Elements software . Grayscale images were merged and pseudo-colored using Adobe Photoshop CS6 . To quantify microglial NF-κB nuclear localization, 10 random fields from each coverslip (2–3 coverslips per treatment group) were imaged. An observer who was blinded to the treatment groups tabulated the percentage of YFP-positive cells with concentrated nuclear NF-kB labeling. To quantify BMEC NF-κB nuclear localization, three random fields from each coverslip or transwell (two to three coverslips or transwells per treatment group) were imaged. The percentage of DAPI-labeled nuclei that were co-labeled with NF-κB was tabulated by a blinded observer. For occludin immunostaining, transwell inserts containing BMEC were fixed in ice cold 100% ethanol for 30 min, blocked for 30 min in PBS + 3% FBS, and then incubated in rabbit anti-occludin antibody  at 4 °C overnight. The next day, cells were incubated in goat anti-rabbit 488 (Life Technologies) for 2 h at room temperature and then mounted with Fluoromount-G with DAPI. Cells were imaged with a DM4000 B fluorescent microscope  equipped with a DFC340 FX camera (Leica).Mixed glia, primary microglia, and BMEC were processed for ICC 24–48 h after they were seeded onto poly-t tests or ANOVA followed by Bonferroni-corrected t tests. For the SIM-A9 time course experiment, groups were compared by two way ANOVA (time × treatment) followed by Bonferroni-corrected t tests. Differences were considered significant when p < 0.05.Data are expressed as the mean ± SEM for each group. Statistical analyses were performed using GraphPad Prism 5 for Mac OS X. Groups were compared by Student’s Il1r1 and Myd88 gene expression in BMEC, mixed glia, enriched astrocytes, and primary microglia that were harvested from WT mice and in SIM-A9 cells  in cultured BMEC from WT mice, but not in MyD88KO endothelial cells  and inducible nitric oxide synthase (Nos2) in WT but not MyD88KO BMEC  and P-selectin (encoded by the Selp gene), both of which promote leukocyte extravasation. We observed large increases in Icam1 (24-fold) and Selp (56-fold) mRNAs in WT but not MyD88KO endothelial cells in response to IL-1β , Cxcl10, Nos2, and Ptgs2 mRNA expression in mixed glia and astrocyte-enriched cultures derived from WT mice  increased by IL-1β treatment in WT but not MyD88KO microglia  and Cxcl10 (2.0-fold) mRNAs. Nos2 was the only gene that was highly upregulated (7.8-fold) by IL-1β in WT microglia. Similarly, IL-1β elicited small but statistically significant increases in the expression of inflammatory genes in the microglial cell line SIM-A9 , but we did not detect increased luminescence in response to IL-1β , we observed nuclear NF-κB in virtually all the cells after LPS treatment, but only rarely in microglia that were treated with IL-1β Fig. . Nfkbia lia Fig. . Expresslia Fig. , c. In W-1β Fig. .Fig. 2MiIl1b, Il6, Tnf, Nfkbia, Cxcl10, Nos2, and Ptgs2 gene expression in response to LPS  or TNFα (a MyD88-independent cytokine). Primary microglia and SIM-A9 cells exhibited pronounced increases in Il1b, Il6, Tnf, Nfkbia, Cxcl10, Ptgs2, and Nos2 gene expression , but did not increase luminescence in the underlying NF-κB Luc SIM-A9 cells  when IL-1β was added to the luminal endothelial surface . In another experiment, we added IL-1β to the lower chambers  and observed patterns of endothelial gene expression that were similar to what we observed following luminal IL-1β exposure  and Ptgs2 (threefold) gene expression in response to abluminal IL-1β treatment  endothelial surface facing the upper chamber and the abluminal  endothelial surface facing the lower chamber . BMEC seent Fig. . The mosace Fig. . With thure Fig. . In the ent Fig. . In the Lox/Lox mice are completely resistant to anorexia, weight loss, fever, and reduced locomotor activity in response to icv IL-1β [IL-1β signaling in the CNS plays a critical role in innate immunity and cellular inflammatory responses. To address the question of which cells mediate IL-1β-induced disruptions in behavior, metabolism, and neuroendocrine function in vivo, many groups have genetically manipulated IL-1R1 or MyD88 expression in specific brain cell populations. For example, the Tie2 promoter is commonly exploited to target gene constructs to endothelial cells. We previously demonstrated that Tie2Cre-MyD88cv IL-1β  or to recv IL-1β , other icv IL-1β –35. ThusNfkbia mRNA expression  in the brain microvasculature following central or peripheral injection [Cxcl10, and synthetic enzymes for production of nitric oxide and prostanoids in WT BMEC. Similar transcriptional and secretory profiles in response to IL-1β have also been demonstrated in BMEC in vivo [Endothelial cells are cellular targets for inflammatory mediators and play a role in generating tissue responses to systemic infections. In the non-inflamed brain, cerebrovascular cells are the principal sites of IL-1R1 expression , and disnjection –40, and njection . We also in vivo , 43.Il1r1 mRNA in our primary microglia cultures, we cannot rule out the possibility that this was due to Il1r1 expressed by residual astrocytes (and/or other CNS cells) that were not removed during the trypsinization process. Similarly, Pinteaux et al. (2002) reported a low level of Il1r1 mRNA expression in primary microglia cultures, but could not rule out oligodendrocyte progenitor cell contamination [Il1r1 mRNA was undetectable in SIM-A9 cells, nor was it detected in two other microglial cell lines [Il1b, Tnf, and Il6) gene expression and chemokine (MIP-1α and MIP-1β) secretion in cultured human fetal microglia [Nfkbia mRNA. The functional significance of this is unclear, because we did not detect NF-κB nuclear localization in isolated primary microglia or increased luciferase activity in NF-κB Luc SIM-A9 cells. We also observed small (1.5- to 2-fold) yet statistically significant increases in other inflammatory genes in primary microglia and SIM-A9 cells. The most pronounced effect of IL-1β was on Nos2 gene expression, which was increased 7.8-fold in WT primary microglia . Nos2 is transcriptionally regulated by diverse inflammatory stimuli and is widely accepted as a marker for classical (M1) microglial activation [In response to pathogens or tissue damage, microglia alter their morphology and release pro-inflammatory cytokines and chemokines. Microglia are activated by various inflammatory stimuli, including pathogen-associated molecular patterns  and cytokines  , 45. In mination . Il1r1 mll lines . Althougicroglia , 48. In icroglia . Likewisicroglia . In our tivation , 50. We Astrocytes express IL-1R1 , 46, 51 Nos2 gene expression in primary microglia. However, Nos2 gene expression was significantly reduced in SIM-A9 cells that were exposed to astrocyte-conditioned media, regardless of whether the astrocytes were treated with PBS or IL-1β, suggesting that astrocytes release signals that dampen microglial nitric oxide production. A role for astrocytes in inhibiting microglial Nos2 production has previously been demonstrated in LPS-treated mixed glia cultures [Nos2 responses of primary microglia and SIM-A9 cells could be indicative of a fundamental difference between the two cell types in their ability to respond to inflammatory signals. Alternatively, induction of Nos2 mRNA could be due to residual astrocyte contamination in our primary microglia cultures.We observed that conditioned media from IL-1β-treated astrocytes increased cultures . The difNfkbia mRNA in the absence of BMEC than they did in the presence of BMEC, which suggests that endothelial cells release signals that dampen microglial NF-κB signaling. However, endothelial IL-1β treatment did increase inflammatory gene expression in SIM-A9 cells. When IL-1β was added to the luminal endothelial surface, only Il1b gene expression was induced in the underlying SIM-A9 cells. Adding IL-1β to the abluminal endothelial surface also induced Il1b mRNA expression in SIM-A9 cells, as well as Il6 and Ptgs2 mRNAs. The fact that SIM-A9 cells did not exhibit inflammatory responses to IL-1β in the absence of BMEC provides further confirmation that the SIM-A9 cells were not directly activated by IL-1β, but rather by an endothelial-derived signal. This is consistent with in vivo experiments demonstrating that after peripheral or central IL-1β injection, increased Nfkbia mRNA expression in brain endothelial cells precedes microglial activation [We also investigated the possibility that IL-1β-treated brain endothelial cells relay inflammatory signals to microglia. When all three cell types  were cultured together in transwells, IL-1β induced NF-κB nuclear localization in approximately 50% of microglia. This percentage of microglial activation is comparable to what we observed in IL-1β-treated mixed glia cultures. IL-1β did not induce luciferase activity in NF-κB Luc SIM-A9 cells that were co-cultured with BMEC. In one of our transwell experiments, IL-1β-treated SIM-A9 cells exhibited greater induction of tivation , 40.Given their polarization and position at the blood-brain interface, it is not surprising that brain endothelial cells have the ability to differentially respond to inflammatory signals arriving via the blood vs. locally generated within the CNS. IL-1R1 is expressed on both the luminal and abluminal endothelial cell membranes . In our Il1b synthesis. Finally, examining the role of direct communication between endothelial cells and astrocytes, as well as the involvement of other brain cell populations  in propagating local inflammatory responses in the CNS is a worthy subject for future investigation.Future studies will be devoted to identifying the astrocyte- and endothelial-derived signaling molecules that activate microglia under inflammatory conditions. Our observation that only astrocyte-derived signals induced NF-κB activity in NF-κB Luc SIM-A9 cells is consistent with the hypothesis that astrocytes and endothelial cells use different signaling mediators to activate microglia. Furthermore, it is plausible that BMEC release different signaling molecules depending upon which endothelial membrane is exposed to IL-1β, because the SIM-A9 transcriptional profile differed in response to luminal vs. abluminal IL-1β treatment. Based upon the BMEC transcriptional response to IL-1β, likely candidate molecules for endothelial-microglia communication include IL-6, TNFα, and prostaglandins. With respect to the latter, prostaglandin signaling is essential for IL-1β-induced fever , 58, 59,We have generated a model that proposes how the microglial response to IL-1β likely depends upon an endothelial-derived intermediate, and thus does not require passage of IL-1β into the brain Fig. . In respAdditional file 1: Figure S1.n = 4 per group. