Murine astrocytes produce IL-24 and are susceptible to the immunosuppressive effects of this cytokine

Abstract

Background: 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 glia 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 cytokine IL-19. IL-24, another member of the IL-10 family, has been studied in a number of inflammatory conditions in the periphery and is known to modulate immune cell activity. However, the ability of glia to produce IL-24 remains unclear and the effects of this pleiotropic cytokine on glial immune functions have not been investigated.

Methods: In this study, we have assessed whether primary murine glia produce IL-24 following stimulation and evaluated the effect of this cytokine on the immune responses of such cells. We have utilized RT-PCR and immunoblot analyses to assess the expression of IL-24 and its cognate receptors by astrocytes following challenge with bacteria or their components. Furthermore, we have determined the effect of recombinant IL-24 on astrocyte immune signaling and responses to clinically relevant bacteria using RT-PCR and specific capture ELISAs.

Results: We demonstrate that astrocytes express IL-24 mRNA and release detectable amounts of this cytokine protein in a delayed manner following bacterial challenge. In addition, we have determined that glia constitutively express the cognate receptors for IL-24 and show that such expression can be increased in astrocytes following activation. Importantly, our results indicate that IL-24 exerts an immunosuppressive effect on astrocytes by elevating suppressor of cytokine signaling 3 expression and limiting IL-6 production following challenge. Furthermore, we have demonstrated that IL-24 can also augment the release of IL-10 by bacterially challenged astrocytes and can induce the expression of the potentially neuroprotective mediators, glutamate transporter 1, and cyclooxygenase 2.

Conclusions: The expression of IL-24 and its cognate receptors by astrocytes following bacterial challenge, and the ability of this cytokine to limit inflammatory responses while promoting the expression of immunosuppressive and/or neuroprotective mediators, raises the intriguing possibility that IL-24 functions to regulate or resolve CNS inflammation following bacterial infection in order to limit neuronal damage.

Keywords: Astrocytes; Bacterial infection; Interleukin-10; Interleukin-24; Neuroinflammation.

Fig. 1

IL-24 mRNA expression is induced in the CNS of mice following in vivo bacterial infection. Wild type C57BL/6J mice were uninfected (control, animal numbers 1 and 2) or infected by direct intracranial administration with Streptococcus pneumoniae (1 × 10⁷ bacteria, animal numbers 3–6). At 72 h following infection, whole brain tissue was collected and expression of mRNA encoding for IL-24 was determined by semi-quantitative RT-PCR, and C57BL/6J mouse whole thymus tissue was used as a positive control (+). Relative IL-24 mRNA expression was determined by densitometric analysis and normalized to the level of the housekeeping gene GAPDH, and all data points and the mean are shown

Fig. 2

Primary murine astrocytes express IL-24 following exposure to bacterial ligands for certain toll-like receptors. a Murine astrocytes were either unstimulated or challenged with LPS (5 ng/ml) for 6, 12, or 24 h, and IL-24 mRNA expression was determined by semi-quantitative (top) and real-time quantitative (bottom) RT-PCR. Expression of GAPDH mRNA housekeeping gene product is included and the image shown is representative of at least three independent experiments. Below, real-time RT-PCR data is shown as mean fold increases in product ± the SEM of three independent experiments and an asterisk indicates a statistically significant difference from unchallenged cells (p < 0.05). b Astrocytes were either unchallenged or challenged with TLR ligands; flagellin (50 and 100 ng/mL; FLG), the lipoprotein Pam3Cys (5 and 25 ng/mL; PAM), or dsRNA polyinosinic:polycytidylic acid (0.5 or 1 μg/mL; PIC) for 6 h, and IL-24 mRNA expression was determined by semi-quantitative RT-PCR. Expression of the housekeeping gene GAPDH is shown, and relative IL-24 mRNA expression was determined by densitometric analysis and normalized to unchallenged cells. Murine whole thymus tissue was used as a positive control for IL-24 expression (+). Asterisks denote statistical significance compared to unchallenged cells (p < 0.05)

