Aggelos Banos

Genomic Analysis Reveals a Novel Nuclear Factor-κB (NF-κB)-binding Site in Alu-repetitive Elements

Background: The transcription factor NF-κB is a critical regulator of immune response pathways. Results: 11% of total NF-κB binding events in humans occur at the Alu-κB element present in Alu repeats. Conclusion: NF-κB has a primate-specific function and a role in human evolution. Significance: Repetitive elements expand the repertoire of binding sites to engage new genes into regulatory networks. The transcription factor NF-κB is a critical regulator of immune responses. To determine how NF-κB builds transcriptional control networks, we need to obtain a topographic map of the factor bound to the genome and correlate it with global gene expression. We used a ChIP cloning technique and identified novel NF-κB target genes in response to virus infection. We discovered that most of the NF-κB-bound genomic sites deviate from the consensus and are located away from conventional promoter regions. Remarkably, we identified a novel abundant NF-κB-binding site residing in specialized Alu-repetitive elements having the potential for long range transcription regulation, thus suggesting that in addition to its known role, NF-κB has a primate-specific function and a role in human evolution. By combining these data with global gene expression profiling of virus-infected cells, we found that most of the sites bound by NF-κB in the human genome do not correlate with changes in gene expression of the nearby genes and they do not appear to function in the context of synthetic promoters. These results demonstrate that repetitive elements interspersed in the human genome function as common target sites for transcription factors and may play an important role in expanding the repertoire of binding sites to engage new genes into regulatory networks.

Tregs restrain dendritic cell autophagy to ameliorate autoimmunity

Design of efficacious Treg-based therapies and establishment of clinical tolerance in autoimmune diseases have proven to be challenging. The clinical implementation of Treg immunotherapy has been hampered by various impediments related to the stability and isolation procedures of Tregs as well as the specific in vivo targets of Treg modalities. Herein, we have demonstrated that Foxp3+ Tregs potently suppress autoimmune responses in vivo through inhibition of the autophagic machinery in DCs in a cytotoxic T-lymphocyte–associated protein 4–dependent (CTLA4-dependent) manner. Autophagy-deficient DCs exhibited reduced immunogenic potential and failed to prime autoantigen-specific CD4+ T cells to mediate autoimmunity. Mechanistically, CTLA4 binding promoted activation of the PI3K/Akt/mTOR axis and FoxO1 nuclear exclusion in DCs, leading to decreased transcription of the autophagy component microtubule-associated protein 1 light chain 3&bgr; (Lc3b). Human DCs treated with CTLA4-Ig, a fusion protein composed of the Fc region of IgG1 and the extracellular domain of CTLA4 (also known as abatacept, marketed as Orencia), demonstrated reduced levels of autophagosome formation, while DCs from CTLA4-Ig–treated rheumatoid arthritis patients displayed diminished LC3B transcripts. Collectively, our data identify the canonical autophagy pathway in DCs as a molecular target of Foxp3+ Treg–mediated suppression that leads to amelioration of autoimmune responses. These findings may pave the way for the development of therapeutic protocols that exploit Tregs for the treatment of autoimmunity as well as diseases in which disturbed tolerance is a common denominator.

Involvement of hepatitis B virus X gene (HBx) integration in hepatocarcinogenesis via a recombination of HBx/Alu core sequence/subtelomeric DNA

The significance of hepatitis B virus (HBV) DNA‐based integration in hepatocarcinogenesis is poorly understood. In the present study, we investigated whether the integration of HBV X gene (HBx) is involved in the event. Our finding showed that the integration of HBx fragment (316–462 bp/262–462 bp) was able to transform human immortalized normal liver LO2 cells using a cell model of HBx‐integration. We identified that the recombination, HBx/Alu core sequence/subtelomeric DNA, was required for the transformation, which could be detected in 5 out of 44 clinical HBx‐positive hepatocellular carcinoma tissues. Thus, we conclude that HBx integration is involved in the hepatocarcinogenesis.

Activin-A co-opts IRF4 and AhR signaling to induce human regulatory T cells that restrain asthmatic responses