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT PBS group, #p < 0.001 vs. WT IL-1β group. (PDF 474 kb)Primary mixed glia and enriched astrocyte response to IL-1β is MyD88-dependent. Inflammatory gene expression in A. Primary mixed glia and B. Enriched astrocyte cultures from WT and MyD88KO mice that were treated with PBS or IL-1β (50 ng/mL) for 4 h. Additional file 2: Figure S2.n = 3 per group. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs. PBS group at the same time point. (PDF 466 kb)SIM-A9 cell transcriptional response to IL-1β. Inflammatory gene expression in SIM-A9 cells that were treated with PBS or IL-1β (50 ng/mL) for 1, 2, 4, 8, or 24 h. Gene expression in the IL-1β-treated cells is expressed as a percentage of the mean value for the PBS-treated cells at the same time point. Additional file 3: Figure S3.n = 4 per group. ***p < 0.001 vs. PBS-treated cells C. Inflammatory gene expression in SIM-A9 cells that were treated with PBS or TNFα (50 ng/mL) for 4 h. n = 4 per group. Data are expressed as mean ± SEM. *p < 0.05, ***p < 0.001 vs. PBS-treated cells. (PDF 468 kb)Primary microglia and SIM-A9 cells respond to LPS and TNFα. Inflammatory gene expression in A. WT primary microglia and B. SIM-A9 cells that were treated with PBS or LPS (10 ng/mL) for 4 h. Additional file 4: Figure S4.n = 4 per group. Data are expressed as mean ± SEM. **p < 0.01, ***p < 0.001 vs. PBS group. B. Inflammatory gene expression in SIM-A9 cells that were treated with PBS-treated astrocyte CM (PBS astrocyte CM) or IL-1β-treated astrocyte CM (IL-1β astrocyte CM) for 4 h. To control for direct effects of IL-1β on SIM-A9 cells, empty wells (containing no astrocytes) were treated with PBS or IL-1β for 24 h, and then the media was removed and applied to SIM-A9 cells for 4 h. These SIM-A9 groups are labeled as PBS and IL-1β, respectively. n = 3 to n = 4 per group. Data are expressed as mean ± SEM. ***p < 0.001 vs. PBS astrocyte CM group, #p < 0.05 vs. IL-1β astrocyte CM group, § vs. IL-1β group. (PDF 418 kb)Lack of effect of astrocyte-conditioned media (CM) on SIM-A9 gene expression. A. Inflammatory gene expression in enriched astrocyte cultures that were treated with PBS or IL-1β (50 ng/mL) for 24 h. Additional file 5: Figure S5.Il1b mRNA expression. BMEC were seeded into the upper inserts and SIM-A9 cells were seeded into the lower chambers of transwell plates. PBS or l-NAME (1 mM) was added to both the upper and lower chambers for 1 h, and then PBS or IL-1β (50 ng/mL) was added to the upper chambers for an additional 8 h. Nitrite/nitrate was measured in the supernatant from the upper chambers. n = 6 per group. Data are expressed as mean ± SEM. Bars with different superscripts are statistically different from one another (p < 0.05). (PDF 364 kb)Blockade of nitric oxide synthesis doesn’t alter SIM-A9 response to IL-1β-treated endothelial cells. A. Supernatant nitrite/nitrate levels, and B. SIM-A9


Trpa1−/− mice fail to effectively activate known fear/stress brain centers upon 2MT exposure, despite their apparent ability to smell and learn to fear 2MT. Moreover, Trpa1 acts as a chemosensor for 2MT/TMT and Trpa1-expressing trigeminal ganglion neurons contribute critically to 2MT-evoked freezing. Our results indicate that Trpa1-mediated nociception plays a crucial role in predator odor-evoked innate fear/defensive behaviors. The work establishes the first forward genetics screen to uncover the molecular mechanism of innate fear, a basic emotion and evolutionarily conserved survival mechanism.Innate behaviors are genetically encoded, but their underlying molecular mechanisms remain largely unknown. Predator odor 2,4,5-trimethyl-3-thiazoline (TMT) and its potent analog 2-methyl-2-thiazoline (2MT) are believed to activate specific odorant receptors to elicit innate fear/defensive behaviors in naive mice. Here, we conduct a large-scale recessive genetics screen of ethylnitrosourea (ENU)-mutagenized mice. We find that loss of Trpa1, a pungency/irritancy receptor, diminishes TMT/2MT and snake skin-evoked innate fear/defensive responses. Accordingly,  TMT is a chemical that evokes innate defensive behaviors yet the molecular mechanisms are not well understood. Here the authors perform a large-scale forward genetics screen in mice and identify Trpa1, a pungency/irritancy receptor, as a chemosensor for predator odor-evoked innate fear and defensive behaviors. Innate behaviors are prevalent and advantageous for survival in animal world. An innate behavior has at least three important traits: (1) it is instinctive and does not require learning; even an animal raised in isolation can perform the behavior when exposed to the stimulus for the first time; (2) it is stereotypic; all individuals of the same species will do it the same way every time; (3) it is heritable, passing from generation to generation through genes. However, the genetic bases of innate behaviors are largely unknown.3. Fear can be induced by both innate and learned mechanisms. In Pavlovian fear conditioning paradigms, rodents are trained to freeze in response to a conditioned  stimulus by pairing it with an unconditioned stimulus . On the other hand, laboratory rodents are instinctively afraid of snakes or cats despite being isolated from predators for generations. This innate fear represents an evolutionarily conserved and genetically encoded pro-survival mechanism4. Therefore, we hypothesized that it might be feasible to investigate the molecular mechanism of innate fear using a forward genetics approach.Fear is a basic emotion that triggers characteristic defensive behaviors and physiological responses to promote survival in dangerous situations6. One of the best-studied kairomones is fox odorant 2,4,5-trimethyl-3-thiazoline (TMT), which can also induce instinctive freezing in naive mice6. A potent analog of TMT, 2-methyl-2-thiazoline (2MT), has recently been shown to elicit highly robust freezing response in mice7. It is widely believed that specific odorant receptors are responsible for sensing TMT/2MT to elicit innate fear/defensive behaviors12. Accordingly, genetic lesion of dorsal class II olfactory sensory neurons (OSNs) rendered mice defective for TMT-induced avoidance9. At least two dozens of candidate odorant receptors for TMT have been identified13. Notably, optogenetic activation of Olfr1019-expressing OSNs induces immobility, whereas Olfr1019 knockout mice are partially defective for TMT-induced freezing13. Moreover, other TMT sensing mechanisms seem to exist because the Grueneberg ganglion (GG), which does not typically express odorant receptors, have been implicated in TMT-evoked freezing15. It has also been proposed that the trigeminal system plays a critical role in TMT-evoked freezing behavior17.Predator odors or kairomones, from the fur, skin, urine, feces, and excretory glands of the predators, have been widely used to evoke innate fear/defensive behaviors, such as avoidance and risk assessment behaviors, in laboratory mice20. In particular, Trpa1 is a well-known chemoreceptor for pungent natural compounds, such as cinnamaldehyde, allyl isothiocyanate and mustard oil23, as well as harmful environmental irritants, such as formalin and acrolein (tear gas)26. Accordingly, Trpa1 is highly expressed in the somatosensory systems, such as the trigeminal ganglia (TG), dorsal root ganglia and nodose ganglia, and has important functions in nociception, i.e., sensing harmful and potentially painful stimuli28. Here, we performed a large-scale recessive genetics screen of randomly mutagenized mice using a highly robust 2MT-evoked innate fear assay. We found that the Trpa1-mediated chemosensory system, in addition to classical olfactory system, plays a central role in mediating predator odor-evoked innate fear/defensive behaviors in mice.The transient receptor potential ankyrin 1 (Trpa1), a member of the TRP family of ion channels, has been shown to function as a chemical, mechanical, and temperature sensor29. By contrast, innate fear behaviors are simpler and less variable than learned fear behaviors. Unlike TMT, 2MT is not noxious, is sold cheaply, and elicits robust freezing behavior in mice7. Thus, we selected 2MT to develop a simple and highly robust innate fear assay suitable for high-throughput mouse screening  ranging 50–100%ing Fig. , we showing Fig. . This oping Fig. , making 30. Each ENU-mutagenized G1 founder male was crossed with wild-type females to generate G2 daughters, which were mated back with the G1 male to create a family of 15–50 G3 mice  contained four phenovariants that showed 20–30% freezing rates upon 2MT exposure  to a homozygous mutation in the Trpa1 gene  for abnormal freezing response upon 2MT exposure. In all, 33,069 coding/splicing mutations in 13,828 genes were examined. Approximately 6.5% of all genes were destroyed or damaged sufficiently to cause a phenotype, with the mutant alleles tested three times or more in the homozygous stateene Fig. . This 5′ins Fig. . By contTrpa1 mutation was the sole cause of the observed fearless phenotype, we examined an independently constructed Trpa1 knockout strain32, which removed exons 22–24 encoding the essential pore-forming domain of Trpa1 channel , a 2MT/TMT-related alarm pheromone15, evoked