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1.  Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells 
Science (New York, N.Y.)  2015;348(6238):1031-1035.
Inflammatory CD4+ T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. While selection of self-specific T cells in the thymus limits responses to tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells, and that MHCII+ ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on human colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4+ T cells in the intestine, and suggest that this process is dysregulated in human IBD.
PMCID: PMC4449822  PMID: 25908663
Commensal bacteria; T cell selection; innate lymphoid cell; inflammatory bowel disease
2.  RFX transcription factors are essential for hearing in mice 
Nature Communications  2015;6:8549.
Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.
Inner ear hair cells are non-regenerative mechanosensory cells essential for hearing. Here, with cell-type-specific expression analyses, the authors identify RFX transcription factors as central mediators of their survival during terminal differentiation and thus essential for hearing in mice.
PMCID: PMC4634137  PMID: 26469318
3.  RFX2 Is a Major Transcriptional Regulator of Spermiogenesis 
PLoS Genetics  2015;11(7):e1005368.
Spermatogenesis consists broadly of three phases: proliferation of diploid germ cells, meiosis, and finally extensive differentiation of the haploid cells into effective delivery vehicles for the paternal genome. Despite detailed characterization of many haploid developmental steps leading to sperm, only fragmentary information exists on the control of gene expression underlying these processes. Here we report that the RFX2 transcription factor is a master regulator of genes required for the haploid phase. A targeted mutation of Rfx2 was created in mice. Rfx2-/- mice are perfectly viable but show complete male sterility. Spermatogenesis appears to progress unperturbed through meiosis. However, haploid cells undergo a complete arrest in spermatid development just prior to spermatid elongation. Arrested cells show altered Golgi apparatus organization, leading to a deficit in the generation of a spreading acrosomal cap from proacrosomal vesicles. Arrested cells ultimately merge to form giant multinucleated cells released to the epididymis. Spermatids also completely fail to form the flagellar axoneme. RNA-Seq analysis and ChIP-Seq analysis identified 139 genes directly controlled by RFX2 during spermiogenesis. Gene ontology analysis revealed that genes required for cilium function are specifically enriched in down- and upregulated genes showing that RFX2 allows precise temporal expression of ciliary genes. Several genes required for cell adhesion and cytoskeleton remodeling are also downregulated. Comparison of RFX2-regulated genes with those controlled by other major transcriptional regulators of spermiogenesis showed that each controls independent gene sets. Altogether, these observations show that RFX2 plays a major and specific function in spermiogenesis.
Author Summary
Failure of spermatogenesis, which is presumed to often result from genetic defects, is a common cause of male sterility. Although numerous genes associated with defects in male spermatogenesis have been identified, numerous cases of genetic male infertility remain unelucidated. We report here that the transcription factor RFX2 is a master regulator of gene expression programs required for progression through the haploid phase of spermatogenesis. Male RFX2-deficient mice are completely sterile. Spermatogenesis progresses through meiosis, but haploid cells undergo a complete block in development just prior to spermatid elongation. Gene expression profiling and ChIP-Seq analysis revealed that RFX2 controls key pathways implicated in cilium/flagellum formation, as well as genes implicated in microtubule and vesicle associated transport. The set of genes activated by RFX2 in spermatids exhibits virtually no overlap with those controlled by other known transcriptional regulators of spermiogenesis, establishing RFX2 as an essential new player in this developmental process. RFX2-deficient mice should therefore represent a valuable new model for deciphering the regulatory networks that direct sperm formation, and thereby contribute to the identification of causes of human male infertility.
PMCID: PMC4498915  PMID: 26162102
4.  MHC Class II-Restricted Antigen Presentation by Plasmacytoid Dendritic Cells Drives Pro-Atherogenic T Cell Immunity 
Circulation  2014;130(16):1363-1373.
Plasmacytoid dendritic cells (pDCs) bridge innate and adaptive immune responses and are important regulators of immuno-inflammatory diseases. However, their role in atherosclerosis remains elusive.
