Division of Cardiovascular Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom; Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; Institut National de la Santé et de la Recherche Médicale, Unit 970, Paris Cardiovascular Research Center, Paris, France; Dept of Pharmacy, University of Naples Federico II, Naples, Italy; Institute of Immunobiology, Kantonal Hospital St. Gallen, St. Gallen, Switzerland; Dept of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland; Center for Molecular Medicine, Dept of Medicine, Karolinska University Hospital, Karolinska Institute, Stockholm, Sweden; Dept of Microbiology and Immunology, Columbia University Medical Center, New York; Dept of Pathology, University of Geneva Medical School, Geneva, Switzerland

It is well established that interactions between CD4(+) T cells and major histocompatibility complex class II (MHCII) positive antigen-presenting cells (APCs) of hematopoietic origin play key roles in both the maintenance of tolerance and the initiation and development of autoimmune and inflammatory disorders. In sharp contrast, despite nearly three decades of intensive research, the functional relevance of MHCII expression by non-hematopoietic tissue-resident cells has remained obscure. The widespread assumption that MHCII expression by non-hematopoietic APCs has an impact on autoimmune and inflammatory diseases has in most instances neither been confirmed nor excluded by indisputable in vivo data. Here we review and put into perspective conflicting in vitro and in vivo results on the putative impact of MHCII expression by non-hematopoietic APCs - in both target organs and secondary lymphoid tissues - on the initiation and development of representative autoimmune and inflammatory disorders. Emphasis will be placed on the lacunar status of our knowledge in this field. We also discuss new mouse models - developed on the basis of our understanding of the molecular mechanisms that regulate MHCII expression - that constitute valuable tools for filling the severe gaps in our knowledge on the functions of non-hematopoietic APCs in inflammatory conditions.

Nucleotide-binding oligomerization domain-like receptors (NLRs) are intracellular proteins involved in innate-driven inflammatory responses. The function of the family member NLR caspase recruitment domain containing protein 5 (NLRC5) remains a matter of debate, particularly with respect to NF-κB activation, type I IFN, and MHC I expression. To address the role of NLRC5, we generated Nlrc5-deficient mice (Nlrc5Δ/Δ). In this article we show that these animals exhibit slightly decreased CD8+ T cell percentages, a phenotype compatible with deregulated MHC I expression. Of interest, NLRC5 ablation only mildly affected MHC I expression on APCs and, accordingly, Nlrc5Δ/Δ macrophages efficiently primed CD8+ T cells. In contrast, NLRC5 deficiency dramatically impaired basal expression of MHC I in T, NKT, and NK lymphocytes. NLRC5 was sufficient to induce MHC I expression in a human lymphoid cell line, requiring both caspase recruitment and LRR domains. Moreover, endogenous NLRC5 localized to the nucleus and occupied the proximal promoter region of H-2 genes. Consistent with downregulated MHC I expression, the elimination of Nlrc5Δ/Δ lymphocytes by cytotoxic T cells was markedly reduced and, in addition, we observed low NLRC5 expression in several murine and human lymphoid-derived tumor cell lines. Hence, loss of NLRC5 expression represents an advantage for evading CD8+ T cell-mediated elimination by downmodulation of MHC I levels—a mechanism that may be exploited by transformed cells. Our data show that NLRC5 acts as a key transcriptional regulator of MHC I in lymphocytes and support an essential role for NLRs in directing not only innate but also adaptive immune responses.

