Phenolic 1,3-diketones attenuate lipopolysaccharide-induced inflammatory response by an alternative magnesium-mediated mechanism

Article abstract of DOI:10.1111/bph.13746

Background and Purpose: Toll-like receptor 4 (TLR4) plays a key role in the induction of inflammatory responses both in peripheral organs and the CNS. Curcumin exerts anti-inflammatory functions by interfering with LPS-induced dimerization of TLR4–myeloid

Full text of DOI:10.1111/bph.13746

Phenolic 1,3-diketones attenuate lipopolysaccharide-induced inflammatory
response by an alternative magnesium-mediated mechanism
Morena Zusso1, Giulia Mercanti1, Federica Belluti2, Rita Maria Concetta Di Martino2,
Andrea Pagetta1, Carla Marinelli1, Paola Brun3, Eugenio Ragazzi1, Rita Lo4, Stefano Stifani4,
Pietro Giusti1*, Stefano Moro5
1 Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo
Meneghetti 2, 35131, Padua, Italy
2 Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of
Bologna, Via Belmeloro 6, 40126 Bologna, Italy
3 Department of Molecular Medicine, University of Padua, Via Gabelli 63, 35121 Padua,
Italy
4 Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill
University, Montreal, Quebec, Canada H3A 2B4
5 Molecular Modeling Section, Department of Pharmaceutical and Pharmacological Sciences,
University of Padua, Via Marzolo 5, 35131, Padua, Italy
Running title:
Phenolic 1,3-diketones as anti-inflammatory agents
*Corresponding author:
Pietro Giusti, MD, PhD
Address: Department of Pharmaceutical and Pharmacological Sciences, University of Padua,
Largo E. Meneghetti 2, 35131 Padua, Italy
Tel.: +39 049 827 5076; fax: +39 049 827 5093
E-mail: pietro.giusti@unipd.it
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/bph.13746
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Abstract
Background and Purpose
Toll-like receptor 4 (TLR4) plays a key role in the induction of inflammatory responses both
in peripheral organs and the CNS. Curcumin exerts anti-inflammatory functions by
interfering with LPS-induced TLR4–myeloid differentiation protein-2 (MD-2) dimerization
and suppressing pro-inflammatory mediator release. However, the inhibitory mechanism of
curcumin remains to be defined.
Experimental Approach
Binding of bis-demethoxycurcumin (GG6) and its cyclized pyrazole analogue (GG9), which
lacks the 1,3-dicarbonyl function, to TLR4–MD-2 was determined using molecular docking
simulations. The effects of these compounds on cytokine release and NF-κB activation were
examined by ELISA and fluorescence staining in LPS-stimulated primary microglia. Their
effects on interference with TLR4 dimerization were assessed by immunoprecipitation in
Ba/F3 cells.
Key Results
Both curcumin analogues bound to the hydrophobic region of the MD-2 pocket. However,
only curcumin and GG6, both possessing the 1,3-diketone moiety, inhibited LPS-induced
TLR4 dimerization, activation of NF-κB and secretion of pro-inflammatory cytokines in
primary microglia. Consistent with the ability of 1,3-diketones to coordinate divalent metal
ions, LPS stimulation in a low magnesium environment resulted in decreased pro-
inflammatory cytokine release and NF-κB p65 nuclear translocation in microglia and
decreased TLR4–MD-2 dimerization in BaF3 cells. Curcumin and GG6 also significantly
reduced cytokine output in contrast to the pyrazole analogue GG9.
Conclusions and Implications
These results indicate that molecules like 1,3-diketones, with a structural motif able to
coordinate the magnesium ion, can modulate LPS-mediated TLR4–MD-2 signaling. Taken
together, these studies identify a previously uncharacterized magnesium-mediated
mechanism for LPS-induced inflammatory response.
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Abbreviations
DAPI, 4,6-diamidino-2-phenylindole
Iba1, ionized calcium binding adaptor molecule 1
MD-2, myeloid differentiation protein-2
MOE, Molecular operation environment
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PDB, Protein data bank
rms, Root-mean-square
TLRs, Toll-like receptors
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TARGETS
LIGANDS
LPS
Other protein targets
TLR4
IL-1β
curcumin
TNF-α
These Tables of Links list key protein targets and ligands in this article that are hyperlinked* to
corresponding entries in http://www.guidetopharmacology.org, the common portal for data from
the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in
The Concise Guide to PHARMACOLOGY 2015/16 (a,b,c,d,eAlexander et al., 2015a,b,c,d,e).
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Introduction
Neuroinflammation is a complex defense response of the CNS involved in tissue repair and
induction of a protective immune response. However, if it becomes chronic it can be
destructive to the brain, leading to neuronal cell damage and, ultimately, death associated
with neurodegenerative and psychiatric disorders (Frank-Cannon et al., 2009; Amor et al.,
2010). In neuroinflammation, cellular and molecular immune components such as microglia
and astrocytes, cytokines, complement and pattern-recognition receptors are the critical
players (Shastri et al., 2013). Microglia, the major resident immune cells of the CNS, provide
the first line of defense against invading pathogens or injury-related products (Hanisch and
Kettenmann, 2007). Microglia-mediated defense mechanisms begin with the recognition of
different pathogens through a limited number of pattern-recognition receptors, including
Toll-like receptors (TLRs) which are transmembrane glycoproteins expressed in a number of
CNS cell types, including microglia (Bsibsi et al., 2002; Kawai and Akira, 2010).
