Functional characterization of ferret CCL20 and CCR6 and identification of chemotactic inhibitors

Abstract

CCL20 is currently the only known chemokine ligand for the receptor CCR6, and is a mucosal chemokine involved in normal and pathological immune responses. Although nucleotide sequence data are available for ccl20 and ccr6 sequences from multiple species, the ferret ccl20 and ccr6 sequences have not been determined. To increase our understanding of immune function in ferret models of infection and vaccination, we have used RT-PCR to obtain the ferret ccl20 and ccr6 cDNA sequences and functionally characterize the encoded proteins. The open reading frames of both genes were highly conserved across species and mostly closely related to canine sequences. For functional analyses, single cell clones expressing ferret CCR6 were generated, a ferret CCL20/mouse IgG2a fusion protein (fCCL20-mIgG2a) was produced, and fCCL20 was chemically synthesized. Cell clones expressing ferret CCR6 responded chemotactically to fCCL20-mIgG2a fusion protein and synthetic ferret CCL20. Chemotaxis inhibition studies identified the polyphenol epigallocatechin-3-gallate and the murine γ-herpesvirus 68 M3 protein as inhibitors of fCCL20. Surface plasmon resonance studies revealed that EGCG bound directly to fCCL20. These results provide molecular characterization of previously unreported ferret immune gene sequences and for the first time identify a broad-spectrum small molecule inhibitor of CCL20 and reveal CCL20 as a target for the herpesviral M3 protein.

Isolation, characterization, and functional analysis of ferret lymphatic endothelial cells

“…Chemotaxis assay was performed using murine L1.2 cells engineered to express ferret CCR6 as described (Qin et al, 2013). Briefly, a total of 200,000 of L1.2.fCCR6 responder cells were loaded on top of the porous membrane (ChemoTx ® Disposable Chemotaxis System, pore size 5 µm, NeuroProbe) in triplicate and incubated at 37°C/5% CO 2 for 2 hr in a humid chamber.…”

“…Chemotactic index (CI) was calculated relative to control wells with no chemokine. Chemically synthesized ferret CCL20 (Qin et al, 2013) was used as a control.…”

“…Despite the utility and increasing use of this animal model there is still a major lack of ferret-specific reagents for use in research, despite efforts that have been invested to obtain reagents to enable development of ferret-specific assays at the cellular and molecular levels (Bruder et al, 2010;Camp et al, 2012). Molecular cloning and phylogenetic analysis of ferret immune-related genes provides tools to assess the inflammatory cytokine and chemokine profiles in infected animals and determine their importance in disease progression and/or clearance of infection (Nakata et al, 2008;Qin et al, 2013). The expression of functional TLRs by human LECs (Pegu et al, 2008) has highlighted that the LE could be a target for new vaccine adjuvancy strategies, alongside monocytes and DCs.…”

TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

“…Studies have identified cross-reactive antibodies that recognize populations of ferret leukocytes, such as CD8, CD11␤, CD44 and CD25, and cytokines IFN␥, TNF␣, IL4 and IL8 (Rutigliano et al, 2008;Martel and Aasted, 2009;Pillet et al, 2011). Cloning and sequencing of ferret cytokine genes have enabled molecular approaches targeting the corresponding mRNAs (von Messling et al, 2006;Danesh et al, 2008;Nakata et al, 2008;Ochi et al, 2008;Qin et al, 2013). Expression of cytokine and chemokine genes has been assessed in ex vivo samples following infection of naïve or vaccinated ferrets with influenza virus or SARS-CoV by microarray analysis Fang et al, 2010;Rowe et al, 2010).…”

Regulation of CD4+FOXP3+ T cells by CCL20/CCR6 axis in early unexplained recurrent miscarriage patients

“…Importantly, CCR6 is highly expressed in Tregs, which suppress immune responsiveness (Kleinewietfeld et al, 2005;Frick et al, 2013;Li et al, 2013). Chemokine (C-C motif) ligand 20 (CCL20) is a chemokine ligand known to attract CCR6 + -expressing cells by inducing migration (Marsigliante et al, 2013) and binding to endothelial cells (Qin et al, 2013). Their role in inducing immune tolerance in pregnancy has drawn increased attention recently.…”