Haitao Wen, Ph.D. - US grants

DESCRIPTION (provided by applicant): The incidence of chronic metabolic diseases such as obesity and diabetes has increased dramatically and constitutes one of the major threats to global health. Recent studies have indicated that cell stress response plays an essential role in the pathogenesis of type 2 diabetes (T2D). Oxidative stress and endoplasmic reticulum (ER) stress cooperatively promote cell dysfunction, apoptosis and insulin resistance. While the activators and downstream effects of cell stress have been partially characterized, much is unknown regarding the molecular mechanisms by which the cell stress responses are regulated. Our lab and others have recently characterized the NLR (NBD-LRR) family of proteins, which have been shown to mediate the cell stress response to microbes and environmental stressors. My preliminary data indicates that NLRX1, a mitochondria-localized NLR protein, promotes ER stress response by mediating the generation of mitochondrial reactive oxygen species (mROS). Mechanistically, NLRX1 directly associates with ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) and TRAF6 (TNF receptor-associated factor 6), both of which are important in mitochondrial respiratory chain assembly. NLRX1-deficient (Nlrx1-/-) cells are protected from ER stress-inhibited insulin-PI3K-Akt pathway. Moreover, Nlrx1-/- mice were protected from obesity- induced insulin resistance by high-fat diet (HFD) feeding. Therefore, I hypothesize that NLRX1 mediates mROS generation by facilitating ECSIT-TRAF6 function, which subsequently promotes ER stress, and that the NLRX1-mediated cell stress responses impair insulin signaling in insulin target tissues. I will employ T2D (HFD feeding and leptin-deficient ob/ob mice) animal models to examine the function of NLRX1-mediated cell stress responses in insulin resistance. I will examine how NLRX1 controls ECSIT function and mROS generation. The goal of the proposal is to examine the mechanism of the interaction between NLRX1-mediated oxidative stress and ER stress in promoting cell dysfunction and a defect in insulin signaling. The proposed genetic and biochemical analyses and animal model studies will provide novel insights into the regulation and function of metabolic signaling pathways. Further studies could lead to the identification of new therapeutic targets and ultimately help develop rational, mechanism-based treatment strategies that target obesity and diabetes.

DESCRIPTION (provided by applicant): The incidence of chronic metabolic diseases such as obesity and diabetes has increased dramatically and constitutes one of the major threats to global health. Recent studies have indicated that cell stress response plays an essential role in the pathogenesis of type 2 diabetes (T2D). Oxidative stress and endoplasmic reticulum (ER) stress cooperatively promote cell dysfunction, apoptosis and insulin resistance. While the activators and downstream effects of cell stress have been partially characterized, much is unknown regarding the molecular mechanisms by which the cell stress responses are regulated. Our lab and others have recently characterized the NLR (NBD-LRR) family of proteins, which have been shown to mediate the cell stress response to microbes and environmental stressors. My preliminary data indicates that NLRX1, a mitochondria-localized NLR protein, promotes ER stress response by mediating the generation of mitochondrial reactive oxygen species (mROS). Mechanistically, NLRX1 directly associates with ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) and TRAF6 (TNF receptor-associated factor 6), both of which are important in mitochondrial respiratory chain assembly. NLRX1-deficient (Nlrx1-/-) cells are protected from ER stress-inhibited insulin-PI3K-Akt pathway. Moreover, Nlrx1-/- mice were protected from obesity- induced insulin resistance by high-fat diet (HFD) feeding. Therefore, I hypothesize that NLRX1 mediates mROS generation by facilitating ECSIT-TRAF6 function, which subsequently promotes ER stress, and that the NLRX1-mediated cell stress responses impair insulin signaling in insulin target tissues. I will employ T2D (HFD feeding and leptin-deficient ob/ob mice) animal models to examine the function of NLRX1-mediated cell stress responses in insulin resistance. I will examine how NLRX1 controls ECSIT function and mROS generation. The goal of the proposal is to examine the mechanism of the interaction between NLRX1-mediated oxidative stress and ER stress in promoting cell dysfunction and a defect in insulin signaling. The proposed genetic and biochemical analyses and animal model studies will provide novel insights into the regulation and function of metabolic signaling pathways. Further studies could lead to the identification of new therapeutic targets and ultimately help develop rational, mechanism-based treatment strategies that target obesity and diabetes.

