Characterisation of CXCL14 function and target cells in blood and tissues

Doctoral thesis English OPEN
Collins, Paul J. (2016)
  • Subject: R1

The human chemokine family consists of around 50 peptides that control the migratory patterns and positioning of all leukocytes. One such member of this family is CXCL14. Very highly expressed in many healthy tissues including skin, gut and kidney, loss of CXCL14 expression in chronic inflammatory conditions and certain forms of cancer has led to a proposed role for CXCL14 in immune surveillance at these sites. The function and target cells of CXCL14 are poorly defined however, largely because the identity of its receptor remains unknown. Here, I have combined the evaluation of chemotactic responses toward CXCL14 with detection of putative CXCL14 receptor(s) on the surface of cells using a synthetic, fluorochrome-conjugated CXCL14, to definitively identify CXCL14 target cells in human. Monocytes were identified as the major target cells in peripheral blood, 28.4 ± 6.1% Monocytes migrating toward 1 µM CXCL14 in ex vivo transwell chemotaxis assays compared to 3.01 ± 0.65% toward buffer alone (p=0.0031). Responses to CXCL14 also identified tissue phagocytes extracted from healthy human skin, including an apparently novel population of skin-resident CD14+ cells characterised by lack of CD45 expression. Screening of CXCL14-responsive cells by RNA sequencing for expression of G protein-coupled receptors revealed five major candidates for the CXCL14 receptor, all of which are orphan receptors; GPR35, GPR68, GPR84, GPR141 and GPR183. At present, I am in the process of testing these candidates in functional assays. Finally, I report on a novel ability of CXCL14 to potently synergise with other chemokines, particularly CXCL12. This ‘synergy’ with CXCL12 likely occurs via a direct interaction between CXCL14 and the receptor for CXCL12, CXCR4, which is broadly expressed on immune cells. This work identifies mononuclear phagocytes in blood and tissue as the primary targets for CXCL14, providing new and exciting insights into the role played by CXCL14 in immune surveillance of peripheral tissues.
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    .............................................................................................................................149 Figure 4.12. None of the five orphan receptors mediate chemotaxis toward CXCL14 or binding of AF-CXCL14......................................................................................151 Figure 4.13. Small hairpin RNA-mediated gene silencing. ....................................152 Figure 4.14. Surface expression of CCR2 and the chemotactic response to CCL2 in THP-1 cells is unaffected by PGE2 treatment........................................................154 Figure 5.5. CXCL14 synergises with CXCL12 in the induction of rapid cellular responses.............................................................................................................174 Figure 5.6. CXCL14 also synergises with the CCR7 ligands CCL21 and CCL19 in the induction of chemotactic responses in primary human T cells...............................175 Figure 5.7. CXCL14 synergises with the CCR7 ligands CCL21 and CCL19 in the induction of chemotactic responses in primary human B cells...............................176 Figure 5.8. CXCL14 synergises with CCL21 and CCL19 in the induction of chemotactic responses in 300.19 cells stably transfected with CCR7. ..................177 Figure 5.9. CXCL14 synergises with the CXCR5 ligand CXCL13 on primary B cells.

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