Jenny P-Y Ting - US grants

Class II major histocompatibility antigens are important in T-B cell interaction and lymphocyte activation/differentiation. These antigens are constitutively expressed by resting B cells but not T cells. Interestingly, T cells infected with the Type I human leukemia virus (HTLV-I) express class II antigens concurrent with the onset of tumorgenicity. A major goal of this proposal is to understand how HTLV-I viral product can activate HLA-DR genes. Another major goal is to examine the molecular mechanisms that result in DR expression in transformed B cell lines and normal primary B cells. We have obtained preliminary evidence that the transactivating gene product, tat-I, of HTLV-I can trans-activate DR alpha upstream promoters. The precise sequences that are responsive to tat-I activation will be mapped by gene transfer of deletion mutants. The levels (transcriptional, post-transcriptional, or translational) at which tat-I is regulating these sequences will also be determined. Previous studies in our laboratory have shown that there are two regulatory elements important for DR expression in transformed B cells. Both of these bind to nuclear proteins. In this proposal, a structure-function analysis will be performed for these two elements. In addition, in vitro transcriptional assays will be used to directly assess if the proteins that bind to these elements have functional roles. Finally, and most importantly, untransformed primary B cells will be examined to deermine if they use identical regulatory pathways as transformed B cells. These experiments are intimately related to the process of leukemogenesis. The induction of DR by HTLV-I product represents a model system to examine the regulation of a cellular gene by viral products that may be oncogenic in nature. Studies of transformed and normal B cells will allow us to determine if transformation results in altered pathways of gene regulation.

As one of the primary ligands for the T cell receptor, the Class II major histocompatibility antigens are uniquely suited to control both the differentiation and activation of T cells. Differences in Ia expression can confer unique capabilities to delete self-reactive T cells in the thymus, or to initiate lymphokine cascades in the periphery. Both of these important for immune surveillance of tumors, infectious agents and soluble antigens. Therefore our ability to modulate the immune response to a variety of antigens ultimately relies on our understanding of class II gene regulation. The goal of this proposal is study the coordinate regulation of multiple class II genes among themselves and with the invariant chain gene. Accumulative evidence suggests that class II genes and invariant gene share some homologous elements. Conversely, some class II genes also have unique regulatory elements. All of the regulatory elements can interact with nuclear proteins which are presume transcription factors. The objectives of this proposal are to determine if DNA-binding proteins that regulate one class II gene (DRalpha) also regulates various other class II genes, as well as the invariant chain gene. To achieve this, relevant DNA binding proteins will also be used. The ability of each DNA binding protein with specificity for the DRalpha gene to control multiple class II genes as well as the invariant chain gene in a coordinate fashion will be assessed. This type of functional analysis will definitely determine if class II genes are coordinately regulated due to shared specificity for DNA-binding proteins.

Class II Major Histocompatibility Complex (MHC) gene products control the acquisition of mature T cell repertoire, serve as restriction elements for CD+ T cells, ,and are receptors for differentiation signals. These multiple functions of class II MHC antigens place them in a crucial role during immune regulation. Hence our abilities to modulate the immune system ultimately will rely on the ability to modulate class II MHC gene expression. This is of pertinence to a large number of diseases with an immune component such as autoimmune, inflammatory and neoplastic diseases. The focus of this proposal is class II MHC gene regulation in lymphoid cell lines and primary blood lymphocytes. In the past few years, we and others have delineated an array of cis-acting elements important for class II gene expression and identified proteins that bind to these elements. The aims of this proposal are to address several issues that are germaine to our further understanding of class II MHC gene regulation. Accordingly, our aims are as follows: (1) To analyze the functions of proteins with specificity for two conserved elements (X and Y) found in all class II MHC promoters. This will be achieved by in vitro transcription, co-transfection or trans-dominant .suppressive mutations. (2) To determine if proteins binding to the X and Y elements interact. This is based on previous studies from our group showing that the stereospecific alignment of X and Y on the DNA helix is important for their function, implicating interactions among proteins that bind to these elements. This will be achieved by electron microscopic examination, immunochemical procedures, affinity chromatography and chemical crosslinking. (3) To determine the regulation of DRA gene in human blood T lymphocytes activated by different ligands to express class II antigens. Preliminary studies in the laboratory suggest that different activation pathways (such as engagement of the T cell receptor vs. mitogen stimulation) require different sets of regulatory elements. Similar studies will be further extended to DR- expressing T lymphocytes from inflammatory autoimmune rheumatoid arthritis to determine if their promoter element usage is similar to that of known ligands. These goals will further our comprehension of class II gene regulation at the molecular and biologic levels.

Class II Major Histocompatibility Complex (MHC) gene products control the acquisition of mature T cell repertoire, serve as restriction elements for CD+ T cells, ,and are receptors for differentiation signals. These multiple functions of class II MHC antigens place them in a crucial role during immune regulation. Hence our abilities to modulate the immune system ultimately will rely on the ability to modulate class II MHC gene expression. This is of pertinence to a large number of diseases with an immune component such as autoimmune, inflammatory and neoplastic diseases. The focus of this proposal is class II MHC gene regulation in lymphoid cell lines and primary blood lymphocytes. In the past few years, we and others have delineated an array of cis-acting elements important for class II gene expression and identified proteins that bind to these elements. The aims of this proposal are to address several issues that are germaine to our further understanding of class II MHC gene regulation. Accordingly, our aims are as follows: (1) To analyze the functions of proteins with specificity for two conserved elements (X and Y) found in all class II MHC promoters. This will be achieved by in vitro transcription, co-transfection or trans-dominant .suppressive mutations. (2) To determine if proteins binding to the X and Y elements interact. This is based on previous studies from our group showing that the stereospecific alignment of X and Y on the DNA helix is important for their function, implicating interactions among proteins that bind to these elements. This will be achieved by electron microscopic examination, immunochemical procedures, affinity chromatography and chemical crosslinking. (3) To determine the regulation of DRA gene in human blood T lymphocytes activated by different ligands to express class II antigens. Preliminary studies in the laboratory suggest that different activation pathways (such as engagement of the T cell receptor vs. mitogen stimulation) require different sets of regulatory elements. Similar studies will be further extended to DR- expressing T lymphocytes from inflammatory autoimmune rheumatoid arthritis to determine if their promoter element usage is similar to that of known ligands. These goals will further our comprehension of class II gene regulation at the molecular and biologic levels.

As one of the primary ligands for the T cell receptor, the Class II major histocompatibility antigens are uniquely suited to control both the differentiation and activation of T cells. Differences in Ia expression can confer unique capabilities to delete self-reactive T cells in the thymus, or to initiate lymphokine cascades in the periphery. Both of these important for immune surveillance of tumors, infectious agents and soluble antigens. Therefore our ability to modulate the immune response to a variety of antigens ultimately relies on our understanding of class II gene regulation. The goal of this proposal is study the coordinate regulation of multiple class II genes among themselves and with the invariant chain gene. Accumulative evidence suggests that class II genes and invariant gene share some homologous elements. Conversely, some class II genes also have unique regulatory elements. All of the regulatory elements can interact with nuclear proteins which are presume transcription factors. The objectives of this proposal are to determine if DNA-binding proteins that regulate one class II gene (DRalpha) also regulates various other class II genes, as well as the invariant chain gene. To achieve this, relevant DNA binding proteins will also be used. The ability of each DNA binding protein with specificity for the DRalpha gene to control multiple class II genes as well as the invariant chain gene in a coordinate fashion will be assessed. This type of functional analysis will definitely determine if class II genes are coordinately regulated due to shared specificity for DNA-binding proteins.

Multiple sclerosis (MS) is a debilitating central nervous system (CNS) disease that has features of autoimmunity. It has been reported that brain lesions of MS contain reactive microglia that express the immune response antigen, Ia. Ia antigens in this disease as well as in most other autoimmunities are generally thought to serve a conventional role in stimulating T cells. More recently we have begun to use the twitcher mouse as a model to study Ia in another demyelinating disease. Twitcher is a genetically-inherited demyelinating model caused by a defect in the galactosyl-ceramidase gene which results in defective galactosyl-ceramide breakdown. With time, oligodendrocytes become dysfunctional resulting in subsequent demyelination. The onset of severe demyelination is coincidental with the hyperexpression of Ia in the brains of these mice, yet T cells are not prominent in the brains of severely afflicted animals. This provides an excellent animal model to elucidate the function of CNS Ia that may be distinct from its conventional role as a T cell stimulating molecule. To directly examine the role of CNS Ia hyperexpression in the twitcher disease, we have produced Ia negative, twitcher transgenic mice. These mice lack Ia expression and also have the defective galactosyl-ceramidase gene. Our results show that the lack of Ia expression resulted in decreased neuropathology, significantly lower numbers of microglia in the CNS, and decreased twitching compared to Ia-positive counterparts. Together these results suggest that Ia is an exacerbating factor in this demyelinating model. The goals of this proposal are: (l) To determine the role of CNS Ia in other demyelinating as well as nondemyelinating neurodegenerative disease models. (2) To better define the role of T cells in the twitcher mice either by the use of reagents to deplete T cells or by the use of CD4-knockout mice. (3) To determine if Ia serves as a signalling molecule that activates brain microglia to synthesize cytokines that may be detrimental to oligodendrocytes. (4) To examine the effects of immunotherapeutic agents known to downregulate class II MHC expression on disease progression in the twitcher mice. These studies are pertinent to the Program Announcement because they will provide: (l) new demyelinating models to study the role of CNS Ia hyperexpression, (2) information on the interactions of activated glial cells and oligodendrocytes, and (3) the rationale for and the testing of specific immunotherapies that may alleviate demyelination.

