Edited by: Xuping Li, Houston Methodist Research Institute, United States
Reviewed by: Hermona Soreq, Hebrew University of Jerusalem, Israel; Tommaso Angelone, Università della Calabria, Italy
†These authors have contributed equally to this work as co-first authors
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Inflammatory responses contribute to the pathogenesis of various neurological diseases, and microglia plays an important role in the process. Activated microglia can differentiate into the pro-inflammatory, tissue-damaging M1 phenotype or the anti-inflammatory, tissue-repairing M2 phenotype. Regulating microglia differentiation, hence limiting a harmful response, might help improve the prognosis of inflammation-related nervous system diseases. The present study aimed 1. to observe the anti-inflammatory effect of lipoxin A4 (LXA4) on the inflammatory response associated to lipopolysaccharide (LPS)-induced microglia activation, 2. to clarify that LXA4 modulates the activation and differentiation of microglia induced by LPS stimulation, 3. to determine whether LXA4 regulates the activation and differentiation of microglia through the Notch signaling pathway, 4. to provide a foundation for the use of LXA4 for the treatment of inflammatory related neurological diseases. To construct a model of cellular inflammation, immortalized murine BV2 microglia cells were provided 200 ng/ml LPS. To measure the mRNA and protein levels of inflammatory factors (interleukin [IL]-1β, IL-10, and tumor necrosis factor [TNF]-α) and M1 and M2 microglia markers (inducible nitric oxide synthase [iNOS], cluster of differentiation [CD]32, arginase [Arg]1, and CD206), we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), immunofluorescence, or flow cytometry. To determine the mRNA and protein levels of Notch signaling components (Notch1, Hes1, and Hes5), we performed qRT-PCR and western blot. LXA4 inhibits the expression of Notch1 and Hes1 associated with M1 type microglial differentiation and decreases the M1 type microglia marker iNOS and related inflammatory factors IL-1β and TNF-α. Moreover, LXA4 upregulates the expression of the M2-associated Hes5, as well as the expression of the M2 microglia marker Arg1 and the associated inflammatory factor IL-10. These effects are blocked by the administration of the γ-secretase inhibitor DAPT, a specific blocker of the Notch signaling pathway. LXA4 inhibits the microglia activation induced by LPS and the differentiation into M1 type with pro-inflammatory effect, while promoting the differentiation to M2 type with anti-inflammatory effect. LXA4 downregulates the inflammatory mediators IL-1β, TNF-α, and iNOS, while upregulating the anti-inflammatory mediator IL-10, which acts through the Notch signaling pathway.
Numerous studies have shown that neuroinflammation plays an important role in the occurrence and development of central nervous system disorders such as ischemic stroke, Alzheimer’s disease, and Parkinson’s disease, and is associated to every stage of the disease process(
Microglia plays an essential role in innate immunity, homeostasis, and neurotropic support in the central nervous system (
In the central nervous system, the Notch signaling pathway is involved in dynamic changes at the cellular level which reflect into the regulation of the nervous system, which in turn plays an important role in the activation and differentiation of microglia (
Lipoxin is an endogenous anti-inflammatory lipid medium. It is released only in small amount under physiological conditions, but its synthesis is significantly increased under pathological conditions in response to inflammatory stimuli. It acts as a modulator of the inflammatory process, exerting anti-inflammatory and pro-inflammatory effects (
The purpose of this study is to clarify the modulatory effect of LXA4 on the inflammatory response associated to LPS-induced microglia activation, with a focus on the regulatory role of LXA4 on the Notch signaling pathway.
