Sulforaphane rescues amyloid-β peptide-mediated decrease in MerTK expression through its anti-inflammatory effect in human THP-1 macrophages

Abstract

Background: Mer tyrosine kinase (MerTK) activity necessary for amyloid-stimulated phagocytosis strongly implicates that MerTK dysregulation might contribute to chronic inflammation implicated in Alzheimer's disease (AD) pathology. However, the precise mechanism involved in the regulation of MerTK expression by amyloid-β (Aβ) in proinflammatory environment has not yet been ascertained.

Methods: The objective of this study was to determine the underlying mechanism involved in Aβ-mediated decrease in MerTK expression through Aβ-mediated regulation of MerTK expression and its modulation by sulforaphane in human THP-1 macrophages challenged with Aβ1-42. We used protein preparation, Ca2+ influx fluorescence imaging, nuclear fractionation, Western blotting techniques, and small interfering RNA (siRNA) knockdown to perform our study.

Results: Aβ1-42 elicited a marked decrease in MerTK expression along with increased intracellular Ca2+ level and induction of proinflammatory cytokines such as IL-1β and TNF-α. Ionomycin A and thapsigargin also increased intracellular Ca2+ levels and production of IL-1β and TNF-α, mimicking the effect of Aβ1-42. In contrast, the Aβ1-42-evoked responses were attenuated by depletion of Ca2+ with ethylene glycol tetraacetic acid. Furthermore, recombinant IL-1β or TNF-α elicited a decrease in MerTK expression. However, immunodepletion of IL-1β or TNF-α with neutralizing antibodies significantly inhibited Aβ1-42-mediated downregulation of MerTK expression. Notably, sulforaphane treatment potently inhibited Aβ1-42-induced intracellular Ca2+ level and rescued the decrease in MerTK expression by blocking nuclear factor-κB (NF-κB) nuclear translocation, thereby decreasing IL-1β and TNF-α production upon Aβ1-42 stimulation. Such adverse effects of sulforaphane were replicated by BAY 11-7082, a NF-κB inhibitor. Moreover, sulforaphane's anti-inflammatory effects on Aβ1-42-induced production of IL-1β and TNF-α were significantly diminished by siRNA-mediated knockdown of MerTK, confirming a critical role of MerTK in suppressing Aβ1-42-induced innate immune response.

Conclusion: 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 therapeutics.

Keywords: Alzheimer’s disease; Aβ1-42; Innate immune response; MerTK; Sulforaphane.

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The authors declare that they have no competing interests.

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Figures

Fig. 1

MerTK expression in human THP-1 macrophages is decreased by treatment with Aβ1-42. To measure MerTK expression in response to Aβ1-42 stimulation, THP-1 cells were incubated with either the vehicle only (−) or increasing amounts of Aβ1-42 for 8 h in serum-free RPMI 1640 medium supplemented with glucose (0.5%). a Total cell lysates were examined for MerTK protein via immunoblot. Aβ1-42 decreased MerTK in a dose-dependent manner. b THP-1 cells were also incubated with cycloheximide (CHX, 2 μM), actinomycin D (ActD, 100 nM), or Aβ1-42 (10 μM) for 8 h. Western blot analyses were conducted to determine the effect of actinomycin D or cycloheximide on MerTK expression. Levels of β-actin were examined to ensure equal amounts of total protein as a loading control. Results are representative of three independent experiments. c, d Quantitative analyses of a and b showing levels of MerTK protein. All data are presented as means ± SEM (n = 3). *P < 0.05, **P < 0.01, versus vehicle-treated samples. ActD, actinomycin; Aβ, amyloid-β; CHX, cyclohexamide, MerTK, Mer tyrosine kinase

Fig. 2

Aβ1-42-mediated reduction of MerTK expression is associated with increased intracellular Ca2+ level compared to treatment with ionomycin A or thapsigargin. a Intracellular Ca2+ images were obtained by Fluo-3AM method at 8 h after treatment with either the vehicle only (−), ionomycin A (4 μM), thapsigargin (2 μM), or 10 μM Aβ1-42 in THP-1 macrophages. b Histogram showing the ratio of intracellular Ca2+ levels to vehicle-treated group. c THP-1 cells were incubated with either the vehicle only (−), ionomycin A (4 μM), thapsigargin (2 μM), or Aβ1-42 (10 μM) for 8 h. d Densitometric quantification of analyses of c showing levels of MerTK protein. e THP-1 cells were pretreated with EGTA (0.5 mM) for 30 min followed by incubation with either the vehicle only (−) or Aβ1-42 (10 μM) for 8 h. Total cell lysates were examined for MerTK via immunoblot. MerTK expression was decreased when intracellular Ca2+ levels were increased by ionomycin A, thapsigargin, or Aβ1-42. In contrast, the Aβ1-42-mediated decrease of MerTK protein was attenuated by depletion of Ca2+ with EGTA. Results are representative of three independent experiments. f Densitometric quantification of analyses of e showing levels of MerTK protein. All data are presented as means ± SEM (n = 3). **P < 0.01, versus vehicle-treated samples; ##P < 0.01, compared to Aβ1-42-treated samples. Aβ, amyloid-β; EGTA, ethylene glycol trtraacetic acid; MerTK, Mer tyrosine kinase

