Dysregulation of neutrophil oxidant production and interleukin-1-related cytokines in granulomatosis with polyangiitis.

OBJECTIVES: Neutrophils play a key role in ANCA-associated vasculitis, both as targets of autoimmunity and facilitators of vascular damage. In granulomatosis with polyangiitis (GPA), data regarding the production of reactive oxygen species (ROS) in neutrophils are unclear. Further, recent data suggests that ROS production could have an anti-inflammatory effect through the regulation of the inflammasome and IL-1-related cytokines. We aimed to analyse the ROS production in neutrophils from patients with GPA and investigate its association with IL-1-related cytokines and the autoantigen proteinase 3 (PR3). METHODS: Seventy-two GPA patients with disease flare were included in the NEUTROVASC prospective cohort study. ROS production was evaluated in whole blood of patients with active GPA and compared with the same patients in remission or healthy controls. Associations between ROS production, PR3 membrane expression on neutrophils, serum levels of IL-1-related cytokines as well as inflammasome-related proteins were analyzed. RESULTS: We observed a robust defect in ROS production by neutrophils from patients with active GPA compared with healthy controls, independent of glucocorticoid treatment. Serum levels of IL-1-related cytokines were significantly increased in GPA patients, particularly in patients with kidney involvement, and levels of these cytokines returned to normal after patients achieved remission. Further, inflammasome-related proteins were significantly dysregulated in the cytosol of neutrophils as well as the serum from GPA patients. CONCLUSION: Our data suggests that ROS production and regulation of the inflammasome in neutrophils from patients with GPA are disturbed and may be a potential therapeutic target. CLINICAL TRIAL REGISTRATION NUMBER: NCT01862068, clinicaltrials.gov, https://www.clinicaltrials.gov.

The classical NLRP3 inflammasome controls FADD unconventional secretion through microvesicle shedding.

Fas-associated death domain (FADD) is a key adaptor molecule involved in numerous physiological processes including cell death, proliferation, innate immunity and inflammation. Therefore, changes in FADD expression have dramatic cellular consequences. In mice and humans, FADD regulation can occur through protein secretion. However, the molecular mechanisms accounting for human FADD secretion were still unknown. Here we report that canonical, non-canonical, but not alternative, NLRP3 inflammasome activation in human monocytes/macrophages induced FADD secretion. NLRP3 inflammasome activation by the bacterial toxin nigericin led to the proinflammatory interleukin-1ß (IL-1ß) release and to the induction of cell death by pyroptosis. However, we showed that FADD secretion could occur in absence of increased IL-1ß release and pyroptosis and, reciprocally, that IL-1ß release and pyroptosis could occur in absence of FADD secretion. Especially, FADD, but not IL-1ß, secretion following NLRP3 inflammasome activation required extracellular glucose. Thus, FADD secretion was an active process distinct from unspecific release of proteins during pyroptosis. This FADD secretion process required K+ efflux, NLRP3 sensor, ASC adaptor and CASPASE-1 molecule. Moreover, we identified FADD as a leaderless protein unconventionally secreted through microvesicle shedding, but not exosome release. Finally, we established human soluble FADD as a new marker of joint inflammation in gout and rheumatoid arthritis, two rheumatic diseases involving the NLRP3 inflammasome. Whether soluble FADD could be an actor in these diseases remains to be determined. Nevertheless, our results advance our understanding of the mechanisms contributing to the regulation of the FADD protein expression in human cells.

Inflammasome activation: Neutrophils go their own way.

Discussion on a new RIPK3-dependent pathway to cell death and cytokine secretion independent of the canonical necroptosis and inflammasome machinery in neutrophils.

FADD at the Crossroads between Cancer and Inflammation.

