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CD40, a member of the tumor necrosis factor receptor superfamily, plays a key role in both adaptive and innate immunity. Engagement of CD40 with its natural trimeric ligand or with cross-linked antibodies results in disulfide-linked CD40 (dl-CD40) homodimer formation, a process mediated by the cysteine-238 residues of the cytoplasmic tail of CD40. The present study was designed to elucidate the biological relevance of cysteine-238-mediated dl-CD40 homodimers to the expression of CD23 on B cells and to investigate its possible involvement in the innate response. Our results indicate that cysteine-238-mediated dl-CD40 homodimerization is required for CD40-induced activation of PI3-kinase/Akt signaling and the subsequent CD23 expression, as inhibition of dl-CD40 homodimer formation through a point mutation-approach specifically impairs these responses. Interestingly, cysteine-238-mediated dl-CD40 homodimers are also shown to play a crucial role in Toll-like receptor 4-induced CD23 expression, further validating the importance of this system in bridging innate and adaptive immune responses. This process also necessitates the activation of the PI3-kinase/Akt cascade. Thus, our results highlight new roles for CD40 and cysteine-238-mediated CD40 homodimers in cell biology and identify a potential new target for therapeutic strategies against CD40-associated chronic inflammatory diseases.

Mycoplasma arthritidis–derived mitogen (MAM) is a member of the superantigen family that structurally differs from other members while still capable of initiating cognate APC/T cell interaction. In addition to the critical role of MHC class II molecules, it has been suggested that TLR2 and TLR4 may cooperate with MHC class II during MAM-induced responses. In this study, we investigated the direct involvement of TLR2 and TLR4 in MAM binding and presentation to T cells. Our results showed that MAM fails to bind to TLR2- and TLR4-transfected cells. However, coexpression of TLR2 or TLR4 with HLA-DR significantly increases MAM binding and the subsequent T cell activation compared with cells expressing HLA-DR alone. The upregulated MAM binding and activity in HLA-DR/TLR–transfected cells is abrogated by an anti–HLA-DR Ab. Interestingly, we also found that MAM complexed with soluble HLA-DR is capable of binding to both TLR2 and TLR4. The enhancing effect of TLR2 or TLR4 on MAM-induced T cell proliferation was not due to TLR ligand contamination in the MAM preparation. Taken together, these results strongly suggest that binding of MAM to HLA-DR leads to a conformational change in MAM structure allowing its interaction with TLR2 and TLR4 and a better recognition by T cells.

Summary There is increasing interest in periprostatic fat and its influence on prostate cancer aggressiveness. In vitro data suggest that adipose stromal/stem cells (ASCs) can increase production of cytokines and growth factors resulting in invasive growth and metastasis in prostate cancer. The objective of the study was to determine the interaction between 5α-reductase inhibitors (5ARIs) and periprostatic adipose tissue (PPAT) and factors of prostate cancer aggressiveness. In this retrospective study, we identified 61 patients treated with 5ARIs for a period of ≥12 months before undergoing radiation therapy (brachytherapy or external beam radiotherapy). The control group consisted of 117 patients without any exposure to 5ARIs. Prior to being treated, all patients underwent abdominal computed tomography (CT). To measure PPAT, we defined the fat pad anteriorly to the prostate, as well as the intra-abdominal visceral adipose tissue (VAT) and subcutaneous tissue (SAT) at the level of L4/L5. All contours were performed manually. These adipose tissue measurements were correlated with the Cancer of the Prostate Risk Assessment (CAPRA) score using Pearson correlation coefficient. Differences in fat contents were evaluated using Student's t-test. Median time on 5ARIs for the 61 patients was 12 months (range 12–96). Patient on 5ARIs had a significantly (p

CD40, a member of the TNF receptor family, is expressed on a variety of immune and non-immune cells. Its interaction with its ligand, CD154, plays a pivotal role in humoral and cell-mediated immunity. A low level of CD40 is constitutively associated within membrane lipid rafts and, upon engagement, this level is significantly enhanced. In this study, our objective is to evaluate the process of CD40/lipid raft association in terms of the signals required for its initiation and the resulting biological outcomes. Here, we show the CD40/lipid raft association to be independent of PI-3-kinase, Src family kinases and p38 MAPK pathways. Moreover, CD40 lacking its intracellular domain, which is usually required for CD40-mediated signaling, still localizes to lipid rafts upon engagement, confirming that the CD40/lipid raft association is independent of signaling events. As to the biological outcomes of the CD40/lipid raft association, we show that disrupting lipid raft integrity selectively abolishes CD40-mediated Akt phosphorylation. In addition, replacing the transmembrane domain of CD40 with that of CD45 (a protein excluded from lipid rafts) dramatically reduced CD40-mediated Akt phosphorylation and B7.1 upregulation, while not influencing p38, ERK and JNK activation. Together, these findings clarify the requirements for CD40/lipid raft association and the signals triggered upon CD40 engagement by CD154.

