SummaryInterferon α (IFNα) is a prompt and efficient orchestrator of host defense against nucleic acids but upon chronicity becomes a potent mediator of autoimmunity. Sustained IFNα signaling is linked to pathogenesis of systemic lupus erythematosus (SLE), an incurable autoimmune disease characterized by aberrant self-DNA sensing that culminates in anti-DNA autoantibody-mediated pathology. IFNα instructs monocytes differentiation into autoinflammatory dendritic cells (DCs) than potentiates the survival and expansion of autoreactive lymphocytes, but the molecular mechanism bridging sterile IFNα-danger alarm with adaptive response against self-DNA remains elusive. Herein, we demonstrate IFNα-mediated deregulation of mitochondrial metabolism and impairment of autophagic degradation, leading to cytosolic accumulation of mtDNA that is sensed via stimulator of interferon genes (STING) to promote induction of autoinflammatory DCs. Identification of mtDNA as a cell-autonomous enhancer of IFNα signaling underlines the significance of efficient mitochondrial recycling in the maintenance of peripheral tolerance. Antioxidant treatment and metabolic rescue of autolysosomal degradation emerge as drug targets in SLE and other IFNα-related pathologies.

Abstract Forkhead box class O1 (FOXO1) acts as a tumor suppressor in solid tumors. The oncogenic phosphoinositide-3-kinase (PI3K) pathway suppresses FOXO1 transcriptional activity by enforcing its nuclear exclusion upon AKT-mediated phosphorylation. We show here abundant nuclear expression of FOXO1 in Burkitt lymphoma (BL), a germinal center (GC) B-cell–derived lymphoma whose pathogenesis is linked to PI3K activation. Recurrent FOXO1 mutations, which prevent AKT targeting and lock the transcription factor in the nucleus, are used by BL to circumvent mutual exclusivity between PI3K and FOXO1 activation. Using genome editing in human and mouse lymphomas in which MYC and PI3K cooperate synergistically in tumor development, we demonstrate proproliferative and antiapoptotic activity of FOXO1 in BL and identify its nuclear localization as an oncogenic event in GC B-cell–derived lymphomagenesis.

Germinal centers (GC) are the predominant origin of human B cell lymphomagenesis. Transgenic mice in which gene expression is altered specifically in GC B cells have broadened our knowledge about the mechanisms of malignant transformation. However, extensive resources are needed due to the genetic complexity of these mouse models. Thus, bone marrow (BM)-derived chimerism is an attractive approach to study GC B cell derived lymphomagenesis, as it allows for an efficient allocation of resources and reduces the number of animals used.

Background/Objectives In SLE circulating IFNa can induce peripheral blood monocytes to differentiate into dendritic cells with increased MHCII antigen presenting capacity. Monocytes’ differentiation and self-antigen presentation has been shown to depend on autophagy, the catabolic degradation of unnecessary/dysfunctional cellular components and organelles and relies on the cooperation of autophagosomes with lysosomes. Our aim was to delineate the role of autophagy in shaping the autoreactive phenotype of SLE monocytes upon IFNa signalling and to dissect the underlying mechanism. Materials/Methods Serum and peripheral blood CD14+ monocytes were isolated from active, newly diagnosed or off-treatment SLE patients (n = 20) and age and sex matched healthy donors (n = 18). Autophagy, both spontaneous and following incubation with SLE serum (10%) or rhIFNa (104U/ml), was assessed by immunoblotting and confocal microscopy for LC3 and P62. ATG5 and P62 mRNA levels were assessed by QPCR. Autophagy induction was inhibited by wortmanin or 3MA and autophagy completion was inhibited by hydroxychloroquine. Rapamycin and LPS were used as autophagy inducers. Healthy monocytes were treated with SLE serum or rIFNa and their antigen presenting capacity was assessed by flow cytometry for HLA-DR, CD86 and CD40. Alterations in autophagolysosomal pH were assessed by confocal microscopy (Lysotracker Red-DND99). ROS production was measured using DCFHDA and mitochondrial polarisation was assessed by MitotrackerRed CMXRos and JC1. Results SLE monocytes exhibited increased levels of Atg5 mRNA (3 ± 0.5 fold increase, p < 0.05) and lipidated LC3B protein (p < 0.005) compared to healthy controls. Notably, autophagosomal P62 degradation was defective in SLE and this coincided with defective lysosomal acidification. Treatment of healthy monocytes with SLE serum or rIFNa recapitulated the abnormalities observed in SLE monocytes as for ATG5 transcription (p < 0.005), LC3B lipidation (p < 0.05), defective autophagosomal P62 degradation (p < 0.05) and defective lysosomal acidification. Importantly, in both SLE monocytes and healthy monocytes treated with rIFNa, mitochondria were found hyperpolarized and ROS production was increased. Enhanced antigen presenting capacity upon SLE serum or IFNa treatment was autophagy dependent as evidenced by autophagy manipulation experiments. Conclusions Monocytes from SLE patients display enhanced autophagy induction, driven by soluble factors such as IFNa. Autophagy completion is disrupted and this coincides with the presence of uncleared hyperpolarized mitochondria, increased cellular oxidative status and defective lysosomal acidification. Under these conditions, the antigen presenting capacity of human monocytes is increased and depends on autophagic alterations. Further studies will address whether deregulated autophagy/mitophagy completion is involved in the generation of an endogenous source of self antigens and whether this leads to SLE characteristic autoimmune phenotype of monocytes.