Methods and Results
Here, we used genetic approaches to investigate the role of pDCs in atherosclerosis. Selective pDC deficiency in vivo was achieved using CD11c-Cre × Tcf4−/flox BM transplanted into Ldlr−/− mice. Compared to control Ldlr−/− chimeric mice, CD11c-Cre × Tcf4−/flox mice had reduced atherosclerosis levels. To begin to understand the mechanisms by which pDCs regulate atherosclerosis, we studied chimeric Ldlr−/− mice with selective MHCII deficiency on pDCs. Significantly, these mice also developed reduced atherosclerosis compared to controls without reductions in pDC numbers or changes in conventional DCs. MHCII-deficient pDCs showed defective stimulation of ApoB100-specific CD4+ T cells in response to native LDL, whereas production of IFN-α was not affected. Finally, the athero-protective effect of selective MHCII deficiency in pDCs was associated with significant reductions of pro-atherogenic T cell-derived Ifn-γ and lesional T cell infiltration, and was abrogated in CD4+ T cell-depleted animals.
This study supports a pro-atherogenic role for pDCs in murine atherosclerosis and identifies a critical role for MHCII-restricted antigen presentation by pDCs in driving pro-atherogenic T cell immunity.
PMCID: PMC4428652  PMID: 25223984
antigen; atherosclerosis; immune system; low-density lipoprotein; Plasmacytoid Dendritic cell; T cell; Interferon gamma
5.  NLRC5 Exclusively Transactivates MHC Class I and Related Genes through a Distinctive SXY Module 
PLoS Genetics  2015;11(3):e1005088.
MHC class II (MHCII) genes are transactivated by the NOD-like receptor (NLR) family member CIITA, which is recruited to SXY enhancers of MHCII promoters via a DNA-binding “enhanceosome” complex. NLRC5, another NLR protein, was recently found to control transcription of MHC class I (MHCI) genes. However, detailed understanding of NLRC5’s target gene specificity and mechanism of action remained lacking. We performed ChIP-sequencing experiments to gain comprehensive information on NLRC5-regulated genes. In addition to classical MHCI genes, we exclusively identified novel targets encoding non-classical MHCI molecules having important functions in immunity and tolerance. ChIP-sequencing performed with Rfx5−/− cells, which lack the pivotal enhanceosome factor RFX5, demonstrated its strict requirement for NLRC5 recruitment. Accordingly, Rfx5-knockout mice phenocopy Nlrc5 deficiency with respect to defective MHCI expression. Analysis of B cell lines lacking RFX5, RFXAP, or RFXANK further corroborated the importance of the enhanceosome for MHCI expression. Although recruited by common DNA-binding factors, CIITA and NLRC5 exhibit non-redundant functions, shown here using double-deficient Nlrc5−/−CIIta−/− mice. These paradoxical findings were resolved by using a “de novo” motif-discovery approach showing that the SXY consensus sequence occupied by NLRC5 in vivo diverges significantly from that occupied by CIITA. These sequence differences were sufficient to determine preferential occupation and transactivation by NLRC5 or CIITA, respectively, and the S box was found to be the essential feature conferring NLRC5 specificity. These results broaden our knowledge on the transcriptional activities of NLRC5 and CIITA, revealing their dependence on shared enhanceosome factors but their recruitment to distinct enhancer motifs in vivo. Furthermore, we demonstrated selectivity of NLRC5 for genes encoding MHCI or related proteins, rendering it an attractive target for therapeutic intervention. NLRC5 and CIITA thus emerge as paradigms for a novel class of transcriptional regulators dedicated for transactivating extremely few, phylogenetically related genes.
Author Summary
Major histocompatibility complex class I (MHCI) molecules are central to immunity and immunological disorders, and constitute a major obstacle in organ transplantation. It is therefore vital to gain insight into the regulation of their expression. NLRC5 was recently found to regulate MHCI gene transcription. However, we lack a thorough understanding of its target gene specificity and mechanism of action. Our work addresses these questions, delineating the unique consensus sequence required for NLRC5 recruitment and pinpointing conserved features conferring its specificity. Furthermore, through genome-wide analyses, we confirm that NLRC5 regulates classical MHCI genes and identify novel target genes, all encoding non-classical MHCI molecules exerting an array of functions in immunity and tolerance. We thereby demonstrate that NLRC5 exclusively transactivates genes of the MHCI pathway, rendering it an attractive target for future therapeutic intervention. The most striking feature of NLRC5 is its restricted and highly focused transcriptional activity, which has been described so far only for one related factor, CIITA. NLRC5 and CIITA therefore emerge as prototypes for a novel kind of extremely specific transcriptional regulator.
