Nunzio Bottini - US grants

DESCRIPTION (provided by applicant): A missense single-nucleotide polymorphism, C1858T in the PTPN22 gene is primarily associated with a wide range of human autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, and juvenile idiopathic arthritis. The PTPN22 gene encodes the lymphoid tyrosine phosphatase LYP, which is expressed only in white blood cells and acts as a gatekeeper of T lymphocyte activation. The molecular mechanism by which LYP tempers T lymphocyte activation involves the formation of a complex between LYP and the negative regulatory kinase Csk. We reported that the autoimmune-predisposing LYP-W620 variant cannot bind Csk, and more recently we found that the same variant is a gain-of-function form of the phosphatase. The increased inhibition of TCR signaling by LYP- W620 may lead to weaker signaling and therefore a failure to delete autoreactive T cells during thymic selection and/or insufficient activity of regulatory T cells. We hypothesize that a specific small-molecule inhibitor of LYP might be useful to revert the effects of LYP-W620 at the central and/or peripheral level, and prevent the emergence, or reappearance, of autoreactive T cells. Such an inhibitor would be widely applicable, both in helping to elucidate the biological role(s) of LYP and as a therapeutic in the treatment of many human autoimmune disorders. Unfortunately, there is currently no LYP assay available that is highly sensitive, continuous, and amenable to HTS. In the present proposal we will carry out all the preliminary work needed to begin a screening of NIH molecular libraries for inhibitors of LYP, and in particular we will (1) set up a reliable assay of LYP activity and (2) optimize it for HTS.

[unreadable] DESCRIPTION (provided by applicant): We recently discovered that a single nucleotide polymorphism in the human lymphoid tyrosine phosphatase LYP (encoded by the PTPN22 gene) is associated with type 1 diabetes, an autoimmune disease that arises from T lymphocyte-mediated destruction of insulin-producing B-cells in the pancreas (Nature Genetics 36, 337-338). LYP is expressed only in white blood cells and acts as a gatekeeper of T lymphocyte activation. The molecular mechanism by which LYP tempers T lymphocyte activation involves the formation of a complex between LYP and the negative regulatory kinase Csk. We have found that the autoimmune- disposing LYP variant, LYP-W620, cannot bind to Csk and our new preliminary data show that LYP-W620 regulates TCR signaling in a different way than the more common LYP-R620. We hypothesize that LYP genetic polymorphism alters the threshold of T cell activation, thereby predisposing carriers to a misdirected immune response against autoantigens in pancreatic islets or other tissues. In this proposal we wish to generate mouse models carrying T cell-specific expression of the 2 genetic variants of the phosphatase and perform their phenotypic analysis. This will allow us to gain important insights about the effect of the polymorphism on TCR signaling, T cell development and differentiation, and the mechanism of association between the R620W polymorphism and type 1 diabetes. The new mouse models will then be available for further immunological analyses and for studies aimed at validating new pharmacologic approaches to disease prevention or intervention in carriers of the LYP-W620 variant. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Decreased signaling through the T cell receptor at the thymic level causes rheumatoid arthritis in the SKG mouse -which carries a mutation of ZAP-70 able to impair the activation of the kinase- as well as in human patients carrying sporadic mutations of ZAP-70. We reported that the low molecular weight protein tyrosine phosphatase (LMPTP) is a positive regulator of T cell receptor signaling. In T cells LMPTP is able to dephosphorylate the tyrosine kinase ZAP-70 on the negative regulatory Tyr-292, and increase ZAP-70 activation after TCR engagement. In this proposal we will test the hypothesis that increased expression of LMPTP at the thymic level under control of the LCK proximal promoter is able to rescue the arthritis phenotype of SKG mice. As a result of our experiments we expect to confirm the relevance of LMPTP as a modulator of ZAP-70 and T cell receptor signaling, and validate the LMPTP as a candidate gene/genetic modifier in rheumatoid arthritis. [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Protein tyrosine phosphatases (PTP) are emerging drug targets for the prevention and/or treatment of cancer, diabetes, autoimmune diseases and other common human ailments. High throughput screening (HTS) for PTP inhibitors is currently performed using enzyme-based assays and simple non-peptide phosphotyrosine (pTyr) analogs, such as para-nitrophenylphosphate (p-NPP) and 6,8-difluoro-4-methylumbiliferyl phosphate (DiFMUP). An obvious limitation of these universal substrates is their lack of enzyme specificity. Also at present none of the available PTP substrates is amenable to detection of intracellular PTP activity, thus precluding the development of cell-based PTP assays. There is an urgent need for new specific PTP assays, suitable to both enzyme-based and cell-based HTS for small molecule PTP inhibitors. We recently generated a novel fluorescent pTyr analog, phosphocoumaryl-amino-propionic acid (pCAP), which can be incorporated into peptides. Peptides containing pCAP are excellent and specific PTP substrates. pCAP-based PTP assays are highly sensitive and direct, and can be used to detect intracellular PTP activity. In this proposal we will optimize pCAP-peptides as substrates for novel, specific PTP assays suitable to enzyme-based and cell-based HTS of PTP inhibitors. We will show that pCAP-peptides have better specificity profiles than DiFMUP when used as PTP substrates in enzyme-based assays. We will also show that specificity of pCAP peptide can be further improved using a library-screening approach. Highly specific pCAP-peptides will then be used to validate an innovative cell-based PTP assay and optimized for HTS of small molecule PTP inhibitors. We will use the lymphoid tyrosine phosphatase (LYP) as our model PTP as our recent work renders LYP a novel drug target for human autoimmunity. We predict that our new cell-based PTP assay will provide a revolutionary paradigm in HTS for PTP inhibitors in both the academic and industrial setting. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): A missense single-nucleotide polymorphism in the PTPN22 gene causing an R620W substitution in the PTPN22 protein is one of the strongest genetic risk factors for rheumatoid arthritis. No consensus model has yet emerged on how PTPN22 increases risk of rheumatoid arthritis. As PTPN22 is a negative regulator of signaling through the T cell receptor, most studies to date have focused on the effect of the polymorphism on adaptive immune cell signaling. Here we wish to explore the concept that PTPN22-W620 impinges on the pathogenesis of rheumatoid arthritis through an action on myeloid cell signaling as well. Recent studies from our laboratory revealed a novel function of PTPN22 as a promoter of type 1 interferon release after engagement of myeloid cell toll-like receptors. Within this pathway, rheumatoid arthritis- predisposing PTPN22-W620 behaves as a loss-of-function variant, and macrophages and dendritic cells expressing PTPN22-W620 display a defect in toll-like receptor-induced type 1 interferon production. Type 1 interferon production by myeloid cells triggers several immunosuppressive mechanisms that protect from rheumatoid arthritis in mouse models. These include IL-27-mediated inhibition of arthritogenic Th17 cell differentiation and IL-1Ra-mediated suppression of inflammation in the rheumatoid joint. Here we hypothesize that loss of function of PTPN22 in dendritic cells and macrophages predisposes to rheumatoid arthritis by reducing type 1 interferon-dependent immunosuppressive mechanism. We will test our hypothesis by exploring whether loss of function of PTPN22 promotes myeloid cell-dependent differentiation of arthritogenic Th17 cells (Aim 1) and IL-1 mediated joint inflammation (Aim 2). In Aim 3 we will assess whether loss-of-function of PTPN22 or carriage of PTPN22-W620 impairs the immunoregulatory ability of human myeloid cells. By defining immunological mechanisms that are defective in carriers of the PTPN22-W620 variant, our study will suggest new strategies for personalized therapy of rheumatoid arthritis in genetically predisposed individuals.

ABSTRACT The objective of this renewal grant proposal is to perform preclinical validation of an inhibitor of the low molecular weight protein tyrosine phosphatase (LMPTP) as a therapeutic treatment for obesity-associated diabetes. Diabetes caused by insulin resistance is a major cause of obesity-associated morbidity. The currently available anti-diabetic treatments are often insufficient to maintain glycemic control in type 2 diabetes patients; thus there is a major unmet medical need for agents that lower insulin resistance. Targeting tyrosine phosphatases that inhibit insulin signaling by dephosphorylating the insulin receptor (IR) is considered a potential strategy for treating type 2 diabetes by sensitizing the cellular response to insulin. The tyrosine phosphatase LMPTP inhibits insulin signaling by dephosphorylation of the activation motif of the IR. In humans, genetic polymorphisms encoding low LMPTP activity associate with lower glycemic levels. We discovered that LMPTP is a key promoter of obesity-induced insulin resistance by inhibiting IR phosphorylation in the liver. Mice carrying global and liver- specific LMPTP deletion gain comparable weight to control littermate mice when fed a high-fat diet, however display substantially improved glucose tolerance and lower fasting insulin levels. During the previous grant funding cycle, through a screening of the Molecular Libraries Probe Production Centers Network chemical library followed by an extensive hit-to-lead optimization campaign, we exploited unique structural features of LMPTP to generate a new class of inhibitors that is orally bioavailable, exclusively selective for LMPTP over other tyrosine phosphatases, and lowers insulin resistance and restores glucose tolerance in obese diabetic mice. Our long- term goal is to advance an LMPTP inhibitor to the clinic as a therapeutic option for patients with type 2 diabetes. Thus here we apply for continued grant funding to collect preclinical efficacy and safety data and perform chemical optimization in order to generate a candidate for investigational new drug-enabling studies. We will accomplish this objective by pursuing 3 Specific Aims: 1) validation of the LMPTP inhibitor lead efficacy in human hepatocytes and in mouse models of obesity-induced diabetes; 2) validation of the LMPTP inhibitor lead safety in mice; 3) generation of an optimized LMPTP inhibitor lead through iterative cycles of structure-guided medicinal chemistry.

In the last five years, dozens of new genes have been identified that increase or decrease the risk of autoimmune diseases. Many laboratories are now trying to determine how certain autoimmune-predisposing or -protective haplotypes (i.e. combinations of gene polymorphisms) affect the expression, splicing, and function of the encoded protein. However, the current approach, based on studying primary cells from individuals carrying different haplotypes, often gives unclear answers. This grant will try to validate a novel approach to functional genetics of autoimmunity, which is complementary to that for primary cells. Our strategy is to assess functional differences between autoimmune-predisposing and protective genetic variations by directly studying full-length gene haplotypes transfected in human cells. The full-length gene will be carried on a bacterial artificial chromosome (BAC) and manipulated before transfection in order to carry the desired variations. The result will be a series of cell clones that carry different full-length gene haplotypes. The clones will then be subjected to a variety of functional studies. We already have a prototype of the system in-hand, and here we apply for the funding needed to validate our approach and optimize it for large-scale application. Thus we will focus on two well-known autoimmunity genes relevant for T cell function, PTPN22 and IL2RA, and use Jurkat T cells as a model cell line. In Aim 1 we will use our prototype assay to assess the functional effect of gene variations in PTPN22 and IL2RA. In Aim 2 we will experiment with long-PCR-based approaches in the attempt to achieve cloning of haplotypes into BACs directly from patient genomic DNA. This will eliminate the need for gene manipulation in order to obtain the desired haplotypes. Our approach is novel and simple, and does not require collection of primary cells. Once validated and optimized, it can be applied to all autoimmunity genes using a variety of cell lines, and even broadly applied to functional genetics studies of complex human diseases.

ABSTRACT Fibroblast-like synoviocytes (FLS) are joint-lining non-hematopoietic cells that in rheumatoid arthritis (RA) contribute to local joint inflammation and damage. There is interest in discovering FLS-directed therapeutic agents that could be combined with current immunosuppressant disease-modifying anti-rheumatic agents (DMARDs). This renewal proposal focuses on understanding the mechanism of action and regulation of the tyrosine phosphatase PTPRS, which plays a critical role in FLS migration and invasion. In the first cycle of this grant, we have shown that PTPRS is an important regulator of FLS aggressiveness during RA and developed an approach to modulate PTPRS function in rheumatoid FLS. Our studies showed that on the surface of RA FLS, PTPRS is kept in an inactive state through specific binding to the heparan sulfate (HS)- proteoglycan syndecan-4 through a mechanism called the proteoglycan switch. Treatment of RA FLS with a decoy fragment of PTPRS encompassing its two most extracellular proteoglycan-binding immunoglobulin domains (called Ig1&2) causes detachment of PTPRS from syndecan-4. This leads to PTPRS-dependent inhibition of migration and invasiveness via dephosphorylation of the PTPRS substrate ezrin. In vivo administration of Ig1&2 reverses arthritis in multiple mouse models via a non-immunological mechanism. We also find that PTPRS expression is inhibited on RA FLS by tumor necrosis factor alpha, suggesting that PTPRS expression is regulated by joint inflammation. In the second cycle of the grant, we would like to deepen our knowledge of the mechanism of action and regulation of PTPRS in FLS and RA. Here we propose to a series of mechanistic studies in primary human FLS and mouse models of RA aimed at understanding the regulation of PTPRS expression in RA FLS (Aims 1) and how Ig1&2 and PTPRS regulate FLS-induced inflammation in arthritis (Aim 2). We will also use biochemical, structural, and cellular biology approaches to understand the key molecular determinants for PTPRS regulation by syndecan-4 (Aim 3). Our long-term goal remains to understand the biology of PTPRS in RA FLS, which will help to complete the validation of the PTPRS-regulated pathway as a therapeutic target for RA.

ABSTRACT Systemic sclerosis is an autoimmune disease, characterized by progressive fibrosis of the skin and internal organs. There is currently no FDA-approved agent to prevent the progression of fibrosis in systemic sclerosis. The pathogenesis of systemic sclerosis involves a complex interplay of abnormalities of the immune system, blood vessels and fibroblasts. One well-studied mechanism of fibrosis in systemic sclerosis consists of the excessive activation of the transforming growth factor beta (TGFbeta) signaling pathway in fibroblasts, which leads to excessive collagen deposition and transformation of fibroblasts in alpha-smooth muscle actin- expressing myofibroblasts. Our laboratory focuses on protein tyrosine phosphatases, enzymes that control signal transduction by removing phosphate from phosphorylated tyrosines, thus balancing the action of protein tyrosine kinases. The role of tyrosine phosphatases in systemic sclerosis has remained mostly unaddressed. This project stems from the observation that a tyrosine phosphatase called PTP4A1 is overexpressed in dermal fibroblast from systemic sclerosis patients and plays a pro-fibrotic function in fibroblasts ex vivo and in vivo. Mechanistically, PTP4A1 promotes TGFbeta signaling by forming a complex with SRC that inhibits basal SRC auto-phosphorylation and degradation. The objectives of this grant proposal are to dissect the molecular details of the PTP4A1-SRC complex (Aim 1), and to validate PTP4A1 as a key player in SSc fibrosis via experimentation in mice (Aim 2) and further assessment of SSc clinical specimens (Aim 3). The long-term goal is to validate PTP4A1 and/or its downstream pathway as possible targets to prevent and treat fibrosis in systemic sclerosis.

