Rick A. Wetsel - US grants

The fifth component of complement, C5, is the precursor for the important phlogistic molecule, C5a, a potent chemotatic agent. Sera from C5-deficient individuals lack bactericidal activity and have severely impaired ability to induce chemotaxis. Deficiency of C5 in the mouse has been recognized since the early 1960s and this model has been used to demonstrate the importance of C5 in the initiation of pulmonary inflammation. Improved methods for the analysis of protein synthesis by cells in culture and the isolation and characterization of cDNA for mouse C5 have allowed evaluation of the molecular basis of this protein deficiency. We have described both quantitative and qualitative differences between C5-sufficient (C5S) and C5-deficient (C5D) mice in the protein synthesized, in the C5 mRNA in cytoplasm and nuclei, and in the organization of the C5 gene. The experiments outlined in this proposal will extend these early studies to examine the defects in the C5 deficiency. We will define the structural abnormalities in the protein synthesized by the C5D cells that interfere with the secretion of the protein. cDNA libraries will be prepared from C5S and C5D mRNA and the C5 specific cDNA will be isolated and characterized to analyze the mRNA sequence abnormality(ies) responsible for production of the abnormal C5 protein. In addition, cosmid libraries will be prepared from C5S and C5D DNA and C5 specific clones will be isolated and characterized to analyze the structural abnormalities in the C5D gene responsible for the abnormalities in the C5D mRNA. Finally, C5S and C5D genomic clones will be transfected into mouse L-cells and the expression of the genes in these cells will be studied to examine the potential role of the C5D cells in producing the abnormalities in protein secretion. The transfected cells may be useful as a reagent for subsequent studies on the role of RNA processing in producing the C5 deficiency state.

The fifth component of complement (C5) is a serum glycoprotein that mediates important inflammatory and cytolytic processes. Sera from C5-deficient individuals lack bactericidal activity and have severely impaired ability to induce chemotaxis. The recent isola- tion and characterization of a full-length cDNA for mouse C5, have thus far allowed us to demonstrate: (a) C5D mice synthesize small amounts (10-20% of normal) but do not secrete single chain intracellular C5 protein; (b) C5D mRNA was quantitatively (lO-fold less) and qualitatively (6.0 and 6.5 kb species in cytoplasm) different from sufficient (C5S) mRNA; (c) restriction fragment length polymorphisms (Hind III and Pvu II) between the C5S and C5D genes correlated with the protein deficiency. The experiments outlined in this proposal will extend these preliminary findings in the mouse and will initiate a parallel study regarding C5 deficiency in humans. We will define the structural abnormalities in the protein synthesized by the C5D cells that interfere with the secretion of the protein. cDNA libraries will be prepared from C5S and C5D mRNA and the C5 specific cDNA will be isolated and characterized to analyze the mRNA sequence abnormality(ies) responsible for production of the abnormal C5 protein. In addition, the full-length C5D cDNAs will be employed in in vitro and in vivo translational systems to determine which C5D mRNA is translated into the non-secreted C5D protein. Genomic libraries (cosmid or YAC) will be prepared from C5S and C5D DNA and C5 specific clones will be isolated and characterized to analyze the structural abnormalities in the C5D gene responsible for the abnormalities in the C5D mRNA. The C5S and C5D genomic clones will be transfected into mouse L-cells and the expression of the genes in these cells will be studied to examine the potential role of the C5D cells in producing the abnormalities in protein secretion. Finally, the defects in the human C5 gene which cause the C5 protein deficiency will be determined from restriction fragment length polymorphisms which correlate with the disease. Also, C5S and C5D genes will be isolated and characterized for structural defects which are ultimately responsible for the protein deficiency.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The fifth component of complement (C5) is a glycoprotein that, after proteolytic activation into C5a and C5b polypeptides, mediates important inflammatory and cytolytic processes. The small C5 activation fragment, C5a, is a very potent phlogistic molecule which, upon binding to the C5a-Receptor (C5a-R), mediates contraction of smooth muscle, vasodilation, and chemotaxis and secretion of granular enzymes from many inflammatory cells; moreover, C5a plays a role in augmenting humoral and cell mediated immune responses. The large C5 activation fragment, C5b, initiates the assembly of the C5b-9 membranolytic attack complex that is directly responsible for complement-mediated target cell cytolysis of bacterial and viral pathogens. Sera from homozygous C5-deficient (C5D) individuals lack bactericidal activity and have a severely impaired ability to induce chemotaxis; as a result, C5D individuals have a propensity for severe recurrent bacterial infections particularly to Neisserial species, including meningitis and extragenital gonorrhea. In certain pathological conditions, damage of healthy tissue