PMCID: PMC4374748  PMID: 25811463
6.  Anti-CD79 Antibody Induces B cell Anergy That Protects Against Autoimmunity 
B cells play a major role in the pathogenesis of many autoimmune disorders including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and type I diabetes mellitus, as indicated by the efficacy of B cell-targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell-targeted therapy, anti-CD20 monoclonal antibody, are contingent upon long-term depletion of peripheral B cells. Here, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell antigen receptor. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAb are do not require cell depletion, but rather act by inducing an anergic-like state. Thus, we describe a novel B cell-targeted approach predicated on the induction of B cell anergy.
PMCID: PMC3941979  PMID: 24442438
7.  Lymph node stromal cells acquire peptide–MHCII complexes from dendritic cells and induce antigen-specific CD4+ T cell tolerance 
The Journal of Experimental Medicine  2014;211(6):1153-1166.
LNSCs present peptide–MHCII complexes acquired from DCs to CD4+ T cells and induce T cell dysfunction by preventing their proliferation and survival.
Dendritic cells (DCs), and more recently lymph node stromal cells (LNSCs), have been described to tolerize self-reactive CD8+ T cells in LNs. Although LNSCs express MHCII, it is unknown whether they can also impact CD4+ T cell functions. We show that the promoter IV (pIV) of class II transactivator (CIITA), the master regulator of MHCII expression, controls endogenous MHCII expression by LNSCs. Unexpectedly, LNSCs also acquire peptide–MHCII complexes from DCs and induce CD4+ T cell dysfunction by presenting transferred complexes to naive CD4+ T cells and preventing their proliferation and survival. Our data reveals a novel, alternative mechanism where LN-resident stromal cells tolerize CD4+ T cells through the presentation of self-antigens via transferred peptide–MHCII complexes of DC origin.
PMCID: PMC4042642  PMID: 24842370
8.  HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus 
PLoS Genetics  2014;10(9):e1004577.
Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.
Author Summary
Cilia are small, specialized projections extending from a cell's surface that play key sensory and sometimes motility functions, such as generating fluid flow for clearing airways or sperm propulsion necessary for male fertility. Ciliary motility is defective in the inherited disease, Primary Ciliary Dyskinesia (PCD). Although the basic cilium blueprint has been elaborated on during evolution, many of the core genes involved in building or maintaining functional cilia have been conserved. We have used the comparatively simple fruit fly, which has motile cilia on only a handful of touch-sensitive sensory cells, to identify genes involved in ciliary motility and which are therefore candidate genes for causing PCD. We show here that when one such gene (CG31320/HEATR2) is disrupted in either flies or in human PCD patients, cilia form but they cannot move. We show this protein stays in the cytoplasm, where it is acts like a flexible scaffold stabilizing and facilitating interactions during the assembly of large multi-component ciliary motor complexes needed to power cilia movement.
PMCID: PMC4168999  PMID: 25232951
9.  Extensive remodeling of DC function by rapid maturation-induced transcriptional silencing 
Nucleic Acids Research  2014;42(15):9641-9655.
The activation, or maturation, of dendritic cells (DCs) is crucial for the initiation of adaptive T-cell mediated immune responses. Research on the molecular mechanisms implicated in DC maturation has focused primarily on inducible gene-expression events promoting the acquisition of new functions, such as cytokine production and enhanced T-cell-stimulatory capacity. In contrast, mechanisms that modulate DC function by inducing widespread gene-silencing remain poorly understood. Yet the termination of key functions is known to be critical for the function of activated DCs. Genome-wide analysis of activation-induced histone deacetylation, combined with genome-wide quantification of activation-induced silencing of nascent transcription, led us to identify a novel inducible transcriptional-repression pathway that makes major contributions to the DC-maturation process. This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation. The repressed genes function in pivotal processes—including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis—underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function. Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression.
