- Email: [email protected]
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Abstract / Cytokine 63 (2013) 243–314
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236 Computational model for mechanistic investigation of negative feedback in Interleukin-17 receptor signaling
238 Homeostatic IL23R signaling limits Th17 responses through IL22-mediated containment of commensal microbiota
Robert P. Sheehan a, Abhishek Garg b, Sarah L. Gaffen b, James R. Faeder a, a Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA, b Department of Medicine, Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, USA
Vincent Feng-Sheng Shih a, Jennifer Cox a,1, Noelyn Kljavin b, Hart Rardin a, Michael Reichelt c, Linda Rangell c, Lauri Diehl c, Wenjun Ouyang a, Nico Ghilardi a, a Department of Immunology, USA, b Department of Molecular Biology, USA, c Department of Pathology, USA
IL-17 is a proinflammatory cytokine that promotes autoimmunity and protects against certain pathogens. Computational modeling of signaling pathways is a powerful tool to help understand the complexities of these dynamic processes. Here, we present a detailed computational model of signaling downstream of the Interleukin-17 receptor (IL-17R). To manage the complexity of the system we use a rulebased modeling approach, in which signaling proteins are modeled as structured objects and rules describe their biochemical interactions. The model includes 19 molecules and 46 reaction rules, which expand to a reaction network with 168 distinct chemical species – complexes and phosphoforms of the basic molecules – which are connected through a network of 5145 unidirectional reactions. The model is defined and simulated using the BioNetGen software. Dynamical behavior of the network is simulated using ordinary differential equations. This model encompasses major signaling components downstream of IL-17R, including the adaptor molecule Act1 and signaling intermediates TRAF6, TAB, TAK1, and IKK, which are activated through site-specific ubiquitination and phosphorylation. These components work in concert to activate NF-jB, which in turn promotes the production of pro-inflammatory cytokines and chemokines that are critical for controlling infections by extracellular pathogens. NF-jB also promotes production of A20, a deubiquitinating enzyme that inhibits NF-jB activation in multiple settings. We recently showed that A20 acts as a negative regulator of the IL-17 signaling pathway, and here by modeling A20 interactions with TRAF6 and IKK, we are able to recapitulate experimentally-observed oscillations in NF-jB–dependent A20 expression at both the mRNA and protein levels. Further expansion of the model will enable us to develop and explore novel hypotheses about the role of A20 and other potential regulators of IL-17-mediated signaling.
Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. Disruption of the physiological barriers that prevent these organisms from disseminating outside their assigned niches leads to inflammatory responses. Using segmented filamentous bacteria (SFB) as a sentinel species, we demonstrate that the IL-23/IL-22 pathway dynamically and selectively modulates the relative abundance of certain commensals in the adult animal. Genetic or pharmacological inactivation of the pathway results in defective barrier function, leading to systemic microbial dissemination and enhanced TH17 responses in distal tissues. Furthermore, we demonstrate suppression of TH17 responses in wild-type animals upon activation of the IL-22 pathway. Our data therefore suggests that IL-23/IL-22 pathway is of critical importance for normal immune homeostasis through the maintenance of mucosal barrier function.