occurs after C5 activation as a result of C5b-9 mediated lysis of non-target (bystander) cells and proteolytic destruction by enzymes released from neutrophils recruited and activated by C5a. For example, tissue damage mediated by C5 activation polypeptides is strongly implicated in the pathogenesis of pulmonary, and myocardial diseases, atherosclerotic lesions, and certain central nervous system disorders, including Alzheimer disease. Furthermore, C5 activation polypeptides play a critical role in the hyperactive rejection of discordant xenografts. The broad long-term goal of this study is to understand and thereby selectively modulate the inflammatory and cytolytic effects resulting from C5 activation. Molecular knowledge of the structural-functional aspects of complement C5 activation and the receptor-cellular interactions that are mediated by the C5a cleavage polypeptide are of paramount importance in achieving this goal. This is especially true as the discovery of C5a-R expression by non-myeloid tissue cells have indicated that the biological functions mediated by C5a are even more pleiotropic than originally assumed. Our immediate objectives are to: 1) determine the molecular genetic and structural basis of human C5 deficiency by DNA, RNA, and protein studies of seven different C5- deficient kindred, 2) determine functionally important binding domains in C5 by extensive mapping studies with monoclonal antibodies and synthetic peptides, 3) determine tissue cells expressing the C5a- receptor by in situ hybridization and immunohistochemistry studies, and 4) determine the effects that selected cytokines have on C5a-R expression using appropriate tissue derived cells, and 5) initiate investigations into the biological functions mediated by the C5a-R on tissue cells by examining expression of acute phase proteins, adhesion molecules involved in neutrophil emigration, and HLA class I and class II molecules involved in antigen presentation.

[unreadable] DESCRIPTION (provided by applicant): One of the major biological consequences of complement activation is the generation of three small cationic peptides C3a, C4a, and C5a, collectively referred to as complement anaphylatoxins. The complement anaphylatoxins mediate numerous biological functions by binding to seven transmembrane G-protein coupled receptors expressed on specific target cells. The acute and chronic overproduction of the two most potent anaphylatoxins, C3a and C5a, is considered to be a major contributor to the pathogenesis of numerous diseases, including rheumatoid arthritis, sepsis, tissue ischemic injury, acute respiratory distress syndrome, multiple system organ failure, and atopic asthma. The complement anaphylatoxins are regulated by carboxypeptidases, which generate their much less active desArg derivatives, C3adesArg and C5adesArg. Historically, CPN, the carboxypeptidase expressed constitutively in plasma, was thought to be the sole carboxypeptidase regulator of complement anaphylatoxins. Recently, two other carboxypeptidases, CPR and CPM, which are expressed in the serum and on epithelial cells (lung and kidney), respectively, have been proposed as additional carboxypeptidase regulators of C3a and C5a. During the past few years studies in our laboratory as well as in others have revealed that the complement anaphylatoxins C3a and C5a in addition to their traditional phlogistic properties are significant modulators of CD4+ Th1 and Th2 effector functions in allergic and infectious disease. The goal of this research program is to increase our understanding of the regulation and biological functions that the complement anaphylatoxins and their receptors mediate in inflammation, immunity, and T cell responses in relevant infectious diseases. By employing C3aR, C5aR, CPN, and CPM "knock-out" mice generated in our laboratory, we propose to major goals: 1) to delineate and evaluate the overall physiological significance of CPN, CPR, and CPM in regulating biological responses mediated by C3a, C5a, bradykinin, and other important inflammatory molecules, and 2) delineate cellular interactions and molecular mechanisms by which the complement anaphylatoxin receptors, C3aR and C5aR/CD88 modulate T-cell effector functions, which are important in the pathogenesis of Listeria monocytogenes. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The objective of this research proposal is to delineate the overall contribution and potential mechanisms that complement utilizes to mediate the pathogenesis of allergic lung disease. The specific aims of this proposal are driven by the central hypothesis that complement activation products regulate key features of allergen-induced airway disease, including airway hyperresponsiveness (AHR) and acute airway inflammation. The results of this proposal will facilitate the evaluation of complement as a possible therapeutic target in the treatment of asthma. An allergen-induced model of pulmonary allergy in mice with specific complement deficiencies will be used to identify the complement pathways, activation fragments, and receptors that are potentially important in mediating the pathogenesis of allergic lung disease. The complement-deficient animals that are subjected to the model will be examined for attenuation of pathological and physiological hallmarks of asthma, including AHR, airway mucus