PMCID: PMC4150779  PMID: 25104025
10.  Characterization of a surrogate murine antibody to model anti-human CD3 therapies 
mAbs  2013;5(4):555-564.
Fc-modified anti-human CD3ε monoclonal antibodies (mAbs) are in clinical development for the treatment of autoimmune diseases. These next generation mAbs have completed clinical trials in patients with type-1 diabetes and inflammatory bowel disease demonstrating a narrow therapeutic window. Lowered doses are ineffective, yet higher pharmacologically-active doses cause an undesirable level of adverse events. Thus, there is a critical need for a return to bench research to explore ways of improving clinical outcomes. Indeed, we recently reported that a short course of treatment affords synergy, providing long-term disease amelioration when combining anti-mouse CD3 and anti-mouse tumor necrosis factor mAbs in experimental arthritis. Such strategies may widen the window between risk and benefit; however, to more accurately assess experimentally the biology and pharmacology, reagents that mimic the current development candidates were required. Consequently, we engineered an Fc-modified anti-mouse CD3ε mAb, 2C11-Novi. Here, we report the functional characterization of 2C11-Novi demonstrating that it does not bind FcγR in vitro and elicits little cytokine release in vivo, while maintaining classical pharmacodynamic effects (CD3-TCR downregulation and T cell killing). Furthermore, we observed that oral administration of 2C11-Novi ameliorated progression of remitting-relapsing experimental autoimmune encephalitis in mice, significantly reducing the primary acute and subsequent relapse phase of the disease. With innovative approaches validated in two experimental models of human disease, 2C11-Novi represents a meaningful tool to conduct further mechanistic studies aiming at exploiting the immunoregulatory properties of Fc-modified anti-CD3 therapies via combination therapy using parenteral or oral routes of administration.
PMCID: PMC3906309  PMID: 23751612
Monoclonal antibody; anti-CD3; oral antibody; EAE; pharmacodynamics; pharmacokinetics; T cell activation
11.  Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis 
PLoS ONE  2014;9(1):e86844.
Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4+ T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4+ T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4+ T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4+ T cells and forkhead box P3 (FoxP3)+ regulatory T (Treg) cells. IFN-γ produced mainly by CD4+ T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.
PMCID: PMC3904943  PMID: 24489792
12.  TREM-1 Deficiency Can Attenuate Disease Severity without Affecting Pathogen Clearance 
PLoS Pathogens  2014;10(1):e1003900.
Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1−/− mice are viable, fertile and show no altered hematopoietic compartment. In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Trem1−/− mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1−/− mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1−/− mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1−/− mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1+/+ controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.
Author Summary
Triggering receptor expressed on myeloid cells-1 (TREM-1) is an immune receptor expressed by myeloid cells that has the capacity to augment pro-inflammatory responses in the context of a microbial infection. While a TREM-1-amplified response likely serves the efficient clearance of pathogens, it also bears the potential to cause substantial tissue damage or even death. Hence, TREM-1 appears a possible therapeutic target for tempering deleterious host-pathogen interactions. However, in models of bacterial sepsis controversial findings have been obtained regarding the requirement of TREM-1 for bacterial control - depending on the overall degree of the TREM-1 blockade that was achieved. In order to conclusively investigate harmful versus essential functions of TREM-1 in vivo, we have generated mice deficient in Trem1. Trem1−/− mice were subjected to experimentally-induced intestinal inflammation (as a model of a non-infectious, yet microbial-driven disease) and also analysed following infections with Leishmania major, influenza virus and Legionella pneumophila. Across all models analysed, Trem1−/− mice showed substantially reduced immune-associated disease. We thus describe a previously unanticipated pathogenic role for TREM-1 also during a parasitic and viral infection. Importantly, our data suggest that in certain diseases microbial control can be achieved in the context of blunted inflammation in the absence of TREM-1.