Antigen presentation by class II major histocompatibility antigens (MHC) require not only the HLA-DR surface molecules, but also antigens processing molecules, DMA/DMB encoded within the HLA-D region, as well as the Ii chain protein encoded on chromosome 5. Class II mhc molecule. DMA/DBA and Ii are tightly linked in their function and most relevant to this proposal in their transcriptional control. In addition, the regulation of this group of molecules by IFN-gamma represents a novel pathway distinct from other IFN-gamma inducible systems. The purpose of this proposal is to continue the studies on the coordinate regulation of these genes, with specific emphasis on the IFN-gamma inducible regulation of this group of genes by the shared regulatory elements. W, X and Y, and by the newly described transactivator, Class II transactivator (CIITA). All class II MHC, Ii and DMA/DMB promoters contain shared regulatory elements which mediate majority of the transcriptional activities. Previous studies from our group have shown that IFN-gamma induced a gradual loading of transcription factors onto these elements. Using the in vivo footprint analysis, our laboratory has shown that binding of the NF-Y transcription factors at the Y (also a CCAAT) element is a critical event for subsequent binding at the X elements upon IFN-gamma induction. The first Aim will determine if loading at the Y element is a key step in the IFN-gamma induction of the Ii promoter, which is much more complicated and contains two separate authentic Y elements. In addition to the importance of NF-Y in IFN-gamma induced promoter loading. Our group has also found that CIITA, a potent positive regulatory identified by complementation cloning of DR-negative cell line, may also be involved in in vivo promoter loading as revealed by studies of G3B, a mutant cell line selectively defective in the IFN-gamma induction of CIITA. A more indepth analysis of this role of CIITA in promoter loading is studied in Aim 2. Published results have also shown that CIITA not only regulates class II MHC genes, but also the Ii land DMB genes. Analysis of other IFN-gamma inducible genes that are regulated by CIITA will be studies in SAim 3. Finally, identification of the basic defect in G3A, which leads to the lack of CIITA induction by IFN-gamma will be identified by complementation cloning.

Description (adapted from applicant's abstract): The primary purpose of this proposal is to examine the role of class II MHC antigen and its associated molecules (H-2M and Ii) in allogeneic and/or xenogeneic transplantation involving keratinocytes and islet cells. These studies stem from the applicants' observation that keratinocytes isolated from class II MHC defective mice failed to prime for an allogeneic response from the host. These studies have motivated an examination of the role of other proteins in allogeneic transplant rejection or acceptance. Specifically, the investigators wish to test three mice strains: mice defective in the IA(b)[beta]gene, the H-2M molecule (which is necessary for peptide loading onto class II MHC proteins) and CIITA, a master transcriptional regulator essential for trans-activation of class II MHC genes, Ii chain, and the H-2M genes. Using keratinocytes and islet cells derived from these three mice, the applicants wish to test their acceptance as allografts. In addition, they wish to test if pre- transplantation with keratinocytes that are defective in class II MHC antigens can result in tolerance induction which can enhance the successful implant of allogeneic islet cells. Accordingly, the following aims will be pursued. First, immunologic priming by IA(b)[beta](-/-), H-2M(-/-), and CIITA(-/-) keratinocytes will be compared. The capacity of cells isolated from these animals to prime an allo- and xenoresponse will be assessed. Secondly, the acceptance of islet allografts from IA(b)[beta](-/-), H-2M(-/-), and CIITA(-/-) animals will be compared to grafts from wild type controls. The capacity to restore normal glycemia will be examined. Finally, the failure of keratinocytes from IA(b)[beta](-/-) allografts suggests that these cells may have caused immunologic tolerance. Direct studies to examine if this is the case are planned. An extension of these experiments will be to determine if mice transplanted with IA(b)[beta](-/-) keratinocytes are more tolerant of I(A(b)[beta](-/-) allogeneic islet cells. Similarly, if the experiments with H-2M(-/-) or CIITA(-/-) are encouraging, they also will be tested to determine if they can induce prolonged acceptance of islet cells. Together these experiments should shed light on the biological function of class II antigens as well as molecules involved in class II presentation in transplants that are relevant to a large proportion of patients with severe unhealed wounds, burns, or diabetes.

Class II Major Histocompatibility Complex (MHC) gene products control the acquisition of mature T cell repertoire, serve as restriction elements for CD+ T cells, ,and are receptors for differentiation signals. These multiple functions of class II MHC antigens place them in a crucial role during immune regulation. Hence our abilities to modulate the immune system ultimately will rely on the ability to modulate class II MHC gene expression. This is of pertinence to a large number of diseases with an immune component such as autoimmune, inflammatory and neoplastic diseases. The focus of this proposal is class II MHC gene regulation in lymphoid cell lines and primary blood lymphocytes. In the past few years, we and others have delineated an array of cis-acting elements important for class II gene expression and identified proteins that bind to these elements. The aims of this proposal are to address several issues that are germaine to our further understanding of class II MHC gene regulation. Accordingly, our aims are as follows: (1) To analyze the functions of proteins with specificity for two conserved elements (X and Y) found in all class II MHC promoters. This will be achieved by in vitro transcription, co-transfection or trans-dominant .suppressive mutations. (2) To determine if proteins binding to the X and Y elements interact. This is based on previous studies from our group showing that the stereospecific alignment of X and Y on the DNA helix is important for their function, implicating interactions among proteins that bind to these elements. This will be achieved by electron microscopic examination, immunochemical procedures, affinity chromatography and chemical crosslinking. (3) To determine the regulation of DRA gene in human blood T lymphocytes activated by different ligands to express class II antigens. Preliminary studies in the laboratory suggest that different activation pathways (such as engagement of the T cell receptor vs. mitogen stimulation) require different sets of regulatory elements. Similar studies will be further extended to DR- expressing T lymphocytes from inflammatory autoimmune rheumatoid arthritis to determine if their promoter element usage is similar to that of known ligands. These goals will further our comprehension of class II gene regulation at the molecular and biologic levels.

OVERALL DESCRIPTION (adapted from applicant's abstract): The long-term and overall goal of this program project is to use molecular genetic as well as immunological approaches to enhance the acceptability of cellular transplants. Specifically, the applicants will focus on two models of transplantation: keratinocyte and islet transplants. The former is important for a large number of patients suffering from unhealed wounds as well as severe burns, while the second has increasingly been considered for treating diabetic patients. However, both procedures are frequently plagued by graft rejection. The central goal of these three projects is to determine if tolerance can be induced using a combination of genetic manipulations and gene therapy approaches. Accordingly, the first project plans to examine the role of class II MHC genes as well as associated antigen presenting molecules in triggering immune response against keratinocyte as well as islet cell transplants. Specifically, gene knockout (GKO) mice bearing defects in various stages of class II antigen presentation will be used as donor tissues to assess the role of these molecules in both allogeneic and xenogeneic transplants. In addition, cells derived from GKO mice will be tested for the capacity to induce tolerance. In a second project, focus will be placed on the NOD mouse model with specific emphasis on the utilization of "immune deviation" to enhance tolerance and to increase transplant acceptance. Specifically, the generation of T cells that are tolerant to pancreatic antigens will be tested. In addition, a gene therapy approach is proposed to use deoxyribonucleic acid (DNA) vaccines to introduce pancreatic genes to tolerize animals, in order to enhance the acceptance of islet cell transplants. In the final project, the focus is a model where tolerance against non-specific peptides can lead to specific tolerance of transplanted tissues. Focus will be placed on peptides that bind class I MHC molecules which are frequently a major culprit in transplant rejection. Together, these three projects address different aspects of induction tolerance to two specific cellular transplants. Significant collaborations and synergy are planned.

transplant rejection; MHC class II antigen; enzyme activity; cyclic AMP; kidney transplantation; gene expression; genetic strain; prostaglandins; heart transplantation; phosphorylation; homologous transplantation; gene targeting; genetically modified animals; laboratory mouse;

DESCRIPTION (provided by applicant): MHC class II molecules are expressed in the central nervous system (CNS) during a number of neurologic disorders and demyelinating disease. In a majority of these diseases, T cells are generally absent in the affected CNS. To address the importance of class II MHC in CNS disorders, we have characterized two models of demyelination that exhibit class II MHC hyper-expression on microglial cells. Characterizations of these disease models show that the deletion of class II MHC caused the alleviation of overt symptoms, reduced neuropathology/demyelination, reduced microglial activation/proliferation and reduced production of inflammatory cytokines. Most intriguingly, the role of class II MHC is unaffected by the absence of T cells in RAGb mice, but dependent on an intact class II MHC cytoplasmic tail. This caused us to propose that class II may serve an in vivo role in signal transduction, distinct from its conventional biologic role in antigen presenting. The Aims are: 1.Analyze a transgenic mouse strain, H-2M for its response to cuprizone treatment. If conventional antigen processing is not important, then the deletion of H-2M should not affect cuprizone-induced demyelination. 2.Analyze how class II MHC and T cells affect the remyelination process. Recent studies in our laboratory have demonstrated that inflammatory cytokines are necessary for optimal remyelination. The presence of MHC class II causes enhanced cytokine expression, therefore it is timely to determine the role of MHC during remyelination, and if lymphocytes are involved in this process. 3. Determine novel mediators of class II MHC-mediated signaling. We will determine if recently discovered mediators of class II signaling, cell activation and proliferation are affected in microglia/macrophages. 4. Identify genes that are activated upon class II MHC engagement. in a microglial-macrophage line by Affy metrix screening, and assess the status of these genes in the cuprizone model.

The class II transactivator (CIITA) is a transcriptional master regulator of class II MHC genes, discovered by complementation cloning of an HLA-D-negative mutant cell line. Defective CIITA leads to a type of severe immunodeficiency in humans, the Group A, type II Bare Lymphocyte Syndrome (BLS). CIITA controls the expression of conventional class II MHC genes, as well as DM, Doalpha and Ii genes. It is a potent activator which causes the activation of class II MHC promoters by up to 100 fold. It controls promoters which contain class II MHC promoter elements, W, X and Y, yet it is not a DNA-binding protein. The mechanism by which CIITA activates class II MHC promoters and its novel target genes has been, and continues to be, the focus of this proposal. Experiments performed during the last grant period have shown that CIITA operates by interaction with the DNA- binding transcription factors, NF-Y and RFX, which control class II MHC promoters. Additionally, CIITA opens class II MHC promoters in vivo as determined by genomic footprinting. The purpose of the first half of this proposal is to understand how CIITA control class II MHC genes at the molecular level. The first two Aims continue to investigate how CIITA alters gene expression, and they are: (1) To further examine a HAT-like domain within CIITA which is associated with histone acetylase activity; (2) to determine if CIITA alters other aspects of chromatin opening by studying changes in intact cells. The purpose of the second half of the proposal is to understand if CIITA controls other genes, how this control is executed, and if these target genes have relevant biologic functions. During the last grant period, we show that CIITA may control other genes and biologic processes as revealed by differential gene screen analysis and by comparing primary cells obtained from CIITA gene knockout mice with wildtype controls. To continue this line of inquiry, the last two Aims are (3) to understand the molecular mechanism by which CIITA controls non-class II MHC genes; and (4) determine if these genes are relevant in antigen presenting cells.

