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Citation: Polini, B.; Ricardi, C.;
Bertolini, A.; Carnicelli, V.;
Rutigliano, G.; Saponaro, F.; Zucchi,
R.; Chiellini, G. T1AM/TAAR1
System Reduces Inflammatory
Response and β-Amyloid Toxicity in
Human Microglial HMC3 Cell Line.
Int. J. Mol. Sci. 2023,24, 11569.
https://doi.org/10.3390/
ijms241411569
Academic Editor: Masashi Tanaka
Received: 25 May 2023
Revised: 7 July 2023
Accepted: 13 July 2023
Published: 17 July 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Article
T1AM/TAAR1 System Reduces Inflammatory Response and
β-Amyloid Toxicity in Human Microglial HMC3 Cell Line
Beatrice Polini 1, *, Caterina Ricardi 1, Andrea Bertolini 1, Vittoria Carnicelli 1, Grazia Rutigliano 2,
Federica Saponaro 1, Riccardo Zucchi 1and Grazia Chiellini 1, *
1Department of Pathology, University of Pisa, 56100 Pisa, Italy; c.ricardi@student.unisi.it (C.R.);
a.bertolini@student.unisi.it (A.B.); vittoria.carnicelli@unipi.it (V.C.); federica.saponaro@unipi.it (F.S.);
riccardo.zucchi@unipi.it (R.Z.)
2Institute of Clinical Sciences, Imperial College London, London SW7 2AZ, UK;
grazia.rutigliano.gr@gmail.com
*Correspondence: beatrice.polini@farm.unipi.it (B.P.); grazia.chiellini@unipi.it (G.C.)
Abstract:
Microglial dysfunction is one of the hallmarks and leading causes of common neurodegen-
erative diseases (NDDs), including Alzheimer’s disease (AD) and Parkinson’s disease (PD). All these
pathologies are characterized by aberrant aggregation of disease-causing proteins in the brain, which
can directly activate microglia, trigger microglia-mediated neuroinflammation, and increase oxidative
stress. Inhibition of glial activation may represent a therapeutic target to alleviate neurodegeneration.
Recently, 3-iodothyronamine (T1AM), an endogenous derivative of thyroid hormone (TH) able to
interact directly with a specific GPCR known as trace amine-associated receptor 1 (TAAR1), gained
interest for its ability to promote neuroprotection in several models. Nevertheless, T1AM’s effects
on microglial disfunction remain still elusive. In the present work we investigated whether T1AM
could inhibit the inflammatory response of human HMC3 microglial cells to LPS/TNF
α
or
β
-amyloid
peptide 25–35 (A
β
25–35) stimuli. The results of ELISA and qPCR assays revealed that T1AM was
able to reduce microglia-mediated inflammatory response by inhibiting the release of proinflam-
matory factors, including IL-6, TNF
α
, NF-kB, MCP1, and MIP1, while promoting the release of
anti-inflammatory mediators, such as IL-10. Notably, T1AM anti-inflammatory action in HMC3 cells
turned out to be a TAAR1-mediated response, further increasing the relevance of the T1AM/TAAR1
system in the management of NDDs.
Keywords:
neurodegeneration; microglia; inflammation; neuroprotection; trace amine-associated
receptors type 1 (TAAR1); 3-iodothyronamine
1. Introduction
Microglial cells, the brain-resident immune cells, play a pivotal role in regulating the
cellular processes involved in the development of neuronal networks and the homeostasis
maintenance of the central nervous system (CNS) [
1
]. Notably, in response to abnormal
microenvironment factors, including, for example, stress, aging, injuries, infections, and
hypoxia–ischemia, microglia can assume different activation states, from neuroprotective
to neurodestructive, the equilibrium of which may contribute to the onset and outcome
of neuroinflammatory and neurodegenerative mechanisms [
2
]. Indeed, dysfunction of
microglia is one of the hallmarks and leading causes of common neurodegenerative diseases
(NDDs), such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease
(HD), and amyotrophic lateral sclerosis (ALS) [
3
–
7
]. All these pathologies are characterized
by aberrant aggregation of disease-causing proteins in the brain [
8
], which can directly
activate microglia, trigger microglia-mediated neuroinflammation, and increase oxidative
stress [9].
Under physiological conditions microglial cells maintain a ramified cell shape, defined
as “resting state” (Figure 1), and vigilantly monitor and protect neuronal function. In
Int. J. Mol. Sci. 2023,24, 11569. https://doi.org/10.3390/ijms241411569 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023,24, 11569 2 of 15
the event of brain injury, infection, or neurodegeneration, microglia adopt a phagocytic
phenotype, defined as “M1 state”, in which the cell attains an amoeboid morphology, and
may secrete a wide variety of neurotoxic factors, including proinflammatory cytokines,
proteinases, and reactive oxygen species (ROS), leading to exacerbated damage and neu-
ronal death [
10
]. Accumulating evidence suggests that the initial classical activation of
microglia is followed by a secondary alternative activation, defined as “M2 state”, which is
important for neuronal cell repair, tissue remodeling, and suppression of inflammation [
11
]
(Figure 1). Since chronic inflammatory activation of microglia is correlated with common
NDDs, functional modulation of microglial phenotypes could represent a valid therapeutic
strategy [10,12,13].