hypersecretion, elevated IgE levels and lung eosinophils. The Th2 cytokines (IL-4, IL-5, IL-13) that have been proposed to play a pivotal role in asthma will also be examined for altered expression. In addition to the murine experimental allergic model, studies with human T-cells as well as other leukocytes isolated from patients with allergic lung disease will be used to examine potentially altered complement mediated cellular responses in asthma. Four specific aims are proposed to accomplish the research goals: 1) to examine the importance of each complement activation pathway in eliciting the asthma associated responses in an allergen-induced model of pulmonary allergy, 2) to determine how the complement anaphylatoxin receptors (C3aR and C5aR) affect the asthma associated responses in an allergen-induced model of pulmonary allergy, 3) to determine how the fifth complement component (C5) affects the asthma associated responses in an allergen-induced mouse model of pulmonary allergy, and 4) to determine the biological effects of the complement anaphylatoxins (C3a and C5a) in regulating T-cell mediated responses in asthma. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The objective of this research proposal is to evaluate the mouse complement C4b binding protein (C4BP) "knock-out" mouse as an in vivo model to delineate the overall contribution of human C4BP in adaptive immunity. The aims of this proposal are driven the the central hypothesis that C4BP on binding CD40 expressed on B cells mediates many important biological responses important in immune regulation, including immunoglobulin class switching in response to T-dependent antigens, and proliferation and rescue of germinal center B cells. Our recent generation of a C4BP "knock-out" mouse provides a unique opportunity to evaluate the physiological impact of C4BP as a significant contributor to adaptive immunity. The results from this proposal will significantly advance the understanding of how the innate and adaptive immune systems interact in providing a robust immune response. Moreover, these studies will also provide basic information that will facilitate a better understanding of B-cell maturation and immunoglobulin production, and thereby provide significant insights into the pathogenesis of autoimmune diseases such as systemic lupus erythematosus as well as IgE associated allergic diseases, including atopic asthma. To accomplish the overall objective of the proposal, three aims are proposed. Aim one will evaluate the binding interactions between mouse C4BP and CD40 expressed on mouse B cells. Aim two will examine lymphocyte development in C4BP-deficient (-/-) mice compared to wild-type mice. Aim three will determine the antibody response to T-dependent and T-independent antigens in C4BP-deficient (-/-) mice compared to wild-type mice, as well as to examine germinal center formation in C4BP-deficient (-/-) mice compare to wild- type mice. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cellular transplantation of lung stem/progenitor cells represents a potential therapeutic approach for a variety of inherited lung diseases. Crucial to the success of such a therapeutic strategy is that the transplanted cells and their progeny are corrected (both genotypically and phenotypically) for the disease-causing mutation and that the transplanted cells do not elicit an immune response in the recipient. Recently developed methodologies for generating induced pluripotent stem (iPS) cells and zinc finger nuclease (ZFN)-mediated genome editing make possible, in principle: a) the generation of autologous, patient-specific iPS cells carrying inherited genetic mutations;b) specific correction of the responsible genetic mutation in chromosomal DNA of the autologous iPS cells, and c) in vitro differentiation of iPS cells into various cell types required for cell transplantation therapy. We propose to employ this approach to generate corrected, autologous iPS cells for patients with either Surfactant Protein B (SP-B) Deficiency or Cystic Fibrosis (CF). For SP-B deficiency we will further generate corrected lung progenitor cells (lung alveolar epithelial type II cells;ATII) with the potential for cellular therapy of patients with this disease. This potentially high-impact two year project brings together the complementary expertise of two highly qualified principal investigators: one (Brian R. Davis) experienced in the generation/characterization of iPS cells together with ZFN-mediated genome editing, the other (Rick A. Wetsel) experienced in directed, in vitro differentiation of human pluripotent stem cells to lung progenitor cells and transplantation of such progenitor cells into mouse lung. This novel convergence of methodologies represents a paradigm applicable to cellular therapy of other single-gene caused diseases of the lung, heart, and blood systems. PUBLIC HEALTH RELEVANCE: The objective of this project is to generate corrected, patient-specific lung progenitor cells for cell transplantation therapy of patients with inherited lung disease. It represents a novel convergence of methodologies for generation of autologous, pluripotent stem cells from somatic cells, efficient site-specific correction of genetic mutations in chromosomal DNA, and directed differentiation of pluripotent cells to cell types appropriate for transplantation.

