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CS17-1. Cytokine pathways regulating the innate immune response to mycobacteria
Cytokine 56 (2011) 107–108
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Concurrent Session 17: Host-Pathogen Interaction 2
Host resistance to Mycobacterium tuberculosis (Mtb) has been shown to require the induction of cytokines during the innate response to the pathogen. An important player in this protective response is IL-1 which provides a key Myd88 dependent signal responsible for limiting early mycobacterial growth. Interestingly, both IL-1a and IL-1020b play essential roles in this pathway and processing of the latter cytokine in vivo does not require caspase-1 dependent inflammasome activation. In contrast, Type 1 IFNs which are induced in vitro and in vivo preferentiallyby virulent Mtb isolates appear to promote rather than restrict infection. We have recently shown that these two major pathways intersect with Type 1 IFN suppressing IL-1 production in human and mouse macrophages in vitro as well as in vivo in infected mice. In vivo in mouse lung the targets of this suppression are two distinct populations of myeloid cells that display either inflammatory monocyte or dendritic cell markers and simultaneously co-produce both IL-1 species. Surprisingly, in addition to type I IFN, CD4 T cell derived IFN-c also suppresses IL-1a and IL-1b production in vivo but specifically in the inflammatory monocyte population. These innate and adaptive IFN regulatory loops may have evolved to mutually benefit the bacteria and host by simultaneously inhibiting IL-1 dependent control of infection while limiting immunopathology. Supported by the intramural research program of the NIAID, NIH, USA
The innate immune system provides the first line of defence against viruses and its activation results in the production of interferons (IFNs), proinflammatory cytokines and chemokines that combat the infection and activate adaptive immunity. During their evolution viruses have developed mechanisms to counteract these defences, and as such can be useful tools to study and better understand the immune system. Such research may also lead to the design of novel therapeutics and more efficacious vaccines. Vaccinia virus (VACV), a large DNA virus used as the vaccine to eradicate smallpox, expresses many proteins that modulate the host response to its benefit. Protein C6 is one such example. C6 is predicted to be a member of the family of nine VACV Bcl-2 proteins, many of which have immunomodulatory functions and inhibit innate immune signalling cascades. C6 was shown to be a small intracellular protein expressed early during infection that acts as a broad inhibitor of IFNb production by targeting the IRF3, but not NF-jB, innate immune signalling pathway. As such, the expression of C6 was found to block IRF3-dependent gene expression and protein production in cells infected with Newcastle disease virus. The level at which C6 abrogates the IRF3 pathway was determined by reporter gene assay to be downstream, or at the level of the TBK1 complex. Consistent with this, C6 was found to bind TANK, SINTBAD and NAP1, important adaptor molecules involved in IFNb activation. The binding site for C6 on TANK was determined to be the N-terminal coiled-coil region, a domain that is shared amongst the three TBK1 adaptor proteins. Deletion of C6 from VACV had no effect on replication and spread in vitro, but significantly attenuated the virus in two murine models of infection. These results demonstrate C6 is a VACV immunomodulator and acts as a virulence factor in vivo.
doi:10.1016/j.cyto.2011.07.415
doi:10.1016/j.cyto.2011.07.417
CS17-2 Regulation of the Actin Modifier Gelsolin by Protein Kinase R Enforces Basal Innate Immune Defense anthony sadler, Aaron T. Irving, Die Wang, Noga Kozer, Andrew H.A. Clayton, Bryan R.G. Williams, MIMR, CCR, Monash University
CS17-4 Human TLR8 is activated upon recognition of Borrelia burgdorferi RNA in the phagosome of human monocytes Jorge L. Cervantes, Carson J. La Vake, Juan C. Salazar, Departments of Pediatrics and Immunology, University of Connecticut Health Center, Farmington, CT and Connecticut Children’s Medical Center, Hartford, CT, USA
CS17-1 Cytokine pathways regulating the innate immune response to mycobacteria A. Sher, K. Mayer-Barber, A. Novikov, B. Andrade, D. Barber, K. Shenderov, C. Feng, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, 20892. USA
Primary resistance to pathogens is reliant on the innate immune response. This innate resistance depends upon both basal and inducible defenses. To date, research has focused upon conspicuous inducible innate immune responses. In contrast to this resistance via cytokine induction, basal defense mechanisms are less evident. Here we show that the antiviral protein kinase R (PKR) inhibits the key actin-modifying protein gelsolin (GSN) to regulate actin dynamics and control cytoskeletal cellular functions under homeostatic conditions. Through this mechanism, PKR controls fundamental innate immune, actin-dependent processes that include membrane ruffling and endocytosis. Significantly, PKR counteracts viral entry into the cell. These findings identify a new layer of host resistance, showing that the regulation of actin-modifying proteins during the innate immune response bolsters first-line defense against intracellular pathogens. Moreover, these data provide proof of principle for a concept in which the cell cytoskeleton could be targeted to elicit broad antiviral protection doi:10.1016/j.cyto.2011.07.416
