How is the immune system affected by the exposure to perfluorooctanesulfonate (PFOS)
Peter Kjellén, Orebro Life Science Center

Specific goals The aim of my proposed research plan is to elucidate how exposure to perfluorooctanesulfonate (PFOS) affects that the immune system. Specifically, the project will examine if PFOS affects the immune cells that regulate inflammation. Cell- lines, advanced proteomics, molecular biology and cell biology methods will be used to obtain the specific goals, which are to: " Elucidate and clarify how the function of T-lymphocytes is affected after the exposure to PFOS. " Elucidate and clarify the intra-cellular signal transduction pathways that are induced by PFOS in T-lymphocytes. Overview of the research area Perfluorooctanesulfonate (PFOS) Perfluorooctanesulfonyl fluoride based compounds have been used in a wide variety of consumer products. These compounds degrade to perfluorooctanesulfonate (PFOS), a persistent metabolite that accumulates in tissues of humans and wildlife 1, 2. Fluorinated compounds have been produced for use as surface and stain protectors for products such as coated cookware, carpets, upholstery, leather, and paper packing including fast food wrappers. These compounds are now widely found in humans and wildlife 1, 3. PFOS has for example been detected in birds, fish, and mammal tissues from the Canadian Artic, Baltic and Mediterranean Seas, and the Great Lakes of North America, and in polar bears from Alaska, Greenland and Canada 3. PFOS has also been identified in human individuals and pooled human serum samples from the United States, Japan and several other countries 1, 4. PFOS has been shown to have harmful effects on cell membranes and communication between cells 5, 6. PFOS severely decreases postnatal survival of neonatal rats and mice 7. However, research about the effect that PFOS has on the immune system has so far been ignored. This project proposes to do research of how PFOS influences the function of the immune system. The project will focus on how PFOS affects the activation and function of T-cell lymphocytes. Activation of T-cells Activation of T-cells by extra-cellular stimuli triggers a cascade of intra-cellular events including phosphorylation and subsequent activation of enzymes. These intra-cellular events, termed the signal transduction pathway, are crucial for the function of T-cells. T-cells get activated by the presentation of antigen, complexed with the major histocompatibility complex (MHC) expressed on antigen presenting cells (APCs), to the T-cell receptor (TCR) (Fig. 1). T-cells can also get activated in vivo by adding antibodies (anti-CD3) specific for the TCR. Additional signals from co-stimulatory receptors, e.g. CD28, are required for efficient subsequent activities such as cell division, cell survival, secretion of cytokines and cytotoxicity. Alternatively, T-cells can be activated by chemical substances, e.g. by phorbol myristate acetate (PMA) plus ionomycin, that also induce signaling pathways in T-cells and the production of cytokines as outlined in Figure 1. In both cases the intra-cellular enzymes called the mitogen-activated protein kinases (MAPKs) have crucial roles for the function of the T-cell. MAPK p38 and signal transduction pathways in T-cells Mitogen-activated protein kinases (MAPKs) are ubiquitously expressed in mammalian cells and transduce signals in response to growth factors, pro-inflammatory cytokines and stress 8. MAPKs are divided into three main groups, i.e. the extra cellular signal-regulated protein kinase (Erk), c-Jun N-terminal kinase/ stress-activated protein kinases (Jnk/SAPK) and p38 9. Four isoforms of p38 ( , , and ) exist. The p38 isoform has been shown to be crucially linked to the production of pro-inflammatory cytokines. Little is known about how TCR-mediated signaling is coupled to the activation of the MAPK p38 in mammalian cells. Typically the MAPK p38 is activated by environmental stress such as ultraviolet radiation and osmotic shock and by pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-2 and IL-17 10. Figure 1.The MAPKKK/MAPKK signaling pathways induced in CD4+ T-cells after engagement of the T-cell receptor (TCR) by antigen presented on the major histocompatibility complex-II (MHC-II). Initially, the TCR gets autophosphorylated