- Email: [email protected]
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Abstract / Cytokine 63 (2013) 243–314
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219 STAT3 induces proapoptotic signals in chronic lymphocytic leukemia (CLL) cells
221 Post-transcriptional regulation of interferon gamma in memory CD8+ T cells
Uri Rozovski, David Harris, Ping Li, Zhiming Liu, Wu Ji Yuan, Michael Keating, Zeev Estrov, Department of Leukemia, MD Anderson Cancer Center, Houston, TX, United States
Fiamma Salerno a, Sander Engels a, Wanqi Zhao a, Deborah Hodge b, Howard A. Young b, Jan Paul Medema c, Monika C. Wolkers a, a Department of Hematopoiesis, Sanquin Research, Amsterdam, The Netherlands, b Laboratory for Experimental Immunology, Cancer and Inflammation Program, Center of Cancer Research, National Cancer Institute at Frederick, USA, c Laboratory of Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), Amsterdam, The Netherlands
Usually, ligand-receptor interactions induce STAT3 tyrosine phosphorylation. Unlike in normal cells, in CLL STAT3 is constitutively phosphorylated on serine 727 residues regardless of clinical characteristics, cytogenetic abnormalities or treatment status. Posphoserine STAT3 activates STAT3-regulated genes (Hazan-Halevy et al., 2010), including STAT3 and the antiapoptotic genes Bcl2, Bcl-XL, Mcl-1, and survivine. Because CLL is a heterogeneous disease and because STAT3-shRNA induced apoptosis of CLL cells, we postulated that high STAT3 protein levels and low apoptosis rates typify CLL cells of patients with high CLL counts. Using western immunoblotting we analyzed STAT3 and Caspase3 protein levels of CLL cells from 64 patients with high (N = 32) and low (N = 32) peripheral blood CLL-cell counts. Indeed, STAT3 levels were high in cells from patients with high counts. However, CLL cells from patients with high counts expressed high levels of total and cleaved Caspase3, cleaved Caspse3 levels markedly increased when STAT3 reached a threshold level, and the spontaneous apoptosis rate of cells from patients with high counts was high as assessed by Annexin V/PI flow cytometry analysis. Furthermore, several apoptotic-pathway genes such as Caspase3, Calpain9, MAPK8, KRAS, PLCc-2, and PKC were overexpressed in high count CLL cells and pathway analysis showed upregulation of apoptosis signaling pathway. Because CLL cells harbor high levels of STAT3 and the Caspase3 gene promoter harbors c-interferon activation sequence (GAS)-like element, we sought to determine whether STAT3 activates the Caspase3 gene. Chromatin immunoprecipitation (ChIP) studies showed that STAT3 antibodies co-immunoprecipitated Caspase3 DNA, and infection of CLL cells with lentiviral STAT3-shRNA downregulated Caspase3 mRNA and protein levels, suggesting that STAT3 activates the Casapse3 gene and upregulates Caspase3 mRNA and protein levels. Taken together, our data suggest that whereas at low levels posphoserine STAT3 is protective, at high levels phosphorylated STAT3 activates the production of Caspase3 protein and induces apoptosis of CLL cells.
Memory CD8+ T cells are pivotal to protect us from recurring infections. They prevent pathogen spreading at early stages of reinfection through rapid release of effector molecules. Strikingly, while resting memory T cells express high levels of cytokine transcripts, the production of the corresponding proteins is blocked unless T cells are reactivated. This indicates that translation of cytokine mRNA is strictly regulated in memory T cells. Here, we investigate how the production of interferon gamma (IFN-c) is controlled in memory T cells. IFN-c is a pro-inflammatory cytokine that is required to control microbial infections, however, aberrant expression is associated with immunopathology. By using reporter gene analysis we demonstrate that IFN-c production is tightly regulated via a 30 untranslated region (30 UTR)-dependent mechanism in CD8+ T cells both in vitro and in vivo. We observed that the highly conserved AU-rich elements (AREs) within the first 253 nucleotides of the IFN-c 30 UTR govern this mechanism. Importantly, we show that the regulation of IFN-c through AREs is maintained in CD4+ and CD8+ T cells and is highly conserved in human and mouse. Moreover, germ-line deletion of the ARE-containing region within the IFN-c 30 UTR abrogates both post-transcriptional and translational regulation, as determined by an increased stability of IFN-c mRNA and hyper-responsiveness of memory T cells. Taken together, these findings indicate that AU-rich elements within the IFN-c 30 UTR comprise a crucial component that keeps memory T cell responses in check and is essential for a protective, yet balanced immune response. Unraveling the molecular mechanisms that govern this tight control of cytokine production will help design strategies to manipulate impaired T cell responses.
