Averil Ma - US grants

Interleukin-2 (IL-2/-/-) deficient transgenic mice regularly develop severe, persistent colitis, which resembles ulcerative colitis in humans. The selective inflammation of the colon suggests IL-2 plays a critical role in regulating mucosal immune responses. We have recently demonstrated that this disease is dependent on mature T, but not B lymphocytes. Thus, dysregulated T lymphocytes are likely to mediate colitis in both these mutant strains. Because IL-2 has previously been shown to program activated T cells to undergo programmed cell death, we postulated that the absence of IL-2/IL-2 alpha receptor signaling leads to a defect in programmed cell death (PCD) of activated T lymphocytes. Preliminary studies suggest that activation induced cell death (AICD) is indeed compromised in both these strains. As AICD is important for the normal regulation of the duration of immune responses, this defect may allow activated mucosal T cells to cause persistent colonic inflammation. In this application, we propose to confirm and extend our preliminary findings defining the role of T cells in mediating colitis in these animals, compare the propensity of mucosal and systemic lymphocytes from IL-2\-\- mice to undergo PCD, and study the expression of proteins known to regulate PCD. These studies should not only lead to a better understanding of how IL-2 regulates mucosal immune responses, but may also lead to important insights into the pathogenesis of human IBD.

[unreadable] DESCRIPTION (provided by applicant): The homeostatic maintenance of memory T cells constitutes the cellular basis of immunological memory. Memory CD8+ T cells proliferate to maintain lineages of antigen specific T cells for months or years, thereby protecting hosts against recurrent infections with microbial pathogens such as viruses. Recent studies from our lab indicate that IL-15 and its high affinity receptor IL-15Ra are essential for the proliferative maintenance of these cells. Surprisingly, we found that IL-15Ra expression on RAG-1 independent hematopoietic cells (i.e., accessory cells), rather than CD8+ T cells, support memory CD8+ T cells. We have also shown that these accessory cells use IL-15Ra to trans-present IL-15 to IL-2/15RP expressing CD8+ T cells in vivo. Finally, our most recent studies suggest that IL-15 and IL-15Ra need to be coordinately expressed by the same accessory cells to trans-present IL-15 to memory CD8+ T cells. These surprising findings prompt us to consider an entirely new cell biological mechanism for IL-15Ra's actions. Specifically, IL-15 may not be a secreted, soluble cytokine under physiological conditions. Rather, IL-15 and IL-15Ra may associate with each other within accessory cells and emerge on the cell surface to transpresent IL-15 to memory CD8+ T cells. Thus, we may need to think about IL-15 and IL-15Ra as a cell surface bound ligand, more similar to a co-stimulatory molecule. To test this hypothesis, we propose to: (1) determine the specific cell types that use IL-15Ra to support memory CD8+ T cells in vivo; (2) determine the intracellular kinetics and association of IL-15Ra and IL-15 in accessory cells; and (3) determine the structural requirements of IL-15Ra important for supporting memory CD8+ T cell homeostasis in vivo. These experiments should reveal novel insights into the cellular and cell biological mechanisms by which IL-15Ra supports lymphoid homeostasis in vivo. The immunological importance of our studies is threefold. First, they redefine the physiological concept of "homeostatic space," which can now be refocused upon cells that bear IL-15Ra and support memory CD8+T cells through "trans-presentation." Secondly, our studies provide the first evidence that IL-15/IL-15Ra complexes on the surface of accessory cells may function as surface bound ligands, indicating that memory CD8+ T cells must make intimate contact with IL-15Ra expressing accessory cells for their homeostasis. Thirdly, they suggest that IL-15 may bind to IL-15Ra prior to exiting accessory cells, raising entirely novel concepts about cytokine biology. Our studies are highly relevant to the regulation of the duration of immune responses, and to immunological memory. Future attempts to either enhance antiviral vaccine effectiveness or to suppress persistent inflammation in autoimmune conditions may benefit greatly from the mechanistic insights we are gaining into IL-15 biology. [unreadable] [unreadable] [unreadable]

