Although it is commonly accepted that FOXP3 and Tbet expression identifies Treg and Th1 cells, respectively, expression of a single transcription factor is not adequate to characterize the Th phenotype of CD4+ T-cells (Oestreich and Weinmann, 2012). Difco Laboratories, Detroit, MI, USA). Immediately thereafter, and again 48 h later on, the mice received an i.p. injection of 500 ng toxin (Sigma) in 100 L of PBS. The animals were examined daily for excess weight loss and disability, and were clinically graded by investigators, unaware of organizations treatments, as follows: 0 shows no indications; 0.5, partial loss of tail tonicity; 1, paralysed tail; 2, ataxia and difficulty in righting; 3, paralysis of the hind limbs and/or paresis of the forelimbs; 4, tetraparalysis; 5, moribund or death. Pharmacological treatments The H4 receptor antagonist JNJ7777120 (Johnson &Johnson, San Diego, CA, USA) was dissolved in 1% ethanol in physiological saline to give a final dose of 10 mgkg?1 JNJ7777120 or vehicle in 100 L per TCS 359 mouse, and administration daily i.p. injections for the entire duration of the experiment (up to 28 days after immunization). Mice were randomly assigned to two different experimental organizations: mice that received daily injections of either JNJ7777120 or vehicle beginning at D10 p.i. and were killed at D28 p.i., and mice that received daily injections of either JNJ7777120 or vehicle beginning at D10 p.i. and were killed at D18 p.i. Neuropathological evaluations At the time of killing, the mice were anaesthetized with pentobarbital (65 mgkg?1, i.p.). The spinal cord was removed from the column and fixed in 4% (v/v) paraformaldehyde in PBS and consequently paraffin-embedded. Transverse sections (5m solid) were cut and placed on glass slides. Serial sections were stained with haematoxylin and eosin (H&E) or Luxol fast blue (LFB)-cresyl violet. Immunohistochemistry Sections were subjected to antigen retrieval by microwave incubation in 10 mM NaCcitrate buffer (pH 6.0) and subsequently immunostained. Briefly, sections were incubated over night at 4C with the primary antibody in the optimized operating dilution prepared in 0.1 M PBS (pH 7.4) with Triton X-100 (0.3%) and BSA (5 mgmL?1). The following primary antibodies were used: anti-neuronal specific nuclear protein (NeuN; 1:1000 dilution, Chemicon International, Temecula, CA, USA) to visualize neurons, anti-glial fibrillary acidic protein (GFAP; 1:500 dilution, DakoCytomation, Glostrup, Denmark) to detect TCS 359 astrocytes, Iba1 (1:100 dilution, Wako Chemicals, Neuss, Germany) to detect microglia; anti-IFN (1:100 dilution, BioLegend, Aachen, Germany). On the second day, the sections were incubated for 1 h with the secondary antibody prepared in 0.1 M PBS plus BSA (1 mgmL?1) and immunostaining was visualized with antibodies conjugated with Cy3 (Jackson ImmunoResearch, Suffolk, UK) and Alexafluor 488 (Molecular Probes, Eugene, OR, USA). Sections were coverslipped in Vectastain fluoromount with DAPI (Vector Laboratories, Burlingame, CA, USA). An Olympus BX40 microscope coupled to analySISB Imaging Software (Olympus, Milan, Italy) was used to acquire representative images. Cells Cells were isolated from lymph nodes (LNs), spleen and spinal cord, and analysed for proliferative response and phenotype as previously explained (Gourdain 0.05. Open in a separate window Number 2 Effect of the H4 receptor antagonist JNJ7777120 on post-EAE immune response. (A) proliferation of T lymphocytes isolated from LNs at D28 p.i., cells were incubated for 72 h with two doses of MOG35C55. Proliferation CTNND1 was evaluated by thymidine incorporation measured during the last 12 h of tradition. Data are indicated as CPM (mean CPM stimulated cells C mean CPM background), = 3 per group. (B) Circulation cytometric analysis of cell distribution in LN at D28 p.i., cells freshly isolated from LN of three mice per group were labelled with monoclonal antibodies (CD3+, T lymphocytes; CD11b+, macrophages and NKs; CD11c+, dendritic cells; CD4+, T helper lymphocytes). All labelled cells were tested for surface manifestation of H4R. (C) anti-MOG35C55 antibodies titrated by solid phase elisa in individual sera of EAE-induced mice collected at D28 p.i., = 3 per group. CTR, sera of non-immunized mice. Treatment having a H4 receptor antagonist raises swelling and demyelination in the spinal cord of EAE mice Infiltration of autoreactive immune cells into the CNS results in swelling of CNS parenchyma and demyelination of motoneurons with consequent paralysis. Following EAE induction, both JNJ7777120- and vehicle-treated mice experienced distinct areas of immune cell infiltration in the spinal cord as exposed by H&E staining (Number 3A, left panels). However, TCS 359 JNJ7777120-treated mice experienced visually more infiltrates than vehicle-treated mice. The degree of myelin loss was visually assessed by LFB staining, which revealed larger plaques of demyelination at the site.