Background
Alzheimer’s disease (AD), a progressive age-related disease, is the most common neurodegenerative disorder primarily affecting the elderly population over the age of 60 years. Amyloid-β (Aβ) deposition, neurofibrillary tangle formation, and neuroinflammation are major pathogenic mechanisms that lead to neocortical and hippocampal atrophy, memory dysfunction, and cognition decline in AD patients [
1]. Chronic neuroinflammation including reactive astrocytes, activated microglia, and enhanced cytokine load elicited by the accumulation and subsequent deposition of Aβ within the brain plays a critical role in the initiation and progression of AD [
2,
3]. Its important role in AD has been highlighted by a succession of genetic studies identifying numerous immune mediators such as triggering receptor expressed on myeloid cells (TREM) and others linked to elevated AD risk [
4,
5]. Up to date, available therapeutic agents are only able to slow down the progression of AD with limited benefits.
Tyro3, Axl, and Mer tyrosine kinase (MerTK) (TAM) family receptor tyrosine kinases (RTKs) together with their ligands Gas6 and PROS1 are negative feedback regulators that can reduce inflammation in tissue macrophages [
6,
7]. Aberrant expression and dysregulated activation of TAM family members have demonstrated that TAM receptors are both controllers of microglial physiology and potential targets for therapeutic intervention for a variety of central nervous system (CNS)-related disorders, including AD [
8,
9]. In fact, earlier report has described that Gas6, a ligand of TAM receptors, has a protective role in Aβ-induced apoptosis [
10]. Tyro3 overexpression in HEK293 cells stably expressing a mutant amyloid precursor protein has been linked to decreased Aβ accumulation while Tyro3 partial knockdown in vivo is associated with increased amyloid plaque formation [
11]. Besides, TAM receptors play pivotal roles in adult hippocampal neurogenesis. The loss of these receptors can cause comprised neurogenesis in the dentate gyrus of adult hippocampus [
12]. Notably, a recent study has demonstrated that induction of MerTK expression in plaque-associated macrophages consequently licensed their phagocytic activity and promoted plaque clearance in murine models of AD [
13]. This strongly suggests that Mer receptors play a vital role in rapid reduction of plaque burden. In addition, MerTK activity necessary for amyloid-stimulated phagocytosis strongly implicates that MerTK dysregulation might contribute to chronic inflammation indicated in AD pathology. However, the precise mechanism involved in the regulation of MerTK expression by Aβ1-42 in proinflammatory environment has not yet been ascertained.
Among naturally occurring dietary phytochemicals, isothiocyanate sulforaphane derived from cruciferous vegetables such as broccoli has received considerable attention as an alternative candidate for AD therapy due to its safety, efficacy, and blood–brain barrier penetration [
14]. Indeed, sulforaphane can ameliorate the cognitive function of Aβ-induced AD acute mouse models [
15] and decreased locomotor activity in mice with AD-like lesions [
16]. Moreover, sulforaphane protects the brain from Aβ-induced oxidative cell death via activating nuclear factor erythroid 2-related factor 2 (NRF2) signaling cascade [
17] which induces cytoprotective proteins including heme oxygenase-1 in the CNS [
14]. Recently, we have reported that sulforaphane possesses anti-inflammatory activity against Aβ peptide via signal transducer and activator of transcription-1 (STAT-1) dephosphorylation and activation of NRF2/HO-1 cascade in human THP-1 macrophages [
18]. Nonetheless, direct evidence indicating that sulforaphane can regulate Aβ1-42-induced effect on MerTK expression during inflammatory responses has not been reported. In addition, the potential mechanism of sulforaphane involved in the modulation of MerTK expression in human microglia-like THP-1 cells is currently unclear.
Therefore, the objective of this study was to determine the underlying mechanism involved in the Aβ-mediated regulation of MerTK expression and its modulation by sulforaphane in human THP-1 macrophages in vitro. Results of the present study indicated that Aβ1-42 could downregulate the level of MerTK protein via increasing intracellular Ca2+ level and NF-κB activation, thereby overproducing interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α), which could act as negative feedback regulators of MerTK expression. Notably, these effects of Aβ1-42 can be significantly reversed by sulforaphane in human THP-1 macrophages. Moreover, small interfering RNA (siRNA)-mediated knockdown of MerTK diminished sulforaphane’s anti-inflammatory effect on Aβ1-42-mediated induction of IL-1β and TNF-α, implicating a critical role of MerTK in the negative regulation of Aβ1-42-induced innate immune response. Collectively, these findings implicate that targeting of MerTK with phytochemical sulforaphane as a mechanism for preventing Aβ1-42-induced neuroinflammation has potential to be applied in AD treatment strategies.
