interleukin-8 and resiquimod

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system-the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1β and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1β, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49-0.61) and between neutrophils and cytokine responses to R848 (0.38-0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.

Topics: Adaptive Immunity; Animals; Cattle; Enzyme-Linked Immunosorbent Assay; Imidazoles; Immunity, Innate; Immunologic Tests; Interleukin-1beta; Interleukin-6; Interleukin-8; Lipopolysaccharides; Neutrophils; Pathogen-Associated Molecular Pattern Molecules; Time Factors

Lactoferrin is an abundant glycoprotein in bovine milk that has immunomodulatory effects on human cells. Bovine lactoferrin (LF) binds lipopolysaccharides (LPS) with high affinity and is postulated to act via TLR4-dependent and -independent mechanisms. It has been shown that LF modulates differentiation of human monocytes into tolerogenic dendritic cells. However, in a previous study, we showed that LPS also mediates differentiation into tolerogenic dendritic cells (DC). Since LF binds LPS with high affinity, it remains to be investigated whether LF or LPS is mediating these effects. We, therefore, further investigated the LPS-independent effect of LF on differentiation of human monocytes into dendritic cells (DC). Human monocytes were isolated by magnetic cell sorting from freshly isolated PBMCs and cultured for six days in the presence of IL-4 and GM-CSF with or without LF or proteinase K treated LF to generate DC. These immature DC were stimulated for 48 h with LPS or Poly I:C + R848. Cell surface marker expression and cytokine production were measured by flow cytometry. DC differentiated in the presence of LF produced higher IL-6 and IL-8 levels during differentiation and showed a lower expression of CD1a and HLA-DR. These LFDCs showed to be hyporesponsive towards TLR ligands as shown by their semi-mature phenotype and reduced cytokine production. The effect of LF was abrogated by proteinase K treatment, showing that the functional effects of LF were not mediated by LPS contamination. Thus, LF alters DC differentiation and dampens responsiveness towards TLR ligands. This study indicates that LF can play a role in immune homeostasis in the human GI tract.

Topics: Animals; Cattle; Cell Differentiation; Dendritic Cells; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Imidazoles; Interleukin-6; Interleukin-8; Lactoferrin; Ligands; Lipopolysaccharides; Monocytes; Phenotype; Poly I-C; Toll-Like Receptor 4

PGE2 is a lipid mediator abundantly produced in inflamed tissues that exerts relevant immunoregulatory functions. Dendritic cells (DCs) are key players in the onset and shaping of the inflammatory and immune responses and, as such, are well known PGE2 targets. By contrast, the precise role of human DCs in the production of PGE2 is poorly characterized. Here, we asked whether different ligands of Toll-like receptors (TLRs), a relevant family of pathogen-sensing receptors, could induce PGE2 in human DCs. The only active ligands were LPS (TLR4 ligand) and R848 (TLR7-8 ligand) although all TLRs, but TLR9, were expressed and functional. While investigating the molecular mechanisms hindering the release of PGE2, our experiments highlighted so far oversight differences in TLR signalling pathways in terms of MAPK and NF-κB activation. In addition, we identified that the PGE2-limiting checkpoint downstream TLR3, TLR5, and TLR7 was a defect in COX2 induction, while TLR1/2 and TLR2/6 failed to mobilize arachidonic acid, the substrate for the COX2 enzyme. Finally, we demonstrated the in vivo expression of PGE2 by myeloid CD11c(+) cells, documenting a role for DCs in the production of PGE2 in human inflamed tissues.

Topics: Blotting, Western; Cells, Cultured; Dendritic Cells; Dinoprostone; Electrophoresis, Polyacrylamide Gel; Humans; Imidazoles; Immunohistochemistry; Interleukin-8; Lipopolysaccharides; Real-Time Polymerase Chain Reaction; Toll-Like Receptor 4; Toll-Like Receptor 7; Toll-Like Receptor 8

The innate immune system plays a critical role in protecting neonates against infections early in life and Toll-like receptors (TLRs) are key components of innate immune recognition of pathogens. This study examined the effects of age and stimulation with a TLR 7/8 agonist (R848) on TLR8 mRNA expression by foal neutrophils during the first month of life. We also examined the effects of R848 stimulation on mRNA expression of interleukin (IL)-6 and IL-8 at 1 and 14 days of life. We observed that TLR8 mRNA was constitutively expressed (P<0.05) at all ages examined, and its expression did not change upon stimulation with R848. Stimulation with R848 resulted in significantly increased (P<0.05) expression of IL-6 and IL-8 mRNA at both 1 and 14 days of life. Our results demonstrate that foal neutrophils express constitutive levels of TLR8 mRNA. The increased expression of IL-6 and IL-8, both downstream products of the TLR 7/8 signaling pathway, following stimulation with R848 suggests that additional experiments to confirm the signaling pathway by which R848 stimulates foal neutrophils, and to investigate its ability to stimulate functional responses of foal neutrophils, are merited.

