Toll-like Receptor 7/8 (TLR7/8)

Resiquimod (R-848) causes circulating T cells (including TH2 effectors) that are specific to haptens and allergens to produce IFN-γ and even lose their capacity to produce IL-4 [2]. In BrdU incorporation experiments, resiquimod (R848) increases the number of BrdU-positive cells and, in a dose-dependent manner, boosts PBL proliferation. The reporter of NF-κB activity, luciferase, significantly increased (3.5-fold) in cells treated with R848 [3].

Rats and mice can be utilized as models for immune-mediated cardiac tissue injury and cytokine production with Resiquimod in animal modeling. In SPF chickens, the intramuscular injection of Resiquimod (R-848) at a dose of 50 μg/bird dramatically increased the production of IFN-α, IFN-β, IFN-γ, IL-1β, IL-4, iNOS, and MHC-II genes [1].

BrdU incorporation assay[3]
For BrdU incorporation assay, R848 (1 µg/ml), CQ (10 µM), CQ plus R848 or PBS were added to PBL in a 6-well tissue culture plate as described earlier (2 × 106 cells/well). The cells were incubated at 22 °C for 48 h, and cellular proliferation was detected with BrdU Cell Proliferation Assay Kit according to the manufacturer's instructions. The assay was performed three times.

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Luciferase reporter assay[3]
For luciferase assay, FG-9307 cells were transfected with the firefly NF-κB–specific luciferase reporter vector pNFκB-Met-Luc2 (Clontech, Mountain View, CA, USA) as described previously (Chi et al., 2013). pNFκB-MetLuc2 is designed to monitor the activation of the NF-κB signal transduction pathway directly from the cell culture medium. The vector contains an NF-κB enhancer element located upstream of the minimal TA promoter (PTA). Located downstream of PTA is a secreted-luciferase gene. Binding of transcription factors to the NF-κB enhancer element allows MetLuc to be expressed and secreted into the surrounding medium. pNFκB-MetLuc2 has been demonstrated to work in fish system (Lauksund et al., 2009). Transfection efficiency was monitored by co-transfection with the pSEAP2 control vector, which constitutively expresses the human secreted enhanced alkaline phosphatase (SEAP). Then the cells were treated with R848 (1 µg/ml), CQ (10 µM), CQ plus R848 or PBS and incubated at 22 °C for 24 h. The culture medium of the transfectants was then analyzed for luciferase activity and SEAP activity using Luciferase Assay Kit and the Great EscAPe™ SEAP Chemiluminescence Detection Kit, respectively. The assay was performed three times.

MTT assay to determine cellular proliferation[3]
To prepare PBL, blood was collected from the caudal veins of flounder and diluted 1:5 with L-15 medium. The diluted blood was placed on top of 61% Percoll and centrifuged at 400 × g for 30 min. The layer of PBL was recovered and washed three times with PBS. The cells were distributed in 96-well tissue culture plates (5 × 105 cells/well) in L-15 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were treated with different concentrations (0, 0.175, 0.25, 0.5, 1, 2, 4, 8, and 16 µg/ml) of R848 for 48 h. For inhibition of lysosomal acidification, cells were incubated with 10 µM CQ for 1 h before R848 treatment. After treatment, 20 µl of 5 mg/ml MTT {3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide} was added to the plate. The plate was incubated at 22 °C for 4 h, and 200 µl dimethyl sulfoxide was added to the plate to dissolve the reduced formazan. The plate was then read at 490 nm with a microplate reader. To determine the effect of Myd88 inhibition on R848-induced cell proliferation, the Myd88 inhibitor Pepinh-MYD (RQIKIWFQNRRMKWKK-RDVLPGTCVNS-NH2) and the control peptide Pepinh-Control (RQIKIWFQNRRMKWKK-SLHGRGDPMEAFII-NH2) were added to PBL at the concentration of 50 µM, and the plate was incubated at 22 °C for 6 h. After incubation, the cells were treated with R848 and subjected to MTT assay as above. To determine the effect of NF-κB inactivation on R848-induced cell proliferation, BAY-11-7082, an irreversible inhibitor of IκB-α phosphorylation, was added to the cells at the concentration of 1 µM, and the plate was incubated at 22 °C for 1 h. After incubation, the cells were treated with R848 and subjected to MTT assay as earlier. All experiments were performed three times.

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Apoptoses assay[3]
PBL prepared earlier were distributed in 12-well tissue culture plates (1 × 106 cells/well) in L-15 medium containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. R848 (1 µg/ml), CQ (10 µM), CQ plus R848 or PBS were added to the cells, and the cells were incubated at 22 °C for 12 h or 48 h. After incubation, the cells were washed twice with cold PBS. The washed cells were treated with FITC-conjugated annexin V and propidium iodide (PI) by using annexin V-FITC and PI Cell Apoptosis Detection Kit according to the manufacturer's instructions. The cells were then subjected to flow cytometry using a FACSort Flow Cytometer equipped with FlowJo software (Tree Star Inc, Ashland OR) for data analysis. To determine the effect of Myd88 inhibition on apoptosis, Pepinh-MYD and Pepinh-Control were added to PBL at the concentration of 50 µM. After incubation at 22 °C for 6 h, the cells were treated with R848 and subjected to annexin V/PI assay as earlier. To determine the effect of NF-κB inactivation on R848-induced anti-apoptosis, BAY-11-7082 was added to the cells at the concentration of 1 µM. After incubated at 22 °C for 1 h, the cells were treated with R848 and subjected to annexin V/PI assay as earlier. All experiments were performed three times.

