Intracellular BH3 Profiling Reveals Shifts in Antiapoptotic Dependency in Human B Cell Maturation and Mitogen-Stimulated Proliferation.
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ABSTRACT:
SUBMITTER: Dai J
PROVIDER: S-EPMC5821561 | biostudies-literature | 2018 Mar
REPOSITORIES: biostudies-literature
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Intracellular BH3 Profiling Reveals Shifts in Antiapoptotic Dependency in Human B Cell Maturation and Mitogen-Stimulated Proliferation.
Dai Joanne J Luftig Micah A MA
Journal of immunology (Baltimore, Md. : 1950) 20180122 5
Apoptosis is critical to B cell maturation, but studies of apoptotic regulation in primary human B cells is lacking. In this study, we sought to better understand the mechanisms of apoptotic regulation in normal and activated B cells. Using intracellular BH3 profiling, we defined the Bcl2 dependency of B cell subsets from human peripheral blood and tonsillar lymphoid tissue as well as mitogen-activated B cells. We found that naive and memory B cells were BCL-2-dependent, whereas germinal center ...[more]
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Project description:UnlabelledApoptosis is a tightly regulated process that plays a crucial role in the removal of virus-infected cells, a process controlled by both pro- and antiapoptotic members of the Bcl-2 family. The proapoptotic proteins Bak and Bax are regulated by antiapoptotic Bcl-2 proteins and are also activated by a subset of proteins known as BH3-only proteins that perform dual functions by directly activating Bak and Bax or by sequestering and neutralizing antiapoptotic family members. Numerous viruses express proteins that prevent premature host cell apoptosis. Vaccinia virus encodes F1L, an antiapoptotic protein essential for survival of infected cells that bears no discernible sequence homology to mammalian cell death inhibitors. Despite the limited sequence similarities, F1L has been shown to adopt a novel dimeric Bcl-2-like fold that enables hetero-oligomeric binding to both Bak and the proapoptotic BH3-only protein Bim that ultimately prevents Bak and Bax homo-oligomerization. However, no structural data on the mode of engagement of F1L and its Bcl-2 counterparts are available. Here we solved the crystal structures of F1L in complex with two ligands, Bim and Bak. Our structures indicate that F1L can engage two BH3 ligands simultaneously via the canonical Bcl-2 ligand binding grooves. Furthermore, by structure-guided mutagenesis, we generated point mutations within the binding pocket of F1L in order to elucidate the residues responsible for both Bim and Bak binding and prevention of apoptosis. We propose that the sequestration of Bim by F1L is primarily responsible for preventing apoptosis during vaccinia virus infection.ImportanceNumerous viruses have adapted strategies to counteract apoptosis by encoding proteins responsible for sequestering proapoptotic components. Vaccinia virus, the prototypical member of the family Orthopoxviridae, encodes a protein known as F1L that functions to prevent apoptosis by interacting with Bak and the BH3-only protein Bim. Despite recent structural advances, little is known regarding the mechanics of binding between F1L and the proapoptotic Bcl-2 family members. Utilizing three-dimensional structures of F1L bound to host proapoptotic proteins, we generated variants of F1L that neutralize Bim and/or Bak. We demonstrate that during vaccinia virus infection, engagement of Bim and Bak by F1L is crucial for subversion of host cell apoptosis.
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Project description:BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.
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Project description:Background: It has been shown that intracellular pathogens hijack DC functions to evade immune defense mechanisms. In this study, we investigated the responses of human monocyte derived DCs to four intracellular bacteria, Tropheryma whipplei, Coxiella burnetii, Brucella abortus and Orientia tsutsugamushi, responsible for human infectious diseases and known to infect myeloid cells. Methods: Whole genome microarrays were assessed to define common and specific transcriptionnal responses to bacterial pathogen. Bacterial pathogen ability to affect DC maturation was assessed by measuring lymphoproliferation and endocytosis as well as phenotypic maturation markers (CD80, CD83, CD86 and HLA-DR) expression. Results: We found that Coxiella burnetii, Orientia tsutsugamushi and Brucella abortus induced DC maturation assessed through decreased endocytosis ability, triggering lymphoproliferation, surface expression of HLA class II molecules and phenotypic changes, whereas Tropheryma whipplei did not induce DC maturation. As revealed by microarray analysis, the response of DCs to these bacteria consisted of a core associated with the maturation of DCs and signatures specific for each pathogen. The core response represented 10% of genes modulated in response to pathogens and consisted of general cellular processes including nucleotide binding, protein transport, cell fraction, protein kinase, cell cycle, mitochondrial membrane and cytoskeleton. The specific transcriptional signature induced by C. burnetii is associated with the communication between innate and adaptive immune cells and DC maturation. B. abortus signature specifically involved arachidonic acid and lipooxygenase pathways and O. tsutsugamushi signature involved type I and type III IFN responses. Conclusion: This study demonstrates that intracellular bacteria use multifaceted pathways to induce DC maturation which may lead to unadapted immune response. The understanding of these pathways may be useful to improve our knowledge of bacterial recognition by the immune system but also intracellular bacterial diseases. IL4/GM-CSF monocyte-derived dendritic cells were stimulated by T. whipplei, B. abortus, C. burnetii, O. tsutsugamushi and LPS during 6 hours. Common and specific signatures were determined by comparison with uninfected DCs. moDCs (5.10^6) were plated in 6 well plates and stimulated with bacteria or LPS for 6 hours, and total RNA was extracted using the RNeasy minikit (Qiagen, adresse CA) and DNase treatment. The Agilent-014850 4X44k Human Whole Genome microarrays (Agilent Technologies, CA) representing 44,000 probes were used as recently described. Reverse transcription, sample labeling and hybridization were performed according to protocols specified by the manufacturer (One-Color Microarray-Based Gene Expression Analysis). Three samples per experimental condition were included in the analysis.