Project Summary/Abstract Sepsis is the most common cause of mortality in many intensive care units and is responsible for more than 250,000 deaths in the United States annually. Microbial infection and trauma are the most common causes of sepsis. Sepsis is characterized by an exaggerated innate immune response leading to a cytokine storm. Recent studies suggest that activation of the innate immune cells causes vigorous metabolic changes towards increased glucose utilization. Elevated glucose metabolism is also a common feature in the initial state of sepsis. However, the role of glucose metabolism reprogramming in the regulation of innate immune function and its relevance to sepsis is poorly understood. In this Proposal, we aim to study the role of two individual glucose metabolism pathways in microbial sepsis, the hexosamine biosynthesis pathway (HBP) and the pentose phosphate pathway (PPP). Our preliminary studies revealed essential roles of HBP-associated O- GlcNAc (O-linked ?-N-acetylglucosamine) signaling and PPP in antagonizing inflammatory response and bacterial spreading, respectively. We further identified nuclear factor E2-related factor-2 (Nrf2) as a critical mediator of both HBP and PPP pathways. Therefore, promoting the activities of HBP and PPP pathways through pharmacological activation of Nrf2 may represent a promising therapeutic regimen for treating microbial sepsis. We hypothesize that 1) HBP-associated O-GlcNAc signaling inhibits the innate immune activation through O-GlcNAcylation of RIPK3 (receptor-interacting serine/threonine kinase 3); 2) PPP is required for macrophage bacterial killing and host survival in sepsis by mediating caspase-1 activation; 3) Genetic and pharmacological activation of these glucose metabolism pathways is effective in the treatment of microbial sepsis. Cecal ligation and puncture-induced polymicrobial sepsis model will be employed to examine the role and functions of glucose metabolism pathways. We will test whether dimethyl fumarate (DMF) treatment plays a protective role in sepsis-induced mortality. The goal of the proposal is to examine the function and mechanism of two glucose metabolism pathways on macrophage bacterial killing and inflammation, both of which are key determinants of host survival. Results of these studies will provide novel insights into the regulation and function of glucose metabolism signaling, which can potentially lead to the identification of new therapeutic targets in the treatment of microbial sepsis.

Project Summary/Abstract: Role of O-GlcNAc Transferase-Mediated Innate Immune Response in Colonic Inflammation and Tumorigenesis Ulcerative colitis is a common form of inflammatory bowel disease that affects about 1 person per 600 in the U.S. Patients suffering from colitis demonstrate a significantly increased risk of colorectal cancer (CRC). A common feature of colitis and CRC is enhanced activation of immune signaling and inflammatory cytokine production. Studies of immune system metabolism (immunometabolism) in recent years have identified a tight link between metabolic reprogramming and inflammation. It has been shown that activation of immune cells is accompanied by robust metabolic changes towards increased glucose utilization. However, the role of individual glucose metabolism pathways in colonic inflammation and tumorigenesis remains unknown. The hexosamine biosynthesis pathway is a unique glucose metabolism pathway that leads to the generation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). O-linked GlcNAc (O-GlcNAc) transferase (OGT) is a key enzyme that utilizes UDP-GlcNAc to modify its target proteins, a process called O-GlcNAcylation. In this Proposal, we aim to study the role of O-GlcNAc signaling in innate immune activation of colonic inflammation and tumorigenesis. Our preliminary studies revealed increased OGT and decreased cullin 3 (Cul3) E3 ubiquitin ligase expression in inflamed colon tissue during chemically-induced colitis. Deletion of OGT or Cul3 in myeloid cells attenuated or exacerbated disease severity during colitis, respectively. The underlying mechanism may involve O-GlcNAcylation-mediated inhibition of signal transducer and activator of transcription 3 (STAT3), a central transcription factor in the regulation of intestinal inflammation and tumorigenesis. Through microarray and biochemical analyses, our recent study (JEM, In Press) identified Cul3 as a negative regulator of OGT expression and STAT3 O-GlcNAcylation by inhibiting nuclear factor E2-related factor-2 (Nrf2). Our studies further revealed lysine-63 (K63)-linked ubiquitination of OGT mediated by TNF receptor associated factor 6 (TRAF6), a crucial E3 ubiquitin ligase regulating immune signaling. Based on the known effects of STAT3 in colitis and CRC, and the well-established cross-regulation between O-GlcNAcylation and phosphorylation, we propose that altered signaling through a Cul3-OGT axis leading to aberrant STAT3 O- GlcNAcylation plays an essential role in innate immune activation during colitis and associated colon cancer. We hypothesize that 1) increased OGT expression and STAT3 O-GlcNAcylation in myeloid cells exacerbates colonic inflammation and inflammation-driven colon cancer; 2) myeloid-derived Cul3 negatively regulates OGT expression and promotes activation of a STAT3-IL-10 axis; and 3) TRAF6-mediated OGT K63 ubiquitination is important for STAT3 O-GlcNAcylation. We will employ both a chemically-induced model and the IL-10 deficiency model in mice to investigate acute colitis and inflammation-driven colon cancer. It is our expectation that this work will bring about a better understanding of metabolic regulation of intestinal inflammation and tumorigenesis and contribute to the generation of new strategies for the treatment of these diseases.

