Wild-type BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA). H4R deficient mice were generated by Lexicon Genetics (Woodlands Park, TX) as previously described . Mice were housed in community cages on a 12-h light cycle and fed mouse chow and water ad libitum. All procedures were performed according to the internationally accepted guidelines for the care and use of laboratory animals in research and were approved by the local Institutional Animal Care and Use Committee. The JNJ 7777120 and JNJ 28307474 (H4R antagonists) were synthesized as previously described [4, 14]. In all cases H4R antagonists were delivered p.o. in 20 % hydroxy-propyl-β-cyclodextran. Lipopolysaccharide (E.coli 0111:B4), methylpalmitate (MP; stock 200 mg/ml in 5 % glucose solution containing 0.2 % Tween-20) and galactosamine (GaIN) were purchased from Sigma-Aldrich, LLC, (St. Louis, MO). Clodronate liposomes (CL) were purchased from ClodronateLiposomes.org (Amsterdam, Netherlands) in a stock solution of around 5 mg/ml in PBS. The stock was further diluted in PBS to 1 mg/ml just before administration and tail vein injection at ~200 μl/mouse. The TNF ELISA was obtained from R&D Systems, Inc. (Minneapolis, MN). Low endotoxin RPMI media was from Sigma-Aldrich Co. LLC. (St. Louis, MO).
In vivo LPS model
Female wild-type or H4R-deficient mice were administered vehicle, JNJ 7777120 or JNJ 28307474 p.o. Thirty minutes later, the mice received an i.p. injection of 20 μg of LPS in PBS. The dose of LPS was selected based on pilot experiments and is consistent with previous reports . Two hours later blood was collected and serum cytokines were measured via ELISA. To assess the activation of NFκB and AP-1, nuclear and cytoplasmic fractions of whole liver tissue sections were prepared using Nuclear Extraction Kit (Active Motif, Carlsbad, CA) with the manufacturer’s protocol. Five μg of proteins from each fraction of individual samples were analyzed for the phosphorylation and/or nuclear translocation of NFκB and AP-1 transcription factors utilizing TransAM NFκB Family Kit (Active Motif, Carlsbad, CA) and TransAM AP-1 Family Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol.
Kupffer cell depletion
For Kupffer cell depletion, the mice were treated by tail vein injection with either vehicle, 2 g/kg MP or 10 mg/kg CL (the vehicle for CL was empty liposomes in PBS). Forty-four hours after treatment with MP or CL 20 μg of LPS in PBS was administered by i.p. Two hours post LPS injection, animals were euthanized and blood collected via cardiac puncture. Serum TNF levels were quantitated by ELISA. Livers were collected in RNAlater reagent (Ambion, Inc., Austin, TX) and total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN, Inc., Valencia, CA). In addition, blood RNA was extracted using QIAamp RNA Blood Mini Kit (QIAGEN, Inc. Valencia, CA). The RNA was reverse-transcribed to cDNA using the GeneAmp Gold RNA PCR Core Kit (Applied Biosystems, Carlsbad, CA). The TNF message levels were detected using commercially available TaqMan primer/probe sets (Applied Biosystems, Carlsbad, CA) with 7500 Real Time PCR System (Applied Biosystems, Carlsbad, CA). The data is presented as the fold-induction compared to naïve animals.
To assess macrophage depletion, liver samples were fixed in 10 % formalin for 24 h, processed, paraffin embedded, sectioned at 5 μm thickness and mounted on the slides. The slides were deparaffinized and hydrated in PBS followed by blocking the endogenous peroxide with 3 % hydrogen peroxide. Incubation with a solution of 3 % trypsin was used to enhance the binding of the primary antibody. Avidin and biotin blocks (Vector laboratories, Burlingame, CA) were used to avoid nonspecific labeling with primary antibody. To avoid nonspecific reaction with the secondary antibody, slides were pretreated with 10 % normal goat serum before incubation with primary antibody. Rat anti-mouse F4/80 at a concentration of 1:50 (AbD Serotec, Raleigh, NC) as primary antibody and goat anti-rat biotinlated IgG as secondary antibody (Cat#AP183b, Chemicon International, Inc., Temecula, CA) at 1:2,000 concentration were used. Normal rat IgG at 1:50 concentration (Cat# CBL606, Chemicon International, Inc., Temecula, CA) was used as a negative control. The immunoreactivity was visualized by ABC reagent (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAKO, Carpinteria, CA) followed by counterstaining with Mayer’s hematoxylin. A Nikon E800 light microscope equipped with a Q Image camera was used to capture images. Images were measured by Image Pro Plus software 7.0 (MediaCybernetics, Inc., Bethesda, MD). Positively stained, centro-lobularly located macrophages (Kupffer cells) were automatically counted in the randomly chosen five hot fields under the ×100 magnification objective lens using the cell-count feature of Image Pro Plus 7.0 software. The mean value of the cell counts in five fields was calculated, defined as cell count per field.