CS17-3 Vaccinia Virus Protein C6 is a Virulence Factor that Binds TBK1 Adaptor Proteins and Inhibits Activation of IRF3 Rebecca P Sumner a,b, Ren H a, Unterholzner L b, Bowie Ag b, Smith Gl a, a Section of Virology, Faculty of Medicine, Imperial College London, St Mary’s Campus, Norfolk Place, London W2 1PG, UK, b School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland
doi:10.1016/S1043-4666(11)00647-8
Phagocytosed Borrelia burgdorferi (Bb), the spirochetal bacterium which causes Lyme disease, induces innate immune signals in human monocytes which include transcription of IFN-b. We previously showed that in response to phagocytosed Bb, TLR8 cooperates with TLR2 in the induction of NF-jB mediated cytokines, while TLR8 is self-amplifying and solely responsible for induction of IFN-b, through IRF-7. In order to determine if borrelial RNA could serve as the pathogen associated molecular pattern (PAMP) responsible for inducing type I IFNs, herein we stimulated highly purified human monocytes with borrelial RNA. R848 (a TLR7/8 ligand), 3M-002 (a TLR8 ligand), and LPS (a TLR4-ligand) were used as controls. We observed that delivery of Bb RNA into the monocyte, like 3M-002, elicits transcription of IFN-b. Using confocal microscopy we show that in human monocytes, TLR8 colocalizes with internalized Bb-RNA bound to its fluorescent delivery vehicle polyethilenimine (PEI) and in both early (EEA1) and late endosomes (LAMP-1). Using HEK.293 cells stably transfected with human TLR-8, along with an NFk-B reporter, we then demonstrate that Bb RNA induces activation of NF-kB. TLR8 also colocalizes with delivered Bb RNA in this cell line as well. Initially associated with recognition of ssRNA of viral origin, we now demonstrate for the first time that phagosomal TLR8 activation occurs upon recognition of bacterial RNA. We conclude that human TLR8 is able to recognize Bb RNA as a ligand responsible for eliciting type I IFN signals in human monocytes. doi:10.1016/j.cyto.2011.07.418
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Concurrent Session 17: Host-Pathogen Interaction 2
Host resistance to Mycobacterium tuberculosis (Mtb) has been shown to require the induction of cytokines during the innate response to the pathogen. An important player in this protective response is IL-1 which provides a key Myd88 dependent signal responsible for limiting early mycobacterial growth. Interestingly, both IL-1a and IL-1020b play essential roles in this pathway and processing of the latter cytokine in vivo does not require caspase-1 dependent inflammasome activation. In contrast, Type 1 IFNs which are induced in vitro and in vivo preferentiallyby virulent Mtb isolates appear to promote rather than restrict infection. We have recently shown that these two major pathways intersect with Type 1 IFN suppressing IL-1 production in human and mouse macrophages in vitro as well as in vivo in infected mice. In vivo in mouse lung the targets of this suppression are two distinct populations of myeloid cells that display either inflammatory monocyte or dendritic cell markers and simultaneously co-produce both IL-1 species. Surprisingly, in addition to type I IFN, CD4 T cell derived IFN-c also suppresses IL-1a and IL-1b production in vivo but specifically in the inflammatory monocyte population. These innate and adaptive IFN regulatory loops may have evolved to mutually benefit the bacteria and host by simultaneously inhibiting IL-1 dependent control of infection while limiting immunopathology. Supported by the intramural research program of the NIAID, NIH, USA
The innate immune system provides the first line of defence against viruses and its activation results in the production of interferons (IFNs), proinflammatory cytokines and chemokines that combat the infection and activate adaptive immunity. During their evolution viruses have developed mechanisms to counteract these defences, and as such can be useful tools to study and better understand the immune system. Such research may also lead to the design of novel therapeutics and more efficacious vaccines. Vaccinia virus (VACV), a large DNA virus used as the vaccine to eradicate smallpox, expresses many proteins that modulate the host response to its benefit. Protein C6 is one such example. C6 is predicted to be a member of the family of nine VACV Bcl-2 proteins, many of which have immunomodulatory functions and inhibit innate immune signalling cascades. C6 was shown to be a small intracellular protein expressed early during infection that acts as a broad inhibitor of IFNb production by targeting the IRF3, but not NF-jB, innate immune signalling pathway. As such, the expression of C6 was found to block IRF3-dependent gene expression and protein production in cells infected with Newcastle disease virus. The level at which C6 abrogates the IRF3 pathway was determined by reporter gene assay to be downstream, or at the level of the TBK1 complex. Consistent with this, C6 was found to bind TANK, SINTBAD and NAP1, important adaptor molecules involved in IFNb activation. The binding site for C6 on TANK was determined to be the N-terminal coiled-coil region, a domain that is shared amongst the three TBK1 adaptor proteins. Deletion of C6 from VACV had no effect on replication and spread in vitro, but significantly attenuated the virus in two murine models of infection. These results demonstrate C6 is a VACV immunomodulator and acts as a virulence factor in vivo.