The thymus ensures the generation of a diverse repertoire of T lymphocytes capable of mounting immune responses directed against pathogens while avoiding autoimmune attacks directed at self-antigens. The thymic medulla provides a specialized microenvironment dedicated to purging the T-cell repertoire of potentially hazardous self-reactive specificities, and thus plays a pivotal role in preventing the development of autoimmune disorders. Medullary thymic epithelial cells (mTECs) play a pivotal role in establishing T-cell tolerance via their unique capacity to express thousands of peripheral tissue-restricted self-antigens. Conversely, formation of the medulla is controlled by the development of single-positive (SP) thymocytes. However, the precise identities of the thymocyte subset and cytokines involved in this process remain poorly documented. By means of knock-out mice lacking CD4+ or CD8+ SP thymocytes, T cell receptor (TCR)-transgenic mice and in vitro reaggregated thymic organ culture experiments, we have studied the respective contributions of SP CD4+ and CD8+ thymocytes in medulla formation. We have also studied by using fetal thymic organ culture (FTOC) experiments the role of three members of the tumor necrosis factor (TNF) superfamily, namely lymphotoxin (LT), RANK ligand (RANKL) and CD40 ligand (CD40L), in mTEC development and homeostasis. We found that SP CD4+ thymocytes are indispensable and sufficient for inducing proper medulla formation. In contrast, CD8+ SP thymocytes are much less efficient and dispensable for sustaining this process. Furthermore, TCR reactivity of SP CD4+ thymocytes with self-antigens expressed by mTECs is compulsory for mTEC development and homeostasis. Importantly, we found that these antigen-dependent interactions with mTECs induce the expression of LTa in autoreactive CD4+thymocytes and the RANKL receptor in mTECs. By using FTOC experiments, we found that of the three individual TNF stimuli (LT, RANKL and CD40L) only the LT induces a significant increase in mTEC cellularity. Remarkably, a synergistic increase in mTEC numbers was observed when LT was combined with RANKL and CD40L. Therefore, LTa expression by autoreactive CD4+thymocytes controls mTEC cellularity by acting in synergy with RANKL and CD40L. Our study demonstrates that antigen-dependent interactions between SP CD4+ thymocytes and mTECs finely regulate the expression of LTa in autoreactive CD4+ thymocytes and RANK in mTECs, thereby completing the signaling axes that are critical for mTEC development and homeostasis, processes pivotal for T-cell tolerance induction.

Si les roles fonctionnels de diverses cellules immunitaires infiltrant des tissus enflammes sont assez bien compris, par contre, etonnamment, on connait bien moins la capacite des cellules non hematopoietiques residant dans des tissus, a moduler l'activite biologique des cellules immunitaires immigrantes, et donc le resultat de la reponse immunitaire. La presentation des antigenes, dans le contexte des molecules du CMH de classe II (CMHII) a la surface des cellules presentatrices d'antigenes (CPA) professionnelles a une sous- population de lymphocytes T, est cruciale pour le developpement des reponses immunitaires protectives specifiques de l'antigene. En general, l'expression de CMHII est reservee aux CPAs. Toutefois, au cours des pathologies inflammatoires specifiques d'organe, telles que l'auto-immunite ou la maladie inflammatoire de l'intestin, l'expression de CMHII est egalement induite par la cytokine interferon (IFN)-y sur des cellules non hematopoietiques qui resident dans des tissus enflammes. Les consequences de ce phenomene sont encore peu comprises. Dans cette etude, nous avons utilise une souche de souris genetiquement modifiees, qui n'a pas la capacite d'induire l'expression de CMHII sur les cellules non hematopoietiques, mais a maintenu la regulation normale d'expression de CMHII sur les cellules hematopoietiques. Nous avons applique ces souris a differents modeles d'inflammation intestinale et a un modele de maladie qui imite la maladie auto-immune de l'inflammation du muscle cardiaque (myocardite) chez l'homme. Nous avons pu montrer que, au cours de l'inflammation intestinale, l'expression du CMHII nonhematopoietique, ou encore l'expression du CMHII par les cellules epitheliales de l'intestin, confere une protection contre la maladie, en reduisant les cellules immunitaires inflammatoires et en augmentant les cellules Τ regulatrices anti-inflammatoires. Ces resultats pourraient expliquer l'echec des