LPS, a well-characterized pathogen-associated molecular pattern present on the outer
membrane of Gram-negative bacteria, is a potent activator of a variety of mammalian cell
types (Ulevitch and Tobias, 1995). Among TLRs, TLR4 is the major LPS receptor and is a
component of LPS signaling (Poltorak et al., 1998; Hoshino et al., 1999; Park and Lee,
2013). The cellular response to LPS requires specific interaction of LPS with the co-receptor
myeloid differentiation protein-2 (MD-2), which is associated with the extracellular domain
of TLR4 (Shimazu et al., 1999; Nagai et al., 2002; Park et al., 2009). After LPS binding, the
TLR4–MD-2 complex dimerizes and a signal is then propagated by recruitment of
intracellular signaling adaptor proteins leading to activation of transcription factor NF-κB.
The latter controls the final immune response through the synthesis of inflammatory
molecules, such as cytokines (e.g., TNF-α, IL-1β and IL-6), chemokines, enzymes and
reactive oxygen and nitrogen species (Bryant et al., 2010). As a consequence, the binding of
an antagonist to the extracellular domain would prevent receptor dimerization and,
consequently, intracellular signaling activation.
Identification of molecules capable of inhibiting LPS-mediated TLR4–MD-2 complex
activation and understanding how these molecules modulate TLR4–MD-2-mediated signaling
represents an attractive strategy for attenuating the severity of various CNS inflammatory
conditions. Recently, a number of small molecules have been identified as TLR4 antagonists
by a variety of in silico, in vitro and in vivo assays. These natural and synthetic compounds,
structurally unrelated to LPS, proved to directly bind to the MD-2 pocket, antagonize the
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cellular response to LPS and improve various inflammatory conditions (Park et al., 2012).
Among them is curcumin, the main bioactive component in the rhizome of the turmeric plant
(Curcuma longa), a safe and a highly pleiotropic molecule endowed with a wide range of
beneficial activities, including anti-inflammatory, anti-tumor, anti-oxidative and
cardiovascular protective effects (Hatcher et al., 2008), and which interacts with diverse
molecular targets (Gupta et al., 2013). In particular, the anti-inflammatory properties of
curcumin are linked to numerous mechanisms (He et al., 2015), including its ability to bind
MD-2 (Gradišar et al., 2007) and inhibit dimerization of the TLR4–MD-2 complex (Youn et
al., 2006) which is required for inflammatory intracellular signaling. However, the inhibitory
mechanism of curcumin remains to be fully understood. Despite the fact that curcumin is an
electrophilic thiol-reactive compound containing a bis-α, β-unsaturated 1,3-diketone moiety
which can react with cysteine residues through a Michael reaction, it binds MD-2 in
proximity of Cys133 residue, without the formation of a covalent adduct (Gradišar et al.,
2007).
In the present study, two curcumin derivatives were synthesized and used to probe the anti-
inflammatory mechanism of curcumin at the molecular level. In particular, bis-
demethoxycurcumin (GG6, 1E,4Z,6E-5-hydroxy-1,7-bis-4-hydroxyphenylhepta-1,4,6-trien-
3-one) and its pyrazole-based analogue lacking the 1,3-dicarbonyl function [GG9, 4,4'-
(1E,1'E)-(1H-pyrazole-3,5-diyl-bis-ethene-2,1-diyl)diphenol] were compared to curcumin in
relation to their anti-inflammatory effects and binding mechanisms to the TLR4–MD-2
complex. Only 1,3 diketones suppressed neuroinflammation, with the ability of the 1,3
diketone moiety to bind divalent cations such as the magnesium ion playing an important role
in inhibiting LPS-induced TLR4–MD-2 complex activation and subsequent inflammatory
response.
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Materials and methods
Reagents and materials
Unless otherwise specified, all reagents were from Sigma-Aldrich (Milan, Italy). Tissue
culture media, antibiotics and fetal calf serum (FCS) were obtained from Invitrogen (San
Giuliano Milanese, Italy). LPS (Ultra-Pure LPS-EB from E. Coli, 0111:B4 strain) was
purchased from InvivoGen (InvivoGen Europe, Toulouse, France). The Ultra-Pure LPS-EB
preparation used here only activates TLR4. Falcon tissue culture plasticware was purchased
from BD Biosciences (SACCO srl, Cadorago (CO), Italy).
Synthesis of curcumin analogues
GG6 and GG9 analogues were synthesized according to previously reported procedures
(Scheme 1). Spectroscopic data were consistent with their chemical structures and in good
agreement with pubblished data (Di Martino et al., 2016; Ishida et al., 2002). GG6 was
prepared according to the Pabon reaction (Pabon, 1964) in which 2,4-pentandione was first
reacted with B2O3 and the obtained boric complex then condensed with 4-
hydroxybenzaldehyde in the presence of n-tributhylborate. Acidic hydrolysis allows for
removal of the boron complexation and obtention of the desired derivative. GG6 was then
reacted with hydrazine hydrate to obtain the pyrazole analogue GG9.
