The epidermis is the outermost layer of the skin and the body’s primary barrier to external pathogens; however, the early epidermal immune response remains to be mechanistically understood. We show that the chemokine CXCL14, produced by epidermal keratinocytes, exhibits robust circadian fluctuations and initiates innate immunity. Clearance of the skin pathogen Staphylococcus aureus in nocturnal mice was associated with CXCL14 expression, which was high during subjective daytime and low at night. In contrast, in marmosets, a diurnal primate, circadian CXCL14 expression was reversed. Rhythmically expressed CXCL14 binds to S. aureus DNA and induces inflammatory cytokine production by activating Toll-like receptor (TLR)9-dependent innate pathways in dendritic cells and macrophages underneath the epidermis. CXCL14 also promoted phagocytosis by macrophages in a TLR9-independent manner. These data indicate that circadian production of the epidermal chemokine CXCL14 rhythmically suppresses skin bacterial proliferation in mammals by activating the innate immune system.

CXCL14 is a primordial chemokine that plays multiple roles in tumor suppression, autoimmune arthritis, and obesity-associated insulin resistance. However, the underlying molecular mechanisms are unclear. Here, we show that CXCL14 transports various types of CpG oligodeoxynucleotide (ODN) into the endosomes and lysosomes of bone marrow-derived dendritic cells (DCs), thereby activating Toll-like receptor 9 (TLR9). A combination of CpG ODN (ODN2395) plus CXCL14 induced robust production of IL-12 p40 by wild-type, but not Tlr9-knockout, DCs. Consistent with this, ODN2395-mediated activation of DCs was significantly attenuated in Cxcl14-knockout mice. CXCL14 bound CpG ODN with high affinity at pH7.5, but not at pH6.0, thereby enabling efficient delivery of CpG ODN to TLR9 in the endosome/lysosome. Furthermore, the CXCL14-CpG ODN complex specifically bound to high affinity CXCL14 receptors on DCs. Thus, CXCL14 serves as a specific carrier of CpG DNA to sensitize TLR9-mediated immunosurveillance.

Some CXC chemokines, including CXCL14, transport CpG oligodeoxynucleotides (ODNs) into dendritic cells (DCs), thereby activating TLR9. The molecular basis of this noncanonical function of CXC chemokines is not well understood. In this study, we investigated the CpG ODN binding and intracellular transport activities of various CXC chemokines and partial peptides of CXCL14 in mouse bone marrow–derived dendritic cells. CXCL14, CXCL4, and CXCL12 specifically bound CpG ODN, but CXCL12 failed to transport it into cells at low dose. CXCL14 N-terminal peptides 1–47, but not 1–40, was capable of transporting CpG ODN into the cell, resulting in an increase in cytokine production. However, both the 1–47 and 1–40 peptides bound CpG ODN. By contrast, CXCL14 peptides 13–50 did not possess CpG ODN binding capacity or transport activity. The chimeric peptides CXCL12 (1–22)–CXCL14 (13–47) bound CpG ODN but failed to transport it. These results suggest that amino acids 1–12 and 41–47 of CXCL14 are required for binding and intracellular transport of CpG ODN, respectively. We found that an anti-CXCL14 Ab blocked cell-surface binding and internalization of the CpG ODN/CXCL14 complex. On the basis of these findings, we propose that CXCL14 has two functional domains, one involved in DNA recognition and the other in internalization of CXCL14-CpG DNA complex via an unidentified CXCL14 receptor, which together are responsible for eliciting the CXCL14/CpG ODN-mediated TLR9 activation. These domains could play roles in CXCL14-related diseases such as arthritis, obesity-induced diabetes, and various types of carcinoma.

Cancer immunotherapeutic CpG oligodeoxynucleotide (ODN) is an agonist for TLR9 and a potent inducer of inflammatory cytokines and type I interferon. Clinical trials of CpG ODNs highlight the urgent need for effective TLR9 agonist CpG ODNs in humans. Here, we developed a highly potent CpG ODN, A602, which induces antitumor immune responses in combination with CXCL14. A602 induced secretion of interferon-α by human peripheral blood mononuclear cells. In mouse macrophages, dendritic cells, and human plasmacytoid dendritic cell lines, CXCL14 enhanced cellular uptake of A602, thereby promoting TLR9-mediated immune responses. Importantly, A602 exhibited strong antitumor activity in syngeneic mouse models of colorectal cancer–derived CT26, B lymphoma-derived A20, and melanoma-derived B16F10 cells. Because the antitumor effect of A602 against B16F10 cells was negated in Cxcl14 knockout mice, endogenously expressed CXCL14 is required for the A602-mediated tumor suppression. Thus, modulation of CXCL14 during the A602-induced immune responses shall unveil an innovative new approach for the antitumor immune therapy.

Latrophilin2 (Lphn2) is an adhesion-class of G protein-coupled receptor with an unknown function in development. Here, we show that Xenopus laevis lphn2 (Xlphn2) is involved in the migration and differentiation of neural crest (NC) cells and placode patterning in Xenopus laevis embryos. Although Xlphn2 mRNA was detected throughout embryogenesis, it was expressed more abundantly in the placode region. Morpholino antisense oligonucleotide-mediated knockdown of Xlphn2 caused abnormal migration of NC cells, irregular epibranchial placode segmentation, and defective cartilage formation. Transplantation of fluorescently-labeled NC regions of wild-type embryos into Xlphn2 morpholino-injected embryos reproduced the defective NC cell migration, indicating that Xlphn2 regulates the migration of NC cells in a non-cell autonomous manner. Our results suggest that Xlphn2 is essential for placode patterning and as a guidance molecule for NC cells.