Topics: Age Factors; Animals; Animals, Newborn; Gene Expression Regulation; Horses; Imidazoles; Immunity, Innate; Interleukin-6; Interleukin-8; Linear Models; Male; Neutrophils; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Toll-Like Receptor 8

This study analysed the immune response in the intestinal tract of ducks infected with low-pathogenic avian influenza viruses compared with ducks treated orally with R848, a synthetic Toll-like receptor 7 (TLR7) agonist. Influenza virus infection induced a type I interferon (IFN)-dependent immune response characterized by the expression of Mx transcripts in the ileum at levels that were proportional to viral load. Mx transcripts were detected in differentiated enterocytes from influenza virus-infected ducks. By contrast, in R848-treated ducks, Mx transcripts were detected solely in intraepithelial round cells of haematopoietic origin. An increase was detected in the number of intraepithelial TLR7-positive cells and intraepithelial IFN-α-producing cells in influenza virus-infected ducks, albeit to a lower level than in R848-treated ducks. IFN-γ expression was also upregulated in the intestine of influenza virus-infected and R848-treated ducks. Finally, interleukin (IL)-1β and IL-8 transcripts were expressed at high levels in R848-treated ducks but were not increased in influenza virus-infected ducks. These findings suggest that a type I IFN-mediated immune response in enterocytes and the activation of IFN-γ-secreting cells contribute to the control of influenza virus replication in the duck intestine.

Topics: Animals; Ducks; Enterocytes; Ileum; Imidazoles; Immunohistochemistry; Influenza A Virus, H7N1 Subtype; Influenza in Birds; Interferon-alpha; Interferon-gamma; Interleukin-1beta; Interleukin-8; Microscopy; Toll-Like Receptor 7

Excessive oxidative stress has been reported to be generated in inflamed tissues and contribute to the pathogenesis of inflammatory lung diseases, exacerbations of which induced by viral infections are associated with toll-like receptor (TLR) activation. Among these receptors, TLR8 has been reported as a key receptor that recognizes single-strand RNA virus. However, it remains unknown whether TLR8 signaling is potentiated by oxidative stress. The aim of this study is to examine whether oxidative stress modulates TLR8 signaling in vitro.. Human peripheral blood neutrophils were obtained from healthy non-smokers and stimulated with TLR 7/8 agonist imidazoquinoline resiquimod (R848) in the presence or absence of hydrogen peroxide (H2O2). Neutrophilic responses including cytokine release, superoxide production and chemotaxis were examined, and the signal transduction was also analyzed.. Activation of TLR8, but not TLR7, augmented IL-8 release. The R848-augmented IL-8 release was significantly potentiated by pretreatment with H2O2 (p < 0.01), and N-acetyl-L-cysteine reversed this potentiation. The combination of H2O2 and R848 significantly potentiated NF-kB phosphorylation and IkBalpha degradation. The H2O2-potentiated IL-8 release was suppressed by MG-132, a proteosome inhibitor, and by dexamethasone. The expressions of TLR8, myeloid differentiation primary response gene 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were not affected by H2O2.. TLR8-mediated neutrophilic responses were markedly potentiated by oxidative stress, and the potentiation was mediated by enhanced NF-kB activation. These results suggest that oxidative stress might potentiate the neutrophilic inflammation during viral infection.

Topics: Acetylcysteine; Chemotaxis, Leukocyte; Cytokines; Flow Cytometry; Humans; Hydrogen Peroxide; Imidazoles; Inflammation; Interleukin-8; Neutrophils; Oxidative Stress; Pancreatic Elastase; Phosphorylation; Reference Values; Signal Transduction; Toll-Like Receptor 7; Toll-Like Receptor 8; Transcription Factor RelA; Virus Diseases

Plasmacytoid dendritic cells (pDCs) are unique with respect to their capacity to produce unsurpassed amounts of IFN-alpha and coexpress TLR7 and TLR9, mediating IFN-alpha production. Although TLRs are critical receptors of innate immunity, little is known about the immunological effects of TLR7/TLR9 costimulation. We have analyzed the effects of TLR7/TLR9 costimulation on IFN-alpha production by leukocytes and pDCs. Our experiments revealed that both synthetic (resiquimod and loxoribine) and natural (ssRNA40) TLR7 ligands abrogate CpG-A- and CpG-C-oligodeoxynucleotide (ODN)-induced IFN-alpha production by human leukocytes. Because TLR7 ligands themselves represent important IFN-alpha inducers, we demonstrated that substimulatory TLR7 ligand concentrations significantly inhibited CpG-A-induced IFN-alpha. Delayed addition of TLR7 ligands still resulted in complete suppression of CpG-A-ODN-induced IFN-alpha production, suggesting that the inhibition is unlikely to be caused by a kinetic uptake advantage. Unlike for CpG-A and CpG-C, TLR7 ligands did not inhibit CpG-B-ODN-induced IFN-alpha production. Experiments with purified human pDCs demonstrated that the inhibitory effects of TLR7/TLR9 costimulation were mediated directly by pDCs. Suppression of IFN-alpha production was not related to increased cell death and was also detectable in enriched mouse pDCs. Analyses of pDCs suggested that the TLR7 signal regulates the outcome of TLR7 ligand/CpG-A-ODN costimulation and can either inhibit (IFN-alpha) or promote (IL-8/CD40) cytokine and surface marker expression. Our data reveal for the first time a strong inhibitory effect of TLR7 stimulation on IFN-alpha production induced by CpG-A- and CpG-C-ODNs. These findings provide novel insight into the effects of TLR7/TLR9 costimulation and may support the development of novel TLR9 inhibitors.