Scheme 1. General synthetic method for GG6 and GG9.
Molecular modeling studies
Target structures
The crystallographic structure of the human TLR4-human MD-2-E. coli LPS ternary complex
was retrieved from the Protein Data Bank (PDB code: 3FXI) (Park et al., 2009).
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Assessment of crystallographic structure quality was performed with the Structure
Preparation tool of the Molecular Operation Environment program (MOE, version 2014.09;
Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC,
Canada, H3A 2R7, 2015). Critical structural issues (such as missing or poorly resolved
atomic data, anomalous topological properties present in amino acid units as well as
anomalous bonding patterns of non-amino acid units) were fixed when necessary. Hydrogen
atoms were added and their appropriate protonation state fixed using the Protonate3D tool as
implemented in the MOE program. To minimize contacts between hydrogen atoms, the
structures were subjected to Amber99 force field (Cornell et al., 1995) minimization until the
root-mean-square (rms) of the conjugate gradient was <0.1 kcal mol-1 Å-1 keeping the heavy
atoms fixed at their crystallographic positions.
Molecular docking protocol
Curcumin and its analogues were built using the “Builder” module of MOE and each
compound was docked into the presumptive binding site (LPS binding site) using flexible
MOE-Dock methodology. The purpose of MOE-Dock is to search for favorable binding
configurations between a small, flexible ligand and a rigid macromolecular target. Searching
is conducted within a user-specified 3D docking box, using the “tabù search” (Baxter et al.,
1998) protocol and the MMFF94 force field (Halgren, 1996). Charges for ligands were
imported from the MOPAC program (Stewart, 1993) output files. MOE-Dock performs a
user-specified number of independent docking runs (50 in the present case) and writes the
resulting conformations and their energies to a molecular database file. The resulting
ligand/protein complexes were subjected to MMFF94 all-atom energy minimization until the
rms of conjugate gradient was <0.1 kcal mol-1 Å-1. GB/SA approximation (Wojciechowski
and Lesyng, 2004) was used to model the electrostatic contribution to the free energy of
solvation in a continuum solvent model. Interaction energy values were calculated as the
energy of the complex minus the energy of the ligand, minus the energy of the protein: 
Einter= E(complex)-(E(L) + E(Protein)).
Cell cultures
Studies involving animals were carried out in accordance with the ARRIVE guidelines
(Kilkenny et al., 2010; McGrath and Lilley, 2015). All animal-related procedures were
performed in accordance with National Institutes of Health guidelines for the care and use of
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laboratory animals and those of the Italian Ministry of Health (D.L. 26/2014), and were
approved by the Institutional Animal Care and Use Committee of the University of Padua
(958/2016-PR). One-day-old Sprague Dawley rat pups (CD strain) of both sexes were rapidly
decapitated, minimizing suffering, discomfort or stress. Primary microglial cells were
isolated from mixed glial cell cultures prepared from cerebral cortex, as previously described
(Skaper et al., 2012). Briefly, upon reaching confluence (7-10 days after isolation) microglia
adhering to the astroglial monolayer were dislodged by shaking (200 r.p.m. for 1 h at 37°C),
re-suspended in high-glucose DMEM supplemented with 2 mM L-glutamine, 10% heat-
inactivated FCS, 100 U ml-1 penicillin, 100 μg ml-1 streptomycin and 50 μg ml-1 gentamicin,
and plated on uncoated plastic wells at a density of 1.25 x 105 cells cm-2. Cells were allowed
to adhere for 45 min and then washed to remove non-adhering cells. After a 24-h incubation
period, the medium was replaced with either a serum-free medium or a low-magnesium
solution containing 2.5 mM CaCl2, 4.7 mM KCl, 1.2 mM KH2PO4, 118 mM NaCl, 25 mM
NaHCO3, 0.1 mM MgSO4 and 11 mM glucose. Purity of the cultures was confirmed by
immunocytochemistry using a primary polyclonal antibody against ionized calcium binding
adaptor molecule 1 (Iba1, 1:800, Wako Chemicals USA Inc., Richmond, VA, USA). Ninety-
seven percent of the cells were Iba1 immunopositive. Murine Ba/F3 cells, an IL-3 dependent
pro-B cell line, stably expressing human TLR4-GFP, human TLR4-Flag, human MD-2-Flag
and human CD14 were kindly provided by Dr. Kensuke Miyake (University of Tokyo,
Tokyo, Japan). Cells were cultured in RPMI1640 medium, supplemented with 10% FCS, 100
µM 2-mercaptoethanol and recombinant murine IL-3 (~ 70 U ml-1) (Saitoh et al., 2004). Cells
were maintained at 37°C in a humidified atmosphere containing 5% CO2/95% air.