DESCRIPTION (provided by applicant): Following infection with the gram positive bacterium Listeria monocytogenes (Lm), innate immune responses are rapidly triggered and are essential for host survival. Despite the importance of the innate immune system in fighting an Lm infection, little is known about the role of complement. There is now strong evidence that the complement anaphylatoxins, C3a and C5a, are potent modulators of T-cell effector functions in allergic diseases, including asthma. However, the biological relevance of C3a/C5a modulation of T-cell responses to bacterial pathogens remains largely unknown. In addition, there is very little understanding of how the anaphylatoxins impact CD8+ T-cell responses that are essential for host clearance of intracellular bacteria such as Lm. The long term goal of this research program is to delineate important biological pathways mediated by the complement anaphylatoxins in innate and adaptive immune responses so that appropriate therapeutic strategies can be developed that will abrogate the deleterious hyper-inflammatory effects of C3a and C5a without impairing the host immune response to infectious disease. These studies are driven by the central hypothesis that the anaphylatoxins on binding their specific receptors mediate numerous biological functions that are critical for the initiation, perpetuation, and regulation of pathways required for robust innate as well as adaptive immune responses. It is also hypothesized that the deleterious and potentially lethal effects that can result from the acute or chronic release of these extremely potent phlogistic molecules are regulated both systemically and locally by specific carboxypeptidases (CPN & TAFI) and by the putative decoy receptor C5L2R. The central hypothesis will be tested in the current application by two major specific aims that will employ novel combinations of C3aR, C5aR, C5L2R, and carboxypeptidase KO mice in a model of systemic Lm infection. Aim 1 will delineate cellular and molecular mechanisms by which the C3a and C5a anaphylatoxins and their specific receptors (C3aR and C5aR, respectively) modulate innate and adaptive immunity in response to Lm infection. Aim 2 will delineate how carboxypeptidase (CPN & TAFI) regulation and C5L2R binding impact anaphylatoxin modulation of innate and adaptive immunity in response to Lm infection. The proposed research provides a unique and powerful in vivo system to delineate comprehensively numerous innate and T-cell immunomodulatory pathways that are mediated by the complement anaphylatoxins. Moreover, the data from the proposed studies will provide novel and important data regarding complement's role in the host response to Lm, an important NIAID category B priority intracellular pathogen.

