Panayotis Verginis

High-density lipoprotein attenuates Th1 and Th17 autoimmune responses by modulating dendritic cell maturation and function

Aberrant levels and function of the potent anti-inflammatory high-density lipoprotein (HDL) and accelerated atherosclerosis have been reported in patients with autoimmune inflammatory diseases. Whether HDL affects the development of an autoimmune response remains elusive. In this study, we used apolipoprotein A-I–deficient (apoA-I−/−) mice, characterized by diminished circulating HDL levels, to delineate the role of HDL in autoimmunity. ApoA-I−/− mice exhibited increased severity of Ag-induced arthritis compared with wild-type mice, and this was associated with elevated Th1 and Th17 cell reactivity in the draining lymph nodes. Furthermore, reconstituted HDL (rHDL) attenuated IFN-γ and IL-17 secretion by Ag-specific T cells upon stimulation of draining lymph nodes in vitro. The suppressive effects of rHDL were mediated through modulation of dendritic cell (DC) function. Specifically, rHDL-treated DCs demonstrated an immature phenotype characterized by downregulated costimulatory molecules, the release of low amounts of proinflammatory cytokines, and failure to promote T cell proliferation in vitro. The mechanism of action involved the inhibition of NF-κB nuclear translocation and the decrease of Myd88 mRNA levels by rHDL. Finally, modulation of DC function by rHDL was critically dependent on the presence of scavenger receptor class B type I and ATP Binding Cassette Transporter A1, but not the ATP Binding Cassette Transporter G1. These findings reveal a novel role of HDL in the regulation of adaptive inflammatory responses through suppression of DC function that could be exploited therapeutically in autoimmune inflammatory diseases.

Neutrophil extracellular traps exacerbate Th1-mediated autoimmune responses in rheumatoid arthritis by promoting DC maturation.

Aberrant formation of neutrophil extracellular traps (NETs) is a key feature in rheumatoid arthritis (RA) and plays a pivotal role in disease pathogenesis. However, the mechanism through which NETs shape the autoimmune response in RA remains elusive. In this study, we demonstrate that inhibition of peptidylarginine deiminases activity in collagen‐induced arthritis (CIA) mouse model significantly reduces NET formation, attenuates clinical disease activity, and prevents joint destruction. Importantly, peptidylarginine deiminase 4 blocking markedly reduces the frequency of collagen‐specific IFN‐γ‐producing T helper 1 (Th1) cells in the draining lymph nodes of immunized mice. Exposure of dendritic cells (DCs) to CIA‐derived NETs induces DC maturation characterized by significant upregulation of costimulatory molecules, as well as elevated secretion of IL‐6. Moreover, CIA‐NET‐treated DCs promote the induction of antigen‐specific Th1 cells in vitro. Finally, NETs from RA patients show an increased potential to induce the maturation of DCs from healthy individuals, corroborating the findings obtained in CIA mouse model. Collectively, our findings delineate an important role of NETs in the induction and expansion of Th1 pathogenic cells in CIA through maturation of DCs and reveal a novel role of NETs in shaping the RA‐autoimmune response that could be exploited therapeutically.

De Novo–Induced Self-Antigen–Specific Foxp3+ Regulatory T Cells Impair the Accumulation of Inflammatory Dendritic Cells in Draining Lymph Nodes

Foxp3+ regulatory T cell (Treg)-based immunotherapy holds promise for autoimmune diseases. However, this effort has been hampered by major caveats, including the low frequency of autoantigen-specific Foxp3+ Tregs and lack of understanding of their molecular and cellular targets, in an unmanipulated wild-type (WT) immune repertoire. In this study, we demonstrate that infusion of myelin in WT mice results in the de novo induction of myelin-specific Foxp3+ Tregs in WT mice and amelioration of experimental autoimmune encephalomyelitis. Myelin-specific Foxp3+ Tregs exerted their effect both by diminishing Ag-bearing inflammatory dendritic cell (iDC) recruitment to lymph nodes and by impairing their function. Transcriptome analysis of ex vivo–isolated Treg-exposed iDCs showed significant enrichment of transcripts involved in functional properties of iDCs, including chemotaxis-related genes. To this end, CCR7 expression by iDCs was significantly downregulated in tolerant mice and this was tightly regulated by the presence of IL-10. Collectively, our data demonstrate a novel model for deciphering the Ag-specific Foxp3+ Treg-mediated mechanisms of tolerance and delineate iDCs as a Foxp3+ Treg cellular target in unmanipulated mice.