Int.J.Mol.Sci.2023,24,115692of17
Underphysiologicalconditionsmicroglialcellsmaintainaramifiedcellshape,de‐
finedas“restingstate”(Figure1),andvigilantlymonitorandprotectneuronalfunction.
Intheeventofbraininjury,infection,orneurodegeneration,microgliaadoptaphagocytic
phenotype,definedas“M1state”,inwhichthecellaainsanamoeboidmorphology,and
maysecreteawidevarietyofneurotoxicfactors,includingproinflammatorycytokines,
proteinases,andreactiveoxygenspecies(ROS),leadingtoexacerbateddamageandneu‐
ronaldeath[10].Accumulatingevidencesuggeststhattheinitialclassicalactivationof
microgliaisfollowedbyasecondaryalternativeactivation,definedas“M2state”,which
isimportantforneuronalcellrepair,tissueremodeling,andsuppressionofinflammation
[11](Figure1).Sincechronicinflammatoryactivationofmicrogliaiscorrelatedwithcom‐
monNDDs,functionalmodulationofmicroglialphenotypescouldrepresentavalidther‐
apeuticstrategy[10,12,13].
Figure1.Diagramofmicrogliaactivation.Restingmicrogliamayturnintodistinctphenotypes,
namely,M1phenotypeandM2phenotype.M1classicalstatereleasesproinflammatorycytokines
andcytotoxicsubstances,includingIL‐1ß,IL‐6,TNFα,andROS,inducingneurologicaldamage.On
theotherhand,theM2alternativestateproducesanti‐inflammatorycytokines,suchasIL‐10,able
toinhibittheproductionofproinflammatorycytokinesbymicroglia,thusexertinganeuroprotec‐
tiveroleintheCNS.
Recently,3‐iodothyronamine(T1AM),anendogenousthyroidhormone(TH)deriv‐
ative,abletointeractdirectlywithaspecificG‐proteincoupledreceptorknownastrace
amine‐associatedreceptor1(TAAR1)[14,15],hasgainedinterestforitsabilitytopromote
neuroprotectiveeffectsinseveralmodels,includingseizure‐relatedexcitotoxicdamage,
alteredautophagy,amyloidosis,andOGD‐inducedsynapticdysfunction[16–19],andto
efficientlycrosstheblood–brainbarrier(BBB)[20].TAAR1isanestablishedregulatory
proteinexpressedandbroadlydistributedinthebrainmonoaminergicsystems[21],
whichincludethedopaminergicsystem,thenoradrenergicsystem,andtheserotonergic
system[22].Collectively,allthesesystemsplayimportantrolesinmood,cognition,emo‐
tion,reward,learning,aention,andmotoractivity[23],andtheirdysregulationisasso‐
ciatedwithavarietyofneurologicalandneurodegenerativedisorders,severalofwhich
areamongtheleadingcausesofdeathanddisabilityworldwide[24].Recentstudiesre‐
vealedthatpharmacologicallytargetingTAAR1withintheCNShasresultedinsuccessful
clinicaltrialsforthetreatmentofschizophrenia,depression,addiction,andNDDs[25–
28].Morerecently,TAA R1expressionandfunctionalityinimmunesystemregulationand
Pro‐inflammatory
phenotype
Microglia(resting)
Pro‐inflammatory
stimuli
Anti‐inflammatory
stimuli
Anti‐inflammatory
phenotype
IL‐1β,IL‐6,TNF ,ROS
IL‐10
Inhibition
Figure 1.
Diagram of microglia activation. Resting microglia may turn into distinct phenotypes,
namely, M1 phenotype and M2 phenotype. M1 classical state releases proinflammatory cytokines
and cytotoxic substances, including IL-1ß, IL-6, TNF
α
, and ROS, inducing neurological damage. On
the other hand, the M2 alternative state produces anti-inflammatory cytokines, such as IL-10, able to
inhibit the production of proinflammatory cytokines by microglia, thus exerting a neuroprotective
role in the CNS.
Recently, 3-iodothyronamine (T1AM), an endogenous thyroid hormone (TH) deriva-
tive, able to interact directly with a specific G-protein coupled receptor known as trace
amine-associated receptor 1 (TAAR1) [
14
,
15
], has gained interest for its ability to promote
neuroprotective effects in several models, including seizure-related excitotoxic damage,
altered autophagy, amyloidosis, and OGD-induced synaptic dysfunction [
16
–
19
], and to effi-
ciently cross the blood–brain barrier (BBB) [
20
]. TAAR1 is an established regulatory protein
expressed and broadly distributed in the brain monoaminergic systems [
21
], which in-
clude the dopaminergic system, the noradrenergic system, and the serotonergic
system [22]
.