DESCRIPTION (provided by applicant): Cardiovascular disease such as atherosclerosis is caused by imbalanced lipid metabolism and represents a leading death cause in United States. Epidemiological studies showed that patients with systemic autoimmune diseases exhibit a higher incidence of atherosclerosis. Conversely, hyperlipidemia has been known to accelerate autoimmune diseases in humans and in animal models. However, there is a considerable gap in our understanding how atherosclerosis impacts the development of the autoimmunity, and vice versa. Our long-term goal is to elucidate novel cross-regulatory mechanisms governing the pathogenesis of atherosclerosis and related autoimmune diseases, which will lead to the development of novel therapeutic interventions for the treatment of these devastating diseases. The primary objective of this R01 application is to investigate the critical cross-regulation between atherosclerosis and autoimmune T cell responses with specific emphasis on Th17 responses. It is our central hypothesis that proatherogenic conditions promote autoimmune Th17 responses through T cell-intrinsic mechanisms which in turn accelerate atherosclerosis. The rationale is that identifying the cross-regulatory mechanisms will enable us to gain multi-disciplinary insights into the pathogenesis of the diseases. Guided by strong preliminary data, this hypothesis will be tested through two specific aims: 1) Determine the role of proatherogenic condition driven Th17 responses in the development of autoimmunity and atherosclerosis; 2) Determine cell-intrinsic regulation of oxidized LDL signaling in Th17 cells. Under the first aim, animal models of autoimmune lupus and atherosclerosis will be used to examine the role of proatherogenic condition-driven Th17 cells in the pathogenesis of these diseases. Under the second aim, biochemical and genetic tools will be used to dissect molecular mechanism for cell intrinsic function of oxidized LDL signaling in promoting Th17 lineage commitment. Our approach is innovative, because it employs interdisciplinary concepts and unique powerful genetic tools to examine mutual pathogenic regulation between atherosclerosis and autoimmune T cell responses by placing hyperlipidemia as an autoimmune mediator. The proposed research is highly significant, because it is anticipated to substantially advance and expand our understanding of how cardiovascular and immune systems cross-talk during the development of atherosclerosis and autoimmunity. Ultimately, such knowledge has the potential to lead to the development of novel immunologic and pharmacologic strategies for the treatment of atherosclerosis and associated autoimmune diseases.

ABSTRACT Circadian (24-hour) rhythms are an essential part of human biology and physiology. A growing number of studies have shown that disruption of our biological clock is detrimental to health, with night and rotating shift workers at substantially increased risk of developing numerous disease pathologies, including cancer, diabetes, obesity, and chronic inflammatory bowel disease. Thus, an understanding of the mechanisms by which circadian disruption are linked to disease development would be of great benefit to a growing percentage of the population subjected to circadian disruption of various forms. Impaired circadian rhythms and gastrointestinal inflammation are directly associated with several leading digestive tract disorders, including inflammatory bowel disease (IBD). While not well studied in a circadian context, complement activation and the complement anaphylatoxins, C3a and C5a, have been implicated in immune dysfunction are tightly linked to the development of numerous diseases, including asthma, cancer, diabetes, and inflammatory bowel disease. Our preliminary data indicate that the complement anaphylatoxins, which are phlogistic peptides with critical roles in host defense and the immune response, may provide the link between circadian disruption and vulnerability to diseases, including gastrointestinal disease. These data indicate that the complement anaphylatoxins (at the level of the peptides themselves as well as their specific receptors) are under direct circadian control in vivo and provide circadian modulation of intestinal lymph exchange in vivo. The overall hypothesis of this application is that the regulation of inflammation and immunity by the complement system (largely via the complement activation anaphylatoxin peptides) is greatly affected by disruption of the 24-hour circadian clock leading to dysregulation of the immune response and normal lymphatic function. Th circadian dependent complement mediated dysfunction of inflammation and immunity in turn leads to increased disease pathologies, including but not limited to the development of digestive disorders such as IBD. In support of this hypothesis, novel preliminary data are presented showing that C5a modulates the cellular content and T-cell polarization in gut lymphoid tissue in a circadian dependent manner. In addition, the expression of the complement anaphylatoxin receptors, C3aR and C5aR1, is deficient in Peyer?s patches in a model of circadian-dependent peripheral arrhythmicity. These data strongly suggest that the C3a/C3aR and C5a/C5aR1 axis is a novel and important mechanism by which circadian gating of the host lymphatic immune response occurs, and that it is a previously unknown yet important link between circadian disruption and disease pathologies, including the development of inflammatory bowel disease. Using circadian mutant models, genetic and environmental manipulation of the complement system, state of the art imaging, and molecular/bio-analytical tools we will delineate the mechanisms by which the complement anaphylatoxins modulate intestinal lymphatics in a circadian manner.