- Email: [email protected]
75
Abstract / Cytokine 63 (2013) 243–314 coding polymorphism regulating risk for MS and other autoimmune diseases, and we observe that this polymorphism affects strength of signaling. Our findings indicate that IL-7R mutations drive T-ALL, whereas polymorphisms that increase signaling promote autoimmunity, implicating IL-7R and Jak1 as therapeutic targets in these diseases. http://dx.doi.org/10.1016/j.cyto.2013.06.077
75 Interferon Regulatory Factor 4 (IRF4) mediates IFN-b and k1 expression upon TLR4 and MR challenge in human alternatively activated macrophages Ashraf El Fiky, Roger Perreault, Gwendolyn J. McGinnis, Ronald L. Rabin, Laboratory for Immunobiochemistry, Division of Bacterial, Parasitic and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, United States Food and Drug Administration, Bethesda, MD 20892, United States Alternative pathway of macrophage activation (AAM) sustains or enhances the inflammation associated with chronic asthma. Using the THP-1 cell line, we have successfully established a model of ‘‘classically” and ‘‘alternatively” activated macrophages (THP-CAM and THP-AAM, respectively), each of which expresses the appropriate signature cytokines and chemokines. We demonstrate that in response to TLR4 challenge by LPS, THP-AAM had attenuated IFN a/b pathway genes including STAT1/2, MyD88, Mx1, CXCL10, OAS1, and ISG1. In addition, THP-AAM poorly expressed IFN-b, IFN-k1, and specific subtypes of IFN-a coupled with reduced ability to drive NF-jB driven luciferase reporter, compared to their CAM counterparts. On the other hand, THP-AAM preferentially expressed both mannose receptor (MR) and IRF4. THP-AAM exhibit an MR-dependent expression of IFN-b, k1, and IL-10 upon binding to house dust mite protein Dermatophagoides pteronyssinus (Derp1). This binding and subsequent cytokine signature expression is compromised upon MR silencing or blocking MR with mannan. IRF4 is critical for expression of key AAM genes including MR and STAT6, and for AAM reduced phagocytosis of Staphylococcus aureus. IRF4 can regulate expression of IFN-b and IFN-k1 in response to TLR4 and MR challenge by LPS or Derp1 respectively, pointing to a potential role of IRF4 in mediating the AAM phenotype. Taken together, our data demonstrate that THP-AAM is a critical tool to investigate cytokines and chemokines expression profile, in particular IFN a/b pathway genes and type I and III IFN subtypes, in this phenotype. In addition, we demonstrate that IRF4 plays a critical role in mediating AAM response to bacterial and allergen challenge. These findings may provide the basis of a novel IRF4 or MR targeted therapy to decrease allergic.
261
77 The immune system cannot generate immunological memory during infection with the Lyme disease agent B. burgdorfer R.A. Elsner, S.W. Barthold, N. Baumgarth, Graduate Group in Microbiology and the Center for Comparative Medicine, University of California-Davis, Davis, CA, USA In vertebrates including humans, mice and dogs, the bacteria Borrelia burgdorferi (Bb) causes a chronic, non-resolving infection known as Lyme disease, which requires antibiotic treatment to clear the bacteria. Re-infections are common in endemic regions. Similarly, mice can be re-infected with the same strain of Bb, implying a lack of functional immune responses. The mechanisms underlying this lack of effective short and long-term immunity to Bb are unknown. Using a mouse model of Bb-infection we show that infection with Bb produces strong T-dependent and T-independent serum antibodies, characterized by the unusual continued presence of IgM. Remarkably, both T-dependent and T-independent antibodies disappear rapidly when infection is controlled by antibiotic treatment and Bb-specific memory B cells could not be recovered. Thus, maintenance of Bb-specific humoral responses requires ongoing infections. Histological and flow cytometric examination of germinal centers, birthplaces of long-term humoral immunity, demonstrate their induction within 2 weeks of a primary infection and the presence of germinal center follicular helper T and B cells. However, the apparent normal induction of germinal centers is followed by their rapid and global collapse in multiple lymphoid organs by day 45. To determine whether the lack of memory formation is due to the nature of the Bb-antigens or is a sign of Bb-infection-mediated immune suppression, we vaccinated mice with influenza virus during an ongoing Bb-infection. Remarkably, in Bb infected mice the early antibody response to this unrelated antigen was skewed towards increased IgM production compared to that in non-infected mice, and influenza specific IgG responses were strongly reduced. Together our data demonstrate that Bb infection suppresses the development of long-lived antibody production and immunological memory formation and indicates that Bb may achieve this by suppressing the function and/or causing the rapid and global collapse of germinal centers. Supported by NIH AI073911 and T32 AI060555. http://dx.doi.org/10.1016/j.cyto.2013.06.080