reduced freezing response in Trpa1−/− mice than in Trpa1+/− mice  of 2MT to further investigate the role of Trpa1 in other innate fear/defensive behaviors. As expected, Trpa1−/− mice displayed a significant deficit in low-dose 2MT-evoked freezing, avoidance, flight, and risk assessment behaviors as compared to Trpa1+/− littermates  and avoidance (average 3.9 vs. 29.6%), and exhibited less flight (average 0.6 vs. 8 times) and risk assessment (average 2.4 vs. 23.5 times) behaviors , including sniffing, moving, biting, and tearing the snake skin , which communicates with downstream brain regions, such as the ventral periaqueductal gray (vPAG), to mediate innate and learned freezing behaviors36. Accordingly, we observed that 2MT-evoked c-fos induction was greatly diminished in the CeA, vPAG, and the paraventricular nucleus (PVN) of hypothalamus in Trpa1−/− brains relative to Trpa1+/− brains  of Trpa1+/− and Trpa1−/− mice, including the dorsal region of OB that was specifically activated by TMT15  to examine whether 2MT activated the Trpa1-expressing TG neurons in vivo. Notably, 2MT exposure elicited strong nuclear c-Fos signals in specific TG neurons of heterozygous, but not homozygous, ice Fig. . Consistins Fig. . Double ure Fig. . Approxiure Fig. , but theure Fig. . The Fosc-fos expression in the Sp5C region was abolished in the lesion side as compared to the control side, confirming that TG neurons acted upstream of Sp5C neurons  mice died within a couple of days after the operation. Thus, we performed unilateral TG lesion (ulTGx) instead in wild-type mice Fig. . In ulTGons Fig. . Althougons Fig. .Fig. 7Tr+ TG neurons, we bilaterally injected adeno-associated virus expressing Trpa1 (AAV-Trpa1) into the TG of Trpa1−/− mice  2MT-evoked innate freezing as compared to AAV-GFP-injected mice  and snake skin-evoked innate freezing and other fear/defensive behaviors. Alternatively, it is possible that Trpa1 deficiency causes wiring/developmental defects in TG. However, this possibility is unlikely because TG injection of AAV-Trpa1 rescued 2MT-evoked innate freezing behaviors. Moreover, in contrast to Trpc2 that is expressed in almost all vomeronasal neurons, Trpa1 is expressed in only a small subset (~10%) of TG neurons, and thus cannot function as a housekeeping molecule for overall TG functions. Collectively, these studies suggest that mice use novel chemoreceptors as well as conventional odorant receptors to detect potentially dangerous chemicals in the environment, including kairomones from predators, to induce innate fear/defensive responses.The innate ability to detect chemical warning signals from predators is absolutely essential for species survival. We are just beginning to understand the molecular mechanisms by which preys detect a variety of predator scents. The trace amine-associated receptor 4 (Taar4), acts as the primary receptor responsible for elicitation of avoidance behavior in response to 2-phenylethylamine, isolated from carnivore urines48. Through unbiased forward genetics screening, we identified that Trpa1 deficiency resulted in diminished 2MT/TMT-evoked innate freezing responses. Whereas unilateral TG lesion significantly attenuated 2MT-evoked freezing in wild-type mice, bilateral TG injection of AAV-Trpa1 partially rescued 2MT-evoked innate freezing in Trpa1−/− mice. Our results, coupled with previous studies39, establish that both the trigeminal and olfactory systems play important roles in 2MT/TMT-evoked innate fear/defensive behaviors. It should be noted that Trpa1 might function in multiple sensory systems. We cannot exclude the possibility that an unidentified subset of Trpa1-expressing olfactory neurons may play a partial role in the predator odor-evoked innate fear/defensive responses. For example, Trpa1 may act as a chemosensor in the GG neurons that have been implicated in TMT-evoked innate freezing15. Future studies are needed to identify other types of tissues/neurons where Trpa1 functions and delineate the neural circuitries that underlie thiazolines-evoked innate fear/defensive responses.It has long been a subject of debate whether TMT is a bona fide predator odor or simply a pungent odor, and whether the olfactory or trigeminal system mediates TMT-elicited innate fear/defensive behaviors51. Thus, this type of learned fear is actually built on a pain-induced innate fear circuit53. In principle, predator odor-mediated activation of the Trpa1 nociceptive pathway should instinctively warn the mice of imminent dangers and trigger emergency responses to promote survival. Our studies provide a compelling molecular logic to explain why predator odor-evoked innate fear/defensive behaviors are genetically hardwired.In almost all of learned fear assays, animals are trained to fear a neutral stimulus by pairing it with an aversive stimulus, such as electric footshock or LiCl injection that causes pain or discomfort54. Emotions define the essence of being human and are instinctive drivers of behaviors3; and yet little is understood about the molecular bases of emotions. Although rapid progress has been made in mapping the neural circuitries of innate and learned fear58, the molecular circuits underlying fear, particularly the core signaling pathways that mediate various fear/defensive responses, remain poorly understood. Our results substantiate the utility of a forward genetics approach to investigate the molecular mechanism of innate fear and perhaps other innate behavioral paradigms.Forward genetics screening in mice represents one of the most effective approaches to understanding the genetic basis of human physiology and disease59 and bred with two pedigree structures at UTSWMC as previously described30. Mice were at least 14 weeks old at the start of fear screening. B6;129P-Trpa1tm1Kykw/J  and B6.129X1-Trpv1tm1jul/J mice  mice were purchased from JAX. It should be noted that although Trpa1−/− and Trpv1−/− mice have a mixed genetic background, the phenotypes of Trpa1−/− mice are consistent with those of homozygous Trpa1 ENU mutant mice in C57BL/6J background. All experiments were repeated independently for at least two times. We do not employ any method of randomization to determine how samples/animals were allocated. For all animal experiments, we chose at least n = 6 for each sample group to ensure adequate statistical power. The experimenters were blind to the genotypes of mice when performing the experiments and analyzing the results.Animal protocols used in this study were approved by the International Institute for Integrative Sleep Medicine at University of Tsukuba, Japan, as well as by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center (UTSWMC) at Dallas, USA. Mice were housed in groups of 4–5 and maintained on a 12-h light-dark schedule (lights on at 06:00) with ad libitum access to food and water. C57BL/6J mice were purchased from Jackson Laboratory , and mutagenizedele-In-One Mouse Tail Direct Lysis Buffer  at 55 °C overnight. The lysate was heated at 90 °C for 10 min to inactivate proteinase, cooled down, and centrifuged at 12,000 × g for 2 min. Genotyping of Trpa1 and Trpv1 knockout mice were carried out by PCR using the primers designed by JAX. For genotyping the Trpa1 fearless (frl) ENU mutant allele, the point mutation of Trpa1 gene was detected using the derived cleaved and amplified polymorphic sequence (dCAPS) method. Briefly, genomic DNA was amplified using 5′-TGGTAGAATACCTCCCCGAGTGCATGAATG-3′ and 5′-TGTGAAGAGCATTCATTCAGC-3′, which produced a BsmI restriction enzyme recognition site specific for the mutant Trpa1 allele. The PCR products were digested with BsmI and resolved by 3.5% agarose gel electrophoresis.For mouse genotyping, mouse tails were cut and lysed in 100 µl All7. A mouse was considered to be freezing if no movement was detected for ≥2 s. We used C57BL/6J male mice to systematically optimize the 2MT-evoked innate fear assay by varying the mouse age, 2MT dose and assay duration. Moreover, we used C57BL/6J males exclusively for the majority of behavioral experiments. However, we assayed both male and female mice from the ENU G3 mutant pedigrees during forward genetics screening. In general, mice of at least 14 weeks of age were individually introduced into an open test cage . A small (2 × 2 cm) filter paper containing 10 µl (1.05 × 10−4 mole) 2MT, 35 µl (2.98 × 10−4 mole) TMT, or 50 µl SBT (3.49 × 10−4 mole) was placed at a corner of test cage for 15–20 min. Mouse freezing behavior was analyzed as described above.Odorants 2MT and 2MO were purchased from Tokyo Chemical Industry , whereas TMT was initially purchased from Contech  and later chemically synthesized using the synthetic scheme described below. For comparison of innate freezing behavior in response to different odors, a constant concentration of test odorant-containing gas (10 ppm vol/vol) was produced using a calibration gas generator  and pumped into a sealed test cage (31.5 × 19.5 × 13 cm) via a tygon tube. A volume of odorant gas equal to that of the test cage was pumped into the test cage prior to the introduction of each mouse. Odor presentation was performed in a chemical fume hood. Mouse behaviors were recorded with a digital video camera  connected to a computer running the FreezeFrame software . The videos were analyzed by FreezeFrame to calculate the freezing rateG3 mice were pre-genotyped at all mutation sites using ampliseq panels and Ion Torrent sequencing. Mice were evaluated by pedigree for phenotypic anomalies using the 2MT-evoked innate fear assay alongside wild-type controls. Raw data (freezing rate) were entered into the Linkage Analyzer program, which tested the statistical association between assay performance and genotype for all mice at every mutation site in every pedigree, using dominant, additive, and recessive models of inheritance. The data were visualized in the form of a Manhattan plot in Linkage Explorer (−log(P) vs. chromosomal location).Trpa1+/+, Trpa1+/frl, Trpa1frl/frl mice