Corso di Laurea Specialistica in Biotecnologie Molecolari aa 2006/2007 Presentazione di Immunologia Molecolare INTERFERON GAMMA.

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1 Corso di Laurea Specialistica in Biotecnologie Molecolari aa 2006/2007 Presentazione di Immunologia Molecolare INTERFERON GAMMA Valentina Grosso

2 The Inteferons 1957: Isaacs and Lindenmann discovered a substance that protected cells from viral infection Interferon (IFN) - Found only in vertebrates; small proteins aa ranging from 19 to 45 kda, modified by glycosylation; - Prototypes for the clinical development of other cytokines; - Their metabolism and excretion take place mainly in the liver and kidneys. They rarely pass the placenta and the blood brain barrier; - Synthesized when induced by viral or other challenges by specialized cells, subsequently secreted, and bind to highly specific receptors on target cells. This specific interaction with its receptors causes the signal transduction to the nucleus and thus regulates the production of about 50 different genes; - Interferons are a group of cytokines with important activities: - immunomodulatory - antiviral α leukocytes - antiangiogenic HPLC β - antiproliferative fibroblasts - antitumor γ immune cells: - NK, - T lymphocytes (CD4+, CD8+ cells)

3 Types of interferons Takaoka A. et al., Interferon signalling network in innate defence, Cellular Microbiology, 8:907, 2006

4 IFN-γ : - strongly produced by activated T cells, macrophages or NK cells; - drives expression of MHC class II on antigen presenting cells; - increases chemokine secretion; - activates macrophages, lymphocytes and endothelial cells; - promotes the differentiation of naive helper T cells into Th1 cells; - activates polymorphonuclear leukocytes (PMN) and cytotoxic T cells and increases the cytotoxicity of NK cells. Bowers W., Microbiology and Immunology on-line, Immunology chapter 12 fig.8

5 The structure of human IFN-γ The IFN-γ gene is located on chromosome 12 and codes for a 17 kda protein, which then undergoes posttranslational glycolysis converting the IFN-γ to a kda glycoprotein. IFN-γ exists as noncovalentlylinked homodimers; the others are monomers. The protein shares no significant homology with IFN-β or the various IFN-α family proteins. IFN-γ is approximately 64% and 4% identical in its aa sequence to canine and mouse IFN-γ, respectively, and is highly species specific. http: //

6 Evolution of IFN-γ IFN-γ has been maintained as a single gene with presumably constant function and evolutionary pressure No homolog of IFN-γ has been observed in frogs and in fish genomes, implying that type II IFN function became essential during tetrapod evolution, though its origin may be considerably earlier. Pestka S. et al., Interferons, interferon-like cytokines, and their receptors, Immunological R eviews, 202:8, 2004

7 During infectious challenge: APC Chemokines: MIP1α (macrophage-infiammatory protein-1α) attract NK cells to the site of inflammation Cytokines: IL-12 and IL-18 potentiate IFNγ secretion by NK or T cells induce IFNγ secretion polarise T cell development towards a Th1 phenotype in sites of inflammation produce IFNγ activation macrophages IFNγ IS A PRO-INFLAMMATORY CYTOKINE, INTIMATELY INVOLVED IN THE INNATE AND ACQUIRED IMMUNE RESPONSES

8 IFNγ is a pro-inflammatory cytokine, intimately involved in the innate and acquired immune responses: IFNγ constitutes a warning signal produced by the host, and participates in an amplification loop to rapidly alert neighbouring cells, as well as effector cells of the innate immune system, to possible infection. IFNγ signalling: - upregulation of pathogen recognition; - induction of an antiviral state; - inhibition of cellular proliferation and modulation of apoptosis; - acquisition of microbicidal effector functions. One of the most important functions of IFNγ is to sensitise macrophages to activation by challenge with pathogen products. Indeed, IFNγ was originally named macrophage - activating factor.

