These data suggest that migratory DC differentiation in the peripheral tissues might be impaired if the activation

signals reach the monocyte precursors before their commitment to the DC differentiation pathway. Our data support this hypothesis by showing that activation of early MoDC precursors leads to inflammatory cytokine and chemokine production but the cells, at early stage of DC differentiation, have a limited ability to modulate their chemokine receptor expression required for lymph node homing. The cytokine producing ability of the developing inflammatory MoDCs can be terminated by the functional exhaustion before the cells differentiate to mature Osimertinib purchase DCs capable of reprogramming their chemokine receptor profile. Early activation of developing MoDCs may thus set the threshold of DC migration to LNs, thereby limiting the continuous transfer of inflammatory signals to T lymphocytes.

Monocytes were isolated from buffy coats by Ficoll gradient centrifugation (Amersham Biosciences) and magnetic cell separation using anti-CD14–conjugated microbeads (Miltenyi Cytoskeletal Signaling inhibitor Biotech). The purified cells were cultured at a density of 2×106 cells/mL in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FCS (Invitrogen), 75 ng/mL GM-CSF (Gentaur) and 100 ng/mL IL-4 (Peprotech EC). For DC activation we used the TLR ligands LPS, CL075, HKSA, Zymosan,

Pam3Cys or poly(I:C), all from Invivogen Resveratrol or soluble CD40L, INF-γ, TNF, IL-1 or IL-6 from Peprotech. Polyclonal IL-10 neutralizing antibodies and the goat isotype control Abs were purchased from R&D Systems. RNA was isolated from MoDCs precultured with or without 5 ng/mL LPS for 2 days using TRI reagent (Invitrogen) followed by a second purification on RNeasy coloumns coupled with DNase I treatement (Qiagen) according to the manufacturer’s recommendations. The extracted samples were labeled with Cy5, hybridized on Illumina Whole Genome HT12 microarrays, according to the manufacturer’s instructions. After scanning, bead-level data was converted into bead-summary data using the Illumina BeadStudio software. Prior to normalization, array probes were annotated using their sequence and converted to unique nucleotide identifiers (nuIDs). Background signal was assessed and corrected using the intensity signal from the control probes present on the array, and then quantile normalization was performed with the aid of the lumi R package 35. Microarray data has been submitted to the Array Express repository (accession number: E-MTAB-658). Differentially expressed genes were calculated using the Rank Product algorithm 36. differentially expressed genes were called significant when their corrected p-value (percentage of false positives) was equal to or lower than 0.05.