Resting B cell dependence on the B cell antigen receptor (BCR) has been attributed solely to its signaling competence. We have recently shown that the presence of the BCR is vital for endoplasmic reticulum (ER) homeostasis and mitochondrial function both in primary B cells and Burkitt lymphoma (BL) cell lines. Unexpectedly, in BL Ramos cells this role has been shown to be independent of BCR signals from the cell membrane. Now, we provide evidence that also in mouse resting B cells ER-resident immunoglobulin (Ig) heavy chains control ER homeostasis, calcium storage and mitochondrial homeostasis through ER-mitochondria contacts. These findings demonstrate that BCR expression shapes cellular fitness already at the stage of assembly in the ER and uncover a new layer of B cell dependence on the production of Ig heavy chains in the ER. Sensing protein expression in the ER, with counterselection of compromised cells, might well operate also in other differentiated cells.

RIF1 is a multifunctional protein that promotes immunoglobulin (Ig) isotype diversification. Whether RIF1 plays additional roles in adaptive immunity is unknown. In this study, we showed that Rif1 expression is upregulated following mature B cell activation, while its deficiency skewed the transcriptional profile of activated B cells towards plasmablasts (PBs) and plasma cells (PCs). Additionally, RIF1 ablation resulted in increased PB formation ex vivo and enhanced terminal differentiation into PCs upon immunization. Therefore, RIF1 serves as a cell identity gatekeeper during late B cell differentiation, providing an additional layer of control in the establishment of humoral immunity.

No abstract

SLE is a prototypical nucleic acid-triggered autoimmune disease during which pathogenic, IgG anti-dsDNA autoantibodies form glomerular and vascular deposits and cause tissue damage. Recent studies suggest that deregulated lysosomal degradation of cytoplasmic DNA may result in its accumulation and sensing by intracellular receptors culminating in antiDNA autoimmunity. In SLE, circulating IFNαinduces peripheral blood monocytes to differentiate into inflammatory DCs, a process implemented through autophagy and mitophagy. Mitochondrial DNA – a DNA of bacterial origin – is a known cytoplasmic danger signal but it has never been explored as a potential source for the triggering of DNA autoimmunity. We sought to explore this possibility for SLE autoimmunity in humans.Serum and peripheral blood monocytes were isolated from active, newly diagnosed or off-treatment SLE patients and matched healthy donors. IFNα signalling was induced by 10% SLE serum or rIFNα. Autophagy was assessed by QPCR, immunoblotting and confocal microscopy for Atg5, LC3 and P62. Autoreactivity was assayed in MLRs and assessed by ELISA for IL6 and TNFα and flow cytometry for HLADR, CD86 and Tcell CFSE dillution. Autophagolysosomal pH, mtROS production, mitochondrial polarisation and mtDNA accumulation were assessed by confocal microscopy and flow cytometry using specific fluorescent dyes (Lysotracker RedDND99, mitoSOX, JC1, MitotrackerRed CMXRos and picogreen respectively).Freshly isolated SLE monocytes and healthy monocytes exposed to SLE serum or rIFNαdisplay autophagy induction (Atg5 and LC3II increase) but defective autophagolysosomal degradation (increase in autophagosomal P62). Lysosomalalkalinization (decreased Lysotracker intensity P < 0.005) coincided with increased mtROS production (3fold higher mitoSOX intensity P < 0.05) leading to accumulation of damaged, oxidising mitochondria (increase in JC1 aggregates, 4fold higher Mitotracker intensity P < 0.005) and increased survival of mtDNA inside cytoplasmic compartments fused with lysosomes (picogreen-lysotrackercolocalisation). mtROS scavenging (mitoTEMPO) and inversion of autophagy completion (rapamycin) rescued the autoreactive phenotype of IFNαeducated monocytes in all cases.This study unveils a molecular mechanism that leads to the generation of a pool of immunogenic DNA autoantigens in SLE. IFNα mediated mitophagy deregulation leads to accumulation of oxmtDNA, a defect that can be targeted in order to avoid the development of the anti-DNA inflammatory response that drives SLE.