Cell viability assay
Microglial cell viability was evaluated by a quantitative colorimetric method utilizing the
metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Mosmann, 1983). Briefly, cells were seeded at the density of 45,000 cells/well in 96-well
plates and treated with test compounds. After 6 h incubation, the medium was removed and
the cells incubated with MTT (0.18 mg ml-1) for 4 h at 37°C. The formazan crystals were
solubilized with DMSO. The plates were then analyzed on a microplate reader (Victor2
Multilabel Counter, Wallac, Cambridge, MA, USA) using a test wavelength of 570 nm and a
reference wavelength of 630 nm, and the results expressed as percentage viability relative to
LPS-treated cultures.
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Cytokine determination
Primary microglia were pre-treated for 1 h with increasing concentrations of test compounds
and then stimulated with 100 ng ml-1 Ultra-Pure LPS-EB for an additional 6 h. At the end of
incubation, culture medium was collected and IL-1β and TNF-α assayed using commercially
available ELISA kits (Antigenix America, Huntington Station, NY, USA), according to the
manufacturer’s instructions. Cytokine concentrations (pg ml-1) in the medium were
determined by reference to standard curves obtained with known amounts of IL-1β or TNF-α
and the results expressed as percentage relative to LPS-stimulated cultures.
Immunofluorescence
Microglia were grown on coverslips in 12-well plates and pre-treated for 1 h with 10 µM
curcumin, GG6 or GG9 and then stimulated with 100 ng ml-1 Ultra-Pure LPS-EB for an
additional 90 min. Cells were fixed with 4% paraformaldehyde (pH 7.4, for 15 min at room
temperature) and subsequently blocked with 5% normal goat serum/0.1% Triton X-100 in
PBS for 1 h (Zusso et al., 2012). Cells were incubated with a mouse anti-p65 antibody (NF-
κB p65, 1:500; Santa Cruz Biotechnology Santa Cruz, CA, USA) followed by an Alexa Fluor
555-conjugated anti-mouse secondary antibody (1:1000; Invitrogen). Nuclei were stained
with 4,6-diamidino-2-phenylindole (DAPI, Sigma) and coverslips were mounted on
microscope slides with Fluoromount-G mounting medium (Fisher Scientific, Milan, Italy).
Fluorescent images for p65 staining were captured with a confocal laser-scanning microscope
(Zeiss LSM 800; Carl Zeiss AG, Oberkochen, Germany) and analyzed with ZEN 2.0 imaging
software (Carl Zeiss AG). ImageJ software (National Institutes of Health, Bethesda, MD,
USA) was used to profile the fluorescence emission intensity of single cells.
Western blotting
Microglial cells were pre-treated for 1 h with 10 µM curcumin, GG6 or GG9 and then
stimulated with 100 ng ml-1 Ultra-Pure LPS-EB for an additional 60 min. Cells were lysed on
ice with NP40 cell lysis buffer (Life Technologies, San Giuliano Milanese, Italy), containing
protease and phosphatase inhibitors (Sigma-Aldrich), and centrifuged at 13,000 g for 10 min
at 4°C. Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and
electrotransferred onto nitrocellulose membranes. The membranes were blocked with
phosphate-buffered saline containing 0.1% Triton X100 and 3% nonfat dry milk and probed
overnight with rabbit anti-IκBα (1:500; Cell Signaling Technology, Beverly, MA, USA),
rabbit anti-NF-κB p65 phospho S536 (p-p65; 1:2000; abcam, Cambridge, UK) or mouse anti-
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β-actin (1:25000; Sigma-Aldrich). Following incubation with appropriate horseradish
peroxidase-conjugated secondary antibodies, the immunoreactive bands were visualized with
ECL substrate (#RPN2106; GE Healthcare Amersham, Buckinghamshire, UK) using the
VersaDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). The band intensities
were quantified by densitometric analysis using ImageJ software. Band quantification was
first normalized to β-actin and then percentage was calculated versus LPS-stimulated cells
Data obtained from densitometric analyses were normalized to account for unequal loading of
samples across the lanes on a gel and for differences in transfer efficiency across a blot.
Immunoprecipitation
Ba/F3 cells stably expressing human TLR4-GFP, human TLR4-Flag, human MD-2-Flag and
human CD14 (80 x 106 cells/condition) were pre-treated for 1 h with curcumin, GG6 or GG9
(50 µM) and then stimulated with 250 ng ml-1 LPS for 30 min (Youn et al., 2006). Protein
extracts were prepared as described previously (Saitoh et al., 2004). The samples were
immunoprecipitated with mouse anti-GFP antibody (#NB600-597; Novus, Littleton, CO) for
16-18 h at 4°C. The recovered immune complex was resolved using 10% SDS-PAGE and
electrotransferred to a nitrocellulose membrane. The membrane was probed overnight with
either mouse anti-Flag antibody (#F3165; Sigma-Aldrich, St. Louis, MO) or chicken anti-
GFP antibody (#06-896; Millipore, Temecula, CA). Thereafter, the membrane was exposed
to horseradish peroxidase-conjugated secondary antibody for 1 h and reactive bands were
detected with the ECL system. Images were analyzed using ImageJ software and TLR4-Flag
expression was normalized to TLR4-GFP levels. Results are expressed as percentage relative
to LPS-stimulated cells.