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Quantitative real-time reverse transcription-PCR (qRT-PCR)[3]
Total RNA was extracted from PBL with the RNAprep Tissue Kit. One microgram of total RNA was used for cDNA synthesis with M-MLV reverse transcriptase. qRT-PCR was carried out in an Eppendorf Mastercycler using the SYBR ExScript qRT-PCR Kit as described previously (Zheng and Sun, 2011). Melting curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The expression level of the target gene was analyzed using comparative threshold cycle method with beta-actin as the internal reference (Zhang et al., 2013). The experiment was performed three times.

R848 and chloroquine (CQ) were resuspended in PBS to 200 µg/ml and 100 µM respectively. Japanese flounder (average 11.6 g) were divided randomly into four groups and administered by intramuscular (i.m.) injection with 50 µl R848, CQ, R848 plus CQ, or PBS. At 24 h post-administration, the fish were challenged by intraperitoneal (i.p.) injection with 50 µl megalocytivirus that had been suspended in PBS to 1 × 107 copies/ml. At 3 d, 5 d, and 7 d post-challenge, kidney and spleen were collected from the fish (4 fish/time point), and the viral amounts in the tissues were determined by absolute quantitative real time PCR as reported previously (Zhang et al., 2012). The experiment was performed three times[3].

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Although the solitary adjuvant potential of R-848 is well established in mammals, such reports are not available in avian species hitherto. Hence, the adjuvant potential of R-848 was tested in SPF chicken in this study. Two week old chicks were divided into four groups (10 birds/group) viz., control (A), inactivated Newcastle disease virus (NDV) vaccine prepared from velogenic strain (B), commercial oil adjuvanted inactivated NDV vaccine prepared from lentogenic strain (C) and inactivated NDV vaccine prepared from velogenic strain with R-848 (D). Booster was given two weeks post primary vaccination. Humoral immune response was assessed by haemagglutination inhibition (HI) test and ELISA while the cellular immune response was quantified by lymphocyte transformation test (LTT) and flow cytometry post-vaccination. Entire experiment was repeated twice to check the reproducibility. Highest HI titre was observed in group D at post booster weeks 1 and 2 that corresponds to mean log2 HI titre of 6.4 ± 0.16 and 6.8 ± 0.13, respectively. The response was significantly higher than that of group B or C (P<0.01). LTT stimulation index (P ≤ 0.01) as well as CD4(+) and CD8(+) cells in flow cytometry (P<0.05) were significantly high and maximum in group D. Group D conferred complete protection against virulent NDV challenge, while it was only 80% in group B and C. To understand the effects of R-848, the kinetics of immune response genes in spleen were analyzed using quantitative real-time PCR after R-848 administration (50 μg/bird, i.m. route). Resiquimod significantly up-regulated the expression of IFN-α, IFN-β, IFN-γ, IL-1β, IL-4, iNOS and MHC-II genes (P<0.01). In conclusion, the study demonstrated the adjuvant potential of R-848 when co-administered with inactivated NDV vaccine in SPF chicken which is likely due to the up-regulation of immune response genes[1].

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Dissolved in saline; 50 nmol; i.p. injection

Wild-type mice, TLR7-deficient mice, and MyD88-deficient mice

[1]. Sachan S, et al. Adjuvant potential of resiquimod with inactivated Newcastle disease vaccine and its mechanism of action in chicken. Vaccine. 2015 Aug 26;33(36):4526-32.
[2]. Brugnolo F, et al. The novel synthetic immune response modifier R-848 (Resiquimod) shifts human allergen-specific CD4+ TH2 lymphocytes into IFN-gamma-producing cells. J Allergy Clin Immunol. 2003 Feb;111(2):380-8.
[3]. Zhou ZX, et al. Immune effects of R848: evidences that suggest an essential role of TLR7/8-induced, Myd88- and NF-κB-dependent signaling in the antiviral immunity of Japanese flounder (Paralichthys olivaceus). Dev Comp Immunol. 2015 Mar;49(1):113-20

C17H22N4O2

314.38

314.17428

C, 64.95; H, 7.05; N, 17.82; O, 10.18

144875-48-9

Resiquimod-d5;2252319-44-9;Resiquimod;144875-48-9

White to light yellow solid

2.15

86.19

CC(O)(C)CN1C(COCC)=NC2=C1C3=CC=CC=C3N=C2N

BXNMTOQRYBFHNZ-UHFFFAOYSA-N

InChI=1S/C17H22N4O2/c1-4-23-9-13-20-14-15(21(13)10-17(2,3)22)11-7-5-6-8-12(11)19-16(14)18/h5-8,22H,4,9-10H2,1-3H3,(H2,18,19)

1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.95 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (6.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)