Collectively, all these systems play important roles in mood, cognition, emotion, reward,
learning, attention, and motor activity [
23
], and their dysregulation is associated with
a variety of neurological and neurodegenerative disorders, several of which are among
the leading causes of death and disability worldwide [
24
]. Recent studies revealed that
pharmacologically targeting TAAR1 within the CNS has resulted in successful clinical
trials for the treatment of schizophrenia, depression, addiction, and NDDs [
25
–
28
]. More
recently, TAAR1 expression and functionality in immune system regulation and immune
cell activation has become a topic of emerging interest [
29
]; nevertheless, so far, few studies
have examined the role of TAAR1 in CNS-resident neuroimmune cells [
30
,
31
]. Notably,
a recent report highlighted the expression of TAAR1 in macrophages/microglia bordering
multiple sclerosis (MS) lesions [
32
], supporting TAAR1 as a novel pharmacological target
in cells directly implicated in neuroinflammation.
Int. J. Mol. Sci. 2023,24, 11569 3 of 15
On these premises, we directed our attention to examining the potential of the
T1AM/TAAR1 signaling system in regulating brain neuroinflammatory responses. Using
HMC3 human microglia cells as an
in vitro
model [
33
], we demonstrated, for the first time,
that T1AM inhibits LPS/TNF
α
-induced inflammatory response through the activation of
TAAR1. Since it is well known that fibrillar amyloid-
β
(A
β
) peptides play an important
role in microglial activation in AD [
34
], we also showed that the T1AM-TAAR1 signaling
pathway was able to protect against A
β
-induced cytotoxic and inflammatory responses in
HMC3 cells, and that TAAR1 stimulation can inactivate microglial NF-κB signaling.
Even though still at a preliminary level, the results of our study highlight the ability of
T1AM to extensively modulate microglia-mediated neuroinflammation, providing further
insight into its possible therapeutic application for the prevention of neurodegeneration.
2. Results
2.1. T1AM Decreased the Inflammatory Phenotype of LPS/TNFα-Stimulated HMC3 Human
Microglial Cells
As previously reported [
35
–
37
], exposure of human microglial clone 3 (HMC3) cells
to LPS/TNF
α
stimulus for 24 h resulted in a significant increase in proinflammatory IL-6
release in cell culture media, while no effect on anti-inflammatory IL-10 levels was observed
as compared with control cells. In the present study, HMC3 microglial cell line was used as
an in vitro model to investigate the ability of T1AM to prevent neuroinflammation.
Dose–response experiments were carried out by exposing HMC3 cells to pretreat-
ment with increasing concentrations (0.1, 1, and 10
µ
M) of T1AM for 1 h, followed by
LPS/TNF
α
treatment for 24 h. ELISA assays on cell culture media revealed that T1AM
causes a significant (p< 0.05) dose-dependent reduction of IL-6 levels as compared to
LPS/TNF
α
-treated cells (Figure 2A). A significant (p< 0.05) dose-dependent increase in
IL-10 levels was also observed in the same set of experiments (Figure 2B), suggesting that
T1AM pretreatment may temper hyperinflammation. Moreover, gene expression analysis
revealed that pretreatment with 1 or 10
µ
M T1AM significantly decreases the expression of
inflammatory-response-related genes in LPS/TNF
α
-treated cells, including the monocyte
chemoattract protein-1 (MCP1), the macrophage inflammatory protein-1 (MIP1), and the
transcription factor NF-kB (Figure 3).
Int.J.Mol.Sci.2023,24,115694of17
Figure2.Releaseofpro‐andanti‐inflammatoryinterleukins,IL–6(A)andIL–10(B),inducedby
differentconcentrationsofT1AM.Datarepresentmeans±S.E.M.fromthreeindependentexperi‐
ments(n=3),performedinduplicate.Statisticalanalysiswasperformedbyordinaryone‐way
ANOVAfollowedbyDunne’smultiplecomparisontest.Foreachexperimentalconditionase‐
lectedspecificpaernhasbeenused.
.
Figure3.T1AMdecreasestheexpressionofinflammatory‐response‐relatedgenesstimulatedby
LPS/TNFαtreatmentinHMC3cells.(A)monocytechemoaractprotein–1(MCP1),(B)macrophage
inflammatoryprotein–1(MIP1),and(C)transcriptionfactorNF‐kB.Datarepresentmeans±S.E.M.
fromthreeindependentexperiments(n=3),performedinduplicate.Statisticalanalysiswasper‐
formedbyordinaryone‐wayANOVAfollowedbyDunne’smultiplecomparisontest.Foreach
experimentalconditionaselectedspecificpaernhasbeenused.
Inaddition,T1AMwasnontoxictomicroglialcellswhenusedat0.1,1,and10µM
concentrationsfor24h(Figure4).
0
100
200
300
400
500
IL-10 [pg/ml]
LPS/TNF
- + + + +
T1AM (µM)
- -
0.1 1 10
0.0031
0.0027
AB
0
250
500
750
1000
1250
1500
IL-6 [pg/ml]
LPS/TNF
- + + + +
T1AM (µM)
- -
0.1 1 10
0.0021
0.0048
0.0078
0.0037
0
2
4
6
8
10
MCP1 expression levels
(2
-
Ct
)
LPS/TNF
- + + +
T1AM (µM)
- -
1
10
0.0071
0.018
0.0029
0
2
4
6
8
MIP1 expression levels
(2
-
Ct
)
LPS/TNF
- + + +
T1AM (µM)
- -
1
10
0.0028 0.008
0.0015
0
1
2
3
4
NFKB expression levels
(2
-
Ct
)
LPS/TNF
- + + +
T1AM (µM)
- -
1
10
0.0078 0.032
0.0081
AB C
Figure 2.