Significance Here, we demonstrate that the cytokine activin-A instructs the differentiation of human IL-10–producing type 1 regulatory T (Tr1)-like cells that exhibit strongly suppressive functions against allergen-induced naive and effector CD4+ T-cell responses. In addition, we show that activin-A induces the activation of interferon regulatory factor (IRF4), which, along with aryl hydrocarbon receptor (AhR) and its binding partner, AhR nuclear translocator, forms a tripartite transcription factor complex that is essential for the differentiation and effector functions of human Tr1 cells. Importantly, administration of human activin-A–induced Tr1 cells in a humanized model of asthma confers protection against cardinal disease manifestations in preventive and therapeutic regimes. Collectively, our studies unravel a biological function for activin-A in the generation of suppressive human Tr1 cells that may be exploited for the control of allergic diseases. Type 1 regulatory T (Tr1) cells play a pivotal role in restraining human T-cell responses toward environmental allergens and protecting against allergic diseases. Still, the precise molecular cues that underlie their transcriptional and functional specification remain elusive. Here, we show that the cytokine activin-A instructs the generation of CD4+ T cells that express the Tr1-cell–associated molecules IL-10, inducible T-Cell costimulator (ICOS), lymphocyte activation gene 3 protein (LAG-3), and CD49b, and exert strongly suppressive functions toward allergic responses induced by naive and in vivo-primed human T helper 2 cells. Moreover, mechanistic studies reveal that activin-A signaling induces the activation of the transcription factor interferon regulatory factor (IRF4), which, along with the environmental sensor aryl hydrocarbon receptor, forms a multipartite transcriptional complex that binds in IL-10 and ICOS promoter elements and controls gene expression in human CD4+ T cells. In fact, IRF4 silencing abrogates activin-A–driven IL10 and ICOS up-regulation and impairs the suppressive functions of human activin-A–induced Tr1-like (act-A–iTr1) cells. Importantly, using a humanized mouse model of allergic asthma, we demonstrate that adoptive transfer of human act-A–iTr1 cells, both in preventive and therapeutic protocols, confers significant protection against cardinal asthma manifestations, including pulmonary inflammation. Overall, our findings uncover an activin-A–induced IRF4-aryl hydrocarbon receptor (AhR)–dependent transcriptional network, which generates suppressive human Tr1 cells that may be harnessed for the control of allergic diseases.

Micro-RNA analysis of renal biopsies in human lupus nephritis demonstrates up-regulated miR-422a driving reduction of kallikrein-related peptidase 4

BACKGROUND Aberrancies in gene expression in immune effector cells and in end-organs are implicated in lupus pathogenesis. To gain insights into the mechanisms of tissue injury, we profiled the expression of micro-RNAs in inflammatory kidney lesions of human lupus nephritis (LN). METHODS Kidney specimens were from patients with active proliferative, membranous or mixed LN and unaffected control tissue. Micro-RNAs were quantified by TaqMan Low Density Arrays. Bioinformatics was employed to predict gene targets, gene networks and perturbed signaling pathways. Results were validated by transfection studies (luciferase assay, real-time PCR) and in murine LN. Protein expression was determined by immunoblotting and immunohistochemistry. RESULTS Twenty-four micro-RNAs were dysregulated (9 up-regulated, 15 down-regulated) in human LN compared with control renal tissue. Their predicted gene targets participated in pathways associated with TGF-β, kinases, NF-κB, HNF4A, Wnt/β-catenin, STAT3 and IL-4. miR-422a showed the highest upregulation (17-fold) in active LN and correlated with fibrinoid necrosis lesions (β = 0.63, P = 0.002). In transfection studies, miR-422a was found to directly target kallikrein-related peptidase 4 (KLK4) mRNA. Concordantly, KLK4 mRNA was significantly reduced in the kidneys of human and murine LN and correlated inversely with miR-422a levels. Immunohistochemistry confirmed reduced KLK4 protein expression in renal mesangial and tubular epithelial cells in human and murine LN. CONCLUSIONS KLK4, a serine esterase with putative renoprotective properties, is down-regulated by miR-422a in LN kidney suggesting that, in addition to immune activation, local factors may be implicated in the disease.

Stochastic responses are not left to pure "chance".

Many coregulated genes assemble in multigene complexes via stochastic inter- and intrachromosomal interactions. In this issue, Fanucchi et al. report that chromatin loop formation governs hierarchical cotranscription within a multigene complex.

Dendritic cells conditioned by activin A–induced regulatory T cells exhibit enhanced tolerogenic properties and protect against experimental asthma

Background: Previously, we demonstrated that regulatory T (Treg) cells induced by the cytokine activin‐A suppress TH2‐mediated allergic responses and linked airway disease. Still, the effects of activin‐A–induced regulatory T (Act‐A‐iTreg) cells on the regulation of dendritic cell (DC)–driven allergic inflammation remain elusive. Objective: Here we investigated whether Act‐A‐iTreg cells can modulate DC responses and endow them with enhanced tolerogenic functions. Methods: Using adoptive cell transfer studies in mouse models of allergic airway disease, we examined the effects of Act‐A‐iTreg cells on DC phenotype, maturation status, and TH2 cell priming potential. Genome‐wide gene expression profiling characterized the transcriptional networks induced in tolerogenic DCs by Act‐A‐iTreg cells. The ability of DCs conditioned by Act‐A‐iTreg cells (Act‐A‐iTreg cell–modified DCs) to protect against experimental asthma, and the mechanisms involved were also explored. Results: Act‐A‐iTreg cell–modified DCs exhibited a significantly impaired capacity to uptake allergen and stimulate naive and TH2 effector responses on allergen stimulation in vivo accompanied by markedly attenuated inflammatory cytokine release in response to LPS. Gene‐profiling studies revealed that Act‐A‐iTreg cells dampened crucial TH2‐skewing transcriptional networks in DCs. Administration of Act‐A‐iTreg cell–modified DCs ameliorated cardinal asthma manifestations in preventive and therapeutic protocols through generation of strongly suppressive forkhead box P3+ Treg cells. Finally, programed death protein 1/programmed death ligand 1 signaling pathways were essential in potentiating the generation of DCs with tolerogenic properties by Act‐A‐iTreg cells. Conclusion: Our studies reveal that Act‐A‐iTreg cells instruct the generation of a highly effective immunoregulatory circuit encompassing tolerogenic DCs and forkhead box P3+ Treg cells that could be targeted for the design of novel immunotherapies for allergic disorders.