http://dx.doi.org/10.1016/j.cyto.2013.06.222 http://dx.doi.org/10.1016/j.cyto.2013.06.224 220 Fibrinogen/CR3 signaling induces demyelination by promoting Th1 cell differentiation and peripheral macrophage recruitment into the CNS Jae Kyu Ryu a, Kim M. Baeten a, Mark A. Petersen a,b, Sara G. Murray a, Anke MeyerFranke a, Dimitrios Davalos a, Catherine Bedard a, Thomas Prod’homme c, Israel F. Charo a, Hans Lassmann d, Jay L. Degen e, Scott S. Zamvil c, Katerina Akassoglou a,c, a Gladstone Inst., Univ. of California, San Francisco, San Francisco, CA, United States, b Department of Pediatrics, Univ. of California, San Francisco, San Francisco, CA, United States, c Department of Neurology, Univ. of California, San Francisco, San Francisco, CA, United States, d Centre for Brain Research, Medical University of Vienna, Vienna, Austria, e Pediatrics, Univ. of Cincinnati, Cincinnati, OH, United States Blood–brain barrier (BBB) disruption, which allows the leakage and deposition of plasma proteins in the central nervous system (CNS), is a hallmark of multiple sclerosis (MS), a chronic autoimmune disease of the CNS characterized by activation of innate and adaptive immune responses. However, the contribution of increased vascular permeability to the development of autoimmunity remains unclear. Here we show that the plasma protein fibrinogen, the physiological ligand of complement receptor 3 (CR3) that is abundant in early and late MS lesions, can spontaneously induce inflammatory demyelination by triggering encephalitogenic T cell responses and macrophage recruitment into the CNS. By a single stereotactic injection in myelinated areas of healthy brain or spinal cord, we demonstrate that fibrinogen induces microglial activation, followed by infiltration of T cells and peripheral macrophages, leading to the development of demyelinating lesions. Demyelination upon fibrinogen injection is reduced in MHCII/ and lymphocyte-deficient Rag2/ cc/ mice. Furthermore, co-culture of fibrinogen-treated bone-marrow-derived macrophages with T cells showed that fibrinogen specifically regulates TH1 cell differentiation. In accordance, studies in myelin oligodendrocyte glycoprotein-specific (2D2) mice revealed that fibrinogen-induced inflammatory demyelination depends upon the recruitment of encephalitogenic T-cells. Genetic depletion or pharmacologically blocking of CR3 attenuated TH1 cell differentiation, chemokine gene expression, peripheral macrophage recruitment, and inflammatory demyelination upon fibrinogen injection. Our data suggest that fibrin/CR3 signaling is an upstream regulator of the immune attack on the CNS by encephalitogenic T cells and peripheral macrophages. Our study identifies fibrinogen as a molecular link between increased vascular permeability and CNS autoimmunity, and introduces fibrinogen-induced encephalomyelitis (FIE) as an innate immune-driven animal model for MS. http://dx.doi.org/10.1016/j.cyto.2013.06.223
222 Cytokine changes in coinfected subjects with Plasmodium and intestinal parasites in naturally exposed populations to malaria Juan C. Sánchez-Arcila a, Marcelle Marcolino de França a, Daiana de Souza Perce da Silva b, Rodrigo Nunes Rodrigues da Silva c, Cesarino J. Lima Aprígio d,e, Mariana Pinheiro Alves Vasconcelos f, Dalma M. Banic b, Josué da Costa Lima Júnior a, Joseli de Oliveira Ferreira a, a Laboratório de Imunoparasitologia, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil, b Laboratório de Simulídeos e Oncocercose, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil, c Departamento de processamento final, Biomanguinhos/Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil, d Laboratório de Quimioterapia/Fiocruz, Porto Velho, Rondônia, Brazil, e Universidade Federal de Rondônia, Porto Velho, Rondônia, Brazil, f Instituto de Infectologia Emilio Ribas, São Paulo, São Paulo, Brazil The geographic distribution of the Plasmodium and intestinal parasites are overlapped over the world, therefore malaria co-infection with intestinal parasites are common in tropical regions of the planet. The immune response against Plasmodium is characterized by a Th1 profile. On the other hand, helminth infections induce a strong immunoregulatory and Th2 response that can inhibit the inflammatory response against malaria, with possible consequences for clinical disease. The aim of our study was to evaluate the occurrence of alterations in epidemiological and hematological parameters and cytokine levels in individuals co-infected with malaria and intestinal parasites and in individuals with single infections. The blood of 280 volunteers living in the city of Porto Velho, Rondônia, (Brazil) was collected for parasite examination, automated blood cell count and to obtain the plasma used in cytokine and chemokine quantification. Stool samples were collected for detection of intestinal parasites. We also compared individuals from endemic areas infected with Malaria (M), Intestinal Parasites (IP), Coinfected (M + IP), Exposed but not infected (E) and non-exposed individuals (CTR). No differences were observed in the epidemiological parameters of the groups. We found a Th1 and Th2 cytokine pattern in M and IP but with different types of cytokines involved (increased TNF-a, IL-2, IL-10, IL-13 in M, and increased IFN-c, IL-12p70, IL-4, IL-7 in PI). We observed a mixed Th1/Th2 pattern in M + IP, but with more Th1 cytokines (increased IFN-c, TNF-a, IL-2, IL-10, IL-4). Cytokine production in IP individuals was less variable compared with M and M + IP individuals. Overall M + IP individuals had cytokine pattern similar with M group. Additionally, parasitemia was no different between M and M + PI groups. Our results indicate that cytokine profile in coinfected individuals is highly influenced by malaria infection, but slight changes in cytokine patters in M + IP could be attributed to intestinal parasites infection.
