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Contents lists available at ScienceDirect
Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi 4 5 3
Regulation of bitter taste responses by tumor necrosis factor
7
Pu Feng 2, Masafumi Jyotaki 2, Agnes Kim 1, Jinghua Chai, Nirvine Simon, Minliang Zhou, Alexander A. Bachmanov, Liquan Huang, Hong Wang ⇑
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Monell Chemical Senses Center, 3500 Market St., Philadelphia, PA 19104, USA
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a r t i c l e
i n f o
Article history: Received 20 January 2015 Received in revised form 23 March 2015 Accepted 1 April 2015 Available online xxxx Keywords: Cytokine TNF Inflammation Taste-related behavior Taste buds
a b s t r a c t Inflammatory cytokines are important regulators of metabolism and food intake. Over production of inflammatory cytokines during bacterial and viral infections leads to anorexia and reduced food intake. However, it remains unclear whether any inflammatory cytokines are involved in the regulation of taste reception, the sensory mechanism governing food intake. Previously, we showed that tumor necrosis factor (TNF), a potent proinflammatory cytokine, is preferentially expressed in a subset of taste bud cells. The level of TNF in taste cells can be further induced by inflammatory stimuli. To investigate whether TNF plays a role in regulating taste responses, in this study, we performed taste behavioral tests and gustatory nerve recordings in TNF knockout mice. Behavioral tests showed that TNF-deficient mice are significantly less sensitive to the bitter compound quinine than wild-type mice, while their responses to sweet, umami, salty, and sour compounds are comparable to those of wild-type controls. Furthermore, nerve recording experiments showed that the chorda tympani nerve in TNF knockout mice is much less responsive to bitter compounds than that in wild-type mice. Chorda tympani nerve responses to sweet, umami, salty, and sour compounds are similar between TNF knockout and wild-type mice, consistent with the results from behavioral tests. We further showed that taste bud cells express the two known TNF receptors TNFR1 and TNFR2 and, therefore, are potential targets of TNF. Together, our results suggest that TNF signaling preferentially modulates bitter taste responses. This mechanism may contribute to taste dysfunction, particularly taste distortion, associated with infections and some chronic inflammatory diseases. Ó 2015 Elsevier Inc. All rights reserved.
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1. Introduction
49
Taste is the sensory system for detecting nutrients and potentially harmful substances in food and drink and, therefore, plays important roles in guiding food intake. Among the five basic taste modalities, sweet and umami tastes detect sugars and amino acids, respectively, and are generally preferred. Bitter taste recognizes toxins and noxious compounds and elicits avoidance behavior. Acids and salts are detected by sour and salt taste mechanisms. Recent research has made rapid progress in understanding taste receptors and signaling pathways, particularly for sweet, umami, and bitter tastes (Breslin and Huang, 2006; Chandrashekar et al.,
50 51 52 53 54 55 56 57 58
⇑ Corresponding author. Tel.: +1 267 519 4773. E-mail addresses: [email protected] (P. Feng), [email protected] (M. Jyotaki), [email protected] (A. Kim), [email protected] (J. Chai), n.simon.uchs@gmail. com (N. Simon), [email protected] (M. Zhou), [email protected] (A.A. Bachmanov), [email protected] (L. Huang), [email protected] (H. Wang). 1 Present address: Drexel University College of Medicine, Philadelphia, PA 19129, USA. 2 These authors contribute equally to this work.
2006; Liman et al., 2014). What remain largely unclear, however, are the regulatory mechanisms that modulate taste responses or taste bud structure under diverse physiological and pathological conditions. Inflammation is likely one of such regulatory mechanisms. Many diseases with underlying inflammation, such as infections and autoimmune ailments, are associated with taste alterations (Bromley and Doty, 2003; Pribitkin et al., 2003; Schiffman, 1983). Taste alterations can occur as taste loss (lacking or reduced taste reception) or taste distortion (e.g. persistent bitter or metallic taste in the mouth) (Brand, 2000; Bromley, 2000). In animal models, induced inflammation has been shown to affect taste responses and taste bud structure (Cavallin and McCluskey, 2005; Cohn et al., 2010; Phillips and Hill, 1996). How inflammation exerts its effects on taste reception or taste bud structure has not been fully elucidated. Inflammation is an immune response to infection, tissue damage, and stress. In addition to its roles in regulating immunity and tissue repair, inflammation can strongly affect metabolism and food intake (Forsythe et al., 2008; Hotamisligil, 2006). The various effects of inflammation are often mediated by