Magnesium assessment
Magnesium assessment in DMEM and in the low magnesium solution was performed using
an atomic absorbance spectrophotometer (Varian AA Duo 55 b/240, Agilent Technologies,
Santa Clara, CA, USA) with a Ca/Mg lamp, air/acetylene flame and detection wavelength of
285.2 nm. Absorbance readings were obtained via integrated duplicate measurement and
compared with a standard, calibrated curve.
Statistical analysis
Data and statistical analysis comply with the recommendations on experimental design and
analysis in pharmacology (Curtis et al., 2015). Data were blindly analyzed using GraphPad
software, version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA) and expressed as mean
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± SEM of at least 5 independent experiments. Individual data points were based on a
technical duplicate to ensure the reliability of single values. As raw values varied between
experiments and the variability could obscure the treatment effect, data were expressed as
percentage of control cells or LPS treatment, taken as baseline of each independent
experiment. Data were analyzed by means of Kruskal-Wallis one-way analysis of variance
followed by post-hoc Dunn's test for multiple comparisons (Siegel and Castellan Jr., 1988) vs
LPS treatment, using p-adjustment according to Bonferroni's method (Pohlert, 2014) or by
one-way ANOVA with Dunnett's post-hoc test when appropriate. A p value of less than 0.05
was deemed statistically significant. Post-hoc tests were carried out only when F value from
analysis of variance was <0.05 and there was no significant variance in homogeneity.
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Results
Effects of curcumin analogues on pro-inflammatory cytokine release by LPS-stimulated
microglia
Stimulation of microglia with TLR agonists, including LPS for TLR4, induces a pro-
inflammatory process through the increased secretion of cytokines (Lehnardt, 2009).
Inhibition of LPS-induced release of IL-1β and TNF-α was initially used to examine the anti-
inflammatory effects of the curcumin analogues GG6 and GG9. Cells were exposed to
increasing concentrations (1-10 μM) of curcumin (positive control), GG6 or GG9 for 1 h and
then stimulated with LPS for 6 h to induce an inflammatory response. Supernatants were
collected and subjected to ELISA to measure IL-1β and TNF-α concentration. Unstimulated
cells released low or undetectable amounts of IL-1β and TNF-α which remained unchanged
after treatment with the three compounds (data not shown). LPS treatment markedly induced
release of IL-1β and TNF-α (taken as 100%; dashed lines in Figure 1A and B) that was
significantly suppressed by curcumin (see also Mercanti et al., 2014) and by its 1,3-diketone
analogue GG6. LPS-induced release of IL-1β was reduced to about 50% of LPS level by 10
μM of both curcumin and GG6 (45.9  3.7% and 58.3  8.4%, respectively) (Figure 1A).
TNF-α release was also significantly inhibited by 10 μM curcumin and GG6 (52.9  8.3%
and 63.0  2.9%, respectively) (Figure 1B), demonstrating that GG6 has effects similar to the
parent molecule. In contrast GG9, the pyrazole-based analogue, failed to inhibit LPS-induced
release of either IL-1β or TNF-α (Figure 1A and B), indicating that the 1,3-diketone moiety is
crucial for anti-inflammatory activity. None of the three compounds compromised cell
viability at the concentrations tested (1-10 μM) (Figure 1C), indicating that the observed
decrease in IL-1β and TNF-α release induced by curcumin and GG6 did not result from
cytotoxicity.
Effects of curcumin analogues on NF-κB activation in LPS-stimulated microglia
In an LPS-induced inflammatory response, activation of the transcription factor NF-κB
through the translocation of its p65 subunit from the cytosol to the nucleus is considered a
key event that controls the expression of a large number of cytokines and other immune
response genes (Ghosh and Hayden, 2008). To investigate whether the studied compounds
reduced LPS-induced pro-inflammatory cytokine release by preventing NF-κB activation, the
subcellular distribution of p65 subunit was studied by immunofluorescent staining in
unstimulated and LPS-stimulated microglia. In unstimulated control cells p65 was mainly
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distributed in the cytoplasm with only minor nuclear staining (Figure 2A; 24.1  2.1 % of
nuclear p65, Figure S1). This distribution remained unchanged after treatment with curcumin,
GG6 and GG9 (10 µM) (Figure 2B-D and Figure S1). A 90-min exposure to LPS increased
nuclear translocation of p65 (Figure 2E; 74.8  11.4% of nuclear p65, Figure S1), indicating
NF-κB activation. Pre-treatment with curcumin and GG6 strongly attenuated LPS-induced
nuclear localization of p65 (Figure 2F and G; 31.7  2.3% and 29.6  5.2% of nuclear p65,
Figure S1). In contrast, pre-treatment with GG9 did not show this effect (Figure 2H and
Figure S1), confirming the structure-activity relationship observed in inhibition of cytokine
release.
To further examine the effect of curcumin and GG6 on NF-κB activation, IκBα degradation
and phosphorylation of p65, both required for NF-κB transcriptional activity (Christian et al.,
2016), were evaluated. Both curcumin and GG6 significantly decreased IκBα degradation
and phosphorylation of p65 induced by a 60-min challenge with LPS (Figure 2I).