DESCRIPTION (provided by applicant): Fibroblast-like synoviocytes (FLS) are key players in mediating inflammation and joint destruction in rheumatoid arthritis (RA). There is an increased level of attention to this cell type as the possible target of a new generation of anti-RA therapie, which would be used in combination with immunomodulators to help control disease without increasing immune-suppression. The behavior of FLS is controlled by multiple interconnected signal transduction pathways. Several of these pathways involve reversible phosphorylation of proteins on tyrosine residues, which is the result of the balanced action of protein tyrosine kinases (PTKs) and phosphatases (PTPs). PTKs are well-known to be key mediators of FLS growth and invasiveness, and more recently, they are emerging as promising drug targets for RA. On the other hand, almost no work has been done on the PTPs in FLS. This grant application focuses on a transmembrane PTP called PTPRS. PTPRS is expressed at low or undetectable levels in hematopoietic cells but we find it to be highly expressed in FLS. The extracellular domain of PTPRS binds to proteoglycans in the extracellular matrix and binding to different proteoglycans results in differences in the intracellular functions of the phosphatase. This PTPRS-mediated mechanism of regulation of intracellular signaling by the extracellular matrix is called the proteoglycan switch. We find that the proteoglycan switch regulates in a PTPRS-dependent way the adhesion and invasiveness of FLS. We also find that interfering with the proteoglycan switch in vivo leads to decreased severity of arthritis in a mouse model. Our working hypothesis is that PTPRS is a key regulator of RA FLS destructive behavior. The objectives of this project are to establish PTPRS as a key regulator of extracellular matrix-induced signals in FLS and provide proof of principle that the proteoglycan switch is a drug target for RA. In Aim 1 we will clarify whether PTPRS and the proteoglycan switch affect various aspects of FLS pathophysiology that are relevant to the pathogenesis of RA In Aim 2 we will identify the substrate of PTPRS that is affected by engagement of the proteoglycan switch and look into the signaling pathways controlled by PTPRS in a proteoglycan-dependent way In Aim 3 we will provide proof of principle that targeting PTPRS helps control RA activity in vivo and can be used as a combination therapy with anti-TNF therapy. The results of this project will shed light on the relationship between extracellular matrix composition and intracellular signaling in FLS. The disease-relevant long-term goal is to validate a novel approach to FLS targeted combination therapy for RA.

ADMINISTRATIVE CORE ABSTRACT The Microenvironment in Arthritis Resource Center (MARC) is a Rheumatic Diseases Research Resource- based Center located in San Diego that will support research efforts of local investigators studying joint biology in rheumatic diseases. To ensure that the resources available in the MARC are optimally utilized by the local community, the Center will be managed by an Administrative (ADM) Core. This Core will be responsible for providing Center leadership, managing the activities of the MARC Resource Cores, facilitating communication between the MARC Resource Cores and their users, and bringing to the field both new investigators and new collaborative research efforts. Aim 1 of the ADM Core is to coordinate and oversee the activities of the three MARC Resource Core facilities. The ADM Core will manage the Resource Cores, which includes organization of recharge services offered by the Cores and financial oversight of services and internal research activities as well coordinate all the reporting and evaluation Core activities. Aim 2 of the ADM Core is to engage and facilitate the investigative efforts of a large research base. The ADM Core will act as the primary conduit for communication between the MARC and the scientific researchers in the community. The Core will not only be responsible for disseminating information about the Center and its resources to the local investigators, but also for evaluation of the services and assessing feedback of the users. Aim 3 of the ADM Core is to support an Enrichment Program designed to spark the interest of local investigators in research on rheumatic diseases of the joint by providing vouchers, Pilot and Feasibility grants, a seminar series and a local arthritis conference. Through these efforts, the ADM Core will be essential for enabling the MARC to fulfill its vision of expanding and deepening the scientific investigation of joint biology and arthritis being conducted by the San Diego research community.

JOINT CELL ISOLATION CORE ABSTRACT The Joint Cell Isolation (JCI) Core of the Microenvironment in Arthritis Resource Center (MARC) will facilitate research in the San Diego community focused on the biology of the joint at the tissue and single cell level. The JCI Core will serve as a local resource for technologies such as laser capture microdissection (LCM) and flow sorting from joint tissues. No other shared resource in the San Diego area offers this type of expertise or service. The JCI Core will be led and managed by faculty and staff with extensive experience in the application of these technologies to research on rheumatologic diseases. The Core will provide a series of operator- assisted services for LCM, tissue disaggregation, and flow sorting from human and mouse joint tissues. The Core will also help with experimental design, collaborate with investigators to tailor protocols to the needs of the individual researcher, and engage in its own internal research to develop new cutting-edge methodologies for investigation into the joint microenvironment. The Aims of this Core are 1) to assist investigators in the design of experiments to isolate tissue fragments and/or single cells from human or mouse joints, 2) to assist investigators in the execution of experiments involving isolation of cells in situ from joint tissue histologic sections via LCM, and 3) to assist investigators in the execution of experiments involving disaggregation of joint tissue and sorting of specific cellular populations. The Core will also facilitate interactions with other shared resources to help investigators with needs related to the workflow for these experiments, such as procurement of synovium or cartilage samples, histological preparation of tissues, and obtaining samples that can be analyzed by transcriptomics or other technologies like chromatin accessibility. By providing services and expertise, the MARC JCI Core will enable researchers in the local community to use techniques previously unavailable to them and will add a new dimension to their research programs.