Initially described as an adaptor molecule for death receptor (DR)-mediated apoptosis, Fas-associated death domain (FADD) was later implicated in nonapoptotic cellular processes. During the last decade, FADD has been shown to participate and regulate most of the signalosome complexes, including necrosome, FADDosome, innateosome, and inflammasome. Given the role of these signaling complexes, FADD has emerged as a new actor in innate immunity, inflammation, and cancer development. Concomitant to these new roles, a surprising number of mechanisms deemed to regulate FADD functions have been identified, including post-translational modifications of FADD protein and FADD secretion. This review focuses on recent knowledge of the biological roles of FADD, a pleiotropic molecule having multiple partners, and its impact in cancer, innate immunity, and inflammation.

Modulatory role of the anti-apoptotic protein kinase CK2 in the sub-cellular localization of Fas associated death domain protein (FADD).

The Fas associated death domain protein (FADD) is the key adaptor molecule of the apoptotic signal triggered by death receptors of the TNF-R1 superfamily. Besides its crucial role in the apoptotic machinery, FADD has proved to be important in many biological processes like tumorigenesis, embryonic development or cell cycle progression. In a process to decipher the regulatory mechanisms underlying FADD regulation, we identified the anti-apoptotic kinase, CK2, as a new partner and regulator of FADD sub-cellular localization. The blockade of CK2 activity induced FADD re-localization within the cell. Moreover, cytoplasmic FADD was increased when CK2ß was knocked down. In vitro kinase and pull down assays confirmed that FADD could be phosphorylated by the CK2 holoenzyme. We found that phosphorylation is weak with CK2α alone and optimal in the presence of stoichiometric amounts of CK2α catalytic and CK2ß regulatory subunit, showing that FADD phosphorylation is undertaken by the CK2 holoenzyme in a CK2ß-driven fashion. We found that CK2 can phosphorylate FADD on the serine 200 and that this phosphorylation is important for nuclear localization of FADD. Altogether, our results show for the first time that multifaceted kinase, CK2, phosphorylates FADD and is involved in its sub-cellular localization. This work uncovered an important role of CK2 in stable FADD nuclear localization.

Fas-associated death domain protein and adenosine partnership: fad in RA.

Inflammation is the principal hallmark of RA. Different pathways are implicated in the production of pro-inflammatory cytokines, the bona fide mediators of this inflammation. Among them are the TNF pathway and the IL-1 receptor/Toll-like receptor (IL-1R/TLR4) pathway. One of the potential negative regulators of IL-1R/TLR4 signalling is the Fas-associated death domain protein (FADD), which is the pivotal adaptor of the apoptotic signal mediated by death receptors of the TNF family. FADD can sequester myeloid differentiation primary response gene 88 (MyD88), the common adaptor of most TLRs, and hence hinder the activation of nuclear factor κB (NF-κB), the downstream transcription factor. We recently described a new regulatory mechanism of FADD expression, via the shedding of microvesicles, mediated by adenosine receptors. Interestingly, adenosine is found in high concentrations in the joints of RA patients and has been largely reported as a regulator of inflammation. This review discusses the possible link that could exist between the adenosine-dependent regulation of FADD in the inflammatory context of RA and the potential role of FADD as a therapeutic target in the treatment of RA. We will see that the modulation of FADD expression may be a double-edged sword by increasing apoptosis and at the same time limiting NF-κB activation.

FADD: a regulator of life and death.

FAS-associated protein with death domain (FADD) is the key adaptor protein transmitting apoptotic signals mediated by the main death receptors (DRs). Besides being an essential instrument in cell death, FADD is also implicated in proliferation, cell cycle progression, tumor development, inflammation, innate immunity, and autophagy. Recently, many of these new functions of FADD were shown to be independent of DRs. Moreover, FADD function is dictated by protein localization and phosphorylation state. Thus, FADD is a crucial and unique controller of many essential cellular processes. The full understanding of the networks dictating the ultimate function of FADD may provide a new paradigm for other multifaceted proteins.

Dramatic efficacy improvement of a DC-based vaccine against AML by CD25 T cell depletion allowing the induction of a long-lasting T cell response.