DESCRIPTION (Applicant's Abstract): A critical step in inciting intestinal inflammation is the elaboration of proinflammatory factors such as TNF. TNF activates NF-kappa-B and JNK signaling pathways, which induce the expression of other genes in a pro-inflammatory cascade. While it is clear that dysregulated expression of TNF can cause intestinal inflammation, it is not clear how TNF signals are normally down-regulated or controlled in vivo. To better understand the molecular mechanisms by which TNF signals are regulated, the investigator is studying A20, a novel molecule thought to regulate cellular responses to TNF. The PI has generated A20 deficient (A20-/-) mice and these mice develop spontaneous bowel inflammation, which is dramatically worsened after exposure to TNF. These remarkable physiological problems are associated with molecular defects in regulating TNF induced NF-kappa-B activity and cellular resistance to programmed cell death (PCD). This compelling preliminary data highlights A20's pivotal role in terminating TNF responses in vivo. It also provides a unique opportunity to interrogate the molecular mechanisms by which such control is normally achieved. Thus, the investigator proposes to: (1) delineate the role of A20 in regulating both spontaneous and TNF induced intestinal inflammation; (2) determine both the requirement and the contribution of A20-/-lymphocytes to mediating intestinal activation in these mice; (3) define the relative contributions of nonlymphoid (myeloid) hematopoietic cells versus non-hematopoietic cells; and (4) define the role of A20 in regulating TNF induced NF-kappa-B and PCD responses in intestinal tissue. Understanding both the cellular and molecular mechanisms by which A20 regulates TNF responses will provide unique insights into how TNF induced inflammation is normally terminated, and may also lead to novel therapies for human IBD patients.

[unreadable] DESCRIPTION (provided by applicant): A critical step in regulating innate immune responses is the elaboration of pro-inflammatory factors such as TNF. TNF activates NF-kB and JNK signaling pathways, which induce the expression of other genes in a pro-inflammatory cascade. While it is clear that deregulated expression of TNF can cause inflammation, it is less clear how TNF signals are normally controlled in vivo. To better understand the mechanisms by which TNF signals are regulated and how such processes affect inflammation, we are studying A20, a novel molecule thought to regulate cellular responses to TNF. We have found that A20 is broadly expressed in multiple cell types, including both adaptive and innate immune cells, as well as nonhematopoietic cells. We have generated A20 deficient (A20-/-) mice, and these mice develop severe spontaneous inflammation and premature death. Preliminary cellular and genetic studies suggest that innate immune cells are severely affected by A20 deficiency in a cell-autonomous fashion. In addition, A20-/- cells display multiple defects in regulating TNF signals, indicating that A20 may be the first molecule identified to be essential for the termination of both TNF induced NF-kB activity and JNK activity. These observations highlight A20's pivotal roles in terminating TNF responses in vivo. It also provides a unique opportunity to interrogate the cellular and molecular mechanisms by which such control is normally achieved. Thus, we propose to: (1) determine the roles of A20 in regulating the homeostasis and function of innate immune cells; (2) determine the molecular mechanism by which A20 regulates NF-kB activity, and (3) determine the molecular mechanisms by which A20 regulates JNK activity. Understanding both the cellular and molecular mechanisms by which A20 regulates TNF responses will provide unique insights into how inflammatory responses are normally terminated, and how A20 regulates cellular activation and proliferation in vivo.