traitements d'anti-IFN-γ dans les maladies intestinales inflammatoires chez l'homme. En revanche, dans la myocardite auto-immune, nos resultats indiquent que la presentation d'antigenes par les cellules non hematopoietiques du coeur est necessaire pour l'apparition de la pathologie cardiaque, comme nos souris sont resistantes a la maladie. Toutefois, cela n'est pas du a un defaut d'activation des lymphocytes T, car les lymphocytes Τ des souris mutantes sont parfaitement capables de promouvoir la maladie apres le transfert adoptif dans des animaux de type naturel. Nos resultats suggerent que, durant les maladies inflammatoires specifiques d'organe, la presentation d'antigene par des cellules non hematopoietiques module et contribue au resultat de la reponse immunitaire d'une maniere opposee, conferant soit la protection contre la maladie ou sa promotion. Nos resultats pourraient ouvrir la voie a des therapies qui prennent en compte la contribution de la presentation d'antigenes par les cellules non hematopoietiques, au cours des maladies inflammatoires specifiques d'organe. - Les molecules du CMH de classe II (CMHII) sont fondamentales pour la presentation des antigenes aux lymphocytes Τ CD4+, car elles permettent le developpement des reponses immunitaires specifiques de l'antigene. Il est largement admis que l'expression de CMHII est reservee aux cellules presentatrices d'antigenes (CPA). Cependant, dans des conditions inflammatoires, l'expression de CMHII est en principe egalement induite par l'interferon (IFN)-y sur les cellules non hematopoietiques, telles que les cellules epitheliales et les cardiomyocytes. Une controverse existe jusqu'a present au sujet de la fonction de cette presentation d'antigenes non professionnelle, pour savoir si elle favorise la tolerance ou l'immunite dependante des lymphocytes Τ in vivo. Pour repondre a cette question, nous avons teste des souris qui ne sont pas capables d'induire l'expression du CMHII sur les cellules non hematopoietiques (souris PIV-/- K14 CIITA Tg) parmi differents modeles murins de pathologies inflammatoires, a savoir les modeles de vaccination pour induire des reponses specifiques d'antigenes des lymphocytes B, plusieurs modeles de colite et un modele de myocardite auto-immune experimental (EAM). Pour cela, nous avons administre a ces souris un modele de colite attenuee, induite par une infection chronique a Helicobacter hepaticus et par l'administration d'anticorps monoclonaux bloquant le recepteur de l'interleukine (IL)-10 (anti-IL-10R). Dans ce systeme, nous avons pu observer que l'expression abrogee de CMHII a aggrave la colite bacterienne, soit par les cellules non hematopoietiques, soit exclusivement par les cellules epitheliales intestinales (CEI) dans un autre modele murin (souris plV_fl/fl vil-Cre Tg). Ce phenotype du colon a ete associe a une augmentation des frequences de cellules immunitaires innees, de lymphocytes Th1 CD4+, et d'expression des cytokines et de chimiokines pro-inflammatoires, y compris l'IFN-γ. Notamment, l'expression defectueuse de CMHII non hematopoietique a egalement reduit les cellules Τ regulatrices (Treg) Forkhead box P3 (FoxP3)+, sans influencer les frequences des cellules innees lymphoides et des cellules Th17. Ces resultats suggerent un role tolerogene de CEIs CMHII+ qui contribue a l'homeostasie immunitaire intestinale. En revanche, dans le modele d'EAM, les souris ayant subi une ablation de CMHII non hematopoietique etaient resistantes a l'induction de la maladie, alors que la progression de la pathologie cardiaque, dans les souris de type naturel ou heterozygotes, a ete accompagnee par une regulation positive de l'expression de CMHII du myocarde. Cependant, l'inflammation cardiaque pourrait etre transferee de maniere adoptive depuis des souris amorcees PIV-/- K14 CIITA Tg vers des souris de type naturel, indiquant l'absence de defaut intrinseque d'amorcage des cellules T CD4+ dans notre modele de souris. Ces observations impliquent un role a jouer pour des cellules CMHII+ non hematopoietiques residentes du coeur, dans la promotion active de ΙΈΑΜ. En conclusion, nos resultats, provenant de diverses pathologies inflammatoires specifiques d'organes, suggerent un role complexe et divergent, soit tolerogene, soit immunogene/ pathologique, pour l'expression de CMHII non hematopoietique au cours des pathologies inflammatoires. L'expression non professionnelle de CMHII semble influencer le resultat des reponses immunitaires en fonction de differents facteurs, tels que le tissu cible, le(s) type(s) de