GSE128735
BET bromodomain inhibitor iBET151 impedes human ILC2 activation and prevents experimental allergic lung inflammation
Group 2 innate lymphoid cells (ILC2) increase in frequency in eczema and allergic asthma patients, and thus represent a new therapeutic target cell for type-2 immune-mediated disease. The bromodomain and extra-terminal (BET) protein family of epigenetic regulators are known to support the expression of cell cycle and pro-inflammatory genes during type-1 inflammation, but have not been evaluated in type-2 immune responses.  We isolated human ILC2 and examined the capacity of the BET protein inhibitor, iBET151, to modulate human ILC2 activation following IL-33 stimulation. iBET151 profoundly blocked expression of genes critical for type-2 immunity, including type-2 cytokines, cell surface receptors and transcriptional regulators of ILC2 differentiation and activation.  Furthermore, in vivo administration of iBET151 during experimental mouse models of allergic lung inflammation potently inhibited lung inflammation and airways resistance in response to cytokine or allergen exposure.  Thus, iBET151 effectively prevents human ILC2 activation and dampens type-2 immune responses. Overall design: Lymphocytes were enriched from 150 ml human peripheral blood using lymphoprep (Axis-Shield) according to the manufacturer's instructions. The collected PBMC fraction was washed with PBS/2% FCS followed by red blood cell lysis with ACK solution for 5 min at room temperature. To pre-enrich ILCs, cells were stained with biotinylated anti-CD3, CD14 and B220 in the presence of TruStain FcX (anti-CD16/CD32) followed by depletion with M280 streptavidin beads (Invitrogen). Subsequently, the enriched fraction was stained with CD45-AF488, CD127-PE-Cy7, CD117-PE, CRTH2-BV421, a lineage cocktail (APC-streptavidin, CD8a, CD11b, CD11c, FceRIa, CD123, CD20, CD56, CD71) and Draq7-viability dye, followed by sorting of ILCs as indicated on an Influx FACS. ILC2s designated as Draq7–(live cells)CD45+Lin–(streptavidin-APC, CD8a, CD11b, CD11c, FceRIa, CD123, CD20, CD56, CD71)CD127+CRTH2+ cells, were purified by flow cytometry from the peripheral blood of 3 individual donors and cultured separately. Purified human ILC2s were grown in media containing IL-2 and IL-7 (both at 10 ng/ml) with IL-33 (10 ng/ml) added to the cultures every alternate week.  ILC2s were then rested for 3 days in IL-2 and IL-7-containing media (no IL-33) before being divided and added to the following culture conditions: IL-2/IL-7, +/-IL33 (all at 10 ng/ml), +/- 125 nM iBET151 for 24 hours. Cells were pelleted and RNA prepared for RNAseq analysis.