TLR4 enhances histamine-mediated pruritus by potentiating TRPV1 activity
© Min et al.; licensee BioMed Central Ltd. 2014
Received: 4 August 2014
Accepted: 7 August 2014
Published: 21 August 2014
Recent studies have indicated that Toll-like receptor 4 (TLR4), a pathogen-recognition receptor that triggers inflammatory signals in innate immune cells, is also expressed on sensory neurons, implicating its putative role in sensory signal transmission. However, the possible function of sensory neuron TLR4 has not yet been formally addressed. In this regard, we investigated the role of TLR4 in itch signal transmission.
TLR4 was expressed on a subpopulation of dorsal root ganglia (DRG) sensory neurons that express TRPV1. In TLR4-knockout mice, histamine-induced itch responses were compromised while TLR4 activation by LPS did not directly elicit an itch response. Histamine-induced intracellular calcium signals and inward currents were comparably reduced in TLR4-deficient sensory neurons. Reduced histamine sensitivity in the TLR4-deficient neurons was accompanied by a decrease in TRPV1 activity. Heterologous expression experiments in HEK293T cells indicated that TLR4 expression enhanced capsaicin-induced intracellular calcium signals and inward currents.
Our data show that TLR4 on sensory neurons enhances histamine-induced itch signal transduction by potentiating TRPV1 activity. The results suggest that TLR4 could be a novel target for the treatment of enhanced itch sensation.
KeywordsToll-like receptor Itch Sensory neurons
Chronic itch is a pathological hallmark of atopic dermatitis and other infectious skin diseases. Recent studies on single nucleotide polymorphisms implicate genes encoding pattern-recognition receptors such as CD14 and toll-like receptors (TLRs) in the development and severity of atopic dermatitis (AD) ,. TLRs are type I transmembrane receptors expressed on innate immune cells that detect pathogen infection or tissue damage. TLR activation of innate immune cells leads to expression of inflammatory mediators including cytokines, chemokines, leukotriene molecules, and histamine ,. Some of these inflammatory mediators excite sensory neurons and may trigger transmission of itch signals. Therefore, itch sensations observed during skin diseases and infections are generally attributed to sensory neuronal activation by inflammatory mediators released from innate immune cells such as mast cells or keratinocytes .
Recently, some pathogen-recognition receptors including TLR3, 4, and 7 were also found to be expressed on sensory neurons . These findings imply that itch signals can also be generated if these TLRs were expressed on itch-specific sensory neurons and activated by pathogen-derived molecules. This possibility is supported by a recent study showing that TLR3 activation on a subpopulation of dorsal root ganglia (DRG) sensory neurons elicits inward currents and action potential . The study found that TLR3 is required for maximal pruritic effects of histamine as well as non-histaminergic pruritogens. In another study, Liu et al. found that TLR7 stimulation by imiquimod, a synthetic TLR7 agonist, directly excites sensory neurons, leading to itch-specific signal generation . However, the same group later reported that TLR7-mediated sensory neuron activation by microRNA elicit pain rather than itch signals . We have also reported the pruritogenic function of imiquimod; however, this was not mediated by TLR7 . Thus, the pruritogenic function of TLR7 on DRG sensory neurons is controversial.
In addition to TLR3 and 7, TLR4 is reported to be expressed in certain DRG and trigeminal ganglia sensory neurons . This finding suggests a role for this receptor in sensory transmission, but this has not been formally studied. Therefore, we investigated the function of sensory neuron TLR4 in itch signal transmission. We found that TLR4 contribute to histamine-induced itch signal transduction at least partly by potentiating TRPV1 activity.