http://dx.doi.org/10.1016/j.bbi.2015.04.001 0889-1591/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Feng, P., et al. Regulation of bitter taste responses by tumor necrosis factor. Brain Behav. Immun. (2015), http:// dx.doi.org/10.1016/j.bbi.2015.04.001
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P. Feng et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx
inflammatory cytokines, a group of signaling proteins that are highly induced during inflammatory responses. A number of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-6, and interferons, are pleiotropic and play important parts in regulating immunity, metabolism, and food intake (Cannon, 2000; Dantzer, 2001; Plata-Salaman, 1998). Our studies have found that several inflammation-associated cytokines are preferentially expressed in taste bud cells compared to nontaste lingual epithelial cells (Cohn et al., 2010; Feng et al., 2012, 2014a,b; Kim et al., 2012; Wang et al., 2007), suggesting that these cytokines may have special functions in the peripheral taste system. In particular, we have found that TNF is specifically expressed in a subset of taste bud cells even in healthy mice (Feng et al., 2012; Kim et al., 2012). Immunocolocalization experiments showed that TNF is colocalized with the sweet and umami taste receptor subunit T1R3, indicating that TNF is expressed specifically in the sweet and umami taste bud cells (Feng et al., 2012). Moreover, TNF expression level and its secretion in taste buds can be further augmented by inflammation, such as lipopolysaccharide (LPS)-induced inflammation (Cohn et al., 2010; Feng et al., 2012; Kim et al., 2012). TNF was thought to be produced primarily by macrophages, but it is also produced by a broad variety of cell types including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, fibroblasts, adipocytes, and neurons (Hotamisligil et al., 1993; Niu et al., 2009; Walsh et al., 1991). TNF is known to activate a variety of cellular signaling pathways that are important not only for fighting against certain pathogens but also for regulating stress responses and metabolism (Cabal-Hierro and Lazo, 2012; Silke, 2011; Wajant et al., 2003). TNF contributes to behavioral changes associated with various illnesses (i.e. sickness behavior) which include fatigue, malaise, depression, and anorexia. It has been shown that administration of recombinant TNF induces significant reduction in food intake both in rodents and in humans (Bernstein et al., 1991; Michie et al., 1989; Spiegelman and Hotamisligil, 1993). How TNF regulates food intake remains incompletely understood. Both peripheral and brain mechanisms are likely involved (Bernstein et al., 1991; Dantzer, 2001; Plata-Salaman, 1998). The taste system plays an important role in regulating food intake. Considering the specific expression of TNF in taste bud cells, it is conceivable that TNF may be involved in modulating taste responses under physiological and pathological conditions. In this study, we used TNF knockout mice and their wild-type controls to investigate the role of TNF in the taste system. We conducted gustatory nerve recording and taste behavioral testing using these mice. Our results show that TNF-deficient mice are significantly less responsive to bitter compounds than control mice, whereas their responses to sweet, umami, sour, and salty compounds did not differ significantly from those of control mice. Our results suggest that TNF is involved in the regulation of bitter taste reception.
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2. Materials and methods
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2.1. Animals
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TNF knockout mice (stock number 005540) and wild-type control mice (C57BL/6J, stock number 000664) were purchased from the Jackson Laboratory (Bar Harbor, ME) and then bred and maintained at the Monell Chemical Senses Center. Generation of TNF knockout mice was described by Pasparakis et al. (1996). In these TNF knockout mice, the first coding exon (including the ATG translation initiation codon) and a portion of the first intron of the TNF gene were deleted. The mutant mice have been backcrossed to C57BL/6J genetic background for ten generations. All mice were
133 134 135 136 137 138 139 140
housed at the Monell Chemical Senses Center animal facility under a 12 h/12 h light/dark cycle. Mice were given free access to standard rodent food (8604 Teklad rodent diet, Harlan Laboratories) and water except during the periods of taste behavioral tests (described below). 4–10 months old mice were used for all the experiments described below. Age and gender matched wild-type and TNF-knockout mice were included for the experiments. All procedures were performed according to protocols approved by the Monell Chemical Senses Center Institutional Animal Care and Use Committee.