PMCID: PMC3894224  PMID: 24453980
13.  NOX1 is responsible for cell death through STAT3 activation in hyperoxia and is associated with the pathogenesis of Acute Respiratory Distress Syndrome 
Reactive oxygen species (ROS) contribute to alveolar cell death in Acute Respiratory Distress Syndrome (ARDS) and we previously demonstrated that NOX1-derived ROS contributed to hyperoxia-induced alveolar cell death in mice. The study investigates whether NOX1 expression is modulated in epithelial cells concomitantly to cell death and associated to STAT3 signaling in the exudative phase of ARDS. In addition, the role of STAT3 activation in NOX1-dependent epithelial cell death was confirmed by using a lung epithelial cell line and in mice exposed to hyperoxia. NOX1 expression, cell death and STAT3 staining were evaluated in the lungs of control and ARDS patients by immunohistochemistry. In parallel, a stable NOX1-silenced murine epithelial cell line (MLE12) and NOX1-deficient mice were used to characterize signalling pathways. In the present study, we show that NOX1 is detected in alveolar epithelial cells of ARDS patients in the exudative stage. In addition, increased alveolar epithelial cell death and phosphorylated STAT3 are observed in ARDS patients and associated with NOX1 expression. Phosphorylated STAT3 is also correlated with TUNEL staining. We also confirmed that NOX1-dependent STAT3 activation participates to alveolar epithelial cell death. Silencing and acute inhibition of NOX1 in MLE12 led to decreased cell death and cleaved-caspase 3 induced by hyperoxia. Additionally, hyperoxia-induced STAT3 phosphorylation is dependent on NOX1 expression and associated with cell death in MLE12 and mice. This study demonstrates that NOX1 is involved in human ARDS pathophysiology and is responsible for the damage occurring in alveolar epithelial cells at least in part via STAT3 signalling pathways.
PMCID: PMC3925898  PMID: 24551274
NOX1; ARDS; cell death; hyperoxia; STAT3; ROS
14.  Antigen Recognition By Autoreactive Cd4+ Thymocytes Drives Homeostasis Of The Thymic Medulla 
PLoS ONE  2012;7(12):e52591.
The thymic medulla is dedicated for purging the T-cell receptor (TCR) repertoire of self-reactive specificities. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process because they express numerous peripheral tissue-restricted self-antigens. Although it is well known that medulla formation depends on the development of single-positive (SP) thymocytes, the mechanisms underlying this requirement are incompletely understood. We demonstrate here that conventional SP CD4+ thymocytes bearing autoreactive TCRs drive a homeostatic process that fine-tunes medullary plasticity in adult mice by governing the expansion and patterning of the medulla. This process exhibits strict dependence on TCR-reactivity with self-antigens expressed by mTECs, as well as engagement of the CD28-CD80/CD86 costimulatory axis. These interactions induce the expression of lymphotoxin α in autoreactive CD4+ thymocytes and RANK in mTECs. Lymphotoxin in turn drives mTEC development in synergy with RANKL and CD40L. Our results show that Ag-dependent interactions between autoreactive CD4+ thymocytes and mTECs fine-tune homeostasis of the medulla by completing the signaling axes implicated in mTEC expansion and medullary organization.
PMCID: PMC3531460  PMID: 23300712
15.  The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development 
PLoS Genetics  2012;8(3):e1002606.
The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.
Author Summary
The Corpus Callosum is the major brain commissure that links the two cerebral hemispheres in mammals. Absence or reduction of the corpus callosum is the most frequent brain malformation observed at birth in humans and leads to cognitive and behavioural deficits. Agenesis of the Corpus Callosum is frequently observed in ciliopathies, a group of human diseases due to defects in cilia assembly or function. However, the cellular origin of this brain malformation in these syndromes remains elusive. RFX3 transcription factor is a key regulator of ciliogenesis in mouse. Here, we show that the Rfx3 mutant mouse shows impaired Corpus Callosum formation. By transplantation experiments, we demonstrate that this is due to defective distribution between the two hemispheres of a transient neuronal population (guidepost neurons) required for routing callosal axons. We show that this abnormal distribution is due to altered FGF8 signalling at early stages of brain development. Our observations show that small but focused signalling defects resulting in specific alterations in the distribution of guidepost neurons are responsible for corpus callosum agenesis.
