Derek Abbott - US grants

DESCRIPTION (provided by applicant): The human body has evolved mechanisms to detect pathogenic organisms and to initiate an appropriate initial immune response against these organisms. Given the diversity of pathogens, a remarkable feature of these innate immunity-signaling pathways is their ability to tailor a response to a particular pathogen. Innate immune responses are initiated at the cell membrane by a family of receptors collectively termed the Toll-like Receptors (TLRs). Downstream signaling pathways activated by the TLRs include the ERK, JNK, p38 and NF- Kappabeta signaling modules. Activation of these signaling pathways then causes cytokine and chemokine release. There is a remarkable specificity to this immune response such that the cytokines released are tailored to the pathogen destined to be eradicated. Although critical, this specificity in signal transduction is poorly understood. The adapter protein kinase RIP2 may mediate a portion of this specificity. RIP2 has been shown to be involved in the proximal TLR signaling pathway as RIP2-null mice show defects in ERK, JNK, p38 and NF-kappaB signaling and subsequent deficiencies in cytokine release in response to LPS from gram-negative bacteria and PGN from gram-positive bacteria. Although RIP2 contains a serine-threonine kinase domain and it readily autophosphorylates, no in vivo RIP2 substrates have been identified. In preliminary studies, we have began to identify the preferred peptide phosphorylation sequence of RIP2. This initial data was used to help identify the adapter protein, TRAF6, as a phosphorylation substrate of RIP2. The central hypothesis of this grant is that as an essential component of the TLR pathway, RIP2's kinase activity may underly the specificity seen in innate immune signaling pathways. This grant application aims to define the preferred peptide phosphorylation sites of RIP2, to map the phosphorylation site of TRAF6 and to identify the physiologic significance of RIP2's phosphorylation of TRAF6.