Release of pro- and anti-inflammatory interleukins, IL–6 (
A
) and IL–10 (
B
), induced by dif-
ferent concentrations of T1AM. Data represent means
±
S.E.M. from three independent experiments
(n= 3), performed in duplicate. Statistical analysis was performed by ordinary one-way ANOVA
followed by Dunnett’s multiple comparison test. For each experimental condition a selected specific
pattern has been used.
Int. J. Mol. Sci. 2023,24, 11569 4 of 15
Int.J.Mol.Sci.2023,24,115694of17
Figure2.Releaseofpro‐andanti‐inflammatoryinterleukins,IL–6(A)andIL–10(B),inducedby
differentconcentrationsofT1AM.Datarepresentmeans±S.E.M.fromthreeindependentexperi‐
ments(n=3),performedinduplicate.Statisticalanalysiswasperformedbyordinaryone‐way
ANOVAfollowedbyDunne’smultiplecomparisontest.Foreachexperimentalconditionase‐
lectedspecificpaernhasbeenused.
.
Figure3.T1AMdecreasestheexpressionofinflammatory‐response‐relatedgenesstimulatedby
LPS/TNFαtreatmentinHMC3cells.(A)monocytechemoaractprotein–1(MCP1),(B)macrophage
inflammatoryprotein–1(MIP1),and(C)transcriptionfactorNF‐kB.Datarepresentmeans±S.E.M.
fromthreeindependentexperiments(n=3),performedinduplicate.Statisticalanalysiswasper‐
formedbyordinaryone‐wayANOVAfollowedbyDunne’smultiplecomparisontest.Foreach
experimentalconditionaselectedspecificpaernhasbeenused.
Inaddition,T1AMwasnontoxictomicroglialcellswhenusedat0.1,1,and10µM
concentrationsfor24h(Figure4).
0
100
200
300
400
500
IL-10 [pg/ml]
LPS/TNF
- + + + +
T1AM (µM)
- -
0.1 1 10
0.0031
0.0027
AB
0
250
500
750
1000
1250
1500
IL-6 [pg/ml]
LPS/TNF
- + + + +
T1AM (µM)
- -
0.1 1 10
0.0021
0.0048
0.0078
0.0037
0
2
4
6
8
10
MCP1 expression levels
(2
-
Ct
)
LPS/TNF
- + + +
T1AM (µM)
- -
1
10
0.0071
0.018
0.0029
0
2
4
6
8
MIP1 expression levels
(2
-
Ct
)
LPS/TNF
- + + +
T1AM (µM)
- -
1
10
0.0028 0.008
0.0015
0
1
2
3
4
NFKB expression levels
(2
-
Ct
)
LPS/TNF
- + + +
T1AM (µM)
- -
1
10
0.0078 0.032
0.0081
AB C
Figure 3.
T1AM decreases the expression of inflammatory-response-related genes stimulated by
LPS/TNF
α
treatment in HMC3 cells. (
A
) monocyte chemoattract protein–1 (MCP1), (
B
) macrophage
inflammatory protein–1 (MIP1), and (
C
) transcription factor NF-kB. Data represent means
±
S.E.M.
from three independent experiments (n= 3), performed in duplicate. Statistical analysis was per-
formed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test. For each
experimental condition a selected specific pattern has been used.
In addition, T1AM was nontoxic to microglial cells when used at 0.1, 1, and 10
µ
M
concentrations for 24 h (Figure 4).
Int.J.Mol.Sci.2023,24,115695of17
Figure4.MTTassayperformedwithdifferentconcentrationsofT1AM.(A)EffectofT1AMon
HMC3Cellviability.(B)EffectofT1AMontheviabilityofLPS/TNFα–treatedcells.Datarepresent
means±S.E.M.fromthreeindependentexperiments,performedintriplicate(n=3).Statisticalanal‐
ysiswasperformedbyordinaryone‐wayANOVAfollowedbyTukey’smultiplecomparisontest.
Foreachexperimentalconditionaselectedspecificpaernhasbeenused.
2.2.T1AMUptakeandMetabolisminHMC3HumanMicroglialCells
T1AMisknowntobetransportedinsidecellsandrapidlytransformedinto3‐iodo‐
thyroaceticacid(TA1)[20,38,39].
Liquidchromatographywithtandemmassspectrometry(LC–MS/MS)wasusedto
measureT1AMandTA1levelsinHMC3cells.AfterincubatingHMC3cellpreparations
with0.1µMT1AMfor5,15,30,and60minat37°C,thecellculturemediawerecollected,
andcelllysateswerepreparedaccordingtoapreviouslyreportedprocedure[40].