Effects of curcumin analogues on TLR4 dimerization
TLR4 dimerization is one of the initial steps involved in activation of the TLR4-mediated
signaling pathway. To investigated whether curcumin analogues could inhibit this step, Ba/F3
cells stably transfected with human TLR4-GFP, TLR4-Flag, MD-2-Flag and CD14 (Saitoh et
al., 2004) were used. Cells were pre-incubated with curcumin, GG6, GG9 or vehicle
(untreated control) for 1 h prior to LPS stimulation for 30 min. To detect TLR4 dimerization,
TLR4-GFP was immunoprecipitated and co-precipitation of TLR4-Flag was probed with
anti-Flag antibody (Figure 3). TLR4 dimerization increased in response to LPS stimulation
and, as previously demonstrated in this cell line (Youn et al., 2006), curcumin reduced LPS-
induced dimerization (Figure 3A and B). Importantly, the 1,3-diketone derivative GG6 also
markedly decreased this effect. In contrast, and in agreement with the effects on inhibition of
cytokine release and NF-κB activation, the pyrazole analogue GG9 did not affect
dimerization of TLR4, confirming a key role for the 1,3-diketone moiety in the first step of
the inflammatory cascade (Figure 3A and B).
The binding mode of curcumin analogues to MD-2
To explore the observed structure-activity relationship of curcumin and its analogues in their
ant-inflammatory effects, molecular docking simulations were carried out to characterize the
putative binding mode of these molecules to the LPS binding site of MD-2. In agreement
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with previous studies curcumin (Wang et al., 2015), as well as GG6 and GG9 could be
accommodated into the large hydrophobic binding pocket of MD-2 occupying a relevant
portion of the LPS binding site (Figure 4) while assuming different binding modes. Among
all binding modes generated for each ligand, the energetically more stable were those that
showed important interactions with MD-2, such as hydrogen bonding, engaging residues
Arg90, Glu92, and Tyr102. These binding modes are well conserved in both curcumin
analogues. As shown in Figure 5, curcumin (panel A), GG6 (panel B) and GG9 (panel C) are
located into the LPS binding site of MD-2 guaranteeing the same cavity occupancy and the
same interaction networks.
Interestingly, the crystallographic structure coded by PDB as 3FXI, in proximity to entrance
of the LPS binding site of MD-2, showed a metal ion at the interface between the MD-2-LPS
complex to TLR4, either directly or indirectly through water molecules, as shown in (Park et
al., 2009). The original crystallographic study suggested this to be Mg2+, whose role at that
time was thought to be dispensable for LPS binding and receptor dimerization (Park et al.,
2009). New molecular docking simulations were performed by using a sampling grid large
enough to include the metal ion. These studies showed that the presence of a 1,3-diketone
moiety, as well as its enolate form, allows the appearance of an alternative binding mode
close to magnesium ion (Figure 6). On the contrary, the pyrazole analogue GG9 lacked this
behavior, thus confirming its inability to coordinate metal ions.
Effect of a low magnesium environment on inflammatory response
To examine the effect of magnesium on the inflammatory response, microglial cells cultured
in a low magnesium environment (0.1  0.05 mM, determined by atomic absorbance
spectroscopy) were exposed to curcumin, GG6 or GG9 for 1 h and then stimulated with LPS
for 6 h. Cell viability as well as low basal levels of IL-1β and TNF-α were not significantly
affected by a low extracellular magnesium concentration (Figure 7A-C). Under these
conditions, however, LPS-induced release of both cytokines was significantly decreased
compared to control conditions (DMEM; magnesium concentration, 0.9  0.1 mM).
Specifically, IL-1β and TNF-α released in the supernatant were reduced to 25.1 ± 5.9% and
37.9 ± 7.0%, respectively (Figure 7D and E, white bars). Treatment with curcumin and GG6
brought about a marked concentration-dependent reduction in IL-1β and TNF-α release -
unlike GG9 (Figure 7D and E, gray bars). Furthermore, the diminished cytokine release
observed in a low magnesium environment correlated well with decreased NF-κB p65
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nuclear translocation in microglia (Figure 8A and B) and suppression of TLR4 dimerization
in Ba/F3 cells (Figure 8C).
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Discussion
Curcumin possesses well-known anti-inflammatory activities, and numerous studies suggest
that MD-2 may be the direct target of curcumin in its inhibition of LPS signaling (Youn et
al., 2006; Gradišar et al., 2007; He et al., 2015). However, the exact interaction between
curcumin and MD-2 is not completely understood. Several curcumin analogues have recently
been synthesized with the aim to learn more about curcumin mechanism of action and to
establish structure-activity relationships. Although the 1,3-diketone moiety in the curcumin
structure has been considered responsible for its chemical instability (Oliveira et al., 2015)
and some mono-carbonyl analogues of curcumin displayed anti-inflammatory effects in vitro
and in vivo (Zhang et al., 2014; Zhang et al., 2015), this structural motif seems to be
important for inhibition of LPS-mediated TLR4–MD-2 activation/dimerization (Youn et al.,
2006). Here, we approached the question of this chemical moiety’s involvement in the
microglial anti-inflammatory response by using two curcumin derivatives: bis-
demethoxycurcumin (GG6) and its cyclized pyrazole analogue (GG9), lacking the 1,3-
dicarbonyl function. Non-cytotoxic concentrations of curcumin and GG6 suppressed, in a
similar manner, LPS-induced inflammatory response in primary cortical microglia. Previous
studies have suggested that prevention of inflammatory cascade by curcumin may be linked
to its ability to inhibit LPS-induced dimerization of TLR4 (Youn et al., 2006). GG6, sharing
with curcumin the 1,3-diketone moiety, suppressed this effect, whereas the pyrazole-based
analogue GG9 was unable to inhibit LPS-induced pro-inflammatory response in microglia
and TLR4 dimerization.