[unreadable] DESCRIPTION (provided by applicant): As humans, we are continuously exposed to pathogens. Our innate immune system must be able to differentiate pathogenic from nonpathogenic organisms, and it must be able to tailor an immune response to respond to that pathogenic organism. This problem is particularly acute at mucosal surfaces, an area of the body in which the surface cells are in direct contact with bacteria, fungi and viruses. A number of inflammatory disorders, including Crohn's Disease, are initiated at these mucosal surfaces when the initial innate immune response is not adequately down-regulated after the pathogen is eradicated. In this grant, we study the mechanisms that control this down-regulation at mucosal surfaces. We have found that a key anti-inflammatory protein, A20, is phosphorylated and activated by the central kinase in the NF-?B signaling pathway (IKK2). We mapped the site of phosphorylation and have shown that it is required for full A20 inhibitory activity. We generated a phospho-specific antibody against this site, and we have shown that this phosphorylation occurs in vivo in response to a number of inflammatory stimuli. Our central hypothesis is that the IKK-dependent phosphorylation of A20 leads to a novel feedback mechanism to inhibit the NF-?B response such that too much inflammation does not occur at mucosal surfaces. Failure of IKK to phosphorylate A20 may lead to inflammatory pathology such as that seen in Crohn's Disease. This grant is designed to test this hypothesis. Mucosal immunity regulates the initial immune response to a variety of viral, bacterial and fungal pathogens. Dysregulation of mucosal immunity is an initiating event in a variety of inflammatory disorders including Inflammatory Bowel Disease, Asthma, Pyelonephritis and a number of primary immunodeficiencies. Understanding how this dysregulation occurs will have relevance both for understanding the pathophysiology of chronic inflammatory diseases and for preventing this dysregulation from occurring after exposure to pathogens. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE Mucosal immunity regulates the initial immune response to a variety of viral, bacterial and fungal pathogens. Dysregulation of mucosal immunity is an initiating event in a [unreadable] variety of inflammatory disorders including Inflammatory Bowel Disease, Asthma, Pyelonephritis, and a number of primary immunodeficiencies. Understanding how this dysregulation occurs will have relevance both for understanding the pathophysiology of chronic inflammatory diseases and for preventing this dysregulation from occurring after exposure to pathogens. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Upon pathogen exposure, the innate immune system initiates a cytokine response such that the adaptive immune system is tailored to eradicate that pathogen (4, 11, 24). Defects in the innate immune signaling pathways result in patient susceptibility to infectious disease (4, 11, 24, 26). IRAK4 (IL-1 Receptor Associated Kinase 4) is a key signaling molecule that links extracellular pathogen exposure to cytokine transcription and release, and recently, a series of unrelated children who develop recurrent pyogenic infections have been found to have mutations in the IRAK4 gene (6, 15, 16, 22, 25). The mutations in IRAK4 cause C-terminal truncations and result in either the absence of the kinase domain of the molecule or the absence of the total IRAK4 protein (6, 15, 16, 22, 25). IRAK4 is part of a signaling pathway linking the Toll-like Receptors to the NF?B pathway and the MAP Kinase pathways. Mice genetically deleted for IRAK4 show an inability to respond to a variety of pathogen-associated molecules including LPS, peptidoglycan, viral RNA and bacterial DNA. In addition, while showing a normal TNF response, these mice cannot respond to IL-1 (30-33). Surprisingly, patients with mutated IRAK4 show a subtly different phenotype than that found in the mouse as these patients are predominantly susceptible to recurrent gram-positive pyogenic bacterial infections (6, 15, 16, 22, 25, 26). While IRAK4's kinase activity plays a clear role in these children's immunodefiency, the role of IRAK4's kinase activity in TLR signaling is unclear (13, 14, 18, 19, 27). Conflicting data has been published on the requirement of IRAK4 kinase activity for TLR responses and IRAK4's in vivo substrates and kinetics of activation are unknown. Given the importance of IRAK4 in mediating innate immunity, and given the fact that children with immunodeficiency caused by an IRAK4 mutation all show a deletion in the kinase domain, understanding the kinetics, spatial localization, and specificity of IRAK4's kinase activity will be important. It will also be important to determine the features that cause differences in IRAK4's role in gram-positive and gram-negative infections. The central hypothesis of this grant application is that IRAK4's kinase activity is responsible for effective signaling responses to gram-positive organisms and that differences in this kinase activity (in regards to activity and spatial localization) may underlie the infectious pathology in children with IRAK4 deficiencies. This grant aims to develop a novel in vivo signaling reporter such that IRAK4's kinase activity can be studied in vivo. If successful, this pilot grant will lead to insights regarding IRAK4's role in immunodeficiencies and will generate reagents that will help carry the work forward in animal models. PUBLIC HEALTH RELEVANCE: People with immunodeficiency syndromes are highly susceptible to infection with bacteria, fungi and viruses. They do not respond normally to these agents, and their immune systems cannot defend them against these pathogens. A mutation in a gene called IRAK4 gives rise to a newly discovered immunodeficiency syndrome. Patients with mutations in IRAK4 develop numerous recurrent infections to fever-causing bacteria. 43% of these patients die in childhood. It is important to understand the function of IRAK4 so that we can better understand the cause of this immunodeficiency syndrome and ultimately, better treat this immunodeficiency syndrome. This grant aims to develop novel reagents with which to study IRAK4 so that this immunodeficiency syndrome can be better understood.

DESCRIPTION (provided by applicant): Aberrant NOD2 signaling causes granulomatous inflammatory disease. Patients with loss-of-function NOD2 alleles are prone to the development of Crohn's disease, an inflammatory disorder of the gastrointestinal tract. In contrast, patients with gain-of-function NOD2 mutations develop Early Onset Sarcoidosis (EOS), an inflammatory disorder characterized by noncaseating granulomas that cause lung, liver and eye damage. The fact that both loss-of-function polymorphisms and gain-of-function mutations both cause inflammatory diseases is likely due to the fact that NOD2 functions as a rheostat to help maintain normal immunologic homeostasis. This rheostat function begins upon bacterial invasion of the cell whereupon NOD2 binds to a breakdown product of bacterial peptidoglycan. This activates NOD2 such that it can modulate the innate immune system to help tailor the adaptive immune response to eradicate the offending pathogen. Either too much or too little NOD2 activation can be deleterious, and this imbalance is central to the development of inflammatory disease. NOD2 and its obligate kinase RIP2 are part of a positive regulatory circuit in which intracellular bacterial recognition causes the NOD2:RIP2 complex to be activated. In addition to stimulating autophagy, bacteriocidal activity, MHC Class II presentation and MAPK activation, the NOD2:RIP2 complex activates NF-?B. Both NOD2 and RIP2 are NF-?B regulated genes, and as such, their activation causes a positive feedback loop in which activation of NOD2:RIP2 stimulates further activation and further inflammation. Additionally, NOD2 and RIP2 expression are stimulated by a variety of mediators of inflammation, including TNF and IFN. Given this, in the prior granting period, we hypothesized that inhibiting this positive regulatory circuit might be efficacious in treating inflammatory disease. We were successful in identifying nanomolar inhibitors of RIP2's kinase activity. Despite this, a troubling fact remains: We still don't know what the kinase activity of RIP2 is doing in the cell. Some studies have shown that the kinase activity is dispensable for NOD2 activity while others have shown that it's essential. Our work has helped clarify this as we showed that RIP2 was misclassified as a serine-threonine kinase. It is actually a dual specificity kinase, meaning that t phosphorylates serines, threonines and tyrosines. Our own work has shown that inhibition of RIP2 attenuates the acute NOD2 inflammatory response. While my lab has found that RIP2's kinase activity helps regulate NF-?B, we don't know its role in regulating other NOD2-driven responses like autophagy or MAPK signaling. The uncertainty regarding RIP2's kinase activity takes on added importance given the interest of pharmaceutical companies in inhibiting RIP2 in inflammatory diseases like sarcoidosis, asthma, IBD and inflammatory arthritis. Understanding the kinase activity is essential if the goal is to inhibit RIP2 in inflammatory disease and to then determine efficacy and response in those diseases. This knowledge is also essential to predict outcomes of RIP2 inhibition in inflammatory disease. This grant application aims to answer these key questions.