AsshowninTabl e1,T1AMwasrapidlytakenupbyHMC3cellsandcatabolizedto
TA1,indicatingthatinHMC3cells,amineoxidasesweremetabolicallyactive.Notably,
theproductofT1AMcatabolism,TA1,afterformingwasreleasedfromcells,anddetected
inthecollectedcellculturemedia,showingincreasingconcentrationsovertime.InHMC3
celllysates,theconcentrationofT1AMappearedtoremainconstantovertimeatvalues
correspondingtoapproximately25%oftheadministereddose,whereasonlyanegligible
amountofTA1 wasfound.
Tab le1.LC–MS/MSmeasurementofT1AMandTA1levelsinHMC3cells
Time
(min)
MediumCellLysates
T1AM(nM)TA1(nM)T1AM(nM)TA1(nM)
0N.D.N.D.N.D.N.D.
564.1±2.700.31±0.02026.0±0.280.06±0.01
1557.2±5.400.66±0.2024.9±0.740.12±0.02
3056.7±8.101.33±0.1527.0±0.300.14±0.02
6053.4±3.603.81±0.8024.5±0.810.23±0.04
ConcentrationsofT1AMandTA1inmediumandcelllysatesafter0,5,15,30,and60minofinfusion.
Datarepresentmean±SEM,n=3pergroup,andareexpressedasnM.T1AMorTA1 contentswere
measuredinmediumandlysateHMC3cells,whichwereincubatedfor0,5,15,30,and60minwith
T1AM(0.1µM).N.D.,notdetectable.
0
20
40
60
80
100
120
Cell viability (% vs. CTRL)
T1AM (µM)
-
0.1 1 10
AB
0
20
40
60
80
100
120
Cell viability (% vs. CTRL)
LPS/TNF
- + + +
T1AM (µM)
- -
1 10
Figure 4.
MTT assay performed with different concentrations of T1AM. (
A
) Effect of T1AM
on HMC3 Cell viability. (
B
) Effect of T1AM on the viability of LPS/TNF
α
–treated cells. Data
represent
means ±S.E.M
. from three independent experiments, performed in triplicate (n= 3). Statis-
tical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison
test. For each experimental condition a selected specific pattern has been used.
2.2. T1AM Uptake and Metabolism in HMC3 Human Microglial Cells
T1AM is known to be transported inside cells and rapidly transformed into 3-iodothyroacetic
acid (TA1) [20,38,39].
Liquid chromatography with tandem mass spectrometry (LC–MS/MS) was used to
measure T1AM and TA1 levels in HMC3 cells. After incubating HMC3 cell preparations
with 0.1
µ
M T1AM for 5, 15, 30, and 60 min at 37
◦
C, the cell culture media were collected,
and cell lysates were prepared according to a previously reported procedure [40].
As shown in Table 1, T1AM was rapidly taken up by HMC3 cells and catabolized to
TA1, indicating that in HMC3 cells, amine oxidases were metabolically active. Notably, the
Int. J. Mol. Sci. 2023,24, 11569 5 of 15
product of T1AM catabolism, TA1, after forming was released from cells, and detected in
the collected cell culture media, showing increasing concentrations over time. In HMC3
cell lysates, the concentration of T1AM appeared to remain constant over time at values
corresponding to approximately 25% of the administered dose, whereas only a negligible
amount of TA1 was found.
Table 1. LC–MS/MS measurement of T1AM and TA1 levels in HMC3 cells.
Time (min) Medium Cell Lysates
T1AM (nM) TA1 (nM) T1AM (nM) TA1 (nM)
0 N.D. N.D. N.D. N.D.
5 64.1 ±2.70 0.31 ±0.020 26.0 ±0.28 0.06 ±0.01
15 57.2 ±5.40 0.66 ±0.20 24.9 ±0.74 0.12 ±0.02
30 56.7 ±8.10 1.33 ±0.15 27.0 ±0.30 0.14 ±0.02
60 53.4 ±3.60 3.81 ±0.80 24.5 ±0.81 0.23 ±0.04
Concentrations of T1AM and TA1 in medium and cell lysates after 0, 5, 15, 30, and 60 min of infusion. Data
represent mean
±
SEM, n= 3 per group, and are expressed as nM. T1AM or TA1 contents were measured in
medium and lysate HMC3 cells, which were incubated for 0, 5, 15, 30, and 60 min with T1AM (0.1
µ
M). N.D.,
not detectable.
2.3. Trace Amine-Associated Receptor TAAR1 Is Involved in T1AM-Mediated Anti-Inflammatory
Response of Microglial Cells
The evidence that T1AM was able to suppress the response of microglia cells to
inflammatory stress prompted us to extend our investigation to its mechanism of action.
It is known from the literature that T1AM is a high-affinity ligand for TAAR1 [
14
], and
TAAR1 was demonstrated to mediate T1AM’s protective effect against synaptic plasticity
abnormalities in a mouse model of AD [
41
]. Therefore, we explored whether TAAR1 could
also be involved in T1AM-mediated anti-inflammatory response of HMC3 cells.
First, expression of TAAR1 in HMC3 microglial cells was assessed by qPCR analysis.
As shown in Figure 5, resting microglial cells showed TAAR1 expression, and no changes
were observed after 24 h treatment with LPS/TNFα.