cellule(s) non hematopoietique(s) participante(s) et l'origine de l'inflammation. Nos resultats pourraient potentiellement ouvrir la voie a des applications therapeutiques, qui tiennent compte de la contribution de la presentation d'antigenes par des CPAs non professionnelles, au cours de l'inflammation specifique d'organe. - MHC class II (MHCII) molecules are fundamental for the presentation of antigens to CD4+ Τ cells, allowing the development of antigen-specific immune responses. It is widely accepted that MHCII expression is restricted to antigen-presenting cells (APC). However, under inflammatory conditions, MHCII expression is typically also induced by interferon (IFN)-y on nonhematopoietic cells such as epithelial cells and cardiomyocytes. So far, it remains controversial whether this nonprofessional antigen-presentation function promotes CD4+ Τ cell-dependent tolerance or immunity in vivo. To address this issue, we utilised mice which lack inducible MHCII expression on nonhematopoietic cells (pIV-/- K14 CIITA Tg mice) in different mouse models of inflammatory pathologies, namely immunisation models to induce antigen-specific Β cell responses, various colitis models and a model of experimental autoimmune myocarditis (EAM). In an attenuated model of colitis induced by chronic Helicobacter hepaticus infection and treatment with anti-interleukin (IL)-10 receptor (anti-IL-10R) monoclonal blocking antibody, we observed that abrogated MHCII expression by nonhematopoietic cells or, in an alternative tamoxifen-inducible mouse model (plV_fl/fl vil-Cre Tg mice), exclusively by intestinal epithelial cells (IEC), exacerbated bacterial-driven colitis, which was associated with increased colonic frequencies of innate immune cells, CD4+ Th1 cells and expression of proinflammatory cytokines and chemokines, including IFN-γ. Notably, defective nonhematopoietic MHCII expression also resulted in reduced Forkhead box P3 (FoxP3)+ regulatory Τ (Treg) cells without influencing innate lymphoid cell (ILC) and Th17 cell frequencies. These findings suggest a tolerogenic role of MHClT lECs to contribute to intestinal immune homeostasis. In contrast, in the EAM model, mice ablated of nonhematopoietic MHCII were resistant to disease induction, whereas progression of cardiac pathology in WT and heterozygous control mice was accompanied by upregulation of myocardial MHCII expression. However, cardiac inflammation could be adoptively transferred from primed pIV-/- K14 CIITA Tg mice into WT mice, indicating no intrinsic defect of CD4+ Τ activation in our mouse model. These observations imply a role for MHCIT heart-resident nonhematopoietic cells in actively promoting EAM. In conclusion, our findings from different organ-specific inflammatory pathologies suggest a complex and diverging role - either tolerogenic or immunogenic/ pathologic - for nonhematopoietic MHCII expression during inflammatory pathologies: Nonprofessional MHCII expression appears to influence the outcome of immune responses depending on 7 factors such as the target tissue, participating non hematopoietic cell type(s) and the origin of inflammation. Our findings may potentially open the way to therapeutic applications taking into account the contribution of antigen presentation by nonprofessional, tissue-resident APCs during organ-specific inflammation.

N-cadherin/LRP5 interaction, indicating that this interaction implies the last 62 AA of N-cadherin. Consistent with this finding, transfection with Δ62 N-cadherin restored the defective β-catenin transcriptional activity induced by full length N-cadherin overexpression in osteoblasts. Furthermore, transfection of osteoblasts with Δ153 or Δ114 N-cadherin enhanced β-catenin transcriptional activity in the presence or absence of Wnt3a. We also found that transfection with Δ62 N-cadherin increased alkaline phosphatase (ALP) activity and osteoblast gene expression (Runx2, ALP, Col1A1) by 2-30-fold, as determined by qPCR analysis. These results reveal that LRP5 interacts with the last 62 amino acids of N-cadherin. Our data also indicate that deletion of the Δ62 domain not only abolishes N-cadherin-LRP5 interaction but also promotes osteoblast differentiation mediated by Wnt/ β-catenin signalling in murine osteoblasts. These results not only identify the N-cadherin domain that physiologically interacts with LRP5 in vivo, but also provides a novel potential molecular target to promote β-catenin-mediated osteoblast differentiation. This work was sponsored in part by the FP7 Program Talos.