and total RNA was extracted by Trizol . Total cDNA was synthesized from 1 µg RNA using the High Capacity cDNA Reverse Transcription kit , according to manufacturer’s protocol. For qPCR, cDNA template was mixed with 2 × iTaq™ universal SYBR® Green supermix  and primers in 384-well plate. The qPCR experiments were performed on C1000 TouchTM Thermal Cycler and CFX384 Real-Time System (Biorad). The amplification curves as well as Ct value were analyzed by Bio-rad CFX Manager. All the assays were performed in quadruplicate and ΔCt value was calculated compared to Pgk1. The results were processed and plotted by Prism 7.0 . The following primers were used: Trpa1-F: 5′-TGCTGACATCCTCCTGAACA-3′ and Trpa1-R: 5′-ACATCCTGGGTAGGTGCTACT-3′; Trpv1-F: 5′-CGAGGATGGGAAGAATAACTCACTG-3′ and Trpv1-R: 5′-GGATGATGAAGACAGCCTTGAAGTC-3′; Pgk1-F: 5′-AACCTCCGCTTTCATGTAGAG-3′ and Pgk1-R: 5′-GACATCTCCTAGTTTGGACAGTG-3′. We observed a faint band below the predicted PCR product in Trpa1+/frl and Trpa1frl/frl samples in Fig. Trpa1 caused skipping of exon 15, resulting in a frame-shift and introduction of a premature stop codon as shown in Fig. TG were dissected from Trpa1+/− and Trpa1−/− mice were individually habituated in a new and open test cage (Ancare N10HT 19.1 × 29.1 × 12.7 cm) for 15 min. We excluded the mice showing persistent immobility during habituation for analysis. After habituation, a small filter paper laced with low dose (1.05 × 10−6 mole) 2MT was presented at a corner of the cage for 5 min. Mouse behaviors were recorded with a digital video camera  from the top and with an iPhone from the side. The following fear-like and defensive behaviors are measured: The videos from the digital camera were analyzed to generate (1) freezing rate by the FreezeFrame and (2) avoidance curve by customized Matlab program. The iphone videos were analyzed to measure the (3) total investigation time when the mouse sniffed within a 1 mm distance from the filter paper; (4) times of the risk assessment behavior (stretch-attend posture)35; and (5) times of flight (turned around and escape) after sniffing the filter paper. For snake skin-evoked innate fear assay, test mice were single housed in the home cage with food/water grid and bedding for overnight. A piece of molted snake skin (2 cm × 16 cm) was introduced into a corner of the cage (do not remove the cover or the food/water grid during the test). The videos were recorded using an IPad from the side and analyzed by one experienced technician.12. Trpa1+/− and Trpa1−/− male mice were individually housed in the home cage and habituated in the test environment overnight. The experiment was performed between 8:00 and 10:00 am. Odor exposure was performed by quietly dropping a piece of filter paper containing water or 2MT  into the home cage (19.1 × 29.1 × 12.7 cm). After odor exposure for 20 min, blood samples were collected and centrifuged at 1000 × g for 15 min at 4 °C. The supernatant was collected and the plasma concentration of corticosterone was determined using an ELISA kit according to manufacturer’s protocols .The plasma level of stress hormone corticosterone was measured as previously describedTrpa1+/− and Trpa1−/− male mice were individually placed in the periphery of a novel open field environment (44 × 44 × 30 cm) in a dimly lit room and allowed to explore for 5 min. The animals were monitored from above by a digital video camera connected to a computer running video tracking software  to determine the time, distance moved and number of entries into two areas: the periphery  and the center (14 cm × 14 cm). The open field arenas were wiped and allowed to dry between mice. Data were analyzed with the unpaired Student’s t-test.Trpa1+/− and Trpa1−/− male mice were individually placed in the center of a black plexiglass elevated plus maze  elevated 31 cm in a dimly lit room and allowed to explore for 5 min. The animals were monitored from above by a video camera connected to a computer running video tracking software  to determine time spent in the open and closed arms, time spent in the middle, and the number of entries into the open and closed arm. The apparatus was wiped and allowed to dry between mice. Data were analyzed with the unpaired Student’s t-test.Trpa1+/− and Trpa1−/− male mice were individually placed into a chamber equipped with a metal grid floor connected to a scrambled shock generator . After 1 min, the mice received a series of footshocks (2 s each) with increasing intensity. The initial shock intensity was 0.05 mA and the amplitude was increased by 0.05 mA for each consecutive footshock with 15 s intershock interval. The first shock intensity that each animal displayed each behavior  was recorded. Once the animal displayed all three behaviors, it was removed from the chamber.Trpa1+/− and Trpa1−/− males were individually placed in the chamber. After 2 min, the mice received three tone-shock pairings . The following day, memory of the context was assessed by placing the mice into the same chambers and freezing was measured automatically by the Med Associates software. Forty-eight hours after training, memory for the white noise cue was measured by placing the mice in a chamber with altered floor and walls, different lighting, and a vanilla smell. Freezing was measured for 3 min, then the noise cue was turned on for an additional 3 min and freezing was measured.For fear conditioning, 9 with minor modifications using 14–19-week-old Trpa1−+/ and Trpa1−/− male mice. In brief, mice were habituated to a clean cage (17.5 × 10 × 15 cm) for 15 min, and then a filter paper (2 × 2 cm) with 20 μl of distilled water was presented for 3 min. This presentation of distilled water was repeated four times with 1-min intervals. On the fifth trial, a filter paper scented with 2MT (2.1 × 10−11 or 2.1 × 10−10 mole) was presented for 3 min. Mouse behaviors was recorded with a digital camera , and the total investigation time, defined as nasal contact within a 1 mm distance to the filter paper, was measured during 3 min exposure.The experiment was performed as described12 with minor modifications. In brief, fear conditioning was conducted in a conditioning chamber  located in a sound-attenuating box equipped with a fan, which allowed continuous removal of the odor. An odor delivery apparatus  was modified to separately deliver two odors. Trpa1 mutant mice were individually placed into the conditioning chambers. After 3 min of free exploration, one of the two odors, anisole  or eugenol  was delivered into the conditioning chamber for 30 s. The animals were exposed to six anisole presentations randomly intermingled with six eugenol presentations and separated by 4 min inter-trial intervals in the conditioning session. The 2 s of 0.4 mA electric footshocks were delivered from the metal-rod floor and overlapped only with the last 2 s of anisole presentations, but not with eugenol presentations. 1 × 10−3 mole eugenol and anisole were used for each animal. At the following day, Trpa1 mutant mice were individually placed into the test cages . After 10 min habituation (no odor), each mouse was presented sequentially with a plain filter paper (2 × 2 cm) for 10 min, a filter paper scented with eugenol (1.25 × 10−4 mole) for 10 min, and a filter paper scented with anisole (1.25 × 10−4 mole) for 10 min. Mouse behaviors were recorded and the freezing rates were quantified by FreezeFrame as described above. For 2MT/TMT-based learned fear assays, Trpa1−/− mice were used for fear conditioning according to the same protocol except that 1 × 10−3 mole 2MT or 1.5 × 10−4 mole TMT was used. Mouse behaviors were similarly analyzed except that each mouse was exposed to filter paper scented with 1.25 × 10−4 mole eugenol, 2.7 × 10−4 mole 2MT, or 4.0 × 10−4 mole TMT, respectively.The dual odor-based fear conditioning assays were performed with 12–14-week-old male mice as previously described7. Briefly, mice were habituated in a test cage for 2 h. A piece of filter paper scented with 15 µl water or 2MT (1.57 × 10−4 mole) was presented for a 30-min period. Mice were anesthetized with gaseous isoflurane and perfused with ice-cold 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The brains with the trigeminal nerve were dissected and fixed overnight in 4% PFA in PBS at 4 °C. The fixed brains were dehydrated in a graded ethanol and xylene series and then embedded in paraffin using an automated system . Coronal sections with a thickness of 5 µm were prepared using an automatic slide preparation system . ISH was performed using an automated system , according to the manufacturer’s protocols. The digoxigenin (DIG)-labeled Fos probe (129-bp to 537-bp and the 543-bp to 1152-bp regions of the gene) was diluted in 1:200 and hybridized at 74 °C for 3 h using a RiboMap Kit (Roche). The slides were then incubated with biotin-conjugated anti-DIG antibody  at 37 °C for 28 min. The probe was detected using the Ventana BlueMap Kit  at 37 °C for 6 h, and counterstained with a Red counterstain kit (Roche) at 37 °C for 4 min. Images were scanned using a NanoZoomer virtual microscope system , converted to gray scale, and signal intensities were quantified using Adobe Photoshop (n = 6–8 for each region). Anatomical locations were determined according to the mouse brain atlas60.All procedures were followed as previously describedacII and NotI and subcloned into pTrpa1 to generate pTrpa1-P2A-mCherry.To make the pTrpa1-P2A-mCherry construct, we amplified from mouse cDNA library the full-length Trpa1 open reading frame (ORF) by PCR with primers 5′-AAATTTCTCGAGATGAAGCGCGGCTTGAGGAG-3′ and 5′-AAATTTCCGCGGAAAGTCCGGGTGGCTAATAG-3′. The PCR product was cut with XhoI and SacII and subcloned into pEGFP-N1 to make pTrpa1. The P2A-mCherry fragment was amplified by PCR with primers 5′-AAATTTCCGCGGGGAAGCGGAGCTACTAACTT-3′ and 5′-AAATTTGCGGCCGCTTACTTGTACAGCTCGTCCATGCCG-3′. The P2A-mCherry fragment was cut with SWe mutated a number of cysteines of mouse Trpa1 to serines in the