Murine astrocytes are responsive to the pro-inflammatory effects of IL-20

Abstract

Glia are key regulators of inflammatory responses within the central nervous system (CNS) following infection or trauma. We have previously demonstrated the ability of activated astrocytes to rapidly produce pro-inflammatory mediators followed by a transition to an anti-inflammatory cytokine production profile that includes the immunosuppressive cytokine interleukin (IL)-10 and the closely related cytokines IL-19 and IL-24. IL-20, another member of the IL-10 family, is known to modulate immune cell activity in the periphery and we have previously demonstrated that astrocytes constitutively express the cognate receptors for this cytokine. However, the ability of glia to produce IL-20 remains unclear and the effects of this pleiotropic cytokine on glial immune functions have not been investigated. In this study, we report that primary murine and human astrocytes are not an appreciable source of IL-20 following challenge with disparate bacterial species or their components. Importantly, we have determined that astrocyte are responsive to the immunomodulatory actions of this cytokine by showing that recombinant IL-20 administration upregulates microbial pattern recognition receptor expression and induces release of the inflammatory mediator IL-6 by these cells. Taken together, these data suggest that IL-20 acts in a dissimilar manner to other IL-10 family members to augment the inflammatory responses of astrocytes.

Keywords: Astrocytes; Bacterial infection; Interleukin-10; Interleukin-20; Neuroinflammation.

FIGURE 1:

Murine and human astrocytes fail to express significant levels of IL-20 mRNA or protein, either constitutively or following bacterial stimulation. Primary murine (Panels A and B) and human (Panel C) astrocytes (3 X 10⁵ cells per well) were untreated (0) or challenged with N. meningiditis (Nm), S. aureus (Sa) or S. pneumoniae (Sp) at the indicated MOI or the inflammatory neuropeptide substance P (SP; 5 nM) or bacterial LPS (5 ng/ml). Panel A: At 6 hours post challenge, IL-20 mRNA expression was determined by semi-quantitative RT-PCR. Expression of GAPDH mRNA housekeeping gene product is included and the image shown is representative of at least three independent experiments. For comparison purposes, IL-20 mRNA expression was also determined in primary murine microglia at 8 hrs following challenge with N. meningiditis (Nm), S. aureus (Sa) or S. pneumoniae (Sp) at a MOI of 10:1 bacteria to cells, and RAW264.7 murine macrophage-like cells (R) and primary murine macrophages (M) stimulated with LPS (5 ng/ml). A blank PCR reaction control is also shown (−). Panel B: At 10 hrs, cell medium was collected and analyzed for IL-20 protein content by immunoblot analysis. Expression of an irrelevant protein is shown as a loading control (lc) and recombinant IL-20 (40 pg) is shown as a positive control (+). The relative IL-20 expression was determined by densitometric analysis, normalized to untreated cells, and shown graphically below. Data is expressed as the mean +/− the SEM of 5 independent experiments and no statistically significant differences were observed from untreated cells. Panel C: At 24 hrs, IL-20 protein release by primary human astrocytes (hAst) or an immortalized human microglial cell line (hMic) was determined by immunoblot analysis and expression of an irrelevant protein is shown as a loading control (lc). Relative IL-20 expression was determined by densitometric analysis, normalized to untreated cells, and shown graphically below. Data is expressed as the mean +/− the SEM of 3 independent experiments and asterisk indicates a statistical significance compared to unchallenged cells (p < 0.05).

FIGURE 2:

Primary astrocytes are functionally responsive to the proinflammatory effects of IL-20. Panel A: Murine astrocytes were unstimulated or exposed to recombinant IL-20 (10, 30, or 100 ng/ml) for 2 hours and levels of mRNA encoding TLR4 were determined by semi-quantitative RT-PCR. Expression relative to densitometric analysis of house-keeping gene GAPDH expression is shown graphically below. Panels B and C: Cells were unstimulated or stimulated with 10, 30, or 100 ng/ml IL-20 for 2 or 4 hours prior to total RNA isolation. Pro-inflammatory cytokine gene expression was analyzed by semi-quantitative RT-PCR for IL-6 (Panel B) and IL-1β (Panel C). Densitometric analysis was performed and gene expression relative to GAPDH levels are shown below. Panel D: Cells were unstimulated or stimulated with recombinant murine IL-20 (3, 10, 30, or 100 ng/ml) for 12 hours and a specific capture ELISA was used to determine the level of IL-6 release. Asterisk denotes statistical significance compared to unchallenged cells (n = 11; p < 0.05).