The recent discovery of TLRs on DRG sensory neurons suggests that these pattern-recognition receptors may function in transduction and/or transmission of sensory signals. Studies thus far implicated TLR3 and TLR7 in itch signal transmission. In this study, we have investigated the roles of TLR4 in sensory transmission and found that TLR4 is required for optimal histamine-induced itch signal transduction through regulating TRPV1 activity. Such role of TLR4 in itch signal transduction is distinct from other TLR members. The activation of TLR3 by poly(IC) treatment directly excited sensory neurons and induced a scratch response . However, TLR4 stimulation by LPS did not directly excite sensory neurons or induce significant scratch response. These data suggest that, unlike TLR3, TLR4 signaling alone in sensory neurons does not elicit itch signal. Instead, histamine-induced and chloroquine-induced itch responses were severely compromised in TLR4 KO mice, suggesting TLR4 is required for optimal itch signal transmission stimulated by these pruritogens. Although a reduction in histamine-induced and chloroquine-induced itch responses were also observed in TLR3 KO mice, the mechanisms are different . In our study, in the absence of TLR4, histamine-induced and chloroquine-induced calcium signals in sensory neurons are severely compromised. These data suggest that the itch phenotype we observed in TLR4 KO mice came from itch signal transduction in sensory neurons. However, TLR3 is not required for pruritogen-induced itch signal transduction in the sensory neurons. Rather, TLR3 contributes to synaptic transmission of the itch signal and central sensitization at the spinal cord level. In addition, TLR3 is expressed mainly in small-sized TRPV1+ neurons. However, TLR4 expression was not limited to small-sized neurons but was also detected in large-sized neurons. TLR4 expression was not limited to TRPV1+ neuron population either. These data suggest that, unlike TLR3, TLR4 expression is not limited to itch-specific sensory neurons. Nonetheless, the subpopulation of TRPV1+ neurons that co-express TLR4 is likely to mainly contribute to histamine-induced itch signal transduction. Currently, the involvement of TLR7 in itch signal transmission is under debate. Imiquimod, a synthetic TLR7 agonist, was found to trigger inward currents and action potentials in TLR7+ sensory neurons and induce an itch-specific response . However, it was recently reported that TLR7 activation in sensory neurons elicits pain . We found that the pruritogenic effects of imiquimod are TLR7-independent, but rather it activates sensory neurons by affecting IP3R and potassium channel ,.
In our study, TLR4 regulates histamine-induced and chloroquine-induced itch by distinct mechanisms. TLR4 potentiates histamine responsiveness by increasing TRPV1 basal activity. However, TLR4 does not affect TRPA1 activity. Instead, TLR4 expression increased MrgprA3 expression. Crosstalk between TLR4 and TRPV1 signals has been documented . In this study, TLR4 activation increased capsaicin-induced intracellular calcium signals and inward currents. However, we did not observe significant differences in calcium signals after LPS treatment (data not shown). Our results are consistent with a report that ultrapure LPS, in contrast to standard-grade LPS, fails to induce neuronal excitability . Instead, our data on decreased TRPV1 activity in TLR4 KO neurons indicate that TLR4 expression potentiates TRPV1 activity. It is not clear how TLR4 enhances TRPV1 activity. A recent study shows that TLR7 can enhance TRPA1 activity by direct physical interaction . This suggests a possibility that TLR4 may also enhance TRPV1 activity by direct interaction, which needs to be tested in the future investigation.
TRPV1 is not only associated with itch signals but also involved in pain signals. In our study, we observed capsaicin-induced paw licking was also slightly reduced in TLR4 KO mice (data not shown). This result suggests that the effect of neuronal TLR4 might not be specific to itch signal, but might be involved in pain transmission as well. Of note, nerve-injury induced mechanical allodynia and hyperalgesia are severely compromised in TLR4 KO mice . In this study, TLR4 involvement was interpreted mainly from the perspective of spinal cord microglia activation. Considering our data, it needs to be re-evaluated the putative contribution of sensory neuron TLR4 to the neuropathic pain development.
Itch susceptibility to pruritogens or bacterial/viral infections depends on genetic background. For instance, polymorphisms in genes for the TLR family and associated receptors are implicated in itch-associated diseases such as AD . Our study found a putative mechanism how TLR4 expression affects itch signal transduction. In addition to polymorphisms, TLR4 expression is regulated in diseases such as bacterial infection and tissue damage. In innate immune cells, TLR4 is upregulated by various inflammatory mediators such as TNF-α and IL-1β. Whether sensory neuron TLR4 is also upregulated by these inflammatory mediators is unknown. If it is, inflammatory mediators secreted during the innate immune response in the skin might regulate itch sensitivity by upregulating TLR4 expression in sensory neurons.