78 Therapeutic inhibition of Gp130/Jak/Stat3-dependent cytokine signaling suppresses WNT-dependent colon cancer formation
http://dx.doi.org/10.1016/j.cyto.2013.06.078
Matthias Ernst, Toby J. Phesse, Stefan Thiem, Michael Buchert, Cell Signaling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia
76 ADAM17 is activated upon cellular senescence and contributes to the secretory profile of senescent cells by ectodomain shedding
The initiating mutations in the majority of sporadic and familial colorectal cancers (CRC) occur in the tumor suppressor gene APC and result in nuclear accumulation of b-catenin associated with aberrant activation of the WNT pathway. Meanwhile, a tightly controlled burst of WNT signalling is required for regeneration of the intestinal epithelium after injury and ensures homeostasis. Since identification of therapeutic WNT pathway inhibitors remains challenging, we explored whether genetic interference with cytokine-dependent gp130/Jak/Stat3 signaling affects the intestine’s response to oncogenic WNT signaling. In situations of chronic inflammation and excessive secretion of interleukin- (IL) 6-family cytokines, we have previously shown that gp130 engagement simultaneously activates Stat3 and the mTOR complex 1, and that the mTorc1-specific inhibitor RAD001 potently suppressed initiation and progression of colitis-associated CRC (Thiem et al., J Clin Invest (2013) 123:767). Here we describe that partial genetic suppression of either Stat3 expression, or of its gp130 receptor-dependent activation, prevents tumorigenesis in mouse models of sporadic and of inherited CRC. Likewise, therapeutic administration of the Jak-kinase inhibitors effectively blocked de novo tumor initiation and progression in these models. Surprisingly, gp130/Jak/Stat3 pathway inhibition selectively impaired the growth of mutant APC mouse tumors and human CRC cell xenografts without affecting homeostasis of the non-neoplastic intestinal epithelium. We identify Stat3-dependent Bmi1 induction and the resulting repression of p16 and p21 as the mechanism by which the gp130/Jak/Stat3 pathway modulates the cellular response to aberrant WNT signaling. Meanwhile, a large proportion human CRC harbours coinciding WNT- and STAT3 gene activation signatures. Interference with the gp130/Jak/Stat3 pathway is therefore readily exploitable for the therapeutic treatment of CRC.
Timo Effenberger, Jan von der Heyde, Kareen Bartsch, Athena Chalaris, Stefan RoseJohn, Björn Rabe, Institute of Biochemistry, Medical Faculty, University of Kiel, Germany Senescence is the irreversible growth arrest following cellular stressors such as DNA damage. It has become increasingly apparent that senescence represents a crucial tumor suppressor mechanism restricting the proliferation of cells at risk for neoplastic transformation. By secreting numerous cytokines, growth factors as well as proteases – a phenomenon that has been termed Senescence – Associated Secretory Phenotype (SASP) – senescent cells generate a strong proinflammatory environment likely promoting the development of chronic inflammatory diseases and – paradoxically – cancer. The supernatant of senescent cells contains various SASP factors, that are primarily synthesized by the cell as membrane-bound proteins and only afterwards are converted into their soluble counterpart by – as yet unknown – posttranslational means. Here, we show that upon induction of cellular senescence the metalloprotease ADAM17 is activated and releases Amphiregulin and TNFRI from the surface of human cancer cells as well as primary cells by ectodomain shedding. ADAM17 activation involves phosphorylation of its cytoplasmic tail, which is mediated by MAP kinase p38. In addition, these findings have been confirmed in primary embryonic fibroblasts derived from ADAM17 deficient mice suggesting that ADAM17mediated shedding of SASP components is relevant across species. However, mechanisms governing the SASP are complex, since elevated levels of ICAM-1 are due to increased microparticle release rather than proteolytic ectodomain shedding. http://dx.doi.org/10.1016/j.cyto.2013.06.079