pTrpa1-P2A-mCherry plasmid using PfuUltra HF DNA polymerase . The primers for site-directed mutagenesis were listed below:C31S-F: 5′-GGGAAGACATGGACTCCTCCAAGGAATCCTTTAAGGTGGA-3′C31S-R: 5′-AAGGATTCCTTGGAGGAGTCCATGTCTTCCCCGACGCC-3′C45S-F: 5′-TTGAAGGAGATATGTCTAGATTAGAAGACTTCATCAAGAACC-3′C45S-R: 5′-GAAGTCTTCTAATCTAGACATATCTCCTTCAATGTCCACCTTAAAG-3′C66S-F: 5′-AGGATGAAAATCTCTCTCCTCTGCATCACGCAGCAGCA-3′C66S-R: 5′-GCGTGATGCAGAGGAGAGAGATTTTCATCCTCATATTTG-3′C89S-F: 5′-TCAATGGTTCTTCGTCTGAAGTGCTGAATATAATGGATGG-3′C89S-R: 5′-ATATTCAGCACTTCAGACGAAGAACCATTGATGATCAGTTC-3′C364S-F: 5′-TGAATTTGCTCCTCTCTAAAGGTGCCAAAGTAGACATAAAAGATC-3′C364S-R: 5′-ACTTTGGCACCTTTAGAGAGGAGCAAATTCACAATGTTCC-3′C415S-F: 5′-AAGACAATGACGGATCCACACCTCTCCATTATGCCTGTAG-3′C415S-R: 5′-TAATGGAGAGGTGTGGATCCGTCATTGTCTTCATCCATCA-3′C422S-F: 5′-CCTCTCCATTATGCCTCTAGGCAGGGGGTTCCTGTCTCTG-3′C422S-R: 5′-GGAACCCCCTGCCTAGAGGCATAATGGAGAGGTGTG-3′C541S-F: 5′-ATACTAACTTGAAATCCACAGACCGACTAGATGAAGAAGG-3′C541S-R: 5′-TCTAGTCGGTCTGTGGATTTCAAGTTAGTATCAAGAATGA-3′C622S-F: 5′-TCCAAGCAATCGATCTCCAATCATGGAGATGGTAGAATACCTC-3′C622S-R: 5′-TCTCCATGATTGGAGATCGATTGCTTGGAGAATTATGAGTG-3′C633S-F: 5′-AATACCTCCCCGAGTCCATGAAAGTTCTTTTAGATTTCTG-3′C633S-R: 5′-AAAAGAACTTTCATGGACTCGGGGAGGTATTCTACCATCT-3′C642S-F: 5′-TTCTTTTAGATTTCTCCATGATACCTTCCACAGAAGACAA-3′C642S-R: 5′-GTGGAAGGTATCATGGAGAAATCTAAAAGAACTTTCATGC-3′C666S-F: 5′-TCAAGTATCTCCAATCCCCATTATCCATGACCAAAAAAGTAGC-3′C666S-R: 5′-GTCATGGATAATGGGGATTGGAGATACTTGAAATTATACTC-3′C714S-F: 5′-TACTCATGAAATGGTCTGCCTATGGATTCAGAGCCCATATGA-3′C714S-R: 5′-CTGAATCCATAGGCAGACCATTTCATGAGTAAGTACTCCCT-3′C859S-F: 5′-AAAGGTTTGAGAACTCTGGAATTTTCATTGTTATGTTGGAGG-3′C859S-R: 5′-ACAATGAAAATTCCAGAGTTCTCAAACCTTTGAAGATACAGTAGG-3′C1041-F: 5′-CAAACATTGACACATCCTTGGAAATGGAAATATTGAAACAG-3′C1041-R: 5′-ATTTCCATTTCCAAGGATGTGTCAATGTTTGGTACTTCTTG-3′amHI and KpnI, subcloned into the pKH3 plasmid, and verified by sequencing. A second PCR was performed to amplify HA-Trpa1 from pKH3-Trpa1 using primers: 5′-AAATTTCTCGAGATGTACCCATACGATGTTCC-3′ and 5′-AAATTTCCGCGGAGCGTAATCTGGAACATCGT-3′, cut with XhoI and SacI, and subcloned into pTrpa1-P2A-mCherry to replace the untagged Trpa1 sequence. Site-directed mutagenesis was performed on the pHA-Trpa1-P2A-mCherry construct using the same primers as listed above.To construct HA-tagged wild-type and mutant Trpa1 plasmids, mouse Trpa1 ORF was amplified by PCR using primers: 5′-AAATTTGGATCCAAGCGCGGCTTGAGGAGGAT-3′ and 5′-AAATTTGGTACCAAAGTCCGGGTGGCTAATAG-3′. The PCR product was digested with B°C with 5% CO2. We regularly tested HEK293T cells for potential mycoplasma contamination using MycoAlertTM mycoplasma detection kit (Lonza). For authentication, we renewed HEK293T cells regularly by purchasing new cell stocks from the RIKEN BRC Cell bank. We ensured that cells were healthy by passaged for a maximum of 3 weeks.HEK293T cells (RCB2202) were obtained from the RIKEN BRC Cell bank and were cultured in DMEM media containing 10% FBS  and 1% Penicillin-Streptomycin at 37 5 HEK293T cells were seeded into a 10-cm dish and passaged at 48 h before reaching confluency. UV-sterilized 96-well sensoplates with glass bottoms  were coated with 25 µg/ml Poly-D Lysine  in 40 µl calcium free Hank’s Balanced Salt Solution  for 2 h. The wells were washed twice with fresh DMEM, loaded with 100 µl DMEM containing 6.5 × 103 cells, and incubated for 18 h. For cell transfection, we mixed 5 µl Opti-MEM  with 0.2 µl Fugene 6 Transfection Reagent  and 60 ng plasmid per well. For plasmids lacking mCherry marker, cells were co-transfected with 30 ng test plasmid and 30 ng mCherry plasmid per well. After 30 min incubation, 100 µl DMEM without antibiotics was added, gently mixed, and transferred to 96 wells followed by incubation for 24 h.For calcium imaging, approximately 6.5 × 102PO4, 0.34 mM Na2HPO4, 5.6 mM Glucose, 4.2 mM NaHCO3, 20 mM HEPES, 12.4 µM Probenecid , 0.1% BSA, pH 7.2). After sonication for 7 min, 100 µl of 4 µM fura-2 AM solution was added to each well of the cell plate and incubated at 37 °C for 50 min. On the other hand, different 4× ligand solutions were made in calcium imaging buffer  and sonicated for 3 min. The cell plate was washed twice with calcium buffer followed by addition of 75 µl calcium buffer per well. Both the cell plate and ligand plate were incubated in the Hamamatsu calcium imaging chamber at 30 °C for 40 min to de-protect the ester form of fura-2 AM prior to calcium imaging.A 4 µM fura-2 AM  solution was made in modified HBSS  dissolved in calcium imaging buffer with 0.2% DMSO, which was vortexed immediately before adding 2MT.Calcium imaging was performed on the Hamamatsu IMACS2 machine using a 20× lens (Olympus UPLSAPO 20× ZDC) and the IMACS imaging software (Version 1.008). After adding 25 µl of 4× ligand solution to the corresponding well in the cell plate, the fluorescence emission was continuously monitored for 5 min. The 340/380 nm fluorescence ratio (F) for each sample was normalized to that (F0) of the control sample: typically, vehicle-treated HEK293T cells that were transfected with the same plasmid(s). The IMACS2 machine automatically recorded the 340/380 nm ratio of mCherry-expressing cells. During post-imaging analysis, the mCherry-positive cells with abnormal or unhealthy morphology were excluded. The average fluorescent ratio of all mCherry-positive cells was measured at every 2 s interval. The quantitative results of calcium imaging for wild-type and mutant Trpa1 proteins were graphed with Prism 7.0 . For confirmation of the expression and functionality of various Trp channels, control compounds were used to stimulate Ca1H and 13C NMR spectra data were obtained with JEOL JNM-ECS 400 instruments. Chemical shifts (δ) are quoted in parts per million using tetramethylsilane (δ = 0 ppm) as the reference for 1H NMR spectroscopy, and CDCl3 (δ = 77.0 ppm) for 13C NMR spectroscopy. Reagents and solvents were purchased from the following commercial suppliers: Tokyo Chemical Ind., Sigma-Aldrich, Inc., Kanto Chemical Co., Inc., Wako Pure Chemical Ind., Ltd., and Nacalai Tesque. Thin-layer chromatography was performed on Merck Kieselgel 60 F254 (0.25 mm) plates. Synthetic schemes of TMT (3a)[3b) are shown in Supplementary Fig. 8a and the methods are described here:All distillations were carried out with a Büchi Glass Oven B-585 Kugelrohr.  TMT (3a) and alkySynthesis of 2,4,5-trimethyl-2,5-dihydrothiazole : To a stirring mixture of 3-mercapto-2-butanone  and acetoaldehyde  was added NH3 gas at 0 ˚C over a period of 2 h (until the reaction which releases water and heat is finished). After being diluted with diethyl ether, the inorganic phase was separated. The organic phase was washed with brine, dried over Na2SO4, and concentrated at pressures above 400 MPa. The residual oil was distillated with Kugelrohr distillation apparatus  to afford 3a as a 43:57 mixture of conceivable isomers .Minor isomer of3a. 1H NMR  δ 5.61–5.52 -S, 1H), 4.37–4.20 -S, 1H), 2.06  = N, 3H), 1.56 , 1.46 . 13C NMR  δ 172.22, 75.53, 56.55, 25.24, 21.72, 17.71.Major isomer of3a.1H NMR  δ 5.52–5.44 -S, 1H), 4.37–4.20 -S, 1H), 2.06  = N, 3H), 1.58 , 1.51 . 13C NMR  δ 172.01, 75.82, 56.78, 26.24, 22.62, 17.68.Synthesis of 2-(but-3-yn-1-yl)-4,5-dimethyl-2,5-dihydrothiazole : To a solution of 4-pentyn-1-ol  in CH2Cl2 (15 ml), trimethylamine (10 ml), DMSO (6 ml), and SO3 pyridine complex  were added at 0 ˚C, and the mixture was stirred at same temperature for 3 h. After being neutralized by 1.0 M aq. hydrochloric acid, the mixture was extracted with chloroform, washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residual oil was distillated with Kugelrohr distillation apparatus  to afford 4-pentyn-1-ol .1, 474 µl, 4.9 mmol) and 2b  was added NH3 gas at 0 ˚C over a period of 2 h (until the reaction which releases water and heat is finished). After being dissolved the white precipitation with ethyl acetate, the inorganic phase was separated. The organic phase was washed with brine, dried over Na2SO4, and concentrated at reduced pressure. The residual oil was distillated with Kugelrohr distillation apparatus  to afford the titled compound as a 3:7 mixture of conceivable isomers .To a stirring mixture of 3-mercapto-2-butanone  δ 5.67–5.62 -S, 1H), 4.28–4.21 -S, 1H), 2.38–2.33 , 2.18–2.09 , 2.06–2.05 =N, 3H), 1.97–1.95 , 1.96–1.86 , 1.46 . 13C NMR  δ 172.92, 83.48, 79.44, 68.93, 55.85, 37.43, 21.64, 17.68, 15.76.Major isomer of3b. 1H NMR  δ 5.59–5.54 -S, 1H), 4.28–4.21 -S, 1H), 2.38–2.33 , 2.18–2.09 , 2.06–2.05 =N, 3H), 1.97–1.95 , 1.96–1.86 , 1.51 . 13C NMR  δ 172.74, 83.48, 79.79, 68.98, 56.05, 38.12, 22.41, 17.68, 15.86.62 and the method is described here. To a suspension of 2-aminoethanethiol hydrochloride  in dry toluene (125 ml) under argon, a solution of triisobuthylaluminium  was added dropwise at room temperature. After refluxing for 30 min, methyl DL-2-methylbutyrate  was added dropwise. After further refluxing for 2 h, the mixture was diluted with 125 ml of toluene, cooled to room temperature, and quenched with MeOH (50 ml). The mixture was stirred at room temperature for 30 min, and then a sat. aq. Rochelle’s salt (100 ml), sat. aq. NaHCO3 (100 ml), and brine (100 ml) were successively added and stirred for 1 h. After being diluted with diethyl ether, the inorganic phase was separated. The organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residual oil was distillated with Kugelrohr distillation apparatus  to afford 6 .SBT was synthesized as previously reported1H NMR  δ 4.21 , 3.24 , 2.68–2.58 , 1.74–1.45 , 1.19 , 0.92 .13C NMR  δ 176.92, 64.38, 40.93, 33.11, 28.64, 18.84, 11.78.5 HEK293T cells were seeded into a 12-well plate and incubated in DMEM medium containing 10% FBS and 1% Penicillin/Streptomycin at 37 °C for 18 h. For transient transfection, 1 µg of pHA-Trpa1-P2A-mCherry construct was mixed with 3 µl Fugene 6 in 60 µl OptiMEM per 12 well. After 24 h of transfection, the cells were washed with 500 µl calcium solution , and incubated with 300 µM alkynyl-TMT in calcium solution at 30 °C for 6 min. After