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2.2. Reagents
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All taste compounds used in behavioral and electrophysiological tests were purchased from Sigma (St. Louis, MO). Rabbit polyclonal antibody against mouse ecto-nucleoside triphosphate diphosphohydrolase 2 (ENTPDase2) was purchased from Centre de Recherche (Quebec, Canada) (Bartel et al., 2006). Rabbit polyclonal antibodies against phospholipase C-b2 (PLC-b2, sc-206) (Clapp et al., 2001) and gustducin (sc-395), goat polyclonal antibodies against the voltage-gated potassium channel KCNQ1 (sc-10646) (Wang et al., 2009) and TNFR1 (sc-1069), and a blocking peptide (sc-1069p) for the anti-TNFR1 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A purified rabbit polyclonal antibody against neural cell adhesion molecule (NCAM) was purchased from Millipore (Billerica, MA). Purified goat polyclonal antibodies against carbonic anhydrase 4 (Chandrashekar et al., 2009) and TNFR2 and a blocking antigen (recombinant mouse sTNFR2, 426-R2-050) for the anti-TNFR2 antibody were purchased from R&D Systems (Minneapolis, MN). Dylight-649 (or Dylight-488)-conjugated donkey anti-rabbit or donkey anti-goat antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
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2.3. Taste behavioral tests
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Two-bottle preference tests were conducted as previously described (Bachmanov and Beauchamp, 2008; Bachmanov et al., 2001, 2002; Wang et al., 2009). Briefly, TNF-deficient and wildtype mice were individually caged. For the first 2 days, mice were familiarized with the two drinking bottles, both containing deionized water. For the following days, mice were presented with two drinking bottles: one contained deionized water and the other a taste solution. The positions of the bottles were switched after 24 h to minimize positional effect. The volume of consumed liquid from each bottle was recorded at 24 and 48 h. Each concentration of a taste compound was tested for 48 h. The taste compounds were tested in the following order: NaCl (37.5, 75, 150, 300, and 600 mM), quinine hydrochloride (QHCl) (0.003, 0.01, 0.03, 0.1, and 0.3 mM), Saccharin (0.0625, 0.25, 1, 4, and 16 mM), inosine50 -monophosphate (IMP) (0.3, 1, and 3 mM), and citric acid (1, 3, and 10 mM). Each mouse in the experiment was tested with all the above listed compounds. Between the testing of two different compounds, mice received deionized water in both drinking tubes for at least 3 days. During the experiment mice had free access to food. TNF-deficient mice and wild-type mice were tested at the same time in parallel. Taste preference scores were calculated for each animal by dividing the volume of consumed taste solution during the 48 h test period by the total volume of fluid intake during the same 48 h test period (i.e. preference score = intake of taste solution/(intake of taste solution + intake of water)). The average preference scores were then calculated for each group. By this calculation formula, preference scores between 0.5 and 1 indicate that mice prefer the taste solution over water, whereas preference scores between 0 and 0.5 indicate that mice avoid the taste solution. 8–16 male mice (4–8 months old) in each group were used.
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Please cite this article in press as: Feng, P., et al. Regulation of bitter taste responses by tumor necrosis factor. Brain Behav. Immun. (2015), http:// dx.doi.org/10.1016/j.bbi.2015.04.001
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Brief-access tests were performed using the Davis MS-160 mouse gustometer (Dilog Instruments, Tallahassee, FL) as previously described (Kim et al., 2012). Briefly, mice were water-deprived for 22.5 h before 30 min training sessions and test sessions for aversive taste compounds (bitter, salty, and sour compounds). Mice were food- and water-restricted (1 g of food and 1.5 ml of water) for 23.5 h before test sessions for appetitive taste compounds (sweet and umami compounds). Water and food restrictions were used to motivate mice to lick the taste solutions presented during the short test periods and were comparable to published protocols for this type of tests (Glendinning et al., 2002). In each test session, mice were tested with three different concentrations of each taste compound along with a water control. Water and taste compounds were randomly presented to mice following random presentation schemes generated by the computer software. Inter-presentation interval was 10 s. The maximum wait for the first lick was 120 s. Lick time limit was 5 s, which was the time from the first lick until the shutter closed. So, for each presentation, mice had maximum 5 s direct contact (or lick) time with the presented solution or water. The session time limit was 30 min. The following taste compounds were tested: QHCl (0.03, 0.3, and 3 mM), NaCl (0.1, 0.6, and 1 M), and citric acid (3, 10, and 100 mM), sucrose (0.1, 0.2, and 0.6 M), IMP (1, 10, and 30 mM). Each mouse was tested with all the compounds. TNF-deficient mice and wild-type mice were tested at the same time in parallel. After each session, mice received a recovery day with free access to food and water for 24 h. Taste stimulus to water lick ratios were calculated by dividing the number of licks for taste compounds by the number of licks for water presented in the same test session. Lick ratios less than 1 indicate avoidance behavior to the taste solution, and lick ratios more than 1 indicate preference behavior. 8–16 TNF-deficient mice and wild-type control mice (all male, 6– 10 months old) were included in this experiment. The same sets of mice were used for two-bottle preference tests and then for brief-access tests with a 3-week interval between the two testing procedures to minimize the possible effects of prior experience on subsequence testing.
