Characterization of the Molecular Mechanisms That Bridge

Investigators

Abstract

The overall goal of our research program is to investigate key endogenous factors that bridge innate and adaptive immune responses and to apply this knowledge to advance product development and patient safety. To this end we have focused on cytokine pathways critical for the function of macrophages and dendritic cells. Currently our research program comprises three projects with both distinct and overlapping components. The following are descriptions of each of these projects: Molecular Mechanisms of the Activation of Myeloid Antigen Presenting Cells. Our laboratory, along with others, has demonstrated that myeloid antigen presenting cells (APC) have many functions previously assigned only to T helper-1 (Th1) cells. For example, macrophages and dendritic cells make interferon gamma (IFN-g) in certain settings soon following immune activation, allowing sentinel immune defenses to occur in the absence of specific recognition by T cells. We have also demonstrated that activated APC express the transcription factors, STAT4 and T-bet, initially described as lymphoid factors that promote IFN-g production. STAT4 is required for IL-12-dependent IFN-g production by APC, and T-bet mRNA levels correspond well with the levels of APC-expressed IFN-g mRNA. Each of these factors is not expressed in resting cells, but induced to high levels following various types of activating signals, including IFN-g. In this manner, both STAT4 and T-bet are central to positive feedback loops in which their expression levels are upregulated by a cytokine that they in turn induce. The primary goal of this laboratory is to investigate other roles of these two key transcription factors in the function of antigen presenting cells using a multi-pronged approach as follows: (1) Experiments with gene targeted mice: Various APC populations are being isolated from wild type, Stat4 -/-, and Stat6 -/- mice (deficient in IL-4 signaling) and evaluated for their costimulatory function (ability to induce T cell proliferation, IFN-g production, IL-4 production). Methods of adoptive transfer of APC are being developed to determine whether differences observed in vitro result in functional consequences in vivo, including responses to intracellular infection. (2) Overexpression models: Methods are being developed to overexpress Stat4 and T-bet in APC using transfection and retroviral transduction. Differences in costimulatory function will be evaluated as described in approach #1. Moreover, T-bet and Stat4 transgenic mice are being generated with a construct driven by an MHCII promoter, in which APC will overexpress these molecules. These animal models will allow evaluation of costimulatory function ex vivo and in vivo. Another goal of the laboratory is to re-evaluate the functional significance of APC production of interferon gamma. We hypothesize that it may play a role in sentinel defenses. This question will be addressed through adoptive transfer of wild type APC into IFNg -/- recipients and the use of various infection models. Moreover, we are actively collaborating with two laboratories with overlapping interests in IL-12 signal transduction, in patients with genetic immunodeficiencies (Dr. Steven Holland) and at the level of signal transduction (Dr. John O'Shea). Characterization of Host Factors that Modulate the Effects of Anthrax Lethal Toxin. Although it is known at a molecular level that anthrax lethal toxin (LT) acts to cleave MAPKKs, its exact role in the overall pathogenesis of anthrax infection is unknown. There is a consensus that LT has at least one indisputable action: LT lyses macrophages at threshold concentrations. However, data regarding the in vivo action of LT on macrophages is lacking. Additionally, significant controversies remain unresolved regarding the in vitro effects of LT. For example, it has been reported that IL-1 and TNF-a produced by macrophages in response to LT are critical mediators underlying its lethal effects, while other investigators have reported that LT may inhibit rather than induce pro-inflammatory cytokine production by macrophages. Regardless of the underlying reason(s) for this discrepancy, it is clear that establishing where and when LT acts will be essential in predicting the efficacy of therapies that target this pathway. In this regard, it has not been established whether cytokine or anti-cytokine therapies can attenuate the action of LT in vivo or whether these potential therapies could augment host responses to infection. With the scientific tools that are currently available (e.g., gene-targeted mice), we can now establish definitively whether TNF-a and IL-1 pro-inflammatory cytokine pathways are required for LT action in vitro and in vivo. Elucidation of the involvement of other cytokine pathways is also warranted. For example, while promoting cellular immunity, the IL-12/Stat4 signal transduction pathway down-regulates other immune responses, including the deleterious responses to endotoxin in mouse models. Therefore, it is conceivable that modulation of Stat4 signaling could decrease the systemic shock response to various bacterial toxins, including anthrax LT. In addition, it is unknown whether cytokines that down-regulate certain myeloid APC functions (e.g., IL-4 and IL-10) or those that promote cell survival (e.g., M-CSF) can attenuate the response of macrophages to LT. Moreover, it is unknown whether concomitant exposure of macrophages to anthrax edema toxin (ET), which would be expected in natural infections, affects LT actions. To address these questions, we have obtained E. coli expression vectors for the three anthrax toxin factors (lethal factor, edema factor, and protective antigen) from Dr. John Collier. In collaboration with Dr. Kathleen Clouse and Dr. Steven Kozlowski in DMA, who has experience in the area of protein production in E. coli, we intend to produce toxin components in large-scale quantities with high purity. It will be essential for us to produce endotoxin-free components. Through the use of blocking antibodies and gene-targeted mice, we will determine the role of the above-mentioned cytokine pathways in LT action. Also, we will investigate whether anthrax ET has effects on LT action in vitro and in vivo. Finally, we will ascertain whether toxin action is attenuated by inhibitory cytokines. One of the major goals of our studies will be to determine the effect of cytokine and anti-cytokine therapies when the toxins are used in vivo. If we can prove that certain cytokines play a role in toxin activity, we will next investigate whether existing cytokine or anti-cytokine therapies are efficacious in attenuating responses to anthrax LT. Next, we plan to confirm these findings using live infection models in collaboration with outside investigators who have access to adequate containment facilities (currently unavailable at CBER). Another important goal of this project is to optimize bioassays for anthrax LT activity. The bioassays that currently exist measure the effect of LT on murine cell line viability. We are currently investigating the endogenous factors that render certain cell lines sensitive to anthrax lethal toxin. In addition, we are developing a bioassay the uses a human cell line that produces pro-inflammatory cytokines in response to anthrax LT. Together these initiatives will provide critical information necessary to improve the bioassays necessary to evaluate anthrax infection therapeutics. Investigating Genetic Variation in the Immunoglobulin J Polypeptide. The immunoglobulin J polypeptide (IMJ) is unique from an evolutionary standpoint. In humans and other higher organisms, it is required for functional polymeric antibodies (IgM and IgA). These antibody classes are essential for both the initial adaptive B cell immune response, but also for mucosal immunity. Interestingly, IMJ is also present in animals with no known adaptive immune responses, indicating that it might represent a link between innate and adaptive immunity. As an essential component for polymeric immunoglobulins, this molecule represents an important area of interest for the Division of Monoclonal Antibodies. However, very little has been investigated regarding genetic polymorphisms that might exist in humans. Polymorphisms that result in coding changes to the amino acid sequence of IMJ would have important implications for the generation of immunogenicity. In a study designed to advance patient safety, we are currently sequencing the IMJ gene in 100 chromosomes from normal controls. If we find evidence of genetic variation, we will next determine whether these changes have functional and clinical significance. This project incorporates FY2002 projects 1Z01BO002009-01 and 1Z01BO002012-02.