epiglucan and resiquimod

In the absence of lymphocytes, rag1-/- mutant zebrafish develop protective immunity to bacteria. In mammals, induction of protection by innate immunity can be mediated by macrophages or natural killer (NK) cells. To elucidate potential responsive cell populations, we morphologically characterized lymphocyte-like cells (LLCs) from liver, spleen and kidney hematopoietic tissues. In fish, these cells include NK cells and Non-specific cytotoxic cells (NCCs). We also evaluated the transcriptional expression response of select genes that are important indicators of NK and macrophage activation after exposure to specific TLR ligands. The LLC cell populations could be discriminated by size and further discriminated by the presence of cytoplasmic granules. Expression levels of mx, tnfα, ifnγ, t-bet and nitr9 demonstrated dynamic changes in response to intra-coelomically administered β glucan (a TLR2/6 ligand), Poly I:C (a TLR3 ligand) and resiquimod (R848) (a TLR7/8 ligand). Following TLR 2/6 stimulation, there was a greater than 100 fold increase in ifnγ in liver, kidney and spleen and moderate increases in tnfα in liver and kidney. TLR3 stimulation caused broad up regulation of mx, down-regulation of tnfα in kidney and spleen tissues and up regulation of nitr9 in the kidney. Following TLR 7/8 stimulation, there was a greater than 100 fold increase in ifnγ in liver and kidney and t-bet in liver. Our gene expression findings suggest that LLCs and macrophages are stimulated following β glucan exposure. Poly I:C causes type I interferon response and mild induction of LLC in the kidney and R-848 exposure causes the strongest LLC stimulation. Overall, the strongest NK like gene expression occurred in the liver. These differential effects of TLR ligands in rag1-/- mutant zebrafish shows strong NK cell-like gene expression responses, especially in the liver, and provides tools to evaluate the basis for protective immunity mediated by the innate immune cells of fish.

Topics: Animals; beta-Glucans; Gene Expression Regulation; Homeodomain Proteins; Imidazoles; Immunity, Innate; Lymphocytes; Macrophages; Organ Specificity; Poly I-C; Toll-Like Receptors; Zebrafish; Zebrafish Proteins

Emerging evidence indicates that innate immunodeficiency syndromes are linked to mutations in innate receptors and to specific infections. X-linked lymphoproliferative syndrome type-2 (XLP-2) is associated with deficiency in X-linked inhibitor of apoptosis protein (XIAP), with poorly understood molecular mechanisms. Here we showed that XIAP deficiency selectively impaired B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-mediated innate responses to dectin-1 ligands but did not affect responses to various Toll-like receptor agonists. Consequently, Xiap(-/-) mice became highly vulnerable on Candida albicans infection. The compromised early innate responses led to the persistent presence of C albicans and inflammatory cytokines in Xiap(-/-) mice. Furthermore, priming of Xiap(-/-) mice with the dectin-1 ligand curdlan alone resulted in XLP-2-like syndromes. Restoration of dectin-1-induced Rac1 activation and phagocytosis by resolvin D1, but not up-regulation of nuclear factor-κB, rescued Xiap(-/-) mice from C albicans lethal infection. Therefore, development of XLP-2 in XIAP-deficient patients could be partly due to sustained inflammation as a consequence of defective BCL10-dependent innate immunity toward specific pathogens. Importantly, our results suggest the potential therapeutic value of resolvin D1 in the treatment of XLP-2 and innate immunodeficiency syndromes.

Topics: Adaptor Proteins, Signal Transducing; Animals; B-Cell CLL-Lymphoma 10 Protein; beta-Glucans; Candida albicans; Candidiasis; ErbB Receptors; Genetic Diseases, X-Linked; Humans; Imidazoles; Immunity, Innate; Inhibitor of Apoptosis Proteins; Lectins, C-Type; Lipopeptides; Lipopolysaccharides; Lymphoproliferative Disorders; Lysine; Lysophospholipids; Macrophages; Mice; NF-kappa B; Phagocytosis; Poly I-C; Protein Binding; Receptors, Antigen, T-Cell; Toll-Like Receptors; Tumor Necrosis Factor-alpha; Ubiquitination

Toll-like receptors (TLRs), which recognize a wide range of microbial pathogens and pathogen-related products, play important roles in innate immunology. Macrophages have a variety of TLRs, and pathogen binding to TLR resulted in the activation of macrophages. R-848, an immune response modifier, is an analog of imidazoquinoline derivative and binds to an endosome-localized TLR to exert an anti-viral response on leukocytes. In the present study, we verified that co-treatment of R-848 with other TLR agonists would enhance immune response. The culture supernatant of Aureobasidium pullulans (A. pullulans, which contains predominantly soluble β-glucan), which binds to cell membrane-localized TLR, and to C-type lectin receptor Dectin-1, was treated together with R-848 to THP-1 macrophages. Compared to R-848 treatment alone, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented TNF-α and IL-12p40 cytokine expression. Next, we investigated whether or not apoptotic cell uptake would be increased by co-treatment of R-848 with A. pullulans culture supernatant. To detect engulfed apoptotic cells, we induced apoptosis in human lymphoma Jurkat cells by 5-fluorouracil and stained them with fluorescent dye 5(6)-carboxytetramethylrhodamine (TAMRA), whereas THP-1 macrophage was labeled with fluorescein isothiocyanate-anti-CD14 and determined the percentage increase in TAMRA-positive THP-1 macrophages by flow cytometric assay. Since R-848 or A. pullulans treatment alone stimulated THP-1 macrophages to induce phagocytosis, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented phagocytosis of apoptotic Jurkat cells. These results suggest that the activation of several different innate immune receptor pathways may enhance the immune response of R-848 significantly.

Topics: Ascomycota; beta-Glucans; Carcinogens; Cell Line, Tumor; Fungal Polysaccharides; Gene Expression Regulation; Humans; Imidazoles; Interleukin-12 Subunit p40; Jurkat Cells; Lectins, C-Type; Phagocytosis; Tetradecanoylphorbol Acetate; Toll-Like Receptors; Tumor Necrosis Factor-alpha