Retinal Muller Glia Initiate Innate Response to Infectious Stimuli via Toll-Like Receptor Signaling

Ocular surgeries and trauma predispose the eye to develop infectious endophthalmitis, which often leads to vision loss. The mechanisms of initiation of innate defense in this disease are not well understood but are presumed to involve retinal glial cells. We hypothesize that retinal Muller glia can recognize and respond to invading pathogens via TLRs, which are key regulators of the innate immune system. Using the mouse retinal sections, human retinal Muller cell line (MIO-M1), and primary mouse retinal Muller cells, we show that they express known human TLR1-10, adaptor molecules MyD88, TRIF, TRAM, and TRAF6, and co-receptors MD2 and CD14. Consistent with the gene expression, protein levels were also detected for the TLRs. Moreover, stimulation of the Muller glia with TLR 2, 3, 4, 5, 7 and 9 agonists resulted in an increased TLR expression as assayed by Western blot and flow cytometry. Furthermore, TLR agonists or live pathogen (S. aureus, P. aeruginosa, & C. albicans)-challenged Muller glia produced significantly higher levels of inflammatory mediators (TNF-α, IL-1β, IL-6 and IL-8), concomitantly with the activation of NF-κB, p38 and Erk signaling. This data suggests that Muller glia directly contributes to retinal innate defense by recognizing microbial patterns under infectious conditions; such as those in endophthalmitis

TLR agonists induced expression and secretion of inflammatory mediators in Muller glia.

<p>MIO-M1 cells were stimulated with indicated TLR ligands. After 4 h, total RNA was extracted, reverse transcribed, and amplified using specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029830#pone-0029830-t001" target="_blank">Table 1</a>), with GAPDH as the control. PCR products were separated by electrophoresis and stained with ethidium bromide (<b>A</b>). The band intensities of PCR products were quantified by densitometric analysis and presented as relative band intensity of cytokines vs GAPDH. (<b>B</b>). In another experiment, MIO-M1cells were challenged with TLR agonists for 8 h and secretion of indicated cytokine/chemokine were measured by ELISA (<b>C</b>). The amount of cytokines was normalized with protein concentration of cell lysate (picograms per milligram cell lysate). One-way ANOVA was performed for statistical analysis of induced expression of TNF-α (∧<i>p</i><0.01), IL-1β (^δ<i>p</i><0.05), IL-6(^δ<i>p</i><0.05), and IL-8 (^#<i>p</i><0.05) in control vs various TLR agonist stimulated cells. Similar analysis was performed for ELISA data (*<i>p</i><0.05). The data shown are cumulative of three experiments performed in duplicate.</p

List of human primers used for RT-PCR analysis.

<p>List of human primers used for RT-PCR analysis.</p

Primary retinal Muller glia express TLRs and are responsive to TLR agonist challenge.

<p>The cultured primary retinal Muller glia isolated from neonatal pups of C57BL/6 mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029830#s4" target="_blank">methods</a> for detail) were analyzed for the expression of mRNAs encoding Muller cells markers using RT-PCR (<b>A</b>) or for protein expression using immunostaining (<b>B</b>). The expression of TLRs 1–9 was assessed by RT-PCR (<b>C</b>) and flowcytometry (<b>D</b>). To determine whether the expressed TLRs are functional, primary Muller glia were stimulated with indicated TLR agonists and secretion of mouse IL-6 and MIP-2 was quantitated by ELISA (<b>E</b>). The indicated statistical differences are comparisons of unstimulated (control) vs TLR agonist stimulated cells: *<i>p</i><0.05 (one-way ANOVA).</p

List of mouse primers used for RT-PCR analysis.

<p>List of mouse primers used for RT-PCR analysis.</p

In vivo and in vitro expression of TLRs in retinal Muller glia.

<p>(<b>A</b>) Eyes from C57BL/6 mice were enucleated and embedded in OCT, and cryo-sections were stained with indicated TLR antibodies. Double immunofluorescence labeling was performed using anti-TLR 2, 3, 4, 5, 7 and 9 (green) antibodies and anti-vimentin (red) antibody, a marker for Muller glia cells. TLRs expression co-localizes to Muller glia, as the Muller cell bodies and radial running processes were immunopositive. (<b>B</b>) To assess the expression of TLRs, other co-receptors, and adaptor molecules, RNA extraction followed by RT-PCR was performed on cultured human retinal Muller glia cell line (MIO-M1) and human monocyte cell line (THP-1), as a positive control. Results shown are representative of two experiments with similar results. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Magnification 20×.</p

Summary of retinal Muller glial innate response to infectious stimuli.

<p>(<b><i>1</i></b>) Retinal Muller cells are specialized glial cells, which span through the entire thickness of the retina. Under normal conditions, they express Toll-like receptors (TLRs) at the basal level. (<b><i>2</i></b>) During trauma or intraocular surgeries, the microbial pathogens or their products (e.g. LPS) gain access to vitreous cavity, where they activate Muller glial expressed TLRs. (<b><i>3</i></b>) Binding of TLRs by its specific ligand (e.g. LPS for TLR4, <i>S. aureus</i> for TLR2 or bacterial CpG DNA by TLR9) leads to up-regulation of TLRs and activates pro-inflammatory signaling cascade, leading to secretion of various inflammatory cytokines (IL-6, TNF-α), chemokines (IL-8), and antimicrobial peptides (LL-37). (<b><i>4</i></b>) Muller glia released chemoattractants, such as IL-8 diffuse across retinal blood vessels and drive PMN infiltration into the retina. While initiating phagocytosis to eliminate the invading microbial pathogens, infiltrated PMNs also release chemoattractants through TLR activation by localized bacterial products to perpetuate PMN infiltration. The TLR-mediated secretion of antimicrobial peptides (LL-37) can directly kill the invading pathogens. Thus, the Muller glia possesses the ability to sense infectious stimuli and initiate innate responses via the action of TLR-signaling.</p

Muller glia expresses and secretes proinflammatory mediators in response to live pathogen challenge.

<p>MIO-M1 cells were stimulated with <i>S. aureus</i> (Gram-positive bacteria), <i>P. aeruginosa</i> (Gram-negative bacteria), and <i>C. albicans</i> (fungi) for 4 h. At the end of the incubation period, cells were used for RNA extraction followed by RT-PCR (<b>A</b>), whereas the culture supernatant was used for measurement of TNF-α (<b>B</b>), and IL-6 (<b>C</b>). The indicated statistical differences are comparisons of unstimulated (control) vs TLR agonist stimulated cells: *<i>p</i><0.05 (one-way ANOVA).</p

Induced TLR expression in Muller glia challenged with TLR ligands.

<p>MIO-M1 cells were left untreated (C) or stimulated with indicated TLR ligands Pam2Cys (10 µg/ml), Poly(IC) (10 µg/ml), LPS (10 µg/ml), Flagellin (250 ng/ml), Poly(dT) (10 µM/ml), and ODN (10 µg/ml) for 4 h. Cells were lysed for Western blot analysis using specific anti-TLR and β-actin (control) antibodies (<b>A</b>). The band intensity was quantified by densitometric analysis and presented as relative band intensity of TLR vs β-actin (<b>B</b>). Induced expression of TLRs was further confirmed by immunostaining using indicated anti-TLR antibodies (<b>C</b>) and for quantitation, cells were stained for FACS analysis and presented as mean fluorescent intensity (MFI) of respective TLR positive cells (<b>D</b>). The data shown are representative of duplicate experiments. The indicated p values (<i>t</i>-test) are comparisons of unstimulated vs TLR agonist stimulated cells. Magnification 20×.</p