DESCRIPTION: (provided by applicant) The "Class II MHC Gene Control and Disease Relevance" conference will focus on recent advances in class II MHC gene control, and its applicability to multiple immunologic-diseases. This is a timely conference because many recent advances have been made in the regulation of class II MHC expression. In addition, the applicability of this knowledge to manipulate diseases where class II MHC plays an important role seems imminently feasible. A meeting that merges interests in class II gene control and disease control is long overdue since the last meeting that was somewhat relevant to these topics was held in Madrid in 1996. The field of class II MHC gene control has undergone an explosion of knowledge, which is relevant to a wide variety of clinical diseases, including autoimmunity, microbial infections, transplantation, immunodeficiency, immunologic and neurologic-inflammatory diseases and cancer development and therapy, to name a few prominent examples. All the major transcriptional regulators of class II MHC genes are now identified. Several of the gene products are missing in different subgroups of the Bare Lymphocyte Syndrome or in vitro generated mutant cell lines. These include the RFX-5, RFXAP, RFXB-RFXANK factors. In addition, a master switch that controls all class II MHC genes, the class II transactivator (CIITA) has been isolated, and has high relevance in many diseases. The mechanism of how class II MHC genes are controlled are becoming increasingly clear, where CIITA serves as a focal point for other transcription factors. Interfering with these interacting sites provide great potentials for manipulating class II MHC gene expression, and disease outcome. This meeting is intended to bring immunologists in two divergent areas (diseases or disease models and MHC gene control) to stimulate new approaches to control a divergent number of diseases where MHC plays a critical role.

Description (provided by applicant): The class II transactivator (CIITA) is a master transcriptional regulator of all class II MHC genes. It is not a DNAbinding transcription factor, but functions through interactions with DNAbinding transcription factors which recognize the class II MHC promoter. During the last granting period, we showed that CIIT A null (CIIT A-/-) mice ack class II MHC expression in virtually all cells. The elimination of CIITA caused donor cardiac grafts to survive twice as long as grafts lacking the class II MHC gene, A-beta. These experiments suggest that CIITA may provide an intervening point for prolonging graft survival. In the last granting period, we also found that the control of CIITA expression and regulation is extremely complex. The composite results from the field indicate that CIITA expression is governed by at least three promoters, resulting in the production of three isoforms. These promoters as well as isoforms have tissue- and cell- specific expression pathways. Based on these previous findings, this proposal plans to address three seminal issues: (1) What are the in vivo functions and expression profiles of each of these isoforms, and how does each contribute to graft rejection? This is being addressed by producing transgenic mice bearing various CIITA isoforms. (2) What are the molecular targets of each of these isoforms? Do they have different effects on global gene expression? This is being addressed by Affymetrix gene array analysis. (3) What are the biochemical and biophysical natures of these three isoforms? Can this information assist us in the design of small peptides that may block the function of CIITA, and therefore enhance the survival of allogeneic grafts?

Inflammation plays a crucial role in both acute pneumonitis and chronic pulmonary fibrosis induced by irradiation. Immune intervention represents a viable approach to alleviate damages associated with radiation-induced injury. However to devise a successful strategy, it is necessary to understand the roles of key innate immune receptors and sensors during radiation. This understanding is required to for the rational selection of appropriate immune modulators for treating radiation-associated damages. During the last grant period, one of our goals was to focus on key immunologic regulators and their role in radiation-induced damage. These regulators included (a) MyD88 (myeloid differentiation factor 88), the common adaptor molecule for Toll-like receptors (TLR) and (b) NLRP3, a member of the NBD-LRR (NLR) gene family that is now recognized as a key component of the inflammasome complex that controls caspase-1, IL-1ß and IL-18. The TLR and NLR pathways respectively are considered by some as extracellular and intracellular pattern recognition sensors important in the control of innate immunity and inflammation. In contrast to our initial expectation that MyD88 and NLRP3 would exacerbate inflammation, we and other labs have increasingly found that during tissue injury, both molecules serve as homeostatic factors important for tissue repair. In specific, we found that MyD88 and NLRP3 protect against injury sustained upon radiation damage in the lung, and both gene products are critical for minimizing fibrosis and restoring lung function. This is consistent with reports showing that flagellin, a TLR5 agonist, can protect against pulmonary radiation. These data suggest a paradigm shift in how we should view MyD88 and NLRP3 in the context of pulmonary radiation and suggest that both pathways are beneficial in attenuating the damage caused by radiation injury. Hence therapeutic agents that can activate these pathways might protect against radiation damage. Furthermore, since the TLR and NLR pathways are also required for protect host response during infection, these two molecules might protect against a dual exposure to radiation and infectious microbes associated with pandemic and opportunistic infections. Accordingly the Aims are: (1) to assess the use of non-flagellin TLR and NLR agonists as deliverable therapeutics for pulmonary radiation-induced injury; (2) to assess the mechanisms by which MyD88 and NLRP3 serve as protective factors during pulmonary radiation; and (3) to determine the roles of MyD88 and NLRP3 during dual exposure to radiation and infectious agents of immense public health impact, influenza virus and K. pneumoniae.

Transplantation acceptance or rejection is determined by the cumulative effects of innate as well as adaptive responses to the foreign graft, however, the role of innate immunity in transplant rejection had been less studied than adaptive immunity. A focus on innate immunity is timely due to the discovery of Toll-like receptors (TLR's) in mammalian cells, which has revolutionized the field. The focus of this proposal is a member of a new family of genes called the CATERPILLAR family. Members are defined by shared motifs, including the caspase activation and recruitment domain (CARD), transcriptional enhancement domain, purine-binding domain, pyrin, and leucine rich domain. Others have also called this the NALP or NOD family. One of the CATERPILLAR genes, which we call Monarch-1, is only expressed by myeloid and monocytic cells. Its expression in primary monocytes is abrogated upon TLR activation through various agonists. When over-expression in non-monocytic/myeloid cell lines, Monarch-1 represses NF-kB/AP-1 activation. Thus it may be a negative regulator of inflammatory responses. In transplantation, negative regulators may be important for enhancing transplant acceptance. The goal fo this proposal is to further explore the role of Monarch-1 in myeloid/monocytic cells, and in cardiac and kidney transplant rejection. The Aims are: (1) To produce a large number of myeloid-monocytic cell lines which lack Monarch-1 expression by the RNA interfenence (RNAi) technology. (2) To delineate the role of Monarch-1 in these cell lines in the activation of NF-KB and AP-1, in cytokine synthesis, and in overall gene expression profile. (3) To characterize Monarch-1 expression during kidney and cardiac transplantation. (4) To characterize the role of Monarch-1 in kidney and cardiac transplanttation by producing and using mice that lack the Monarch-1 gene.

DESCRIPTION: Periodontal diseases are associated with pathogenic bacteria which colonize the subgingival area. Inflammatory responses mediated by cells of the innate immune system are critical determinants of periodontal diseases. One of the most prominent bacteria is Porphyromonas gingivalis (Pg). Periodontitis is thought to result from host tissue injury caused by the response of cells of the innate immune system, namely monocytes/macrophages and granulocytes. In recent years, the Toll-like receptors (TLR) have rapidly emerged as a dominant route by which the innate immune system recognizes microbial pathogens. TLR activation requires a host of intracellular adaptor proteins proximal to the TLRs, including MyD88, TRAF6, MD-2 and TRAM. More recently, we have identified a family of proteins that further modulate TLR signaling, called the CATERPILLER proteins. Two of these, Monarch-1 and CIAS/cryopyrin, are novel inhibitors of NF-kappaB, AP-1 and cytokine production. Nevertheless, the function of these two proteins is dependent on a modifier protein ASC (Apoptotic Speck protein with a CARD), which subverts their negative function, and causes a pro-inflammatory phenotype. The purpose of this application is to understand the roles of TLRs, their adaptors, CATERPILLER proteins and ASC during a Pg infection. Comparisons of host response to Pg vs. E. coli will be made to assess if Pg elicits unique host responses. Accordingly, the Aims are: 1) to determine which TLR molecules and their downstream mediators are important to signal host responses to Pg vs. E. coli. This will be achieved by using RNA interference technology to target human TLRs and TLR adaptor genes; we will determine if the removal of these genes causes alterations in host response to Pg as measured by cytokine responses, and by gene expression profile. 2) Monocytic/macrophage cell lines that lack Monarch-1 will be similarly tested as in 1). Since Monarch-1 appears to be an inhibitory molecule of the NF-kB and AP-1 pathway, and can modulate cytokine production, we posit that the absence of Monarch-1 may favor a more vigorous pro-inflammatory response. 3) Cell lines that lack or overexpress CIAS/cryopyrin, another modulator of inflammatory response will be similarly tested. 4) Cell lines with reduced ASC will also be tested. Since ASC can overcome the negative regulatory function of Monarch-1 and CIAS, ASC may enhance immune response to Pg to contain the infection.