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2.4. Gustatory nerve recording
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Chorda tympani nerve recording was carried out as previously described (Kim et al., 2012). Briefly, under pentobarbital anesthesia (50–60 mg/kg of body weight, i.p.), the trachea of each animal was cannulated, and the mouse was then fixed in the supine position with a head holder. The right chorda tympani nerve was exposed at its exit from the lingual nerve, cut near its entrance to the bulla, and was placed on a platinum wire recording electrode. An indifferent electrode was positioned nearby in the wound. Neural responses resulting from chemical stimulations of the tongue were fed into an amplifier (Grass Instruments, West Warwick, RI), monitored on an oscilloscope and an audio monitor. Whole-nerve responses were integrated with a time constant of 1.0 s and recorded using a computer for later analysis using a PowerLab system (PowerLab/sp4; AD Instruments, Colorado Springs, CO). For taste compound stimulation, the tongue was enclosed in a flow chamber, and solutions were delivered into the chamber by gravity flow. The following solutions were used as stimuli: 0.1–20 mM QHCl, 0.1–20 mM denatonium benzoate, 0.003–0.5 mM cycloheximide, 3–300 mM MgSO4, 10–1000 mM sucrose, 0.3–20 mM saccharin, 10–1000 mM monopotassium glutamate (MPG) with or without 0.5 mM IMP, 0.1–10 mM IMP, 1–100 mM citric acid, 0.01–10 mM HCl, 10–1000 mM NaCl with or without 100 lM amiloride, and 0.1 M NH4Cl. To analyze nerve responses to each stimulus, the magnitudes of integrated responses at 5, 10, 15, 20, and 25 s after stimulus onset were measured and averaged. The relative response magnitude for each stimulus was calculated
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3
against the response magnitude to 0.1 M NH4Cl, and this value was used for statistical analysis and for plotting dose–response curves. Eight wild-type mice (6 males and 2 females) and seven TNF-deficient mice (5 males and 2 females), 4–8 months old, were used for nerve recordings.
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2.5. mRNA in situ hybridization
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Digoxigenin (DIG)-labeled sense and antisense cRNA probes corresponding to the coding region of mouse TNFR1 and TNFR2 were synthesized using the DIG RNA labeling kit (Roche Applied Science). Fresh-frozen taste sections (10 lm/section) from C57BL/ 6J mice (4–6 months old) were attached to clean glass slides. Sections were then fixed with 4% paraformaldehyde and processed for in situ hybridization as previously described (Wang et al., 2007). Hybridizations were performed at 72 °C overnight with DIG-labeled probes in 50% formamide, 5 SSC, 5 Denhardt’s solution, 250 lg/ml yeast RNA, and 500 lg/ml sperm DNA. Sections were washed 3 times at 72 °C with 0.2 SSC. Hybridized DIG-labeled cRNA was detected immunologically with an alkaline–phosphatase-conjugated anti-DIG antibody and standard chromogenic substrates 4-Nitro Blue tetrazolium chloride (NBT, Roche Applied Science). Images were taken using a Nikon fluorescence microscope. In all the experiments, hybridizations to antisense and sense probes were performed in parallel to verify the specificity of hybridization signals.
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2.6. Immunohistochemistry
291
Tissue preparation and immunofluorescent staining procedures were described previously (Feng et al., 2012; Wang et al., 2007). All mice used for this procedure were 4–6 months old. Briefly, excised mouse tongue tissues were fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h on ice and then cryoprotected in 20% sucrose/PBS solution at 4 °C overnight and embedded in mounting medium. Tissues were sliced into 10-lm-thick sections using a Microm HM 500 OM cryostat (Thermo Scientific Microm, Walldorf, Germany). Purified goat polyclonal antibodies against TNFR1 and TNFR2 (see Section 2.2) were used to detect the expression of TNFR1 and TNFR2 in taste tissues. Two control experiments for TNFR1 and TNFR2 immunostaining were conducted. In one control experiment, primary antibodies against TNFR1 and TNFR2 were omitted in the procedure. In the second control experiment, antibodies against TNFR1 and TNFR2 were preincubated with their corresponding blocking antigens before adding to tissue sections. To investigate what types of taste bud cells express TNFR1 or TNFR2, double immunostaining was carried out using rabbit antibodies against taste-cell-type markers and goat antibodies against TNFR1 or TNFR2. Antibodies to the following taste-cell-type markers were used: ENTPDase2 (1:500), PLC-b2 (1:1000), and NCAM (1:300). Dylight-649-conjugated donkey anti-rabbit and Dylight-488-conjugated donkey anti-goat secondary antibodies were used. For immunostaining of taste tissue sections from wild-type and TNF knockout mice, antibodies to KCNQ1 (1:1000), PLC-b2 (1:1000), gustducin (1:1000), and carbonic anhydrase 4 (1:500) were used. Secondary antibodies were Dylight-649-conjugated donkey anti-goat and Dylight-488-conjugated donkey anti-rabbit antibodies. Fluorescent images were acquired using Leica Sp2 confocal microscope.