These observations highlight the fundamental role of the 1,3 diketone moiety in the anti-
inflammatory effects of curcumin and GG6, by directly suppressing LPS-induced initiation
of receptor signaling. This structural information prompted us to characterize the binding
mode of GG6 and GG9 to the LPS binding site on MD-2. Molecular modeling studies
showed that, as for curcumin (Wang et al., 2015), both analogues could bind the hydrophobic
region of the MD-2 pocket, leading to a stable complex via interactions with Arg90, Glu92
and Tyr102 residues, all of which are conserved between human, mouse and rat (Shimazu et
al., 1999; Akashi et al., 2000; Winnall et al., 2011). Considering the size of the LPS binding
cavity, it is also not surprising that these compounds may assume different poses inside this
binding site. Based on the above considerations, however, it is not possible to explain the
remarkable decrease in anti-inflammatory activity of the pyrazole analogue GG9, compared
to the 1,3-diketone analogue GG6.
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As previously reported a metal ion, probably Mg2+, is present at the interface between the
MD-2–LPS complex and TLR4. As EDTA was unable to block LPS binding and receptor
dimerization (Park et al., 2009) the Mg2+ presence as been considered superfluous. Diketones
efficiently coordinate divalent metal ions (including copper, nickel, cobalt, zinc and
magnesium) (Vyas et al., 2013). Conceivably, the presence of a metal ion like Mg2+ could
affect docking simulations facilitating ligand movement from inside the LPS binding cavity
of MD-2 closer to the metal coordination sphere, where possible in terms of complex
stability. Molecular docking simulations including Mg2+ confirmed that only the presence of a
1,3-diketone moiety allowed the appearance of poses close to the metal ion, confirming the
inability of the pyrazole analogue GG9 to coordinate metal ions. This computational
evidence led us to hypothesize that the anti-inflammatory activity of 1,3-diketone derivatives
is associated with their ability to coordinate Mg2+ which could in turn affect proper assembly
of the TLR4–MD-2–LPS ternary complex. Exposing microglial to a low magnesium
environment significantly decreased LPS-induced IL-1β and TNF-α release and NF-κB
nuclear translocation, compared to normal magnesium conditions. Moreover, curcumin and
GG6 markedly reduced cytokine output in contrast to GG9, suggesting further that the
capability of the 1,3 diketone moiety to coordinate divalent cations, such as magnesium, may
play a critical role in anti-inflammatory responses. These findings contrast with previous
studies in both rodent and human cells, which suggested a link between magnesium
deficiency and inflammation and, conversely, showed that elevated magnesium limited the
inflammatory response by inhibiting NF-κB activation and pro-inflammatory cytokine
production (Lin et al., 2010; Lee et al., 2011; Sugimoto et al., 2012; Gao et al., 2013). This
discrepancy may reflect differences in experimental conditions, such as the length of
exposure to a low magnesium environment or LPS concentration. Sugimoto and colleagues
showed a decreased cytokine production induced by magnesium supplementation with LPS
concentration (50 pg ml-1) too low to activate all cells. In contrast high LPS concentrations,
similar to those used here, abrogated the magnesium effect (Sugimoto et al., 2012).
The mechanism(s) by which magnesium exerts its effect on inflammatory responses is poorly
understood. As a known antagonist of some calcium ion channels (Sonna et al., 1996),
magnesium may counteract the toxic consequences of excessive calcium entry into immune
cells, including microglia (Lin et al., 2010; Gao et al., 2013). Other potential cellular
mechanisms concerning the relationship between magnesium status and inflammation have
been considered, such as ion effects on NMDA receptors, release of substance P and
activation of NF-κB (Mazur et al., 2007). Furthermore, whether magnesium functions via an
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intra- or extracellular mechanism has been debated (Mazur et al., 2007; Gao et al., 2013). The
present findings reveal a previous unrecognized direct link between a low magnesium
environment and LPS-mediated TLR4 activation, by showing inhibition of TLR4–MD-2
complex dimerization in LPS-stimulated Ba/F3 cells exposed to a low Mg2+ concentration.
These results provide evidence for a new and alternative mechanism for the LPS-mediated
activation of TLR4, suggesting that molecules like 1,3-diketones, which possess a structural
motif able to coordinate Mg2+, could modulate LPS-mediated TLR4–MD-2 signaling, an
important component of immune responses in the nervous system (Kigerl et al., 2014). In a
broader framework, neuroinflammation and microglial activation may also involve activation
of multiple TLRs by exogenous and endogenous ligands in a context-dependent manner (Li
et al., 2016). Furthermore, the TLR4–MD-2 complex and its dimerization process may not be
the exclusive targets through which curcumin and its 1,3-diketone analogue may modulate
neuroinflammation. Even so, the mechanism proposed in this study can provide a starting
point for the design and development of novel agents targeting the MD-2 protein, applied to
neuropathologies in which prolonged TLR4–MD-2 activation is known to exacerbate disease
conditions.