PS2:38 Comparative tissue transcriptome analysis by next-generation sequencing reveals novel pathways that characterise genetic susceptibility and developmental biology in systemic lupus erythematosus (sle)

Purpose Next-generation RNA-sequencing was applied to investigate SLE pathogenesis through a comparative transcriptomic analysis of a peripheral lymphoid organ (spleen) and end-organ tissues (kidneys, brain) of lupus-prone and healthy mice. Methods NZB/W-F1 lupus-prone mice were sacrificed at the pre-puberty, pre-autoimmunity and nephritic stage of the disease. Age-matched C57/BL6 mice were used as controls. Spleen, kidneys and brain were removed and total RNA was extracted. Paired-end RNA-sequencing was performed with Illumina HiSeq 2000 platform. Relative expression levels of transcripts and differentially expressed genes (DEG) (FC >1.5, p<0.05) were calculated. Functional enrichment analysis was performed with IPA, RNEA and gprofiler. Results To investigate SLE developmental biology, a comparative analysis between the same organs of the same model at different stages of the disease was performed. In the spleen, brain and kidneys of NZB/W-F1 mice, 277, 6 and 8 DEG at the pre-puberty vs pre-autoimmunity stage; 212, 6 and 8 DEG at the nephritic vs pre-puberty stage; and 15, 6 and 2 DEG at the nephritic vs pre-autoimmunity stage were identified, respectively. In the brain, kidneys and spleen, hierarchical clustering revealed 178, 1012 and 2105 genes respectively that were deregulated in at least 1 of 3 stages. Clusters were subjected to functional enrichment analysis. In the brain, genes were mainly downregulated and enriched for metabolic pathways (glycolysis/TCA cycle/Pentose-Phosphate pathway), whereas overexpressed genes were enriched for Jak/Stat signalling pathway. In kidneys, one of the biggest cluster points towards metabolic pathways, particularly to lipid metabolism. In the spleen, DEG were mainly overexpressed. Of note, early genes were particularly enriched in cell-cycle processes; intermediate genes in membrane-related and extracellular matrix functions; and late genes in inflammatory and immune response pathways. To investigate SLE genetic susceptibility, a comparative analysis of the same organ of lupus-prone vs healthy mice at different stages of the disease was performed. In addition to immune response pathways (interferon signalling/antigen-presentation), DEG were involved in canonical pathways such as the phagosome, platelet activation, epithelial adherence junction signalling and the extrinsic prothrombin activation pathway. Conclusions We identified novel stage-specific tissue-dependent pathways involved in immune response, and tissue injury and response in SLE. Validation is in progress.

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.

Autophagy orchestrates the regulatory program of tumor-associated myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) densely accumulate into tumors and potently suppress antitumor immune responses, promoting tumor development. Targeting MDSCs in tumor immunotherapy has been hampered by lack of understanding of the molecular pathways that govern MDSC differentiation and function. Herein, we identify autophagy as a crucial pathway for MDSC-mediated suppression of antitumor immunity. Specifically, MDSCs in patients with melanoma and mouse melanoma exhibited increased levels of functional autophagy. Ablation of autophagy in myeloid cells markedly delayed tumor growth and endowed antitumor immune responses. Notably, tumor-infiltrating autophagy-deficient monocytic MDSCs (M-MDSCs) demonstrated impaired suppressive activity in vitro and in vivo, whereas transcriptome analysis revealed substantial differences in genes related to lysosomal function. Accordingly, autophagy-deficient M-MDSCs exhibited impaired lysosomal degradation, thereby enhancing surface expression of MHC class II molecules, resulting in efficient activation of tumor-specific CD4+ T cells. Finally, targeting of the membrane-associated RING-CH1 (MARCH1) E3 ubiquitin ligase that mediates the lysosomal degradation of MHC II in M-MDSCs attenuated their suppressive function, and resulted in markedly decreased tumor volume followed by development of a robust antitumor immunity. Collectively, these findings depict autophagy as a molecular target of MDSC-mediated suppression of antitumor immunity.