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2.7. Statistical analysis
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Data from nerve recording experiments and taste behavioral tests were first compiled using Microsoft Excel. For statistical analyses, repeated measures two-way ANOVA with post hoc t tests
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Please cite this article in press as: Feng, P., et al. Regulation of bitter taste responses by tumor necrosis factor. Brain Behav. Immun. (2015), http:// dx.doi.org/10.1016/j.bbi.2015.04.001
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were performed using Statistica (Dell Software, Aliso Viejo, CA) or Statcel (OMS, Tokyo, Japan). p-Values
No. of Pages 11, Model 5G
16 April 2015 Brain, Behavior, and Immunity xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi 4 5 3
Regulation of bitter taste responses by tumor necrosis factor
7
Pu Feng 2, Masafumi Jyotaki 2, Agnes Kim 1, Jinghua Chai, Nirvine Simon, Minliang Zhou, Alexander A. Bachmanov, Liquan Huang, Hong Wang ⇑
8
Monell Chemical Senses Center, 3500 Market St., Philadelphia, PA 19104, USA
6
9 10 1 5 2 2 13 14 15 16 17 18 19 20 21 22 23 24
a r t i c l e
i n f o
Article history: Received 20 January 2015 Received in revised form 23 March 2015 Accepted 1 April 2015 Available online xxxx Keywords: Cytokine TNF Inflammation Taste-related behavior Taste buds
a b s t r a c t Inflammatory cytokines are important regulators of metabolism and food intake. Over production of inflammatory cytokines during bacterial and viral infections leads to anorexia and reduced food intake. However, it remains unclear whether any inflammatory cytokines are involved in the regulation of taste reception, the sensory mechanism governing food intake. Previously, we showed that tumor necrosis factor (TNF), a potent proinflammatory cytokine, is preferentially expressed in a subset of taste bud cells. The level of TNF in taste cells can be further induced by inflammatory stimuli. To investigate whether TNF plays a role in regulating taste responses, in this study, we performed taste behavioral tests and gustatory nerve recordings in TNF knockout mice. Behavioral tests showed that TNF-deficient mice are significantly less sensitive to the bitter compound quinine than wild-type mice, while their responses to sweet, umami, salty, and sour compounds are comparable to those of wild-type controls. Furthermore, nerve recording experiments showed that the chorda tympani nerve in TNF knockout mice is much less responsive to bitter compounds than that in wild-type mice. Chorda tympani nerve responses to sweet, umami, salty, and sour compounds are similar between TNF knockout and wild-type mice, consistent with the results from behavioral tests. We further showed that taste bud cells express the two known TNF receptors TNFR1 and TNFR2 and, therefore, are potential targets of TNF. Together, our results suggest that TNF signaling preferentially modulates bitter taste responses. This mechanism may contribute to taste dysfunction, particularly taste distortion, associated with infections and some chronic inflammatory diseases. Ó 2015 Elsevier Inc. All rights reserved.
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
47 48
1. Introduction
49
Taste is the sensory system for detecting nutrients and potentially harmful substances in food and drink and, therefore, plays important roles in guiding food intake. Among the five basic taste modalities, sweet and umami tastes detect sugars and amino acids, respectively, and are generally preferred. Bitter taste recognizes toxins and noxious compounds and elicits avoidance behavior. Acids and salts are detected by sour and salt taste mechanisms. Recent research has made rapid progress in understanding taste receptors and signaling pathways, particularly for sweet, umami, and bitter tastes (Breslin and Huang, 2006; Chandrashekar et al.,
50 51 52 53 54 55 56 57 58
⇑ Corresponding author. Tel.: +1 267 519 4773. E-mail addresses: [email protected] (P. Feng), [email protected] (M. Jyotaki), [email protected] (A. Kim), [email protected] (J. Chai), n.simon.uchs@gmail. com (N. Simon), [email protected] (M. Zhou), [email protected] (A.A. Bachmanov), [email protected] (L. Huang), [email protected] (H. Wang). 1 Present address: Drexel University College of Medicine, Philadelphia, PA 19129, USA. 2 These authors contribute equally to this work.
