interleukin-8 and cytidylyl-3--5--guanosine

The innate immune system recognizes bacterial DNA as a nonself to induce rapid immune activation. TLR9 recognizes synthetic oligodeoxynucleotides (ODNs) and bacterial DNA containing unmethylated CpG dinucleotides in the context of specific base sequences (CpG-DNA). Here, we demonstrate that phosphorothioate backbone CT-ODN (PS-CT-ODN), a derivative of phosphorothioate backbone CpG-DNA (PS-ODN) with CT sequences substituted for the CG sequences, stimulates IL-8 promoter activation and gene expression. Furthermore, we identified an immunostimulatory phosphodiester bond CT-ODN (PO-CT-ODN) from Staphylococcus aureus chromosomal DNA and found that the PO-CT-ODN induces cytokine production in a TLR9-dependent manner when encapsulated with a proper liposome. Our experimental analyses also demonstrate that the immunostimulatory PO-CT-ODN can act as an adjuvant for the induction of Ag-driven IgG production. Further investigation of the functional role of PO-CT-ODN may support the future application of PO-CT-ODN in immunotherapeutics.

Topics: Adjuvants, Immunologic; Animals; B-Lymphocytes; Base Sequence; Cell Line; Cytokines; Dinucleoside Phosphates; DNA, Bacterial; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Immunization; Immunoglobulin G; Interleukin-8; Liposomes; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Phosphorothioate Oligonucleotides; Promoter Regions, Genetic; Staphylococcus aureus; Toll-Like Receptor 9

During intramammary infections pathogen associated molecular patterns (PAMPs) induce an inflammatory response, recognized clinically as mastitis. Recognition of PAMPs by mammary cells leads to the production of the pro-inflammatory cytokines, TNF-alpha and IL-1beta. These cytokines augment the secretion of various chemokines that are responsible for directing the host cellular immune response, and consequently the outcome of infection. Previous research has shown that gram-negative and gram-positive bacteria elicit different types of innate immune responses. The purpose of this study, therefore, was to characterize the expression of various chemokine genes in bovine mammary gland explants in response to lipopolysaccharide (LPS), peptidoglycan (PTG) combined with lipotechoic acid (LTA), and CpG oligodeoxynucleotide (CpG-ODN) 2135 representing gram-negative bacteria, gram-positive bacteria, and bacterial DNA, respectively, to determine if these PAMPs induce different chemokine gene expression patterns. Explants from 3 Holstein cows were cultured with 10 microg/mL of LPS, LTA + PTG, or CpG-ODN 2135 for 6 and 24 h. Total RNA was extracted and the expression of CXCL8, MCP-1, MCP-2, MCP-3, MIP1-alpha, and RANTES genes was measured by real-time polymerase chain reaction (RT-PCR). Lipopolysaccharide significantly induced MCP-1, MCP-2, and MCP-3 expression, and slightly increased CXCL8 gene expression. The combined PAMPs, LTA + PTG, on the other hand, significantly induced MCP-1 gene expression, and slightly increased MCP-3 expression. No significant expression differences for any of the chemokine genes were observed in explants stimulated with CpG-ODN 2135. These results demonstrate that PAMPs associated with different mastitis-causing pathogens induce chemokine-specific gene expression patterns that may contribute to different innate immune responses to bacteria.

Topics: Animals; Cattle; Cattle Diseases; Chemokine CCL2; Chemokine CXCL6; Dinucleoside Phosphates; Female; Gene Expression Regulation; Inflammation; Interleukin-8; Lipopolysaccharides; Mammary Glands, Animal; Oligodeoxyribonucleotides; Peptidoglycan; Teichoic Acids; Toll-Like Receptor 9