9 IFNγ system dysfunction Patients with inactivating mutations of the human IFNGR1 or IFNGR2 chains and IFNγ -/- and IFNGR1 -/- mice showed no developmental defects, and their immune system appeared to develop normally, but these patients show deficiencies in natural resistance to bacterial, parasitic, and viral infections. Infants with deficient production of IFNγ exhibited decreased neutrophil mobility and NK cell activity, highlighting the importance of IFNγ in the inflammatory response and immunoregulation. His function is significant in tumor surveillance and also as IFN-γ is vitally implicated in the regulation of immune response, its production can lead to autoimmune disorders.

10 IFNγ and development of Th1 response IFNγ is a major product of Th1 cells and influences naive CD4+ cell differentiation toward a Th1 phenotype. During the primary response to antigen, IL-12 and IFNγ coordinate the link between pathogen recognition by innate immune cells and the induction of specific immunity, by mediating a positive feedback loop to amplify the Th1 response. LPS recognise - macrophages, - DCs - neutrophils IL12 production IFNγ secretion in - naive CD4+ T cells - NK cells positive feedback inhibits IL-4 secretion by Th2 populations Important in initiation or stabilization of the Th1 response

11 T-bet is required for Th1 lineage commitment T-bet is a member of the T-box family of transcription factors, induced in early developing Th1 cells and absent in developing Th2 cells; T-bet is required for IFNγ production and lineage commitment of CD4+ T cells. 1. CD4+ T cells purifed from the lymph nodes of T-bet +/+, T-bet +/-, and T-bet -/- mice; 2. Stimulated the cells with anti-cd3 and anti-cd28; 3. Measured IFNγ production during primary stimulation. A marked decrease in IFNγ production by T-bet -/- CD4+ T cells was observed, even in the presence of IL-12, a potent inducer of IFNγ production T-bet controls immediate cytokine production from naive CD4+ T cells, and also profoundly affects long-term T helper differentiation. In the absence of T-bet, CD4+ T cells fail to differentiate into the Th1 lineage and promote to a Th2 fate. Thus T-bet not only induces Th1 development but also actively suppresses Th2 differentiation. Szabo S.J. Et al, Distinct Effects of T-bet in TH1 Lineage Commitment and IFN- γ Production in CD4 and CD8 T Cells, Science, 295:338, 2002

12 IFNγ: promoter or inhibitor of Th1 responses? The initial activation/expansion phase of CD4+ T-cell responses is followed by a death phase during which most (90%) effector cells are eliminated. This is a complex process that likely involves multiple mechanisms, including: - Fas-FasL and (TNF) TNFRI/II interactions, - cytokine withdrawal, - nitric oxide, - tryptophan catabolism, - Tim-3-galectin-9 pathway, - IFNγ -mediated death IFNγ have different roles in regulating Th1 responses depending on the stage of the response and the activation state of CD4+ T cells; At the initiation of a response: IFNγ enhances the differentiation of Th1 cells to generate effectors capable of providing protection against infection. Th1 response transitions from effector to memory phase: IFNγ mediates the death of Th1. This type of negative regulation later in the response could be serve to minimize the potential risk of autoreactivity that would be associated with sustaining a high frequency of activated Th1 effector cells. Foulds K.E. et al., Th1 memory: implications for vaccine development, Immunological R eviews, 211:58, 2006

13 Schroder K. et al., Interferon-γ :an overview of signals, mechanisms and functions, Journal of Leukocyte Biology, 75:163, 2004 The IFNγ induced antiviral state The IFN system regulates innate and adaptive immunity to viral infection. Although types I and II IFN are crucial in the immediate cellular response to viral infection, the immunomodulatory activities of IFNγ become important later in the response in coordinating the immune response and establishing an antiviral state for longer term control.

14 Schroder K. et al., Interferon-γ :an overview of signals, mechanisms and functions, Journal of Leukocyte Biology, 75:163, 2004 Immunomodulation and leukocyte trafficking IFNγ orchestrates the trafficking of specific immune cells to sites of inflammation up-regulating expression of - adhesion molecules: ICAM-1, VCAM-1; - chemokines: IP-10, MCP-1, MIG, MIP-1/, RANTES TNF-α and IL-1 synergistically regulate many of these molecules.

