Transient Receptor Potential Cation Channel Subfamily M Member 8 channels mediate the anti‐inflammatory effects of eucalyptol

CFA‐induced skin inflammation/paw swelling

CFA was injected (10 μL) into the plantar surface of the right hind paw. Saline was injected in control mice. Paw volumes were measured with a plethysmometer (IITC, Woodland Hills, CA, USA) immediately before (0 h, V_before) and 2, 4, 24 and 48 h (V_after) after CFA injection. Changes in paw volume were shown as % increase in paw volume and calculated as follows: % increase in paw volume = (V_after − V_before)/V_before. For therapy, eucalyptol (30, 100 or 300 mg·kg⁻¹), ibuprofen (30 mg·kg⁻¹) or vehicle (corn oil) were injected i.p. (25 μL·10 g⁻¹ body weight injection volume) 30 min before CFA injection, and then again at 6, 24, 30 and 48 h after CFA injection for a total of five times for each group.

von Frey hair analysis of mechanical allodynia in CFA‐injected mice

Mice were habituated for 45 min to the wire mesh screen surface before testing. Paw withdrawal thresholds were determined using a series of von Frey filaments (0.008 to 6.00 g) pressed against the plantar surface of the hind paw in ascending order beginning with the finest fibre, following standard procedures (Chaplan et al., 1994; Liu et al., 2013b). The minimum force (g) that caused the mouse to withdraw its hind paw away from the filament was considered as the withdrawal threshold. For each paw, a von Frey hair was applied five times at 10 s intervals. The threshold was determined when paw withdrawal was observed in more than three out of five applications. A withdrawal response was considered valid only if the hind paw was removed completely from the platform. If the paw withdrawal response was ambiguous, the application was repeated. The test was performed 2 h after the third eucalyptol treatment injection or 26 h after the CFA paw injection. Behavioural tests were performed by an experimenter blinded to experimental conditions.

Skin sample collection and ELISA

Mice were killed 3 h after the last eucalyptol treatment. The skin of the CFA or saline‐treated paw was collected by a 4 mm biopsy punch (Miltex Inc., PA, USA), weighed and immediately frozen in liquid nitrogen. Tissue was homogenized using a Bullet Blender (NextAdvance, Averill Park, NY, USA) in 50 mM Tris‐base (pH 7.4) and 150 mM NaCl with protease inhibitors (Roche, Indianapolis, IN, USA) and 0.2% Triton‐X100 for 25 min at full speed. Then the samples were centrifuged at 10 000 g for 15 min at 4°C. The supernatant was tested by ELISA for IL‐1β, TNF‐α and IL‐6 (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.

LPS‐induced pulmonary inflammation

Mice received a single intranasal dose of 10 μg LPS in 40 μL sterile saline under sevoflurane anaesthesia. Controls received saline only. For therapy, eucalyptol was injected i.p. at 200 mg·kg⁻¹ in corn oil as vehicle (25 μL·10 g⁻¹ injection volume). Treated mice received four i.p. injections: one 12 h before LPS administration, a second dose 1 h before LPS, a third dose 12 h after LPS and a fourth dose 1 h before killing. Mice were killed for sample collections 24 h after LPS administration, a time point where the acute lung inflammation (ALI) peaks (Hirano, 1997; Santos et al., 2006). Control animals received vehicle (corn oil) at the same intervals above.

Collection and analysis of bronchoalveolar lavage (BAL) fluid

BAL was performed by cannulation of the trachea and gentle instillation/aspiration (three times) of 1.0 mL of PBS with 0.1% BSA and protease inhibitors (Roche, Indianapolis, IN) as published before (Caceres et al., 2009). The lavage fluid was centrifuged at 1500 × g for 5 min, and the supernatant was frozen at −80°C for later assessment of cytokines and myeloperoxidase (MPO) activity. The cell pellet was treated with red‐blood‐cells lysing buffer (BD Biosciences, San Diego, CA, USA), washed and resuspended in 200 μL of PBS. Total cell counts were determined with a haematology analyser (Scil Vet ABC, Gurnee, IL, USA), spun onto cytoslides (Cytospin 3, Shandon Inc, Pittsburg, PA, USA) and stained with Diff‐Quick (Dade‐Behring Inc., Newark, DE, USA). Differential cell counts were obtained by microscopic counting of a minimum of 200 cells per slide and using standard morphological and staining criteria.

