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Contains fulltext : 248786.pdf (Publisher’s version ) (Open Access), Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Contains fulltext : 285287.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 282219.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 248786.pdf (Publisher’s version ) (Open Access), Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Contains fulltext : 282219.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 248786.pdf (Publisher’s version ) (Open Access), Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Contains fulltext : 206946.pdf (publisher's version ) (Open Access), The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 5 (Stx5) in mammals and its ortholog Sed5p in Saccharomyces cerevisiae mediate anterograde and retrograde endoplasmic reticulum (ER)-Golgi trafficking. Stx5 and Sed5p are structurally highly conserved and are both regulated by interactions with other ER-Golgi SNARE proteins, the Sec1/Munc18-like protein Scfd1/Sly1p and the membrane tethering complexes COG, p115, and GM130. Despite these similarities, yeast Sed5p and mammalian Stx5 are differently recruited to COPII-coated vesicles, and Stx5 interacts with the microtubular cytoskeleton, whereas Sed5p does not. In this review, we argue that these different Stx5 interactions contribute to structural differences in ER-Golgi transport between mammalian and yeast cells. Insight into the function of Stx5 is important given its essential role in the secretory pathway of eukaryotic cells and its involvement in infections and neurodegenerative diseases.

Contains fulltext : 202589.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, Immune-cell activation by inflammatory stimuli triggers the transcription and translation of large amounts of cytokines. The transport of newly synthesized cytokines to the plasma membrane by vesicular trafficking can be rate-limiting for the production of these cytokines, and immune cells upregulate their exocytic machinery concomitantly with increased cytokine expression in order to cope with the increasing demand for trafficking. Whereas it is logical that trafficking is rate-limiting for regulated secretion where an intracellular pool of molecules is waiting to be released, the reason for this is not obvious for constitutively secreted cytokines, such as interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha). These constitutively secreted cytokines are primarily regulated at the transcriptional and/or translational level but mounting evidence presented here shows that cells might also increase or decrease the rate of post-Golgi cytokine trafficking to modulate their production. Therefore, in this Hypothesis, we ask the question: why is there a need to limit the trafficking of constitutively secreted cytokines? We propose a model where cells monitor and adjust their production rate of cytokines by sensing the intracellular level of cytokines while they are in transit to the plasma membrane. This self-regulation of cytokine production could prevent an overshooting response of acute-phase cytokines, such as IL-6, IL-12 and TNF-alpha, upon acute infection.

Contains fulltext : 201259.pdf (publisher's version ) (Open Access)

Contains fulltext : 206946.pdf (publisher's version ) (Open Access), The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 5 (Stx5) in mammals and its ortholog Sed5p in Saccharomyces cerevisiae mediate anterograde and retrograde endoplasmic reticulum (ER)-Golgi trafficking. Stx5 and Sed5p are structurally highly conserved and are both regulated by interactions with other ER-Golgi SNARE proteins, the Sec1/Munc18-like protein Scfd1/Sly1p and the membrane tethering complexes COG, p115, and GM130. Despite these similarities, yeast Sed5p and mammalian Stx5 are differently recruited to COPII-coated vesicles, and Stx5 interacts with the microtubular cytoskeleton, whereas Sed5p does not. In this review, we argue that these different Stx5 interactions contribute to structural differences in ER-Golgi transport between mammalian and yeast cells. Insight into the function of Stx5 is important given its essential role in the secretory pathway of eukaryotic cells and its involvement in infections and neurodegenerative diseases.

Contains fulltext : 202589.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, Immune-cell activation by inflammatory stimuli triggers the transcription and translation of large amounts of cytokines. The transport of newly synthesized cytokines to the plasma membrane by vesicular trafficking can be rate-limiting for the production of these cytokines, and immune cells upregulate their exocytic machinery concomitantly with increased cytokine expression in order to cope with the increasing demand for trafficking. Whereas it is logical that trafficking is rate-limiting for regulated secretion where an intracellular pool of molecules is waiting to be released, the reason for this is not obvious for constitutively secreted cytokines, such as interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha). These constitutively secreted cytokines are primarily regulated at the transcriptional and/or translational level but mounting evidence presented here shows that cells might also increase or decrease the rate of post-Golgi cytokine trafficking to modulate their production. Therefore, in this Hypothesis, we ask the question: why is there a need to limit the trafficking of constitutively secreted cytokines? We propose a model where cells monitor and adjust their production rate of cytokines by sensing the intracellular level of cytokines while they are in transit to the plasma membrane. This self-regulation of cytokine production could prevent an overshooting response of acute-phase cytokines, such as IL-6, IL-12 and TNF-alpha, upon acute infection.

Contains fulltext : 201259.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, Immune-cell activation by inflammatory stimuli triggers the transcription and translation of large amounts of cytokines. The transport of newly synthesized cytokines to the plasma membrane by vesicular trafficking can be rate-limiting for the production of these cytokines, and immune cells upregulate their exocytic machinery concomitantly with increased cytokine expression in order to cope with the increasing demand for trafficking. Whereas it is logical that trafficking is rate-limiting for regulated secretion where an intracellular pool of molecules is waiting to be released, the reason for this is not obvious for constitutively secreted cytokines, such as interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha). These constitutively secreted cytokines are primarily regulated at the transcriptional and/or translational level but mounting evidence presented here shows that cells might also increase or decrease the rate of post-Golgi cytokine trafficking to modulate their production. Therefore, in this Hypothesis, we ask the question: why is there a need to limit the trafficking of constitutively secreted cytokines? We propose a model where cells monitor and adjust their production rate of cytokines by sensing the intracellular level of cytokines while they are in transit to the plasma membrane. This self-regulation of cytokine production could prevent an overshooting response of acute-phase cytokines, such as IL-6, IL-12 and TNF-alpha, upon acute infection.

Contains fulltext : 191566.pdf (publisher's version ) (Open Access)

Contains fulltext : 200162.pdf (publisher's version ) (Open Access), Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-alpha secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.

Contains fulltext : 190876pos.pdf (postprint version ) (Open Access)

Contains fulltext : 200162.pdf (publisher's version ) (Open Access), Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-alpha secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.

Contains fulltext : 200162.pdf (publisher's version ) (Open Access), Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-alpha secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.

Contains fulltext : 190876pos.pdf (postprint version ) (Open Access)

Contains fulltext : 191566.pdf (publisher's version ) (Open Access)

Contains fulltext : 190876pos.pdf (postprint version ) (Open Access)

Contains fulltext : 191566.pdf (publisher's version ) (Open Access)

Contains fulltext : 200162.pdf (publisher's version ) (Open Access), Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-alpha secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.

Contains fulltext : 177737.pdf (publisher's version ) (Open Access), SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Forster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood-derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution.

Contains fulltext : 177737.pdf (publisher's version ) (Open Access), SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Forster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood-derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution.