Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report here that bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides induce murine B cells to proliferate and secrete immunoglobulin in vitro and in vivo. This activation is enhanced by simultaneous signals delivered through the antigen receptor. Optimal B-cell activation requires a DNA motif in which an unmethylated CpG dinucleotide is flanked by two 5' purines and two 3' pyrimidines. Oligodeoxynucleotides containing this CpG motif induce more than 95% of all spleen B cells to enter the cell cycle. These data suggest a possible evolutionary link between immune defence based on the recognition of microbial DNA and the phenomenon of 'CpG suppression' in vertebrates. The potent immune activation by CpG oligonucleotides has implications for the design and interpretation of studies using 'antisense' oligonucleotides and points to possible new applications as adjuvants.

Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polyclonal B-cell (3,4,(7)(8)(9)11). Recent work from our laboratory suggested that this B-cell stimulation was mediated by a 6-base nt motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (12). This motif is expressed nearly 20 times more frequently in bacterial than vertebrate DNA (12)(13)(14).In addition to B-cell activation, the innate immune response to bacterial infection is characterized by the production of immunomodulatory cytokines (15,16). These include interleukin (IL) 6, which contributes to T-and B-cell activation (17-20), IFN-y, which (25)(26)(27).The concentration of cytokine in culture supernatants was determined by using a modification of the assay system described above. Supernatants were serially diluted in PBS/1% bovine serum albumin and incubated on anti-cytokine-coated microtiter plates for 2 hr. Biotinylated anti-cytokine antibody followed by avidin-conjugated alkaline phosphatase were used in a colorimetric assay to detect the presence of bound cytokine (27).Cell Purification. Mononuclear cells were incubated with biotinylated anti-CD4, anti-CD8, anti-CD11, anti-CD14, or

Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA. B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immunestimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA. In type 12 genomes, the distribution of CpG-f lanking bases is similar to that predicted by chance. However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15-to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5 side or a G on the 3 side. Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs. Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing. These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.

Research over the last decade has revealed that CYP11A1 can hydroxylate the side chain of vitamin D3 at carbons 17, 20, 22 and 23 to produce at least 10 metabolites, with 20(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3, 17,20(OH)2D3 and 17,20,23(OH)3D3 being the main products. However, CYP11A1 does not act on 25(OH)D3. The placenta, adrenal glands and epidermal keratinocytes have been shown to metabolize vitamin D3 via this CYP11A1-mediated pathway that is modified by the activity of CYP27B1, with 20(OH)D3 (the major metabolite), 20,23(OH)2D3, 1,20(OH)2D3, 1,20,23(OH)3D3 and 17,20,23(OH)3D3 being detected, defining these secosteroids as endogenous regulators/natural products. This is supported by the detection of a mono-hydroxyvitamin D3 with the retention time of 20(OH)D3 in human serum. In new work presented here we demonstrate that the CYP11A1-initiated pathways also occurs in Caco-2 colon cells. Our previous studies show that 20(OH)D3 and 20,23(OH)2D3 are non-calcemic at pharmacological doses, dependent in part on their lack of a C1α hydroxyl group. In epidermal keratinocytes, 20(OH)D3, 20(OH)D2 and 20,23(OH)2D3 inhibited cell proliferation, stimulated differentiation and inhibited NF-κB activity with potencies comparable to 1,25(OH)2D3, acting as partial agonists on the VDR. 22(OH)D3 and 20,22(OH)2D3, as well as secosteroids with a short or no side chain, showed antiproliferative and prodifferentiation effects, however, with lower potency than 20(OH)D3 and 20,23(OH)2D3. The CYP11A1-derived secosteroids also inhibited melanocyte proliferation while having no effect on melano-genesis, and showed anti-melanoma activities in terms of inhibiting proliferation and the ability to grow in soft agar. Furthermore, 20(OH)D3 and 20,23(OH)2D3 showed anti-fibrosing effects in vitro, and also in vivo for the former. New data presented here shows that 20(OH)D3 inhibits LPS-induced production of TNFα in the J774 line, TNFα and IL-6 in peritoneal macrophages and suppresses the production of proinflammatory Th1/Th17-related cytokines, while promoting the production of the anti-inflammatory cytokine IL-10 in vivo. In summary, CYP11A1 initiates new pathways of vitamin D metabolism in a range of tissues and products could have important physiological roles at the local or systemic level. In the skin, CYP11A1-derived secosteroids could serve both as endogenous regulators of skin functions and as excellent candidates for treatment of hyperproliferative and inflammatory skin disorders, and skin cancer. This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’.