- Email: [email protected]
123
272
Abstract / Cytokine 63 (2013) 243–314
TCR repertoire is regulated sequentially by three major factors: VDJ recombination, thymic selection, and peripheral homeostasis. Using mice with Lck-cre-driven conditional deletion of PP4 locus, we show that protein phosphatase 4 (PP4), the product of PP4 gene, is essential for the development of mature T cells, such that thymocyte development is completely blocked at the CD4 CD8 stage in the absence of PP4 protein. Further studies of PP4’s functions during late thymocyte development and in mature T cells are, however, prevented by this severe developmental block. To circumvent this limitation, we introduced CD4-cre transgene into mice carrying the conditional PP4 allele (CD4cre-PP4f/f mice). FACS analyses and mixed bone marrow chimera experiments showed that thymocyte development was retarded at multiple stages in CD4cre-PP4f/f mice. The introduction of ab TCR transgene could partially rescue thymocyte maturation, suggesting that PP4 may be important for the generation of TCR-expressing cells. Recently PP4 was shown to regulate cH2AX during DNA repair, which shared many components with VDJ recombination. Western and immunofluorescence analyses of PP4-deficient thymocytes, however, did not reveal any defect in H2AX phosphorylation. To further study the roles of PP4 in TCR generation, we resorted to an inducible VDJ recombination system that utilized vAbl-transformed cell lines. Results from this inducible system showed that the inhibition of PP4 did not cause defects in RAG expression or DNA repair, implying that VDJ recombination per se was not tightly regulated by PP4. Instead, PP4 inhibitor significantly enhanced the induction of apoptosis in cells with on-going VDJ recombination; interestingly, ATM antagonized the effects of PP4 in this context. Our studies thus reveal a novel role of PP4 in thymocytes maturation and TCR repertoire formation, in which the survival window of thymocytes with active VDJ recombination may be critically regulated by PP4.
studies demonstrated that the CD4+ Th17-associated cytokines IL-23 and IL-17 are crucial for host defense in OPC; however, the mechanism of defense is incompletely understood. IL-23 deficiency is associated with decreased induction of signals involved in neutrophil regulation. In contrast to this indirect evidence of a role for neutrophils in resistance to OPC, humans with isolated neutropenia are infrequently susceptible to this disease. We hypothesized that IL-17 mediates host defense against OPC primarily through neutrophil recruitment. To explore this, mice deficient in neutrophil recruitment by knockout (KO) of a key chemokine receptor, CXCR2, were subjected to OPC. CXCR2 KO mice were susceptible to OPC without fungal dissemination. In contrast, IL-23 KO and IL-17 receptor A (IL-17RA) KO mice depleted of neutrophils by treatment with a monoclonal antibody had an incremental increase in susceptibility to OPC without dissemination. Analysis of tongue tissue by flow cytometry showed a robust infiltrate of CD11b+Ly-6Glow cells in mice treated with neutrophildepleting antibody, indicating that this cell population is ineffective or inhibitory to host defense. Neutrophil-depleted C57BL/6 mice showed a bimodal response, wherein most mice were fully resistant to disease while 14% became profoundly ill with local and disseminated disease. Use of low-dose anti-Gr-1 antibody, a potent but less specific neutrophil-depleting agent, resulted in profound susceptibility of C57BL/6 mice to OPC. We conclude that neutrophils can play a role in host defense from OPC, but IL-17 mediates defense primarily through other mechanisms. Detailed understanding of local host defense and interaction between immune cells is likely to aid in the understanding of susceptibility to disease with selective immunomodulatory agents and lead to the discovery of biomarkers of disease risk. http://dx.doi.org/10.1016/j.cyto.2013.06.127
http://dx.doi.org/10.1016/j.cyto.2013.06.125 125 The Sin3a repressor complex regulates the balance between the activities of different STAT proteins 123 IL-33 drives biphasic IL-13 production for noncanonical type 2 immunity against hookworms Li-Yin Hung a,1, Ian P. Lewkowich b,1, Lucas A. Dawson b, Jordan Downey b, Yanfen Yang b, Dirk E. Smith c, De’Broski R. Herberta a, a Division of Experimental Medicine, University of California, San Francisco, CA 94110, United States, b Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, United States, c Department of Inflammation Research, Amgen, Seattle, WA 91320, United States Parasitic helminths are a major cause of chronic human disease, affecting more than 3 billion people worldwide. Host protection against most parasitic helminths relies upon Type 2 cytokine production, but the mechanisms that regulate interleukin (IL) 4 and 13 production from CD4+ T helper 2 cells (TH2) and innate lymphoid type 2 cells (ILC2s) remain incompletely understood. The epithelial cell-derived cytokines IL-25 and IL-33 promote Type 2 responses, but the extent of functional redundancy between these cytokines is unclear and whether Type 2 memory relies upon either IL-25 or IL-33 is unknown. Herein, we demonstrate a pivotal role for IL33 in driving primary and anamnestic immunity against the rodent hookworm Nippostrongylus brasiliensis. IL-33–deficient mice have a selective defect in ILC2–derived IL-13 during both primary and secondary challenge infections but generate stronger canonical CD4+ T helper 2 cells responses (IL-4, IgE, mast cells, and basophils) than WT controls. Lack of IL-13 production in IL-33–deficient mice impairs resistin-like molecule beta (RELMb) expression and eosinophil recruitment, which are two mechanisms that eliminate N. brasiliensis parasites from infected hosts. Thus, IL33 is requisite for IL-13 but not IL-4–driven Type 2 responses during hookworm infection.