2006; Liman et al., 2014). What remain largely unclear, however, are the regulatory mechanisms that modulate taste responses or taste bud structure under diverse physiological and pathological conditions. Inflammation is likely one of such regulatory mechanisms. Many diseases with underlying inflammation, such as infections and autoimmune ailments, are associated with taste alterations (Bromley and Doty, 2003; Pribitkin et al., 2003; Schiffman, 1983). Taste alterations can occur as taste loss (lacking or reduced taste reception) or taste distortion (e.g. persistent bitter or metallic taste in the mouth) (Brand, 2000; Bromley, 2000). In animal models, induced inflammation has been shown to affect taste responses and taste bud structure (Cavallin and McCluskey, 2005; Cohn et al., 2010; Phillips and Hill, 1996). How inflammation exerts its effects on taste reception or taste bud structure has not been fully elucidated. Inflammation is an immune response to infection, tissue damage, and stress. In addition to its roles in regulating immunity and tissue repair, inflammation can strongly affect metabolism and food intake (Forsythe et al., 2008; Hotamisligil, 2006). The various effects of inflammation are often mediated by
http://dx.doi.org/10.1016/j.bbi.2015.04.001 0889-1591/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Feng, P., et al. Regulation of bitter taste responses by tumor necrosis factor. Brain Behav. Immun. (2015), http:// dx.doi.org/10.1016/j.bbi.2015.04.001
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P. Feng et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx
inflammatory cytokines, a group of signaling proteins that are highly induced during inflammatory responses. A number of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-6, and interferons, are pleiotropic and play important parts in regulating immunity, metabolism, and food intake (Cannon, 2000; Dantzer, 2001; Plata-Salaman, 1998). Our studies have found that several inflammation-associated cytokines are preferentially expressed in taste bud cells compared to nontaste lingual epithelial cells (Cohn et al., 2010; Feng et al., 2012, 2014a,b; Kim et al., 2012; Wang et al., 2007), suggesting that these cytokines may have special functions in the peripheral taste system. In particular, we have found that TNF is specifically expressed in a subset of taste bud cells even in healthy mice (Feng et al., 2012; Kim et al., 2012). Immunocolocalization experiments showed that TNF is colocalized with the sweet and umami taste receptor subunit T1R3, indicating that TNF is expressed specifically in the sweet and umami taste bud cells (Feng et al., 2012). Moreover, TNF expression level and its secretion in taste buds can be further augmented by inflammation, such as lipopolysaccharide (LPS)-induced inflammation (Cohn et al., 2010; Feng et al., 2012; Kim et al., 2012). TNF was thought to be produced primarily by macrophages, but it is also produced by a broad variety of cell types including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, fibroblasts, adipocytes, and neurons (Hotamisligil et al., 1993; Niu et al., 2009; Walsh et al., 1991). TNF is known to activate a variety of cellular signaling pathways that are important not only for fighting against certain pathogens but also for regulating stress responses and metabolism (Cabal-Hierro and Lazo, 2012; Silke, 2011; Wajant et al., 2003). TNF contributes to behavioral changes associated with various illnesses (i.e. sickness behavior) which include fatigue, malaise, depression, and anorexia. It has been shown that administration of recombinant TNF induces significant reduction in food intake both in rodents and in humans (Bernstein et al., 1991; Michie et al., 1989; Spiegelman and Hotamisligil, 1993). How TNF regulates food intake remains incompletely understood. Both peripheral and brain mechanisms are likely involved (Bernstein et al., 1991; Dantzer, 2001; Plata-Salaman, 1998). The taste system plays an important role in regulating food intake. Considering the specific expression of TNF in taste bud cells, it is conceivable that TNF may be involved in modulating taste responses under physiological and pathological conditions. In this study, we used TNF knockout mice and their wild-type controls to investigate the role of TNF in the taste system. We conducted gustatory nerve recording and taste behavioral testing using these mice. Our results show that TNF-deficient mice are significantly less responsive to bitter compounds than control mice, whereas their responses to sweet, umami, sour, and salty compounds did not differ significantly from those of control mice. Our results suggest that TNF is involved in the regulation of bitter taste reception.
130
2. Materials and methods
131
2.1. Animals
132
TNF knockout mice (stock number 005540) and wild-type control mice (C57BL/6J, stock number 000664) were purchased from the Jackson Laboratory (Bar Harbor, ME) and then bred and maintained at the Monell Chemical Senses Center. Generation of TNF knockout mice was described by Pasparakis et al. (1996). In these TNF knockout mice, the first coding exon (including the ATG translation initiation codon) and a portion of the first intron of the TNF gene were deleted. The mutant mice have been backcrossed to C57BL/6J genetic background for ten generations. All mice were
133 134 135 136 137 138 139 140
housed at the Monell Chemical Senses Center animal facility under a 12 h/12 h light/dark cycle. Mice were given free access to standard rodent food (8604 Teklad rodent diet, Harlan Laboratories) and water except during the periods of taste behavioral tests (described below). 4–10 months old mice were used for all the experiments described below. Age and gender matched wild-type and TNF-knockout mice were included for the experiments. All procedures were performed according to protocols approved by the Monell Chemical Senses Center Institutional Animal Care and Use Committee.