washing again with calcium solution, the cells were lysed with lysis buffer  pH 8.0, supplemented with complete™, Mini, EDTA-free protease inhibitor cocktail ) for 20 min at room temperature. The lysate was centrifuged at 12,000 × g for 3 min to pellet cell debris and the protein concentration was measured by BCA assay .Approximately 1 × 104 solution was mixed with 2 µl of 2.5 mM tris(3-hydroxypropyltriazolylmethyl)amine , followed by addition of 1 µl of 1.25 mM azide-PEG3-biotin conjugate  and 1 µl of 50 mM tris(2-carboxyethyl)phosphine . The 4 µl mixture of CuSO4/THPTA complex was then mixed with 44 µl cell lysates  by vortexing and incubated at room temperature for 2 h with constant agitation. Afterwards, equal amount of protein samples (~20–40 µg) were diluted with RIPA buffer  containing protease inhibitor cocktail and incubated with 15 µl of streptavidin agarose  for 2 h at room temperature with constant rotating. After washing the streptavidin beads three times with 150 µl RIPA buffer, the biotin-conjugated proteins were eluted with 1× SDS loading buffer with 50 mM DTT at 95 °C for 10 min. The eluted proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and immunoblotted with rabbit anti-HA antibody  or mouse anti-β-actin antibody .For click chemistry reaction, 2 µl of 50 mM CuSO−4 mole) was placed into the home cage for 30 min. After the filter paper was removed, test mouse was allowed to recover in the home cage for another 30 min. Mice were then rapidly anesthetized with pentobarbital  and sequentially perfused by PBS and 4% PFA in PBS. Trigeminal ganglions were dissected and fixed for 24 h in the same fixative solution and stored at 4 °C, and then cryoprotected with 30% sucrose (wt/vol) in PBS for 48 h. The tissues were put in the Tissue-Tek O.C.T compound (Sakura Finetek), and 30-μm-thick coronal sections were cut on a cryostat . For c-Fos IHC, the floating TG sections were washed three times with PBS for 5 min, incubated with 0.3% Triton X-100 in 0.01 M PBS for 15 min, and washed again with PBS for three times. The slides were treated with 1% H2O2/PBS for 30 min and washed three times with PBS. After incubating in 0.3% of BSA/0.25% Triton-X-100/ PBS for 1 h, the sections were incubated with rabbit anti-c-Fos antibody  at 4 °C overnight. After washing six times with PBST, the sections were incubated with biotinylated rabbit anti-goat IgG  for 2 h at room temperature and followed by PBS washing for three times. The sections were incubated with HRP-Streptavidin  for 1 h at room temperature. After washing with PBS three times, the slides were stained in DAB Peroxidase (HRP) Substrate Kit (VECTOR) in the dark until the color developed. The reaction was stopped by PBS washing and the sections were mounted onto glass slides for microscopy. All images were acquired using the Zeiss LSM700 confocal microscope with a 10× objective lens (NA = 0.45) under the AxioVision4.8 software. The nuclear c-Fos + neurons were counted in all sections from the same TG. Representative images shown in the figures were chosen from a similar region of the trigeminal ganglion based on morphology.Mice were single housed and habituated in the test environment 24 h before the experiment. A small filter paper containing 15 μl of water or 2MT -treated distilled deionized water (ddH2O) and RNase-free reagents. The DNA templates for making sense or antisense Trpa1 probes were generated using PCR primers: 5′-GCCTAATACGACTCACTATAGGCGGCTTGAGGAGGATTCTG-3′ and 5′-TCTGTGAAGCAGGGTCTCCT-3′; or 5′-GCGGCTTGAGGAGGATTCTG-3′ and 5′-GCCTAAGACTCACTATAGGGTCTGTGAAGCAGGGTCTCCT-3′. The RNA probes labeled by digoxygenin-UTP were generated by in vitro transcription by T7-RNA labeling kit (Roche). The floating TG sections were washed with 1× PBS for 10 min and treated with PFA  for 15 min, then the slides were reduced by treatment of 0.3% of sodium borohydride and washed with PBS 10 min. Tissue slides were treated with 0.3% Triton X-100 (vol/vol) in PBS for 15 min followed by twice of PBS washing. The slides were then treated with 0.75% glycine for 15 min followed by PBS washing and acetylated by adding 1/400 vol of acetic anhydride (vol/vol) in PBS for 15 min, followed by PBS washing two times. Afterward, the tissue sections were incubated in the hybridization solution  without probes for 1 h at 55 °C, followed by incubation in the hybridization solution with the Trpa1 antisense probe for 16–18 h at 60 °C. After hybridization, slides were washed with 2× SSC/50% formamide/0.1% N-lauroylsarcosine for 20 min at 50 °C followed by treatment with 5 μg/ml RNaseA in PBS at 37 °C for 30 min. The slides were then washed with 2× SSC/0.1% N-lauroylsarcosine for 30 min at 37 °C, followed by washing twice with 0.2% SSC/0.1% N-lauroylsarcosine for 30 min at 37 °C. The slides were rinsed with TS 7.5 for 5 min and incubated with 1% blocking reagent (Roche) diluted in TS7.5 for 1 h. After blocking, the slides were incubated with 1:2000 Anti-DIG-AP antibody  for 16–18 h at 4 °C. Next day, the sections were washed three times in TS7.5/0.1% Tween 20 for 20 min each and were incubated in TS9.5 for 5 min. Finally, the slides were incubated in 1:50 diluted NBT/BCIP solution (Roche) in TS9.5 in the dark until the color was developed. After staining was stopped by PBS washing, c-Fos IHC staining was performed as described above.For double n = 8 each) as described above. After testing, mice were presented with filter papers spotted with 2MT, sacrificed, and c-fos mRNA expression was analyzed. Lesioned areas were verified by the histological examination of coronal sections.Mice were anesthetized with pentobarbital  and mounted on a stereotaxic apparatus . The skin was opened, the skull was exposed, and three holes were made over the right hemisphere using a dental drill. An electrode was lowered into the trigeminal ganglion  in both the sham and the unilateral lesion (ulTGx) groups. In the ulTGx group, a constant current (2 mA) was applied to the trigeminal ganglion for 15 s. The electrode was removed and the skin was closed. After recovery for 8 days, 2MT-evoked freezing behavior was measured for the sham and ulTGx mice /ml for AAV-Trpa1 and ~1.0 × 1014 viral genomes (VG)/ml for AAV-GFP) at a flow rate of 70 nl/min. Coordinates for injection are: A-P, +0.9 mm; M-L, ±1.4 mm; D-V, −6.5 mm from bregma. Both AAV-GFP and AAV-Trpa1-injected Trpa1−/− mice were subjected to 2MT-evoked innate fear assay 2 months after virus administration.Mice were deeply anesthetized with 2% isoflurane and positioned on stereotaxic apparatus . Small holes were drilled into the skull and injections of AAV-GFP or AAV-Trpa1 were performed bilaterally using a Hamilton 0.5 μL Neuros Model 7000.5KH syringe with 33GA needles  with 800 nl of virus solution .Values are expressed as mean ± SEM. Data were tested for Gaussian distribution and variance and it fits the normal distribution pattern. Between different groups, we tested their similarity with Levene’s test. For Figs. We claim all relevant data are available from the authors with reasonable request.Supplementary InformatioPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


Immunoglobulin A (IgA) plays an important role in protecting our mucosal surfaces from viral infection, in maintaining a balance with the commensal bacterial flora, and in extending maternal immunity via breast feeding. Here, we report an additional innate immune effector function of human IgA molecules in that we demonstrate that the C-terminal tail unique to IgA molecules interferes with cell-surface attachment of influenza A and other enveloped viruses that use sialic acid as a receptor. This antiviral activity is mediated by sialic acid found in the complex N-linked glycans at position 459. Antiviral activity was observed even in the absence of classical antibody binding via the antigen binding sites. Our data, therefore, show that the C-terminal tail of IgA subtypes provides an innate line of defense against viruses that use sialic acid as a receptor and the role of neuraminidases present on these virions.  •Heterosubtypic IgA1 or IgA2 antibodies neutralize virus much more potently than IgG1•Sialic acid in IgA’s C-terminal tail competes with viral receptor binding•This may represent an innate line of defense against viral pathogens Vertebrate IgA molecules possess a conserved N-linked glycosylated C-terminal tail. Maurer et al. show that sialic acid found in the complex glycosylation of the C-terminal tail of human IgA1 inhibits sialic-acid-binding viruses and, therefore, may constitute an additional line of innate immunity. Vaccination against influenza A viruses relies on the induction of strain-specific neutralizing immunoglobulin G (IgG) and has to be repeated annually. Rare heterosubtypic antibodies, i.e., antibodies that are able to neutralize multiple strains and subtypes of influenza virus, can be found in most individuals  but currImmunoglobulin A (IgA) exists as monomeric molecules in serum or as dimeric secretory IgA on mucosal surfaces. Although roughly three quarters of the daily antibody production  are of the IgA isotype , IgA defAll Ig isotypes contain complex N-linked glycosylation: while about 10% of the N-linked glycans in IgG1 contain sialic acid  over 90%Escherichia coli  1.12  and 3.1 in vitro was assessed. While all IgG subtypes had comparable neutralizing activities, both IgA subtype monomers were 10- to 1,000-fold more potent than IgG1, depending on the mAb and isolate tested  or with 2-3,6,8,9-specific neuraminidase A from Arthrobacter ureafaciens (NA A). Both neuraminidase treatments resulted in reduced neutralizing antibody titers compared to the untreated IgA1 molecule, indicating that removal of sialic acid—in particular, that of α2–3-linked sialic acid—sufficed to reduce the potency of the IgA1 molecules considerably. The neuraminidase treatment also slightly reduced the neutralizing activity of the virtually unsialylated mAb 3.1 IgG1 molecules, suggesting additional