OVERALL ABSTRACT We will establish a Rheumatic Diseases Research Resource Center at the University of California, San Diego (UCSD). The theme of our Microenvironment in Arthritis Resource MARC (MARC) is to facilitate translational research focused on the joint microenvironment in arthritis. Limited knowledge of in situ joint biology and pathogenic mechanisms in rheumatic diseases has prevented development of novel biomarkers and therapies that target cellular interactions within joint structures. The MARC will address this gap by stimulating interest in the joint microenvironment among a large interdisciplinary group of well-funded scientists from multiple institutions in San Diego. The goal of the MARC is to provide the San Diego scientific community with a comprehensive local framework to plan, perform and analyze a large variety of joint microenvironment studies using invasive and non-invasive technology and foster novel, collaborative, and interdisciplinary arthritis- relevant research. The first two Aims of the Center are to facilitate studies on tissue microenvironment by providing access to (Aim 1) cutting-edge joint imaging technology and approaches to isolate single cells and tissue from joints and to (Aim 2) a dedicated computational service focusing on integration of diverse multi- dimensional datasets. Aim 3 is to lower the barrier for new investigators to enter the field and to foster collaboration between rheumatic disease-focused scientists across San Diego. This will be accomplished via an Enrichment Program, which includes vouchers for core services, pilot and feasibility grant funding, and creation of a new arthritis seminar series and a regional arthritis-focused symposium to stimulate collaboration. The San Diego science community has a deeply rooted combination of creativity, dynamism, collaborative attitude and focus on translation. Thus, our Center will be uniquely positioned to facilitate the novel science that will be transformative for the diagnosis and treatment of rheumatic diseases.

ABSTRACT The objective of this grant application is to understand how loss-of-function genetic variants of the PTPN2 gene -encoding the T cell-protein tyrosine phosphatase (TC-PTP)- enhance risk of rheumatoid arthritis (RA). PTPN2 is ubiquitous, and very highly expressed in immune cells and is a critical negative regulator of Janus kinases and signal transducers and activators of transcription downstream multiple cytokine receptors. In order to model the mechanism of action of PTPN2 autoimmunity-associated variants in RA, we assessed mice carrying Ptpn2 haploinsufficiency (Ptpn2+/- mice), which causes a loss of expression of PTPN2 comparable to the human PTPN2 RA-risk variants. We found that in the SKG RA model- characterized by CD4 T cell-driven disease- partial loss of function of PTPN2 caused significant enhancement of arthritis severity. By leveraging conditional Ptpn2 haploinsufficiency and fate-mapping mice, we showed that the phenotype of SKG.Ptpn2+/- mice is due to enhanced inflammation-induced FoxP3+ regulatory T cell (Treg) instability, a process known to lead to conversion of peripheral FoxP3+ Treg into pathogenic FoxP3- ?exTreg? expressing interleukin-17 (IL-17). We have evidence that the enhanced conversion of Ptpn2+/- Tregs into IL-17-producing ?exTreg? is due to increased STAT3 phosphorylation after stimulation with IL-6 and potentially other inflammation-induced factors. Here we apply for funding to further understand the mechanism of action of PTPN2 in Treg instability and the pathogenesis of RA via mouse immunology and cell signaling studies. In Aim 1 and 2 we will elucidate the mechanism and topology of enhanced inflammation-induced instability and pathogenicity of SKG.Ptpn2+/- Treg. In Aim 3 we will assess whether overexpression of PTPN2 in Treg can reverse the Treg and arthritis phenotype induced by Ptpn2+/- in SKG mice. Our long-term goal is to acquire knowledge of PTPN2 functional genetics to enable the discovery of personalized and non-immunosuppressive therapies for RA patients carrying genetic PTPN2 risk variants.