DESCRIPTION (provided by applicant): Tuberculosis is a global health threat, and the emergence of Multiply Drug Resistant (MDR) Mycobacterium tuberculosis (Mtb) strains makes the situation even more alarming. Because of the potential bioterror threat it poses, MDR Mtb is classified as a Class C priority pathogen by NIAID. Means to enhance host response to vaccines and to Mtb infection are central to the control of a catastrophic MDR TB infection. A thorough understanding of the immune response, specifically the role innate and adaptive immunity plays in host protection against Mtb, should facilitate improvement of vaccines for MDR and drug-sensitive Mtb. The focus of this proposal is to study Mtb and determine the roles of TLR and their adaptors, and a new class of genes (CATERPILLER/NOD) that modulates the outcome of TLR activation. The focus on human primary cells is of special relevance to enhancing immunity to Mtb in humans. To understand the interplay between TLR, CATERPILLER and Mtb: 1. We have used, and will continue to use the small interference RNA (siRNA) technology to target human TLR and TLR adaptors for reduced or ablated expression. We will determine if the removal of TLRs and/or their adaptors causes alterations in host responses to Mtb. This will be studied in transformed cell lines and primary macrophage/dendritic cells. Critical data will be reproduced using MDR TB. If a specific adaptor of TLR activation is found to be important, analysis of mice lacking that adaptor will be studied in the future. 2. Preliminary data indicate that Mtb causes the suppression of Monarch-1 gene expression, a negative regulator of NF-KB/AP-1 activation and proinflammatory responses. In reverse, we also found that Monarch-1 inhibits the TLR and NF-kappaB pathways, leading to a reduction of inflammatory cytokines during Mtb infection. We will profile innate and adaptive immune genes in cells containing siRNA targeting Monarch-1 expression to assess if this protein is a negative regulator of host response to Mtb in cell lines and primary cells. In addition, we will use Monarch-1-null mice to assess how the removal of Monarch-1 alters host response. Crucial experiments will be reproduced with MDR TB. 3. Finally, ASC is a modifier of Monarch-1 function. Its removal in monocytic cells resulted in enhanced cytokine response in response to Mtb. It is also thought to control apoptosis in bacteriainfected macrophages. This Aim will examine the role of ASC in host response by studying cell lines and primary cells during an Mtb infection. Crucial experiments will be reproduced with MDR TB. If ASC alters host response to Mtb, future experiments will be planned to study an ASC-null mouse.

DESCRIPTION (provided by applicant): CIITA (class II transactivator) is the master transcriptional activator regulator of both classical and nonclassical (DM and DOa) MHC-II genes. Mutations within this gene form the genetic basis for the immunodeficiency, type II group A Bare Lymphocyte Syndrome (BLS). CIITA functions by interacting with transcription factors that directly bind the MHC-II promoter such as RFX/CREB and NF-Y. CIITA is an early step of promoter loading, and is required for subsequent epigenetic changes including the recruitment of histone acetylases and methylases to MHC-II promoters. In addition to MHC-II, we recently identified Plexin A1 (Plxna1) as a novel gene that is also regulated by CIITA. This was accomplished by profiling cDNA isolated from primary dendritic cells (DC) obtained from CIITA-deficient mice versus wildtype mice. Plexins comprise a large gene family and are considered the receptor for semaphorin family members. They were initially identified in neurons as important for neuronal guidance and axonal growth and serve as either retractive or attractive signals to guide axonal extension. We use short-hairpin RNA (shRNA) to block Plxna1 in DCs and show that Plxna1 is important for antigen-specific T cell stimulation. Plxna1 shRNA blocks the ability of DCs to stimulate T cells by >80%. Plxna1 is not involved in peptide processing or antigenic-peptide binding. Preliminary data suggest it functions in Rho but not cdc42 or Rac activation, and is important in actin polarization. Additional data suggest that Semaphorin 6D (Sema6D) is a candidate T cell ligand for Plxna1 on DC. We elect to study three major aspects of the Plxna1 and Sema6D pair in primary DC and T cells: (1) The transcriptional regulation of Plxna1 by CIITA, by other transcription factors and by histone modifying enzymes; (2) the functional effects of Plxna1 on the Rho GTPase pathway; and (3) the interaction of Sema6D on T cells and PlxnA1 on DC, and the T cell signaling pathway that is activated upon Sema6D engagement. PUBLIC HEALTH RELEVANCE: This project focuses on two new molecules, Plexin-A1 and Semaphorin 6D, that control T lymphocyte activation. Plexin-A1 and Semaphorin 6D are important for the optimal activation of T cells by dendritic cells. T lymphocyte activation is central in immunology, and is important in autoimmunity, infectious diseases, vaccine development among other clinically-relevant issues.

We recently discovered the CATERPILLER family which share structural similarities with the NB-LRR (nucleotide-binding, leucine-rich repeat) super-family of disease resistance (R) proteins that constitutes the plant immune system. In the animal kingdom, this family is also known as NOD or NLR. The clinical importance of this family is underscored by the genetic linkage of family members to a number of immunologic disorders. Among the human gene family members, several of these appear to mediate negative regulatory function in controlling an overzealous inflammatory response. Most notable is the Monarch-1 protein which blocks the function of NF-kB inducing kinase (NIK). Inhibition of NIK reduces the expression of an array of chemokines with relevance in asthma. Gene profiling of induced sputum from mildly asthmatic individuals suggests that the Monarch-1 gene is reduced in these individuals relative to controls, supporting the inhibitory role of this gene during inflammation. Another group of family members regulates IL-1 production. Most notable among these is cryopyrin which mediates formation of the inflammasome complex upon stimulation with a number of inducers. The inflammasome complex is required for procaspase 1 processing to mature caspase 1. In turn, caspase-1 is required for the processing of pro-IL-1 and pro-IL-18 to their mature forms. IL-1 and IL-18 are respectively important in inflammation and TH2 skewing. Cryopyrin is also important in mediating macrophage necrosis which exacerbates inflammation. Thus there are compelling reasons to believe that Monarch-1 and cryopyrin have crucial roles in asthma, however there is no in vivo data to indicate that this is the case. Furthermore we have shown that both of these proteins are ATP-binding proteins, and they exhibit ATPase activity, thus providing ways to modulate their function, which might be important leads to drug discovery. The goals of this proposal are: (1) To study the relevance of Monarch-1 in three animal models of asthma (OVA-induced, endotoxin, and house dust mite and delineate the biochemical effects of Monarch-1 in vivo and ex vivo. (2) To study the relevance of cryopyrin and a cryopyrin-adaptor (ASC) in asthma. (3) To study and identify factors which modulate the nucleotide-binding properties and ATPase function of Monarch-1 and cryopyrin.

2 Dimensional Gel Electrophoresis; APS Brand of Allopurinol; Abscission; Adenock; Affect; Allo-Puren; Allozym; Allural; Aloprim; Aloral; Alositol; Anoprolin; Anzief; Appendix; Apulonga; Apurin; Apurol; Azupharma Brand of Allopurinol; BASF Brand of Allopurinol; Bacteria; Bacterial RNA; Binding; Binding (Molecular Function); Bleminol; Blood Coagulation Factor IV; Bloxanth; Boehringer Mannheim Brand of Allopurinol; Boots Brand of Allopurinol; CSIF; CSIF-10; Ca++ element; Calcium; Caplenal; Cell Communication and Signaling; Cell Line; Cell Lines, Strains; Cell Signaling; Cell-Death Protease; CellLine; Cellidrin; Cells; Coagulation Factor IV; Complex; Cosuric; Cytokine Synthesis Inhibitory Factor; Cytokine formation-inhibiting factor (mouse clone F115 protein moiety reduced); DIF; Dabroson; Development; Dorsch Brand of Allopurinol; EC 2.7; Electrophoresis, Gel, 2-D; Electrophoresis, Gel, 2D; Electrophoresis, Gel, Two-Dimensional; Embarin; Epidropal; Excision; Extirpation; Factor IV; Family; Family member; Flagellin; Foligan; Future; Geapur; Gene Family; Gene Targeting; Genus Mycobacterium; Gichtex; Glaxo Wellcome Brand of Allopurinol; Hamarin; Hennig Brand of Allopurinol; Henning Berlin Brand of Allopurinol; Hexanurat; ICE-like protease; IL-10; IL10; IL10A; Immune response; Immunity; Immunity, Innate; Immunity, Native; Immunity, Natural; Immunity, Non-Specific; Infection; Inflammatory Response; Interleukin 1 Signal Transducer; Interleukin 10 Precursor; Interleukin-10; Intracellular Communication and Signaling; Ketanrift; Ketobun-A; Kinases; L-Leucine; LMP1-Associated Protein 1; Ledopur; Leucine; Leucine, L-Isomer; Ligands; Lopurin; Lysuron; MEFV gene product; MGC[{..}]45012; Mass Spectrum; Mass Spectrum Analysis; Mediator; Mediator of Activation; Mediator of activation protein; Membrane; Merckle Brand of Allopurinol; Miniplanor; Modeling; Molecular; Molecular Interaction; Monarch; Multipharma Brand of Allopurinol; Mycobacterium; Myelogenous; Myeloid; Names; Natural Immunity; Nektrohan; Nicholas Brand of Allopurinol; Outcome; Pan Quimica; Pasteurella pestis; Pathogenicity Factors; Pattern; Pattern Recognition; Pattern Recognition/Display/Analysis; Pattern recognition receptor; Phosphotransferases; Photometry/Spectrum Analysis, Mass; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Proteins; Proteoglycan; Purines; Quelling; RNA Interference; RNA Silencing; RNA Silencings; RNA, Bacterial; RNAi; Range; Receptor Activation; Receptor Protein; Remid; Removal; Rh?'ne-Poulenc Rorer Brand of Allopurinol; Riball; Roche Brand of Allopurinol; Role; Sequence-Specific Posttranscriptional Gene Silencing; Signal Transduction; Signal Transduction Systems; Signaling; Site; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Structure; Surgical Removal; Suspendol; TAD Brand of Allopurinol; TIL4; TLR protein; TLR2; TLR2 gene; TNF; TNF A; TNF gene; TNF receptor-associated factor 2; TNF receptor-associated factor 6; TNFSF2; TRAF-6 Protein; TRAF2; TRAF2 gene; TRAF6; TRAP3; Takanarumin; Targetings, Gene; Teichoic acid, lipo-; Toll-Like Receptor Pathway; Toll-like receptors; Toll/Interleukin 1 Receptor-Like 4 Gene; Transphosphorylases; Tumor Necrosis Factor Gene; U937 Cells; Urbol; Uricemil; Uripurinol; Urosin; Urtias; Vaccination; Vermiform Appendix; Viral; Virulence; Virulence Factors; Wellcome Brand of Allopurinol; Work; Xanturat; Y. pestis; Y.pestis; Yersinia; Yersinia pestis; Zyloprim; Zyloric; biological signal transduction; caspase; cultured cell line; cystein protease; cystein proteinase; cysteine endopeptidase; cytokine; fungus; gene product; gepepharm Brand of Allopurinol; host response; immunoresponse; inhibitor; inhibitor/antagonist; interest; lipoarabinomannan; lipoteichoic acid; macrophage; marenostrin; membrane structure; novel; pathogen; purine; pyrin; receptor; resection; response; small molecule; social role