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Abstract / Cytokine 63 (2013) 243–314
http://dx.doi.org/10.1016/j.cyto.2013.06.225
223 A novel type I interferon antagonist has in vivo efficacy in a model of SIV infection in rhesus macaques Netanya G. Sandler a, Richard T. Zhu a, Jacob D. Estes b, Eli Boritz a, Srinivas Rao a, Jeffrey D. Lifson b, Doron Levin c, Gideon Schreiber c, Jerome A. Langer d, Daniel C. Douek a, a Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA, b AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA, c Department of Biological Chemistry, Weizmann Inst., Rehovot, Israel, d Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Type I interferons (IFNs) are critical for the innate defense against viruses. However, long-term or chronic production of Type I IFNs may promote autoimmune diseases such as systemic lupus erythematosus (SLE; lupus). Data also suggest that Type I IFN may contribute to the persistence of some viral infections. There is a need for robust Type I IFN antagonists for understanding the in vivo role(s) of Type I IFNs, and to potentially block IFN when it causes or exacerbates a pathologic condition. We previously described an interferon analogue, ‘‘IFN-Ant1” (IFN-a2[Arg120Glu]8CT) that was derived from human IFN-a2. IFN-Ant1 lacks most biological activity and acts in vitro as a partial competitive antagonist. We now report that IFN-Ant1 functions in vivo in primates. The efficacy of this antagonist was tested in rhesus macaques (N = 6) challenged with simian immunodeficiency virus (SIVmac251), and treated during the acute infection stage (4 weeks) with daily injections of IFN-Ant1 or placebo. Macaques were analyzed for IFN-related and immunological markers during this acute phase (4 weeks) and then for an additional 6 months. Treatment of the macaques with the antagonist during the acute phase of SIV infection was well tolerated and led to significant changes in several immune and virologic parameters. Among these: (1) macaques treated with IFN-Ant1 had significantly higher serum viral loads than placebo-treated; and (2) treated animals rapidly progressed to AIDS. Various molecular and immune parameters were monitored during the acute and chronic phases of the infection. These data contribute to our understanding of the role of endogenous Type I IFN during SIV infection of macaques, and provide evidence for efficacy of the antagonist in primates. http://dx.doi.org/10.1016/j.cyto.2013.06.226
224 Resistance to respiratory syncytial virus replication is STAT1 and STAT2 dependent, but I interferon independent Marvin Sandoval a, Troy Cline b, Jian-Da Lin c, Sergei V. Kotenko c, Russell K. Durbin a, Joan E. Durbin a, a Department of Pathology, New York University School of Medicine, New York, NY, USA, b Department of Infectious Disease, St. Jude’s Children’s Research Hospital, Memphis, TN, USA, c Department of Biochemistry, New Jersey Medical School, Newark, NJ, USA Despite the known ability of type I IFNs to inhibit virus replication, we and others noted that mice lacking the IFN-ab Receptor (IFNAR/) did not show increased sensitivity to either influenza A virus (IAV) or respiratory syncytial virus (RSV) infection. An explanation for this seemingly contradictory result was offered by the discovery of type III or IFN-k, a unique family of cytokines that bind a distinct receptor but can also promote interferon stimulated gene factor 3 (ISGF3) formation and the transcriptional upregulation of IFN-stimulated genes. The presence of robust IFN-k induction in IAVinfected IFNAR/ mice can explain their resistance to infection, but a distinct protective mechanism is at work in RSV-infected IFNAR/ animals. In the absence of the IFNAR, no type III IFN RNA or protein can be detected in RSV-infected animals, nor is there any evidence of ISGF3 formation or upregulation of the ISGF3 dependent gene, Mx1. Nonetheless, we observe enhanced virus susceptibility to RSV in mice lacking either STAT1 or STAT2. We therefore conclude from our studies that (1) triggering of IFN-k synthesis is pathogen dependent: induction of IFN-k by RSV, but not by IAV, is dependent on functional type I IFN signaling, and (2) resistance to RSV is mediated by a novel STAT1/STAT2 containing complex. http://dx.doi.org/10.1016/j.cyto.2013.06.227
