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NFIL3 Mutations Alter Immune Homeostasis and Sensitise For Ann Rheum Dis: first published as 10.1136/annrheumdis-2018-213764 on 14 December 2018. Downloaded from Basic and translational research EXTENDED REPORT NFIL3 mutations alter immune homeostasis and sensitise for arthritis pathology Susan Schlenner,1,2 Emanuela Pasciuto,1,2 Vasiliki Lagou,1,2 Oliver Burton,1,2 Teresa Prezzemolo,1,2 Steffie Junius,1,2 Carlos P Roca,1,2 Cyril Seillet,3,4 Cynthia Louis,3 James Dooley,1,2 Kylie Luong,3,4 Erika Van Nieuwenhove,1,2,5 Ian P Wicks,3,4 Gabrielle Belz,3,4 Stéphanie Humblet-Baron,1,2 Carine Wouters,1,5 Adrian Liston1,2 Handling editor Josef S ABSTRact Key messages Smolen Objectives NFIL3 is a key immunological transcription factor, with knockout mice studies identifying functional ► Additional material is Homozygous NFIL3 mutations identified in roles in multiple immune cell types. Despite the importance ► published online only. To view monozygotic twins with juvenile idiopathic please visit the journal online of NFIL3, little is known about its function in humans. arthritis. (http:// dx. doi. org/ 10. 1136/ Methods Here, we characterised a kindred of two Enhanced susceptibility to arthritis induction in annrheumdis- 2018- 213764). monozygotic twin girls with juvenile idiopathic arthritis at ► Nfil3-knockout mice. 1 the genetic and immunological level, using whole exome Department of Microbiology NFIL3 loss in patients and mice is associated sequencing, single cell sequencing and flow cytometry. ► and Immunology, KUL - with elevated production of IL-1 . University of Leuven, Leuven, Parallel studies were performed in a mouse model. β Knockdown of NFIL3 in healthy macrophages Belgium Results The patients inherited a novel p.M170I in NFIL3 ► 2VIB Center for Brain and drives IL-1β production. Disease Research, Leuven, from each of the parents. The mutant form of NFIL3 Belgium demonstrated reduced stability in vitro. The potential 3Walter and Eliza Hall Institute contribution of this mutation to arthritis susceptibility was 6 of Medical Research, Parkville, demonstrated through a preclinical model, where Nfil3- of natural killer (NK) cells, with similar func- Victoria, Australia tions in other innate lymphoid cells,7 and the 4 deficient mice upregulated IL-1β production, with more Department of Medical Biology, + 8 severe arthritis symptoms on disease induction. Single cell CD8α dendritic cell subset. Within T cells, Nfil3 University of Melbourne, 9 sequencing of patient blood quantified the transcriptional enhances the Th2 lineage while suppressing the Parkville, Victoria, Australia 10 5Department of Pediatrics, dysfunctions present across the peripheral immune system, Th17 lineage. The net effect of these changes is University Hospitals Leuven, converging on IL-1β as a pivotal cytokine. the spontaneous development of colitis, a process Leuven, Belgium 11 Conclusions NFIL3 mutation can sensitise for arthritis dependent on microflora and T cell activation. development, in mice and humans, and rewires the Less is known about the function of NFIL3 in Correspondence to innate immune system for IL-1 over-production. humans. Expression of NFIL3 is reduced in patients Dr Adrian Liston, Professor β 12 Carine Wouters and Dr with Crohn’s disease and ulcerative colitis, and in Stéphanie Humblet-Baron, vitro gene silencing of NFIL3 in T cells and B cells 13 Department of Microbiology and promotes self-reactivity. These results suggest that http://ard.bmj.com/ Immunology, KUL - University of INTRODUCTION NFIL3 plays a key immune homeostatic role in Leuven, Leuven 3000, Belgium; humans; however, genetically deficient patients are adrian. liston@ vib. be, Juvenile idiopathic arthritis (JIA) is the most carine. wouters@ uzleuven. be, common of the childhood rheumatic diseases. required to understand the in vivo function. stephanie. humbletbaron@ vib- JIA is characterised as juvenile-onset persistent Here, we have characterised monozygotic twins kuleuven. be arthritis with no defined cause. A high degree of with JIA and inflammatory complications, who harbour homozygous mutations in NFIL3. Parallel Received 13 May 2018 clinical heterogeneity is observed within the JIA on September 24, 2021 by guest. Protected copyright. Revised 17 November 2018 group of diseases, thought to reflect a diversity in studies in mice confirm the arthritogenic potential of Accepted 19 November 2018 genetic and environmental factors and mechanistic NFIL3-deficiency, with Nfil3 knockout mice showing drivers. JIA shows similarities to adult autoimmune enhanced susceptibility to arthritis induction. Mech- diseases, and, indeed, genome-wide association anistic analysis identified elevated production of studies identify a strong overlap in the common IL-1β and TNFα by myeloid cells in the