Topics: Animals; CD40 Antigens; Dendritic Cells; Guanosine; Humans; Imidazoles; Interferon-alpha; Interleukin-8; Ligands; Mice; Oligodeoxyribonucleotides; Signal Transduction; Toll-Like Receptor 7; Toll-Like Receptor 9

Toll-like receptors (TLRs) are important pattern recognition molecules that activate the nuclear factor (NF)-kappaB pathway leading to the production of antimicrobial immune mediators. As keratinocytes represent the first barrier against exogenous pathogens in human skin, we investigated their complete functional TLR1-10 expression profile. First, reverse transcription-polymerase chain reaction (PCR) analysis revealed a very similar pattern of TLR mRNA expression when comparing freshly isolated human epidermis and cultured primary human keratinocytes. Thus, further experiments were carried out with primary keratinocytes in comparison with the spontaneously immortalized human keratinocyte cell line HaCaT. The quantitative expression of TLR1-10 mRNA in real-time PCR of primary human keratinocytes and HaCaT cells was analysed. Both cell types constitutively expressed TLR2, TLR3, TLR5, and to a lesser extent TLR10. TLR4 was only found in HaCaT cells, TLR1 to a higher degree in primary keratinocytes. In line with this, LPS induced mRNA expression of CD14 and TLR4 only in HaCaT cells. After stimulation with various TLR ligands, the NF-kappaB-activated chemokine interleukin-8 (IL-8) was measured. In primary keratinocytes and HaCaT cells the TLR3 ligand poly (I:C) was the most potent stimulator of IL-8 secretion. The TLR ligands peptidoglycan, Pam3Cys and flagellin which bind to TLR2, TLR1/TLR2 heterodimer, and TLR5, respectively, also induced IL-8 secretion, whereas no IL-8 was induced by LPS, R-848, loxoribine and cytosine guanine dinucleotide-containing oligodeoxynucleotide. A corresponding pattern was found in the RelA NF-kappaB translocation assay after ligand stimulation of primary keratinocytes. These studies provide substantial evidence for a functional TLR expression and signalling profile of normal human keratinocytes contributing to the antimicrobial defence barrier of human skin.

Topics: Biological Transport; Cell Culture Techniques; Cell Line, Transformed; Dendritic Cells; Epidermis; Gene Expression; Guanosine; Humans; Imidazoles; Interleukin-8; Keratinocytes; Ligands; Lipopolysaccharides; Membrane Glycoproteins; NF-kappa B; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stimulation, Chemical; Toll-Like Receptor 1; Toll-Like Receptor 10; Toll-Like Receptor 2; Toll-Like Receptor 3; Toll-Like Receptor 4; Toll-Like Receptor 5; Toll-Like Receptors

TLRs are involved in innate cell activation by conserved structures expressed by microorganisms. Human T cells express the mRNA encoding most of TLRs. Therefore, we tested whether some TLR ligands may modulate the function of highly purified human CD4+ T lymphocytes. We report that, in the absence of APCs, flagellin (a TLR5 ligand) and R-848 (a TLR7/8 ligand) synergized with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent stimuli (anti-CD2 mAbs or IL-2) to up-regulate proliferation and IFN-gamma, IL-8, and IL-10 but not IL-4 production by human CD4+ T cells. No effect of poly(I:C) and LPS, ligands for TLR3 and TLR4, respectively, was detected. We also observed that CD4+CD45RO+ memory T cell responses to TLR ligands were more potent than those observed with CD4+CD45RA+ naive T cells. Moreover, among the memory T cells, CCR7- effector cells were more sensitive to TLR ligands than CCR7+ central memory cells. These data demonstrate for the first time a direct effect of TLR5 and TLR7/8 ligands on human T cells, and highlight an innate arm in T cell functions. They also suggest that some components from invading microorganisms may directly stimulate effector memory T cells located in tissues by up-regulating cytokine and chemokine production.

Topics: CD4-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Flagellin; Humans; Imidazoles; Immunologic Memory; Interferon-gamma; Interleukin-10; Interleukin-2; Interleukin-8; Leukocyte Common Antigens; Ligands; Lymphocyte Activation; Membrane Glycoproteins; Receptors, CCR7; Receptors, Cell Surface; Receptors, Chemokine; RNA, Messenger; T-Lymphocyte Subsets; Toll-Like Receptor 3; Toll-Like Receptor 4; Toll-Like Receptor 5; Toll-Like Receptor 7; Toll-Like Receptors; Up-Regulation