DESCRIPTION (provided by applicant): NK cells protect hosts against viral pathogens as well as transformed cells. IL-15 is thought to play a critical role in NK cell development, and may mediate these effects through binding to its high affinity receptor, IL-15Ra. By generating and characterizing mice deficient for IL-15Ra, and analyzing radiation bone marrow chimera derived from these mice, we have obtained data suggesting that IL-15Ra plays critical roles in regulating mature NK cell homeostasis and NK cell activation, as well as NK cell development. Surprisingly, the mechanism by which IL-15Ra performs these functions may occur in a non-cell autonomous fashion, i.e., IL-15Ra expression on cells other than NK cells regulate NK cell functions. Accordingly, we propose to investigate the mechanism(s) by which IL-15Ra regulates NK cell function and development. Specifically, we will determine the roles of IL-15Ra in supporting: (1) NK cell development; (2) mature NK cell homeostasis; and (3) NK cell activation. These experiments should reveal novel insights into which cells use IL-15Ra to supports NK cells in vivo, and how IL-15Ra performs its unusual molecular function.

? DESCRIPTION (provided by applicant): Inappropriate inflammasome activation contributes to multiple human diseases, including autoimmune, autoinflammatory, and metabolic diseases. Yet the mechanisms by which inflammasomes are suppressed are poorly understood. A20 (or TNFAIP3) is a potent anti-inflammatory protein that regulates ubiquitin dependent signals, and is genetically linked to multiple autoimmune and autoinflammatory diseases in humans. Our recent studies show that A20-deficient macrophages exhibit spontaneous NLRP3 inflammasome activity in response to LPS alone without the need for a second signal (e.g., ATP). We have also discovered that IL1? undergoes ubiquitination. Hence, our central hypothesis is that A20 restricts activating ubiquitination events and inflammasome activity in innate immune cells and protects against inflammatory diseases. To test this hypothesis, we will pursue the following specific aims. First, we will identify how A20 regulates a novel TRIF- RIP1-RIP3-Caspase dependent pathway of NLRP3 inflammasome activation. We will determine the in vivo consequences of this A20 function by analyzing the pathophysiology of mice deficient for both A20 and inflammasome components or the downstream pro-inflammatory cytokines. Second, we will utilize a combination of mass spectrometry and novel A20 knockin mutants to precisely define how A20 regulates inflammasome ubiquitination. We will also determine how RIP3 supports IL1? ubiquitination. Finally, we will identify the E3 ubiquitin ligase(s) that ubiquitinate IL-1? and how the A20 binding partner, TXBP, works with A20 to regulate IL1? ubiquitination. Our studies should define new mechanisms by which ubiquitination and A20 regulate inflammasomes, thereby providing a new potential therapeutic pathway targeting inflammasome- mediated diseases.

DESCRIPTION (provided by applicant): Training creative and productive academicians is a critical mission of gastroenterology programs. Emerging discoveries in related fields ranging from human genetics to molecular and cellular biology, microbiology and epidemiology have had major impacts on the understanding of pathophysiology of gastrointestinal diseases. The UCSF Division of Gastroenterology has been a leader in gastrointestinal research for over 40 years. In addition, allied Divisions and Departments at UCSF augment the training program by providing complementary and synergistic training experiences. During the past funding cycle, we have renewed our selection and training procedures to optimize the success of our trainees. The enhanced pool of applicants and trainees in our program attests to the success of our efforts. In this application, we propose to continue our training of innovative and creative academic investigators in gastroenterology.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Toll like receptors (TLRs) on eukaryotic cells mediate the recognition of microbial molecules and regulate host responses to microbial pathogens. Proper regulation of TLR signals not only restricts the intensity and duration of inflammatory responses, but may also maintain immune homeostasis in the intestine, where a plethora of microbial pathogens co-exist with host cells. Recent studies from our labs indicate that A20 is a novel protein that is required for terminating TLR induced signals on myeloid cells and for preventing excessive inflammatory responses in vivo. Our preliminary data suggest that A20 is essential for restricting TLR signals in vivo. Moreover, our findings suggest that A20 may be a novel enzyme that performs this critical function by directly modulating the ubiquitylation status of TLR signaling proteins such as TRAF6. These modifications may cause both de-activation and degradation of TLR signaling proteins. These exciting preliminary findings provide us with unique opportunities to test our central hypothesis that A20 regulates ubiquitylation of signaling proteins, TLR signals, and immune homeostasis. This application represents a synergistic effort between the genetic and cellular expertises of the Ma lab and the biochemical expertise of the Pickart lab to determine: (i) whether A20 is essential for regulating TLR responses in mice;(ii) whether A20 regulates ubiquitylation of critical TLR signaling proteins;and (iii) how A20 recognizes ubiquitylated proteins and may function as both a de-ubiquitylating enzyme and an E3. In the latter approach, mass spectroscopic analysis will be essential for understanding how A20 modifies ubiquitylated proteins. Selected TLR signaling proteins will be examined as putative substrates, and both A20's de-ubiquitylating and ubiquitylating functions will be assessed on candidate substrates. Results from these studies promise to yield significant insights into how A20 links the regulation of ubiquitylation with disease.