Project Summary/Abstract Sepsis is the most common cause of death in intensive care units and represents a major burden to the US health care system. Microbial infection and trauma are the most common triggers of acute systemic inflammatory response that eventually leads to end organ failure and mortality in sepsis. Mitochondria, a highly metabolically active organelle, have been shown to play an essential role in the innate immune function and inflammatory response. Robust changes in mitochondrial metabolism (mito-metabolism) occur during clinical and experimental sepsis. However, the signaling mechanism leading to alterations in mito-metabolism and its functional consequence on the pathogenesis of sepsis are poorly understood. In this Proposal, we aim to study the detrimental effects of metabolic abnormalities mediated by mitochondrial calcium signaling on the innate immune function during microbial sepsis. Our preliminary studies identified the mitochondrial calcium uniporter (MCU), a key calcium channel for mitochondrial calcium uptake, as an essential regulator of bacterial killing and septic inflammation. We found that genetic ablation of MCU resulted in improved phagosomal bacterial killing and less interleukin 1? (IL-1?) secretion due to elevated LC3-associated phagocytosis (LAP). Mechanistically, MCU inhibits the assembly of LAP complex by promoting mitochondrial metabolite acetyl- coenzyme A (acetyl-CoA) generation via the pyruvate dehydrogenase (PDH). Therefore, blockade of MCU or PDH function may represent a promising therapeutic regimen for treating microbial sepsis. The goal of the proposal is to examine the function and mechanism of mitochondrial calcium signaling-mediated mito- metabolism on phagosomal bacterial killing and inflammation, both of which are key determinants of host survival during microbial sepsis. We hypothesize that 1) decreased acetyl-CoA generation in Mcu-deficient macrophages promotes LAP formation via protein acetylation-dependent mechanism; 2) enhanced LAP formation promotes phagosome member repair mechanism to limit excessive inflammasome-mediated IL-1? cleavage; 3) pharmacological inhibition of PDH by CPI-613 is effective in the treatment of microbial sepsis. Cecal ligation and puncture-induced polymicrobial sepsis model will be employed to examine the role and functions of MCU-mediated acetyl-CoA metabolism. We will test whether PDH inhibition by CPI-613 plays a protective effect on sepsis-induced mortality, as well as sepsis-induced immunosuppression. Results of these studies will provide novel insights into the regulation and function of mito-metabolism, which can potentially lead to the identification of new therapeutic targets in the treatment of microbial sepsis.

Project Summary/Abstract Sepsis is the most common cause of death in many intensive care units and represents a major burden to the US health care system. Despite advances in intensive care technology and mechanical ventilator support, pharmacological options for sepsis are limited, which reflects an insufficient understanding of host-dependent mechanisms that underlie this pathophysiological disorder. A wealth of evidence from recent clinical and experimental sepsis studies indicates that a prolonged immunosuppressive status, due to profound cell death and dysfunction of lymphocytes, is a critical determinant of sepsis-elicited mortality. Therefore, restoration of lymphocyte cell survival and functions by blocking immune inhibitory molecule(s) may represent a promising therapeutic regimen for treating sepsis. In this Proposal, we aim to study the role and mechanism of a previously unrecognized immune inhibitory molecule called SUSD2 (sushi domain containing 2) in promoting sepsis-induced immunosuppression. Through an unbiased gene profiling assay, our previous study has identified a cell surface molecule SUSD2 whose high expression correlated with an immunosuppressive phenotype in an experimental cancer model. In this Proposal, we observed elevated Susd2 expression in T lymphocytes in experimental septic animals and patients with sepsis compared to non-septic controls. Genetic deletion of SUSD2 (Susd2?/?) resulted in a significantly improved animal survival and attenuated apoptosis of T lymphocytes in the cecal ligation and puncture (CLP)-induced polymicrobial sepsis model. Mechanistically, our preliminary studies discovered an inhibitory effect of SUSD2 on interleukin-2 receptor (IL-2R) signaling, a well- established pathway essential for T cell survival and effector functions. The goal of the proposal is to examine the causal effect of SUSD2 on cell death and dysfunction of T lymphocytes in microbial sepsis. We hypothesize that 1) elevated SUSD2 expression leads to diminished IL-2-dependent cell survival and effector functions in T lymphocytes, resulting in a sustained immunosuppressive state and worse disease outcome in sepsis; 2) enhanced activation of STAT5 (signal transducer and activator of transcription 5) and BATF (basic leucine zipper ATF-like transcription factor) signaling maintains cell survival and effector functions in Susd2?/? T cells post sepsis; 3) SUSD2 blockade reverses sepsis-induced cell death and dysfunction of T lymphocytes. Single-cell RNA sequencing analysis of circulatory immune cells will be performed to examine the inhibitory effect of SUSD2 on T cell response post sepsis at the single-cell level. We will test whether treatment with a neutralizing anti-SUSD2 antibody reverses dysfunctional T cells isolated from septic patients. Results of these studies will provide both experimental and clinical evidence to support a promoting function of SUSD2 on sepsis-induced immunosuppression, which can potentially lead to the development of new approach for sepsis treatment.