141
2.2. Reagents
151
All taste compounds used in behavioral and electrophysiological tests were purchased from Sigma (St. Louis, MO). Rabbit polyclonal antibody against mouse ecto-nucleoside triphosphate diphosphohydrolase 2 (ENTPDase2) was purchased from Centre de Recherche (Quebec, Canada) (Bartel et al., 2006). Rabbit polyclonal antibodies against phospholipase C-b2 (PLC-b2, sc-206) (Clapp et al., 2001) and gustducin (sc-395), goat polyclonal antibodies against the voltage-gated potassium channel KCNQ1 (sc-10646) (Wang et al., 2009) and TNFR1 (sc-1069), and a blocking peptide (sc-1069p) for the anti-TNFR1 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A purified rabbit polyclonal antibody against neural cell adhesion molecule (NCAM) was purchased from Millipore (Billerica, MA). Purified goat polyclonal antibodies against carbonic anhydrase 4 (Chandrashekar et al., 2009) and TNFR2 and a blocking antigen (recombinant mouse sTNFR2, 426-R2-050) for the anti-TNFR2 antibody were purchased from R&D Systems (Minneapolis, MN). Dylight-649 (or Dylight-488)-conjugated donkey anti-rabbit or donkey anti-goat antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
152
2.3. Taste behavioral tests
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Two-bottle preference tests were conducted as previously described (Bachmanov and Beauchamp, 2008; Bachmanov et al., 2001, 2002; Wang et al., 2009). Briefly, TNF-deficient and wildtype mice were individually caged. For the first 2 days, mice were familiarized with the two drinking bottles, both containing deionized water. For the following days, mice were presented with two drinking bottles: one contained deionized water and the other a taste solution. The positions of the bottles were switched after 24 h to minimize positional effect. The volume of consumed liquid from each bottle was recorded at 24 and 48 h. Each concentration of a taste compound was tested for 48 h. The taste compounds were tested in the following order: NaCl (37.5, 75, 150, 300, and 600 mM), quinine hydrochloride (QHCl) (0.003, 0.01, 0.03, 0.1, and 0.3 mM), Saccharin (0.0625, 0.25, 1, 4, and 16 mM), inosine50 -monophosphate (IMP) (0.3, 1, and 3 mM), and citric acid (1, 3, and 10 mM). Each mouse in the experiment was tested with all the above listed compounds. Between the testing of two different compounds, mice received deionized water in both drinking tubes for at least 3 days. During the experiment mice had free access to food. TNF-deficient mice and wild-type mice were tested at the same time in parallel. Taste preference scores were calculated for each animal by dividing the volume of consumed taste solution during the 48 h test period by the total volume of fluid intake during the same 48 h test period (i.e. preference score = intake of taste solution/(intake of taste solution + intake of water)). The average preference scores were then calculated for each group. By this calculation formula, preference scores between 0.5 and 1 indicate that mice prefer the taste solution over water, whereas preference scores between 0 and 0.5 indicate that mice avoid the taste solution. 8–16 male mice (4–8 months old) in each group were used.
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Brief-access tests were performed using the Davis MS-160 mouse gustometer (Dilog Instruments, Tallahassee, FL) as previously described (Kim et al., 2012). Briefly, mice were water-deprived for 22.5 h before 30 min training sessions and test sessions for aversive taste compounds (bitter, salty, and sour compounds). Mice were food- and water-restricted (1 g of food and 1.5 ml of water) for 23.5 h before test sessions for appetitive taste compounds (sweet and umami compounds). Water and food restrictions were used to motivate mice to lick the taste solutions presented during the short test periods and were comparable to published protocols for this type of tests (Glendinning et al., 2002). In each test session, mice were tested with three different concentrations of each taste compound along with a water control. Water and taste compounds were randomly presented to mice following random presentation schemes generated by the computer software. Inter-presentation interval was 10 s. The maximum wait for the first lick was 120 s. Lick time limit was 5 s, which was the time from the first lick until the shutter closed. So, for each presentation, mice had maximum 5 s direct contact (or lick) time with the presented solution or water. The session time limit was 30 min. The following taste compounds were tested: QHCl (0.03, 0.3, and 3 mM), NaCl (0.1, 0.6, and 1 M), and citric acid (3, 10, and 100 mM), sucrose (0.1, 0.2, and 0.6 M), IMP (1, 10, and 30 mM). Each mouse was tested with all the compounds. TNF-deficient mice and wild-type mice were tested at the same time in parallel. After each session, mice received a recovery day with free access to food and water for 24 h. Taste stimulus to water lick ratios were calculated by dividing the number of licks for taste compounds by the number of licks for water presented in the same test session. Lick ratios less than 1 indicate avoidance behavior to the taste solution, and lick ratios more than 1 indicate preference behavior. 8–16 TNF-deficient mice and wild-type control mice (all male, 6– 10 months old) were included in this experiment. The same sets of mice were used for two-bottle preference tests and then for brief-access tests with a 3-week interval between the two testing procedures to minimize the possible effects of prior experience on subsequence testing.
