Haribabu Bodduluri - US grants

The Chemokines are a diverse gene family of chemotactic cytokines that regulate the migration and activation of leukocytes by interacting with cell-surface G-protein coupled receptors. They play a major role in the pathophysiology of many inflammatory disorders such as rheumatoid arthritis, asthma and other lung diseases. Recently, chemokine receptors CCR5 or CXCR4 were identified as essential co-receptors for the entry of human immunodeficiency virus HIV-1 into CD4 positive cells. While CCR5 is the target for the entry of primary viruses CXCR4 may be important in the progression to AIDS from asymptomatic infection. We have developed novel cellular models for understanding the molecular mechanisms of regulation of chemokine receptors. Chemokine receptors including CXCR4 are functionally expressed in a rat basophilic leukemia cell line (RBL-2H3) which displays many leukocyte activities. These studies have provided evidence that chemokine receptors cross-regulate each other's functions at multiple levels. The overall objective of this proposal is to delineate the pathways of CXCR4 signaling, desensitization and internalization using the RBL-2H3 model system. The role of phosphorylation in signaling and desensitization of the receptors will be determined in RBL-cells stably expressing epitope-tagged native or mutated CXCR4. The structural elements on the receptors that regulate internalization will be identified. Interaction of CXCR4 receptors with T-tropic HIV-1 envelope glycoprotein (GP120) and the peptides derived from it will be studied. The mechanisms by which other chemokine receptors and adhesion molecules regulate CXCR4 function and vice versa will be investigated. Cross regulation of chemokine receptors along with differential expression may be responsible for selective leukocyte accumulation in pathological conditions and cell type specificity in viral infection. Understanding the mechanism by which CXCR4 is regulated will provide novel targets and better rationale for therapeutic intervention in HIV-1 infected individuals and in other inflammatory disorders.

The chemokines are a family of structurally related peptides that interact through cell surface G-protein coupled receptors in leukocytes to mediate diverse biological and biochemical activities such as adhesion, directed migration and activation. They play a major role in the pathophysiology of many inflammatory disorders. Chemokine receptors CCR5 or CXCR4 were identified as essential co-receptors for the entry of human immunodeficiency virus HIV-1 into CD4 positive cells. While CCR5 is the target for the entry of primary viruses CXCR4 may be important in the progression to AIDS from asymptomatic infection. The overall objective of the parent proposal is to delineate the pathways of CXCR4 signaling, desensitization and internalization using the RBL-2H3 cells stably co-expressing epitope-tagged native or mutated CXCR4 along with human CD4. In this AIDS-FIRCA, Dr. Sozzani proposes to utilize many reagents (epitope-tagged, green fluorescent protein tagged native and mutated CXCR4 DNAs and transfected RBL cell lines) developed for the parent proposal to determine the role of MAPKs on cPLA2 activation by the ligand SDF-1 and the relevance of this pathway to leukocyte chemotaxis using both pharmacological and biochemical approach. The ability of the HIV-1 envelope glycoproteins (gp120) to induce second messenger formation in CXCR4-expressing RBL-2H3 will be investigated. Activation of MAPKs, and cPLA2 will be evaluated and correlated with the ability of gp120 proteins to induce cell migration. The parent laboratory is utilizing and HIV-1 infection permissive human astroglioma cell line, U87, transfected with human CD4 and native or mutated CXCR4 to determine the role of the signaling evens and internalization in HiV-1 infection. The studies on cPLA2 and MAPKs in RBLs will provide a basis for extension of the same to human cell lines in the parent laboratory and to determine the role of these pathways in HIV-1 infection.

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Neutrophils are found in large numbers in rheumatoid synovium and have been suggested to be involved in clinical signs of inflammation and pain associated with RA. Neutrophil activation during host defense and inflammation is mediated by G-protein coupled receptors (GPCRs) for chemoattractants. Leukotriene B4 (LTB4), a potent chemoattractant for neutrophils activates its receptor (BLT-1) to mediate diverse physiological effects in neutrophils. A second LTB4 receptor (BLT-2) with distinct antagonist specificity and tissue distribution was recently described. GPCRs are regulated by receptor phosphorylation leading to desensitization as well as down regulation. We will test the hypothesis that LTB4 acting through the high affinity receptor BLT-1 mediates its effects on neutrophils in RA, while LTB4 acting through BLT-2 modulates T-lymphocyte activation and function in RA. The goal of the current studies is to develop comprehensive in vivo and in vitro models to determine the role of LTB4 and the relative contributions of BLT-1 and BLT-2 in the development of murine RA. In specific aim 1 we will define the role of BLT-1 in the development and progression of collagen induced arthritis in the BLT-1 deficient mice we have already generated by targeted gene disruption. In specific aim 2 we will use the well-established RBL-2H3 cell model to determine the differences in signaling, desensitization, internalization and antagonist specificity of BLT-1 and BLT-2. These studies take advantage of the novel video microscopy and live cell imaging methods we have recently developed. Leukotrienes are involved in the pathophysiology of many acute and chronic inflammatory diseases such as systemic anaphylaxis, atherosclerosis, RA and asthma. Understanding the precise function of distinct LTB4 receptors in mice deficient in specific receptors and defining the role of these receptors in RA will identify novel targets for therapeutic intervention of RA.