A key component in the systems biology approach to the study of asthmatic disease is the profiling of transcriptome and immune mediators. Over the last decade, our group has published on the utilization of various cDNA profiling technology including microarrays from commercial sources, such as Affymetrix and Agilent, and technology developed custom to the lab, such as representational difference analysis, differential display analysis, and serial analysis of gene expression (SAGE). Recently due to our need to include several novel genes that we found which are not represented on any commercial arrays, we have established our own custom arrays focusing on genes important in immune and oxidative responses. In addition, we have also investigated several gene network- pathway programs to support data analysis. The main goal of this core is to extend our expertise to investigators in this U19 Project, and to provide a userfriendly, one-stop facility for the broad analysis of cDNA and protein arrays relevant to immunity and oxidative stress, two issues of prominence in this proposal. The goals are: 1. To provide validated, sensitive and standardized human and mouse cDNA profiling technology for U19 investigators. 2. To provide help in the usage of broad human and mouse cytokine/chemokine and immune protein arrays. 3. To assist in the bioinformatics analysis of data from the cDNA and protein arrays;the Core will provide access and assistance in using computer programs to analyze the data, and pathway programs to assist in the interpretation of data to understand its biological significance.

A major conceptual advance for innate immunity has been the discovery of Pathogen-associated Molecular Pattern recognition receptors or sensors, a prime example being the toll-like receptor (TLR). More recently, we and others discovered a large family of pathogen sensors in humans and mice, called the NLR (nucleotide-binding domain, leucine-rich repeat containing) family, known previously as CATERPILLER, NACHT-LRR, or NOD-like receptor. An important function of NLR proteins is the formation of a biochemically-defined complex, called the inflammasome, which processes pro-caspase-1 and pro-IL-1b/IL- 18 to their mature forms. In addition to this role, some NLRs have a profound effect on T cell responses. This creates a new paradigm where NLRs can also influence adaptive immunity. Additional studies indicate that NLR proteins mediate type I interferon (IFN) production in response to viruses through an interference of the intracellular pathway mediated by the mitochondria! anti-viral signaling molecule (MAVS, a.k.a. IPS, VISA and CARDIF). This proposal will examine the divergent roles of NLR during flavivirus infection. Flaviviruses such as Dengue (DENV) and West Nile (WNV) have surfaced at the forefront of biodefense and emerging infections. Type I IFN is pivotal in anti-flaviviral host defense, and the intracellular pathway of viral RNA recognition is crucial for this response during DENV and WNV viral infection. Of equal importance, IL-1 Droduction has been observed by several groups as being a crucial outcome of human infection by Flaviviruses. Finally, adaptive immunity is unquestionably of importance to anti-flaviviral host response. However, the link between NLR members to any of these anti-viral host responses has not been explored. In this proposal, we plan to explore the roles of different NLRs in eliciting interferon response, inflammasome :unction, and adaptive immunity upon infection by WNV and DENV.

DESCRIPTION (provided by applicant): This Is a competing continuation for our current training program in immunology that has been continuously funded since 1984. The program is a combined pre- and postdoctoral training program in immunology. Predoctoral students will receive the Ph.D. at the end of the training. Since the faculty have increased we believe that we have both the applicant pool and the training opportunity to support 5 predocs. In addition we are requesting 1 additional slot, to be funded at NC A&T. This training program is primarily based in the Department of Microbiology and Immunology and laboratories are located in the Lineberger Comprehensive Cancer Center, the Department of Dermatology, Department of Neurology, the Dental Research Center (Oral Biology Program), Genetics and Molecular Biology, the Neurosciences Center and the Department of Medicine. The training for both pre- and postdoctoral fellows will take place in those laboratories. This training program is particularly strong in the areas of immunogenetics and regulation of the immune response. The research costs of the trainees are born by the research grants of the training faculty and research performed by the trainees normally appears as a joint author with the training faculty members. Physicians, veterinarians, and Ph.D.s in other disciplines will be recruited as postdoctoral trainees. Serious effort will be made to attract physician trainees by publicity among training programs in internal medicine, and in rheumatology. Postdoctoral training will be individual and, depending on the trainee, may consist of a combination of course work as well as laboratory research. Training may occur in more than a single laboratory. RELEVANCE: We are requesting funds to continue training of immunologists. A unique feature of this proposal is the close partnership with NC A&T for the recruitment of minority scientists into PhD programs. This program will aid in not only development of the next generation of scientists and furthers the future diversity of science.

Immunology The goals of the Immunology Program are to promote basic and translational science focusing on cancer immunobiology and to promote an environment conducive to intra and interprogrammatic collaborations. These goals are achieved by the recruitment and retention of outstanding basic and translational scientists, and the promotion of interactions with clinicians in the NC Cancer Hospital. The 25 members' research is organized into two major themes, innate and adaptive immunity. Within each of the themes are multiple subthemes with the innate immune theme focusing on cancer and inflammation with a spot light on colitis and colorectal cancer and hepatitis and hepatocellular carcinoma. Additional subthemes include dendritic cell biology, interactions between immune response and radiation biology, and novel pattern sensing receptors. The adaptive immune program is divided into three subthemes that include vaccine development, graft-versus-host biology and the role of migratory proteins in the interaction between T and B lymphocytes, tumor cells and the stroma. Research highlights include: (1) completion of the first clinical trial combining standard therapy with vaccine treatment for patients with breast cancer; (2) identification of the first mitochondrial NLR protein that recognizes viral nucleic acid; (3) preclinical development of an IKK inhibitor for the prevention of GvHD; (4) identification of novel pathways in which chemokines interact with migrating stromal cells to promote pulmonary metastasis; (5) new models and Insights into the interaction of innate immune cells as critical mediators of carcinogenesis in the Gl tract; and (6) the development of novel humanized mouse models to explore the interaction between the human immune system and the development of cancer. The program addes value to the Cancer Center by promoting preclinical studies into clinical therapies, forging collaborations between scientists, generating new collaborations between scientists and clinicians via retreats and monthly meetings, and providing funding support for novel translational and basic investigations. The program is led by Dr. Jenny Ting, Ph.D., a leader in innate and molecular immunology and Dr. Jonathan Serody, M.D., a leader in cellular immunology, In vivo imaging and vaccine development. In 2009 the program's 25 members had extramural funding totaling $16.2M (total costs).

ABSTRACT: Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in the United States. Two important risk factors for CRC are a history of chronic colitis or inflammatory bowel disease (IBD) and obesity, both of which are increasing at an alarming rate worldwide. However, the mechanisms linking these diseases to CRC are not well understood and thus there is a pressing need to define them. The NLR (NOD-like receptor or NBD-LRR) is a multi-member gene family that encodes a group of cytosolic proteins that are involved in the intracellular sensing of microbial products as well as damage-associated molecular patterns. NOD2, an NLR family member, has a strong genetic association with Crohns' disease and has been implicated in colitis-associated CRC (CAC). Additionally, NLRs including NOD2 and NLRP6 can affect the microbiome to impact colitis in mice, suggesting that family members are important in maintaining or disrupting the homeostasis of gut microbiome. We and others have shown a role for the inflammasome in models of IBD and CRC. This group of proteins respond to pathogen derived products and can assemble into inflammasomes in conjunction with the key NLR adaptor protein, ASC (apoptotic speck containing protein with a CARD) to activate the IL-1? and IL-18-processing enzyme caspase-1. Caspase-1 activation then leads to the cleavage and maturation of pro-IL-1beta and IL-18. In addition to the analyses of these well-studied inflammasome components in models of IBD and CRC, we and others have shown a strong role for other NLRs which have anti- inflammatory functions (referred to as regulatory NLRs), and can attenuate the clinical outcome in animal models of colitis and inflammation-associated colon carcinoma. Furthermore, recent findings in our lab showed that these regulatory NLRs play a protective role in spontaneous CRC in a genetic model of colon cancer. This proposal plans to examine the roles of inflammasome and regulatory NLRs in human and murine models of gastrointestinal cancer.

The NLR (nucleotide-binding domain, leucine-rich repeat protein, also known as NOD-like receptor) family is a group of cytosolic proteins that detect intracellular microbes. Within this gene family, the N0D2 gene has received the most attention due its genetic association with inflammatory bowel disease (IBD). Furthermore, we and others have shown that inflammasome-associated NLRs such as NLRP3 and NLRP6, as well as inflammasome components can protect against experimental colitis and alter the microbiome, indicating a more expanded relevance of this family with colitis. Most recently a subgroup of NLRs is found to inhibit NFkappaB activation and impact experimental colitis. We and others showed that NLRP12 reduces colitis by suppressing NF-kappaB activation. This indicates for the first time that NLRs can attenuate colitis by negatively impacting NF-kappB. NLRP12 suppresses inflammation by causing the proteasome-mediated degradation of NIK (NF-kappaB inducing kinase), which is critical for non-canonical NF-kappaB activation. Others have shown that NLRP12 negatively impacts the canonical NF-kappaB. Furthermore both groups showed that NLRP12 downregulates MAP kinases, and the presence of NLRP12 attenuates ERK phosphorylation. In addition to NLRP12 we have recently identified NLRC3 as another member that inhibits TLR signaling by reducing K63- ubiquitination of TRAF6 and preventing canonical NF-kappa kappa activation. This inhibition of NF-kappa kappa is accompanied by attenuated LPS response. This project will assess the roles and mechanisms by which these two anti-inflammatory NLRs affect a number of colitis models and profile the expression of these proteins and their downstream effects in samples from colitis patients vs. controls.