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2.4. Gustatory nerve recording
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Chorda tympani nerve recording was carried out as previously described (Kim et al., 2012). Briefly, under pentobarbital anesthesia (50–60 mg/kg of body weight, i.p.), the trachea of each animal was cannulated, and the mouse was then fixed in the supine position with a head holder. The right chorda tympani nerve was exposed at its exit from the lingual nerve, cut near its entrance to the bulla, and was placed on a platinum wire recording electrode. An indifferent electrode was positioned nearby in the wound. Neural responses resulting from chemical stimulations of the tongue were fed into an amplifier (Grass Instruments, West Warwick, RI), monitored on an oscilloscope and an audio monitor. Whole-nerve responses were integrated with a time constant of 1.0 s and recorded using a computer for later analysis using a PowerLab system (PowerLab/sp4; AD Instruments, Colorado Springs, CO). For taste compound stimulation, the tongue was enclosed in a flow chamber, and solutions were delivered into the chamber by gravity flow. The following solutions were used as stimuli: 0.1–20 mM QHCl, 0.1–20 mM denatonium benzoate, 0.003–0.5 mM cycloheximide, 3–300 mM MgSO4, 10–1000 mM sucrose, 0.3–20 mM saccharin, 10–1000 mM monopotassium glutamate (MPG) with or without 0.5 mM IMP, 0.1–10 mM IMP, 1–100 mM citric acid, 0.01–10 mM HCl, 10–1000 mM NaCl with or without 100 lM amiloride, and 0.1 M NH4Cl. To analyze nerve responses to each stimulus, the magnitudes of integrated responses at 5, 10, 15, 20, and 25 s after stimulus onset were measured and averaged. The relative response magnitude for each stimulus was calculated
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against the response magnitude to 0.1 M NH4Cl, and this value was used for statistical analysis and for plotting dose–response curves. Eight wild-type mice (6 males and 2 females) and seven TNF-deficient mice (5 males and 2 females), 4–8 months old, were used for nerve recordings.
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2.5. mRNA in situ hybridization
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Digoxigenin (DIG)-labeled sense and antisense cRNA probes corresponding to the coding region of mouse TNFR1 and TNFR2 were synthesized using the DIG RNA labeling kit (Roche Applied Science). Fresh-frozen taste sections (10 lm/section) from C57BL/ 6J mice (4–6 months old) were attached to clean glass slides. Sections were then fixed with 4% paraformaldehyde and processed for in situ hybridization as previously described (Wang et al., 2007). Hybridizations were performed at 72 °C overnight with DIG-labeled probes in 50% formamide, 5 SSC, 5 Denhardt’s solution, 250 lg/ml yeast RNA, and 500 lg/ml sperm DNA. Sections were washed 3 times at 72 °C with 0.2 SSC. Hybridized DIG-labeled cRNA was detected immunologically with an alkaline–phosphatase-conjugated anti-DIG antibody and standard chromogenic substrates 4-Nitro Blue tetrazolium chloride (NBT, Roche Applied Science). Images were taken using a Nikon fluorescence microscope. In all the experiments, hybridizations to antisense and sense probes were performed in parallel to verify the specificity of hybridization signals.
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2.6. Immunohistochemistry
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Tissue preparation and immunofluorescent staining procedures were described previously (Feng et al., 2012; Wang et al., 2007). All mice used for this procedure were 4–6 months old. Briefly, excised mouse tongue tissues were fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h on ice and then cryoprotected in 20% sucrose/PBS solution at 4 °C overnight and embedded in mounting medium. Tissues were sliced into 10-lm-thick sections using a Microm HM 500 OM cryostat (Thermo Scientific Microm, Walldorf, Germany). Purified goat polyclonal antibodies against TNFR1 and TNFR2 (see Section 2.2) were used to detect the expression of TNFR1 and TNFR2 in taste tissues. Two control experiments for TNFR1 and TNFR2 immunostaining were conducted. In one control experiment, primary antibodies against TNFR1 and TNFR2 were omitted in the procedure. In the second control experiment, antibodies against TNFR1 and TNFR2 were preincubated with their corresponding blocking antigens before adding to tissue sections. To investigate what types of taste bud cells express TNFR1 or TNFR2, double immunostaining was carried out using rabbit antibodies against taste-cell-type markers and goat antibodies against TNFR1 or TNFR2. Antibodies to the following taste-cell-type markers were used: ENTPDase2 (1:500), PLC-b2 (1:1000), and NCAM (1:300). Dylight-649-conjugated donkey anti-rabbit and Dylight-488-conjugated donkey anti-goat secondary antibodies were used. For immunostaining of taste tissue sections from wild-type and TNF knockout mice, antibodies to KCNQ1 (1:1000), PLC-b2 (1:1000), gustducin (1:1000), and carbonic anhydrase 4 (1:500) were used. Secondary antibodies were Dylight-649-conjugated donkey anti-goat and Dylight-488-conjugated donkey anti-rabbit antibodies. Fluorescent images were acquired using Leica Sp2 confocal microscope.
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2.7. Statistical analysis
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Data from nerve recording experiments and taste behavioral tests were first compiled using Microsoft Excel. For statistical analyses, repeated measures two-way ANOVA with post hoc t tests
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were performed using Statistica (Dell Software, Aliso Viejo, CA) or Statcel (OMS, Tokyo, Japan). p-Values