Quantitative analysis of cytokines and chemokines in BAL fluid

Cytokines and chemokines in the BAL fluid were measured using the Luminex xMAP technology. Analyte levels were determined using a Milliplex MAP mouse cytokine/chemokine kit (Millipore, Billerica, MA, USA) following the manufacturer's instructions. Briefly, 50 μL of BALF were added in duplicate to each well of a 96‐well plate. Antibody‐conjugated beads solution was added to each well and incubated in the dark prior to incubation with the biotinylated detection antibody specific for each analyte. Fluorescence intensity was determined by incubation with streptavidin‐phycoerythrin solution. All readings were carried out using a Luminex 200 analyzer (Luminex, Austin, TX, USA). MPO activity in the BAL fluid was assayed using a chlorination assay kit (Cayman Chemical Company, MI, USA).

RNA extraction and cDNA synthesis

After obtaining the BAL, lungs were perfused with PBS, surgically removed and snap‐frozen by immersion in liquid nitrogen. Total RNA was isolated from the tissue homogenates of each sample using the RNeasy Mini Kit 50 (Qiagen, MD, USA). The homogenization of all the tissues in Buffer RLT with 2‐mercaptoethanol was carried out in a NextAdvance bullet blender (max speed, 5 min). cDNA synthesis was performed from 500 ng of RNA right after the RNA isolation following the manufacturer's instructions (High Capacity Reverse Transcription Kit, Applied Biosystems).

Real‐time PCR (qPCR)

TaqMan® Gene Expression Assays from Applied Biosystems (Foster City, CA, USA) were used for expression assessment. About 20 μL reactions contained 10 μL of TaqMan Fast Universal PCR Master Mix (2×), 1 μL of the specific TaqMan assay, 1 μL cDNA and water up to 20 μL. Real‐Time PCR was performed on a LightCycler 480 (Roche, Indianapolis, IN) with cycling parameters of 10 min initial denaturation at 95°C, followed by 45 cycles at 95°C for 15 s and 1 min at 60°C. Each reaction was performed in triplicate and normalized to the endogenous 18S and Act‐b gene expression. The CT value of each well was determined using the LightCycler 480 software, and the average of the triplicates was calculated. The relative quantification was determined by the ΔΔCT method (Livak and Schmittgen, 2001). TaqMan Gene Expression Assays used are as follows: mTrpm8: Mm01299593_m1; mTrpm8: Mm00454566_m1; mTRPV4: Mm00499025_m1; mActb: Mm00607939_s1; 18SrRNA: Mm03928990_g1; TNF‐α: Mm99999068_m1; IL‐1b: Mm00434228_m1; IFM‐g: Mm99999071_m1; and IL‐6: Mm00446190_m1.

Cell culture and calcium influx imaging

HEK293T cells were cultured in DMEM and supplemented with 10% FBS, 100 units·mL⁻¹ penicillin and 0.1 mg·mL⁻¹ streptomycin. Cells were plated on poly‐D‐lysine‐coated 100 mm tissue culture plastic dishes and grown overnight to 60–70% confluency. The cells were transiently transfected with human TRPM8 plasmid DNA (Origene Technologies, Rockville, MD, USA, Cat. RC220615, Genbank NM_024080) using Fugene 6 transfection reagent and Opti‐MEM according to manufacturer's protocols for 16–24 h. Cells were re‐suspended and plated onto poly‐D‐lysine‐coated 96‐well plates at 100 000 cells per well (100 μL per well) and allowed to grow for another 16–24 h prior to the experiments. Cells were maintained as monolayers in a 5% CO2 incubator at 37°C.