The BV2 murine microglia cell line was purchased from the Wuhan University China Culture Collection. BV2 microglia were placed in MEM/EBSS medium containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin and streptomycin, and cultured at 37°C in an incubator with a 95% O2/5% CO2 atmosphere. Every 2–3 days, the cells were washed twice with phosphate-buffered solution (PBS). After adding 1–1.5 ml of 0.125% trypsin, the attached cells were allowed to detach from the surface of the cell cultures at 37°C; the trypsin was neutralized with culture medium, and the cells were transferred into a new flask containing MEM/EBSS medium (supplemented with 10% FBS and 100 U/ml penicillin and streptomycin) and placed in the incubator. When cells grew adherent and the cell body is branched, they were transferred into 24-well plates (105 cells/well for enzyme-linked immunosorbent assay [ELISA], 104 cells/well for immunofluorescence), 6-well plates (2.5 × 105 cells/well for quantitative reverse transcription polymerase chain reaction [qRT-PCR], 5 × 105 cells/well for flow cytometry), or 100-mm culture dishes (1.2 × 106 cells/dish for western blotting).
In this study, we used the following materials: LXA4 (5S,6R,15R-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; Cayman); Minimum essential medium (Eagle) with 2 mM
Cell treatment: BV2 microglia cultured
Experimental grouping:
Part I LXA4 regulates the activation and differentiation of microglia (Results 3.1–3.2).
Control group: cells cultured in serum-free medium containing 0.035% ethanol.
LXA4 group: cells cultured in serum-free medium containing 100 nmol/l LXA4.
Lipopolysaccharide group: cells pretreated with serum-free medium containing 0.035% ethanol for 30 min, after which LPS was added to a final concentration of 200 ng/ml.
LXA4 group + LPS group: cells pretreated with serum-free medium containing 100 nmol/l LXA4 for 30 min, after which LPS was added to a final concentration of 200 ng/ml.
Part II Study on the regulation of Notch signaling pathway by LXA4 (Results 3.3).
LXA4 inhibits the expression of molecules downstream of the Notch signaling pathway.
Grouped with the first part
LXA4 regulates Notch signaling pathway.
Control group: cells cultured in serum-free medium containing 0.035% ethanol.
LPS group: cells pretreated with serum-free medium containing 0.035% ethanol for 30 min, after which LPS was added to a final concentration of 200 ng/ml.
DAPT+LPS group: cells were pretreated with serum-free medium containing 10 μmol/l DAPT for 1 h, after which LPS was added to a final concentration of 200 ng/ml.
LXA4+LPS group: cells pretreated with serum-free medium containing 100 nmol/l LXA4 for 30 min, after which LPS was added to a final concentration of 200 ng/ml.
DAPT+LXA4+LPS group: after pretreatment with DAPT with a final concentration of 10 μM for 1 h, 100 nmol/l LXA4 was added for 30 min, and then added to a final concentration of 200 ng/ml LPS.
The concentrations of IL-1β, IL-10, and TNF-α in cell supernatants were determined by ELISA, according to the ELISA kit manufacturer’s protocol (Shanghai ExCell Biotechnology). BV2 microglia cultured on 24-well plates were treated with LPS for 6 h. Next, the cell supernatants were collected, and the total protein level therein contained was normalized for each sample prior to performing the ELISA measurements for IL-1β, IL-10, and TNF-α.
Total RNA was extracted from BV2 microglia using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the reagent instructions. The concentration of RNA was measured using Nanodrop-1000 (Nanodrop Technologies, United States) and the purity was evaluated by the absorbance ratio at 260 and 280 nm, and the RNA purity was between 1.9 and 2.1. The cDNA was synthesized according to HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Nanjing Vazyme Biotech Biotechnology) reagent and stored at −20°C. The mRNA expression level was detected by real-time fluorescent quantitative PCR using the AceQ® qPCR SYBR® Green Master Mix (Low ROX Premixed) kit. The expression level of the gene of interest was normalized using GAPDH (glyceraldehyde triphosphate dehydrogenase), the CT value represents a real-time fluorescent quantitative PCR value, and the 2−ΔΔCT method was used to calculate relative change analysis data of gene expression. No treatment affected the expression of GAPDH mRNA. The primer sequences used are given in the
Primers used for qRT-PCR.