doi:10.1016/j.cyto.2011.07.415
doi:10.1016/j.cyto.2011.07.417
CS17-2 Regulation of the Actin Modifier Gelsolin by Protein Kinase R Enforces Basal Innate Immune Defense anthony sadler, Aaron T. Irving, Die Wang, Noga Kozer, Andrew H.A. Clayton, Bryan R.G. Williams, MIMR, CCR, Monash University
CS17-4 Human TLR8 is activated upon recognition of Borrelia burgdorferi RNA in the phagosome of human monocytes Jorge L. Cervantes, Carson J. La Vake, Juan C. Salazar, Departments of Pediatrics and Immunology, University of Connecticut Health Center, Farmington, CT and Connecticut Children’s Medical Center, Hartford, CT, USA
CS17-1 Cytokine pathways regulating the innate immune response to mycobacteria A. Sher, K. Mayer-Barber, A. Novikov, B. Andrade, D. Barber, K. Shenderov, C. Feng, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, 20892. USA
Primary resistance to pathogens is reliant on the innate immune response. This innate resistance depends upon both basal and inducible defenses. To date, research has focused upon conspicuous inducible innate immune responses. In contrast to this resistance via cytokine induction, basal defense mechanisms are less evident. Here we show that the antiviral protein kinase R (PKR) inhibits the key actin-modifying protein gelsolin (GSN) to regulate actin dynamics and control cytoskeletal cellular functions under homeostatic conditions. Through this mechanism, PKR controls fundamental innate immune, actin-dependent processes that include membrane ruffling and endocytosis. Significantly, PKR counteracts viral entry into the cell. These findings identify a new layer of host resistance, showing that the regulation of actin-modifying proteins during the innate immune response bolsters first-line defense against intracellular pathogens. Moreover, these data provide proof of principle for a concept in which the cell cytoskeleton could be targeted to elicit broad antiviral protection doi:10.1016/j.cyto.2011.07.416
CS17-3 Vaccinia Virus Protein C6 is a Virulence Factor that Binds TBK1 Adaptor Proteins and Inhibits Activation of IRF3 Rebecca P Sumner a,b, Ren H a, Unterholzner L b, Bowie Ag b, Smith Gl a, a Section of Virology, Faculty of Medicine, Imperial College London, St Mary’s Campus, Norfolk Place, London W2 1PG, UK, b School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland
doi:10.1016/S1043-4666(11)00647-8
Phagocytosed Borrelia burgdorferi (Bb), the spirochetal bacterium which causes Lyme disease, induces innate immune signals in human monocytes which include transcription of IFN-b. We previously showed that in response to phagocytosed Bb, TLR8 cooperates with TLR2 in the induction of NF-jB mediated cytokines, while TLR8 is self-amplifying and solely responsible for induction of IFN-b, through IRF-7. In order to determine if borrelial RNA could serve as the pathogen associated molecular pattern (PAMP) responsible for inducing type I IFNs, herein we stimulated highly purified human monocytes with borrelial RNA. R848 (a TLR7/8 ligand), 3M-002 (a TLR8 ligand), and LPS (a TLR4-ligand) were used as controls. We observed that delivery of Bb RNA into the monocyte, like 3M-002, elicits transcription of IFN-b. Using confocal microscopy we show that in human monocytes, TLR8 colocalizes with internalized Bb-RNA bound to its fluorescent delivery vehicle polyethilenimine (PEI) and in both early (EEA1) and late endosomes (LAMP-1). Using HEK.293 cells stably transfected with human TLR-8, along with an NFk-B reporter, we then demonstrate that Bb RNA induces activation of NF-kB. TLR8 also colocalizes with delivered Bb RNA in this cell line as well. Initially associated with recognition of ssRNA of viral origin, we now demonstrate for the first time that phagosomal TLR8 activation occurs upon recognition of bacterial RNA. We conclude that human TLR8 is able to recognize Bb RNA as a ligand responsible for eliciting type I IFN signals in human monocytes. doi:10.1016/j.cyto.2011.07.418