PMCID: PMC3315471  PMID: 22479201
16.  Novel Murine Dendritic Cell Lines: A Powerful Auxiliary Tool for Dendritic Cell Research 
Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.
PMCID: PMC3491238  PMID: 23162549
dendritic cell line; in vitro research
17.  The Transcription Factor Rfx3 Regulates β-Cell Differentiation, Function, and Glucokinase Expression 
Diabetes  2010;59(7):1674-1685.
Pancreatic islets of perinatal mice lacking the transcription factor Rfx3 exhibit a marked reduction in insulin-producing β-cells. The objective of this work was to unravel the cellular and molecular mechanisms underlying this deficiency.
Immunofluorescence studies and quantitative RT-PCR experiments were used to study the emergence of insulin-positive cells, the expression of transcription factors implicated in the differentiation of β-cells from endocrine progenitors, and the expression of mature β-cell markers during development in Rfx3−/− and pancreas-specific Rfx3-knockout mice. RNA interference experiments were performed to document the consequences of downregulating Rfx3 expression in Min6 β-cells. Quantitative chromatin immunoprecipitation (ChIP), ChIP sequencing, and bandshift experiments were used to identify Rfx3 target genes.
Reduced development of insulin-positive cells in Rfx3−/− mice was not due to deficiencies in endocrine progenitors or β-lineage specification, but reflected the accumulation of insulin-positive β-cell precursors and defective β-cells exhibiting reduced insulin, Glut-2, and Gck expression. Similar incompletely differentiated β-cells developed in pancreas-specific Rfx3-deficient embryos. Defective β-cells lacking Glut-2 and Gck expression dominate in Rfx3-deficent adults, leading to glucose intolerance. Attenuated Glut-2 and glucokinase expression, and impaired glucose-stimulated insulin secretion, were also induced by RNA interference–mediated inhibition of Rfx3 expression in Min6 cells. Finally, Rfx3 was found to bind in Min6 cells and human islets to two well-known regulatory sequences, Pal-1 and Pal-2, in the neuroendocrine promoter of the glucokinase gene.
Our results show that Rfx3 is required for the differentiation and function of mature β-cells and regulates the β-cell promoter of the glucokinase gene.
PMCID: PMC2889767  PMID: 20413507
18.  MHC class II–restricted antigen presentation by plasmacytoid dendritic cells inhibits T cell–mediated autoimmunity 
The Journal of Experimental Medicine  2010;207(9):1891-1905.
Although plasmacytoid dendritic cells (pDCs) express major histocompatibility complex class II (MHCII) molecules, and can capture, process, and present antigens (Ags), direct demonstrations that they function as professional Ag-presenting cells (APCs) in vivo during ongoing immune responses remain lacking. We demonstrate that mice exhibiting a selective abrogation of MHCII expression by pDCs develop exacerbated experimental autoimmune encephalomyelitis (EAE) as a consequence of enhanced priming of encephalitogenic CD4+ T cell responses in secondary lymphoid tissues. After EAE induction, pDCs are recruited to lymph nodes and establish MHCII-dependent myelin-Ag–specific contacts with CD4+ T cells. These interactions promote the selective expansion of myelin-Ag–specific natural regulatory T cells that dampen the autoimmune T cell response. pDCs thus function as APCs during the course of EAE and confer a natural protection against autoimmune disease development that is mediated directly by their ability to present of Ags to CD4+ T cells in vivo.
PMCID: PMC2931160  PMID: 20696698
19.  DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells 
Immunome Research  2010;6:10.
The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs).
Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules.
The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.
PMCID: PMC3000836  PMID: 21092113
20.  Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts 
Extracellular matrix (ECM) turnover is controlled by the synthetic rate of matrix proteins, including type I collagen, and their enzymatic degradation by matrix metalloproteinases (MMPs). Fibrosis is characterized by an unbalanced accumulation of ECM leading to organ dysfunction as observed in systemic sclerosis. We previously reported that proteasome inhibition (PI) in vitro decreases type I collagen and enhances MMP-1 production by human fibroblasts, thus favoring an antifibrotic fibroblast phenotype. These effects were dominant over the pro-fibrotic phenotype induced by transforming growth factor (TGF)-β. Here we investigate the molecular events responsible for the anti-fibrotic phenotype induced in fibroblasts by the proteasome inhibitor bortezomib.