ABSTRACT The objective of this grant application is to explore the plasticity of Th17 in arthritis. Interleukin-17A (IL-17A) producing Th17 are present often in large numbers in the synovium of patients with rheumatoid and psoriatic arthritis. However, targeting of IL17A is generally insufficient to fully control joint inflammation in these conditions. One potential scenario is that in the context of worsening joint inflammation, Th17 undergo conversion into pathogenic IL17A-negative cell populations, collectively called exTh17. The conversion of Th17 into exTh17 has been documented in the context of neuroinflammation and infections, and locally produced IL-7 was described as a key promoter of Th17 plasticity in the lung. However, the occurrence of Th17 plasticity in arthritis and its potential role in perpetuating synovial inflammation remain unknown. We generated a novel fate-mapping mouse model of autoimmune arthritis, which allows to follow the conversion of Th17 into exTh17, and collected preliminary data suggesting that Th17 undergo significant loss of IL17A expression and conversion into exTh17 in the context of synovial inflammation. We also identified candidate exTh17 subpopulations which might contribute to perpetuate joint inflammation despite their loss of IL17A expression. Here we will leverage our mouse model to collect pilot evidence about the immunoregulatory and/or pathogenic role of exTh17 in synovial autoimmune inflammation (Aim 1). Also, we will explore whether IL-7 or other factors produced by synovial fibroblast play a role in inducing conversion of Th17 into exTh17 (Aim 2). Our long-term goal is to leverage knowledge of local immune cell phenotypes at various stages of disease to enable stage-specific and personalized therapies of arthritis to minimize non specific immunosuppression.

PROJECT SUMMARY The objective of this proposal is to determine the mechanism of action of the low molecular weight protein tyrosine phosphatase (LMPTP) in cardiac fibrosis. Cardiac fibrosis is a major contributor to the pathogenesis of heart failure. In cardiac tissue, fibrosis prompts pathological changes that include dilation and hypertrophy, and ultimately leads to heart failure. There are no FDA-approved anti-fibrotic medications for heart failure; therefore, novel agents to alleviate cardiac fibrosis are a major unmet medical need in cardiology. This proposal focuses on the tyrosine phosphatase LMPTP, which is encoded by the ACP1 gene. LMPTP has been considered an inhibitor of signaling through receptor tyrosine kinases by dephosphorylation of tyrosine residues in their activation motifs. In humans, genetic polymorphisms in the ACP1 gene encoding for high LMPTP activity are known to promote myocardial hypertrophy. We previously reported that LMPTP expression is significantly upregulated in hearts of humans with end-stage heart failure. We generated the first LMPTP knockout mice and found that when subjected to blood pressure overload through transverse aortic constriction (TAC), they are protected from cardiac hypertrophy and failure, and develop substantially decreased fibrosis in the heart. We also found that inhibiting LMPTP with a small-molecule chemical inhibitor that we developed leads to reduced cardiac fibrosis, hypertrophy, and failure in TAC-treated mice. Taken together, these findings suggest a novel role for LMPTP as a promoter of cardiac fibrosis and failure. Here we propose a series of mechanistic experiments to elucidate the physiological and molecular mechanisms of action of LMPTP in fibrosis-associated heart failure. We will (Aim 1) demonstrate that LMPTP promotes cardiac fibrosis in multiple mouse models, (Aim 2) determine the cell type by which LMPTP promotes cardiac fibrosis, and (Aim 3) determine the molecular mechanism of action of LMPTP in promoting cardiac fibrosis and failure.