Myeloid-derived suppressor cells and T regulatory cells in tumors: unraveling the dark side of the force

Important conceptual advances in tumor immunology over the last years have shifted the paradigm from focusing on the malignant cell to the importance of host immune components in the design of successful immunotherapies. The immune system, through sophisticated innate and adaptive immune surveillance mechanisms, inhibits the growth and establishment of tumors. However, despite immune surveillance, tumors still escape and grow, mainly as a result of endowed tumor‐induced immunosuppressive circuits. Regulatory T cells (Tregs) and myeloid‐derived suppressor cells (MDSCs) are the major components of these regulatory networks that facilitate tumor immune escape and significantly compromise the efficacy of current immunotherapies. A better understanding of the induction, function, and expansion of these powerful regulatory compartments represents a major challenge on the clinical benefit of current treatments and may foster the design of novel cancer immunotherapies.

Intratumoral accumulation of podoplanin-expressing lymph node stromal cells promote tumor growth through elimination of CD4+ tumor-infiltrating lymphocytes

ABSTRACT The beneficial effects of checkpoint blockade in tumor immunotherapy are limited to patients with increased tumor-infiltrating lymphocytes (TILs). Delineation of the regulatory networks that orchestrate the presence of TILs holds great promise for the design of effective immunotherapies. Podoplanin/gp38 (PDPN)-expressing lymph node stromal cells (LNSCs) are present in tumor stroma; however, their effect in the regulation of TILs remains elusive. Herein we demonstrate that intratumor injection of ex-vivo-isolated PDPN+ LNSCs into melanoma-bearing mice induces elimination of TILs and promotes tumor growth. In support, PDPN+ LNSCs exert their function through direct inhibition of CD4+ T cell proliferation in a cell-to-cell contact independent fashion. Mechanistically, we demonstrate that PDPN+ LNSCs mediate T cell growth arrest and induction of apoptosis to activated CD69+CD4+ T cells. Importantly, LTbR-Ig-mediated blockade of PDPN+ LNSCs expansion and function significantly attenuates melanoma tumor growth and enhances the infiltration and proliferation of CD4+ TILs. Overall, our findings decipher a novel role of PDPN-expressing LNSCs in the elimination of CD4+ TILs and propose a new target for tumor immunotherapy.

Aberrant function of myeloid-derived suppressor cells (MDSCs) in experimental colitis and in inflammatory bowel disease (IBD) immune responses

Abstract Background and aims: Myeloid-derived suppressor cells (MDSCs) encompass a novel population of suppressor cells and a potential candidate for cell-based therapies in inflammatory diseases. Herein, we investigated their immunomodulatory properties in experimental inflammatory colitis and T cell-mediated immune responses in inflammatory bowel disease (IBD) patients. Methods: MDSCs (defined as CD14−HLA−DR−/lowCD33+CD15+) numbers were determined in peripheral blood (PB) from IBD patients. PB MDSC function was assessed in vitro. Experimental colitis was induced upon 2,4,6-trinitrobenzene sulfonic acid (TNBS) treatment and MDSCs were characterized by flow cytometry. The in vivo suppressive potential of bone marrow (BM)-derived MDSCs (BM-MDSCs) was tested by using both depleting and adoptive transfer strategies. Results: MDSCs were enriched in the periphery of IBD patients during active disease. TNBS colitis induced amplification of MDSCs, particularly of the granulocytic (Ly6G+) subset during the effector phase of disease. Of interest, BM-MDSCs potently suppressed CD4+ T cell responses under steady state but failed to control colitis-associated immune responses in vivo. Mechanistically, under the colonic inflammatory milieu MDSCs switched phenotype (decreased proportion of Gr1high and increased numbers of Gr1low) and downregulated CCAAT/enhancer-binding protein beta (CEBPβ) expression, a critical transcription factor for the suppressive function of MDSCs. In accordance with the murine data, human CD33 + CD15+ MDSCs from peripheral blood of IBD patients not only failed to suppress autologous T cell responses but instead enhanced T cell proliferation in vitro. Conclusions: Our findings demonstrate an aberrant function of MDSCs in experimental inflammatory colitis and in IBD-associated immune responses in vitro. Delineation of the mechanisms that underlie the loss of MDSCs function in IBD may provide novel therapeutic targets.