Primer name | Sequence (5′–3′) |
---|---|
GAPDH | F-GGGTGTGAACCACGAGAAAT |
R-CCTTCCACAATGCCAAAGTT | |
Arg1 | F-GACCTGGCCTTTGTTGATGT |
R-CCATTCTTCTGGACCTCTGC | |
iNOS | F-ACGAGACGGATAGGCAGAGA |
R-CACATGCAAGGAAGGGAACT | |
CD206 | F-GGGACTCTGGATTGGACTCA |
R-GCTCTTTCCAGGCTCTGATG | |
CD32 | F-GCTCAAGGAAGACACGGTGA |
R-GTGTAGCTGGCTTGGACCTG | |
TNF-α | F-CCGATGGGTTGTACCTTGTC |
R-AGATAGCAAATCGGCTGACG | |
IL-1β | F-GCTGCTTCCAAACCTTTGAC |
R-AGCTTCTCCACAGCCACAAT | |
IL-10 | F-CCAGTTTTACCTGGTAGAAGTGATG |
R-TGTCTAGGTCCTGGAGTCCAGCAGACTCAA | |
Notch1 | F-GCCTTCGTGCTCCTGTTCTT |
R-CTTCTTGCTGGCCTCTGACA | |
Hes1 | F-TCATGGAGAAGAGGCGAAGG |
R-CGGAGGTGCTTCACAGTCATT | |
Hes5 | F-AGGCCGACATCCTGGAGAT |
R-TCGCTGTAGTCCTGGTGCAG |
BV2 microglia cells were treated with LPS for 4 and 8 h, and the cell culture medium was discarded and washed three times with sterile phosphate buffered saline. A 100:1 mixture of 100 μl of ice-cold cell lysis buffer and protease inhibitor (PMSF) was added to the cell culture, then cells were incubated on ice for 30 min, and the lysate was clarified by spinning for 10 min at 4°C (12,000 rpm), leaving the supernatant for later use. Protein quantification was performed using the micro bicinchoninic acid method. A 5× sodium dodecyl sulfate loading buffer was added, before incubating at 99°C for 5 min. Then the samples were loaded at 15 μg protein/lane on 6 or 12% acrylamide gels and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis for about 1.5 h at 80 mV (stacking gel) and 120 mV (resolving gel). Proteins were then transferred to a polyvinylidene fluoride membrane and blocking was made for 2 h in a 5% non-fat dry milk in Tris base/Tween-buffered saline (TBST). The molecular weights of the iNOS, Notch1, arginase (Arg)1, Hes1, Hes5, and β-tubulin proteins are 131, 120, 35/38, 35, 18, and 55 kDa, respectively. Samples were incubated at 4°C overnight with Rabbit anti-iNOS (1:500), Notch1 (1:500) and mouse anti-Arg1 (1:300), Hes1 (1:500), Hes5 (1:300), β-tubulin (1:1,000) primary antibodies. After washing three times with TBST, the samples were incubated for 1 h at room temperature with a secondary antibody of the IgG family, conjugated with HRP. Enhancement of the antibody reaction using an hypersensitive chemiluminescent (ECL) reagent (Beyotime Biotechnology) allowed for visualizing the protein. Protein bands were quantified using the ImageJ software and the band intensity was normalized to the band intensity of β-tubulin.
To detect the expression of BV2 M1 and M2 microglia biomarkers, we first treated the cells with LPS. After treatment, the cells were washed 3 × 5 min with PBS (pH 7.4), and 4% paraformaldehyde was added for 30 min at room temperature, after which the cells were again washed 3 × 5 min with PBS. The membranes were disrupted with 0.5% Triton X-100 (PBS configuration) for 5 min. After washing three times with PBS for 5 min each, we added PBS containing 2% bovine serum albumin and 10% goat serum, and the plates were sealed at 37°C for 45 min. The following antibodies were added overnight at 4°C: rabbit anti-iNOS (1:200), rabbit anti-CD32 (1:200), rabbit anti-Arg1 (1:100), and rabbit anti-CD206 (1:100). After washing 3 × 5 min with PBS, we added a fluorescently labeled goat anti-rabbit IgG secondary antibody (1:500) for 1 h at room temperature. The cells were again washed 3 × 5 min with PBS, and counterstained in the dark with a mixture of 1 ml DAPI (1 mg/ml) + 1 ml H2O. The cells were incubated at 37°C for 10 min, washed 3 × 5 min with PBS, observed under an Inverted fluorescence microscope (IX71 Japan OLYMPUS Corp.), and photographed.
