CXCL14 is a primordial CXC-type chemokine that induces migration of immature dendritic cells (DCs), tissue resident macrophages, and natural killer cells. It has been reported that CXCL14 plays multiple roles in tumor suppression, exacerbation of autoimmune arthritis, and induction of obesity-associated insulin resistance. However, underlying molecular mechanisms of these phenomena remain to be elucidated. Although CXCL14 binds to the CXCL12 receptor CXCR4 with high affinity, chemoattractive activity of CXCL14 is much weaker than CXCL12.
In this study, we newly discovered that CXCL14 specifically binds to CpG DNA and activates Toll-like receptor 9 (TLR9), thereby inducing inflammatory cytokines such as IL-6, IL-12 p40, and TNFa in mouse bone marrow-derived dendritic cells (BMDCs). Cell surface expression levels of MHC class-II and CD86 in BMDCs were also enhanced by the combination of CpG DNA and CXCL14. In this experimental setting, CXCL14 interacted with certain classes of CpG DNA, but not with RNA ligands for TLR3 and TLR8. In addition, this CpG DNA-cooperative activity was not present in CXCL8 and CXCL12, excluding a nonspecific interaction between CpG DNA and an alkaline chemokine.
In BMDCs and inguinal lymph node DCs, intracellular transport of a low concentration of CpG DNA was greatly enhanced by the addition of CXCL14. Confocal microscopical analyses revealed that CpG DNA and CXCL14 mainly co-localized in EEA1+ endosome and LAMP1+ lysosomal compartments where TLR9 is present. Furthermore, we demonstrated that CXCL14 binds to CpG DNA in vitro in the neutral pH condition with high affinity (Kd=9.8 nM). This interaction was completely dissociated in pH 6.0, implying that CpG DNA can be released and passed to TLR9 in the endosome and lysosome. Consistent with our hypothesis, induction of IL-12 p40, MHC class-II, and CD86 by the combination of CpG DNA and CXCL14 was not observed in BMDCs derived from TLR9 knockout (KO) mice. Moreover, after systemic administration of CpG DNA, plasma concentration of IL-12 p40 and frequency of MHC-class II+CD11c+CD8+DCs in spleen were significantly decreased in CXCL14-deficient mice when compared to littermate control mice.
Taken together, these results demonstrated that CXCL14 serves as a specific carrier for CpG DNA into conventional DCs for activating TLR9-mediated adaptive immune system. This is also the first demonstration of a DNA sensing function of chemokine. The combination of CXCL14 and CpG DNA would be a promising vaccine adjuvant for enhancing immnunosurveillance against pathogens and malignant cancers.
Tanegashima:Tokyo Metropolitan Institute of Medical Science: Patents & Royalties. Takahashi:Tokyo Metropolitan Institute of Medical Science: Patents & Royalties. Tsuji:Tokushima University: Patents & Royalties. Shigenaga:Tokushima University: Patents & Royalties. Otaka:Tokushima University: Patents & Royalties.
Asterisk with author names denotes non-ASH members.