Memory-Like Inflammatory Responses of Microglia to Rising Doses of LPS: Key Role of PI3Kγ

Abstract

Trained immunity and immune tolerance have been identified as long-term response patterns of the innate immune system. The causes of these opposing reactions remain elusive. Here, we report about differential inflammatory responses of microglial cells derived from neonatal mouse brain to increasing doses of the endotoxin LPS. Prolonged priming with ultra-low LPS doses provokes trained immunity, i.e., increased production of pro-inflammatory mediators in comparison to the unprimed control. In contrast, priming with high doses of LPS induces immune tolerance, implying decreased production of inflammatory mediators and pronounced release of anti-inflammatory cytokines. Investigation of the signaling processes and cell functions involved in these memory-like immune responses reveals the essential role of phosphoinositide 3-kinase γ (PI3Kγ), one of the phosphoinositide 3-kinase species highly expressed in innate immune cells. Together, our data suggest profound influence of preceding contacts with pathogens on the immune response of microglia. The impact of these interactions-trained immunity or immune tolerance-appears to be shaped by pathogen dose.

Keywords: LPS; PI3Kγ; microglia; phagocytosis; tolerance; training; β-glucan.

Figure 1

Cytokine responses of microglia primed with increasing doses of LPS and β-glucan. (A) Schematic view of the two-step approach to induce memory-like inflammatory responses. Primary microglial cells (75,000 cells/well) isolated from neonatal mice were seeded on day 0 and stimulated with increasing doses of LPS (1 fg/ml to 100 ng/ml) or β-glucan (1 fg/ml to 1 μg/ml) on day 1 for 24 h. On day 2, cells were washed up and followed by medium change on day 4. Thereafter, cells were left to rest and restimulated on day 6 with a fixed dose of LPS (100 ng/ml). Six h and 24 h after the second stimulation, samples have been collected and processed for further analysis. (B–F) Opposing inflammatory response of microglia after priming with increasing doses of LPS or β-glucan. Microglial cells were stimulated with increasing doses of LPS (B,C: 1 fg/ml to 100 ng/ml) or β-glucan (D: 1 fg/ml to 1 μg/ml) for 24 h and rechallenged on day 6 with a fixed dose of LPS (100 ng/ml). Supernatants and protein lysates have been collected 24 h after the second stimulus by LPS (100 ng/ml). Cytokine levels (B,D: TNF-α, n = 8; E: IL-6, n = 8; F: IL-10, n = 8) have been analyzed by ELISA (normalized to total protein concentration), whereas protein expression (C: TNF-α, n = 5) has been assayed by Western blotting and quantification (unprimed cells assigned as 1.0). Data are given as scatter dot plots, mean + SEM, ^#p < 0.05, ^#significant differences vs. unprimed condition (gray column). US, unstimulated; UP, unprimed.

Figure 2

Reactive oxygen and nitrogen responses of microglia primed with increasing doses of LPS. Primary microglial cells were stimulated with increasing doses of LPS (1 fg/ml to 100 ng/ml) for 24 h and rechallenged on day 6 with a fixed dose of LPS (100 ng/ml). Supernatants and protein lysates were collected 24 h after the second stimulus by LPS. (A) ROS levels (n = 3, repeated measurements) were measured by the ROS 2′ ,7′-Dichlorodihydrofluorescein-diacetate assay. (B) iNOS (n = 5) were measured by Western blotting and quantification (unprimed cells assigned as 1.0). Data are given as scatter dot plots, mean + SEM, ^#p < 0.05, ^#significant differences vs. unprimed condition (gray column). US, unstimulated; UP, unprimed.

Figure 3

Modification of TLR4 expression and related intracellular signaling in primed microglia. Primary microglial cells were stimulated using the two-step approach with fixed priming doses of LPS [ULP (ultra-low dose, 1 fg/ml) and HP (high-dose, 100 ng/ml)]. RNA samples (6 h) and lysates (24 h) were collected after the second stimulus with LPS (100 ng/ml) and analyzed for (A) TLR4 expression (n = 5), (B) MyD88 expression (n = 5), and (C) HIF-1α expression (n = 3) by real-time PCR normalized to GAPDH representing relative values to unprimed state (assigned as 1.0). (D) Phospho-p65 (n = 5) and (E) PI3Kγ subunit p110γ (n = 5) protein expression were measured by Western blotting and quantified (unprimed cells assigned as 1.0). Data are shown as scatter dot plots, mean + SEM, ^#p < 0.05, ^# significant differences vs. unprimed condition (gray column). US, unstimulated; UP, unprimed.