DESCRIPTION (provided by applicant): NF-kB signaling lies at the center of Immunodeficiency and Inflammatory diseases. A key regulator of NF-kB signal transduction, NEMO, has recently been found to be mutated in an Immunodeficiency Disorder called called Anhidrotic (hypohydrotic) Ectodermal Dysplasia with Immunodeficiency (EDA-ID). Patients with EDA-ID are susceptible to infection with gram-positive organisms, and molecularly, a dysfunctional NF-kB signaling pathway, owing to hypomorphic mutations in nemo is to blame. While the NF-kB pathway is so well-studied that it has become a paradigm for inflammatory signal transduction, this paradigm has shifted in recent years with the recognition that this pathways signal transduction strength and coordination is critically dependent on Lysine-63 (K63)-linked polyubiquitination. NEMO was very recently shown to contain a ubiquitin binding domain that is essential for NF:B signaling, and NEMO, itself, is K63-polyubiquitinated on two sites in response to innate immune stimulation (NOD2 (Crohn's Disease-susceptibility protein) and Toll-like Receptor (TLR) activation). This NEMO ubiquitination is necessary for optimal NF:B signaling (1, 2, 6, 13, 26). The spectrum and location of point mutations of nemo in EDA-ID suggest that biochemically, they may be interfering with ubiquitin binding by NEMO and/or K63-linked polyubiquitination of NEMO in response to innate immune stimuli. In addition, the varied immunologic phenotypes and immunodeficiencies in EDA-ID suggest cell-type specificity in regards to the particular mutations. Lastly, it has been difficult for the field to uncouple ubiquitin binding by NEMO from K63-linked polyubiquitination of NEMO as it relates to function and ultimately, NF:B-induced gene expression. All these difficulties point to the need to systematically evaluate EDA-ID- associated NEMO mutations in regards to ubiquitin binding, K63-linked polyubiquitination of NEMO and NF-kB-associated gene expression. These difficulties also point to a need for an in vivo model of defective NEMO ubiquitination to decipher its role in in vivo NF:B-dependent gene expression and inflammatory and immunodeficiency disorders. The grant application aims to answer these key questions. PUBLIC HEALTH RELEVANCE: The ability to determine the genetics of susceptibility to infectious disease has led to the identification of novel forms of immunodeficiency, and the challenge is to translate these genetic findings into biochemical mechanisms of disease to both better understand the disease and allow better treatment for that disease. NEMO, a protein that is central to the major inflammatory signaling pathway is mutated in a disease called EDA-ID, a disease that is characterized by susceptibility to bacterial infections. This grant aims to determine how NEMO is faulty in EDA-ID and to generate a mouse model that might mimic many of the features of EDA-ID in hopes that this might lead to a better understanding of and better treatments for this disease.

