TLR9 transcriptional regulation in response to double-stranded DNA viruses.

The stimulation of TLRs by pathogen-derived molecules leads to the production of proinflammatory cytokines. Because uncontrolled inflammation can be life threatening, TLR regulation is important; however, few studies have identified the signaling pathways that contribute to the modulation of TLR expression. In this study, we examined the relationship between activation and the transcriptional regulation of TLR9. We demonstrate that infection of primary human epithelial cells, B cells, and plasmacytoid dendritic cells with dsDNA viruses induces a regulatory temporary negative-feedback loop that blocks TLR9 transcription and function. TLR9 transcriptional downregulation was dependent on TLR9 signaling and was not induced by TLR5 or other NF-κB activators, such as TNF-α. Engagement of the TLR9 receptor induced the recruitment of a suppressive complex, consisting of NF-κBp65 and HDAC3, to an NF-κB cis element on the TLR9 promoter. Knockdown of HDAC3 blocked the transient suppression in which TLR9 function was restored. These results provide a framework for understanding the complex pathways involved in transcriptional regulation of TLR9, immune induction, and inflammation against viruses.

Protein kinase cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE. METHODS: We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology. RESULTS: We identified a homozygous missense mutation in PRKCD, encoding protein kinase δ (PKCδ), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype. CONCLUSION: Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE.

Tax protein-induced expression of antiapoptotic Bfl-1 protein contributes to survival of human T-cell leukemia virus type 1 (HTLV-1)-infected T-cells.

Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4(+) T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-x(L), and Bcl-2. Indeed, both Bfl-1 and Bcl-x(L) knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-x(L) in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-x(L) represent potential therapeutic targets for ATLL treatment.

C-terminal residues regulate localization and function of the antiapoptotic protein Bfl-1.

Unlike other antiapoptotic members of the Bcl-2 family, Bfl-1 does not contain a well defined C-terminal transmembrane domain, and whether the C-terminal tail of Bfl-1 functions as a membrane anchor is not yet clearly established. The molecular modeling study of the full-length Bfl-1 performed within this work suggests that Bfl-1 may co-exist in two distinct conformational states: one in which its C-terminal helix alpha9 is inserted in the hydrophobic groove formed by the BH1-3 domains of Bfl-1 and one with its C terminus. Parallel analysis of the subcellular localization of Bfl-1 indicates that even if Bfl-1 may co-exist in two distinct conformational states, most of the endogenous protein is tightly associated with the mitochondria by its C terminus in both healthy and apoptotic peripheral blood lymphocytes as well as in malignant B cell lines. However, the helix alpha9 of Bfl-1, and therefore the binding of Bfl-1 to mitochondria, is not absolutely required for the antiapoptotic activity of Bfl-1. A particular feature of Bfl-1 is the amphipathic character of its C-terminal helix alpha9. Our data clearly indicate that this property of helix alpha9 is required for the anchorage of Bfl-1 to the mitochondria but also regulates the antiapoptotic function Bfl-1.

Synthesis and biological activities of new di- and trimeric quinoline derivatives.

The synthesis of non-peptidic helix mimetics based on a trimeric quinoline scaffold is described. The ability of these new compounds, as well as their synthetic dimeric intermediates, to bind to various members of the Bcl-2 protein anti-apoptotic group is also evaluated. The most interesting derivative of this new series (compound A) inhibited Bcl-x(L)/Bak, Bcl-x(L)/Bax and Bcl-x(L)/Bid interactions with IC(50) values around 25 µM.

Synthesis and biological activities of polyquinoline derivatives: new Bcl-2 family protein modulators.

The synthesis of quinoline derivatives, designed to interact with Bcl-x(L), and to inhibit its interaction with pro-apoptotic partners, is described and their biological effects investigated. We showed that 5 out of 28 synthetized compounds restored cell death of 3T3 cells overexpressing Bcl-x(L) following serum starvation. Active compounds were further characterized for their binding capacity to the anti-apoptotic member of the Bcl-2 family, Bcl-x(L) as well as Bcl-2, Bfl-1 and Mcl-1, and for their pro-apoptotic activities toward lymphoid tumor cells and peripheral blood mononuclear cells. Altogether our results indicate that dimeric, rather than trimeric quinoline derivatives, represent a new attractive class of BH3 mimetics for cancer therapy.

NOD1 cooperates with TLR2 to enhance T cell receptor-mediated activation in CD8 T cells.

Pattern recognition receptors (PRR), like Toll-like receptors (TLR) and NOD-like receptors (NLR), are involved in the detection of microbial infections and tissue damage by cells of the innate immune system. Recently, we and others have demonstrated that TLR2 can additionally function as a costimulatory receptor on CD8 T cells. Here, we establish that the intracytosolic receptor NOD1 is expressed and functional in CD8 T cells. We show that C12-iEDAP, a synthetic ligand for NOD1, has a direct impact on both murine and human CD8 T cells, increasing proliferation and effector functions of cells activated via their T cell receptor (TCR). This effect is dependent on the adaptor molecule RIP2 and is associated with an increased activation of the NF-κB, JNK and p38 signaling pathways. Furthermore, we demonstrate that NOD1 stimulation can cooperate with TLR2 engagement on CD8 T cells to enhance TCR-mediated activation. Altogether our results indicate that NOD1 might function as an alternative costimulatory receptor in CD8 T cells. Our study provides new insights into the function of NLR in T cells and extends to NOD1 the recent concept that PRR stimulation can directly control T cell functions.

µ-Calpain conversion of antiapoptotic Bfl-1 (BCL2A1) into a prodeath factor reveals two distinct alpha-helices inducing mitochondria-mediated apoptosis.

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.