Dendritic cell (DC)-based vaccination is a promising approach to enhance anti-tumor immunity that could be considered for acute myeloid leukemia (AML) patients with high-risk of relapse. Our purpose was to study the efficiency and to optimize the immunogenicity of a DC-based vaccine in a preclinical AML murine model. In this report, C57BL6 mice were vaccinated with DC pulsed with peptides eluted (EP) from the syngeneic C1498 myelomonocytic leukemic cell line in a prophylactic setting. In this model, a natural antileukemic immunity mediated by NK cells was observed in the control unloaded DC-vaccinated group. On the other hand, we showed that the cytotoxic antileukemic immune response induced by vaccination with eluted peptides pulsed-DC (DC/EP), in vitro and in vivo, was mainly mediated by CD4(+) T cells. Treatment with anti-CD25 antibody to deplete CD4(+) CD25(+) regulatory T cells before DC-vaccination dramatically improved the antileukemic immune response induced by immunization, and allowed the development of long-lasting immune responses that were tumor protective after a re-challenge with leukemic cells. Our results suggest that this approach could be successful against weakly immunogenic tumors such as AML, and could be translated in human.

Adenosine receptors control a new pathway of Fas-associated death domain protein expression regulation by secretion.

FADD is the key adaptor transmitting the apoptotic signal mediated by death receptors. We have previously shown that FADD protein expression could be lost in vivo in cancerous cells, in mice and humans, and be used as prognostic factor. Furthermore, loss of FADD could contribute to tumor progression and aggressiveness. However, the mechanism accounting for the loss of FADD was unknown. Using in vitro-cultured mouse organ models, we demonstrated that loss of FADD occurred through a new regulatory pathway of FADD expression by secretion. The secretion of FADD is an active release following shedding of microvesicles derived from the plasma membrane. In our experimental settings, this phenomenon was restricted to 6 of 12 FADD-expressing organs. This process is calcium- and adenosine-dependent. Moreover, we identified the two receptors with low affinity to adenosine, namely A(2B) and A(3) adenosine receptors, as regulators of the FADD secretion process. Furthermore, we showed that modulating A(3) adenosine receptor can convert a nonsecreting organ into a FADD-secreting one. Finally, we reported that mouse FADD release occurred in vivo during tumor disease. These results demonstrate the existence of a new localization site (in microvesicles) and regulatory mechanism (by secretion) of the FADD protein, and the implication of adenosine receptors in this process. These data open a new field of investigation consisting of the possibility to regulate FADD expression via the modulation of adenosine receptors, which constitutes a therapeutic target in diseases in which FADD-mediated signaling is impaired.

Late-onset neutropenia following rituximab results from a hematopoietic lineage competition due to an excessive BAFF-induced B-cell recovery.

Rituximab is used in the treatment of lymphoma and autoimmune diseases, for which late-onset neutropenia (LON) were reported. LON-related mechanisms remain unclear. To obtain insights into the mechanisms, we assessed serum, peripheral blood and bone marrow (BM) samples of a patient with LON. Factors classically associated with neutropenia such as anti-neutrophil antibodies, T-LGL, soluble Fas Ligand were not detectable. We then evaluated the kinetics of various cytokines involved in B-cell and granulocyte homeostasis. We found that LON is related to a lack of granulopoiesis in the BM that coincides with a very high level of BAFF, a strong stimulator of B-cell recovery, and hypothesized a hematopoietic lineage competition due to an excessive B-cell recovery in the BM by promotion of B-cell lymphopoiesis over granulopoiesis within common developmental niches. Assessment of serum BAFF levels following rituximab could detect patients at risk of developing LON.

DC-based vaccine loaded with acid-eluted peptides in acute myeloid leukemia: the importance of choosing the best elution method.