BV2 microglia were seeded at 5 × 105/well and treated according to their experimental group. After treatment, the culture supernatant was discarded, and cells were washed gently with 2 ml of PBS. Then, the PBS was aspirated and the adherent cells were digested with 1 ml of trypsin; 1 ml of MEM/EBSS medium was used to stop the digestion. The cells were centrifuged (1,000 rpm, 5 min, 4°C), washed twice with PBS, and resuspended at 1 × 105 cells/ml. To label the cells, we added PE/Cy5 anti-mouse CD16/CD32 monoclonal antibody (≤0.25 μg/106 cells, 100 μl) and Alexa Fluor® 488-labeled anti-mannose receptor antibody (1:500) for 20 min at room temperature in the dark, washed the cells twice with PBS, and resuspended them in 300 μl PBS. The cell surface expression of CD16/CD32 and CD206 was detected using a FACS Calibur flow cytometer (BD Company), and the data were analyzed using FlowJo software.
All data are expressed as mean ± SD. Statistical analysis was performed using SPSS 21.0 statistical software. One-way analysis of variance (ANOVA) was used for comparison between groups, and pairwise comparison between sample means was performed using the Bonferroni method. Statistical significance was set at
The expression of IL-1β, TNF-α and IL-10 mRNA was determined by qRT-PCR 6 h after the corresponding treatment. LPS induced a significant increase in IL-1β and TNF-α mRNA in BV2 microglia as compared to the control group (
LPS-induced microglia activation and release of inflammatory factors; After 6 h of LPS stimulating
Eight hours after the corresponding treatment, the ELISA method was used to detect the protein expression levels of IL-1β, TNF-α and IL-10. As compared to the control group, LPS induced a significant increase in IL-1β and TNF-α protein levels in BV2 microglial culture supernatants, and the difference was statistically significant (
Therefore, LXA4 inhibited the genes and protein expression of M-1 microglia-associated inflammatory factors IL-1β and TNF-α, and upregulated the expression of IL-10, an inflammatory factor associated with M2 microglia.
The expression of iNOS, cluster of differentiation (CD)32, Arg1 and CD206 mRNA was detected via qRT-PCR 6 h after the corresponding treatment. As compared to the control group, the relative expression of iNOS and CD32 mRNA in the LPS group was significantly increased (
Quantitative RT-PCR analysis of iNOS, CD32, Arg1, CD206 mRNA levels in the BV2microglia 6 h after LPS treatment.
Each group was tested 8 h after the corresponding treatment. The levels of iNOS, Arg1, CD32 and CD206 proteins were determined by Western blot, cellular immunofluorescence and flow cytometry.
Western blot analysis showed that the expression of iNOS protein in LPS group was significantly increased as compared to the control group (
Western blot analysis of the effect of LXA4 on iNOS and Arg1.
Cellular immunofluorescence showed that BV2 microglia cells were activated in response to LPS treatment, the cell body became larger and rounder, the protrusion decreased and became thicker, and the morphology appeared as amoeba-like. LXA4 pretreatment weakened the response to LPS in terms of morphological changes (
Immunofluorescence images showing the BV2 microglia after LPS stimulates which were labeled with iNOS antibody, with LXA4, the expression of iNOS is decreased. Green fluorescence indicated iNOS positive cells, while blue fluorescence indicated DAPI-labeled nuclei. Scale bar: 20 μm.
Immunofluorescence images showing the BV2 microglia after LPS stimulates which were labeled with CD32 antibody, with LXA4, the expression of iNOS is decreased. Green fluorescence indicated CD32 positive cells, while blue fluorescence indicated DAPI-labeled nuclei. Scale bar: 20 μm.