http://dx.doi.org/10.1016/j.cyto.2013.06.239
237 Human BDCA2+ BDCA3int dendritic cells are a novel functional subtype of classical plasmacytoid dendritic cell Tiffany Shih b, Jihong Dai a, Patricia Fitzgerald-Bocarsly a,b, a Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry New Jersey-New Jersey Medical School, Newark, NJ, USA, b University of Medicine and Dentistry New JerseyGraduate School of Biomedical Sciences, Newark, NJ, USA Dendritic cells (DCs) are a heterogeneous cell population critical in regulating immune responses to pathogens and self-antigens. Human peripheral blood DCs have been broadly characterized as plasmacytoid DC (BDCA2+), myeloid DC1 (BDCA1+) and myeloid DC2 (BDCA3+) subsets. Our study aimed to understand whether BDCA3+ DCs from human peripheral blood, the putative functional equivalent to murine CD8a+ DCs, constitute a homogeneous population of myeloid DC2 or if there are BDCA2+BDCA3+DCs that are functionally related to pDC. We constructed multi-color panels to phenotype lineageneg PBMC for expression of BDCA2 and BDCA3 by flow cytometric analysis. Three different lineageneg DC subsets from the total circulating PBMC were observed: BDCA2+/3neg (0.21%), BDCA2+/3int(0.22%), and BDCA2neg/3int/ hi (0.17%). To assess the functional significance of the BDCA2+/3int DCs, we stimulated PBMC with TLR9 agonists CpG-A or HSV-1 and stained for intracellular IFN-a and TNF-a in cells expressing BDCA2 and/or BDCA3. At 6hrs, the stimulated BDCA2+/3int DC subset yielded more IFN-a+ and TNF-a+ cells than the other 2 subsets. By 8hrs, the % IFN-a+ was similarly higher for BDCA2+/3 and BDCA2+/3int DC subsets than for BDCA2 /3int/hi DCs. All three DC subsets produced IFN-l1/3 upon 8hr HSV-1 stimulation, but more of the BDCA2+/3 and BDCA2+/3int DCs expressed IFN-l1/3 than BDCA2 /3int/hi DCs. IRF7, the master regulatory transcription factor of IFN production in pDCs, was constitutively expressed by the BDCA2+/3neg and BDCA2+/3int subsets but not by the BDCA2neg subset. Uptake (‘‘nibbling”) of cell-associated membrane from live virus-infected cells induces pDCs to produce IFN-a and mature. Both BDCA2+/3neg and BDCA2+/3int DCs preferentially nibbled HSV-infected Raji cells (with nibbling by BDCA2+/3int > BDCA2+/3neg DCs), while the BDCA2neg/3int/hi DC subset efficiently nibbled both uninfected and infected cells to a similar extent. Together, these results suggest that BDCA2+/3int DCs exhibit major functions associated with classical pDCs and are phenotypically and functionally distinct from BDCA2neg/3int/ hi DCs.
http://dx.doi.org/10.1016/j.cyto.2013.06.240
http://dx.doi.org/10.1016/j.cyto.2013.06.241
239 Dynamic nuclear trafficking of STAT5 regulated by an unconventional nuclear localization signal Ha Youn Shin, Nancy C. Reich, Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA As a regulated transcription factor, precise cellular localization of STAT5 is essential for physiological processes that include hematopoiesis, liver metabolism, and mammary gland development. Conventional nuclear localization signals consist of short stretches of basic amino acids. We provide evidence that STAT5 nuclear import is dependent on an unconventional nuclear localization signal that functions within the conformation of an extensive coiled-coil domain. Both in vitro binding and in vivo functional assays reveal that STAT5 nuclear import is mediated by the importin-a3/b1 system independent of STAT5 activation by tyrosine phosphorylation. The integrity of the coiled-coil domain is essential for STAT5 transcriptional induction of the b-casein gene following prolactin stimulation, as well as its ability to synergize with the glucocorticoid receptor. The glucocorticoid receptor accumulates in the nucleus in response to prolactin, and this nuclear import is dependent on STAT5 nuclear import. STAT5 continually shuttles in and out of the nucleus, and live cell imaging demonstrates STAT5 nuclear export is mediated by both Crm1-dependent and Crm1-independent pathways. A Crm1-dependent nuclear export signal was identified within the STAT5 amino terminus. These findings provide insight to fundamental mechanisms that regulate STAT5 nuclear trafficking and cooperation with the glucocorticoid receptor, and provide a basis for clinical intervention of STAT5 function in disease. http://dx.doi.org/10.1016/j.cyto.2013.06.242
240 Characterization of leukocyte glucocorticoid receptor translocation in response to physical stress Maria Y. Shiu a, Shawn G. Rhind b, Alex P. Di Battista a, Ruth A. Lanius c, Donald J. Richardson c,d, Rakesh Jetly e, a Graduate Program, University of Toronto, Toronto, ON, Canada, b Defence Research & Development (DRDC) Canada Toronto, Toronto, ON, Canada, c Dept of Psychiatry, Schulich School of Medicine, Western University, London, ON, Canada, d Parkwood Operational Stress Injury Clinic, St. Joseph’s Health Care, London, ON, Canada, e Canadian Forces Health Services, Directorate of Mental Health, Dept of National Defence, Canada Physical stress provides an easily manipulated experimental model that allows for the study of interactions between the neuroendocrine and immune systems. Severe exercise elicits marked alterations in circulating stress hormones, causing shifts in leukocyte mobilization and function both during and after exercise. Circulat-