15 Antitumoral Activity of Interferon-γ 1. Tumor antigen-carrying cancer cells are recognized by: - NK - DC - CD4+/CD8+ cells - macrophages 2. Begin acute immune response cascade in which all together cooperate to destroy foreign tissue structures. 3. IFNγ with TNF-α, trigger pro-apoptotic and anti-proliferative enzyme systems including: - degradation of tryptophan by indoleamine (2,3)-dioxygenase (IDO) - production of ROS and of NO which is oxidized by ROS superoxide anion to cytocidal peroxynitrite (ONOO-). Brandacher G. et al., Antitumoral Activity of Interferon-γ Involved in Impaired Immune Function in Cancer Patients, Current Drug Metabolism, 7:599, 2006

16 Indoleamine 2,3 dioxygenase (IDO) IDO is rate-limiting enzyme in the degradation of the essential amino acid tryptophan via the kynurenine pathway to form N-formyl-kynurenine, which is subsequently converted to niacin. Thereby, growth of microbes and tumor cells is affected, because tryptophan deprivation limits protein biosynthesis. IDO is expressed, particularly by IFNγ, in multiple cell types: - fibroblasts - macrophages - DC - trophoblasts - epithelial cells For many years has been considered as an innate defence mechanism limiting growth of viruses, bacteria, intracellular pathogens and malignant cells by withdrawing tryptophan from the microenvironment. Recently, it has been shown that activation of IDO is also critically involved in the regulation of immune responses. If the immune system is unable to eliminate the malignant tumor cells, immune activation may persist, which causes overproduction of IFN-γ. One consequence for these detrimental IFN-γ effects might be increased tryptophan depletion, which can affect T-cell responses and thus contribute to the development of immunodeficiency in cancer patients.

17 When the acute immune response cascade has failed to completely destroy foreign tissue structures, the anti-proliferative enzyme systems (the degradation of tryptophan by IDO, the production of ROS, of NO and ONOO-) are chronically up-regulated by IFNγ together with TNF-α. Moreover, cancer cells stimulated by the cytokines themselves become a relevant source of tryptophan degradation. As a consequence also metabolism of normal cells more and more begins to suffer from symptoms linked with IFNγ over-expression namely anemia, cachexia, depression, immunodeficiency and higher risk of infection and sepsis. Although IFN-γ is a central pro-inflammatory cytokine, evidence increasingly indicates that it is also involved in the development of tumor immune escape, tolerance induction and impaired immune functions by activating a negative feedback loop. Brandacher G. et al., Antitumoral Activity of Interferon-γ Involved in Impaired Immune Function in Cancer Patients, Current Drug Metabolism, 7:599, 2006

18 BIBLIOGRAFIA Pestka S. et al., Interferons, interferon-like cytokines, and their receptors, Immunological Reviews, 202:8, 2004 Parmar S. et al., Interferons: mechanisms of action and clinical applications, Curr Opin Oncol, 15:431, 2003 Takaoka A. et al., Interferon signalling network in innate defence, Cellular Microbiology, 8:907, 2006 Ghosh S. et al., Interfering with interferons in inflammatory bowel disease, Gut, 55:1071, 2006 Platanias L.C., Mechanisms of type-i- and type-ii-interferon-mediated signalling, Nature Reviews Immunology, 5:375, 2005 Schroder K. et al., Interferon-γ :an overview of signals, mechanisms and functions, Journal of Leukocyte Biology, 75:163, 2004 Schroder K. et al., Signal integration between IFN γ and TLR signalling pathways in macrophages, Immunobiology, 211:511, 2006 Szabo S.J. Et al, Distinct Effects of T-bet in TH1 Lineage Commitment and IFN- γ Production in CD4 and CD8 T Cells, Science, 295:338, 2002 Foulds K.E. et al., Th1 memory: implications for vaccine development, Immunological Reviews, 211:58, 2006 Bogdan C. et al., Production of interferon- γ by myeloid cells fact or fancy?, TRENDS in Immunology, 27:282, 2006 Brandacher G. et al., Antitumoral Activity of Interferon-γ Involved in Impaired Immune Function in Cancer Patients, Current Drug Metabolism, 7:599, 2006