Lipopolysaccharide Preconditioning Induces an Anti-inflammatory Phenotype in BV2 Microglia

Abstract

Increasing evidence indicates that endotoxin tolerance is an essential immune-homeostatic response to repeated exposure to lipopolysaccharide (LPS) that induces a state of altered responsiveness in macrophage, resulting in repression of pro-inflammatory gene expression and increased expression of factors that mediate the resolution of inflammation. In this study, quantitative real-time polymerase chain reaction and Western blot for M1 and M2 markers were performed to characterize phenotypic changes of BV2 microglia. We found that the cytokine and chemokine expression during endotoxin tolerance were mostly similar to those found during M2 polarization. We further examined the expression of M1 and M2 markers in CD11b⁺ BV2 by double immunofluorescent staining. The expression of M2 markers (CD206) increased, whereas the expression of M1 (CD54) markers reduced during endotoxin tolerance. Moreover, expression of different transcription factor, known for their function in the regulation of pro- and anti-inflammatory reaction, was also different. Our data demonstrate that repeat LPS treatment activates a differentiation program that leads to microglial polarization toward M2-like phenotype.

Keywords: Alternative activation; Anti-inflammatory; Endotoxin tolerance; Mice; Microglia.

Fig. 1

Expression of cytokine and chemokine in LPS-tolerant BV2 microglia. a BV2 microglia were challenged with or without LPS (10 ng/ml) in a single dose (Medium/LPS) or two doses at a 2 h interval (10–100 ng/ml LPS) and treated for 2 and 6 h (for mRNA), and 6 and 18 h (for protein) as indicated. b BV2 microglia were collected, and TNF-α, IL-β, IL-6, PTGS2, Arginase 1, Fizz 1, IL-4, and IL-10 mRNA were detected by qPCR. Data are representative of more than three independent experiments and are shown as relative mRNA. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Chemokine profile (CXCL9, CXCL10, CXCL11, CCL2, CCL17, and CCL22) and cell surface marker (CD54 and CD206) expression responses during inflammation and endotoxin tolerance. BV2 microglial cells were treated with a single 2- or 6-h LPS (10 ng/ml) challenge, or endotoxin tolerance with a 2- or 6-h second LPS (100 ng/ml) stimulus (LPS/LPS). Gene expression of chemokine and surface marker was analyzed using qPCR. Results are shown as mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Analysis of pro-inflammatory and anti-inflammatory protein expression during endotoxin tolerance. BV2 microglial cells were, respectively, treated with 100 or 10–100 ng/ml LPS for 0, 6, and 18 h. After the indicated time periods, the culture supernatants and lysate of cells were subject to ELISA for IL-1β and IL-10 (a) and Western analysis for PTGS1, PTGS2, HO-1, p-Smad2, and p-Smad3 (b) proteins. c Integrated band densities were obtained by scanning using a densitometer. The blots were reprobed with anti-β-actin to show equal loading. Each treatment was executed in triplicates. Each experiment was repeated at least three times. Results are shown as mean ± SEM. *p < 0.05

Fig. 4

CD206 and CD54 expression in BV2 during endotoxin tolerance (LPS/LPS) and inflammation (medium/LPS). Double fluorescence staining is performed using BV2 marker CD11b (green, FITC) and polyclonal antibody against CD206 and CD54 (red, TRITC). Pseudocolor in yellow indicates colocalization. Nucleus of BV2 was stained with DAPI. Scale bar 25 μm (Color figure online)

Fig. 5

Expression of transcription factor in single and repeat LPS-treated BV2. LPS-induced BV2 microglial cells were treated for different times. The levels of NF-κB, AP-1 (a, c), KLF4, and PPARγ (b, d) were detected by Western blotting. Each blot was representative of at least three independent experiments. All values were expressed as mean ± SEM. *p < 0.05

Fig. 6

Model of the BV2 microglial neuroprotection during endotoxin tolerance. BV2 microglia can adopt distinct phenotypes in response to single LPS or repeat LPS stimulation, which elicit pro-inflammatory or neuroprotection responses. The pro-inflammatory phenotype includes the production of TNF-α, IL-β, IL-6, PTGS1, PTGS2, CXCL9, CXCL10, CXCL11, and CD54. The reparative phenotype is characterized by expression of Arginase 1, Fizz 1, IL-4, and IL-10, and CCL2, CCL17, CCL22, CD206, and HO-1. These phenotypes are directly linked to the distinct function of microglia. TF, transcription factor