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Activation of Plasmacytoid Dendritic Cells and B Cells with Two Structurally Different Toll-Like Receptor 7 Agonists

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Activation of Plasmacytoid Dendritic Cells and B Cells with Two Structurally Different Toll-Like Receptor 7 Agonists Received: 3 September 2019 | Revised: 17 March 2020 | Accepted: 18 March 2020 DOI: 10.1111/sji.12880 HUMAN IMMUNOLOGY Activation of plasmacytoid dendritic cells and B cells with two structurally different Toll-like receptor 7 agonists Olof Berggren | Pascal Pucholt | Cane Amcoff | Lars Rönnblom | Maija-Leena Eloranta Department of Medical Sciences, Rheumatology, Science for Life Laboratory, Abstract Uppsala University, Uppsala, Sweden Synthetic Toll-like receptor (TLR) 7 agonists have been suggested as immune modu- lators in a range of conditions. In contrast, self-derived TLR7 activators, such as Correspondence Maija-Leena Eloranta, Department of RNA-containing immune complexes (RNA-IC), can contribute to autoimmune dis- Medical Sciences, Rheumatology, Uppsala eases due to endogenous immune activation. The exact difference in immune cell University, Uppsala, Sweden. response between synthetic and endogenous TLR7 triggers is only partly known. An Email: [email protected] understanding of these differences could aid in the development of new therapeu- Funding information tic agents and provide insights into autoimmune disease mechanisms. We therefore The study was supported by grants from the compared the stimulatory capacity of two TLR7 agonists, RNA-IC and a synthetic Swedish Rheumatism Association, King Gustaf V’s 80-Year Foundation, Swedish small molecule DSR-6434, on blood leucocytes, plasmacytoid dendritic cells (pDCs) Research Council for Medicine and Health and B cells from healthy individuals. IFN-α, IL-6, IL-8 and TNF levels were meas- and Swedish Society of Medicine and Ingegerd Johansson donation. ured by immunoassays, and gene expression in pDCs was analysed by an expression array. DSR-6434 triggered 20-fold lower levels of IFN-α by pDCs, but higher pro- duction of IL-6, IL-8 and TNF, compared to RNA-IC. Furthermore, IFN-α and TNF production were increased with exogenous IFN-α2b priming, whereas IL-8 synthesis by B cells was reduced for both stimuli. Cocultivation of pDCs and B cells increased the RNA-IC-stimulated IFN-α and TNF levels, while only IL-6 production was en- hanced in the DSR-6434-stimulated cocultures. When comparing pDCs stimulated with RNA-IC and DSR-6434, twelve genes were differentially expressed (log2 fold change >2, adjusted P-value <.05). In conclusion, RNA-IC, which mimics an endog- enous TLR7 stimulator, and the synthetic TLR7 agonist DSR-6434 trigger distinct inflammatory profiles in immune cells. This demonstrates the importance of using relevant stimuli when targeting the TLR7 pathway for therapeutic purposes. 1 | INTRODUCTION between innate and adaptive immunity.1-4 Pattern recognition receptors (PRRs) are expressed by various immune cells to Activated plasmacytoid dendritic cells (pDCs) and B cells are sense conserved pathogen-associated molecular patterns two efficient immune regulators that interact in the interplay (PAMPs) on microorganisms and alert the immune system Berggren and Pucholt contributed equally to the study. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology Scand J Immunol. 2020;91:e12880. wileyonlinelibrary.com/journal/sji | 1 of 10 https://doi.org/10.1111/sji.12880 2 of 10 | BERGGREN ET AL. of a possible threat.5,6 One important PRR family consists of cytokine production and gene expression in pDCs of 594 of Toll-like receptors 1 to 10 (TLR1-10), which upon rec- immune system-related genes. ognition of PAMPs can evoke a powerful inflammatory re- sponse.6 The pDCs and B cells uniquely express TLR7 and TLR9 in their endo-lysosomal compartments where they 2 | MATERIALS AND METHODS sense single-stranded (ss) RNA and double-stranded DNA, respectively.7-10 In addition to natural agonists, TLR7 can be 2.1 | Cell isolation and culture conditions activated by synthetic compounds such as the imidazoquino- lines resiquimod and imiquimod structurally resembling nu- Blood cells were isolated from healthy blood donor buffy cleic acids or by oxoadenine derivatives.10-13 coats (Department of Transfusion Medicine, Uppsala The endosomal TLRs are normally protected from contact University Hospital). Peripheral blood mononuclear cells with endogenous nucleic acids. However, when in complex (PBMC) were isolated by Ficoll-Hypaque (GE Healthcare) with autoantibodies they can, via autoantibody-dependent density-gradient centrifugation. Due to their inhibitory ef- FcγR-mediated uptake or surface Ig-receptors, get access into fect on IFN-α synthesis, CD14-positive monocytes were endosomes of pDCs and B cells.14-16 Recognition of ssRNA depleted from PBMC by magnetic bead separation (CD14 by TLR7 leads to recruitment