loss of activity unrelated to the respective glycosidase activity.To identify the domain of the Ig molecule responsible for this increased potency, chimeric molecules were prepared by exchanging the CH1 the hinge, and the CH2/CH3 domains from IgA1 and IgG1 with each other. The neutralizing activity of these chimeric molecules revealed that, although the hinge region has some impact on the antibody’s potency, the enhanced neutralization phenotype segregated with the CH2-CH3 portion. To address the impact of the C-terminal tail with its additional N-linked glycosylation site, variants of mAb 3.1 IgA1 were prepared in which the glycosylation sites in CH2 and CH3 (Kabat positions 263 and 459) were removed. While the non-glycosylated N263D and N263D/N459D variants expressed well, the N459D did not express. Loss of both the CH2 and CH3 glycosylation, but not of the glycosylation in CH2 alone, abolished the improved neutralizing activity , indicat5GlcNAc2 . The res5GlcNAc2 , and sinTo test whether C-terminal sialylation was interfering with receptor binding, hemagglutination inhibition assays were performed. Although stem-specific heterosubtypic antibodies should not inhibit hemagglutination, IgA1 of both mAb 3.1 and mAb 1.12 prevented hemagglutination by rg-A/Chicken/Vietnam/C58/2004 (H5N3) and A/Puerto Rico/8/1934 (H1N1) . By cont2 preparation (data not shown). Since neither b12 IgG1 nor b12 IgA1 grown in 293S cells, nor b12 IgA2 samples depleted from antibodies using CaptureSelect beads (IgA-CH1 (Hu)) displayed antiviral activity  and A/Puerto Rico/8/1934 (H1N1) was tested. Surprisingly, HIV-1 gp120-specific b12 IgA1 was able to neutralize the avian but not the human isolate, while b12 IgA2 was able to neutralize both viruses . This obactivity A, the obactivity , indicatactivity .Figure 34PY8).To further investigate the potential interaction between the IgA1 and hemagglutinin, we visualized mAb 3.1 bound to recombinant HA from A/Vietnam/1203/2004 (H5N1) by negative stain electron microscopy (EM). The reference-free class averages for HA bound to IgG1 and IgA1 isotypes are shown in The recombinant antibodies used for this study were monomeric antibodies, while in the respiratory track, IgA is primarily found as dimeric secretory IgA. To rule out the possibility that our 293T-cell-expressed IgA displayed atypical glycosylation, we analyzed the glycosylation of human secretory IgA isolated from the saliva of two individuals by high-pressure liquid chromatography (HPLC). It was found that dimeric IgA contained 14.8%–17.3% terminal sialylation, while monomeric IgA was found to contain between 10.5% and 27.4% terminally sialylated complex glycans. α2–3linked sialic acid made up for 1.4%–6.7% of all complex glycans and was primarily found in multiantennary N-linked glycans . The preThe lack of dimerization in our expression system is likely to be attributed to the expression system. In contrast to Chinese hamster ovary (CHO) cells, which tend to produce a higher proportion of dimeric IgA , HEK293Fd affinity interaction  against pathogens using sialic acid as a receptor. However, in contrast to the other proteins, the ability of IgA to attach to virus via the antigen-binding fragment enables it to generate effective local accumulations of sialic acid and can thereby compensate for the poor kinetics of the sialic acid/HA interaction. The importance of IgA’s sialylation is further supported by the observation that, although IgA deficiency is largely asymptomatic in humans, children with this defect are more prone to pseudocroup caused by another sialic-acid binding virus, human parainfluenza 1 virus . Moreovein vitro findings with in vivo data; however, passive immunization of mice with human IgA1 is problematic, as it has been shown to rapidly induce elevated tumor necrosis factor alpha (TNF-α) levels and xenotypic antibodies in the recipient  and rg-A/Chicken/Vietnam/C58/2004 (H5N3), which were propagated on Madin-Darby canine kidney (MDCK) cells (discussed later). rg-A/Chicken/Vietnam/C58/2004 (H5N3) is a 6+1+1 reassortant between the internal genes of A/Puerto Rico/8/1934 (H1N1), the neuraminidase from A/Duck/Germany/1215/1973 (H2N3), and a hemagglutinin from A/Chicken/Vietnam/C58/2004 (H5N1), from which the polybasic cleavage site has been removed. GFP-expressing Newcastle disease virus has been propagated as described in Canis familiaris; adult female cocker spaniel); and Vero kidney cells  were propagated in D10 medium , supplemented with 10% fetal bovine serum , 25,000 U penicillin, 25 mg of streptomycin, and 217 mg GlutaMAX  at 37°C/5%CO2 according to ATCC’s recommendations. FreeStyle 293-F cells  were propagated according to the manufacturer’s instruction.HEK293T and HEK293S , both transfectants of the HEK293 cell line ; MDCK cells , IgA2 (GenBank: AK126352), IgG2 , IgG3 (GenBank: K01313), and IgG4 (GenBank: K01316) and were cloned into pAbVec-IgG1-3.1 a SalI-HindIII cassette. For the corresponding 1.12, b12, C65c6, and 2F5 constructs, the AgeIxSalI containing their V-region cassettes were cloned into the isotype variants of the 3.1 containing pAbVec constructs. The sequence of the original pAbVec-IgG1 can be accessed at GenBank: FJ475055.For the construction of the isotype variants, SalI and HindIII-flanked fragments were synthesized corresponding to the spliced constant regions for IgA1 . Primers used for amplification of the fragments were: GAAA-G-Fw-IgG1-Sall, GAAA-G-Rv-CH1-IgG1, GAAA-AAA-Fw-Hinge-CH3 IgA1, GAAA-AAA-Rv-IgA1-Hindlll, AGGG-A-Fw-IgA1-Sall, AGGG-A-Rv-CH1-IgA1, AGGG-GGG-Fw-Hinge-CH3 IgG1, AGGG-GGG-Rv-IgG1-Hindlll, GGAA-GG-Fw-IgG1-Sall, GGAA-GG-Rv-CH1-Hinge-IgG1, GGAA-AA-Fw-CH2-CH3-IgA1, GGAA-AA-Rv-IgA1-Hindlll, AAGG-AA-Fw-IgA1-Sall, AAGG-AA-Rv-CH1-Hinge-IgA1, AAGG-GG-Fw-CH2-CH3-IgG1, AAGG-GG-Rv-IgG1-Hindlll, AGAA-AG-Fw-IgA1-Sall, AGAA-AG-Rv-CH1-Hinge IgG1, AGAA-AA-Fw-CH2-CH3-IgA1, AGAA-AA-Rv-IgA1-Hindlll, GAGG-GA-Fw-IgG1-Sall, GAGG-GA-Rv-CH1-Hinge-IgA1, GAGG-GG-Fw-CH2-CH3-IgG1, and GAGG-GG-Rv-IgG1-Hindlll, whereby the first four letters indicate the fusion construct separated by a dash from the fragment and followed by the priming direction and the further designations.E. coli. After sequencing of the mini-preparations, maxi-preparations of positive clones were performed using the PureYield Plasmid Maxiprep System . For the generation of the mAb 3.1 variants, the variable region cassette of 1.12 was exchanged for that of 3.1, using SalI and AgeI digestion in all chimeric plasmids.1 μg fusion PCR product and pAbVec-IgG1-1.12 plasmid were digested with Sal l and Hind III, gel purified, quantified, and ligated into the digested backbone using T4 Ligase  for 20 min at room temperature (RT). The ligated product was then transformed into 50 μL chemically competent PCR thermal cycling conditions were as follows: 2 min at 95°C;  × 30; 10 min at 72°C; ∞ at 4°C. Fusion PCR thermal cycling conditions were as follows: fragment assembly for chimeric IgG1/IgA1: 5 min at 95°C; 2 min at 72°C (ramping rate [dT] 0.1°C/s); ∞ at 4°C. Amplification was as follows: 2 min at 95°C;  × 30; 10 min at 72°C; ∞ at 4°C.N-linked glycosylation consensus sequences located at position 263 of CH2 or 459 of CH3 of the IgA1 Fc were removed from the pAbVec-IgA1-3.1 plasmid by site-directed mutagenesis converting the asparagine of the consensus into aspartic acid. To this end, primer pairs QC-N264D-Rv/QC-N264D-Fw and QC-N459D-Rv,/QC-N459D-Fw and the QuikChange II XL Site-Directed Mutagenesis Kit  were used according to the manufacturer’s instructions. For all other specificities, the AgeI-SalI cassette, containing the V region, was exchanged, as described earlier.6 HEK293T or 2.5 × 106 HEK293S cells were seeded into 15-cm tissue culture plates  in 15 mL D10 medium 1 day before transfection. Tissue culture flasks were coated with poly-L-lysin  before seeding HEK293S cells. For the transfections, 21.5 μg of the H- and L-chain-expressing plasmids were diluted in 450 μL of 1 M NaCl and adjusted to a final volume of 1 mL with double-deionised water (ddH2O). In parallel, 180 μL of 1 mg/mL polyethylenimine  were diluted into 1 mL of ddH2O. Both solutions were then mixed together, mixed immediately, and incubated at RT for 20 min. 3 mL of the DNA/NaCl/PEI complex solution was then added to each transfection plate. Cells were incubated at 37°C, 5% CO2, and 95% humidity for 7 hr before transfection medium was exchanged with 30 mL pre-warmed complete D10. After 6 days of incubation at 37°C/5% CO2, supernatants were harvested and clarified by centrifugation at 3,700 relative centrifugal force (rcf) for 15 min and were sterile filtered at 0.2 μm . The filter membrane was washed with the same volume of Protein L (ProtL) binding buffer  and volume adjusted to 1 L with ddH2O.For transfection, either 2.5 × 10All antibodies were affinity purified using Protein L Agarose Beads , according to the manufacturer’s instructions. Abs were eluted by 0.1 M glycine (pH 2.7). The pH was immediately adjusted to a pH of 7 by adding 1 M Tris (pH 8.5). The procedure was repeated once to increase yields.To remove contaminating kappa dimers and free heavy chains, and to perform a buffer exchange, antibodies were additionally purified by size exclusion chromatography. To this end, a Superdex 200 HiLoad 26/60 column  was equilibrated in PBS before the antibody in elution buffer was loaded, and separation was run at 3 mL/min. Antibody fractions were identified according to their in-line absorbance at 280 nm and used for further analysis. The purity of all fractions was assessed by non-reducing SDS-PAGE, followed by subsequent silver staining. Fractions containing the correctly assembled antibodies were pooled. Since molecular ultrafiltration leads to considerable loss of IgA, pooled antibody fractions were transferred into 3.5-kDa MWCO dialysis tubes  and concentrated via reverse osmosis by sprinkling the tubes with 1–3 g of polyethylene glycol 35000 .2O for 25 min. After being washed with water for 25 min, gels were incubated in 50% ethanol for another 25 min. Gels were then incubated in 100 mL ddH2O containing 16.7 mg Na2S2O3 for 1.5 min. Gels were washed three times with ddH2O and incubated with 0.26 g (w/v) AgNO3 and 250 μL of 36% (w/v) formaldehyde for 10 min. After an additional washing step, gels were developed in a solution containing 2 g (w/v) Na2CO3, 42 μL of a 10% (w/v) Na2S2O3 and 42 μL of 36% (w/v) formaldehyde until the protein bands became visible. The reaction was then stopped by the addition of a 5% (w/v) acetic acid solution.Gels were fixed in 100 mL 10% acetic acid, 40% methanol, and 50% ddH10 μL CaptureSelect IgA-CH1 (Hu) Affinity Matrix  was washed two times with 1 mL PBS. The slurry was centrifuged, and the supernatant was discarded. 