Cells plated in 96‐well plates (Krystal black walled plates, Genesee Scientific) were loaded with FLIPR Calcium 6 no wash dye for 2 h (Molecular Devices) according to manufacturer's instructions. In preliminary experiments, we determined that the optimal dye concentration was one‐third of that suggested by the manufacturer. After 2 h, the plate was transferred to a FlexStation III benchtop scanning fluorometer chamber. Fluorescence measurements were performed at 37°C. The cells were excited at 485 nm, and Ca²⁺‐bound FLIPR Calcium 6 emission was recorded at 525 nm at every 1.62 s intervals. After recording baseline fluorescence for 18 s, 50 μL of a 5× concentration of eucalyptol or 2‐OH‐1,8, cineole was added to the cells yielding a final volume of 250 μL per well; the fluorescence was monitored for an additional 100 s. The FLIPR Calcium 6 dye fluorescence was expressed as F_max − F₀, where F_max is the maximum and F₀ is the basal fluorescence measured in each well. The EC₅₀ values and their 95% confidence intervals for eucalyptol and 2‐OH‐1,8 cineole stimulation of calcium influx were determined by non‐linear regression analysis with a three‐parameter logistic equation (Graphpad Prism software).

Data and statistical analysis

The data and statistical analysis in this study comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Data in bar graphs are expressed as means ± SEM. Statistical comparisons were made between groups using Student's t‐test (for comparison between two groups) or one‐way ANOVA (for comparison among ≥3 groups) followed by Tukey's post hoc test, with values of P < 0.05 considered significant.

TRPM8 channels are essential for the anti‐inflammatory effects of eucalyptol in the CFA model

After establishing the efficacious dose for eucalyptol in the CFA model, we compared eucalyptol's anti‐inflammatory and analgesic effects in wild‐type and Trpm8^−/− mice. Trpm8^−/− mice developed a similar degree of hind paw swelling upon CFA injection as wild‐type mice (Figure 2A, B). However, eucalyptol (300 mg·kg⁻¹, i.p.) was completely ineffective for suppressing paw swelling in TRPM8 channel‐deficient mice (Figure 2A, B, P > 0.05). In contrast to eucalyptol, ibuprofen (30 mg·kg⁻¹, i.p.) was equally effective in suppressing paw swelling in wild‐type and Trpm8^−/− mice, suggesting that other anti‐inflammatory pathways remain intact in mice lacking TRPM8 channels (Figure 2B, C). We also compared the weights of skin punch biopsies obtained from CFA‐injected hind paws of wild‐type and Trpm8^−/− mice, excised 50 h after CFA injection and 2 h after the last treatment injection. Both eucalyptol and ibuprofen suppressed the increase in biopsy weights in wild‐type mice, whereas eucalyptol was ineffective (P > 0.05) in skin biopsies from Trpm8^−/− mice (Figure 2D).

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Figure 2

Comparison of the effects of eucalyptol on CFA‐induced paw oedema in wild‐type and Trpm8^−/− mice, with animals treated as in Figure 1A. (A) Representative photographs of CFA‐induced paw oedema in wild‐type (top row) or Trpm8^−/− mice (bottom row) injected i.p. with vehicle (Veh, corn oil) or 300 mg·kg⁻¹ eucalyptol (Euc), taken 50 h after intraplantar CFA injection. (B) Time course of CFA‐induced paw oedema in wild‐type mice, treated with vehicle, eucalyptol (300 mg·kg⁻¹) or the reference drug ibuprofen (Ibu, 30 mg·kg⁻¹). Black arrows indicate the time points of treatment. (C) Time course of CFA‐induced paw oedema in Trpm8^−/− mice. (D) Comparisons of weights of paw biopsies from wild‐type and Trpm8^−/− mice of control group (no CFA), CFA groups treated with vehicle, eucalyptol or ibuprofen respectively. Tissues were collected 50 h after CFA injection from mice tested in Figure 2B, C. n = 6–8 mice per group. ^# P < 0.05, significantly different from control group, *P < 0.05, significantly different from CFA + Veh group, NS, no significance (P > 0.05), one‐way ANOVA.