of the adaptor molecule my- MicroBeads (Miltenyi Biotec)).32 pDCs and B cells were iso- eloid differentiation factor 88 (MyD88), and initiation of a lated from PBMCs using negative selection (pDC Isolation signalling cascade activating the transcription factors inter- kit II and B cell Isolation kit II; Miltenyi Biotec). Purity of feron regulatory factor (IRF) 7 and NF-kB, finally resulting isolated B cells and pDCs was determined by flow cytometry in gene expression of type I interferons (IFN), IL-6, TNF and after staining with anti-CD19 (clone HIB19, BD Biosciences) other proinflammatory cytokines.17-19 and anti-BDCA2 (Miltenyi Biotec) monoclonal antibodies Type I IFN is an important mediator of the first line of an- (mAbs) and was found to be at least 95%. Cells were cultured tiviral defence. The pDCs are specialized to rapidly produce as previously described4 with 0.25 x 104 pDCs alone, or to- large amounts of type I IFN, especially IFN-α, in response gether with 1 x 104 B cells, in volumes of 0.1 mL per well 19,20 to a broad range of viruses and some bacteria. Type I in 96-well plates (Nunc) for 20 hours at 37°C with 5% CO2. IFN also has powerful immunomodulatory effects that can The cells were incubated in the presence of IFN-α2b (500 U/ be deleterious and increase the risk for autoimmune disease mL: IntronA, MSD) and/or monoclonal antibody to CD31 in genetically predisposed individuals.21-24 For example, in (PECAM-1), as indicated. The study was approved by the systemic lupus erythematosus (SLE) a continuous type I IFN Regional Ethics Committee, and written informed consent production by pDCs is triggered by an endogenous stimula- was obtained from all blood donors. tor consisting of nucleic acid containing immune complexes (IC), for example autoantibodies and nucleic acid binding proteins.24-27 In addition, B cells can enhance the IFN-α pro- 2.2 | Cell stimulation duction by pDCs when stimulated by immune complexes consisting of small nuclear ribonucleoproteins and IgG The small molecule TLR7 agonist 6-Amino-2-(butylamino)- isolated from SLE patients.4 We demonstrated earlier that 9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-7,9- blocking of the platelet endothelial cell adhesion molecule dihydro-8H-purin-8-one (DSR-6434, Tocris Bioscience) (PECAM-1/CD31)28 by monoclonal antibodies diminished [EC50 = 7.2 nmol/L]12 was titrated to achieve optimal in- the stimulatory effect of B cells on the RNA-IC stimulated duction of IFN-α and was used at a final concentration of IFN-α production. However, whether CD31 is involved in the 75 nmol/L in subsequent experiments. regulation of other proinflammatory cytokines had not been U1 snRNP particles were purified from HeLa cells as clarified. previously described,33 and SLE-IgG was isolated from two Although many synthetic TLR7 agonists have been inves- individual SLE patients’ serum containing autoantibodies to tigated for their ability to activate immune functions in pDCs SmB, SmD, RNP-A, RNP-C, ribosomal P antigen, histone and B cells,10,29-31 to our knowledge no studies have compared and dsDNA by protein G chromatography. The U1 snRNP the stimulatory effects of synthetic TLR7 agonists and RNA- particles and SLE-IgG were used in cell cultures at final containing immune complexes, which mimic endogenous concentrations of 2.5 μg/mL and 1 mg/mL, respectively.4 To TLR7 activation. Since pDCs and B cells play central roles in exclude that differences in cytokine production triggered by several autoimmune diseases and are potential treatment tar- DSR-6434 and RNA-IC were due to increased cell death, the gets, it is important to clarify how the different types of TLR7 cell viability was analysed by flow cytometry and showed no agonists affect these cells. In the present study, we stimulated difference between the two stimuli. The viability of the pDCs pDCs and B cells, with RNA-IC or a synthetic 8-oxoadenine was >80% after overnight incubation independent of stimuli derivative TLR7 agonist DSR-6434, to compare the pattern (results not shown). BERGGREN ET AL. | 3 of 10 2.3 | Immunoassays healthy individuals with different concentrations of DSR- 6434. The levels of IFN-α, IL-6, IL-8, IL-10 and TNF in IFN-α was analysed in culture supernatants by a dissocia- the cell cultures were analysed with immunoassays after tion-enhanced lanthanide fluoroimmunoassay (DELFIA) 20 hours. as previously described.34,35 The DELFIA assay does not Stimulation with DSR-6434 resulted in low or no IFN-α detect the IFN-α subtype 2b which was used for priming. production by both CD14-depleted PBMC (Figure 1A, Cytokines IL-6, IL-8, IL-10 and TNF-α were analysed using left panel) and PBMC (results not shown). Next, we asked the cytometric bead array (CBA) Human Soluble Protein whether the weak IFN-α production could be enhanced by Flex Set system (BD Biosciences) or ELISA immunoas- exogenous IFN-α (‘priming’).
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