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Objective: Patients with systemic lupus erythematosus (SLE) have an ongoing interferon (IFN) production due to an activation of plasmacytoid dendritic cells (pDCs), which can be triggered to type I IFN synthesis by RNA containing immune complexes (RNA-IC). Considering emerging data suggesting a role of type III IFN in the SLE disease process, we asked if RNA-IC can induce type III IFN production in pDC and how this production can be regulated., Methods: Peripheral blood mononuclear cells (PBMCs) or immune cell subsets were isolated from healthy blood donors or SLE patients and stimulated with IC containing U1 snRNP and SLE-IgG (RNA-IC). Hydroxychloroquine (HCQ) and an interleukin receptor 1-associated kinase 4 inhibitor (IRAK4i) were added to cell cultures. Cytokine mRNA levels were determined with a microarray and protein levels with immunoassays. Single-cell RNA sequencing of pDCs using ddSEQ technology was performed., Results: Type III IFN mRNA and protein was induced in RNA-IC-stimulated pDC-NK and pDC-B cell co-cultures. A subset of activated pDCs (3%) expressed both type III and type I IFN mRNA. IFN-λ2, IFN-α2b, interleukin (IL)-3, IL-6, or granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced IFN-λ1/3 production 2-5-fold. HCQ and an IRAK4i blocked the RNA-IC-triggered IFN-λ1/3 production (p < 0.01). IFN-α2b and GM-CSF increased the proportion of SLE patients producing IFN-λ1/3 in response to RNA-IC from 11 to 33%., Conclusions: Type III IFN production is triggered by RNA-IC in pDCs in a TLR-MyD88-dependent manner, enhanced by NK and B cells as well as several pro-inflammatory cytokines. These results support a contributing role for both type I and type III IFNs in SLE, which needs to be considered when targeting the IFN system in this disease.

Background: In systemic lupus erythematosus (SLE), immune complexes (ICs) containing self-derived nucleic acids trigger the synthesis of proinflammatory cytokines by immune cells. We asked how an interleukin (IL)-1 receptor-associated kinase 4 small molecule inhibitor (IRAK4i) affects RNA-IC-induced cytokine production compared with hydroxychloroquine (HCQ)., Methods: Plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. PBMCs from SLE patients and healthy individuals were depleted of monocytes. Cells were stimulated with RNA-containing IC (RNA-IC) in the presence or absence of IRAK4i I92 or HCQ, and cytokines were measured by immunoassay or flow cytometry. Transcriptome sequencing was performed on RNA-IC-stimulated pDCs from healthy individuals to assess the effect of IRAK4i and HCQ., Results: In healthy individuals, RNA-IC induced interferon (IFN)-α, tumor necrosis factor (TNF)-α, IL-6, IL-8, IFN-γ, macrophage inflammatory protein (MIP)1-α, and MIP1-β production in pDC and NK cell cocultures. IFN-α production was selective for pDCs, whereas both pDCs and NK cells produced TNF-α. IRAK4i reduced the pDC and NK cell-derived cytokine production by 74-95%. HCQ interfered with cytokine production in pDCs but not in NK cells. In monocyte-depleted PBMCs, IRAK4i blocked cytokine production more efficiently than HCQ. Following RNA-IC activation of pDCs, 975 differentially expressed genes were observed (false discovery rate (FDR) < 0.05), with many connected to cytokine pathways, cell regulation, and apoptosis. IRAK4i altered the expression of a larger number of RNA-IC-induced genes than did HCQ (492 versus 65 genes)., Conclusions: The IRAK4i I92 exhibits a broader inhibitory effect than HCQ on proinflammatory pathways triggered by RNA-IC, suggesting IRAK4 inhibition as a therapeutic option in SLE.