Tumor-associated peptides isolated by acid elution are frequently used for therapeutic immunization against various tumors both in mice and in humans. In acute myeloid leukemia (AML), the frequent accessibility of a large tumor burden allows for extraction of peptides from leukemia cells by using either citrate-phosphate (CP) or trifluoroacetic acid (TFA) buffer. To develop an optimal immunotherapeutic protocol for AML patients, we evaluated both in mice and in humans, the immunogenicity of peptides eluted from leukemia cells with the two acids (TFA or CP). Although ex vivo studies in mice showed that both prophylactic immunizations with mature dendritic cells (DC) loaded with TFA-peptides (DC/TFA), or CP-peptides (DC/CP), were able to stimulate specific antileukemia immune responses, only vaccination with DC/TFA was able to prevent leukemia outgrowth. Moreover, in humans, only DC/TFA generated significant antileukemia CD4(+) and cytotoxic CD8(+) T cell responses in vitro. In summary, these data demonstrate that the choice of the acid elution procedure to isolate immunogenic peptides strongly influences the efficacy of the antileukemia immune responses. These finding raise essential considerations for the development of immunotherapeutic protocols for cancer patients. In our model, our results argue for the use of the TFA elution method to extract immunogenic AML-associated peptides.

Characterization and functional consequences of underexpression of clusterin in rheumatoid arthritis.

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.

Autologous peptides eluted from acute myeloid leukemia cells can be used to generate specific antileukemic CD4 helper and CD8 cytotoxic T lymphocyte responses in vitro.

BACKGROUND AND OBJECTIVES: The poor prognosis of acute myeloid leukemia (AML) treated with conventional chemotherapy justifies seeking additional immunotherapeutic approaches to eliminate minimal residual disease. Hence, we evaluated the feasibility of generating in vitro antileukemic immune responses, which would bypass the need for epitope identification and rely on antigen presentation by autologous dendritic cells. DESIGN AND METHODS: Naturally processed peptides were extracted by acid elution from circulating AML cells of six patients at diagnosis. Mature dendritic cells (mDC) were derived from autologous monocytes obtained when the patients were in complete remission, and were loaded with the pool of eluted peptides to stimulate autologous T lymphocytes in vitro. RESULTS: We were able to induce in vitro antileukemic Th1 responses characterized by CD4(+) T-cell proliferation, significant interferon-gamma secretion by both CD4+ and CD8(+) T lymphocytes by recognition of autologous AML cells and generation of cytotoxic CD8(+) T lymphocytes. INTERPRETATION AND CONCLUSIONS: These results demonstrate that naturally processed peptides eluted from AML cells and presented by differentiated autologous mDC could be immunogenic in vitro. Although more in vitro data will be needed to check the safety of such an approach, notably to rule out possible autoimmune adverse effects, these results lay the basis for a potentially effective antileukemic immunotherapy for high-risk AML patients with minimal residual disease.

FADD adaptor in cancer.

FADD (Fas Associated protein with Death Domain) is a key adaptor molecule transmitting the death signal mediated by death receptors. In addition, this multiple functional protein is implicated in survival/proliferation and cell cycle progression. FADD functions are regulated via cellular sublocalization, protein phosphorylation, and inhibitory molecules. In the present review, we focus on the role of the FADD adaptor in cancer. Increasing evidence shows that defects in FADD protein expression are associated with tumor progression both in mice and humans. Better knowledge of the mechanisms leading to regulation of FADD functions will improve understanding of tumor growth and the immune escape mechanisms, and could open a new field for therapeutic interventions.

Absence or low expression of fas-associated protein with death domain in acute myeloid leukemia cells predicts resistance to chemotherapy and poor outcome.