The steady-state mRNA levels of COL1A1, COL1A2, TIMP-1, MMP-1, and MMP-2 were assessed by quantitative PCR in human dermal fibroblasts cultured in the presence of TGF-β, bortezomib, or both. Transient fibroblast transfection was performed with wild-type and mutated COL1A1 and MMP-1 promoters. Chromatin immunoprecipitation, electrophoretic mobility shift assay (EMSA), and DNA pull-down assays were used to assess the binding of c-Jun, SP1, AP2, and Smad2 transcription factors. Immunoblotting and immunofluorescent microscopy were performed for identifying phosphorylated transcription factors and their cellular localization.
Bortezomib decreased the steady-state mRNA levels of COL1A1 and COL1A2, and abrogated SP1 binding to the promoter of COL1A2 in both untreated and TGF-β-activated fibroblasts. Reduced COL1A2 expression was not due to altered TGF-β-induced Smad2 phosphorylation, nuclear translocation, or binding to the COL1A2 promoter. In contrast to collagen, bortezomib specifically increased the steady-state mRNA levels of MMP-1 and enhanced the binding of c-Jun to the promoter of MMP-1. Furthermore, disruption of the proximal AP-1-binding site in the promoter of MMP-1 severely impaired MMP-1 transcription in response to bortezomib.
By altering the binding of at least two transcription factors, c-Jun and SP1, proteasome inhibition results in increased production of MMP-1 and decreased synthesis of type I collagen in human dermal fibroblasts. Thus, the antifibrotic phenotype observed in fibroblasts submitted to proteasome inhibition results from profound modifications in the binding of key transcription factors. This provides a novel rationale for assessing the potential of drugs targeting the proteasome for their anti-fibrotic properties.
PMCID: PMC2888229  PMID: 20429888
21.  MHC Class II Molecules Enhance Toll-Like Receptor Mediated Innate Immune Responses 
PLoS ONE  2010;5(1):e8808.
Major histocompatibility complex (MHC) class II molecules play crucial roles in immune activation by presenting foreign peptides to antigen-specific T helper cells and thereby inducing adaptive immune responses. Although adaptive immunity is a highly effective defense system, it takes several days to become fully operational and needs to be triggered by danger-signals generated during the preceding innate immune response. Here we show that MHC class II molecules synergize with Toll-like receptor (TLR) 2 and TLR4 in inducing an innate immune response.
Methodology/Principal Findings
We found that co-expression of MHC class II molecules and TLR2 or TLR4 in human embryonic kidney (HEK) cells 293 leads to enhanced production of the anti-microbial peptide human-β-defensin (hBD) 2 after treatment with TLR2 stimulus bacterial lipoprotein (BLP) or TLR4 ligand lipopolysaccharide (LPS), respectively. Furthermore, we found that peritoneal macrophages of MHC class II knock-out mice show a decreased responsiveness to TLR2 and TLR4 stimuli compared to macrophages of wild-type mice. Finally, we show that MHC class II molecules are physically and functionally associated with TLR2 in lipid raft domains of the cell membrane.
These results demonstrate that MHC class II molecules are, in addition to their central role in adaptive immunity, also implicated in generating optimal innate immune responses.
PMCID: PMC2808354  PMID: 20098705
22.  New dimensions to CIITA 
Nature immunology  2008;9(7):713-714.
PMCID: PMC2659346  PMID: 18563077
23.  Nucleosome eviction from MHC class II promoters controls positioning of the transcription start site 
Nucleic Acids Research  2009;37(8):2514-2528.