L. Icardi a, R. Mori a, V. Gesellchen a, S. Eyckerman a, J. Verhelst b, K. Vercauteren c, X. Saelens b, P. Meuleman c, K. De Bosscher a, M. Boutros d, J. Tavernier a, a Department of Medical Protein Research, VIB, 9000 Ghent, Belgium, b Department of Molecular Biomedical Research, VIB, 9000 Ghent, Belgium, c Center for Vaccinology, Ghent University and Hospital Ghent University, 9000 Ghent, Belgium, d German Cancer Research Center (DKFZ), Department of Cell and Molecular Biology, Heidelberg, Germany The activity of Signal Transducer and Activator of Transcription (STAT) proteins is regulated by a multitude of posttranslational modifications, which control different aspects of signal transduction. In particular, lysine acetylation and deacetylation has emerged as a critical modification, regulating the activity of all seven STAT members. Unlike tyrosine phosphorylation/dephosphorylation, which mainly acts as an on/off switch of STAT activity, the control exerted by acetylation appears multifaceted and by far more complex, exerting distinct and even opposed regulation to different STAT members. We observed that type I interferons (IFNs) can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide siRNA screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as a novel regulator of STAT activity. Sin3a directly interacts with STAT3 and controls its acetylation status, its nuclear accumulation and DNA binding, strongly influencing the transcription of a subset of STAT3-responsive genes. Interfering with Sin3a expression restores, at least partially, the STAT3 transcriptional blockade observed upon IFN treatment. Strikingly, Sin3a exerts opposed regulation on ISGF3 activity, as it is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. We conclude that Sin3a contributes to the regulation of the balance between the activities of different STAT members, and interfering with its activity or expression may re-direct cytokine signals, favoring the onset of pathological conditions, such as autoimmune syndromes, inflammation and cancer.
http://dx.doi.org/10.1016/j.cyto.2013.06.126 http://dx.doi.org/10.1016/j.cyto.2013.06.128
124 The interleukin-17-mediated regulation and role of neutrophils in resistance to oropharyngeal candidiasis Anna R. Huppler a,b, Nydiaris Hernández-Santos b, Heather R. Conti b, Sarah L. Gaffen b, Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA, b Division of Rheumatology and Clinical Immunology, University of Pittsburgh, PA, USA a
Oropharyngeal candidiasis (OPC) is an opportunistic infection associated with a broad range of human conditions, including infancy, acquired immunodeficiencies such as AIDS, and interleukin-17 (IL-17)-related primary immunodeficiencies. Recent
1
These authors contributed equally to this work.
