Figure 2: Histamine receptors are not involved in the inhibition of stimulated pDC.

(a) Isolated pDC were pretreated for 1 h with various histamine receptors inhibitors, pyridylethylamine (H1R), cimedine (H2R), thioperamide (H3R) and JNJ7777120 and A943931 (H4R) at 50 μM, and then treated with CB before overnight stimulation with Influenza A virus. IFN-α was quantified in the supernatants by Elisa. (b) Mouse MNC (multinucleated cells) were obtained from the spleen using a homogenizer and purified using a 35% isotonic Percoll density gradient (Amersham Biosciences). Spleen MNC were depleted of RBC using red cell lysis buffer (8.3 mg ml⁻¹ NH₄Cl, 1 mg ml⁻¹ KHCO₃, and 3.72 μg ml⁻¹ EDTA put in Mat and Med). Wild type (WT) (n=14) or H4RKO mice (n=10) spleen MNC were pre-incubated with CB (10 μM) and then cultured overnight with Influenza A virus. IFN-α was quantified in the supernatants by ELISA. N=5. (c) and (d) pDC were treated for 24 h with a siRNA H4R (siH4R) or a siRNA Control (siCTR) at 160 nM. Cells were pre-incubated with histamine (c) or CB (d) (10 μM) and then stimulated with HIV. mRNA levels of TRAIL, IFN-α and IFN-β from those cells were then quantified by RT-qPCR. (e) Chemical structures of histamine, dopamine and serotonin. (f) Levels of membrane TRAIL were assessed by flow cytometry on purified pDC after pre-incubation with histamine, dopamine and serotonin then overnight HIV stimulation. (g) mRNA levels of TRAIL and IFN-(α, β) from purified pDC pre-incubated with histamine, CB, dopamine and serotonin and stimulated overnight with HIV, were measured by RT-qPCR and normalized to RPL13A. When not specify, data shown as the means of three independent experiments ±s.e.m. P values (p) were determined using a two-tailed Student’s t test. ***P<0.001, **P<0.01, *P<0.05.