Immunofluorescence images showing the BV2 microglia after LPS stimulates which were not labeled with Arg1 antibody, with LXA4, the expression of Arg1 is expressed at high levels. Green fluorescence indicated Arg1 positive cells, while blue fluorescence indicated DAPI-labeled nuclei. Scale bar: 20 μm.
Immunofluorescence images showing the BV2 microglia after LPS stimulates which were not labeled with CD206 antibody, with LXA4, the expression of Arg1 is expressed at high level. Green fluorescence indicated CD206 positive cells, while blue fluorescence indicated DAPI-labeled nuclei. Scale bar: 20 μm.
CD32 is a M1 microglia surface marker molecule and can be detected by flow cytometry. As shown in
Protein expression levels of CD32 and CD206 were measured by flow cytometry.
Western blot, cellular immunofluorescence and flow cytometry were concordant in indicating that LXA4 inhibited the expression of M1 type biomarkers iNOS and CD32 at the protein expression and transcription levels, and upregulated the M2 type biomarkers Arg1 and CD206. In other words, LXA4 promoted the shift of M1 to M2 microglia.
The relative expression levels of Notch1, Hes1 and Hes5 mRNA were determined by qRT-PCR 3 h after the corresponding treatments were administered. As compared to the control group, the expression of Notch1 and Hes1 mRNA in the LPS group was significantly different (
Quantitative RT-PCR analysis of Notch1, Hes1 and Hes5 mRNA levels in the BV2 microglia.
Each group was performed for 4 h with corresponding treatments and the expression levels of Notch1, Hes1, and Hes5 proteins were determined through Western blot. The expression of Notch1 and Hes1 protein in the LPS group was significantly higher than in the control group (
Western blot analysis of the effect of LXA4 on Notch1, Hes1 and Hes5.
Therefore, LXA4 affected the expression of downstream effector molecules within the Notch signaling pathway at the level of genes and protein; inhibited the expression of Notch1 and Hes1, and upregulated the expression of Hes5.
Each group was performed for 6 h with corresponding treatments and the expression levels of Notch1, Hes1, and Hes5 proteins were determined through Western blot. The levels of Notch1 and Hes1 protein in the LPS group were significantly higher than in the control group (
Western blot analysis of the Notch1, Hes1, Hes5, iNOS and Arg1 effect after DAPT pretreatment.
Therefore, LXA4 downregulated the expression of Notch1 and the downstream effector Hes1 in M1 microglia differentiation (
Each group was performed for 6 h with corresponding treatments and the expression levels of microglia M1 biomarker iNOS and M2 biomarker Arg1 proteins were determined through Western blot. The expression of the iNOS protein increased in the LPS group as compared to the control group (
Each group was performed for 6 h with corresponding treatments and the ELISA method was used to detect the expression levels of M1 microglia-associated inflammatory cytokines IL-1β and TNF-α and M2 microglia-associated inflammatory factor IL-10. As compared to the control group, the expression of IL-1β and TNF-α protein in the LPS group increased (
LPS-induced microglia activation and release of inflammatory factors; After 6 h of LPS stimulating
An increasing number of studies reveal that inflammatory reactions exert a significant influence on the occurrence and development of central nervous system conditions such as ischemic stroke, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis, at multiple stages of the disease process. Inflammation aggravates the damage to nerve cells and tissues, worsening the pathological condition (
The role of microglia in the neuroinflammatory response is important (
Activation and differentiation of microglia involves multiple signaling pathways, such as the Notch signaling pathway, NF-κB, tyrosine protein kinase (JAK) signal transduction/transcriptional activator (STAT), peroxisome proliferator-activator receptor-γ (PPAR-γ) and cAMP response element binding protein (CREB) (
Lipoxins (LXs) are a class of arachidonic acid-derived mediators formed via lipoxygenase-catalyzed reactions, which carry anti-inflammatory and pro-inflammatory properties, and are classified according to the position and conformation of the hydroxyl groups in the molecule. LXA4 and LXB4, and their epimers 15-epi-LXA4 and 15-epi-LXB4, are synthesized only in small amount under physiological conditions. However, their levels significantly rise under various pathological conditions involving inflammatory stimuli, to act as downregulators of the inflammatory process. LXs play a role in anti-inflammatory and pro-inflammatory decline. Some synthetic lipoxins such as LXA4 have exhibited anti-inflammatory effects in experimental studies on respiratory tract infections, lung injury, peritonitis, enteritis, nephritis, gynecological inflammation, and various tumor-related inflammations (
In this study, the immortalized murine microglial cell line BV-2 was used to construct an inflammatory model. BV2 microglia retains many morphological features, phenotypical characterization, and functional characteristics of the microglia, and is consequently an ideal model for studying microglia. We found that after LPS stimulation of BV2 microglia, the microglia cells were activated, and the round, swelling, and thin processes of the cell body retracted from branching to an amoeba-like asset. After pretreatment with LXA4, microglia cells showed small bodies and many branches.