In this study, we have uncovered that TLR4 expression in sensory neurons potentiates histamine-induced itch signal transduction. The histamine-induced scratch response is significantly compromised in TLR4 KO mice. Similarly, histamine-induced intracellular calcium signals and inward currents are reduced TLR4-deficent sensory neurons. As a mechanism for these results, we found that TLR4 expression potentiates histamine-mediated itch by regulating TRPV1 channel activity. Our data suggest that TLR4 could be a novel therapeutic target for treating enhanced itch sensations during inflammatory responses.
Eight-week-old C57BL6 mice were purchased from Daehan Biolink (Eumsung, Korea). TLR4 KO mice of C57BL6 background were generously provided by Dr. S. Akira (Osaka University, Japan). Mice were housed at 23 ± 2°C with a 12-h light–dark cycle and fed food and water ad libitum. All surgical or experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University.
Scratching behavioral assays were performed as previously described . Briefly, mice were placed individually in transparent cages for at least 30 minutes before assays and received pruritogens or LPS by intradermal injection into the rostral back. Numbers of scratching bouts that included only the hind paw were analyzed at 5-minute intervals for 30 minutes.
Primary DRG neuron culture
DRGs collected from 8-week-old mice were incubated in Hank’s balanced salt solution (HBSS) (Welgene, Daegu, Korea) containing 0.33 mg/ml papain (Wothington, Lakewood, NJ, USA) and 0.65 mg/ml L-cysteine (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes at 37°C and then HBSS containing 4 mg/ml collagenase (Roche, Mannheim, Germany) and 5 mg/ml dispase (Invitrogen, Calrsbad, CA, USA) for 10 minutes at 37°C. Samples were washed with DMEM/F12 with 10% (v/v) fetal bovine serum (Invitrogen), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Welgene). The cell suspension was filtered through a 70-μm cell strainer and cultured in poly-D-lysine (Sigma-Aldrich) and laminin-coated culture dish or glass coverslips (Sigma-Aldrich).
Cell culture and transfection
HEK293T cells were maintained in DMEM containing 10% FBS, 100 U/ml ampicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine in a humidified incubator containing 5% CO2. HEK293T cells were transfected using Effectene transfection reagent (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions.
Single-cell RT-PCR was performed as previously described . Briefly, single DRG neuronal cells were collected with a glass micropipette connected to micromanipulator under a microscope and transferred to a PCR tube containing RNase-free water. Digestion with DNase I was performed before reverse transcription for 1 h at 50°C. The cDNA product was used for PCR in 50 μl of PCR buffer containing 0.2 mM dNTPs, 0.2 μM outer primers, 5 μl RT product, and 0.2 μl platinum Taq DNA polymerase (Invitrogen). The protocol was 5 min at 95°C followed by 40 cycles of 40 s at 95°C, 40 s at 60°C, and 40 s at 72°C. Reactions were completed with 7 min of final elongation. The second round of amplification was in reaction buffer (20 μl) containing 0.2 mM dNTPs, 0.2 μM inner primers, 5 μl first-round PCR product, and 0.1 μl platinum Taq DNA polymerase following the same amplification procedure as the first round. The following primers were used: TLR4 outer forward 5′- ATC TGA GCT TCA ACC CCT TG-3′; TLR4 outer reverse 5′- AAT TCC CTG AAA GGC TTG GT-3′; TLR4 inner forward 5′- TCA GAA CTT CAG TGG CTG GA-3′; TLR4 inner reverse 5′- TTG ACT TGT GGA TTT TCA CG-3′; TRPV1 outer forward 5′- CAT GCT CAT TGC TCT CAT GG-3′; TRPV1 outer reverse 5′- AAC CAG GGC AAA GTT CTT CC-3′; TRPV1 inner forward 5′- CAT GGG CGA GAC TGT CAA C-3′; TRPV1 inner reverse 5′- CTG GGT CCT CGT TGA TGA TG-3′; MAP2 outer forward 5′- GAA GAG TTC CAA GGC CCA CTT-3′ MAP2 outer reverse 5′- GCC TGA AAT TTG CCT TTT CC-3′; MAP2 inner forward 5′- CCT GTG CAA TTC CAG CTC AGT-3′; MAP2 inner reverse 5′- CCC CCA TGT GGC ATG AAA TAT3′.