1 Present address: Novo Nordisk Research Center, 530 Fairview Ave N, Seattle, WA 98109, USA.
299
236 Computational model for mechanistic investigation of negative feedback in Interleukin-17 receptor signaling
238 Homeostatic IL23R signaling limits Th17 responses through IL22-mediated containment of commensal microbiota
Robert P. Sheehan a, Abhishek Garg b, Sarah L. Gaffen b, James R. Faeder a, a Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA, b Department of Medicine, Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, USA
Vincent Feng-Sheng Shih a, Jennifer Cox a,1, Noelyn Kljavin b, Hart Rardin a, Michael Reichelt c, Linda Rangell c, Lauri Diehl c, Wenjun Ouyang a, Nico Ghilardi a, a Department of Immunology, USA, b Department of Molecular Biology, USA, c Department of Pathology, USA
IL-17 is a proinflammatory cytokine that promotes autoimmunity and protects against certain pathogens. Computational modeling of signaling pathways is a powerful tool to help understand the complexities of these dynamic processes. Here, we present a detailed computational model of signaling downstream of the Interleukin-17 receptor (IL-17R). To manage the complexity of the system we use a rulebased modeling approach, in which signaling proteins are modeled as structured objects and rules describe their biochemical interactions. The model includes 19 molecules and 46 reaction rules, which expand to a reaction network with 168 distinct chemical species – complexes and phosphoforms of the basic molecules – which are connected through a network of 5145 unidirectional reactions. The model is defined and simulated using the BioNetGen software. Dynamical behavior of the network is simulated using ordinary differential equations. This model encompasses major signaling components downstream of IL-17R, including the adaptor molecule Act1 and signaling intermediates TRAF6, TAB, TAK1, and IKK, which are activated through site-specific ubiquitination and phosphorylation. These components work in concert to activate NF-jB, which in turn promotes the production of pro-inflammatory cytokines and chemokines that are critical for controlling infections by extracellular pathogens. NF-jB also promotes production of A20, a deubiquitinating enzyme that inhibits NF-jB activation in multiple settings. We recently showed that A20 acts as a negative regulator of the IL-17 signaling pathway, and here by modeling A20 interactions with TRAF6 and IKK, we are able to recapitulate experimentally-observed oscillations in NF-jB–dependent A20 expression at both the mRNA and protein levels. Further expansion of the model will enable us to develop and explore novel hypotheses about the role of A20 and other potential regulators of IL-17-mediated signaling.
Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. Disruption of the physiological barriers that prevent these organisms from disseminating outside their assigned niches leads to inflammatory responses. Using segmented filamentous bacteria (SFB) as a sentinel species, we demonstrate that the IL-23/IL-22 pathway dynamically and selectively modulates the relative abundance of certain commensals in the adult animal. Genetic or pharmacological inactivation of the pathway results in defective barrier function, leading to systemic microbial dissemination and enhanced TH17 responses in distal tissues. Furthermore, we demonstrate suppression of TH17 responses in wild-type animals upon activation of the IL-22 pathway. Our data therefore suggests that IL-23/IL-22 pathway is of critical importance for normal immune homeostasis through the maintenance of mucosal barrier function.