Fig. 3

Primary murine glia express IL-24 following bacterial challenge. a Astrocytes were uninfected or infected with Neisseria meningitidis (Nm) at MOI of 1 or 10 bacteria to glia for 6 h prior to RNA collection. IL-24 and GAPDH mRNA expression was determined by semi-quantitative RT-PCR, and the average of three separate experiments is shown as mean relative gene expression as determined by densitometric analysis normalized to the expression of the housekeeping gene GAPDH ± SEM. b Isolated primary murine microglia were uninfected or infected with N. meningitidis, Staphylococcus aureus (Sa), or S. pneumoniae (Sp) (MOI of 10:1 bacteria to microglia) for 8 h prior to RNA isolation. C57BL/6J thymus tissue was used as a positive control. Relative IL-24 gene expression was determined by densitometric analysis normalized to GAPDH gene expression and is depicted as the mean of three individual experiments ± SEM. c Astrocytes were uninfected or infected with N. meningitidis for 24 or 48 h prior to immunoblot analysis of cell medium IL-24 protein content. Expression of an irrelevant protein is shown as a loading control (lc), and the relative IL-24 expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean ± the SEM of three independent experiments. Asterisk indicates a statistical significance compared to unchallenged cells (p < 0.05)

Fig. 4

Primary murine glia constitutively express the IL-22Rα subunit of the Type II receptor for IL-24. a Murine astrocytes were unstimulated or challenged with LPS (1 or 5 ng/ml) for 6 or 24 h, and levels of mRNA encoding IL-22Rα and GAPDH were determined by semi-quantitative RT-PCR. b Astrocytes were uninfected or infected with N. meningitidis (Nm), S. aureus (Sa), or S. pneumoniae (Sp) at MOI of 1, 10, or 50 bacteria to glia for 24 h prior to immunoblot analysis for IL-22Rα expression. Expression of β-actin is shown as a loading control, and relative IL-22Rα expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean ± the SEM of 3 independent experiments, and an asterisk indicates a statistically significant difference from unchallenged cells (p < 0.05). c Murine microglia were uninfected or infected with N. meningitidis (Nm), S. aureus (Sa), or S. pneumoniae (Sp) at MOI of 1, 10, or 50 bacteria to glia for 24 h prior to immunoblot analysis for IL-22Rα expression. Expression of β-actin is shown as a loading control and relative IL-22Rα expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean ± the SEM of three independent experiments

Fig. 5

IL-24 augments the expression of suppressive cytokine signaling components by murine astrocytes and limits inflammatory cytokine release by these cells. a Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 30 min and the presence of phosphorylated STAT3 and STAT1 was determined by immunoblot analysis. Expression of β-actin is shown as a loading control and these immunoblots are representative of two separate experiments. b Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 2 or 4 h, and SOCS3 mRNA expression was determined by semi-quantitative RT-PCR. Expression of the housekeeping gene product GAPDH is shown, and relative SOCS3 expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean ± the SEM of three independent experiments, and an asterisk indicates a statistically significant difference from unchallenged cells at each time point (p < 0.05). c Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 8 h prior to immunoblot analysis for SOCS3 protein expression. Expression of the housekeeping gene β-actin is shown and relative SOCS3 protein expression was determined by densitometric analysis normalized to untreated cells. Asterisks indicate statistically significant differences from unchallenged cells (p < 0.05). d Astrocytes were untreated or treated with IL-24 (0.5, 3, 10, 30, or 100 ng/mL) for 4 h prior to challenge with bacterial LPS (5 ng/mL) or vehicle control for 12 h, and IL-6 secretion was determined by specific capture ELISA. Asterisks indicate a statistically significant difference (p < 0.05) from similarly challenged cells in the absence of IL-24 (n = 3). e Primary astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 4 h prior to being uninfected or infected with Nm for 48 h before cell viability analysis via MTS assay. Data is presented as the mean absorbance ± SEM for three experiments. 0.1% Triton X-100 was used as a positive control, and an asterisk indicates a statistically significant difference from unchallenged cells in the absence of IL-24 (p < 0.05)

Fig. 6

IL-24 increases the expression of anti-inflammatory cytokines and neuroprotective factors by primary murine astrocytes. a Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/ml) for 4 h prior to N. meningitidis infection (Nm; MOI of 10:1 bacteria to each astrocyte) or vehicle control. At 24 or 48 h postinfection, IL-10 protein release was assessed by specific capture ELISA. Asterisks and dagger indicate a statistically significant difference from uninfected cells and similarly challenged cells in the absence of IL-24, respectively (n = 3; p < 0.05). b Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 2 or 4 h, and mRNA expression of GLT-1 was determined by semi-quantitative RT-PCR. Expression of the housekeeping gene product GAPDH is shown, and relative GLT-1 expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean ± the SEM of three independent experiments, and an asterisk indicates a statistically significant difference (p < 0.05) from unchallenged cells at each time point. c Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 2 h and mRNA expression of COX2 was determined by semi-quantitative RT-PCR. Data is expressed as the mean ± the SEM of three independent experiments, and an asterisk indicates a statistically significant difference (p < 0.05) from unchallenged cells