In acute myeloid leukemia (AML), coexpression of death receptors and ligands of the tumor necrosis factor (TNF) receptor/TNF-alpha superfamily on leukemic cells after chemotherapy is not always accompanied by apoptosis, suggesting that the apoptotic death receptor signaling pathway is disrupted. Because Fas-associated protein with death domain (FADD) is the main adaptor for transmitting the Fas, TNF-related apoptosis-inducing ligand receptors, and TNF receptor 1 death signal, expression of FADD was analyzed by Western blot and immunocytochemistry in leukemic cells of 70 de novo AML patients treated with the European Organization of Research and Treatment of Cancer AML-10 randomized trial before initiation of induction chemotherapy. Thirty seven percent of patients (17 of 46) with FADD negative/low (FADD(-/low)) leukemic cells had a primary refractory disease compared with 12% of FADD(+) patients (3 of 24; P = 0.05). FADD(-/low) expression was significantly associated with a worse event-free survival [EFS (P = 0.04)] and overall survival (P = 0.04). In multivariate analysis, FADD(-/low) protein expression was independently associated with a poor EFS and overall survival (P = 0.002 and P = 0.026, respectively). Importantly, FADD(-/low) protein expression predicted poor EFS even in patients with standard- or good-risk AML (P = 0.009). Thus, we identified low or absent expression of the FADD protein in leukemic cells at diagnosis as a poor independent prognostic factor that can predict worse clinical outcome even for patients with standard- or good-risk AML.

Loss of FADD protein expression results in a biased Fas-signaling pathway and correlates with the development of tumoral status in thyroid follicular cells.

Downregulation of proapoptotic molecules like Fas or caspase 8, or upregulation of antiapoptotic molecules like FLICE inhibitory protein has been suggested to be a regulatory mechanism set up by tumor cells to block the death signal received via death receptors. In an in-depth study of the Fas/FasL-signaling pathway in thyroid tumor development, we have demonstrated that tumor cells specifically downregulate the multideath receptor adapter Fas-associated death domain (FADD). The regulation of FADD expression occurred only at the protein level. Furthermore, in the absence of FADD, Fas-signaling resulted in accelerated growth of thyrocytes. Since thyrocytes also acquired FasL expression during tumor development, the absence of FADD protein could lead to greater resistance to numerous death receptor-mediated apoptosis, stimulation of their own proliferation through Fas/FasL interaction, and the capacity to counter-attack the infiltrating lymphocytes.

IL-10 is necessary for FasL-induced protection from experimental autoimmune thyroiditis but not for FasL-induced immune deviation.

Ectopic expression of FasL on thyrocytes confers immune privilege status to the thyroid by inducing apoptosis of Fas-expressing autoimmune effector T cells and anti-thyroglobulin (Tg) immune deviation away from the T1 type. Fas-mediated apoptosis of lymphoid cells leads to rapid production of anti-inflammatory cytokines such as IL-10. On the other hand, cytokines play a crucial role in the immunoregulation and pathology of experimental autoimmune thyroiditis (EAT), and systemic and local administration of IL-10 has a curative effect on EAT. To test the effect of endogenous IL-10 production in EAT, and to find out whether IL-10 production could be involved in FasL-induced protection, EAT was induced in IL-10(-/-) and in IL-10(-/-)xFasL-transgenic CBA/J mice. The results demonstrated that wild-type and IL-10 knockout (KO) animals developed similar EAT. In contrast, lack of endogenous IL-10 abolished the protective effect of FasL. Polymorphonuclear cells were observed significantly more frequently in the inflammatory cell infiltrates from IL-10(-/-)xFasL animals compared to IL-10(-/-) animals, but they were never detected in wild-type or IL-10(+/+)/FasL-transgenic mice. A shift away from T1 response was observed in FasL-transgenic mice irrespective of their IL-10 status, demonstrating that in our model, endogenous IL-10 plays no part in the T1-towards-T2 anti-Tg immune balance induced by FasL. In summary, endogenous IL-10 is not essential in EAT, or for the immune deviation induced by thyroid FasL expression, whereas it is necessary for the immune privilege status of the thyroid conferred by FasL expression on thyrocytes.