Int.J.Mol.Sci.2023,24,115696of17
2.3.TraceAmine‐AssociatedReceptorTAAR1IsInvolvedinT1AM‐MediatedAnti‐Inflamma‐
toryResponseofMicroglialCells
TheevidencethatT1AMwasabletosuppresstheresponseofmicrogliacellstoin‐
flammatorystresspromptedustoextendourinvestigationtoitsmechanismofaction.
ItisknownfromtheliteraturethatT1AMisahigh‐affinityligandforTAAR1[14],
andTAAR1 wasdemonstratedtomediateT1AM’sprotectiveeffectagainstsynapticplas‐
ticityabnormalitiesinamousemodelofAD[41].Therefore,weexploredwhetherTAAR1
couldalsobeinvolvedinT1AM‐mediatedanti‐inflammatoryresponseofHMC3cells.
First,expressionofTAAR1inHMC3microglialcellswasassessedbyqPCRanalysis.
AsshowninFigure5,restingmicroglialcellsshowedTAAR1expression,andnochanges
wereobservedafter24htreatmentwithLPS/TNFα.
Figure5.Real‐timequantificationofTAAR1expressioninHMC3cellsbeforeandafterexposureto
LPS(10µg/mL)/TNFα (50ng/mL)treatmentfor24h.Datarepresentmeans±S.E.M.fromthree
independentexperiments,performedintriplicate(n=3).Statisticalanalysiswasperformedbyor‐
dinaryStudent’st‐test.
Then,weinvestigatedwhetherTAAR1couldplayaroleinourmodelofinflamed
microgliabyusingaTAAR1selectiveantagonist(EPPTB)andaselectiveagonist
(RO5166017)[42].
WeobservedthatT1AM’sprotectiveeffectagainstmicrogliaactivationwasabolished
bycoadministrationofTAAR1selectiveantagonistEPPTB(5nM)(Figure6A,B).Con‐
versely,theadministrationoftheTAAR1agonistRO5166017(1µM)toLPS/TNFα‐stimu‐
latedHMC3cellsmimickedtheeffectsonthereleaseofbothIL‐6andIL‐10previously
observedafteradministeringT1AM(10µM)(Figure6A,B).
HMC3
HMC3 + INF
0.0
0.1
0.2
0.3
TAAR1 mRNA expression
(2
-Ct
)
Figure 5.
Real-time quantification of TAAR1 expression in HMC3 cells before and after exposure
to LPS (10
µ
g/mL)/TNF
α
(50 ng/mL) treatment for 24 h. Data represent means
±
S.E.M. from
three independent experiments, performed in triplicate (n= 3). Statistical analysis was performed by
ordinary Student’s t-test.
Then, we investigated whether TAAR1 could play a role in our model of inflamed mi-
croglia by using a TAAR1 selective antagonist (EPPTB) and a selective agonist (RO5166017) [
42
].
We observed that T1AM’s protective effect against microglia activation was abolished
by coadministration of TAAR1 selective antagonist EPPTB (5 nM) (Figure 6A,B). Conversely,
Int. J. Mol. Sci. 2023,24, 11569 6 of 15
the administration of the TAAR1 agonist RO5166017 (1
µ
M) to LPS/TNF
α
-stimulated
HMC3 cells mimicked the effects on the release of both IL-6 and IL-10 previously observed
after administering T1AM (10 µM) (Figure 6A,B).
Int.J.Mol.Sci.2023,24,115697of17
Figure6.AbilityofT1AMtodecreasetheinflammatoryphenotypeofLPS/TNFα‐stimulatedHMC3
cellsbythemodulationofTAAR1. Barsrepresenttherelease(pg/mL)ofIL–6(A)andIL–10(B)in
thepresenceofthedrugsattheindicatedconcentrations.Datarepresentmeans±S.E.M.frominde‐
pendentexperiments(n=3),performedinduplicate.Statisticalanalysiswasperformedbyordinary
one‐wayANOVAfollowedbyDunne’smultiplecomparisontest.Foreachexperimentalcondition
aselectedspecificpaernhasbeenused.
2.4.3‐IodothyroaceticAcid(TA1)WasNotAbletoDecreasetheInflammatoryPhenotypeof
LPS/TNFα‐StimulatedHMC3HumanMicroglialCells
Since3‐iodothyroaceticacid(TA1),themajorcataboliteofT1AM[43–45],hasbeen
reportedtoberesponsibleforsomeeffectselicitedaftertheadministrationofexogenous
T1AM,wecheckedwhetherTA1administrationcouldalsodecreasetheinflammatory
phenotypeofLPS/TNFα‐stimulatedHMC3cells.WeobservedthatadministrationofTA1
(0.1,1,and10µM)toLPS/TNFα‐stimulatedHMC3cellswasnotabletoproduceanysig‐
nificanteffectonbothIL‐6andIL‐10releasefromcells(Figure7),suggestingthatthede‐
creasedactivationofmicrogliaisexclusivelyduetotheactionofT1AMthroughtheinter‐
actionwiththeTAAR1receptor.