http://dx.doi.org/10.1016/j.cyto.2013.06.081
75 Interferon Regulatory Factor 4 (IRF4) mediates IFN-b and k1 expression upon TLR4 and MR challenge in human alternatively activated macrophages Ashraf El Fiky, Roger Perreault, Gwendolyn J. McGinnis, Ronald L. Rabin, Laboratory for Immunobiochemistry, Division of Bacterial, Parasitic and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, United States Food and Drug Administration, Bethesda, MD 20892, United States Alternative pathway of macrophage activation (AAM) sustains or enhances the inflammation associated with chronic asthma. Using the THP-1 cell line, we have successfully established a model of ‘‘classically” and ‘‘alternatively” activated macrophages (THP-CAM and THP-AAM, respectively), each of which expresses the appropriate signature cytokines and chemokines. We demonstrate that in response to TLR4 challenge by LPS, THP-AAM had attenuated IFN a/b pathway genes including STAT1/2, MyD88, Mx1, CXCL10, OAS1, and ISG1. In addition, THP-AAM poorly expressed IFN-b, IFN-k1, and specific subtypes of IFN-a coupled with reduced ability to drive NF-jB driven luciferase reporter, compared to their CAM counterparts. On the other hand, THP-AAM preferentially expressed both mannose receptor (MR) and IRF4. THP-AAM exhibit an MR-dependent expression of IFN-b, k1, and IL-10 upon binding to house dust mite protein Dermatophagoides pteronyssinus (Derp1). This binding and subsequent cytokine signature expression is compromised upon MR silencing or blocking MR with mannan. IRF4 is critical for expression of key AAM genes including MR and STAT6, and for AAM reduced phagocytosis of Staphylococcus aureus. IRF4 can regulate expression of IFN-b and IFN-k1 in response to TLR4 and MR challenge by LPS or Derp1 respectively, pointing to a potential role of IRF4 in mediating the AAM phenotype. Taken together, our data demonstrate that THP-AAM is a critical tool to investigate cytokines and chemokines expression profile, in particular IFN a/b pathway genes and type I and III IFN subtypes, in this phenotype. In addition, we demonstrate that IRF4 plays a critical role in mediating AAM response to bacterial and allergen challenge. These findings may provide the basis of a novel IRF4 or MR targeted therapy to decrease allergic.
261
77 The immune system cannot generate immunological memory during infection with the Lyme disease agent B. burgdorfer R.A. Elsner, S.W. Barthold, N. Baumgarth, Graduate Group in Microbiology and the Center for Comparative Medicine, University of California-Davis, Davis, CA, USA In vertebrates including humans, mice and dogs, the bacteria Borrelia burgdorferi (Bb) causes a chronic, non-resolving infection known as Lyme disease, which requires antibiotic treatment to clear the bacteria. Re-infections are common in endemic regions. Similarly, mice can be re-infected with the same strain of Bb, implying a lack of functional immune responses. The mechanisms underlying this lack of effective short and long-term immunity to Bb are unknown. Using a mouse model of Bb-infection we show that infection with Bb produces strong T-dependent and T-independent serum antibodies, characterized by the unusual continued presence of IgM. Remarkably, both T-dependent and T-independent antibodies disappear rapidly when infection is controlled by antibiotic treatment and Bb-specific memory B cells could not be recovered. Thus, maintenance of Bb-specific humoral responses requires ongoing infections. Histological and flow cytometric examination of germinal centers, birthplaces of long-term humoral immunity, demonstrate their induction within 2 weeks of a primary infection and the presence of germinal center follicular helper T and B cells. However, the apparent normal induction of germinal centers is followed by their rapid and global collapse in multiple lymphoid organs by day 45. To determine whether the lack of memory formation is due to the nature of the Bb-antigens or is a sign of Bb-infection-mediated immune suppression, we vaccinated mice with influenza virus during an ongoing Bb-infection. Remarkably, in Bb infected mice the early antibody response to this unrelated antigen was skewed towards increased IgM production compared to that in non-infected mice, and influenza specific IgG responses were strongly reduced. Together our data demonstrate that Bb infection suppresses the development of long-lived antibody production and immunological memory formation and indicates that Bb may achieve this by suppressing the function and/or causing the rapid and global collapse of germinal centers. Supported by NIH AI073911 and T32 AI060555. http://dx.doi.org/10.1016/j.cyto.2013.06.080
