AB0171 THE REGULATION AND PHARMACOLOGICAL MODULATION OF IMMUNE COMPLEX INDUCED PRODUCTION OF TYPE III IFN BY PLASMACYTOID DENDRITIC CELLS

Background: The type I Interferons (IFNs) are the most important drivers of the IFN gene signature in Systemic Lupus Erythematosus (SLE). However, both type II and type III IFNs (IFN-λ1-3) can be measured in a proportion of patients with SLE and contribute to the IFN signature. The exact role of type III IFNs in SLE is not completely clear, but serum levels of type III IFN correlate with disease activity and specific organ manifestations, such as arthritis, nephritis and anti-dsDNA antibodies. Type III IFN can be induced in pDCs by TLR9 agonist Oligodinucleotide (ODN) 2216 and many viruses. Whether type III IFN can also be induced in pDCs by nucleic acid containing immune complexes (IC), has, to our knowledge, not been investigated before. Objectives: We asked if RNA containing immune complexes (RNA-IC), which trigger the synthesis of large amounts of IFN-α by plasmacytoid dendritic cells (pDCs), can act as stimuli for type III IFN production, and how this production is regulated by Natural Killer (NK) cells and different cytokines. We also investigated if the type III IFN production could be blocked by hydroxychloroquine (HCQ) and an interleukin receptor 1 associated kinase 4 inhibitor (IRAK4i). Methods: Peripheral blood mononuclear cells (PBMCs) from SLE patients or healthy individuals were used to isolate PDCs and natural killer (NK) cells, or were depleted of monocytes. Cells were stimulated with RNA-IC, and cytokines were measured by immunoassays. mRNA expression in RNA-IC stimulated pDCs and NK cells cells was analyzed with a microarray. The effect of HCQ and IRAK4i on the IFN-λ1/3 production was investigated in pDCs and NK cells from healthy individuals. Results: Type III IFN mRNA expression was strongly upregulated in co-cultures of pDC-NK cells stimulated with RNA-IC. High levels of IFN-λ1/3 and IFN-λ2 (medians 2000 pg/ml and 100 pg/ml) were detected in supernatants from RNA-IC stimulated pDC-NK cell co-cultures. IFN-λ2 enhanced IFN-λ1/3 and IFN-α production by purified pDCs. Interleukin (IL) -3, IL-6, and GM-CSF significantly enhanced IFN-λ1/3 production (4-5 fold) by RNA-IC stimulated pDCs. Monocyte depleted PBMCs and pDC-NK cell co-cultures from 15% and 9% of SLE patients produced IFN-λ1/3 in response to RNA-IC stimulation. Exogenous IFN-α2b and GM-CSF in pDC-NK cell co-cultures increased the proportion of patients responding to RNA-IC stimulation from 9 to 36%. IFN-λ1/3 production by RNA-IC-stimulated pDCs and pDC-NK cells was significantly inhibited by HCQ (by 99% and 93% respectively) and an IRAK4i (by 98% and 96% respectively). Conclusion: pDCs produce both type I and type III IFN in response to RNA containing immune complexes. This is promoted by activated NK cells as well as a number of pro-inflammatory cytokines, including IFN type I and type III, considered important in SLE. Consequently, in order to achieve a proper control the IFN driven autoimmune process in SLE, both type I and type III IFN need to be targeted. In this system of stimulated, co-cultivated pDCs and NK cells, HCQ and an IRAK4 inhibitor blocked the type III IFN production. Disclosure of Interests: None declared