Mice develop robust mechanical allodynia in the CFA model. Using von Frey hair analysis, we tested whether eucalyptol would diminish mechanical allodynia in this model, and whether this effect is altered in Trpm8^−/− mice. Eucalyptol treatment (regimen as above, Figure 1A) strongly reduced paw withdrawal thresholds to a similar degree as ibuprofen, measured 24 h after CFA injection (Figure 3). In Trpm8^−/− mice, eucalyptol was ineffective (P > 0.05), while ibuprofen retained its analgesic effect (Figure 3).

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Figure 3

Effects of eucalyptol on CFA‐induced mechanical allodynia in wild‐type and Trpm8^−/− mice, measured by von Frey hair analysis (administration as in Figure 1A). Testing was performed 26 h after CFA injection, 2 h after the mice received the third treatment of vehicle (Veh, corn oil), eucalyptol (Euc, 300 mg·kg⁻¹) or ibuprofen (Ibu, 30 mg·kg⁻¹). ^# P < 0.05, significantly different from control group; *P < 0.05, significantly different from CFA + Veh group, NS, no significance (P > 0.05), one‐way ANOVA. n = 6–12 mice per group.

CFA also triggers the production of key pro‐inflammatory cytokines. We examined the effects of eucalyptol on cytokine levels in hind paw skin biopsies from the CFA‐treated mice. Using ELISA, we detected markedly increased levels of TNF‐α, IL‐1β and IL‐6 in hind paw skin homogenates of CFA‐treated wild‐type mice. Eucalyptol inhibited the production of these cytokines in wild‐type mice (Figure 4). In Trpm8^−/− mice, CFA injection caused increases in TNF‐α, IL‐1β and IL‐6 to similar levels as observed in wild‐type mice. However, eucalyptol treatment (300 mg·kg⁻¹, i.p.) did not reduce the levels of any of these inflammatory cytokines in TRPM8 channel‐deficient mice (Figure 4, P > 0.05). Ibuprofen significantly reduced the levels of IL‐1β, IL‐6 and TNF‐α in skin homogenates from both wild‐type and Trpm8^−/− mice treated with CFA (Figure 4). Taken together, these results show that TRPM8 channels are crucial for both the anti‐inflammatory and analgesic effects of eucalyptol in the mouse CFA model.

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Figure 4

Influence of eucalyptol treatment on CFA‐induced inflammatory cytokines in hind paw skin of wild‐type and Trpm8^−/− mice. Mice were treated (i.p.) with vehicle (Veh, corn oil), eucalyptol (Euc, 300 mg·kg⁻¹) or ibuprofen (Ibu, 30 mg·kg⁻¹) as in Figure 1A. Hind paw biopsies were collected 50 h after CFA injection, and levels of IL‐1β, IL‐6 and TNF‐α were determined by ELISA in tissue homogenates. ^# P < 0.05, significantly different from control group; *P < 0.05, significantly different from CFA + Veh group, NS, no significance (P > 0.05), one‐way ANOVA. n = 6–12 mice per group.

Mitigation of LPS‐induced pulmonary inflammation by eucalyptol is dependent on TRPM8 channels

Eucalyptol is frequently used to counteract respiratory inflammation and discomfort due to the common cold and other respiratory infections, as well as in asthma and COPD (Bastos et al., 2009). To study the actions of eucalyptol in the respiratory system, we used the mouse LPS model of ALI, a model in which LPS (10 μg in 40 μL of sterile saline) is instilled intranasally to mimic respiratory infections or environmental exposures to endotoxins. Respiratory inflammation was monitored by measurements of leukocyte concentrations in BAL, levels of cytokines in bronchoalveolar lavage fluid (BALF) and whole lung and MPO activity as a measure of neutrophil function. As described earlier (Zhao et al., 2014), eucalyptol (200 mg·kg⁻¹) was injected 12 and 1 h prior to LPS instillation and also 8 and 23 h thereafter, with responses analysed 24 h after (Figure 5A). In wild‐type mice, eucalyptol treatment strongly reduced neutrophil counts in BALF of LPS‐exposed animals (Figure 5B). MPO enzyme activity was also diminished by eucalyptol treatment (Supporting Information Figure S1). Eucalyptol decreased protein concentrations and gene transcription of key pro‐inflammatory cytokines in BALF, including TNF‐α, IFN‐γ, KC, IL‐6 and G‐CSF, measured by ELISA in BALF and quantitative real time PCR of cDNA prepared from whole lungs after BAL and perfusion (Figures 5C and ​and6A).6A). The anti‐inflammatory effects of eucalyptol in this model were absent in Trpm8^−/− mice (Figures 5B, C and ​and66B).