30 μL of b12 IgA2 (9.32 μg) was incubated with 10 μL prewashed CaptureSelect IgA-CH1 (Hu) Affinity Matrix at RT on a rotor wheel for 1 hr. The mix was then centrifuged at 10,000 rcf for 5 min, and the supernatant was transferred to another 10 μL prewashed beads. The process was repeated a total of four times. 21.9 μL from the depleted b12 IgA2 solution was used for neutralization assays.2O [pH 7.6]) overnight. Blots were washed twice with TBS + 0.1% Tween 20 and incubated with a 1:1,000 dilution of biotinylated Maackia Amurensis Lectin II  in TBS + 0.1% Tween 20 or a 1:1,000 dilution of biotinylated Elderberry Bark Lectin  in TBS + 0.1 mM CaCl2, MgCl2, and MnCl2 + 0.1% western blot (WB) blocking reagent + 0.1% Tween 20. After washing six times with TBS + 0.1% Tween 20 for 5 min, streptavidin-horseradish peroxidase (HRP) diluted 1:2,000 in TBS was added and incubated for 30 min. Membranes were washed again six times with TBS + 0.1% Tween 20 for 5 min and rinsed once with TBS. Membranes were developed using chemiluminiscent enhanced chemiluminescence (ECL) substrate and a LAS 4000 Mini chemiluminescent image analyzer .To determine the glycosylation pattern, 1 μg/mL reduced Ab was loaded on a 10% SDS-PAGE gel. After separation, proteins were transferred to a nitrocellulose membrane  using a semi-dry blotting system . Membranes were then blocked with a casein containing western blocking reagent  in Tris-buffered saline . 1 μl neuraminidase was added before the mixture was incubated at 37°C for 1 hr.Neuraminidase S  and neuraminidase A  were used to desialylate IgA1. To this end, 2 μg IgA variants were combined with H2SO4  was used to stop the reaction. The absorbance at 450 nm was then detected in an EnVision Multiplate Plate Reader . Prism 6  was used to perform non-linear regression of logarithmized data to the Hill Equation.The ELISA 384-well plates  were coated with 4 μg/mL recombinant HA from different influenza A viruses in PBS at RT for 1 hr. Plates were then washed 3 times with TBS + 0.1% Tween 20 in an ELx405 Select CW plate washer  and blocked with TBS + 0.1% Tween 20 + 5% milk at RT for another hour. Ab variants (20 μg/mL) were diluted 3-fold in TBS containing 0.1% Tween 20 and 0.1% milk, transferred to the plates, and incubated at RT for 1 hr. After washing three times, goat α-human IgK HRP was used as a detection Ab at a dilution of 1:5,000. The 384-well plates were incubated at RT for another hour before they were washed 3 times. To develop the assay, TMB substrate  was added. After 2 to 4 min, 2 M H2, and 95% humidity overnight. The next day, 3-fold dilutions of the Ab, starting at 40 μg/mL in infectious media, i.e., DMEM supplemented with 0.2% BSA , penicillin/streptomycin (Pen/Strep), Glutamax, and 10 mM HEPES ], were produced and transferred into a white 384-well plate. Virus diluted to an MOI of 4 was added into the wells with the diluted Ab, resulting in an initial mAb concentration of 20 μg/mL and a MOI of 2. The plates were then incubated at 37°C (5% CO2) for 2 hr before the Ab/virus mix was transferred to the corresponding wells of the plates containing the MDCK cells that were washed once with PBS using an ELx405 Select CW plate washer. Infection was then allowed to proceed at 37°C/5% CO2 for another hour before the non-adsorbed virus and Abs were washed away with PBS. Then, 50 μL infection media was added to each well, and plates were incubated at 37°C (5% CO2) for 6 hr. After incubation, plates were fixed with absolute methanol and stained with 2–4 μg/mL fluorescein isothiocyanate (FITC)-labeled mAb HB65 (ATCC HB-65) in PBS + 1% BSA at 4°C overnight. Cells were washed three times with PBS the next day, and 40 μL PBS was added before fluorescence at 505 nm was determined at 16 locations of each well in an EnVision Multilabel Plate Reader . The average values from all measurements in a well were then loaded into Prism 6 software  and normalized against the median values of the control wells, and a non-linear regression of logarithmized data to the Hill Equation was performed.Each well of a black 384-well plate  was seeded with MDCK cells in 50 μL D10 medium. Plates were incubated at 37°C, 5% CO2) for 24 hr. The fluorescence at 505 nm was determined at 16 locations of each well in an EnVision Multilabel Plate Reader. The average value from all measurements in a well were then loaded into Prism 6 software and normalized against the median values of the control well, and a non-linear regression of logarithmized data to the Hill Equation was performed.Six thousand Vero cells (ATCC CCL-81) were seeded in 15 μL medium into each well of a black 384-well plate on the day before the assay. The next day, samples were diluted in a 3-fold dilution series starting from 40 μg/mL in infectious media, which lacked the phenol red dye and only had 5% FBS added. A recombinant Newcastle disease virus expressing GFP with an MOI 4 was added, leading to a final virus concentration of an MOI of 2. After the Ab/virus mix was transferred to cells, then the plates were incubated at 37°C . The same volume of a 1% chicken red blood cell  suspension was then added to each well and incubated at RT for 30 to 60 min until CRBC pellets had formed in the negative control.After quantifying the optimal virus-to-red blood cell (RBC) concentration, 50 μL of serial 2-fold dilutions of mAbs were prepared, starting at a concentration of 200 μg/mL, and mixed with 50 μL of the optimal virus dilution. After 30-min incubation at 4°C, 50 μL of a 0.5% CRBC suspension were added to all columns, resulting in a maximal mAb concentration of 25 μg/mL. Plates were incubated at RT until CRBC pellets could be observed in the negative control.Antibody/HA complexes were formed by incubating 50 μg recombinant HA from A/Vietnam/1203/2004 (H5N1) with 150 μg of antibody in PBS at 4°C overnight. High-molecular-weight complexes were then isolated by size exclusion chromatography on a Superdex 200 Increase 10/300 GL column . Complexes were then added onto carbon-coated copper mesh grids and stained with 1% uranyl formate for 30 s. Samples were imaged on a Tecnai F12 microscope using the automated image acquisition software Leginon , and imaFor the purification of human IgA, two male Caucasian volunteers aged between 30 and 45 years collected between 30 and 35 mL of saliva by repetitive spitting into a 50-ml tube on ice. Saliva was then clarified by centrifugation at 4,000 rpm and 4°C for 20 min. 20 mL of the clarified saliva was mixed with 20 mL of ProtL binding buffer. Saliva IgA was then affinity purified via 1 mL of Protein L slurry and buffer exchanged as described earlier.3 [pH 7.4]) at 37°C for 16 hr. The released glycans were extracted from the gel matrix by washing three times with water and then dried in a SpeedVac Concentrator Plus . The glycan samples were resuspended in 30 μL water, followed by incubation at 80°C for 1 hr in 80 μL labeling mixture containing 3% (w/v) 2-aminobenzoic acid (2-AA), 4.5% w/v sodium cyanoborohydride, 4% w/v sodium acetate trihydrate, and 2% w/v boric acid in methanol. Removal of excess label from the 2-AA fluorescently labeled glycan samples was performed using Speed Amide-2 cartridges .The secretory IgA samples isolated from saliva of two individuals were fractionated by SDS-PAGE Novex NuPAGE Bis-Tris 4%–12% pre-cast gels . After being alternately washed with acetonitrile and water for five times, the excised gel bands were incubated with protein N-glycosidase F  in a reaction buffer , α2-3 neuraminidase from Streptococcus pneumoniae , β1,4-galactosidase from Bacteroides fragilis , and α-L-fucosidase from bovine kidney , β-N-acetylglucosaminidase from Xanthomonas manihotis , and α-mannosidase from Jack bean . Endoglycosidase H (endoH) from Streptomyces picatus  was used for quantification of oligomannose structures. Prior to chromatographic analysis, polyvinylidene difluoride (PVDF) protein-binding membrane plates  were used for the removal of enzymes.The 2-AA labeled glycans were sequentially digested using the following exoglycosidases according to the manufacturers’ instructions: α2-3,6,8 neuraminidase from Analysis of the fluorescently labeled N-linked glycans was performed using hydrophilic interaction chromatography-ultra-performance liquid chromatography (HILIC-UPLC), with the 2-AA-labeled glucose homopolymer ladder  being used as a calibration standard. The following gradient was run on a 2.1 mm × 10 mm (1.7-μm particle size) ACQUITY Ethylene Bridged Hybrid (BEH) glycan column  pre-installed in the Waters ACQUITY UPLC instrument: time = 0 min (t = 0): 22% A, 78% B (flow rate of 0.5 mL/min); t = 38.5: 44.1% A, 55.9% B (0.5 mL/min); t = 39.5: 100% A, 0% B (0.25 mL/min); t = 44.5: 100% A, 0% B; and t = 46.5: 22% A, 78% B (0.5 mL/min), where solvent A was 50 mM ammonium formate (pH 4.4) and solvent B was acetonitrile. Fluorescence was measured using an excitation wavelength of 250 nm and a detection wavelength of 428 nm. Data processing was performed using Empower 3 software. The percentage of abundance of oligomannose- and that of complex-type glycans were calculated by integration of the relevant peak areas before and after EndoH digestion, following normalization.