or phosphorylated by cytoplasmic protein-tyrosine kinases (Lck and Fyn) on the immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylated ITAMs recruits the ZAP-70, which gets phosphorylated and activated by Lck. The linker for activation of T-cells (LAT), which is a substrate for ZAP-70, recruits the phospholipase- C 1 (PLC 1), which is a critical enzyme for T-cell activation. The MAPK p38 gets activated by a MAPK kinase (MKK), which upstream has been activated by a MAPK kinase kinase (MKKK). MAPK p38 and inflammation Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting 1% of the western world population. RA is characterized by a chronic and erosive inflammation of cartilaginous joints leading to a progressive destruction of cartilage and bone 11. The disease is characterized by an over-active immune system and the production of pro-inflammatory substances by immune cells that infiltrate the synovium. The initial events cause the proliferation of synovial fibroblast and further release of inflammatory molecules that eventually will cause degradation of cartilage and joint destruction. The chronic inflammation of the joints is mainly mediated by activated T-cell, macrophages and B-cells where cytokine induction of enzymes like matrix metalloproteinases play a destructive role 11. Several MAPK p38 inhibitors have been shown to block the production of interleukin-1 (IL-1), tumor-necrosis factor- (TNF- ) and other pro-inflammatory cytokines 12 and many reports support that the MAPK p38 plays an important role in the signaling pathway that regulates inflammation 10. Besides the important role in regulating the production of pro-inflammatory cytokines the MAPK p38 controls induction of enzymes such as iNOS (inducible nitric oxide synthase) 13. Induction of nitrogen oxide (NO), which is a mediator of cartilage and bone destruction, is one of the mechanisms that pro-inflammatory cytokines, e.g. IL-1 and TNF, elicit their effects. Blocking the activity of p38 by pyridinyl imidazole (SB 203580) blocks pro-inflammatory cytokine production, NO production and inflammation in animal models for arthritis 13. In summary, the MAPK p38 is a potent regulator of inflammation. Project description This project will examine the ability of PFOS to activate T-cells and to induce the production of pro-inflammatory cytokines. The project will also examine the intra-cellular events, as exemplified in Figure 1, that are triggered by exposing T-cells to PFOS. The main applicant (Peter Kjellen) will be the main supervisor of the project. The project has the following research plan: 1. Setting up a system for studying cell signaling pathways in PFOS exposed T-cells (Estimated time: 6 months) In this part of the project the basic parameters for studying signaling pathways will be set up. The optimal parameters for exposing immune cells (T-cells) to PFOS will be titrated and determined. Protein expression levels and amount of phosphorylation for a small number of signaling molecules involved in the MAPK p38 signaling pathway will be determined. The purpose of this part of the project is to prepare for a large proteomics based screening assay. 2. Using proteomics as a method to identify PFOS induced signaling pathways. (Estimated time: 12 months) The goal here is to simultaneously identify a large amount signaling molecules by a proteomics based assays. Protein expression levels and amount of phosphorylation of several signaling molecules from PFOS exposed T-cells will be determined using a screening service offered by BD Biosciences and R and D systems. The "Proteome Profiler Array" is a tool designed to simultaneously detect the relative phosphorylation and activation of 40-200 different signaling molecules. The project will also use a recently developed protein chip technique to perform the screening. This will be performed in collaboration with Dr Christian Maercker, Resource Center, German Cancer Research Center, Heidelberg and Dr. Mats-Olof Mattsson, Örebro University. These methods present a very effective approach to elucidate the signaling pathways in cells. Identified proteins function and role in signaling pathways will be elucidated using various methods, including over-expression by virus-mediated infection (Lentivirus), co-immunoprecipitation, FACS, western blot, siRNA and kinase assays. 