In vitro and ex vivo LPS stimulation of whole blood
For in vitro stimulation, blood from wild-type and H4R-deficient mice was diluted 1:1.8 with low endotoxin RPMI media containing vehicle (0.4 % DMSO) or 50 μM JNJ 7777120 and incubated for 1 h at 37 °C. After incubation, 10 ng/ml LPS was added and the samples were incubated for 4 h at 37 °C. Cells were centrifuged, supernatants were harvested and TNF levels were measured by ELISA. For the ex vivo stimulation, wild-type mice were dosed p.o. with JNJ 7777120 (1, 5 and 20 mg/kg). Mice were sacrificed 15 min later (the T
max for the plasma levels of JNJ 7777120) and blood collected. Blood was diluted 1:2 with low endotoxin RPMI media containing 10 ng/ml LPS and the samples were incubated for 4 h at 37 °C. Cells were centrifuged, supernatants were harvested and TNF levels were measured by ELISA.
Immunofluorescence for dual F4/80 and TNF staining
Mice received an i.p. injection of 20 μg of LPS (Sigma-Aldrich Co. LLC, St. Louis, MO) or vehicle (PBS) and 2 h later livers, lungs and spleens were harvested and frozen in tissues cassettes with O.C.T. freezing compound. Immunofluorescence detection was carried out by Seventh Wave Laboratories LLC (Chesterfield, MO). Rat anti-mouse F4/80 (monoclonal IgG2b; AbD Serotec) and Dylight 488 mouse anti-rat IgG (green; Thermo Fisher Scientific, Inc. Rockford, IL) was used for F4/80 detection. Rabbit anti-human TNF (polyclonal IgG fraction; Sigma-Aldrich Co. LLC, St. Louis, MO) and Dylight 594 mouse anti-rabbit IgG (red; Thermo Fisher Scientific, Inc. Rockford, IL) was used for TNF detection. The 4′, 6-diamidino-2-phenylindole was used to provide a nuclear counterstain in all the sections. The frozen blocks were sectioned (approximately 6 micrometer thickness) to produce one slide per tissue per mouse, and the sections were immunoreacted to detect TNF and F4/80 Immunofluorescence. A cocktail of rat IgG2b and normal rabbit IgG fraction adjusted to the equivalent protein concentrations as the primary antibodies was substituted for the primary antibody cocktail for the negative control sections.
H4R expression in Kupffer cells
Freshly isolated female mouse and human Kupffer cells were purchased from Celsis In Vitro Technologies (Baltimore, MD). Total RNA was extracted using RNeasy Plus Mini Kit from QIAGEN, Inc. (Valencia, CA). The H4R message levels were detected using commercially available TaqMan primer/probe sets (Applied Biosystems, Carlsbad, CA) with 7500 Real Time PCR System (Applied Biosystems, Carlsbad, CA).
Liver injury model
Female mice were treated with 10 mg/kg LPS + 1 g/kg GaIN in PBS through tail vein injection. One hour before LPS + GaIN treatment, the mice were pretreated with vehicle or 20 mg/kg JNJ 28307474 p.o. Six hours after LPS + GaIN injection, serum was harvested for alanine transaminase (ALT) measurement using ACE Alera system (Alfa Wassermann, West Caldwell, NJ).
Mouse asthma model
A mouse asthma model was run as previously described  with the following modifications. Endotoxin was removed from the OVA by phase separation using Triton X-114 as previously described . Thirty minutes prior to OVA aerosol challenge on day 21 through day 24, animals under light anesthesia were given a 50 μl intranasal dose of PBS or PBS + 1 ng LPS. The JNJ 7777120 was given at 20 mg/kg p.o. 30 min prior to LPS.