126 NOD1-dependent sensing of bacterial outer membrane vesicles and induction of an autophagic response Aaron Irving a, Lorinda Turner b, Hitomi Mimuro c, Chihiro Sasakawa c, Thomas Kufer d, Dana Philpott e, Richard Ferrero b, Maria Kaparakis-Liaskos b, a Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia, b Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Clayton, Victoria, Australia, c Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan, d Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Germany, e Department of Immunology, The University of Toronto, Ontario, Canada
Abstract / Cytokine 63 (2013) 243–314
TCR repertoire is regulated sequentially by three major factors: VDJ recombination, thymic selection, and peripheral homeostasis. Using mice with Lck-cre-driven conditional deletion of PP4 locus, we show that protein phosphatase 4 (PP4), the product of PP4 gene, is essential for the development of mature T cells, such that thymocyte development is completely blocked at the CD4 CD8 stage in the absence of PP4 protein. Further studies of PP4’s functions during late thymocyte development and in mature T cells are, however, prevented by this severe developmental block. To circumvent this limitation, we introduced CD4-cre transgene into mice carrying the conditional PP4 allele (CD4cre-PP4f/f mice). FACS analyses and mixed bone marrow chimera experiments showed that thymocyte development was retarded at multiple stages in CD4cre-PP4f/f mice. The introduction of ab TCR transgene could partially rescue thymocyte maturation, suggesting that PP4 may be important for the generation of TCR-expressing cells. Recently PP4 was shown to regulate cH2AX during DNA repair, which shared many components with VDJ recombination. Western and immunofluorescence analyses of PP4-deficient thymocytes, however, did not reveal any defect in H2AX phosphorylation. To further study the roles of PP4 in TCR generation, we resorted to an inducible VDJ recombination system that utilized vAbl-transformed cell lines. Results from this inducible system showed that the inhibition of PP4 did not cause defects in RAG expression or DNA repair, implying that VDJ recombination per se was not tightly regulated by PP4. Instead, PP4 inhibitor significantly enhanced the induction of apoptosis in cells with on-going VDJ recombination; interestingly, ATM antagonized the effects of PP4 in this context. Our studies thus reveal a novel role of PP4 in thymocytes maturation and TCR repertoire formation, in which the survival window of thymocytes with active VDJ recombination may be critically regulated by PP4.
studies demonstrated that the CD4+ Th17-associated cytokines IL-23 and IL-17 are crucial for host defense in OPC; however, the mechanism of defense is incompletely understood. IL-23 deficiency is associated with decreased induction of signals involved in neutrophil regulation. In contrast to this indirect evidence of a role for neutrophils in resistance to OPC, humans with isolated neutropenia are infrequently susceptible to this disease. We hypothesized that IL-17 mediates host defense against OPC primarily through neutrophil recruitment. To explore this, mice deficient in neutrophil recruitment by knockout (KO) of a key chemokine receptor, CXCR2, were subjected to OPC. CXCR2 KO mice were susceptible to OPC without fungal dissemination. In contrast, IL-23 KO and IL-17 receptor A (IL-17RA) KO mice depleted of neutrophils by treatment with a monoclonal antibody had an incremental increase in susceptibility to OPC without dissemination. Analysis of tongue tissue by flow cytometry showed a robust infiltrate of CD11b+Ly-6Glow cells in mice treated with neutrophildepleting antibody, indicating that this cell population is ineffective or inhibitory to host defense. Neutrophil-depleted C57BL/6 mice showed a bimodal response, wherein most mice were fully resistant to disease while 14% became profoundly ill with local and disseminated disease. Use of low-dose anti-Gr-1 antibody, a potent but less specific neutrophil-depleting agent, resulted in profound susceptibility of C57BL/6 mice to OPC. We conclude that neutrophils can play a role in host defense from OPC, but IL-17 mediates defense primarily through other mechanisms. Detailed understanding of local host defense and interaction between immune cells is likely to aid in the understanding of susceptibility to disease with selective immunomodulatory agents and lead to the discovery of biomarkers of disease risk. http://dx.doi.org/10.1016/j.cyto.2013.06.127
http://dx.doi.org/10.1016/j.cyto.2013.06.125 125 The Sin3a repressor complex regulates the balance between the activities of different STAT proteins 123 IL-33 drives biphasic IL-13 production for noncanonical type 2 immunity against hookworms Li-Yin Hung a,1, Ian P. Lewkowich b,1, Lucas A. Dawson b, Jordan Downey b, Yanfen Yang b, Dirk E. Smith c, De’Broski R. Herberta a, a Division of Experimental Medicine, University of California, San Francisco, CA 94110, United States, b Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, United States, c Department of Inflammation Research, Amgen, Seattle, WA 91320, United States Parasitic helminths are a major cause of chronic human disease, affecting more than 3 billion people worldwide. Host protection against most parasitic helminths relies upon Type 2 cytokine production, but the mechanisms that regulate interleukin (IL) 4 and 13 production from CD4+ T helper 2 cells (TH2) and innate lymphoid type 2 cells (ILC2s) remain incompletely understood. The epithelial cell-derived cytokines IL-25 and IL-33 promote Type 2 responses, but the extent of functional redundancy between these cytokines is unclear and whether Type 2 memory relies upon either IL-25 or IL-33 is unknown. Herein, we demonstrate a pivotal role for IL33 in driving primary and anamnestic immunity against the rodent hookworm Nippostrongylus brasiliensis. IL-33–deficient mice have a selective defect in ILC2–derived IL-13 during both primary and secondary challenge infections but generate stronger canonical CD4+ T helper 2 cells responses (IL-4, IgE, mast cells, and basophils) than WT controls. Lack of IL-13 production in IL-33–deficient mice impairs resistin-like molecule beta (RELMb) expression and eosinophil recruitment, which are two mechanisms that eliminate N. brasiliensis parasites from infected hosts. Thus, IL33 is requisite for IL-13 but not IL-4–driven Type 2 responses during hookworm infection.
