Induction of endogenous IL-10 promotes resolution and tolerance of nitric oxide in microglia

Abstract

Endogenous interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is induced in a timely and coordinated manner to dampen microglia-mediated brain inflammation. However, it remains unclear how it alters the inflammatory process to shape the immune polarization of microglia. This study aimed to investigate the anti-inflammatory mechanisms of endogenous IL-10 in activated and tolerized microglia using in vitro multiple-reconstituted primary brain cell cultures and an in vivo IL-10 knockout (IL-10KO) animal model. Upon a single or repeated lipopolysaccharide (LPS) treatment regimen, the expression levels of the inflammatory factors during the neuroinflammatory/tolerance process were measured by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and Griess reagent assay. ELISA data showed that cell-autonomous induction of endogenous IL-10 occurs in LPS-activated and LPS-tolerized microglia. Furthermore, comparing the LPS-elicited pro-inflammatory factor expressions at different neuroinflammatory stages between the wild-type and IL-10KO groups, our data revealed the failure of negative-feedback suppression of inducible nitric oxide synthesis (iNOS) during immune resolution in the IL-10KO brains. Moreover, LPS-treated IL-10KO microglia increase the supernatant level of nitrite and become overactive during late-stage inflammation, despite no changes in cell number; in contrast, LPS-tolerized IL-10KO microglia fail to program endotoxin tolerance of nitric oxide/inducible nitric oxide synthesis (iNOS). In summary, our data demonstrate that the cell-autonomous induction of endogenous IL-10 in microglia is crucial for mitigating brain immune responses, particularly in the resolution and tolerance of nitric oxide.

Keywords: Endotoxin tolerance; IL-10; Immune resolution; Microglia; Neuroinflammation; Nitric oxide.

Fig. 1

Cell-autonomous induction of endogenous IL-10 in LPS-activated and LPS-tolerized microglia. (A) As an illustrated experimental procedure of endotoxin treatment regimen for the development of the activated and tolerized microglia, the neuron-glia (neurons + astroglia + microglia), mixed-glia (astroglia + microglia), and microglia-enriched cultures were prepared and pre-incubated with a vehicle (activated) or LPS (tolerized) (15 ng/ml) for 6 h. After being replaced with fresh medium for 6 h, LPS (15 ng/ml) was re-added to the cells. (B-D). After receiving the endotoxin treatment regimen (activated versus tolerized) as indicated in (A), supernatant levels of IL-10 in neuron-glia (B), mixed-glia (C), and microglia-enriched (D) cultures were detected, respectively, by ELISA assay 6 and 24 h later. Data are expressed as the mean ± SEM from more than three independent experiments (n ≥ 3) in duplicate with two-way ANOVA with Tukey's multiple comparisons test (6-h group versus 24-h group: ∗∗p < 0.01, ∗∗∗p < 0.001; LPS group versus LPS/LPS group: #p < 0.05, ##p < 0.01, ###p < 0.001).

Fig. 2

Deficiency in IL-10 impairs negative-feedback suppression of inducible nitric oxide synthesis in the inflammatory brain. (A) Schematic of the experimental design for determining the role of endogenous IL-10 in LPS-induced immune resolution of pro-inflammatory factors. The C57/10J (WT) and IL-10KO mice received intraperitoneal LPS (0.3 mg/kg) and saline treatment, followed by measurement of endogenous IL-10 induction in the brain, liver, and serum, and the inflammatory mRNA expression, including TNF-α, IL-1β, COX-2, and iNOS, using ELISA and real-time PCR at different time points as indicated. Finally, the survival rate of these LPS-treated mice was calculated. (B-E) Expression of TNF-α (B), IL-1β (C), COX-2 (D), and iNOS (E) mRNA in the WT and IL-10KO mice's brains after 1, 3, 6, and 12 h of saline (10 WT and 10 IL-10KO) and LPS (12 WT and 12 IL-10KO) treatment was measured by RT-PCR. Data are presented as a fold change relative to the saline-treated control and expressed as the mean ± SEM from three independent experiments performed in triplicate with two-way ANOVA with Tukey's multiple comparisons test (saline group versus LPS group: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; WT group versus IL-10KO group: #p < 0.05, ##p < 0.01, ###p < 0.001).

Fig. 3

IL-10KO microglia increase LPS-induced nitrite production in a time- and dose-dependent manner. (A) Schematic of the experimental design to determine the effects of endogenous IL-10 induction on nitrite production in microglia under LPS challenge. (B, C) Measurement of supernatant nitrite in LPS-treated WT and IL-10KO mixed-glia cultures at different time points (B) or concentrations (C) using a Griess reagent. Data are expressed as the mean ± SEM (n ≥ 3) with two-way ANOVA with Tukey's multiple comparisons test (Saline group versus LPS group: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; WT group versus IL-10KO group: #p < 0.05, ##p < 0.01).

Fig. 4

IL-10 deficiency enhances microglia activation at the late-stage inflammation. (A) Schematic of the experimental design for the study of endogenous IL-10 on microglia number and morphology in normal and inflammatory conditions. (B) The expression of Iba-1, a marker for microglia, in these mixed-glia cells at 24, 48, 72, and 92 h in the presence and absence of LPS (15 ng/ml) was detected by immunocytochemistry staining. Magnification: 200×; scale bar: 100 μm. (C-F) Quantitative data of microglial number (Iba-1-positive cells) in these cells treated with LPS at 24 (C), 48 (D), 72 (E), and 96 (F) hours are shown. Data are expressed as the percentage of the WT saline group (mean ± SEM, each dot represented an individual field from three independent experiments) with two-way ANOVA with Tukey's multiple comparisons test (WT group versus IL-10KO group: ∗∗∗p < 0.001; NS indicates no significant difference). (G–J) The density of Iba-1 immunostaining in these LPS-treated mixed cells at 24 (G), 48 (H), 72 (I), and 96 (J) hours was measured and quantified. Data are shown as integrated intensity and expressed as the percentage of the WT saline group (mean ± SEM, each dot represented an individual field from three independent experiments) with two-way ANOVA with Tukey's multiple comparisons test (WT group versus IL-10KO group: ∗p < 0.05; NS indicates no significant difference).

Fig. 5

IL-10KO microglia fail to program endotoxin tolerance of nitrite. (A) Illustration of experimental procedure for studying IL-10's role in nitrite tolerance in LPS-tolerized microglia. WT and IL-10KO mixed-glia cultures were pre-incubated with a vehicle or LPS (15 ng/ml) for 6 h. After 6 h of replacing it with fresh medium, LPS was re-added to the cultures. (B) After receiving the ET treatment regimen (LPS versus LPS/LPS) as indicated, supernatant levels of IL-10 in WT and IL-10KO mixed-glia cultures were detected 72 h later by ELISA assay. Data are expressed as the mean ± SEM from three independent experiments in duplicate with two-way ANOVA with Tukey's multiple comparisons test (LPS group versus LPS/LPS group: ∗∗∗p < 0.001). (C) Supernatant levels of nitrite in WT and IL-10KO mixed-glia cultures under an endotoxin treatment regimen were detected 72 h later by a Griess reagent. Data are expressed as the mean ± SEM from three independent experiments in duplicate with two-way ANOVA with Tukey's multiple comparisons test (LPS group versus LPS/LPS group: ∗∗∗p < 0.001; WT group versus IL-10KO group: ##p < 0.01, ###p < 0.001; NS indicates no significant difference).