Rationale aim and conculsion Assay IFNa release assay

Rationale, aim and conculsion Assay: IFNa release assay Rationale: Interferon alpha (IFNa) is a main driver of systemic autoimmune diseases such as SLE and myositis, and its overexpression in patients with these diseases is in part caused by autoantibodycontaining immune complexes. Immune complexes containing autoantibodies bound to nucleic acids can stimulate innate cell typs, such as plasmacytoid dendritic cells (p. DC), to secrete IFNa. By binding to cell surface receptors for immune complexes (Fc. R), immune complexes containing in particular RNA are endocytosed and stimulate IFNa production by interacting with toll like receptors (TLR), such as TLR 7. Aim: To investigate whether chemical inhibitory probes can affect INFa secretion induced by autoantibody-containing immune complexes. Conclusions: Chemical probes MK-2206 and skepinone inhibiting kinases AKT and p 38 -MAPK, respectively, reduce INFa secretion. In addition, the chemical probe UNC 0638 targeting the methyltransferase G 9 a and unidentified off target proteins, reduces IFNa secretion. Experimental protocol General protocol: PBMC (Peripheral Blood Mononuclear Cells) are cultured for 24 hours in presence of SLE-derived immune complexes. The effect on IFNa secretion by addition of chemical probes is investigated by ELISA. The frequency of p. DC in the PBMC preparation is investigated by flow cytometry. Cell culture condition: Peripheral blood mononuclear cells (PBMC) are cultured at 2, 5 million/ml, 200µl/well, in triplicates. Culture medium is RPMI supplemented with 10% heat inactivated bovine serum. Cells are pre-incubated with probes (1 m. M or 0, 1 m. M) 30 min before addition of SLE-derived immune complexes (anti-RNP+ Ig. G from SLE sera 100 mg/ml mixed with apoptotic celline U 937, kind gift from Pfizer). Readout: Flow cytometric analysis is performed on freshly isolated PBMC, and include staining of CD 303, CD 11 c, CD 14, CD 3 and a marker for cell viability. Cell culture supernatants are removed after 24 hours and analysed for IFNa using a pan-IFNa specific ELISA. Controls include unstimulated condition (cell culture medium without added immune complexes), stimulated condition with vehicle control only (DMSO), stimulated condition with IRAK 1/4 inhibitor I.

Results INFa release upon 24 hours stimulation with SLE immune complex was investigated in 30 blood donors including 15 patients with systemic lupus erythemtosus (SLE), 8 with idiopathic inflammatory myositis (IIM) and 7 healthy donors. SLE patients were subdivided into hydroxychloroquine-treated and -untreated patients, and IIM patients into those with dermatomyositis (DM) and polymyositis (PM). Immune complex induced variable levels of IFNa with lower levels in SLE patients compared to healthy donors (p<0, 05). Of note, 6 of the 10 SLE patients treated with hydroxychloroquine (HCQ) presented with low IFNa induction (<100 pg/ml) and were removed from further analyses (U 17, U 27, U 39, U 40, U 41). Fig 1 100000 IFNa pg/ml 10000 100 10 8 U 4 2 U 1 7 U 2 1 U 2 4 U 2 6 U 2 7 U 3 9 U 4 0 U 4 1 U 4 U 0 3 1 U 0 6 1 U 0 9 2 U 0 0 2 U 0 3 1 U 0 4 2 U 0 2 2 U 0 5 2 HC 9 12 HC 1 HC 3 1 HC 4 1 HC 5 1 HC 6 1 HC 7 29 8 U 3 8 U 2 U 1 5 1 SLE treated with hydroxychloroquin myositis (DM) myositis (PM) healthy donors IFNa released after 24 h stimulation of PBMC with SLE immune complex. Data are depicted from 30 individual blood donors, as average+SD of biological triplicates.

Although p. DC are considered the most efficient producers of IFNa in blood, the frequency of p. DC in the cell culture was not found to correlate to immune complex induced IFNa levels. There was no difference in frequency of p. DC between groups of blood donors. The six SLE patients treated with hydroxychlorine and with low IFNa induction did not differ in the frequency of p. DC to the rest of the blood donors (Fig 2 b, red dots). Fig 2 25000 IFNa pg/ml 20000 15000 10000 5000 0 Gated on CD 3 -CD 14 - live PBMC 0 0. 5 1 % p. DC 1. 5 2 2. 5 IFNa released after 24 h stimulation of PBMC with SLE immune complex vs the percent of p. DC of CD 3 -CD 14 - live PBMC. Data are depicted from 30 individual blood donors. SLE patients treated with hydroxychloroquin and with <100 pg/ml IFNa induction are marked in red.

A set of 51 probes were tested in 24 blood donors including 9 patients with systemic lupus erythemtosus (SLE), 8 with idiopathic inflammatory myositis (IIM) and 7 healthy donors. The effect on PBMC IFNa secretion after 24 h cell culture in presence of SLE immune complex is expressed normalized to the vehicle control (0, 01% or 0, 001% DMSO). Probes are added at 1 u. M or 0, 1 u. M, as indicated. Pobes with p<0, 07 vs vehicle control are marked with red boxes. Due to constraints in the number of PBMC available from each donor, not all probes were tested in each blood donor. Kinase inh K-Me inh K-Ac inh Fig 3 Log 2 transformed White box: no data available

The results from individual donors of the 9 probes with p<0, 07 vs vehicle control is shown in figure 4. Of these, UNC 0638, MK-2206 and skepinone showed significant inhibitory effects (p<0, 05 and >50% average inhibition). The effects of the different probes did not differ between groups of blood donor. Thus, we confirm AKT and p 38 -MAPK as important players in immune complex induced cellular activation. The inhibitory effect of UNC 0638 may indicate off target interactions, which are presently being investigated. We saw variable but significant inhibitory effect using the IRAK 1/4 inhibitor I at 1 u. M. IFNa induction normalised to DMSO control Fig 4 * 4. 09599999989236 * ** * 2. 04799999994618 1. 02399999997309 0. 511999999986545 0. 25599993273 0. 12799996636 0. 063999983181 0. 031999991591 0. 015999995795 0. 0079999978977 * p<0, 05 vs ** p<0, 01 Probes with >50% average inhibition are marked with red boxes Average inhibition of all tested donors is marked with black dashes Dots represent results from individual blood donors 8 K on in ep sk SC H 7 72 98 e/ p 3 IR A K 4/ ER /A KT M K- 22 06 M /P R 07 C 7 SG UN C 0 63 8/ G 9 T 3 a a 2/ G 9 64 UN C 0 /C -1 NV S NI -5 7/ BR PF EC R 2 0. 0039999989488