A8.28 Reconstituted high density lipoprotein (RHDL) modulates TH1 and TH17 immune responses and pro-inflammatory cytokine production in a murine model of rheumatoid arthritis

Background and Objectives High density lipoprotein (HDL) has a variety of functions which confer protection from cardiovascular and other human diseases. Increased cardiovascular risk is observed in humans with autoimmune disorders such as rheumatoid arthritis (RA), which is associated with either low levels or dysfunctional HDL. Recent evidence suggests a role of HDL in modulating both innate and adaptive immune responses. We sought to investigate the role of rHDL during the development of an autoimmune response in the antigen-induced arthritis (AIA) mouse model. Materials and Methods C57BL/6 mice were subcutaneously immunised with ovalbumin (OVA) in complete Freund’s adjuvant (CFA) and the inguinal lymph nodes (LN) were excised 9 days after the antigenic challenge. The LN cells were cultured in vitro in the presence of varying concentrations of reconstituted HDL (rHDL) in the presence or absence of OVA and OVA-specific immune responses were measured. To assess the effect of HDL on dendritic cell activation and maturation, mouse bone marrow was cultured with GM-CSF to generate dendritic cells (BM-DCs), which were collected, cultured and treated with LPS in the presence or absence of rHDL. Results Presence of rHDL significantly inhibited the proliferation of OVA-primed LN cells in vitro, in a dose-dependent manner, as indicated by IL-2 measurement. Suppressed proliferation accompanied by reduced levels of IFN-γ and IL-17 in culture supernatants indicating that rHDL modulates the induction of Th1 and Th17 effector cells. Finally, rHDL-treated LPS-stimulated BM-DCs secreted significantly lower amounts of pro-inflammatory cytokines such as IL-6 and IL-8 whereas secretion of TNF-α was not affected. Conclusions rHDL exerts a direct immunomodulatory function on T cells in vitro by suppressing their proliferation and the induction of Th1 and Th17 effector cells. Furthermore, rHDL modulate the secretion of pro-inflammatory cytokines by DCs. Ongoing work is focused on the delineation of the mechanism involved in the rHDL-mediated suppression of the immune response both in vitro and in vivo. These data identify rHDL as an important player in the homeostatic regulation of the inflammatory response and a potential therapeutic target for chronic inflammatory diseases.

S4D:7 Next generation sequencing in hematopoietic progenitors of murine sle model reveals aberrant regulation of cebp/a expression

Background and purpose All blood cell lineages that have implicated to the pathogenesis of SLE originate from the Hematopoietic Stem Cells (HSCs). Studying HSCs may help to dissect fundamental immune aberrations in SLE and elucidate the HSC contribution to the pathogenesis of the disease. Materials and methods HSCs were isolated from either healthy C57/BL6 or NZBxNZW/F1 lupus-prone mice bone marrow. The selection markers used are Lin-Sca-1+c-Kit+for LSK compartment that encompasses both long- and short-term HSCs as well as multipotent progenitors (MPP). Flow cytometry cell sorting of LSK was used for enumeration, RNA extraction, qPCR and cell cultures. Paired-end RNA-sequencing analysis was performed by the Illumina HiSeq 2000 platform. Results We found significantly increased numbers of LSK in the BM of lupus NZBxNZW/F1 mice with established disease as compared to pre-diseased NZBxNZW/F1 mice in combination with evidence of them exiting the latent state and progression of cell cycle and aberrant differentiation with skewing towards the myeloid lineage. Transcriptome analysis revealed 800 differentially expressed genes (DEGs) (FC>1.5, q<0.05) in diseased lupus mice compared to pre-diseased with enrichment in transcription factors involved in hematopoiesis, regulation of immune responses and HSC function/homeostasis. We selected Cebpα (FC −0,88) -a master regulator of myeloid differentiation, self-renewal and resistance to stress-induced apoptosis of HSCs- for further investigation. qPCR analysis showed decreased Cebpα expression during the progression of the disease in SLE but increased Cebpα in aged healthy C57/BL6 mice. In vitro stimulation with IFNα decreased Cebpα expression in lupus -but not in healthy LSK. Serum from pre-diseased NZBxNZW/F1 decreased Cebpα expression only in pre-diseased LSK. Experiments to reverse Cebpα downregulation (using lenti-virus and modifiers of the metabolomics) are in progress. Conclusions HSC RNA-seq analysis suggests both intrinsic and extrinsic influences resulting in downregulation of Cebpα in murine lupus. SLE HSCs have pronounced expansion, enhanced proliferation and aberrant differentiation -in part due to the effects of IFNα. Together these results suggest a decreased capacity of lupus HSCs to respond to stressors which may account for the cytopenias and the infections in SLE.

IFNα Impairs Autophagic Degradation of mtDNA Promoting Autoreactivity of SLE Monocytes in a STING-Dependent Fashion

Summary Interferon 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.

A1.23 IFNα mediated deregulation of mitochondrial DNA clearance as an inciting event for the development of SLE autoimmunity

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.