peripheral variants linked to autoimmune susceptibility and blood of NFIL3 patients and the inflamed joints of JIA susceptibility.1 JIA also has similarities to auto- Nfil3-deficient mice. Our results here demonstrate a inflammatory diseases, such as genetic associations link between NFIL3 mutation and restraint of inflam- to innate inflammatory pathways2 and response to matory cytokine production in the myeloid lineage, IL-1β blockade.3 The recent success in identifying contributing to a monogenic form of JIA. © Author(s) (or their 4 monogenic causes of autoinflammatory diseases employer(s)) 2018. Re-use permitted under CC BY. suggests that monogenic causes may also underlie a METHODS Published by BMJ. subset of patients with JIA. Indeed, the association Genetic analysis of systemic JIA with mutations in LACCI5 supports The study was approved by the Ethics Committee To cite: Schlenner S, the potential productivity of this approach in the of UZ Leuven, Belgium, and written informed Pasciuto E, Lagou V, et al. Ann Rheum Dis Epub ahead non-systemic JIA diseases. consent was obtained from the parents of the of print: [please include Day NFIL3 is an important transcription factor in the patients and age-matched healthy individuals. The Month Year]. doi:10.1136/ immune system. Analysis of Nfil3-deficient mice has study was performed in accordance with the modi- annrheumdis-2018-213764 identified a key role for Nfil3 in the development fied version of the Helsinki declaration. Whole Schlenner S, et al. Ann Rheum Dis 2018;0:1–8. doi:10.1136/annrheumdis-2018-213764 1 Ann Rheum Dis: first published as 10.1136/annrheumdis-2018-213764 on 14 December 2018. Downloaded from Basic and translational research exome sequencing was performed as previously described on P1 (0.1%) cover slips to subconfluency. Plasmid transfection was and P2.14 Only coding non-synonymous variants with genotype done using Lipofectamine 3000 according to the manufac- quality >60, gene damage index score of <12 40515 and mean turers protocol (Thermo Fisher). Twenty-four hours after trans- allele frequency of <0.005 in NHLBI GO Exome Sequencing fection, the cells were washed in PBS, fixed in 4% PFA and Project 6500, 1000 Genomes Project (October 2014) or in The permeabilised in 0.1% Triton X-100 (in PBS). After blocking Exome Aggregation Consortium database were considered. in PBS with 2% bovine serum albumine (BSA), 10% donkey serum and 0.1% Triton X-100 for 30 min, cells were stained Human macrophage differentiation with an anti-Flag polyclonal affinity antibody (F7425; Sigma Peripheral blood mononuclear cells (PBMCs) were isolated from Aldrich) for 2 hours, then washed and incubated for 1 hour heparinised blood using lymphocyte separation medium (LSM, with Alexa Fluor 555 donkey-anti-rabbit (A31572; Molecular MP Biomedicals). PBMCs were plated in Poly-D-Lysine coated Probes) antibody as well as DAPI (D1306; Molecular Probes). flask in complete RPMI (Thermo Fisher Scientific) and incu- After washing the cells, they were covered using Fluoromount bated for 2 hours to enrich the monocyte population by plastic (Thermo Fisher). Images were collected on an LSM 510 Meta adherence. Monocytes were differentiated into macrophages for confocal microscope (Ziess) with a 60× immersion objective. 7–10 days in differentiation media containing complete RPMI Quantification of mean fluorescence intensity was measured medium supplemented with recombinant hM-CSF (50 ng/mL) using ImageJ software. Alternatively, 24 hours post-transfec- and hIL-10 (25 ng/mL) (both BioLegend), replacing media every tion, cells were stained with fixable viability dye (eBioscience), 2–3 days during the differentiation period. Macrophages were fixed and stained for human NFIL3 following the eBioscience transfected with either scrambled siRNA (SantaCruz, sc-36869, protocol for flow cytometry analysis. 30 pmol) or three siRNA targeting Nfil3 mRNA (ThermoFisher assay IDs 144020, 115 656 and 115655, 30 pmol each) by nucleofection (Lonza, VPA-1008, Nucleofector program T-016). Arthritis induction in mice C57Bl/6 and Nfil3-/- mice16 were bred and housed under barrier Twenty-four hours post-transfection cells were stimulated with 1 conditions at a specific pathogen-free facility at the Walter and µg/mL lipopolysaccharide (LPS) for 24 hours. Eliza Hall Institute Animal Facility. Eight-to-ten week-old mice were used for all experiments. All procedures were approved by Flow cytometry the Walter and Eliza Hall Institute Animal Ethics Committee. PBMCs were isolated using LSM (MP Biomedicals) from Serum transfer arthritis was induced by injection of arthrito- patients and healthy individuals. For intracellular staining, cells genic serum from 12-week-old progeny of KRN and non-obese were plated with complete RPMI containing phorbol myristate
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