The concept of Pathogen-Associated Molecular Pattern (PAMPs) engaging Pattern Recognition Receptors (PRR) to initiate innate immunity has revolutionized immunology. We first reported the 22- member NLR (Nucleotide-binding and Leucine Rich repeat or NOD-like receptors) family. Most NLRs activate innate immunity, prime examples being NODI and N0D2 which recognize bacterial peptidoglycan and inflammasome NLRs that trigger caspase-1 activation leading to IL-1B and ILI8 maturation. Recently, we and several other groups documented a new NLR subfamily that reduces inflammatory and immune activation which is comprised of NLRP4, NLRP6, NLRP12, NLRC3, NLRC5 and NLRXI. These proteins largely operate by interacting with adaptors and signaling pathways in the innate immune system. In this proposal we will examine the intersection of some of these novel NLRs in regulating host response to a number of NIAID Priority RNA and DNA viruses. We will apply several cutting edge proteomic approaches to assess specific proteomes that are dependent on NLRs during infection with NIAID Priority viral pathogens. These directions are in precise concordance with the RFA which states that emphasis of research proposed in response to this FOA should be in defining novel cellular and molecular immune mechanisms involved in immunity to virus infection. Additionally, the mechanisms by which these NLRs regulate host response are broadly applicable to many viruses of relevance on the Priority Pathogens' list. It supports the overall goal of the Program by (a) investigating the role of novel PRRs as sensors or receptors of viral nucleic acid which affect subsequent innate immune responses to NIAID high priority viral pathogens in human; (b) applying cutting edge quantitative proteomic approaches for the identification of novel paradigm-shifting pathways of pathogen sensing; (c) assessing cross-talk between multiple PRRs; (d) using unique biochemical capabilities that are technically challenging to study the ligand-binding functions of PRRs, and (e) performing experiments with primary human materials. This project will involve extensive collaboration with Projects 2 and 3, as well as Cores A-C.

The core directed by Drs. de Silva and Miley will provide the following services. 1) Cloning of recombinant dengue protein constructs for expression (Aim 1 Miley) 2) Cloning of recombinant influenza protein constructs for expression (Aim 1 Miley) 3) Large scale production and purification of dengue antigens from the 4 serotypes (Aim 1 Miley) 4) Large scale production and purification of influenza antigens (Aim 1 Miley) 5) Assessing the structural integrity of recombinant antigens (Aim 1- desilva) 6) Quality control testing of vaccine antigens (Aim 1-Miley) 7) Growth of hybridomas and purification of monoclonal antibodies (Aim 2-Miley) 8) Testing of immune sera for DENV neutralizing antibodies against the 4 serotypes (Aim 3 desilva) 9) Testing the breadth of neutralizing antibody responses using panels of viruses that cover the genetic diversity of each dengue serotype (Aim 3 deSilva)

The overarching purpose of this U19 Center grant application is to optimize a novel nanoparticle (NP) platform for the delivery of vaccines and vaccine adjuvants. The application is cross-disciplinary and requires expertise in material sciences, immunology, virology and animal models. We will test this platform for two viruses of high-medical need: influenza and Dengue virus. Once optimized, this platform should be adaptable for the delivery of vaccines against a variety of microbial pathogens. The NP technology platform is distinguished by the application of a soft lithography particle molding process called Particle Replication In Non-wetting Templates (PRINT) to produce the particles. This technology was developed by Dr. Joseph DeSimone at the University of North Carolina, who also founded the biotechnology company, Liquidia Technologies. A major advantage of PRINT is that the NPs produced are immunologically-inert and are of precise size, chemistry, porosity, flexibility and shape. Importantly, GMP (Good Manufacturing Practices) quality PRINT-NPs can be fabricated in large quantities by our industrial partner, Liquidia. A standard PRINT-NP has been used to deliver FDA-approved vaccines, with preliminary results demonstrating that this delivery system enhanced immunity compared to soluble vaccine and further provided a dose-sparing effect. This application has three projects based at UNC, supported by four cores that include industry-academia partnerships. All three projects are highly inter-related and have the ultimate goal of enabling an eventual IND application for optimized vaccine/adjuvant biologics. The first project will optimize the PRINT-NP chemistries to enhance biologic efficacy as a vaccine delivery system. The second project will focus on the co-delivery of PAMPs (Pathogen-associated Molecular Patterns) as adjuvants to stimulate anti-viral immunity in mice and appropriate larger animal models. The third project will use a novel humanized mouse system to assess human immune responses to NP-delivered vaccine and adjuvant. The three projects are highly integrated to discover the most optimal PRINT-NP platform needed for vaccine and adjuvant delivery for translation in humans.

This project is focused on the development of a unique GMP-compliant particle molding technology that we have pioneered called PRINT? (Particle Replication in non-wetting templates) as a delivery platform for particulate multivalent vaccines targeting influenza and Dengue viruses. PRINT provides complete control over particle size, shape and chemical composition, and enables the systematic tailoring of antigen proximity to the particle, antigen density and particle size and shape to screen and identify the optimal particle parameters to elicit enhanced immunological responses. The down-selected particle parameters will provide design rules to manufacture influenza and dengue particulate multivalent vaccines to investigate the ability to generate a complete and robust neutralizing antibody response to each antigen. Also, additional Projects within our Program will utilize these multivalent particulate vaccines to address the use of novel adjuvants to modulate the immune response. These particulate vaccines will be evaluated in a humanized mouse model to determine how to prevent infection of homotypic or all 4 dengue virus serotypes. We will combine the learning from each Project to scale-up the most promising multivalent particulate vaccines and advance these to large animal challenge studies for both influenza and dengue. Due to the immediate medical need for multivalent vaccines for influenza and dengue and the cGMP-compliant nature of our particle molding technology, the formulations can be advanced into product development for human trials.

Recent advances have led to the appreciation that Pathogen-associated Molecular Patterns (PAMPs) derived from microbial pathogens have adjuvant activities. A major conceptual advance in innate immunity has been the discovery of Pattern Recognition Receptors (PRR) that recognize these PAMPs. This recognition profoundly shapes adaptive immunity, thereby impacting host responses to pathogens and to vaccines. A plethora of PRR families have now been described. While some PAMPs are known to activate a specific receptor, more in-depth analyses have revealed that many PAMPs activate multiple PRRs or PRR pathways. The overarching purpose of this project is to use a novel nanoparticle (NP) platform to deliver PAMPs that can best activate the innate immune system to shape the desirable adaptive immunity for beneficial, efficacious vaccine outcome. The distinguishing technology platform of this application is the production of NP by a soft lithography particle molding process called Particle Replication In Nonwetting Templates (PRINT), a technology described and further studied in Project 1. A major advantage of PRINT is the fabrication of large quantities of immunologically-inert NPs of precise size, chemistry, porosity and shape of Good Manufacturing Practices (GMP) quality. This proposal plans to co-deliver antigen and PAM adjuvant complexes by PRINT-NP to optimize vaccine responses. This platform of vaccine/adjuvant delivery by NP will be used to test vaccines against viral pathogens of high medical needs. Mechanistic studies are also proposed to understand how the PAMP adjuvants activate the immune system by defining the specific receptors that mediate the effect of a PAMP adjuvant(s). Challenge studies will be performed in appropriate large or larger animal models to enable eventual translation to the clinic.

The Nanoparticle Vaccine Production Core (Core B) will use the proprietary PRINT process to produce nanoparticles which will be provided to the all three research programs for in vitro and vivo usage throughout the project period. The particles will fall into two main categories. First, nanoparticles made from poly(lacticco- glycolic acid) (PLGA), a material used in FDA approved drug carriers and therapeutic devices. An influenza vaccine based on PLGA PRINT particles have previously been evaluated in a clinical study that demonstrated their functionality and showed no safety concerns. Second, particles made from PEG hydrogel materials. The two materials were chosen not only based on safety but because they complement each other in how they can be utilized for vaccine design. PLGA particles will have the vaccine antigen electrostatically adsorbed and adjuvant components associate by either adsorption or encapsulation. The chemical composition of hydrogel particles will allow for antigens and adjuvants to be linked by conjugation either inside or outside of the particles. The Core will initially produce PLGA particles of 12 different sizes and shapes during the first three months of the project, then devote 6 months for developing process methods for manufacture of at least 10 hydrogel particles. For the duration of the project, we will work closely with the research projects to produce particles formulated with antigens, adjuvants and fluorescent molecules for experiments defined in respective project's research plans.

Under Dr. Jenny Ting, Program Director, the Administrative Core supports the scientific and translational goals of this Center of Excellence for Immune Mechanisms of Virus Control (CEIMVC) by providing leadership and day-to-day operational administration. The Administrative Core is responsible for managing, coordinating, and supervising the overall project. It will coordinate administrative/scientific oversight of all research projects, cores, and pilot programs and the activities ofthe external Scientific Advisory Board (SAB). In addition, this Core is responsible for the day-to-day operations and management, including monitoring of expenditures, addressing grant management issues as well as unexpected issues that arise. The primary goal of the Administrative Core is to promote success for all Center programs by managing, coordinating, and supervising all Center activities. This includes research programs, core activities, pilot research projects, financial monitoring and oversight, compliance and regulatory activities, intellectual property and technology development, biosafety and security issues for all personnel, materials and data sharing plans associated with Center programs. The Administrative Core is ultimately responsible for ensuring the successful completion of the Center's mission to study novel immune mechanisms that are generalizable to anti-viral immunity. We will be assisting all personnel associated with the Program in achieving the goals listed in the Program Abstract, specifically in advancing knowledge regarding novel PRRs that are important for nucleic acid sensing and applying this knowledge to the study of NIAID high priority viral pathogens. The Scientific Administrators and the Administrative Staff will work together toward the stated goals.