Figure7.3‐iodothyroaceticacidTA1doesnotdecreasetheinflammatoryphenotypeofLPS/TNFα–
stimulatedHMC3cells.AfterpretreatmentwithTA1 attheindicatedconcentrationsthereleaseof
0
250
500
750
1000
1250
1500
IL-6 [pg/ml]
LPS/TNF - + + + + + +
T
1
AM (µM) - -
10
-
-
10 -
RO5166017 (µM) - - -
0.25 1
-
1
EPPTB (µM)
- - -
-
-
1 1
0.0035 0.0041
0.0045
0.0031
0
200
400
600
IL-10 [pg/ml]
LPS/TNF - + + + + + +
T
1
AM (µM) - -
10
-
-
10 -
RO5166017 (µM) - - -
0.25 1
-
1
EPPTB (µM)
- - -
-
-
1 1
0.0023
0.0031
0.0019
AB
0
50
100
150
200
250
IL-10 [pg/ml]
LPS/TNF
- + + + +
TA
1
(µM) - +
0.1 1 10
AB
0
250
500
750
1000
1250
1500
IL-6 [pg/ml]
LPS/TNF
- + + + +
TA
1
(µM) - +
0.1 1 10
0.0023
Figure 6.
Ability of T1AM to decrease the inflammatory phenotype of LPS/TNF
α
-stimulated HMC3
cells by the modulation of TAAR1. Bars represent the release (pg/mL) of IL–6 (
A
) and IL–10 (
B
)
in the presence of the drugs at the indicated concentrations. Data represent means
±
S.E.M. from
independent experiments (n= 3), performed in duplicate. Statistical analysis was performed by
ordinary one-way ANOVA followed by Dunnett’s multiple comparison test. For each experimental
condition a selected specific pattern has been used.
2.4. 3-Iodothyroacetic Acid (TA1) Was Not Able to Decrease the Inflammatory Phenotype of
LPS/TNFα-Stimulated HMC3 Human Microglial Cells
Since 3-iodothyroacetic acid (TA1), the major catabolite of T1AM [
43
–
45
], has been
reported to be responsible for some effects elicited after the administration of exogenous
T1AM, we checked whether TA1 administration could also decrease the inflammatory
phenotype of LPS/TNF
α
-stimulated HMC3 cells. We observed that administration of TA1
(0.1, 1, and 10
µ
M) to LPS/TNF
α
-stimulated HMC3 cells was not able to produce any
significant effect on both IL-6 and IL-10 release from cells (Figure 7), suggesting that the
decreased activation of microglia is exclusively due to the action of T1AM through the
interaction with the TAAR1 receptor.
2.5. T1AM-TAAR1 System Protects against A
β25–35
-Mediated Release of Proinflammatory Factors
in HMC3 Cells
Studies have revealed that A
β
oligomers activate microglia to secrete proinflammatory
factors, including cytokines, chemokines, complements factors, and a large variety of free
radicals [
46
,
47
]. Suppressing the response of microglia cells to inflammatory stress may
attenuate AD pathology and lessen the disease progression [48,49].
We initially assessed the effects of
β
-amyloid peptide 25–35 (A
β
25–35) [
50
] on HMC3
cells’ viability. The cells were treated with two different concentrations (1 and 10
µ
M)
of A
β
25–35 for 24 or 48 h. A
β
25–35 was found to induce cytotoxicity in HMC3 cells in
a concentration- and time-dependent manner, with a 10
µ
M concentration promoting a 50%
reduction in cell viability after 24 h (Figure 8A). We thus used this condition to evaluate the
modulation of A
β
25–35 cytotoxicity in further experiments. HMC3 cells were treated with
10
µ
M A
β
25–35 in the presence of different concentrations (0.1, 1, and 10
µ
M) of T1AM
for 24 h to determine the effects of T1AM on
β
-amyloid-induced cytotoxicity. In line with
expectations, MTT assays indicated that T1AM was able to protect cells from A
β
-induced
Int. J. Mol. Sci. 2023,24, 11569 7 of 15
cytotoxicity in a concentration-dependent manner, with the identified best concentrations
of 1 and 10 µM being used in further experiments (Figure 8B).
Int.J.Mol.Sci.2023,24,115697of17
Figure6.AbilityofT1AMtodecreasetheinflammatoryphenotypeofLPS/TNFα‐stimulatedHMC3
cellsbythemodulationofTAAR1. Barsrepresenttherelease(pg/mL)ofIL–6(A)andIL–10(B)in
thepresenceofthedrugsattheindicatedconcentrations.Datarepresentmeans±S.E.M.frominde‐
pendentexperiments(n=3),performedinduplicate.Statisticalanalysiswasperformedbyordinary
one‐wayANOVAfollowedbyDunne’smultiplecomparisontest.Foreachexperimentalcondition
aselectedspecificpaernhasbeenused.