225 Inosine-mediated modulation of RNA sensing by innate immune sensors Soroush T. Sarvestani, Michael P. Gantier, Bryan R.G. Williams, Monash Institute of Medical Research, Monash University, Victoria, Australia
RNA-specific adenosine deaminase (ADAR)-mediated adenosine to inosine (A-toI) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of non-self RNA by innate immune sensors, and accordingly investigated the impact of inosine-modified RNA on Toll Like Receptor (TLR) 7/8 sensing. Inosine incorporation into synthetic ssRNA potentiated TNF-a or IFN-a production in human PBMCs, in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs, and was not seen with inosine-containing short dsRNAs, nor with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated mouse Tlr7 activation, as established through the use of Tlr7-deficient cells. There was a correlation between hyper-editing of influenza A viral ssRNA and its ability to stimulate TNF-a, independent of 50 -triphosphate residues. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate sensing of phagocytosed viral RNA by innate immune sensors TLR7/8. http://dx.doi.org/10.1016/j.cyto.2013.06.228
226 The transcription factor IRF8 is required for the development of basophils Haruka Sasaki a, Daisuke Kurotak a, Hideaki Sato a, Herbert C. Morse b, Keiko Ozato c, Tomohiko Tamura a, a Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan, b Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States, c Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, United States Basophils are the rarest granulocytes circulating in the peripheral blood. Although their non-redundant roles in Th2-type immunity and allergic disorders have been clarified, little is known about the pathways and regulators of their development. Interferon Regulatory Factor-8 (IRF8) is a transcription factor known to promote the development of monocytes/macrophages and dendritic cells while inhibiting that of neutrophils. In this report, we show that this transcription factor is required for the development of the basophil lineage. Irf8/ mice display a severe reduction of basophil counts in the bone marrow, peripheral blood and spleen. Bone marrow transfer experiments showed that IRF8 functions in a bone marrow cell-intrinsic manner. Analysis of bone marrow progenitors revealed that the number of granulocyte-committed progenitors (GPs) in Irf8/ mice is comparable to that of wild-type mice, while that of basophil-committed progenitors (BaPs) is diminished. Using IRF8-GFP knock-in mice, we found that GPs but not BaPs and mature basophils express IRF8, suggesting that IRF8 may act in the stage of GPs. Indeed, GPs purified from Irf8/ mice failed to efficiently give rise to basophils in vitro in the presence of IL-3. Taken together, these results uncover a previously unrecognized role of IRF8 in granulocyte development.
http://dx.doi.org/10.1016/j.cyto.2013.06.229
227 IL-27 affects helper T cell response via regulation of PGE2 production by macrophages Yayoi Sato a,b,c, Hiromitsu Hara a, Shinobu Suzuki b, Tsuneyasu Kaisho c, Hiroki Yoshida a, a Division of Molecular and Cellular Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Japan, b Dept. of Molecular & Cellular Biology, Kobe Pharma Research Institute, Nippon Boehringer Ingelheim Co., Ltd., Japan, c Laboratory of Immune regulation, Immunology Frontier Research center, Osaka University, Japan IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. Although IL-27 was initially reported as a Th1 inducer by the induction of IL-12R on naïve CD4+T cells, recently it is revealed that IL-27 acts as an immune suppressor by the induction of Tr1 type T cells, which produce IL-10 and inhibit effector T cells. IL-27 also suppresses function of macrophages and dendritic cells by regulation of cytokine secretion. On the other hand, effects of IL-27 on regulation of inflammation via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrowderived macrophages (BMDMs) from WSX-1 (IL-27Ra) KO mice to find enhanced IFN-g and IL-17A secretion by CD4+T cells as compared with that of control BMDMs.