78 Therapeutic inhibition of Gp130/Jak/Stat3-dependent cytokine signaling suppresses WNT-dependent colon cancer formation
http://dx.doi.org/10.1016/j.cyto.2013.06.078
Matthias Ernst, Toby J. Phesse, Stefan Thiem, Michael Buchert, Cell Signaling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia
76 ADAM17 is activated upon cellular senescence and contributes to the secretory profile of senescent cells by ectodomain shedding
The initiating mutations in the majority of sporadic and familial colorectal cancers (CRC) occur in the tumor suppressor gene APC and result in nuclear accumulation of b-catenin associated with aberrant activation of the WNT pathway. Meanwhile, a tightly controlled burst of WNT signalling is required for regeneration of the intestinal epithelium after injury and ensures homeostasis. Since identification of therapeutic WNT pathway inhibitors remains challenging, we explored whether genetic interference with cytokine-dependent gp130/Jak/Stat3 signaling affects the intestine’s response to oncogenic WNT signaling. In situations of chronic inflammation and excessive secretion of interleukin- (IL) 6-family cytokines, we have previously shown that gp130 engagement simultaneously activates Stat3 and the mTOR complex 1, and that the mTorc1-specific inhibitor RAD001 potently suppressed initiation and progression of colitis-associated CRC (Thiem et al., J Clin Invest (2013) 123:767). Here we describe that partial genetic suppression of either Stat3 expression, or of its gp130 receptor-dependent activation, prevents tumorigenesis in mouse models of sporadic and of inherited CRC. Likewise, therapeutic administration of the Jak-kinase inhibitors effectively blocked de novo tumor initiation and progression in these models. Surprisingly, gp130/Jak/Stat3 pathway inhibition selectively impaired the growth of mutant APC mouse tumors and human CRC cell xenografts without affecting homeostasis of the non-neoplastic intestinal epithelium. We identify Stat3-dependent Bmi1 induction and the resulting repression of p16 and p21 as the mechanism by which the gp130/Jak/Stat3 pathway modulates the cellular response to aberrant WNT signaling. Meanwhile, a large proportion human CRC harbours coinciding WNT- and STAT3 gene activation signatures. Interference with the gp130/Jak/Stat3 pathway is therefore readily exploitable for the therapeutic treatment of CRC.
Timo Effenberger, Jan von der Heyde, Kareen Bartsch, Athena Chalaris, Stefan RoseJohn, Björn Rabe, Institute of Biochemistry, Medical Faculty, University of Kiel, Germany Senescence is the irreversible growth arrest following cellular stressors such as DNA damage. It has become increasingly apparent that senescence represents a crucial tumor suppressor mechanism restricting the proliferation of cells at risk for neoplastic transformation. By secreting numerous cytokines, growth factors as well as proteases – a phenomenon that has been termed Senescence – Associated Secretory Phenotype (SASP) – senescent cells generate a strong proinflammatory environment likely promoting the development of chronic inflammatory diseases and – paradoxically – cancer. The supernatant of senescent cells contains various SASP factors, that are primarily synthesized by the cell as membrane-bound proteins and only afterwards are converted into their soluble counterpart by – as yet unknown – posttranslational means. Here, we show that upon induction of cellular senescence the metalloprotease ADAM17 is activated and releases Amphiregulin and TNFRI from the surface of human cancer cells as well as primary cells by ectodomain shedding. ADAM17 activation involves phosphorylation of its cytoplasmic tail, which is mediated by MAP kinase p38. In addition, these findings have been confirmed in primary embryonic fibroblasts derived from ADAM17 deficient mice suggesting that ADAM17mediated shedding of SASP components is relevant across species. However, mechanisms governing the SASP are complex, since elevated levels of ICAM-1 are due to increased microparticle release rather than proteolytic ectodomain shedding. http://dx.doi.org/10.1016/j.cyto.2013.06.079
http://dx.doi.org/10.1016/j.cyto.2013.06.081