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Figure 5

TRPM8‐dependent suppression of LPS‐induced pulmonary inflammation by eucalyptol. (A) LPS administration and eucalyptol treatment timeline. (B) Comparison of cell differential counts (total cells, neutrophils, lymphocytes and macrophages) in BALF of wild‐type or Trpm8^−/− mice exposed to vehicle (saline) or LPS and injected with treatment vehicle (corn oil) or eucalyptol (200 mg·kg⁻¹). (C) Cytokine levels in BALF of mice in Figure 5B, tested by bead array multiplex analysis. Protein concentrations are shown for TNF‐α, IFN‐g, KC, IL‐6 and G‐CCSF. (n = 4–8 mice per group). *P < 0.05, WT + LPS significantly different from WT + LPS + Eucalyptol.

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Figure 6

Cytokine transcripts analysed by quantitative real time qPCR in cDNA from whole lungs of LPS‐ and vehicle‐exposed mice treated and untreated with eucalyptol. (A) Impaired induction of cytokines in Trpm8^−/− mice treated with eucalyptol before and after intranasal LPS administration. (B) Unaltered expression of cytokines in Trpm8^−/− mice treated with eucalyptol before and after LPS exposure. (n = 4–6 mice per group). *P < 0.05, significantly different as indicated.

The treatment protocol for eucalyptol we used specified two pretreatment injections, administered 12 and 1 h prior to LPS instillation (Zhao et al., 2014). We then tested eucalyptol injections given after LPS instillation for their effects, injecting eucalyptol 1 and 6 h after LPS (200 mg·kg⁻¹ each). This treatment protocol failed to suppress inflammatory responses (Supporting Information Figure S2, P > 0.05).

It remains unclear whether TRPM8 is transcribed and expressed locally in the lung where it may contribute to eucalyptol's anti‐inflammatory effects. Using two quantitative PCR hydrolysis probes covering key sequences of the cDNA of TRPM8 wild‐type and reported variants (Mm01299593_m1 and Mm00454566_m1), we found TRPM8 transcripts to be undetectable in cDNA prepared from mouse whole lungs (Figure 7, Supporting Information Figure S3). In contrast, cDNA of TRPV4, a TRP ion channel expressed in lung epithelial and vascular cells, was readily detectable (Figure 7). cDNA obtained from peripheral sensory ganglia (trigeminal and dorsal root ganglia) contained very high levels of TRPM8 transcripts, confirming the utility of the probes used and the highly selective expression of TRPM8 channels in sensory neurons (Figure 7, Supporting Information Figure S3). Levels of TRPA1 and TRPV1 transcripts in sensory ganglia did not show significant differences between wild‐type and Trpm8^−/− mice (Supporting Information Figure S4, P > 0.05).

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Figure 7

Comparison of TRPV4 and TRPM8 expression in lung and sensory ganglia. Averaged relative quantities (RQ) of TRPV4 and TRPM8 transcript levels were measured by real‐time Taqman PCR of cDNA from whole lung, DRG and TG of wild‐type mice. We used one TRPV4 Taqman probe (mTRPV4: Mm00499025_m1, 25) and two mTRPM8 probes covering different cDNA segments (mTrpm8: Mm01299593_m1, 93 and mTrpm8: Mm00454566_m1, 66). Actin was used as reference gene (n = 4 mice per group).

Supported by grants from the National Heart Lung and Blood Institute (NHLBI, R01HL105635 and R01HL105635‐S1 to S.E.J. and J.B.M.), and the National Institute on Drug Abuse (NIDA, Yale Tobacco Center of Regulatory Science, P50DA036151 Project #1 to S.E.J.) of the National Institutes of Health (NIH) of the United States. Contents are solely the responsibility of the authors and do not necessarily represent the official views of the federal government of the United States.