The discovery of new functions for CD154 and CD40 molecules has expanded our knowledge to claim that CD154 plays well beyond its postulated role in adaptive immunity. Active research in this area has outlined an important role of CD154 and its receptor CD40 in the physiopathology of autoimmunity. CD154/CD40 interactions have been shown to underlay inflammatory events characterizing autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosous, and multiple sclerosis. Besides CD40, three additional receptors were recently discovered for CD154, namely, � IIb� 3, � 5� 1 and Mac-1 integrins. This review gives an overview on CD154 and its receptors, and outlines the function of CD154/CD40 interactions in both normal and autoimmune states. Moreover, the potential usefulness of various CD154-interfering agents in treatment/prevention of autoimmune events is discussed.

Bacterial sensing by intracellular Nod proteins and other Nod-like receptors (NLRs) activates signaling pathways that mediate inflammation and pathogen clearance. Nod1 and Nod2 associate with the kinase Rip2 to stimulate NF-kappaB signaling. Other cytosolic NLRs assemble caspase-1-activating multiprotein complexes termed inflammasomes. Caspase-12 modulates the caspase-1 inflammasome, but unlike other NLRs, Nod1 and Nod2 have not been linked to caspases, and mechanisms regulating the Nod-Rip2 complex are less clear. We report that caspase-12 dampens mucosal immunity to bacterial infection independent of its effects on caspase-1. Caspase-12 deficiency enhances production of antimicrobial peptides, cytokines, and chemokines to entric pathogens, an effect dependent on bacterial type III secretion and the Nod pathway. Mechanistically, caspase-12 binds to Rip2, displacing Traf6 from the signaling complex, inhibiting its ubiquitin ligase activity, and blunting NF-kappaB activation. Nod activation and resulting antimicrobial peptide production constitute an early innate defense mechanism, and caspase-12 inhibits this mucosal antimicrobial response.

International audience; Programmed cell death (apoptosis) is a genetically regulated process of cell elimination essential during development. During development, programmed cell death is involved in the specific shaping of organs, in the elimination of cells having achieved their program, and in regulating the number of cells to differentiate. Tooth development includes these three aspects and was used here as a model to study the control of apoptosis. Bone morphogenetic proteins (BMPs) are currently considered as playing a major role in signaling apoptosis. This apoptosis could be stopped by treatments with a BMP antagonist ("Noggin"). We selected a model system made by a layer-by-layer approach using poly-L-glutamic acid (PlGA) and poly-L-lysine (PlL) films into which BMP4 and/or Noggin have been embedded. Our results indicate that in situ control of apoptosis during tooth differentiation mediated by both BMP4 and Noggin embedded in a polyelectrolyte multilayer film is possible. We show here for the first time that in the presence of BMP4 and Noggin embedded in a multilayered film, we can induce or inhibit cell death in tooth differentiation, and conserve their biological effects.

Caspases are cysteinyl-aspartate-specific proteinases known for their role in apoptosis (cell death or apoptotic caspases) and proinflammatory cytokine maturation (inflammatory caspases). The inflammatory caspases were among the first to be discovered, but only recently have the mechanisms leading to their activation and inhibition begun to be elucidated. In this review, we examine the biochemistry, substrates, and function of this unique family of inflammatory proteases, highlight the most recent findings regarding their regulatory mechanisms, and discuss what remains to be understood about their roles in health and disease.