This investigation capitalized on a complete set of analyses, including qRT-PCR, ELISA, western blot, cell immunofluorescence, and flow cytometry. A first part of the study explored the regulatory role of the LXA4 on the activation and differentiation of the microglia. We found that LXA4 could inhibit gene and protein expression of M1 biomarkers iNOS, CD32 and M1 related inflammatory cytokines IL-1β and TNF-α. Conversely, LXA4 caused upregulation of the expression of M2 biomarkers Arg1 and CD206 and M2 microglia-associated inflammatory factor IL-10. LXA4 can regulate the switch from M1 to M2 microglia and alleviate inflammation. A second part of this study focused on the LXA4 regulation of the Notch signaling pathway. LXA4 could affect the expression of downstream effector molecules of the Notch signaling pathway at both the gene and protein levels: it inhibited the expression of Notch1 and Hes1 related to the differentiation of M1 microglia. Upregulating the expression of Hes5 in association with M2 differentiation suggests that LXA4 promotes the transformation of M1 type to M2 microglia through the Notch signaling pathway.
It was subsequently found that the specific blocker Notch signaling pathway and the regulation of Notch downstream effector molecules Hes1 and Hes5 by LXA4 were blocked after the treatment of γ-secretase inhibitor DAPT. The results suggest that LXA4 regulates the differentiation of microglia through Notch signaling pathway.
Further studies showed that LXA4 decreased the expression of M1 related biomarkers iNOS and the related inflammatory cytokines IL-1β and TNF-α. The upregulation of the expression of Arg1 and the related inflammatory factor IL-10 can also be blocked by the DAPT. Therefore, we finally confirmed that LXA4 can exert its anti-inflammatory effects by regulating the differentiation of microglia through the Notch signaling pathway.
In conclusion, LXA4 inhibited the activation of microglia induced by LPS, promoted the transformation of M1–M2, and reduced the expression of IL-1β, TNF-α, and iNOS, while enhancing the expression of the anti-inflammatory mediator IL-10 through the Notch signaling pathway. This study shows: 1. LPS stimulation induced M1 microglia activation and increased the secretion of pro-inflammatory factors; 2. The Notch1 receptor and downstream effector Hes1 increased, suggesting that microglia differentiated into M1 type; 3. We revealed that LXA4 has desirable anti-inflammatory properties, which is consistent with previous studies (
It should be highlighted that γ-secretase is a key enzyme in the activation of the Notch pathway. DAPT can inhibit the activation of the Notch pathway by inhibiting the γ-secretase. However, the expression of the Notch1 protein was not affected in theory. Therefore, the expression of Hes1 and Hes5 was mainly inhibited after DAPT administration, and the change of Notch1 was not significant. Previous studies have shown that the γ-secretase enzyme blockers affect the Notch signaling pathway and produce a series of side effects at the level of the gastrointestinal tract and hematopoietic system, as well as induce thymocyte damage (
JW and D-hD participated in the design of this study and they both carried out the study and performed the statistical analysis. JW collected important background information and D-hD drafted the manuscript, and JW repeatedly modified the text structure and Details. Q-qL, X-yW, Y-yS, and L-JL participate in the original article and discussion about article writing and revision. All the authors revised and approved final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.