Project 2, headed by Dr. Derek Abbott, will test the alternative hypothesis that exaggerated N0D2 signaling leads to chronic intestinal inflammation. This project will investigate the dysregulation of the Nod2 gene at the molecular level. Lack of coordination between inflammatory signaling pathways influences the development of CD. We recently published that the E3 ubiquitin ligase ITCH, causes K63-linked polyubiquitination of RIP2, and this event downregulates active NOD2:RIP2 complexes. Mice in which Itch is genetically lost (itchy mice) develop inflammatory disease at mucosal surfaces (including intestinal inflammation). We have data showing that ITCH-/- mice develop gastritis, ileitis and colitis and that drugs that inhibit RIP2 tyrosine phosphorylation, such as tarceva and iressa, inhibit the exaggerated N0D2 responses. The central hypothesis of this project is that ITCH downregulates NOD2;RIP2-induced NFDB signaling, and that CD results when this downregulation is lost. Aim 1 will study this ITCH-induced ubiquitination event to determine the biochemistry and physiologic function of ITCH-induced RIP2 ubiquitination. In Aim 2, we will determine the role of tyrosine phosphorylation of R1P2 and the role that pharmacological inhibition of this phosphorylation plays in ITCH-induced R1P2 ubiquitination and N0D2- induced cytokine responses. In Aim 3, we will characterize the Gl inflammation of the Itchy mouse and will determine whether tarceva or iressa can alleviate chronic intestinal inflammation in these mice. Our preliminary data shows that Itchy mice have increased gastrointestinal permeability. This increased permeability allows MDP leakage into the lamina propria and causes prolonged NFkappaB activation to occur. We hypothesize that tarceva and iressa will inhibit exaggerated lamina propria N0D2 activation in the itchy mice and alleviate the chronic inflammation. The overall objective of this project is to determine the biochemistry of the ITCH:RIP2 interaction, the physiologic significance of this interaction, and most importantly, whether this interaction can be inhibited pharmacologically to ameliorate chronic intestinal inflammation.

? DESCRIPTION (provided by applicant): The field of glycomics is a technically challenging field which commonly utilizes specialized reagents and equipment, thereby limiting its widespread adoption in secondary fields of impact. Moreover, many of the advances have emphasized chemical synthesis, macromolecular interactions, and improved mass spectroscopy methodology, which has pushed the field forward in terms of our biochemical understanding but has also done little to lower the barrier of entry for outside investigators. Due to the lack of expansion into other fields, the impact of the glycome on in vivo biology and our knowledge of how the glycome fits into disease mechanisms remains in its nascent phase. To address this gap, we must strive to provide a more complete set of tools for biomedical investigators to explore the influence of the glycome in any biological system or pathway, and we believe that the way forward is to provide facile genetic technology applicable to both murine and human biology without the requirements for specialized expertise. We therefore propose to utilize CRISPR-based molecular biology to create Glycome-Enabled KnockOut (GEKO) targeting technology for the selective ablation of glycome- associated genes across multiple species. The primary deliverables from the proposed GEKO technology are: (1) 100 validated CRISPR targeting constructs targeting 50 genetic loci for the selective ablation of glycome- associated genes, (2) validated targeting sequences for agile application of the CRISPR technology to other systems, and (3) 50 human HEK293 GEKO cell lines with validated knockout of each glycome-associated gene. These tools will enable glycobiologists and non-glycobiologists alike to easily manipulate the glycome through genetics in vitro, ex vivo, and in vivo; thereby providing an inexpensive gateway to explore the influence of glycans on the function of proteins, cells, and tissues in a wide variety of normal and disease contexts ranging from cancer and neurodegeneration to intracellular signaling and immunity.

PROJECT SUMMARY Ubiquitination, innate immunity and inflammatory bowel disease are tightly linked. Dysregulation of ubiquitination pathways causes dysfunction of innate immune signaling which in turn, affects the mucosal immunity of the gastrointestinal tract. In the previous granting cycle, our lab has made important contributions in helping understand this process of dysregulation. Given this, in the next granting period, we will focus on an E3 ligase newly discovered to regulate the NOD:RIP2 signaling pathway. xIAP is a RING-domain containing E3 ubiquitin ligase. Its role in pediatric Crohn's disease was discovered when whole exome sequencing was performed on pediatric patients with severe, steroid-resistant IBD. A significant subset of these patients were found to have both truncating and missense xIAP mutations, and early work showed that patients with mutations in the BIR2 domain of xIAP had deficient NOD:RIP2 signaling. While this work is incredibly important, the published literature is incomplete and contains many inconsistencies. For instance, Crohn's disease-causing xIAP mutations are present throughout the gene and only the mutations in a small region of xIAP (the BIR2 domain) affect NOD:RIP2 signaling. Additionally, while NOD2 polymorphic patients are at an increased risk of developing Crohn's disease, the high level of NOD2 polymorphism in the population (approximately 9%) means that most Crohn's disease- associated NOD2 carriers have normal GI mucosal immunologic homeostasis. In contrast, xIAP mutation- carriers develop a severe form of inflammatory bowel disease before the age of 5 years old that is intractable to current treatments. Lastly, while NOD2 polymorphisms phenotypes are largely restricted to Crohn's disease, xIAP mutation carriers also develop X-linked lymphoproliferative syndrome, a serious life-threatening inflammatory response to cytomegalovirus exposure. For all these reasons, we hypothesize that defective NOD:RIP2 signaling is not the only pathway affected by xIAP mutation and by understanding the pathways affected, we can discover new targets for inflammatory bowel disease treatment. It will be important to characterize genotype:phenotype relationships of xIAP mutation carriers. It will be important to determine the ubiquitination patterns disrupted by xIAP mutations in innate immune and inflammatory signaling, and it will be crucial to determine if inhibition of WT xIAP will be efficacious in the treatment of inflammatory bowel disease.