For DRG staining, adult mice were anesthetized with urethane and perfused with 4% paraformaldehyde and DRGs were collected. DRGs were incubated in 4% paraformaldehyde, cryoprotected in 30% sucrose, and frozen in OCT. DRG sections (12 μm) were obtained by cryostat and mounted on collagen-coated slides. Sections were stained with mouse anti-histamine receptor H1 (Santa Cruz, Dallas, TX, USA) or anti-TRPV1 (Santa Cruz) then incubated for 1 h with Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch, West Groove, PA, USA), and coverslipped with VectaShield medium (Vector Labs, Burlingame, CA, USA).
For immunocytochemistry, DRG neuronal cells or transfected HEK293T cells were seeded onto poly-D-lysine (PDL)-coated coverglass. Cells were fixed in 4% PFA in 0.1 M PBS (pH 7.4) for 15 min. After rinsing in 0.1 M PBS, cells were blocked with 0.1 M PBS containing 5% normal goat serum, 5% fetal bovine serum, 2% bovine serum albumin, and 0.1% Triton X-100 for 1 h at RT. Cells were incubated overnight at 4°C with mouse anti-NeuN (1:1000; Millipore, Billerica, MA, USA), rabbit anti-TLR4 (1:100; Santa Cruz), or mouse anti-HA (1:1000; Cell signaling, Danvers, MA, USA) antibody. Cells were incubated for 1 h at RT with FITC- or Cy3-conjugated secondary antibody and mounted with VectaShield medium.
For live cell staining, DRG neurons were seeded on PDL-coated coverglasses and incubated with 5 μg/ml Alexa Fluor 488-conjugated LPS from Escherichia coli O55:B5 (Molecular Probes, Eugene, OR, USA) for 1 h at 37°C. After rinsing in 0.1 M PBS, coverglasses were mounted with VectaShield medium. Images were captured using a confocal laser scanning microscopy (LSM7 PASCAL; Carl Zeiss, Jena, Germany).
Real-time RT-PCR was performed using SYBR Green PCR Master Mix (ABI, Warrington, UK) as described previously . PCR was performed in duplicate in a total volume of 10 μl containing 10 pM primer, 4 μl cDNA, and 5 μl SYBR Green PCR Master Mix. The mRNA levels of each target gene were normalized to GAPDH mRNA. Fold-induction was calculated using the 2-∆∆CT method as previously described . All real-time RT-PCR experiments were performed at least three times and are presented as mean ± SEM unless otherwise noted.
Primers for real-time RT-PCR were MrgprA3 forward: 5’-CGA CAA TGA CAC CCA CAA CAA-3’; MrgprA3 reverse: 5’-GGA AGC CAA GGA GCC AGA AC-3’; histamine receptor H1 forward: 5’-GGG AAA GGG AAA CAG TCA CA-3’; histamine receptor H1 reverse: 5’-ACT GTC GAT CCA CCA AGG TC-3’; histamine receptor H4 forward: 5’-GGA AGC TAG CCA GGT CAC T-3’; histamine receptor H4 reverse: 5’-CCG TTC TGG AAG TTG AA-3’; TRPV1 forward: 5’-AAG GCT CTA TGA TCG CAG GA-3’; TRPV1 reverse: 5’-CAG ATT GAG CAT GGC TTT GA-3’; GAPDH forward: 5’-AGG TCA TCC AGC TGA ACG-3’; GAPDH reverse: 5’-CAC CCT GTT GCT GTA GCC GTA T-3’.