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219 STAT3 induces proapoptotic signals in chronic lymphocytic leukemia (CLL) cells
221 Post-transcriptional regulation of interferon gamma in memory CD8+ T cells
Uri Rozovski, David Harris, Ping Li, Zhiming Liu, Wu Ji Yuan, Michael Keating, Zeev Estrov, Department of Leukemia, MD Anderson Cancer Center, Houston, TX, United States
Fiamma Salerno a, Sander Engels a, Wanqi Zhao a, Deborah Hodge b, Howard A. Young b, Jan Paul Medema c, Monika C. Wolkers a, a Department of Hematopoiesis, Sanquin Research, Amsterdam, The Netherlands, b Laboratory for Experimental Immunology, Cancer and Inflammation Program, Center of Cancer Research, National Cancer Institute at Frederick, USA, c Laboratory of Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), Amsterdam, The Netherlands
Usually, ligand-receptor interactions induce STAT3 tyrosine phosphorylation. Unlike in normal cells, in CLL STAT3 is constitutively phosphorylated on serine 727 residues regardless of clinical characteristics, cytogenetic abnormalities or treatment status. Posphoserine STAT3 activates STAT3-regulated genes (Hazan-Halevy et al., 2010), including STAT3 and the antiapoptotic genes Bcl2, Bcl-XL, Mcl-1, and survivine. Because CLL is a heterogeneous disease and because STAT3-shRNA induced apoptosis of CLL cells, we postulated that high STAT3 protein levels and low apoptosis rates typify CLL cells of patients with high CLL counts. Using western immunoblotting we analyzed STAT3 and Caspase3 protein levels of CLL cells from 64 patients with high (N = 32) and low (N = 32) peripheral blood CLL-cell counts. Indeed, STAT3 levels were high in cells from patients with high counts. However, CLL cells from patients with high counts expressed high levels of total and cleaved Caspase3, cleaved Caspse3 levels markedly increased when STAT3 reached a threshold level, and the spontaneous apoptosis rate of cells from patients with high counts was high as assessed by Annexin V/PI flow cytometry analysis. Furthermore, several apoptotic-pathway genes such as Caspase3, Calpain9, MAPK8, KRAS, PLCc-2, and PKC were overexpressed in high count CLL cells and pathway analysis showed upregulation of apoptosis signaling pathway. Because CLL cells harbor high levels of STAT3 and the Caspase3 gene promoter harbors c-interferon activation sequence (GAS)-like element, we sought to determine whether STAT3 activates the Caspase3 gene. Chromatin immunoprecipitation (ChIP) studies showed that STAT3 antibodies co-immunoprecipitated Caspase3 DNA, and infection of CLL cells with lentiviral STAT3-shRNA downregulated Caspase3 mRNA and protein levels, suggesting that STAT3 activates the Casapse3 gene and upregulates Caspase3 mRNA and protein levels. Taken together, our data suggest that whereas at low levels posphoserine STAT3 is protective, at high levels phosphorylated STAT3 activates the production of Caspase3 protein and induces apoptosis of CLL cells.
Memory CD8+ T cells are pivotal to protect us from recurring infections. They prevent pathogen spreading at early stages of reinfection through rapid release of effector molecules. Strikingly, while resting memory T cells express high levels of cytokine transcripts, the production of the corresponding proteins is blocked unless T cells are reactivated. This indicates that translation of cytokine mRNA is strictly regulated in memory T cells. Here, we investigate how the production of interferon gamma (IFN-c) is controlled in memory T cells. IFN-c is a pro-inflammatory cytokine that is required to control microbial infections, however, aberrant expression is associated with immunopathology. By using reporter gene analysis we demonstrate that IFN-c production is tightly regulated via a 30 untranslated region (30 UTR)-dependent mechanism in CD8+ T cells both in vitro and in vivo. We observed that the highly conserved AU-rich elements (AREs) within the first 253 nucleotides of the IFN-c 30 UTR govern this mechanism. Importantly, we show that the regulation of IFN-c through AREs is maintained in CD4+ and CD8+ T cells and is highly conserved in human and mouse. Moreover, germ-line deletion of the ARE-containing region within the IFN-c 30 UTR abrogates both post-transcriptional and translational regulation, as determined by an increased stability of IFN-c mRNA and hyper-responsiveness of memory T cells. Taken together, these findings indicate that AU-rich elements within the IFN-c 30 UTR comprise a crucial component that keeps memory T cell responses in check and is essential for a protective, yet balanced immune response. Unraveling the molecular mechanisms that govern this tight control of cytokine production will help design strategies to manipulate impaired T cell responses.