Abstract Infectious bacteria and inflammatory insults can be so toxic to an organism that they require an immediate response. One such response, called pyroptosis, causes an inflammatory cell death that both alerts the immune system to the immediate threat and also ensures a continued inflammatory effort. In classical pyroptosis, Caspase-1 or Caspase-11 (Caspase-4/5 human) cleaves the pore forming protein, Gasdermin D (GSDMD). This cleaved GSDMD then oligomerizes to form a pore in cellular membranes. Gasdermin D pore formation allows the acute release of IL-1 from the cell, while also destroying membrane integrity such that mitochondrial damage and electrolyte imbalances quickly kill the cell. Implicit in this is that should pyroptosis be blocked, either genetically or pharmacologically, neutralization of the pathogen is so important to organismal survival that alternative mechanisms to initiate cytokine release and inflammatory cell death must have evolved. We are only now beginning to understand these compensatory responses and their role in shaping the immune response. Our preliminary data, with support from the preliminary data from the other three projects in this PPG application, will establish that mechanisms of compensation involve both Gasdermin redundancy and alternative protease cleavage events. We hypothesize that these compensatory mechanisms are cell-type specific. We further posit that they influence the timing and amplitude of cytokine release, the timing and inflammatory capacity of the resulting cell death and the in vivo immune response to inflammatory stimuli. The overall hypothesis of this application is that mechanisms to compensate for loss of pyroptosis alter the inflammatory and immunologic response to an inflammatory insult. We further hypothesize that this compensation helps establish myeloid cell homeostasis and that disruption of these compensatory mechanisms influences the pathologic development of Myelodysplasia and subsequent Leukemia progression. The long-term goal of this work is to better understand how pyroptotic compensatory mechanisms influence the inflammatory response and immunologic homeostasis in hopes of better understanding how to manipulate these pathways in disease.

Project Summary Case Western Reserve University School of Medicine (CWRU SOM) has provided integrated MD-PhD training since 1956 with many distinguished alumni, including two Nobel laureates. The NIH-funded CWRU Medical Scientist Training Program (MSTP) was started in 1975 (T32 GM07250) and has 107 current students. Research and clinical training occurs at CWRU and affiliated institutions (University Hospitals Cleveland Medical Center, Cleveland Clinic, MetroHealth Medical Center and the Louis Stokes Veteran?s Administration Medical Center). These sites are well equipped for cutting edge research and state of the art clinical training. The CWRU MSTP curriculum integrates scientific and clinical instruction in all three phases: both PhD and MD course work during the first two years, substantial personalized clinical instruction as well as research during the PhD years, and clinical clerkships, clinical electives and research electives in the final phase. PhD mentors provide individualized research training. Active mentors are well supported by NIH research grants or other funding sources; they also have established research training track records or, in the case of Initial Mentors, will be paired with a Senior Mentor to insure effective mentoring of trainees and to develop the mentoring skills of new trainers. Criteria for acceptance of MSTP students include superior academic performance and evidence of strong commitment and skills for research. Acceptance by the MSTP automatically admits a student to the SOM and any of the 14 MSTP-affiliated PhD programs. All CWRU MSTP students receive full financial support for stipend, tuition and health fees for all phases of the program (8.3 years mean duration), and the program provides laptop computers and travel awards for students to attend national research meetings. Numerous MSTP-specific program activities supplement the PhD and MD curricula. The program emphasizes development of leadership skills through student governance, faculty mentoring, and student-led program activities.