International audience; During development, Bone Morphogenetic Proteins (BMPs) can induce apoptosis, cell growth or differentiation. These different effects are mediated by dimers of two types of BMP-receptors (BMPRs). To identify the responding cells during tooth development and search for possible tissue-or stage-specificities in the receptors involved, the distribution patterns of BMPR-IA, -IB and -II were investigated in the mouse molar, from bud to bell stage. At the bud stage, BMP-2 was suggested to be involved in the formation of an epithelial signaling center, the primary enamel knot (PEK), while BMP-4 would mediate the condensation of the mesenchyme. Immunostaining showed the presence of BMPR-IA and -II in the epithelium instead of BMPR-IB and -II in the mesenchyme. At the cap stage, BMPR-IB was detected in the epithelium but not BMPR-II, suggesting the existence of another type II receptor to form a functional dimer. At the late cap stage in the epithelium, BMP-4, BMPR-IA and -II were restricted to the internal part of the PEK and the stalk: two apoptotic areas. The three proteins were detected in the mesenchyme, showing a strong staining where cusps were about to form. At the late bell stage, BMP-2 or -4 may induce cell differentiation. BMPR-IB and -II were detected in odontoblasts instead of BMPR-IA and -II in ameloblasts. These results provide the first evidence of multiple type I and type II BMP-receptors, expressed in the dental epithelium and mesenchyme at different stages of development, to signal different cellular activities in a time- and tissue-specific way.

International audience; Reciprocal epithelial-mesenchymal interactions control odontogenesis and the cap stage tooth germ mesenchyme specifies crown morphogenesis. The aim of this work was to determine whether this mesenchyme could also control epithelial histogenesis. Dental mesenchyme and enamel organ were dissociated from mouse first lower molars at E14. At this early cap stage, the enamel organ consists of four cell types forming the inner dental epithelium (IDE), primary enamel knot (PEK), outer dental epithelium (ODE) and the stellate reticulum (SR). Pelleted trypsin-dissociated single dental epithelial cells, which had lost all positional information, were reassociated to either dental mesenchyme or dissociated mesenchymal cells and cultured in vitro. Although with different timings, teeth developed in both types of experiments showing a characteristic dental epithelial histogenesis, cusp formation, and the differentiation of functional odontoblasts and ameloblasts. The rapid progression of the initial steps of histogenesis suggested that the cell history was not memorized. The dental mesenchyme, as well as dissociated mesenchymal cells, induced the formation of a PEK indicating that no specific organisation in the mesenchyme is required for this step. However, the proportion of well-formed multicusped teeth was much higher when intact mesenchyme was used instead of dissociated mesenchymal cells. The mesenchymal cell dissociation had consequences for the functionality of the newly-formed PEK.

Intercellular signaling controls all steps of odontogenesis. The purpose of this work was to immunolocalize in the developing mouse molar four molecules that play major roles during odontogenesis: BMP-2, −4, FGF-4, and WNT10b. BMP-2 and BMP-4 were detected in the epithelium and mesenchyme at the bud stage. Staining for BMP-2 markedly increased at the cap stage. The relative amount of BMP-4 strongly increased from E14 to E15. At E15, BMP-4 was detected in the internal part of the enamel knot where apoptosis was intense. In contrast to TGFβ1, BMP-2 and −4 did not show accumulation at the epithelial-mesenchymal junction where the odontoblast started differentiation. When odontoblasts became functional, BMP-2 and BMP-4 were detected at the apical and basal poles of preameloblasts. BMP-2, which induces ameloblast differentiation in vitro, may also be involved physiologically. The decrease in FGF-4 from E14 to E15 supports a possible role for the growth factor in the control of mesenchymal cell proliferation. The relative amount of FGF-4 was maximal at E17. The subsequent decrease at E19 showed correlation with the withdrawal of odontoblasts and ameloblasts from the cell cycle. WNT10b might also stimulate cell proliferation. At E14-15, WNT10b was present in the mesenchyme and epithelium except for the enamel knot, where the mitotic activity was very low. At E19 there was a decreasing gradient of staining from the cervical loop where cells divide to the tip of the cusp in the inner dental epithelium where cells become postmitotic. The target cells for FGF-4 and WNT10b appeared different.