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Acknowledgements
This work was supported by “Progetto di Ateneo”, University of Padua, Italy
(CPDA144389/14 to M.Z.); by a PRIN (20103W4779) Project Grant from MIUR, Italy; and
by the Canadian Institute of Health Research (grants MOP-123270 and MOP-123500 to
S.S.). The computational work coordinated by S.M. received support from the University of
Padua. We thank Dr. Carla Argentini for technical assistance, Dr. Chiara Calore for
assistance with atomic absorption spectroscopy and Dr. Kensuke Miyake for providing the
Ba/F3 cells. We are also very grateful to the Chemical Computing Group for the scientific
and technical partnership. Finally, the authors would like to thank Dr. Stephen D. Skaper for
critical reading of the manuscript.
Author contributions
M.Z., P.G., and S.M. designed research; M.Z., G.M., F.B., R.D.M., C.M., P. B., R.L. and
S.M. performed research; M.Z., A.P., E.R., S.S., P.G. and S.M. analyzed data; M.Z. and S.M.
wrote the paper; all authors read and approved the final manuscript.
Conflict of interest statement
The authors declare no conflict of interest.
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Figure 1. Effects of curcumin, GG6 and GG9 on cytokine release from LPS-stimulated
cortical microglia and cell viability. Microglia were cultured for 24 h in 10% serum-
containing medium, which was replaced with serum-free medium before pre-treatment with
curcumin, GG6 and GG9 (1-10 μM) for 1 h followed by stimulation with 100 ng ml-1 LPS
for 6 h. Supernatants were collected and analyzed for (A) IL-1β and (B) TNF-α release. (C)
At the end of incubation, cell viability was also determined by MTT assay. Results are
expressed as percentage of cytokine release and cell viability relative to LPS-stimulated
microglia. Data are means ± SEM (n = 5). *p < 0.05 versus LPS stimulation (dashed line),
Kruskal–Wallis followed by Dunn's post-hoc test and p-adjustment according to Bonferroni's
method.
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Figure 2. Effects of curcumin, GG6 and GG9 on NF-κB activation in unstimulated and LPS-
stimulated cortical microglia. Microglia were subcultured for 24 h in 10% serum-containing
medium, which was replaced with serum-free medium before stimulation with curcumin
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(Curc), GG6 and GG9 (10 μM)  100 ng ml-1 LPS. Cells were then processed for NF-κB p65
immunostaining. Fluorescence intensity profiles along an ideal oriented line across a
representative cell (yellow arrow in the third line) are shown in the fourth line. Experiments
were performed 5 times and representative confocal images showing subcellular localization
of p65 in unstimulated and LPS-stimulated microglia are shown in (A-D) and (E-H),
respectively. Scale bar, 10 µm. (I) Protein levels of IκBα and phosphorylated p65 (p-p65)
were examined by Western blot. Experiments were performed 5 times and representative
images are shown. Densitometric analysis of Western blots relative to β-actin is expressed as
percentage of LPS-stimulated cells. Data are means ± SEM (n = 5). *p < 0.05 versus LPS
treatment, Kruskal–Wallis followed by Dunn's post-hoc test and p-adjustment according to
Bonferroni's method. Veh, vehicle.
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Figure 3. Effects of curcumin, GG6 and GG9 on LPS-induced TLR4 dimerization. (A)
Ba/F3 cells expressing TLR4-Flag (TLR4F), TLR4-GFP (TLR4G), MD2-Flag and CD14
were pre-treated with 50 μM curcumin (Curc), GG6 or GG9 for 1 h and then stimulated with
250 ng ml-1 LPS for 30 min. Cell lysates were immunoprecipitated with mouse anti-GFP
antibody and immunoblotted with mouse anti-Flag (upper) or chicken anti-GFP (lower)
antibody. Experiments were performed at least 5 times and representative images are shown.
(B) Histograms showing the ratio TLR4-Flag/TLR4-GFP, expressed as percentage of LPS-
stimulated cells. Data are means ± SEM (n = 5). * p < 0.05 versus LPS stimulation, Kruskal–
Wallis followed by Dunn's post-hoc test and p-adjustment according to Bonferroni's method.
IP, immunoprecipitation; IB, immunoblot; Veh, vehicle.
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Figure 4. Overall structure of the human TLR4-MD2 complex derived by the
crystallographic structure 3FXI. Some atoms have been voluntarily omitted. Bottom left:
TLR4 (orange) and MD-2 (magenta) are represented showing their secondary structure. Top
right: the LPS binding cavity of MD-2 is enlarged and shown as the Connolly surface.
Hydrophobic regions of the Connolly surface are colored in green, polar in magenta and
those exposed to solvent in red. Mg2+ is represented by CPK (bronze).
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