Nucleosome depletion at transcription start sites (TSS) has been documented genome-wide in multiple eukaryotic organisms. However, the mechanisms that mediate this nucleosome depletion and its functional impact on transcription remain largely unknown. We have studied these issues at human MHC class II (MHCII) genes. Activation-induced nucleosome free regions (NFR) encompassing the TSS were observed at all MHCII genes. Nucleosome depletion was exceptionally strong, attaining over 250-fold, at the promoter of the prototypical HLA-DRA gene. The NFR was induced primarily by the transcription factor complex that assembles on the conserved promoter-proximal enhancer situated upstream of the TSS. Functional analyses performed in the context of native chromatin demonstrated that displacing the NFR without altering the sequence of the core promoter induced a shift in the position of the TSS. The NFR thus appears to play a critical role in transcription initiation because it directs correct TSS positioning in vivo. Our results provide support for a novel mechanism in transcription initiation whereby the position of the TSS is controlled by nucleosome eviction rather than by promoter sequence.
PMCID: PMC2677874  PMID: 19264803
24.  The NLR gene family: An official nomenclature 
Immunity  2008;28(3):285-287.
Iimmune regulatory proteins such as CIITA, NAIP, IPAF, NOD1, NOD2, NALP1, cryopyrin/NALP3 are members of a family characterized by the presence of a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). Members of this gene family encode a protein structure similar to the NB-LRR subgroup of disease-resistance genes in plants and are involved in the sensing of pathogenic products and the regulation of cell signaling and apoptosis. Several members of this family have been associated with immunologic disorders. NOD2 for instance is associated with both Crohn's disease and Blau syndrome.
A variety of different names are currently used to describe this gene family, its subfamilies and individual genes, including CATERPILLER (CLR), NOD-LRR, NACHT-LRR, CARD, NALP, NOD, PAN and PYPAF, and this lack of consistency has led to a pressing need to unify the nomenclature. Consequently, we collectively propose the family designation NLR (nucleotide-binding domain and leucine-rich repeat containing) and provide unique and standardized gene designations for all family members.
PMCID: PMC2630772  PMID: 18341998
25.  Identification of CIITA Regulated Genetic Module Dedicated for Antigen Presentation 
PLoS Genetics  2008;4(4):e1000058.
The class II trans-activator CIITA is a transcriptional co-activator required for the expression of Major Histocompatibility Complex (MHC) genes. Although the latter function is well established, the global target-gene specificity of CIITA had not been defined. We therefore generated a comprehensive list of its target genes by performing genome-wide scans employing four different approaches designed to identify promoters that are occupied by CIITA in two key antigen presenting cells, B cells and dendritic cells. Surprisingly, in addition to MHC genes, only nine new targets were identified and validated by extensive functional and expression analysis. Seven of these genes are known or likely to function in processes contributing to MHC-mediated antigen presentation. The remaining two are of unknown function. CIITA is thus uniquely dedicated for genes implicated in antigen presentation. The finding that CIITA regulates such a highly focused gene expression module sets it apart from all other transcription factors, for which large-scale binding-site mapping has indicated that they exert pleiotropic functions and regulate large numbers of genes.
Author Summary
Most mammalian transcription factors and transcriptional co-activators are believed to regulate the activities of numerous genes fulfilling multiple functions. This pleiotropic role has recently been confirmed directly for several individual factors by large-scale mapping studies aimed at generating comprehensive catalogues of their binding sites in the genome. Until now, all transcription factors, for which such studies have been performed, were found to regulate hundreds or even thousands of genes. We demonstrate, here, that the transcriptional co-activator CIITA (class II transactivator) is an exception to this rule. CIITA is a key regulator of the immune system because it controls the transcription of genes coding for Major Histocompatibility Complex (MHC) class II molecules, which are cell-surface molecules that present peptide antigens to T lymphocytes. To address the possibility that CIITA might exert more widespread functions, we have performed extensive genome-wide searches to establish a comprehensive list of CIITA-regulated genes. Surprisingly, we found that CIITA regulates only a small number of genes, most of which code for proteins implicated directly or indirectly in MHC-mediated antigen presentation. CIITA is thus remarkably dedicated for the regulation of a unique set of functionally related genes constituting a genetic module devoted to a single biological process.
PMCID: PMC2278383  PMID: 18437201

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