Methods
Materials
Synthetic siRNA for MerTK and nonspecific control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against lamin B1 and MerTK were obtained from Abcam (Cambridge, UK). Anti-NF-κB was obtained from Cell Signaling Technology (Danvers, MA, USA). Sandwich enzyme-linked immunosorbent assay (ELISA) kits for human TNF-α and IL-1β were purchased from BD Biosciences (San Diego, CA, USA). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Sulforaphane and BAY 11-7082 were also acquired from Abcam. Pre-immune IgG was obtained from BioLegend (San Diego, CA, USA). Recombinant proteins of human TNF-α, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) were purchased from R&D Systems (Minneapolis, MN, USA). Actinomycin D (inhibitor of de novo mRNA expression) and cycloheximide (inhibitor of protein synthesis) were obtained from Calbiochem (La Jolla, CA, USA). Ionomycin (Ca2+ ionophore) and thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Anti-β-actin antibody and other chemicals were also acquired from Sigma-Aldrich.
Preparation of Aβ peptides
Aβ1-42 peptide was purchased from American Peptides (Sunnyvale, CA, USA) and prepared before use as described previously [
19,
20]. Aβ1-42 peptide was dissolved in dimethyl sulfoxide at 5 μM to be diluted to 250 μM in double-distilled water before experiments. This preparation mostly contained a monomeric form of Aβ1-42 with very small amounts of dimers and larger oligomers up to 6-mers [
21].
Differentiation of human microglia-like THP-1 cells
Human monocytic cell line THP-1 was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in RPMI 1640 containing 10% heat-inactivated fetal calf serum as described previously [
21,
22]. THP-1 has been widely used as a model of human monocytes/macrophages or microglia due to its functional and morphological similarities, including its capacity for signal transduction pathways as well as its functional differences in distinct species [
22]. Human monocyte-derived macrophages share many phenotypic and functional features with human microglial cells (so-called brain macrophages). Thus, all experiments required THP-1 cells to be differentiated to explore substantial changes in responsiveness during differentiation from monocyte to macrophage. THP-1 cells (10
5 cells/mL) were seeded into 96-well culture plates and incubated with 20 nM phorbol 12-myristate 13-acetate (PMA) for 48 h to become adherent to plastic culture dish and develop morphology of differentiated macrophages most closely resembling microglia as described previously [
21,
22].
Experimental treatment
After washing, adherent THP-1 macrophages were incubated at 37 °C with serum-free RPMI supplemented with 0.5% glucose for 1 h before stimulation by adding Aβ1-42 peptide in the presence or absence of sulforaphane (5 μM). In some experiments, cells were incubated with ionomycin A or thapsigargin to determine the effect of increased intracellular Ca2+ level. To examine the relationship between proinflammatory cytokines and MerTK expression, THP-1 cells were also exposed to recombinant TNF-α, IL-1β, or MCP-1. All concentrations were selected based on the maximal effect of the drug on its specified target. Vehicles were treated identically without Aβ1-42 or pharmacological agents. After stimulation with Aβ1-42 and/or specific agent for an appropriate time, total cell lysates and supernatants were prepared and stored at − 20 °C until use for Western blot analysis as described below. Concentrations of human IL-1β or TNF-α in culture media were also analyzed as described below.
Calcium imaging and fluorescence measurements
To visualize intracellular steady-state Ca
2+ levels, THP-1 cells were stained by adding Fluo-3A in its acetoxymethyl ester form (Fluo-3AM) to culture media at a final concentration of 5 μg/mL throughout Aβ1-42 or vehicle treatment as described previously [
20]. Ca
2+ influx fluorescence images were captured after the indicated treatment. Images were recorded using an inverted microscope (Nikon Eclipse TE300) and analyzed with ImageJ program. An increase of intracellular Ca
2+ level in different cultures was expressed in fold compared to that in vehicle-treated control for each individual experiment.
Nuclear fractionation
Cells were harvested and lysed on ice in 100 μL of lysis buffer A (10 mM Tris-HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl
2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1% NP-40) for 4 min as described previously [
19]. After centrifugation at 3000
g for 10 min, cell pellet was resuspended in 50 μL of extraction buffer B (20 mM HEPES (pH 7.9), 20% glycerol, 1.5 mM MgCl
2, 1 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride), incubated on ice for 30 min, and centrifuged at 13,000
g for 5 min. Nuclear proteins were stored at − 70 °C after determining protein concentration. Nuclear fractions were then subjected to Western blot analysis.
siRNA studies
Transfection of cells with siRNA was performed using Lipofectamine
® 2000 transfection reagent as described previously [
19,
21]. Commercially available human MerTK and negative control siRNA were used for transfection at indicated concentrations. Briefly, at 16 h after transfection, cells were treated with sulforaphane for 30 min prior to treatment with Aβ1-42 for 16 h. Levels of IL-1β or TNF-α in culture supernatant were analyzed using human-specific IL-1β or TNF-α ELISA kit (BD Biosciences).