L. Icardi a, R. Mori a, V. Gesellchen a, S. Eyckerman a, J. Verhelst b, K. Vercauteren c, X. Saelens b, P. Meuleman c, K. De Bosscher a, M. Boutros d, J. Tavernier a, a Department of Medical Protein Research, VIB, 9000 Ghent, Belgium, b Department of Molecular Biomedical Research, VIB, 9000 Ghent, Belgium, c Center for Vaccinology, Ghent University and Hospital Ghent University, 9000 Ghent, Belgium, d German Cancer Research Center (DKFZ), Department of Cell and Molecular Biology, Heidelberg, Germany The activity of Signal Transducer and Activator of Transcription (STAT) proteins is regulated by a multitude of posttranslational modifications, which control different aspects of signal transduction. In particular, lysine acetylation and deacetylation has emerged as a critical modification, regulating the activity of all seven STAT members. Unlike tyrosine phosphorylation/dephosphorylation, which mainly acts as an on/off switch of STAT activity, the control exerted by acetylation appears multifaceted and by far more complex, exerting distinct and even opposed regulation to different STAT members. We observed that type I interferons (IFNs) can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide siRNA screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as a novel regulator of STAT activity. Sin3a directly interacts with STAT3 and controls its acetylation status, its nuclear accumulation and DNA binding, strongly influencing the transcription of a subset of STAT3-responsive genes. Interfering with Sin3a expression restores, at least partially, the STAT3 transcriptional blockade observed upon IFN treatment. Strikingly, Sin3a exerts opposed regulation on ISGF3 activity, as it is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. We conclude that Sin3a contributes to the regulation of the balance between the activities of different STAT members, and interfering with its activity or expression may re-direct cytokine signals, favoring the onset of pathological conditions, such as autoimmune syndromes, inflammation and cancer.
http://dx.doi.org/10.1016/j.cyto.2013.06.126 http://dx.doi.org/10.1016/j.cyto.2013.06.128
124 The interleukin-17-mediated regulation and role of neutrophils in resistance to oropharyngeal candidiasis Anna R. Huppler a,b, Nydiaris Hernández-Santos b, Heather R. Conti b, Sarah L. Gaffen b, Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA, b Division of Rheumatology and Clinical Immunology, University of Pittsburgh, PA, USA a
Oropharyngeal candidiasis (OPC) is an opportunistic infection associated with a broad range of human conditions, including infancy, acquired immunodeficiencies such as AIDS, and interleukin-17 (IL-17)-related primary immunodeficiencies. Recent
1
These authors contributed equally to this work.
126 NOD1-dependent sensing of bacterial outer membrane vesicles and induction of an autophagic response Aaron Irving a, Lorinda Turner b, Hitomi Mimuro c, Chihiro Sasakawa c, Thomas Kufer d, Dana Philpott e, Richard Ferrero b, Maria Kaparakis-Liaskos b, a Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia, b Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Clayton, Victoria, Australia, c Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan, d Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Germany, e Department of Immunology, The University of Toronto, Ontario, Canada