Cells were acquired and analyzed on FACS Canto II by using FACSDIVA software. Real time polymerase chain reaction Comparative mRNA levels of cytokines were assessed in the joints of mice. RNA was extracted by using the thing RNAeasy mini kit in accordance with the instructions of the manufacturer. Generation of cDNA was carried by using the ABI High capacity reverse Inhibitors,Modulators,Libraries transcription system with random hexamer primers in accordance with the protocol of the manufacturer. Amplification of cDNA was performed on an ABI AB7900HT real time polymerase chain reaction machine in a 384 well plate by using TaqMan Gene Expression Assays sets from the ABI inventoried library for all genes and mouse hypoxanthine phosphoribosyl transferase control.

The relative concentration of each gene of interest was calculated by using the method and expressed as relative units by using a WT arthritic mouse as a calibrator Inhibitors,Modulators,Libraries after normalizing against HPRT. Statistical methods Mean, standard deviation, standard error of the mean, and statistical significance were calculated by using GraphPad version 3. For statistical analysis, Inhibitors,Modulators,Libraries a one tailed t test of paired data was used with a 95% confidence interval. SEM was used for pooled experimental data, whereas SD was used in graphs showing Inhibitors,Modulators,Libraries representative experiments. A one tailed Mann Whitney test was used with a 95% confi dence interval for the CIA data. Results Mianserin inhibits cytokine production induced by TLR3, 7, and 9 from murine bone marrow derived macrophages In a previous study, we demonstrated that mianserin could inhibit the activation of TLRs 3, 7, 8, and 9 but not the cell surface TLRs 1 2, 4, and 5 in primary human cells.

Before testing the effects of mianserin in CIA, it was essential to Inhibitors,Modulators,Libraries confirm that mianserin was able to inhibit endosomal TLR activation in murine primary macrophages. done Mianserin inhibited the produc tion of TNF upon activation of TLR7 and 9, but not TLR4, in BMMs. RANTES was used as a readout for TLR3 activation because polyinosinic,polycy tidylic acid is a weak inducer of TNF in BMMs. Upon TLR3 stimulation, mianserin also dose depen dently inhibited the production of RANTES. TLR8 activation was not measured, as the mechanism of activation of this receptor remains unclear at present. Human TLR7 and 8 can be activated by singled stranded RNA or resiquimod, but in murine cells only TLR7 responds to these ligands. Cell viability was not affected by mianserin as assessed by the MTT assay . Mianserin suppresses disease progression in CIA At the same concentration as was previously shown for inhibition of human TLR signaling, mianserin inhibited the spontaneous production of TNF and IL 6 from human synovial membrane cultures.