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Project description:Background: It has been shown that intracellular pathogens hijack DC functions to evade immune defense mechanisms. In this study, we investigated the responses of human monocyte derived DCs to four intracellular bacteria, Tropheryma whipplei, Coxiella burnetii, Brucella abortus and Orientia tsutsugamushi, responsible for human infectious diseases and known to infect myeloid cells. Methods: Whole genome microarrays were assessed to define common and specific transcriptionnal responses to bacterial pathogen. Bacterial pathogen ability to affect DC maturation was assessed by measuring lymphoproliferation and endocytosis as well as phenotypic maturation markers (CD80, CD83, CD86 and HLA-DR) expression. Results: We found that Coxiella burnetii, Orientia tsutsugamushi and Brucella abortus induced DC maturation assessed through decreased endocytosis ability, triggering lymphoproliferation, surface expression of HLA class II molecules and phenotypic changes, whereas Tropheryma whipplei did not induce DC maturation. As revealed by microarray analysis, the response of DCs to these bacteria consisted of a core associated with the maturation of DCs and signatures specific for each pathogen. The core response represented 10% of genes modulated in response to pathogens and consisted of general cellular processes including nucleotide binding, protein transport, cell fraction, protein kinase, cell cycle, mitochondrial membrane and cytoskeleton. The specific transcriptional signature induced by C. burnetii is associated with the communication between innate and adaptive immune cells and DC maturation. B. abortus signature specifically involved arachidonic acid and lipooxygenase pathways and O. tsutsugamushi signature involved type I and type III IFN responses. Conclusion: This study demonstrates that intracellular bacteria use multifaceted pathways to induce DC maturation which may lead to unadapted immune response. The understanding of these pathways may be useful to improve our knowledge of bacterial recognition by the immune system but also intracellular bacterial diseases.
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Project description:Conventional chemotherapy is still of great utility in oncology and rationally constructing combinations with it remains a top priority. Drug-induced mitochondrial apoptotic priming, measured by dynamic BH3 profiling (DBP), has been shown in multiple cancers to identify drugs that promote apoptosis in vivo. We therefore hypothesized that we could use DBP to identify drugs that would render cancers more sensitive to conventional chemotherapy. We found that targeted agents that increased priming of non-small cell lung cancer (NSCLC) tumor cells resulted in increased sensitivity to chemotherapy in vitro. To assess whether targeted agents that increase priming might enhance the efficacy of cytotoxic agents in vivo as well, we carried out an efficacy study in a PC9 xenograft mouse model. The BH3 mimetic navitoclax, which antagonizes BCL-xL, BCL-w, and BCL-2, consistently primed NSCLC tumors in vitro and in vivo. The BH3 mimetic venetoclax, which electively antagonizes BCL-2, did not. Combining navitoclax with etoposide significantly reduced tumor burden compared to either single agent, while adding venetoclax to etoposide had no effect on tumor burden. Next, we assessed priming of primary patient NSCLC tumor cells on drugs from a clinically relevant oncology combination screen (CROCS). Results confirmed for the first time the utility of BCL-xL inhibition by navitoclax in priming primary NSCLC tumor cells and identified combinations that primed further. This is a demonstration of the principle that DBP can be used as a functional precision medicine tool to rationally construct combination drug regimens that include BH3 mimetics in solid tumors like NSCLC.
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Project description:BackgroundNeuroblastoma is a paediatric cancer that is characterised by poor prognosis for chemoresistant disease, highlighting the need for better treatment options. Here, we asked whether BH3-mimetics inhibiting BCL2 proteins may eliminate chemoresistant neuroblastoma cells.MethodsWe utilised cisplatin-adapted neuroblastoma cell lines as well as patient tissues before and after relapse to study alterations of BCL2 proteins upon chemoresistance.ResultsIn a direct comparison of cisplatin-resistant cells we identified a prominent loss of sensitivity to BCL2/BCL-XL inhibitors that is associated with an increase in MCL1 dependency and high expression of MCL1 in patient tumour tissues. Screening of FDA-approved anti-cancer drugs in chemoresistant cells identified therapeutics that may be beneficial in combination with the clinically tested BH3-mimetic ABT263, but no synergistic drug interactions with the selective MCL1 inhibitor S63845. Further exploration of potential treatment options for chemoresistant neuroblastoma identified immunotherapy based on NK cells as highly promising, since NK cells are able to efficiently kill both parental and chemoresistant cells.ConclusionsThese data highlight that the application of BH3-mimetics may differ between first line treatment and relapsed disease. Combination of NK cell-based immunotherapy with BH3-mimetics may further increase killing of chemoresistant neuroblastoma, outlining a new treatment strategy for relapsed neuroblastoma.
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