Electrophoresis and Western blotting
Immunoblotting was conducted as described previously [
19,
20]. Briefly, equal quantities of sample proteins were subjected to 11% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes (GE Healthcare, Buckinghamshire, UK), blocked with 3% milk in Tris-buffered saline-Tween for 0.5 h, and probed with primary antibody diluted with 1% milk and incubated at 4 °C overnight. After incubating with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch), signals were acquired with an enhanced chemiluminescence system. Densitometric values were normalized against levels of β-actin.
ELISA
Differentiated THP-1 cells were treated with a variety of stimuli as indicated, and concentrations of human IL-1β or TNF-α in culture media were evaluated with sandwich ELISA kits (BD Biosciences) in accordance with the manufacturer’s recommendations. Standard curves were obtained using recombinant human IL-1β or TNF-α.
Statistical analyses
Differences between groups were evaluated for statistical significance using one-way ANOVA and Student’s t test. Null hypotheses of no difference were rejected if p value was less than 0.05.
Discussion
Results of the present study indicated that Aβ1-42 elicited a significant decrease in MerTK expression accompanied by increased intracellular Ca2+ level, thereby overproducing IL-1β and TNF-α in human THP-1 macrophages. These effects were mimicked by ionomycin A or thapsigargin and attenuated by depletion of Ca2+ with EGTA. Interestingly, recombinant IL-1β or TNF-α potently decreased MerTK expression whereas immunodepletion of IL-1β or TNF-α with neutralizing antibodies significantly rescued the decrease in MerTK protein level provoked by Aβ1-42 treatment, implicating a negative feedback regulation of MerTK expression by IL-1β and TNF-α. Notably, sulforaphane inhibited Aβ1-42-induced downregulation of MerTK expression by decreasing the overload of intracellular Ca2+ level, ultimately inhibiting IL-1β and TNF-α production. A subsequent mechanistic study revealed that inhibition of NF-κB signaling was involved in the anti-inflammatory effect of sulforaphane as BAY 11-7082, a NF-κB inhibitor, mimicked the aforementioned effect of sulforaphane. Moreover, siRNA-mediated knockdown of MerTK diminished the anti-inflammatory effect of sulforaphane against Aβ1-42, consequently resulting in increased production of IL-1β and TNF-α. These findings suggest that MerTK is indispensable for the negative regulation of innate immune response provoked by Aβ1-42 in human macrophages. Moreover, the anti-inflammatory potential of sulforaphane through inhibiting an Aβ1-42-mediated decrease in MerTK protein level strongly supports the notion that sulforaphane may serve as a potential nutraceutical agent for AD.
Results from the present study clearly provide direct evidences that negative modulation of MerTK expression by Aβ1-42 occurs through increased intracellular Ca
2+ levels, subsequently leading to hypersecretion of IL-1β and TNF-α in human THP-1 macrophages. First, ionomycin A (Ca
2+ ionophore) and thapsigargin (an endoplasmic reticulum Ca
2+ pump inhibitor) increase the intracellular Ca
2+ level and decrease the level of MerTK protein while they increase the production of IL-1β and TNF-α. Second, these Aβ1-42-evoked responses were attenuated by depletion of Ca
2+ with EGTA. These observations indicate that an increase of intracellular Ca
2+ levels resulted in a decrease of MerTK protein level which ultimately released excessive IL-1β and TNF-α upon exposure to Aβ1-42. Thus, this is the first report showing that Aβ1-42 can decrease MerTK expression through intracellular Ca
2+ overload, leading to the excessive production of IL-1β and TNF-α in human macrophages. Interestingly, recombinant IL-1β or TNF-α treatment elicited a decrease in MerTK protein level. Furthermore, immunodepletion of IL-1β or TNF-α with neutralizing antibodies significantly inhibited an Aβ1-42-mediated decrease in MerTK protein level. These findings implicate a negative feedback regulation of MerTK expression by IL-1β and TNF-α in human THP-1 macrophages. Our observation is consistent at least in part with a previous report demonstrating that MerTK is induced in immunosuppressive environments whereas Axl levels are increased by proinflammatory factors [
24]. Interestingly, a more recent study has demonstrated that MerTK is downregulated in classically activated microglial cells while it is one of the proteins mostly upregulated in TGF-β-treated microglia cells [
27].