Calcium response in DRG neurons was measured by single-cell calcium imaging using Fura-2 AM (Invitrogen). Cells were plated on PDL-coated coverglasses and incubated overnight. Cells were incubated for 50 min at RT with 2 μM Fura-2 AM in HBSS containing 25 mM HEPES (pH 7.5) and washed with HBSS-HEPES twice before assays. A baseline reading was taken for 60 s before addition of histamine, chloroquine or capsaicin. After treatment, to test cell viability, 100 mM KCl was added. Intracellular calcium levels were measured by digital video microfluorometry with an intensified charge-coupled device camera (CasCade, Roper Scientific, Trenton, NJ, USA) coupled to a microscope and analyzed with MetaFluor software (Universal Imaging Corp., Downington, PA, USA). Fura-2 AM excitation wavelengths were selected by a Lambda DG-4 monochromator wavelength changer (Shutter Instrument, Novato, CA, USA).
To determine intracellular calcium levels in HEK293T cell population, cells were detached from plates 24 h after transfection and stained with 2 μM Fura-2 AM in Locke’s solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 2.2 mM CaCl2, 5.0 mM HEPES, 10 mM glucose, pH 7.4) for 50 min at 37°C. Cells were then washed twice with Locke’s solution and suspended at 1 × 106 cells/ml for assays. Intracellular calcium levels were monitored with dual excitation at 340 and 380 nm and emission at 500 nm by a spectrofluorophotometer (Shimadzu RF-5301-PC, Shimadzu, Kyoto, Japan). The ratio of emission after 340 nm and 380 nm excitation (340 nm/380 nm) was used for index of intracellular calcium concentration ([Ca++]i.). The net change in [Ca++]i upon drug treatment (Δ ratio (340 nm/38 nm)) was calculated by subtracting basal [Ca++]i from the peak [Ca++]i achieved after exposure to the drug.
Whole cell patch-clamp recording
Cells were plated on PDL-coated cover glasses and incubated in medium for at least 6 h. DRG neuron recordings were performed in HEPES buffer (10 mM HEPES, 150 mM NaCl, 10 mM KCl, 2 mM MgCl2, 5.5 mM glucose, and 22 mM sucrose, pH 7.4). Under an upright microscope, whole-cell patch recordings were obtained from acute-isolated DRG neurons in voltage-clamp mode and switched current-clamp configuration for recordings with an Axopatch 700B (Molecular Devices, Sunnyvale, CA, USA). Pipette resistance ranged from 3 to 6 MΩ. The internal solution was 140 mM K-gluconate, 10 mM HEPES, 7 mM NaCl, 4 mM Mg-ATP, and 0.3 mM Na3-GTP, pH 7.4. Data were analyzed and plotted with pClamp (Molecular Devices).
Western blot assay
For western blots, protein samples were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk in TBST (20 mM Tris, pH 7.4, 0.1% Tween 20, 150 mM NaCl), membranes were incubated with rabbit anti-histamine receptor H1 (1:500, Santa Cruz), or mouse anti-β-actin (1:5000, Sigma-Aldrich) antibodies. Proteins were detected with horseradish peroxidase conjugated secondary antibodies using West Save Gold western blot detection kit (Ab Frontier, Seoul, Korea). Signal was visualized by MicroChemi (DNR Bio-imaging Systems, Jerusalem, Israel).
All data are presented as mean value with SEM. Differences between groups were determined by one-way ANOVA with Tukey’s multiple comparison test or Student’s t-test. Differences were considered significant when p was less than 0.05.
Cluster of differentiation
Dorsal root ganglia
Glyceraldehyde 3- phosphate dehydrogenase
Hank’s balanced salt solution
Histamine receptor H1
Histamine receptor H4
MAS-related G protein-coupled receptor family, member A3
Toll-like receptor 4
Tumor necrosis factor-α
Transient receptor potential cation channel, subfamily A, member 1
Transient receptor potential cation channel, subfamily V, member 1
The work was supported by the National Research Foundation of Korea, funded by the Ministry of Science, ICT, and Future Planning (2013R1A1A2074231), Republic of Korea.
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