The purposes of the project are to investigate the biological roles of members of the chemokine family of cytokines, to understand basic aspects of chemokine receptor signaling, to use chemokine receptors to understand the relationships between the trafficking patterns and broader biological functions of subsets of effector/memory T cells, and to understand the contributions of the chemokine system to infectious and inflammatory/autoimmune disease and cancer. Chemokines and their receptors are critical for leukocyte trafficking, and our experiments are directed to understanding how manipulating the chemokine system could be used to treat diseases in which leukocytes play a critical role. In addition, some chemokine receptors are expressed by cancer cells, and these receptors can potentially be exploited for diagnosis and as targets for therapy. In FY 2016, we have continued to investigate mouse models of skin inflammation that have features of psoriasis. One model involves injection of a cytokine, IL-23, which appears to have a role not only in psoriasis, but also in other immune-mediated diseases, such as Crohns disease. We and others had described that the chemokine receptor CCR6 is expressed by IL-23-dependent T cells that produce the cytokines IL-17 and IL-22. IL-22 and IL-17 are important in producing disease in the mouse psoriasis model, and are thought to be important in causing tissue injury in some autoimmune diseases. We have shown previously that mice lacking CCR6 are resistant to the IL-23-induced disease, but the mechanism has been unclear. We further characterized the origins and types of monocyte/macrophage and dendritic cell subsets that infiltrate the skin and contribute to the psoriasis-like inflammation. We have identified a subset of inflammatory dendritic cells that enter the outer layers of the skin (epidermis), determined that these and other dendritic cells important for the inflammatory response are derived from infiltrating monocytes, and studied how these cells are recruited. We have found that CCR6 supports the recruitment of the monocytes that give rise to monocyte-derived dendritic cells that are important for IL-23-induced dermatitis. In the last year we have investigated this model further. We determined that the initial responding cells are resident gamma/delta T cells that produce IL-17 and that an important activity of the monocyte-derived dendritic cells is their production of inflammatory cytokines. We extended our observations to a second mouse model of psoriasis using a drug that activates the immune system (imiquimod), and which is known to induce psoriasis-like changes after application to the skin of patients. Here we have also found that CCR6 and monocyte-derived cells are required for the psoriasis-like dermatitis. We have also extended our observations to humans by examining lesional skin from patients with psoriasis and have identified monocyte-derived cells in both dermis and epidermis. In the last year we have continued studies of the process whereby effector/memory T cells migrate from the blood, across the layer of endothelial cells that line the inside off the blood vessel, into a site of tissue infection or inflammation. We have characterized subsets of CD8+ (killer) T cells that are particularly efficient at migrating across the endothelium, and we have identified some of the molecular features of the surfaces of these cells, including the combination of chemokine receptors, that make them so efficient. We have continued work on a transcription factor that regulates a number of genes that encode proteins important for the ability of these cells to migrate across endothelium into inflammatory sites. Most recently, we have begun to extend these studies to subsets of human CD4+ (helper) T cells, initially characterizing the relationships among patterns of expression of chemokine receptors, selectin ligands, and transcription factors of interest. In the last year we have completed our analysis of signaling by the chemokine receptor CXCR6. CXCR6 is a chemokine receptor expressed by a variety of leukocytes that we identified initially as a co-receptor for HIV and SIV and that is thought to play a role in the migration of T lymphocytes into inflamed tissue. CXCR6 and other chemokine receptors are G protein coupled receptors (GPCRs), which are polypeptide chains that contain seven helical segments that traverse the cell membrane. CXCR6 has an amino acid sequence unusual for chemokine receptors and/or other class A GPCRs at the cytoplasmic terminus of the third transmembrane helix, a region important for G protein binding, namely aspartic acid (D)126 arginine (R)127 phenylalanine (F)128 isoleucine (I)129 valine (V)130 in place of the typical five amino acid sequence, DRY(tyrosine) L(leucine)/I/V A(alanine). Binding to and activating G proteins inside the cell are critical mechanisms used by GPCRs in order to produce signals that affect the cells behaviors. There are four subfamilies of G proteins, and CXCR6 signals using members of the Gi/o subfamily. By mutating amino acid residues in CXCR6 and expressing and analyzing the mutated proteins in different cell types, we discovered that the atypical F128 and V130 are functionally linked, such that deleterious effects on ligand binding and receptor function in CXCR6-F128Y (phenyalanine changed to tyrosine) were corrected in the double mutant, CXCR6-F128Y/V130A; that these residues and D126 are important for selectivity in the receptors coupling to members of the Gi/o subfamily; that the effects of mutations on ligand binding, receptor function and use of specific Gi/o proteins were cell-type specific; and that, in contrast to the mutant receptors, the wild-type receptor used all available Gi/o proteins independent of cell type. Overall, our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the third transmembrane helix, and suggest that the native sequence allows for preservation of this function across different cellular environments.
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