Dendritic cells (DCs) are innate immune cells that regulate both immune homeostasis as well as antigen driven immune responses. The functions of DCs are highly regulated by signals initiated by Toll-like receptors (TLRs). Consequently, intracellular molecules that regulate TLR signaling pathways are pivotal for regulating host immune responses. We have recently found that A20 is a ubiquitin modifying enzyme that restricts toll-like receptor (TLR) induced signals. We are studying the roles of A20 expression in DCs in regulating immune homeostasis and responses. To accomplish this, we have generated two strains of gene targeted mice: one that disrupts the A20 gene in all cells and one in which a "floxed" allele of A20 has been targeted to the endogenous A20 locus. We have interbred the "floxed" mice with a novel strain of CD11-Cre transgenic mouse line that expresses Cre enzyme with high selectivity and specificity in DCs. These compound A20flox/flox CD11c-Cre mice will thus provide us with a very powerful and novel reagent for studying the role of A20 expression in DCs in regulating immune homeostasis and immune responses. We propose to use these A20flox/flox CD11-cre compound mice, as well as cells derived from both strains of mice, to determine how A20 expression in DCs regulates their responses to TLR ligands in vitro and in vivo. The first aim will focus upon understanding the cell biology and biochemistry underlying A20's roles in regulating TLR signaling in DCs, and will elucidate the cell-autonomous functions of DCs that are regulated by A20. The second aim will utilize A20flox/flox CD11c-Cre mice to determine how A20 expression in DCs regulates their homeostasis and activation in vivo. This second aim will also interrogate how A20 expression in DCs regulates peripheral tolerance and immune responses and prevents autoimmunity. These studies should teach us a great deal about how dendritic cells are normally regulated, and how they may play a major role in preventing autoimmunity and regulating immune responses. They may also provide novel therapeutic targets for controlling inflammation.