http://dx.doi.org/10.1016/j.cyto.2013.06.222 http://dx.doi.org/10.1016/j.cyto.2013.06.224 220 Fibrinogen/CR3 signaling induces demyelination by promoting Th1 cell differentiation and peripheral macrophage recruitment into the CNS Jae Kyu Ryu a, Kim M. Baeten a, Mark A. Petersen a,b, Sara G. Murray a, Anke MeyerFranke a, Dimitrios Davalos a, Catherine Bedard a, Thomas Prod’homme c, Israel F. Charo a, Hans Lassmann d, Jay L. Degen e, Scott S. Zamvil c, Katerina Akassoglou a,c, a Gladstone Inst., Univ. of California, San Francisco, San Francisco, CA, United States, b Department of Pediatrics, Univ. of California, San Francisco, San Francisco, CA, United States, c Department of Neurology, Univ. of California, San Francisco, San Francisco, CA, United States, d Centre for Brain Research, Medical University of Vienna, Vienna, Austria, e Pediatrics, Univ. of Cincinnati, Cincinnati, OH, United States Blood–brain barrier (BBB) disruption, which allows the leakage and deposition of plasma proteins in the central nervous system (CNS), is a hallmark of multiple sclerosis (MS), a chronic autoimmune disease of the CNS characterized by activation of innate and adaptive immune responses. However, the contribution of increased vascular permeability to the development of autoimmunity remains unclear. Here we show that the plasma protein fibrinogen, the physiological ligand of complement receptor 3 (CR3) that is abundant in early and late MS lesions, can spontaneously induce inflammatory demyelination by triggering encephalitogenic T cell responses and macrophage recruitment into the CNS. By a single stereotactic injection in myelinated areas of healthy brain or spinal cord, we demonstrate that fibrinogen induces microglial activation, followed by infiltration of T cells and peripheral macrophages, leading to the development of demyelinating lesions. Demyelination upon fibrinogen injection is reduced in MHCII/ and lymphocyte-deficient Rag2/ cc/ mice. Furthermore, co-culture of fibrinogen-treated bone-marrow-derived macrophages with T cells showed that fibrinogen specifically regulates TH1 cell differentiation. In accordance, studies in myelin oligodendrocyte glycoprotein-specific (2D2) mice revealed that fibrinogen-induced inflammatory demyelination depends upon the recruitment of encephalitogenic T-cells. Genetic depletion or pharmacologically blocking of CR3 attenuated TH1 cell differentiation, chemokine gene expression, peripheral macrophage recruitment, and inflammatory demyelination upon fibrinogen injection. Our data suggest that fibrin/CR3 signaling is an upstream regulator of the immune attack on the CNS by encephalitogenic T cells and peripheral macrophages. Our study identifies fibrinogen as a molecular link between increased vascular permeability and CNS autoimmunity, and introduces fibrinogen-induced encephalomyelitis (FIE) as an innate immune-driven animal model for MS. http://dx.doi.org/10.1016/j.cyto.2013.06.223
222 Cytokine changes in coinfected subjects with Plasmodium and intestinal parasites in naturally exposed populations to malaria Juan C. Sánchez-Arcila a, Marcelle Marcolino de França a, Daiana de Souza Perce da Silva b, Rodrigo Nunes Rodrigues da Silva c, Cesarino J. Lima Aprígio d,e, Mariana Pinheiro Alves Vasconcelos f, Dalma M. Banic b, Josué da Costa Lima Júnior a, Joseli de Oliveira Ferreira a, a Laboratório de Imunoparasitologia, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil, b Laboratório de Simulídeos e Oncocercose, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil, c Departamento de processamento final, Biomanguinhos/Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil, d Laboratório de Quimioterapia/Fiocruz, Porto Velho, Rondônia, Brazil, e Universidade Federal de Rondônia, Porto Velho, Rondônia, Brazil, f Instituto de Infectologia Emilio Ribas, São Paulo, São Paulo, Brazil The geographic distribution of the Plasmodium and intestinal parasites are overlapped over the world, therefore malaria co-infection with intestinal parasites are common in tropical regions of the planet. The immune response against Plasmodium is characterized by a Th1 profile. On the other hand, helminth infections induce a strong immunoregulatory and Th2 response that can inhibit the inflammatory response against malaria, with possible consequences for clinical disease. The aim of our study was to evaluate the occurrence of alterations in epidemiological and hematological parameters and cytokine levels in individuals co-infected with malaria and intestinal parasites and in individuals with single infections. The blood of 280 volunteers living in the city of Porto Velho, Rondônia, (Brazil) was collected for parasite examination, automated blood cell count and to obtain the plasma used in cytokine and chemokine quantification. Stool samples were collected for detection of intestinal parasites. We also compared individuals from endemic areas infected with Malaria (M), Intestinal Parasites (IP), Coinfected (M + IP), Exposed but not infected (E) and non-exposed individuals (CTR). No differences were observed in the epidemiological parameters of the groups. We found a Th1 and Th2 cytokine pattern in M and IP but with different types of cytokines involved (increased TNF-a, IL-2, IL-10, IL-13 in M, and increased IFN-c, IL-12p70, IL-4, IL-7 in PI). We observed a mixed Th1/Th2 pattern in M + IP, but with more Th1 cytokines (increased IFN-c, TNF-a, IL-2, IL-10, IL-4). Cytokine production in IP individuals was less variable compared with M and M + IP individuals. Overall M + IP individuals had cytokine pattern similar with M group. Additionally, parasitemia was no different between M and M + PI groups. Our results indicate that cytokine profile in coinfected individuals is highly influenced by malaria infection, but slight changes in cytokine patters in M + IP could be attributed to intestinal parasites infection.
