Poly-Thymidine Oligonucleotides Mediate Activation of Murine Glial Cells Primarily Through TLR7, Not TLR8

The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS) remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs) or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs) with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS

Neuropeptide Y Has a Protective Role during Murine Retrovirus-Induced Neurological Disease▿

Viral infections in the central nervous system (CNS) can lead to neurological disease either directly by infection of neurons or indirectly through activation of glial cells and production of neurotoxic molecules. Understanding the effects of virus-mediated insults on neuronal responses and neurotrophic support is important in elucidating the underlying mechanisms of viral diseases of the CNS. In the current study, we examined the expression of neurotrophin- and neurotransmitter-related genes during infection of mice with neurovirulent polytropic retrovirus. In this model, virus-induced neuropathogenesis is indirect, as the virus predominantly infects macrophages and microglia and does not productively infect neurons or astrocytes. Virus infection is associated with glial cell activation and the production of proinflammatory cytokines in the CNS. In the current study, we identified increased expression of neuropeptide Y (NPY), a pleiotropic growth factor which can regulate both immune cells and neuronal cells, as a correlate with neurovirulent virus infection. Increased levels of Npy mRNA were consistently associated with neurological disease in multiple strains of mice and were induced only by neurovirulent, not avirulent, virus infection. NPY protein expression was primarily detected in neurons near areas of virus-infected cells. Interestingly, mice deficient in NPY developed neurological disease at a faster rate than wild-type mice, indicating a protective role for NPY. Analysis of NPY-deficient mice indicated that NPY may have multiple mechanisms by which it influences virus-induced neurological disease, including regulating the entry of virus-infected cells into the CNS

pT-ODN stimulation induction of low level CCL2 and CXCL10 production is dependent on Unc93b1.

<p>Primary cortical cells from wildtype C57BL/6 and C57BL/6 Unc93b1 3D mice were cultured and stimulated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022454#pone-0022454-g002" target="_blank">Fig. 2</a>. 5 µM of pT-ODNs, instead of 1 µM pT-ODN, was used in this study since this concentration induced optimal stimulation in cells from C57BL/6 mice in the presence or absence of 20 µM of CL075. Samples for RNA were collected at 6 hps and processed for real-time quantitative RT-PCR analysis. Data were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022454#pone-0022454-g002" target="_blank">Fig. 2</a>. Data are the mean ± SEM of three wells per group and are representative of two replicate experiments. Statistical analysis was completed by one-way ANOVA with Newman-Keuls post test. Asterisks (*) directly above data symbols represent a statistically significant difference compared to media alone controls. Asterisks above lines between two columns indicate a statistically significant difference between the two indicated groups. ** P<0.01, *** P<0.001.</p

CL075, but not pT-ODN induces changes in microglia morphology.

<p>Primary cortical cultures from wildtype mice were stimulated with 20 µM CL075 and/or 1 µM pT-ODN. After 48 hrs, cells were fixed and then stained for the microglia/macrophage marker, Iba1 using an Alexafluor-488 conjugated anti-rabbit antibody for detection. Iba1 positive cells in (A) mock (unstimulated) and (B) pT-ODN-stimulated cultures were primarily elongated with only a few amoeboid cells. In contrast, Iba1 positive cells in (C) CL075 or (D) CL075/pT-ODN stimulated cultures were completely amoeboid in appearance. However, Iba1 positive cells from (E–F) TLR7-deficient mice did not take on the amoeboid appearance after stimulation with (E) CL075 or (F) CL075/pT-ODN. Images are representative of cells in each well and are the results of one of two replicate experiments.</p

pT-ODNs do not alter uptake of TLR7/8 agonists.

<p>Primary cortical cells were grown in 24-well plates and stimulated with rhodamine-labeled CL264 in the presence or absence of 1 µM pT-ODN. At the indicated times, cells were washed 3X with PBS and fluorescence intensity measured (fluorescence units/ml/well). Data are the mean ± SEM of three wells per group and are representative of two replicate experiments. No significant differences were observed using a two-way ANOVA analysis between rhodamine-CL264 and rhodamine-CL264 with pT-ODN.</p

pT-ODNs do not alter endosomal localization of TLR7/8 agonists.

<p>Primary mixed cortical cells were grown on 8-chambered coverglass until semi-confluent. Cells were stimulated with 20 µM of rhodamine-labeled CL264 (left column) or 20 µM of rhodamine-labeled CL264 and 1 µM pT-ODN (right column) along with lyso-tracker green for 30 minutes. Each chamber was washed with media to remove unbound CL264 and analyzed by confocal microscopy. Images are representative of cells within the wells, with cells selected at random for imaging. Cells incubated with pT-ODNs alone or unstimulated cells did not have detectable rhodamine fluorescence (data not shown). Cells were followed for 3 h, with no significant differences noted in agonist localization.</p

TLR7 and TLR8 protein expression on (A) primary mixed cortical cells, (B) purified astrocytes and (C) purified microglia.

<p>Primary cortical cells were generated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022454#s4" target="_blank">materials and methods</a>. Cells were either cultured as a mixed cell population or used to generate >95% pure microglia or astrocyte cultures. At 7–10 days post culture, cells were analyzed for TLR7 or TLR8 expression by flow cytometry using rabbit anti-TLR7 (gray line) or goat anti-TLR8 antibodies (black line). Data are shown in comparison to secondary alone antibody (anti-rabbit 488, gray filled). Isotype controls and rabbit anti-goat 488 antibodies were comparable to goat anti-rabbit staining (data not shown). Data are representative of at least two replicate experiments for each cell population.</p

Primers used for real-time RT-PCR analysis.

<p>Primers used for real-time RT-PCR analysis.</p

CL075/pT-ODN-induced production of (A–D) proinflammatory cytokines and (E–H) chemokines by primary cortical cells is dependent on TLR7.

<p>Primary cortical cultures from wildtype or TLR7-deficient mice were stimulated with 20 µM CL075 and/or 1 µM pT-ODN. Supernatants were collected at 48 hps and analyzed for cytokine and chemokine levels using a cytokine 20-plex multi-plex bead array. Data were calculated as pg/ml using in-plate standard curves. Data are the mean ± SEM of three wells per group and are representative of two replicate experiments. Statistical analysis was completed by One-way ANOVA with a Newman-Keuls post test within each group. Symbols above columns indicate a statistically significant difference compared to media controls. * P<0.05, ** P<0.01, and *** P<0.001 for wildtype mice; # P<0.05, ## P<0.01, and ### P<0.001 for TLR7-deficient mice. Symbols above lines between two columns indicate a statistically significant difference between the two indicated groups.</p