http://dx.doi.org/10.1016/j.cyto.2013.06.239
237 Human BDCA2+ BDCA3int dendritic cells are a novel functional subtype of classical plasmacytoid dendritic cell Tiffany Shih b, Jihong Dai a, Patricia Fitzgerald-Bocarsly a,b, a Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry New Jersey-New Jersey Medical School, Newark, NJ, USA, b University of Medicine and Dentistry New JerseyGraduate School of Biomedical Sciences, Newark, NJ, USA Dendritic cells (DCs) are a heterogeneous cell population critical in regulating immune responses to pathogens and self-antigens. Human peripheral blood DCs have been broadly characterized as plasmacytoid DC (BDCA2+), myeloid DC1 (BDCA1+) and myeloid DC2 (BDCA3+) subsets. Our study aimed to understand whether BDCA3+ DCs from human peripheral blood, the putative functional equivalent to murine CD8a+ DCs, constitute a homogeneous population of myeloid DC2 or if there are BDCA2+BDCA3+DCs that are functionally related to pDC. We constructed multi-color panels to phenotype lineageneg PBMC for expression of BDCA2 and BDCA3 by flow cytometric analysis. Three different lineageneg DC subsets from the total circulating PBMC were observed: BDCA2+/3neg (0.21%), BDCA2+/3int(0.22%), and BDCA2neg/3int/ hi (0.17%). To assess the functional significance of the BDCA2+/3int DCs, we stimulated PBMC with TLR9 agonists CpG-A or HSV-1 and stained for intracellular IFN-a and TNF-a in cells expressing BDCA2 and/or BDCA3. At 6hrs, the stimulated BDCA2+/3int DC subset yielded more IFN-a+ and TNF-a+ cells than the other 2 subsets. By 8hrs, the % IFN-a+ was similarly higher for BDCA2+/3 and BDCA2+/3int DC subsets than for BDCA2 /3int/hi DCs. All three DC subsets produced IFN-l1/3 upon 8hr HSV-1 stimulation, but more of the BDCA2+/3 and BDCA2+/3int DCs expressed IFN-l1/3 than BDCA2 /3int/hi DCs. IRF7, the master regulatory transcription factor of IFN production in pDCs, was constitutively expressed by the BDCA2+/3neg and BDCA2+/3int subsets but not by the BDCA2neg subset. Uptake (‘‘nibbling”) of cell-associated membrane from live virus-infected cells induces pDCs to produce IFN-a and mature. Both BDCA2+/3neg and BDCA2+/3int DCs preferentially nibbled HSV-infected Raji cells (with nibbling by BDCA2+/3int > BDCA2+/3neg DCs), while the BDCA2neg/3int/hi DC subset efficiently nibbled both uninfected and infected cells to a similar extent. Together, these results suggest that BDCA2+/3int DCs exhibit major functions associated with classical pDCs and are phenotypically and functionally distinct from BDCA2neg/3int/ hi DCs.
http://dx.doi.org/10.1016/j.cyto.2013.06.240
http://dx.doi.org/10.1016/j.cyto.2013.06.241
239 Dynamic nuclear trafficking of STAT5 regulated by an unconventional nuclear localization signal Ha Youn Shin, Nancy C. Reich, Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA As a regulated transcription factor, precise cellular localization of STAT5 is essential for physiological processes that include hematopoiesis, liver metabolism, and mammary gland development. Conventional nuclear localization signals consist of short stretches of basic amino acids. We provide evidence that STAT5 nuclear import is dependent on an unconventional nuclear localization signal that functions within the conformation of an extensive coiled-coil domain. Both in vitro binding and in vivo functional assays reveal that STAT5 nuclear import is mediated by the importin-a3/b1 system independent of STAT5 activation by tyrosine phosphorylation. The integrity of the coiled-coil domain is essential for STAT5 transcriptional induction of the b-casein gene following prolactin stimulation, as well as its ability to synergize with the glucocorticoid receptor. The glucocorticoid receptor accumulates in the nucleus in response to prolactin, and this nuclear import is dependent on STAT5 nuclear import. STAT5 continually shuttles in and out of the nucleus, and live cell imaging demonstrates STAT5 nuclear export is mediated by both Crm1-dependent and Crm1-independent pathways. A Crm1-dependent nuclear export signal was identified within the STAT5 amino terminus. These findings provide insight to fundamental mechanisms that regulate STAT5 nuclear trafficking and cooperation with the glucocorticoid receptor, and provide a basis for clinical intervention of STAT5 function in disease. http://dx.doi.org/10.1016/j.cyto.2013.06.242
240 Characterization of leukocyte glucocorticoid receptor translocation in response to physical stress Maria Y. Shiu a, Shawn G. Rhind b, Alex P. Di Battista a, Ruth A. Lanius c, Donald J. Richardson c,d, Rakesh Jetly e, a Graduate Program, University of Toronto, Toronto, ON, Canada, b Defence Research & Development (DRDC) Canada Toronto, Toronto, ON, Canada, c Dept of Psychiatry, Schulich School of Medicine, Western University, London, ON, Canada, d Parkwood Operational Stress Injury Clinic, St. Joseph’s Health Care, London, ON, Canada, e Canadian Forces Health Services, Directorate of Mental Health, Dept of National Defence, Canada Physical stress provides an easily manipulated experimental model that allows for the study of interactions between the neuroendocrine and immune systems. Severe exercise elicits marked alterations in circulating stress hormones, causing shifts in leukocyte mobilization and function both during and after exercise. Circulat-
1 Present address: Novo Nordisk Research Center, 530 Fairview Ave N, Seattle, WA 98109, USA.