The primary goal of the CETR Administrative Core (Core A) is to promote success for all CETR programs including research programs and cores, supplemental research projects, financial monitoring and oversight, compliance and regulatory activities, biosafety and security issues, for all personnel, materials, data and facilities associated with CCTRHIB programs. The Core will work closely with the Scientific Advisory Board to assure that excellent progress is made on all projects, and that innovative supplemental projects are being funded. The Administrative Core is ultimately responsible for ensuring that the mission of this CETR for novel vaccine technology platform for high medical need infectious is met. Under Dr. Jenny Ting, Program Director and Dr. Barbara Vilen, Associate Director, the Administrative Core supports the scientific and translational goals as follows: (1) Execute day-to-day operations and management, including monitoring of expenditures, addressing grant management issues as well as unexpected issues that arise. (2) Managing, coordinating, and supervising Scientific Projects 1-3 and Cores B-D. (3) Coordinate the review activities ofthe advisory committee, both reviews of Projects and Cores and reviews of supplemental applications. (4) Oversee activities of the research programs, core activities, supplemental research projects to assure proper compliance and regulatory activities, biosafety and security issues for all personnel, materials, data publication and resource sharing, and technology transfer/intellectual properties necessary for product development associated with Center programs.

The long-term goal of this project is to develop a highly active tetravalent vaccine to prevent Dengue Virus (DENV) infection using a novel vaccine delivery nanoparticle (PRINT-NP) platform with novel PAMP-based adjuvants. DENV infection has posed a serious global public health problem. No DENV vaccines are currently available, partly due to the lack of efficient vaccine delivery/adjuvants and of robust in vivo models of human vaccine responses and DENVl-4 infection. Due to the significant difference between mouse and human immune systems, humanized mice will play a critical role in evaluating human adjuvants, novel antigens and vaccine delivery platforms. Recent improved humanized mouse models have allowed stable reconstitution of a functional human immune system that have been increasingly used for evaluating and comparing human immune response to vaccination and to DENV infection. The AFC8-hu HSC/Hep/TEC mouse developed in the Su group with both human immune and liver cells is well-suited for evaluating human adjuvants and DENV vaccines and for studying DENV infection. With a stable human immune system from human HSC/TEC transplant, functional human immune cells are developed in all lymphoid/liver organs in AFC8-hu mice, which will provide an improved humanized mouse model for evaluating human vaccine response and DENV infection. Using humanized mouse models, we will model and develop novel PRINT-NP to deliver novel vaccine/adjuvant to human APC that induce efficient human protective immunity in vivo. Thus findings from this project will help to 1) identify top PRINT-NP for delivering human vaccines to induce human immunity; 2) develop novel PAMP-based human adjuvants; 3) define novel DENV antigens to induce neutralizing antibody (nAb); 4) establish the humanized mouse model for studying DENV1-4 infection and evaluating DENV vaccines; and 5) identify top tetravalent DENV vaccines for further clinical development. The PRINT-NP and novel adjuvants platform can also be used in vaccine design against other emerging pathogens.

The overall objective of this core is to provide large-scale immunology and influenza Virology support to enhance the product development activities of the center investigators. The core will provide multiplex cytokine/chemokine profiling, high-throughput humoral response monitoring via antigen-specific ELISA and Surface Plasmon Resonance, and provide comprehensive influenza virology support (viral stocks, titers and neutralization assays). To accomplish this goal the expertise and efforts of Dr. Sempowski's cellular immunology lab and Dr. Ramsburg's viral-immunology lab have been combined. The inclusion on an Immunology/Influenza Virology Core will-provide a common set of platforms for mechanistic analysis of host immune response for all three center investigators. The use of ELISA/SPR will allow for monitoring host response to both experimental vaccines/adjuvants and challenge pathogens. Blood, tissue and culture supernatant inflammatory biomarker analysis will provide further valuable insight into specific host responses which will only enhance the challenge and mechanistic studies proposed by center projects to develop nanopartical vaccines and therapeutics to Flu and Dengue. Lastly, the inclusion of a centralized lab for maintenance of influenza virus strains and tittered stocks for all projects will allow for uniform flu studies across all projects. Moreover, the influenza support lab will work closely with investigators and collaborators to ensure standardized execution of influenza viral titers and neutralizing antibody responses to influenza.

DESCRIPTION (as provided by applicant): A major conceptual advance in innate immunity is the discovery of Pathogen Recognition Receptors (PRR), which profoundly shape adaptive immunity to affect host response to pathogens. Major PRRs crucial for innate immunity against viral pathogens include toll-like receptors and RIG-I like receptors, while the role for NLR (nucleotide-binding leucine rich repeat containing, or NOD-like receptor) family in viral infection is just emerging. In this Program, we will focus on the revelation of novel nucleic acid sensing pathways relevant to multiple NIAID priority pathogens. The Program is comprised of three Projects, each led by an international leader in his/her field. The Program will be performed in a highly collaborative fashion with two Cores which will provide cutting edge proteomics and protein purification capabilities, headed by directors who have contributed seminal work in the field of PRRs. The overarching goals are: * To investigate the role of novel PRRs as receptors of viral nucleic acid which affect subsequent innate immune responses to NIAID high priority viral pathogens in human. * To apply cutting edge quantitative proteomic approaches for the identification of novel paradigm-shifting pathways of pathogen sensing. * To contrast and compare role of PRRs across diverse NIAID high priority human viruses. * To reveal cross-talk between multiple PRRs in host response to NIAID priority human viruses * To investigate intracellular trafficking of viral ligands to sites of recognition by PRRs. * To capitalize on unique biochemical capabilities that are technically challenging to quantify the ligand-binding functions of PRRs. * To maximize opportunities for validating experimental findings with primary human materials. These goals are highly response to the RFA-AI-12-048 and are in precise concordance with the stated purpose of the RFA that emphasis of research proposed in response to this FOA should be in defining novel cellular and molecular immune mechanisms involved in immunity to virus infection. It is also responsive because the pathways explored are broadly relevant to multiple high priority pathogens and will be studied in the context of five high priority RNA and DNA viruses. Finally, the proposed work is responsive to the purpose of the RFA because it specifically expands our understanding of novel PRR interaction with viruses and the accompanied changes in signaling pathways by profiling proteomic modifications in human innate immune cells caused by NIAID priority viral pathogens.

Project 2 Abstract Improving the delivery of immunotherapeutics or immunomodulatory molecules is key to improving cancer vaccines. Nanoparticles (NPs) represent an ideal vehicle for in vivo delivery of cancer immunotherapy or cancer vaccines. We pioneered a nanoparticle molding technology, PRINT® (Particle Replication in Non-wetting Templates), that allows unprecedented control and specification of the physical/chemical properties of NPs. PRINT can produce particles with varied parameters such as shape, size, porosity, and chemical composition and therefore represents an ideal platform to test such parameters to achieve optimized anti-tumor vaccination and immunity. Additionally, we and others have shown the potency of small molecules comprised of PAMPs (Pathogen-associated Molecular Patterns) which engage the pathogen-recognition receptors (PRRs), elicit adjuvant activity, and activate desirable anti- cancer immunity. The use of PRINT to deliver these adjuvants is ideal in that PRINT nanoparticles are taken up by antigen presenting cells, which when activated can elicit strong specific T cell response. Another key finding is that successful therapeutic cancer vaccines must be able to induce a robust response to circumvent the highly immunosuppressive nature of the tumor microenvironment. The most promising results have been obtained by combining potent cancer vaccines that elicit strong anti-tumor immunity with immune modulating agents that help to break immunological tolerance. Nanotechnology represents a promising system to deliver small molecules to the tumor site to more efficiently inhibit immune-tolerance inducing pathways. The goal of this proposal is to address these three key issues.

This application focuses on the central goals of the RFA for Centers for Medical Countermeasures Against Radiation by: (a) identifying countermeasure strategy that mitigates delayed radiation injury, (2) characterizing injury and countermeasures in the susceptible pediatric population, and (3) applying countermeasures that treat radiation and infection combined injury. Multi-organ radiation-induced injury is a major threat during targeted terror attack, and adaptive and innate immunity are increasingly found to play a key role in ·this process. Innate immune receptors collectively referred to as Pathogen Recognition Receptors (PRR) have undergone an explosive discovery phase. Prominent PRR families include the membrane bound Toll-like receptors (TLR) which interact with extracellular ligands and NOD- like receptors which respond to intracellular agonists. These have been extensively studied in infection and inflammatory diseases, but their impact on radiation-induced damage (RID) is just emerging. Post-exposure, radiation not only causes acute injury but also delayed injury such as fibrosis and defective cellular and immune development. We and others have explored the roles of TLRs and NLRs during RID and unexpectedly found that TLRs and NLRs are protective of delayed radiation-induced damage. Common cytokines that are activated by PRR families are IL-1?? and IL-18. Indeed, deletion of IL-1? and/or IL-18 resulted in exacerbated clinical severity in the immune and susceptible organs of radiated mice. In this proposal, we will investigate how cytokines such as IL-1?? protects against delayed radiation-induced damage in solid organs. We will also address the same issue in immune cells. Finally, we will examine if the microbiome is altered in a gene-dependent way in response to radiation exposure.

Project Abstract NLRs (NOD-like receptors) are intracellular sensors, serving divergent functions in the regulation of innate immunity. While the best-studied NLRs exhibit positive function during immunity, we and others documented several NLRs that are anti-inflammatory in nature and these cause reduced inflammatory and immune activation during infection and inflammation. As examples, NLRX1 and NLRC3 serve as brakes of inflammatory response during viral and/or bacterial infection. One of the functions mediated by NLRX1 is that it reduces the RNA sensing pathway. By contrast, another inhibitory NLR, NLRC3, reduces the DNA sensing pathway mediated by STING and attenuates the proliferative pathway mediated by PI3K. Interestingly, two groups have shown that the LRR domain of NLRX1 can bind RNA, although the functional consequence of this finding is less clear. Recently, we have obtained multiple evidence that NLRC3 is an intracellular receptor for nucleic acids as well. Evidence for the direct binding of NLRs to their ligands is of paramount importance because an over-riding issue in the NLR field is whether NLRs are authentic ligand-binding receptors. The association of these NLRs with their putative nucleic acid agonists provides transformative evidence that some NLRs are indeed receptors of nucleic acids. An investigation of the interaction of NLRX1 and NLRC3 with nucleic acids, and how this interaction impacts cellular responses during microbial and non-microbial associated perturbation should be of great significance. In addition, at least two members within the NLR family are authentic transcriptional regulators, namely, CIITA which is a master regulator of class II MHC, and NLRC5 which is a transcriptional regulator of class I MHC in immune cells. We will explore if other NLRs might also represent transcriptional activators.