Fig. 3

Ionomycin A or thapsigargin induced excessive production of proinflammatory cytokines IL-1β and TNF-α in comparison with treatment with Aβ1-42. a, b THP-1 cells were incubated with either the vehicle only (−), ionomycin A (4 μM), thapsigargin (2 μM), or Aβ1-42 (10 μM) for 16 h. c, d THP-1 cells were pretreated with EGTA (0.5 mM) for 30 min followed by incubation with either the vehicle only (−) or Aβ1-42 (10 μM) for 16 h. Levels of IL-1β (a, c) or TNF-α (b, d) in supernatants were quantitated by ELISA. Ionomycin A and thapsigargin mimicked Aβ1-42-mediated effects, increasing the production and release of IL-1β and TNF-α whereas depletion of Ca2+ with EGTA attenuated Aβ1-42-evoked responses. Values are expressed as means ± SEM of at least six independent experiments measured in duplicates. **P < 0.01, versus vehicle-treated samples; ##P < 0.01, compared to Aβ1-42-treated samples. Aβ, amyloid-β; EGTA, ethylene glycol trtraacetic acid; IL-1β, interleukin 1-β; TNF-α, tumor necrosis factor-α

Fig. 4

IL-1β and TNF-α decreased MerTK expression whereas neutralizing antibodies against IL-1β and TNF-α inhibited Aβ1-42-mediated reduction in MerTK expression. a THP-1 cells were incubated with either the vehicle only (−), IL-1β, TNF-α, or MCP-1 (10 ng/mL each) for 8 h. b THP-1 cells were exposed to Aβ1-42 (10 μM) for 8 h in the presence or absence of neutralizing anti-IL-1β, anti-TNF-α antibodies, or preimmune IgG (0.1 μg/mL each). Total cell lysates were examined for MerTK protein via immunoblot as described in Fig. 1. Results are representative of three independent experiments. c, d Densitometric quantification of analyses of a and b showing levels of MerTK protein. All data are presented as means ± SEM (n = 3–5). **P < 0.01, versus vehicle-treated samples; #P < 0.05, compared to Aβ1-42-treated samples. Aβ, amyloid-β; IgG, immunoglobulin G; IL-1β, interleukin 1-β; MCP-1, monocyte chemoattractant protein-1; MerTK, Mer tyrosine kinase; TNF-α, tumor necrosis factor-α

Fig. 5

Sulforaphane decreased intracellular Ca2+ levels and the production of IL-1β and TNF-α provoked by Aβ1-42 insult. a THP-1 cells were pretreated with sulforaphane (5 μM) for 30 min and then exposed to Aβ1-42 for 8 h. Intracellular Ca2+ levels were measured as described in Fig. 2. b, c THP-1 cells were pretreated with sulforaphane (5 μM) for 30 min and then exposed to Aβ1-42 for 16 h. Levels of IL-1β (b) or TNF-α (c) in the supernatants were quantitated by ELISA. Values are expressed as means ± SEM of at least six independent experiments measured in duplicates. **P < 0.01, versus vehicle-treated samples; ##P < 0.01, compared to Aβ1-42-treated samples. Aβ, amyloid-β; IL-1β, interleukin 1-β; TNF-α, tumor necrosis factor-α

Fig. 6

Sulforaphane rescued an Aβ1-42-mediated decrease of MerTK expression through inhibiting NF-κB nuclear translocation. a, b THP-1 cells were pretreated with a sulforaphane (5 μM) or b BAY 11-7082 (20 μM) for 30 min and then exposed to Aβ1-42 for 8 h. MerTK protein in cell lysates was measured as described above. c THP-1 cells were pretreated with sulforaphane (5 μM) or BAY 11-7082 (20 μM) for 30 min and then exposed to Aβ1-42 for 8 h. NF-κB protein in nuclear extracts was measured by Western blotting and normalized against the level of lamin B1. d–f Densitometric analysis of a–c. Values are expressed as means ± SEM of triplicate experiments. **P < 0.01, compared to vehicle-treated samples; ##P < 0.01, compared to Aβ1-42-treated samples. Aβ, amyloid-β; MerTK, Mer tyrosine kinas; NF-κB, nuclear factor kappa B

Fig. 7

MerTK silencing using siRNA decreased anti-inflammatory activities of sulforaphane against Aβ1-42-induced production of IL-1β and TNF-α. a, b THP-1 cells were transfected with 100 ng/mL MerTK siRNA or control siRNA for 16 h. Transfected THP-1 cells were then exposed to Aβ1-42 (10 μM) for 16 h after pretreatment with sulforaphane (5 μM) for 30 min. Levels of IL-1β (a) or TNF-α (b) in culture media were measured as described above. Values are expressed as means ± SEM of at least three independent experiments measured in duplicates. **P < 0.01, compared to Aβ1-42-treated samples; ##P < 0.01, compared to sulforaphane-treated samples. Aβ, amyloid-β; IL-1β, interleukin 1-β; TNF-α, tumor necrosis factor-α; siRNA, small interfering RNA

Fig. 8

Proposed mechanisms for beneficial effects of sulforaphane against decreased MerTK expression following Aβ1-42 insult in human THP-1 macrophages. Sulforaphane attenuates Aβ1-42-induced MerTK reduction through inhibiting intracellular Ca2+ overload and NF-κB signaling as replicated by EGTA and BAY 11-7082. Consequently, sulforaphane rescues a decrease of MerTK expression following Aβ1-42 stimulation, thereby inhibiting overproduction of IL-1β and TNF-α. Interestingly, Αβ1-42-induced IL-1β and TNF-α could act as negative feedback regulators of MerTK expression as confirmed with neutralizing antibodies against IL-1β or TNF-α, implicating that MerTK downregulation and induction of IL-1β and TNF-α by Aβ1-42 stimulation are interdependent. Depletion of MerTK with siRNA significantly suppressed the sulforaphane’s anti-inflammatory activities against Aβ1-42, implicating a pivotal role of MerTK for the negative regulation of the innate immune response elicited by Aβ1-42 in human THP-1 macrophages