Figure 4

PI3Kγ mediates innate immune memory cytokine response of microglia. Primary microglial cells (wild-type •, open columns; PI3Kγ^−/− ■, dark gray columns; PI3Kγ^KD/KD ▴, hatched columns) were primed with fixed priming doses of LPS [ULP (ultra-low dose, 1 fg/ml) and HP (high-dose, 100 ng/ml)] and restimulated on day 6 with a fixed dose LPS (100 ng/ml). Supernatants were collected 24 h after the second stimulation by LPS and the cytokine levels for (A) TNF-α, (B) IL-6, and (C) IL-10 were assayed by ELISA (normalized to total protein concentration). Data are shown as scatter dot plots, mean + SEM, n = 6, ^#p < 0.05, ^#significant differences vs. unprimed condition (UP) and ^§p < 0.05, ^§significant differences vs. microglia derived from wild-type mice. US, unstimulated.

Figure 5

PI3Kγ mediates Akt and Erk signaling in primed microglia. Primary microglial cells (wild-type •, open columns; PI3Kγ^−/− ■, dark gray columns; PI3Kγ^KD/KD ▴, hatched columns) were primed with fixed priming doses of LPS [ULP (ultra-low dose, 1 fg/ml) and HP (high-dose, 100 ng/ml)] and restimulated on day 6 with a fixed dose LPS (100 ng/ml). Lysates were collected 24 h after the second stimulation by LPS and the protein expression of (A) phospho-Akt and (B) phospho-ERK1/2 were assayed by Western blotting and quantified (unprimed cells assigned as 1.0). Data were presented as scatter dot plots, mean + SEM, n = 6, ^#p < 0.05, ^#significant differences vs. unprimed condition (UP) and ^§p < 0.05, ^§significant differences vs. microglia derived from wild-type mice. US, unstimulated.

Figure 6

PI3Kg mediates phagocytic activity of microglial cells after priming with LPS in cell culture and mouse brain. (A,B) Primary microglial cells [wild-type (WT), •, open columns; PI3Kγ^−/− ■, dark gray columns; PI3Kγ^KD/KD ▴, hatched columns] were stimulated using the two-step approach with fixed priming doses of LPS [ULP (ultra-low dose, 1 fg/ml) and HP (high-dose, 100 ng/ml)] and restimulated on day 6 with a fixed dose LPS (100 ng/ml). Twenty-four hours later, cells were incubated with fluorescein isothiocyanate (FITC)-labeled Zymosan A for 1 h. Iba1 was used as a marker of primary microglial cells (red color). DAPI (blue) was used for staining the nucleus of the cells and GFP for labeling FITC-Zymosan particles. Quantification of in vitro phagocytosis (B) (n = 4) was performed as uptake rate of FITC-Zymosan particles per cell (phagocytic index). (C) Mice [wild-type (WT), •, open columns; PI3Kγ^−/− ■, dark gray columns; PI3Kγ^KD/KD ▴, hatched columns] were initially injected with either low-dose LPS (LP, 0.025 mg/kg, i.p.) or high-dose LPS (HP, 10 mg/kg, i.p.) for priming and 3 days later with a subsequent application of 10 mg/kg, i.p. LPS. Administration of FITC-labeled Zymosan particles (9,800 U/ml) was performed 24 h after the second LPS injection. Brains were harvested and processed 24 h later. Iba1 (microglial cells) and GFP (FITC-labeled Zymosan particles) were used as markers. Quantification of phagocytosis (D) was performed as percentage of Iba-1 positive cells per voxel mm³ containing Zymosan particles (n = 7). Data are shown as scatter dot plots, mean + SEM, ^#p < 0.05, ^#significant differences vs. unprimed condition (in vitro) or vs. controls (single injection with 10 mg/kg, i.p. LPS; in vivo); ^§significant differences vs. microglia derived from wild-type mice or WT mice of the same treating group. US, unstimulated; UP, unprimed; LP, low primed.