3. Immunological effects of exposing T-cells to PFOS (Estimated time: 18 months) The effect of PFOS on cell survival, proliferation and cytokine production will be determined. Cell membrane and intracellular activation markers will be analyzed in this part of the project. The ability of PFOS to induce the production of pro-inflammatory cytokines will be determined by the ELISA method. The production of IL-2, IFN- , IL-4, TNF- , IL-10 are examples of cytokines that will be measured in T-cell supernatant culture after exposure to PFOS. The ability of PFOS to induce the expression of the cell surface activation markers, e.g. CD69, and to induce self-programmed cell death (apoptosis) will be examined by FACS. Expertise collaborators and available research equipment The main applicant is employed by Orebro University and has a faculty financed position as assist. Prof. ("forskarassistent"). The applicant has a Med. Dr. Sci degree from the inst. of Medical Inflammation Research, Lund University, supervised by Prof. Rikard Holmdahl who has expertise in inflammatory and autoimmune diseases. Dr. Magnus Engwall, MTM research center, Orebro University will provide expertise help in environmental toxicology. The project will have collaboration with international laboratories. Prof. Bart Sefton, Salk Institute, La Jolla, USA, will be a scientific adviser regarding intra-cellular signal transduction pathway in T-cells. Prof. Sefton was the applicant's post-doc supervisor. Dr Christian Maercker, Resource Center, German Cancer Research Center, Heidelberg is a collaborator in the field of protein micro-array methods. Research equipment to start up this project is available at Orebro University and at laboratories of collaborating researchers. This equipment includes for example facilities for sterile work, cell culture incubators, microscopes, FACS, spectrophotometers and PCR-machines. Relevance of the project This project has the aim to clarify how the exposure of PFOS affects the immune system. Other research groups in the world have not done this to a large extent. The harmful effects of PFOS are very important to explore because it is accumulating in humans and animals around the world. Results from this project will provide the society with a tool to pressure the industries to use less perfluorooctanesulfonyl fluoride based compounds.

References

1. Kannan, K. et al. Perfluorooctanesulfonate and related fluorochemicals in human blood from several countries. Environ Sci Technol 38, 4489-95 (2004). 2. Giesy, J. P. & Kannan, K. Global distribution of perfluorooctane sulfonate in wildlife. Environ Sci Technol 35, 1339-42 (2001). 3. WWF International Arctic Programme, D. c. The tip of the iceberg: Chemical contamination in the Arctic. 4. Taniyasu, S., Kannan, K., Horii, Y., Hanari, N. & Yamashita, N. A survey of perfluorooctane sulfonate and related perfluorinated organic compounds in water, fish, birds, and humans from Japan. Environ Sci Technol 37, 2634-9 (2003). 5. Hu, W. et al. Alterations in cell membrane properties caused by perfluorinated compounds. Comp Biochem Physiol C Toxicol Pharmacol 135, 77-88 (2003). 6. Hu, W. et al. Inhibition of gap junctional intercellular communication by perfluorinated compounds in rat liver and dolphin kidney epithelial cell lines in vitro and Sprague-Dawley rats in vivo. Toxicol Sci 68, 429-36 (2002). 7. Lau, C. et al. Exposure to perfluorooctane sulfonate during pregnancy in rat and mouse. II: postnatal evaluation. Toxicol Sci 74, 382-92 (2003). 8. Zhang, J. et al. p38 mitogen-activated protein kinase mediates signal integration of TCR/CD28 costimulation in primary murine T cells. J Immunol 162, 3819-29 (1999). 9. Pearson, G. et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22, 153-83 (2001). 10. Ono, K. & Han, J. The p38 signal transduction pathway: activation and function. Cell Signal 12, 1-13 (2000). 11. Feldmann, M., Brennan, F. M. & Maini, R. N. Rheumatoid arthritis. Cell 85, 307-10 (1996). 12. Kumar, S., Boehm, J. & Lee, J. C. p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov 2, 717-26 (2003). 13. Badger, A. M. et al. SB 203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production, and inducible nitric oxide synthase in bovine cartilage-derived chondrocytes. J Immunol 161, 467-73 (1998).