CD20 is an attractive therapeutic target given the success of its monoclonal antibody, Rituximab, in the treatment of B-cell malignancies and B-cell-mediated autoimmune diseases. Treatment with Rituximab causes a rapid depletion of B cells and a decrease in disease symptoms. Despite the clinical efficiency of Rituximab, its mechanism of action is not completely understood. In this study, we aimed at further investigating the Rituximab-induced cell death and the factors affecting such responses. Our results indicate that Rituximab-induced cell death depends on the nature of the cells and levels of CD20 expression on the cell surface. Coexpression of CD20 with CD40, a member of the TNF receptor family that is known to be physically associated with CD20 on the cell surface, enhances the apoptotic response induced by Rituximab. Inhibiting the formation of CD40 disulfide-bound-homodimers, a process required for some CD40 signaling, further enhances Rituximab-induced cell death. Cell death induced by anti-CD40 mAb is also upregulated by the presence of CD20, suggesting a bidirectional influence of the CD20/CD40 association. Moreover, treating cells with both anti-CD20 and anti-CD40 antibodies improves the cell death response induced by a single-agent treatment. These results highlight the role of the CD20/CD40 association in triggering B-cell depletion and may pave the way for an alternative more efficient therapeutic strategy in treating B-cell-mediated disorders.

International audience; CD154 (CD40 ligand) is a type II transmembrane protein that belongs to the tumor necrosis factor superfamily. The soluble form of CD154 (sCD154), which results from the shedding of membrane-bound CD154, plays a key role in the production of proinflammatory cytokines and has been linked to various autoimmune and vascular disorders. Therefore, elucidating the mechanisms by which CD154 is released from the cell surface following its interaction with its various receptors is of primordial importance. Using co-culture experiments, we show that CD154 is shed predominantly upon its engagement with CD40. Indeed, only CD40 (both membrane-bound and soluble) and not α5β1 or αMβ2 is involved in the cleavage and release of CD154 from Jurkat E6.1 T-cells. Interestingly, CD154 is cleaved independently of the formation of cell surface CD40 homodimers and independently of its association into lipid rafts. In contrast, we found that the protein kinase C (PKC) signaling family and the matrix metalloproteinases ADAM10 and ADAM17 are intimately involved in this process. In conclusion, our data indicate that CD154 is released from T-cells by ADAM10 and ADAM17 upon CD40 ligation. These findings add significant insights into the mechanisms by which CD154 is down-regulated and may lead to the generation of novel therapeutic targets for the treatment of CD154-associated disorders.

Atherosclerosis, now regarded as a chronic inflammatory disease of the arterial wall, and its clinical manifestations have increasingly been associated with rheumatoid arthritis (RA), supporting the notion that autoimmune diseases and vascular disorders share common etiological features. Indeed, evidence pertaining to this matter indicates that inflammation and its multiple components are the driving force behind the pathogenesis of these disorders. Interestingly, CD154 and its receptors have emerged as major players in the development of RA and atherosclerosis, which raises the possibility that this axis may represent an important biological link between both complications. Indeed, CD154 signaling elicits critical inflammatory responses that are common to the pathogenesis of both diseases. Here, we provide an overview of the traditional and disease-related interrelations between RA and vascular abnormalities, while focusing on CD154 as a potential mediator in the development of atherosclerotic events in RA patients.

Background Increasing evidence suggests a close relationship between systemic inflammation and cancer development and progression. The neutrophil to lymphocyte ratio (NLR) has been shown to be an independent prognostic indicator in various advanced and localized cancers. We investigated the influence of markers of systemic inflammation such as leucocyte counts and metabolic co-morbidities on overall survival (OS) after radiotherapy for localized prostate cancer. Methods We conducted a retrospective study of patients with localized prostate cancer treated with definitive external beam radiotherapy or brachytherapy. Univariate and multivariate cox proportional hazards models were used to investigate the influence of the following factors on OS: age, neutrophil and lymphocyte counts, neutrophil-to-lymphocyte ratio (NLR), Cancer of the Prostate Risk Assessment (CAPRA) score as well as comorbidities associated with inflammation such as cardiac history, diabetes and use of a statin. A stepwise selection of variable based on the Akaike information criterion (AIC) was used for multivariate analysis. Results In total, 1772 pts were included; blood count data was available for 950 pts. Median age was 68 years (44–87). Actuarial 5 years OS and biochemical recurrence-free survival (BRFS) for the 1772 patients were 93 % and 95 %, respectively, with a median follow-up of 44 months (1–156). On univariate analysis, neutrophil count (p = 0.04), cardiac history (p = 0.008), age (p = 0.001) and CAPRA (p = 0.0002) were associated with OS. Lymphocytes, NLR and comorbidities other than cardiac history were not associated with mortality. On multivariate analysis, neutrophil count (HR = 1.18, 95 % CI: 1.017-1.37, p = 0.028), age (HR = 1.06, 95 % CI: 1.01-1.1, p = 0.008) and CAPRA (HR = 1.16, 95 % CI: 1.03-1.31, p = 0.015) were independent predictors of OS. Conclusion Neutrophil count, as a possible marker of systemic inflammation, appear to be an independent prognostic factor for overall mortality in localized prostate cancer. A validation cohort is needed to corroborate these results.