Both MerTK and Axl receptors as markers of alternative activation for anti-inflammatory actions in macrophages can act as negative feedback regulators to reduce inflammation. They are responsible for promoting tissue repair and phagocytosis in tissue macrophages [
6,
7,
28]. In particular, roles for MerTK in mediating phagocytosis and clearance of apoptotic cells have been described in MerTK−/− macrophages [
29] via tethering apoptotic cells to macrophage surface and driving their subsequent internalization [
30]. Nevertheless, a previous study has demonstrated that abundant inflammatory macrophages are principally associated with extracellular deposits of amyloid in the AD brain. These macrophages are unable to efficiently phagocytose or clear plaques from the brain [
2]. More studies have also reported that the interaction of macrophages with plaques elicits the secretion of an array of inflammatory cytokines that can directly suppress phagocytosis [
31]. In addition, the presence of amyloid acts to suppress macrophage phagocytic function [
32]. In this regard, our results clearly indicate that the interaction of infiltrating macrophages upon exposure to amyloid could directly suppress MerTK protein level, which can permit excessive secretion of proinflammatory cytokines such as IL-1β and TNF-α, consequently resulting in phagocytically inactive macrophages. Furthermore, our data demonstrating a negative feedback regulation of the MerTK expression by IL-1β and TNF-α excessively induced upon Aβ1-42 exposure might explain why inflammatory macrophages fail to effectively phagocytize amyloid deposits, leading to progressive accumulation of plaques. Consistently, a recent study has demonstrated that induction of phagocytic receptor MerTK can enhance phagocytic capacity and reduce plaque burden in murine models of AD [
13], strongly supporting results of our study. It is also important to note that MerTK plays a critical role in adult neurogenesis [
12]. In addition, adult mice deficient in microglial MerTK have exhibited marked accumulation of apoptotic cells in neurogenic regions of the CNS [
9].
This is also the first report to demonstrate that sulforaphane can restore the depletion of MerTK and inhibit IL-1β and TNF-α production through decreasing intracellular Ca
2+ overload and attenuating NF-κB activation in human THP-1 macrophages exposed to Aβ1-42. A decrease in intracellular Ca
2+ level caused by sulforaphane treatment is consistent with a recent study demonstrating that sulforaphane could inhibit Ca
2+ overloading induced by methylmercury [
33]. Furthermore, BAY 11-7082, a NF-κB inhibitor, restored the depletion of MerTK and decreased the excessive secretion of IL-1β and TNF-α through attenuating NF-κB signaling in Aβ1-42-stimulated human THP-1 macrophages, mimicking the effect of sulforaphane. Taken together, these data provide direct evidence that the anti-inflammatory mechanism of sulforaphane involved in upregulating MerTK expression is through inhibiting Ca
2+ overloading and attenuating NF-κB signaling in human macrophages exposed to Aβ1-42, at least in part. Results of this study implicate that phytochemical sulforaphane might be used as a preventive and/or therapeutic target for AD management in addition to recently growing evidence suggesting beneficial effects of sulforaphane on Aβ pathology of AD [
16,
18,
25,
26].
It has been reported that sulforaphane can suppress lipopolysaccharide (LPS)-induced IL-1β secretion via inhibiting transcriptional activity of NF-κB [
34]. In addition, sulforaphane can suppress LPS-induced inflammation in mouse peritoneal macrophages through NRF2-dependent pathway [
35]. Our recent study has also shown that sulforaphane possesses anti-inflammatory effects against Aβ1-42 through STAT-1 dephosphorylation and activation of NRF2/HO-1 cascade in human THP-1 macrophages [
18]. These findings together imply that the anti-inflammatory effect of sulforaphane is likely due to its activities against various molecular targets, some of which might be interdependent. Therefore, more work is required to elucidate the mechanisms underlying the intricate signaling crosstalk between NF-κB inactivation and NRF2/HO-1 activation. Importantly, anti-inflammatory activities of sulforaphane at a multitude of molecular targets would be beneficial to the exploitation of multitarget-directed drugs to control AD [
36], given that AD is a multifactorial disease, of which approximately 95% occurs sporadically with unknown origin while 5% may be due to familial AD [
37].
Notably, our finding that depletion of MerTK with siRNA significantly suppressed sulforaphane’s anti-inflammatory activities against Aβ1-42 clearly implicates that MerTK plays a pivotal role in the negative regulation of innate immune response. Our recent study has demonstrated that inhibiting MerTK kinase can enhance inflammatory responses in LPS-induced acute lung injury [
38]. We have also observed that restoring MerTK protein expression by treatment with TNF-α processing inhibitor-0 (TAPI-0) can efficiently prevent the inflammatory cascade during acute lung injury [
39]. Moreover, an earlier study has shown that a lack of Mer inhibitory signal in
merKD mice will lead to elevated and prolonged NF-κB activation, causing excess macrophage activation and TNF-α production [
40]. Taken together, these observations suggest that MerTK is indispensable for the negative regulation of innate immune response provoked by Aβ1-42. In particular, MerTK depletion could contribute to a decrease of Aβ phagocytic activity [
13], increasing Aβ accumulation and chronic inflammation implicated in AD pathology. Further study remains to present the Aβ phagocytosis following MerTK depletion in the macrophages/microglia with sulforaphane to provide important molecular insights into AD and sulforaphane’s therapeutic potential.