DESCRIPTION (provided by applicant): Colon cancer is a leading cause of cancer related deaths in the United States. Exposure to a number of hazardous environmental agents and consumption of the Western diet are known to contribute to the pathogenesis of malignant colon cancer. While chronic inflammation is known to be a major promoter of tumor development the role of gut microflora in contributing to the colon cancer is unknown. Our laboratory has identified leukotriene B4 receptor, BLT1 as a major regulator of inflammation and host response to infections. In a mouse model of spontaneous intestinal cancer (ApcMin/+), we have made the observation that absence of BLT1 greatly enhances intestinal tumor development. This has led to the central hypothesis that Absence of BLT1 enhances colon tumor development by defective immune surveillance and/or altered microbial gut homeostasis. This apparently paradoxical result that loss of a proinflammatory mediator increases inflammation and tumor development suggests that the ever changing micro environment of intestine can have a major influence on the development of colorectal cancer. The current proposal will test this hypothesis in three specific aims. In aim 1, we will examine the mechanisms of leukotriene B4 pathway mediated protection of intestinal cancers in ApcMin/+ mice. Aim 2 will establish the immune functions in tumor bearing mice in the context of BLT1+/+ and BLT1-/- mice. Aim 3 will test the novel concept that defective host response and altered gut microbiota are responsible for rapid development of colon tumors in BLT1-/- mice. These studies will provide a comprehensive model system to examine the interplay of inflammation and infections to the development of colon cancer. Since the mouse ApcMin/+ model is highly related to human colon cancers the result will have direct and immediate impact for the treatment of human colon cancer. PUBLIC HEALTH RELEVANCE: Leukotrienes are involved in the pathophysiology of many acute and chronic inflammatory diseases such as systemic anaphylaxis, asthma, atherosclerosis and arthritis. The novel finding that leukotriene B4-leukotriene B4 receptor1 axis may have a protective function in the development of colon cancer offers a unique opportunity to understand the role of these mediators in tumor development. Understanding the precise function of distinct leukotriene B4 receptors in mice and defining the role of these receptors in host defense and shaping the gut microflora will provide novel approaches to the treatment of colon cancer.

ABSTRACT Exposure to irritant crystals such as crystalline silica (CS) induces inflammation of lungs leading to chronic, irreversible disease-silicosis. Silicosis is incurable due to impaired particle clearance resulting in chronic lung inflammation and may eventually lead to many inflammatory diseases such as arthritis and lung cancer. CS exposure leads to recruitment of inflammatory cells to lungs, of which neutrophils are key mediators of the pathophysiology of silicosis. Our results in mouse models showed that CS-induced pulmonary inflammation is attenuated in leukotriene B4 receptor1 (BLT1-/-) deficient. Extensive in vivo studies in CS exposed mice and ex vivo studies in isolated bone marrow derived mast cells and macrophages suggested an intricate interplay of mediators such as LTB4, IL-1? and neutrophil active chemokines regulate silicosis. Moreover, our results suggest that CS-induced LTB4 production by mast cells sets the pace of sterile neutrophilic inflammation in the lung. Such inflammation is further perpetuated by IL-1? and CXC and CC neutrophil chemokines. The hypothesis of this proposal is that ?CS-induced lipidosome and inflammasome pathways function in consort with chemokines to coordinate sustained chronic sterile inflammation in silicosis?. In the proposed research plan in two specific aims we will perform studies to 1) delineate the molecular links between phagosome, lipidosome and inflammasome pathways in regulating the CS-induced production of neutrophil chemoattractants (LTB4, IL-1? and CXCL1). Signaling intermediates unique to each of these pathways will be identified as they can serve as potential drug targets to block neutrophil recruitment into silicotic lungs. In aim2 we will identify the cellular mechanisms that integrate the lipid/cytokine/chemokine (LTB4, IL-1? and CXCL1) responses into chronic inflammation in vivo. Select inhibitors of the leukotriene pathway will be tested for their efficacy in controlling CS-induced neutrophilic inflammation in preclinical mouse models. This knowledge will facilitate development of immunotherapeutic strategies to fight silicosis.

Biomedical research areas including the study of metagenomics, inflammation and pathogenesis are resource and technology intense. All COBRE-supported investigators require state of-the-art resources to successfully compete with investigators from other resource-rich institutions for NIH support. The primary goal of the Functional Microbiomics Core (FMC) is to establish an integrated service center that functions as a critical resource for all Microbiome, Inflammation and Pathogenicity COBRE investigators. Since all the COBRE projects utilize mouse models to explore the inter-relationships between inflammation and pathogenesis, and the contribution of microbiota to this process, the development of a facility to house and maintain germ-free and gnotobiotic mice is crucial to the FMC. An anaerobic culture facility will support research on gnotobiotic mice that requires specific colonization with fecal transplants or defined anaerobic bacteria. Further, in Aim 2, we propose to establish an integrated metagenomics center for microbiota. This facility will prepare genomic DNA following unified protocols and generate 16S rRNA gene amplicon sequences, and provide bioinformatics support for all microbiota analysis. The FMC will also support all COBRE projects with analysis of their biological samples for inflammatory markers as well as histopathological examination of tissue sections. Education and training of junior investigators and the university wide research community in microbiota research is an important goal of the FMC. Towards this goal, the FMC will organize seminars as well as informal meetings that will serve as a forum for highlighting the scientific and technical advances in the field. Finally, the FMC plans to become an independent service center at UofL to support the needs of all researchers in this area of research