ABSTRACT The discovery of innate immune receptors has revolutionized the field of innate immunity. An unexpected finding is that innate immune receptor are not only located on the cell membrane, but a majority of these are found intracellularly. This allows cells to detect intracellular perturbation from pathogen-associated molecular patterns (PAMPs) or from damage-associated molecular patterns (DAMPs). Our studies have revealed a novel roles of intracellular innate immune receptor in attenuating inflammation and shaping the microbiome during inflammatory bowel disease (IBD). The central hypothesis is that the bi-directional interplay of inhibitory intracellular innate receptors with the microbiome is a major contributing factor in IBD. Our proposal focuses on the intracellular NLR (nucleotide-binding domain, leucine-rich repeat containing protein, or NOD-like receptor) proteins. While NOD2 remains the most prominent in Crohns? disease, we have focused on the role of other NLRs in colitis. We have data to show that several inhibitory NLR proteins strongly mitigate intestinal inflammation by reducing the activation of immune signaling pathway thus preventing an inflammatory cytokine response that is integral to colitis. This in turn can affect both innate and adaptive immune cells, as well as colon epithelial cells. We have also shown that these inhibitory NLRs affect the microbiome, partly by maintaining bacteria that can contain a pro-inflammatory response. Thus the first goal is to understand the bidirectional interaction of inhibitory NLR and the gut microbiota in mitigating gut inflammation in mice by working with Projects 2, 4 and the Animal Models Core A. Another over-arching goal is to assess the translational relevance of results obtained in mice to humans. Working with Project 3 and the Human Tissue and Genomics Core B, we will analyze patient-derived material to verify the significance of our findings in mice to inflammatory bowel disease patient samples.

Abstract The overarching purpose of this proposal is to produce a universal influenza vaccine using a new micro-particle-adjuvant combination that generates dramatic dose-sparing and robust immune enhancement effects. Influenza virus is a recurrent public health threat of international concern. Current influenza viral vaccines are comprised of life attenuated virus, inactivated influenza vaccine or recombinant influenza protein vaccine. The latter usually requires an adjuvant. However, a major challenge is that dominant flu vaccine antigens such as hemagglutinin are highly variable among different influenza strains, and hence new vaccines have to be produced annually. There is a national push for an alternative approach relying on a universal influenza vaccine. This approach involves common antigens that are shared among different influenza strains, but faces a major hurdle in that these antigens are typically weakly immunogenic and requires a strong adjuvant for their efficacy, which has not been achieved to date. Microbial pathogen-associated molecular patterns (PAMP) are small molecules produced by microbes that stimulate the immune system, and these have emerged as strong adjuvants. However the receptors for many PAMPs reside in the cytosol, thus presenting a challenge for delivery. We have used a particle-based delivery system that successfully delivers PAMPs inside the cell to activate their respective receptors. This produces a robust adjuvant effect that does not cause toxicity or systemic inflammation. In the context of hemagglutinin, this microparticle-PAMP combination enhances specific antibody response up to 105 fold over bare antigen, induces a strong T cell response and fully protects infected mice and ferrets. This proposal plans to use this platform in a universal influenza vaccine. To advance this vaccine platform towards pre-IND development, we have a regulatory expert and a toxicologist guiding us throughout the proposal. In addition, a main industrial partner with expertise in particle production and other contract research organizations have been recruited to assist us towards the development of a lead universal vaccine. Thus, this proposal is fully responsive to the RFA-AI-17-042 and is focused on the preclinical development of a robust vaccine candidate that elicits strong T and B cell responses and cross-reacting antibodies to address one of the greatest public health concerns.

Abstract The challenges addressed by this R35 application are multiple. First, colorectal cancer (CRC) remains a leading cancer worldwide that is resistant to many treatments. Two important risk factors for CRC are a history of chronic colitis or inflammatory bowel disease (IBD) and obesity, both of which are increasing at an alarming rate. However, the mechanisms linking these predisposing factors to CRC are not well understood and thus there is a pressing need to elucidate these basic mechanisms. Second, obesity is also a contributing factor to other gastrointestinal cancers, where the role of innate immunity receptors is less well-defined than CRC. Understanding the roles of innate immunity in these other cancers is a high priority. Third, the interaction of host genetics, microbiome, inflammation and cellular transformation is complex, but fundamental to the onset of gastrointestinal cancers. Elucidating this network of interaction is important for devising new approaches for cancer therapy. Fourth, while the roles of adaptive immune molecules and cells have been the main stake of cancer immunotherapy, much less emphasis has been placed on innate immune receptors which may alter adaptive immunity to advance cancer immunotherapy, which should be an attractive strategy to combat cancer. Finally, while studies in animals are important in establishing a foundation, a well-defined plan to translate basic findings to humans remains the ultimate goal and challenge that we will address. The NLR (nucleotide-binding domain, leucine-rich repeat containing proteins, or nucleotide-oligomerization domain receptor) is a multi-member gene family that encodes a group of cytosolic proteins that are involved in the intracellular sensing of microbial products as well as damage-associated molecular patterns. NOD2, an NLR family member, has a strong genetic association with Crohns' disease and has been implicated in colitis- associated CRC. Additionally, NLRs including NOD2 and NLRP12 can affect the microbiome to impact colitis in mice, suggesting that NLR family members are important in maintaining or disrupting the homeostasis of gut microbiome. We and others have shown a role for the inflammasome NLRs in models of colitis and CRC. In addition to our analyses of well-studied inflammasome components in models of colitis and CRC, we have been at the forefront of defining a strong role for other NLRs which have anti-inflammatory functions (referred to as inhibitory NLRs), and can alter the course of inflammation and cancer. This proposal plans to examine the roles of NLRs in humans and in murine models of cancers, to elucidate the complex interaction of NLRs with the microbiome and cellular transformation and to harness these proteins to enhance cancer immunotherapy.

UNC-CH SUBCONTRACT Abstract This application focuses on a central goal of the RFA for Centers for Medical Countermeasures Against Radiation to ?further develop existing as well as novel therapies to minimize tissue damage, hasten tissue recovery, restore normal physiological function, and improve survival.? Multi-organ radiation-induced injury is a major threat during targeted terror attack, and adaptive and innate immunity are increasingly found to play a key role in this process. Innate immune receptors collectively referred to as Pathogen Recognition Receptors (PRR) have undergone an explosive discovery phase. Prominent PRR families include the membrane bound Toll-like receptors (TLR) which interact with extracellular ligands. These have been extensively studied in infection and inflammatory diseases, and their impact on radiation-induced damage has emerged in the last few years. Post-exposure, radiation not only causes acute injury but also delayed injury such as fibrosis and defective cellular and immune development. We and others have explored the roles of TLRs in radiation and unexpectedly found that certain TLRs and their ligands are protective of radiation-induced damage involving both the hematopoietic system as well as the gastrointestinal tissues. In addition to TLR ligands, we have also isolated beneficial microbiota and metabolites from animals that survived lethal radiation, and propose to explore if these microbes and their metabolites can mitigate radiation damage. This proposal will focus on the use and mechanism of TLR ligands, commensal microbes and their metabolites as radiation mitigators that can reduce radiation induced damage.

Abstract The COVID-19 pandemic caused by SARS-CoV-2 has resulted in swift and catastrophic losses of human lives globally. Acute respiratory distress syndrome (ARDS) is one of the most detrimental outcomes of COVID-19 infection that can lead to the rapid deterioration and death of patients. ARDS is primarily caused by the cytokine storm which unleashes a plethora of inflammatory cytokines during the late stages of COVID-19. The master cytokines that are thought to be responsible for much of the damage are interleukin 1 (IL-1), interleukin 6 (IL-6) and tumor necrosis factor (TNF). Currently several clinical trials have already been initiated to test the efficacy of biologic inhibitors to target these pathways. However, in many cases, the mechanism and impact of these cytokines during ARDS are poorly understood. An indepth mechanistic understanding of cytokine induction is important because this understanding will significantly impact the design and success of ARDS treatment. This application focuses on the role and mitigation of the inflammasome complex which leads to the proinflammatory cytokine, IL-1?, in ARDS. The inflammasome is a protein supramolecular structure that leads to caspase 1 activation, which then cleaves pro-IL-1? and pro-IL-18 to mature IL-1? and IL-18. In addition to the release of IL-1? and IL-18, caspase 1 cleaves gasdermin D to cause inflammatory pyroptotic cell death, thus leading to a cascade of cell death and inflammation. The inflammasome is comprised of a receptor or sensor, with the most prominent ones represented by NLRP1, NLRP3, NLRP6, NLRC4 and AIM2. It also includes an adaptor molecule ASC (apoptosis- associated speck-like protein containing a CARD), and the effector caspase-1. Each receptor or sensor can be activated by specific pathogen products called PAMPs or cell damage associated molecules called DAMPs. NLRP3 is the most studied member since it is activated by a large list of stimulators. Studies of other coronavirus such as SARS show inflammasome activation by key viral proteins. Expression data from COVID-19 patients also show dramatic increases of inflammasome sensors in the bronchial alveolar lavage of COVID-19 patients. However the mechanism of inflammasome activation by SARS-CoV-2, especially in the human system, remains unknown. This proposal will identify the viral protein that activates human inflammasome, and further define the specific human inflammasome sensor/receptor that mediates the response. We will then design ways to reduce inflammasome activation during SARS-CoV-2 infection using established therapeutics as well as new approaches to broadly attenuate inflammatory cytokines.