Background: Hyperactive B cells and a continuous interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs) play a key role in systemic lupus erythematosus (SLE). We asked whether the interaction between B cells and pDCs stimulated with RNA-containing immune complexes affects peripheral B cell subsets., Methods: B cells and pDCs were isolated from blood of healthy individuals and stimulated with immune complexes consisting of SLE-IgG and U1snRNP (RNA-IC). Expression of cell surface molecules as well as IL-6 and IL-10 production were determined by flow cytometry and immunoassays. Gene expression profiles were determined by a NanoString nCounter expression array., Results: We found a remarkable increase of double negative CD27-IgD- B cells, from 7% within fresh CD19+ B cells to 37% in the RNA-IC-stimulated co-cultures of B cells and pDCs, comparable to the frequency of double negative B cells in SLE patients. Gene expression analysis of the double negative CD27-IgD- and the CD27+IgD- memory B cells revealed that twenty-one genes were differentially expressed between the two B cell subsets (≥ 2-fold, p<0.001). The, IL21R, IL4R, CCL4, CCL3, CD83 and the IKAROS Family Zinc Finger 2 (IKZ2) showed higher expression in the double negative CD27-IgD- B cells., Conclusion: The interactions between B cells and pDCs together with RNA-containing IC led to an expansion of B cells with similar phenotype as seen in SLE, suggesting that the pDC-B cell crosstalk contributes to the autoimmune feed-forward loop in SLE.

Objectives: Patients with systemic lupus erythematosus (SLE) have an ongoing interferon-α (IFN-α) production by plasmacytoid dendritic cells (pDCs). We investigated whether T cells can promote IFN-α production by pDCs., Methods: Human pDCs were stimulated with immune complexes (ICs) containing U1 small nuclear ribonucleic proteins particles and SLE-IgG (RNA-IC) in the presence of T cells or T cell supernatants. T cells were activated by anti-CD3/CD28 antibodies or in a mixed leucocyte reaction. IFN-α and other cytokines were determined in culture supernatants or patient sera with immunoassays. The effect of interleukin (IL) 3 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) on pDCs was examined by the use of antibodies, and the expression of CD80/CD86 was determined using flow cytometry., Results: Activated T cells and supernatants from activated T cells increased IFN-α production by >20-fold. The stimulatory effect of T cell supernatants was reduced after depletion of GM-CSF (81%) or by blocking the GM-CSF receptor (55%-81%). Supernatant from activated T cells, furthermore, increased the frequency of CD80 and CD86 expressing pDCs stimulated with RNA-IC from 6% to 35% (p<0.05) and from 10% to 26% (p<0.01), respectively. Activated SLE T cells enhanced IFN-α production to the same extent as T cells from healthy individuals and a subset of patients with SLE had increased serum levels of GM-CSF., Conclusions: Activated T cells enhance IFN-α production by RNA-IC stimulated pDCs via GM-CSF and induce pDC maturation. Given the increased serum levels of GM-CSF in a subset of patients with SLE, these findings suggest that activated T cells may upregulate type I IFN production in SLE., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Patients with systemic lupus erythematosus (SLE) display an activated type I IFN system due to unceasing IFN-α release from plasmacytoid dendritic cells (pDCs) stimulated by nucleic acid-containing immune complexes (ICs). NK cells strongly promote the IFN-α production by pDCs; therefore, we investigated surface molecules that could be involved in the pDC-NK cell cross-talk. In human PBMCs stimulated with RNA-containing ICs (RNA-ICs), the expression of the signaling lymphocyte activation molecule (SLAM) family receptors CD319 and CD229 on pDCs and CD319 on CD56(dim) NK cells was selectively increased. Upregulation of CD319 and CD229 on RNA-IC-stimulated pDCs was induced by NK cells or cytokines (e.g., GM-CSF, IL-3). IFN-α-producing pDCs displayed a higher expression of SLAM molecules compared with IFN-α⁻ pDCs. With regard to signaling downstream of SLAM receptors, pDCs expressed SHIP-1, SHP-1, SHP-2, and CSK but lacked SLAM-associated protein (SAP) and Ewing's sarcoma-activated transcript 2 (EAT2), indicating that these receptors may act as inhibitory receptors on pDCs. Furthermore, pDCs from patients with SLE had decreased expression of CD319 on pDCs and CD229 on CD56(dim) NK cells, but RNA-IC stimulation increased CD319 and CD229 expression. In conclusion, this study reveals that the expression of the SLAM receptors CD319 and CD229 is regulated on pDCs and NK cells by lupus ICs and that the expression of these receptors is specifically altered in SLE. These results, together with the observed genetic association between the SLAM locus and SLE, suggest a role for CD319 and CD229 in the SLE disease process.