DESCRIPTION (provided by applicant): Recent studies suggest that inflammatory bowel disease (IBD) results from the disruption of normal host immune cells responses to microbial molecules that can trigger inflammation in the intestine. Dendritic cells (DCs) are specialized immune cells that are highly sensitive to microbes and can potently activate inflammatory cells. As DCs may frequently or tonically sense the presence of microbes in the intestine, their propensity to cause inflammation may be dependent on their intracellular regulation or interpretation of encounters with microbes. Hence, intracellular proteins that regulate signals triggered by microbes may be central to the commitment to overt inflammatory responses. A20 is an enzyme that potently restricts signals from microbial sensing pathways, including Toll-like receptor (TLR), NOD and TNF signals. Thus, our central hypothesis is that A20 expression specifically in DCs preserves immune homeostasis and prevents IBD and IBD-associated arthritis. To test our central hypothesis, we have generated a novel strain of mice, A20FL/FL CD11c-Cre mice, in which A20 is deleted specifically from DCs. Remarkably; our preliminary data with these mice suggest that these mice spontaneously develop colitis, sero-negative arthritis and spondyloarthritis, a stereotypical syndrome in human IBD. We now propose to use these A20FL/FL CD11-cre mice to determine the cellular and molecular mechanisms linking A20 expression in DCs to these provocative phenotypes. Specifically, we will determine whether luminal microbes and T cells are involved in the pathophysiologies by which A20 deficient DCs cause colitis and arthritis (Aim 1). As A20 may restrict intracellular signals in DCs, including MyD88 dependent TLR signals, we will use compound A20FL/FL MyD88FL/FL CD11c-Cre mice to determine which A20 regulated signals in DCs are MyD88-dependent and which signals are MyD88-independent. Studies with these mice will unveil which intracellular DC signals and DC products regulate T cell activation, colitis, and sero-negative arthritis (Aim 2). A20 is a ubiquitn modifying enzyme that regulates ubiquitination of signaling proteins and also binds to A20 Binding Inhibitor of NFkB-1, or ABIN-1. To define the molecular mechanisms by which A20 and ABIN-1 may collaborate to restrict signals in DCs, we have also generated mice lacking ABIN-1 specifically in DCs. We will now use these mice to study how ABIN-1 collaborates with A20 to restrict signaling in DCs and prevent colitis and arthritis (Aim 3).

? DESCRIPTION (provided by applicant): Project Summary/Abstract Psoriasis is a chronic inflammatory, hyperproliferative disease of the skin, whose complex pathophysiology is determined by both environmental factors and genetic susceptibilities. Abnormalities in keratinocyte function, adaptive and innate immune cells, and cytokine production have been implicated in psoriasis. Human genetic studies have strongly linked single nucleotide polymorphisms (SNPs) of the gene TNFAIP3, also known as A20, to psoriasis susceptibility. Subsequent GWAS have replicated these associations. TNFAIP3 SNPs have also been linked to reduced A20 expression and therapeutic responses in patients. Our recent studies indicate that A20 restricts innate immune cells by regulating NF-¿B signals. We have also found that mice expressing reduced amounts of A20 are susceptible to imiquimod-induced psoriasis. Hence, our central hypothesis is that reduced A20 expression leads to perturbed innate immune, cytokine, and T cell functions and increased susceptibility to psoriasis. To test this hypothesis, we propose to first determine the cellular mechanisms by which A20 regulates psoriasis susceptibility using mice lacking or reduced A20 expression in dendritic cells, T cells, and epithelial cells. As we have found that A20 is a novel ubiquitin binding and ubiquitin modifying enzyme that interacts with other ubiquitin binding proteins, we will use novel A20 knock-in mice bearing strategic point mutations as well as mice lacking key A20 binding partners to test the physiological functions of these biochemical activities. These experiments should provide comprehensive and mechanistic insights into how A20 prevents psoriasis, and provide new areas for therapeutic discovery.

Project Summary/Abstract Arthritides are chronic, progressive systemic inflammatory diseases that lead to significant joint damage, pain, dysfunction, and disability. A20, also known as TNFAIP3, is genetically and epigenetically linked to human arthritis, and recent experimental studies implicate A20 in regulating several potential pathophysiological arthritis pathways. Mice bearing A20 deficient dendritic cells or A20 deficient macrophages/neutrophils spontaneously develop arthritis. A20 restricts several inflammatory pathways, including TLR, TNF?, and inflammasome signals. How A20 performs these critical functions and prevents arthritis are poorly understood. A20 has multiple motifs that mediate deubiquitinating, ubiquitin binding, and E3 ubiqutin ligase activities. The goal of this proposal is to understand the biochemical mechanisms by which A20 prevents arthritis. We have generated several knock-in lines of mice with strategic point mutations that abrogate specific biochemical functions of A20. Using these new mouse strains, we will determine which of A20's ubiquitin dependent functions preserve the cellular and molecular pathways that prevent arthritis. The proposed studies will establish a new robust mouse model of spontaneous arthritis based on a human arthritis susceptibility protein.