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Abstract / Cytokine 63 (2013) 243–314
http://dx.doi.org/10.1016/j.cyto.2013.06.225
223 A novel type I interferon antagonist has in vivo efficacy in a model of SIV infection in rhesus macaques Netanya G. Sandler a, Richard T. Zhu a, Jacob D. Estes b, Eli Boritz a, Srinivas Rao a, Jeffrey D. Lifson b, Doron Levin c, Gideon Schreiber c, Jerome A. Langer d, Daniel C. Douek a, a Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA, b AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA, c Department of Biological Chemistry, Weizmann Inst., Rehovot, Israel, d Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Type I interferons (IFNs) are critical for the innate defense against viruses. However, long-term or chronic production of Type I IFNs may promote autoimmune diseases such as systemic lupus erythematosus (SLE; lupus). Data also suggest that Type I IFN may contribute to the persistence of some viral infections. There is a need for robust Type I IFN antagonists for understanding the in vivo role(s) of Type I IFNs, and to potentially block IFN when it causes or exacerbates a pathologic condition. We previously described an interferon analogue, ‘‘IFN-Ant1” (IFN-a2[Arg120Glu]8CT) that was derived from human IFN-a2. IFN-Ant1 lacks most biological activity and acts in vitro as a partial competitive antagonist. We now report that IFN-Ant1 functions in vivo in primates. The efficacy of this antagonist was tested in rhesus macaques (N = 6) challenged with simian immunodeficiency virus (SIVmac251), and treated during the acute infection stage (4 weeks) with daily injections of IFN-Ant1 or placebo. Macaques were analyzed for IFN-related and immunological markers during this acute phase (4 weeks) and then for an additional 6 months. Treatment of the macaques with the antagonist during the acute phase of SIV infection was well tolerated and led to significant changes in several immune and virologic parameters. Among these: (1) macaques treated with IFN-Ant1 had significantly higher serum viral loads than placebo-treated; and (2) treated animals rapidly progressed to AIDS. Various molecular and immune parameters were monitored during the acute and chronic phases of the infection. These data contribute to our understanding of the role of endogenous Type I IFN during SIV infection of macaques, and provide evidence for efficacy of the antagonist in primates. http://dx.doi.org/10.1016/j.cyto.2013.06.226
224 Resistance to respiratory syncytial virus replication is STAT1 and STAT2 dependent, but I interferon independent Marvin Sandoval a, Troy Cline b, Jian-Da Lin c, Sergei V. Kotenko c, Russell K. Durbin a, Joan E. Durbin a, a Department of Pathology, New York University School of Medicine, New York, NY, USA, b Department of Infectious Disease, St. Jude’s Children’s Research Hospital, Memphis, TN, USA, c Department of Biochemistry, New Jersey Medical School, Newark, NJ, USA Despite the known ability of type I IFNs to inhibit virus replication, we and others noted that mice lacking the IFN-ab Receptor (IFNAR/) did not show increased sensitivity to either influenza A virus (IAV) or respiratory syncytial virus (RSV) infection. An explanation for this seemingly contradictory result was offered by the discovery of type III or IFN-k, a unique family of cytokines that bind a distinct receptor but can also promote interferon stimulated gene factor 3 (ISGF3) formation and the transcriptional upregulation of IFN-stimulated genes. The presence of robust IFN-k induction in IAVinfected IFNAR/ mice can explain their resistance to infection, but a distinct protective mechanism is at work in RSV-infected IFNAR/ animals. In the absence of the IFNAR, no type III IFN RNA or protein can be detected in RSV-infected animals, nor is there any evidence of ISGF3 formation or upregulation of the ISGF3 dependent gene, Mx1. Nonetheless, we observe enhanced virus susceptibility to RSV in mice lacking either STAT1 or STAT2. We therefore conclude from our studies that (1) triggering of IFN-k synthesis is pathogen dependent: induction of IFN-k by RSV, but not by IAV, is dependent on functional type I IFN signaling, and (2) resistance to RSV is mediated by a novel STAT1/STAT2 containing complex. http://dx.doi.org/10.1016/j.cyto.2013.06.227