CD40 has an important role in T cell-B cell interaction which rescues B lymphocytes from undergoing apoptosis. However, various studies have demonstrated that CD40 can also play a direct role in the induction of specific cell death and thus in the inhibition of tumour cell proliferation. Our previous studies showed that CD40-mediated cell death was independent of caspases and required no de novo protein synthesis. Knowing that CD40 signaling is mediated by its association with several intracellular effectors, including members of TNFR-associated factors (TRAFs) family, the goal of the present study is to investigate the mechanisms involved in the induction of cell death by CD40. Our data reveals that CD40-mediated cell death required lysosomal membrane permeabilization and the subsequent cathepsin B release. In addition, CD40 homodimer formation, a phenomenon known to be necessary for some CD40-mediated signals, was shown to negatively regulate cell death induced by CD40. Moreover, using HEK293 cells ectopically expressing CD40 deficient in TRAF binding, we showed that CD40-mediated apoptosis occurred in the absence of TRAF2 and TRAF3 association, but was significantly reduced when CD40 was deficient in its TRAF6 binding. Therefore, by outlining the role of lysosomal pathways and intracellular effectors, namely TRAF6 in CD40-mediated cell death, our study identifies new targets for anti-cancer therapy.

International audience; Recent developments in tooth-tissue engineering require that we understand the regulatory processes to be preserved to achieve histomorphogenesis and cell differentiation, especially for enamel tissue engineering. Using mouse first lower molars, our objectives were: (1) to determine whether the cap-stage dental mesenchyme can control dental epithelial histogenesis, (2) to test the role of the primary enamel knot (PEK) in specifying the potentialities of the dental mesenchyme, and (3) to evaluate the importance of positional information in epithelial cells. After tissue dissociation, the dental epithelium was further dissociated into individual cells, re-associated with dental mesenchyme, and cultured. Epithelial cells showed a high plasticity: Despite a complete loss of positional information, they rapidly underwent typical dental epithelial histogenesis. This was stimulated by the mesenchyme. Experiments performed at E13 demonstrated that the initial potentialities of the mesenchyme are not specified by the PEK. Positional information of dental epithelial cells does not require the memorization of their history.

CD40 is a critical regulator of the immune response and a key player in several autoimmune diseases. Accumulating evidence indicates that CD40 is essential for both innate and adaptive immunity. Moreover, it has been reported that CD40 engagement induces the formation of disulfide-linked (dl) CD40 homodimers, albeit the role of this response in B-cell function remains elusive. Here, we report that oxidative stress induces CD40 self-association through cysteine-238 residues independently of ligand binding. However, our results show that the production of ROS downstream of CD40 is independent of its dimerization. Notably, CD40 homodimers are required for full B-cell activation and TLR4-induced CD23 and CD80 expression. In addition, these responses were dependent upon Jak3 association to CD40 and PI-3 kinase activation. Our results highlight the role of CD40 homodimers in innate and adaptive immunity and may lead to the generation of novel biological targets for the treatment of CD40-assciated inflammatory disorders.

Tissue engineering of teeth requires the coordinated formation of correctly shaped crowns, roots, and periodontal ligament. Previous studies have shown that the dental mesenchyme controls crown morphogenesis and epithelial histogenesis during tooth development in vivo, but little is known about the inductive potential of dissociated mesenchymal cells used in ex vivo cultures. A 2-step method is described in which, by using different types of reassociations between epithelial and mesenchymal tissues and/or cells from mouse embryos, reassociations were cultured in vitro before in vivo implantation. In vitro, the reassociated tissues developed and resulted in tooth-like structures that exhibited normal epithelial histogenesis and allowed the functional differentiation of odontoblasts and ameloblasts. After implantation, the reassociations formed roots and periodontal ligament, the latter connected to developing bone. The shape of the crown, initially suspected to depend on the integrity of the mesenchyme, could be modulated by adjusting the number of dissociated mesenchymal cells reassociated with the epithelial compartment. Based on these results, we propose a refined strategy for tooth tissue engineering that may help to eventually generate morphologically defined teeth.