225 Inosine-mediated modulation of RNA sensing by innate immune sensors Soroush T. Sarvestani, Michael P. Gantier, Bryan R.G. Williams, Monash Institute of Medical Research, Monash University, Victoria, Australia
RNA-specific adenosine deaminase (ADAR)-mediated adenosine to inosine (A-toI) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of non-self RNA by innate immune sensors, and accordingly investigated the impact of inosine-modified RNA on Toll Like Receptor (TLR) 7/8 sensing. Inosine incorporation into synthetic ssRNA potentiated TNF-a or IFN-a production in human PBMCs, in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs, and was not seen with inosine-containing short dsRNAs, nor with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated mouse Tlr7 activation, as established through the use of Tlr7-deficient cells. There was a correlation between hyper-editing of influenza A viral ssRNA and its ability to stimulate TNF-a, independent of 50 -triphosphate residues. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate sensing of phagocytosed viral RNA by innate immune sensors TLR7/8. http://dx.doi.org/10.1016/j.cyto.2013.06.228
226 The transcription factor IRF8 is required for the development of basophils Haruka Sasaki a, Daisuke Kurotak a, Hideaki Sato a, Herbert C. Morse b, Keiko Ozato c, Tomohiko Tamura a, a Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan, b Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States, c Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, United States Basophils are the rarest granulocytes circulating in the peripheral blood. Although their non-redundant roles in Th2-type immunity and allergic disorders have been clarified, little is known about the pathways and regulators of their development. Interferon Regulatory Factor-8 (IRF8) is a transcription factor known to promote the development of monocytes/macrophages and dendritic cells while inhibiting that of neutrophils. In this report, we show that this transcription factor is required for the development of the basophil lineage. Irf8/ mice display a severe reduction of basophil counts in the bone marrow, peripheral blood and spleen. Bone marrow transfer experiments showed that IRF8 functions in a bone marrow cell-intrinsic manner. Analysis of bone marrow progenitors revealed that the number of granulocyte-committed progenitors (GPs) in Irf8/ mice is comparable to that of wild-type mice, while that of basophil-committed progenitors (BaPs) is diminished. Using IRF8-GFP knock-in mice, we found that GPs but not BaPs and mature basophils express IRF8, suggesting that IRF8 may act in the stage of GPs. Indeed, GPs purified from Irf8/ mice failed to efficiently give rise to basophils in vitro in the presence of IL-3. Taken together, these results uncover a previously unrecognized role of IRF8 in granulocyte development.
http://dx.doi.org/10.1016/j.cyto.2013.06.229
227 IL-27 affects helper T cell response via regulation of PGE2 production by macrophages Yayoi Sato a,b,c, Hiromitsu Hara a, Shinobu Suzuki b, Tsuneyasu Kaisho c, Hiroki Yoshida a, a Division of Molecular and Cellular Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Japan, b Dept. of Molecular & Cellular Biology, Kobe Pharma Research Institute, Nippon Boehringer Ingelheim Co., Ltd., Japan, c Laboratory of Immune regulation, Immunology Frontier Research center, Osaka University, Japan IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. Although IL-27 was initially reported as a Th1 inducer by the induction of IL-12R on naïve CD4+T cells, recently it is revealed that IL-27 acts as an immune suppressor by the induction of Tr1 type T cells, which produce IL-10 and inhibit effector T cells. IL-27 also suppresses function of macrophages and dendritic cells by regulation of cytokine secretion. On the other hand, effects of IL-27 on regulation of inflammation via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrowderived macrophages (BMDMs) from WSX-1 (IL-27Ra) KO mice to find enhanced IFN-g and IL-17A secretion by CD4+T cells as compared with that of control BMDMs.