2.4.3‐IodothyroaceticAcid(TA1)WasNotAbletoDecreasetheInflammatoryPhenotypeof
LPS/TNFα‐StimulatedHMC3HumanMicroglialCells
Since3‐iodothyroaceticacid(TA1),themajorcataboliteofT1AM[43–45],hasbeen
reportedtoberesponsibleforsomeeffectselicitedaftertheadministrationofexogenous
T1AM,wecheckedwhetherTA1administrationcouldalsodecreasetheinflammatory
phenotypeofLPS/TNFα‐stimulatedHMC3cells.WeobservedthatadministrationofTA1
(0.1,1,and10µM)toLPS/TNFα‐stimulatedHMC3cellswasnotabletoproduceanysig‐
nificanteffectonbothIL‐6andIL‐10releasefromcells(Figure7),suggestingthatthede‐
creasedactivationofmicrogliaisexclusivelyduetotheactionofT1AMthroughtheinter‐
actionwiththeTAAR1receptor.
Figure7.3‐iodothyroaceticacidTA1doesnotdecreasetheinflammatoryphenotypeofLPS/TNFα–
stimulatedHMC3cells.AfterpretreatmentwithTA1 attheindicatedconcentrationsthereleaseof
0
250
500
750
1000
1250
1500
IL-6 [pg/ml]
LPS/TNF - + + + + + +
T
1
AM (µM) - -
10
-
-
10 -
RO5166017 (µM) - - -
0.25 1
-
1
EPPTB (µM)
- - -
-
-
1 1
0.0035 0.0041
0.0045
0.0031
0
200
400
600
IL-10 [pg/ml]
LPS/TNF - + + + + + +
T
1
AM (µM) - -
10
-
-
10 -
RO5166017 (µM) - - -
0.25 1
-
1
EPPTB (µM)
- - -
-
-
1 1
0.0023
0.0031
0.0019
AB
0
50
100
150
200
250
IL-10 [pg/ml]
LPS/TNF
- + + + +
TA
1
(µM) - +
0.1 1 10
AB
0
250
500
750
1000
1250
1500
IL-6 [pg/ml]
LPS/TNF
- + + + +
TA
1
(µM) - +
0.1 1 10
0.0023
Figure 7.
3-iodothyroacetic acid TA1 does not decrease the inflammatory phenotype of LPS/TNF
α
–
stimulated HMC3 cells. After pretreatment with TA1 at the indicated concentrations the release
of IL–6 (
A
) and IL–10 (
B
) from LPS/TNF
α
–stimulated HMC3 cells were examined. Data represent
means
±
S.E.M. from three independent experiments (n= 3), performed in duplicate. Statistical
analysis was performed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison
test. For each experimental condition a selected specific pattern has been used.
Int.J.Mol.Sci.2023,24,115698of17
IL–6(A)andIL–10(B)fromLPS/TNFα–stimulatedHMC3cellswereexamined.Datarepresent
means±S.E.M.fromthreeindependentexperiments(n=3),performedinduplicate.Statisticalanal‐
ysiswasperformedbyordinaryone‐wayANOVAfollowedbyDunne’smultiplecomparisontest.
Foreachexperimentalconditionaselectedspecificpaernhasbeenused.
2.5.T1AM‐TAAR1 SystemProtectsagainstAβ25–35‐MediatedReleaseofProinflammatoryFactors
inHMC3Cells
StudieshaverevealedthatAβoligomersactivatemicrogliatosecreteproinflamma‐
toryfactors,includingcytokines,chemokines,complementsfactors,andalargevarietyof
freeradicals[46,47].Suppressingtheresponseofmicrogliacellstoinflammatorystress
mayaenuateADpathologyandlessenthediseaseprogression[48,49].
Weinitiallyassessedtheeffectsofβ‐amyloidpeptide25–35(Aβ25–35)[50]onHMC3
cells’viability.Thecellsweretreatedwithtwodifferentconcentrations(1and10µM)of
Aβ25–35for24or48h.Aβ25–35wasfoundtoinducecytotoxicityinHMC3cellsinacon‐
centration‐andtime‐dependentmanner,witha10µMconcentrationpromotinga50%
reductionincellviabilityafter24h(Figure8A).Wethususedthisconditiontoevaluate
themodulationofAβ25–35cytotoxicityinfurtherexperiments.HMC3cellsweretreated
with10µMAβ25–35inthepresenceofdifferentconcentrations(0.1,1,and10µM)of
T1AMfor24htodeterminetheeffectsofT1AMonβ‐amyloid‐inducedcytotoxicity.In
linewithexpectations,MTTassaysindicatedthatT1AMwasabletoprotectcellsfromAβ‐
inducedcytotoxicityinaconcentration‐dependentmanner,withtheidentifiedbestcon‐
centrationsof1and10µMbeingusedinfurtherexperiments(Figure8B).
Figure8.MTTassaysinHMC3cellsexposedtoAβ.(A)EffectsonHMC3cells’viabilityoftreatment
withβ–amyloidpeptide25–35(Aβ)at1and10µMfor24or48h.(B)EffectsonHMC3cells’viability
ofpretreatmentwithT1AMat0.1,1,and10µM,followedbyexposureto10µMAβfor24h.Data
representmeans±S.E.M.fromthreeindependentexperiments,performedintriplicate(n=3).Sta‐
tisticalanalysiswasperformedbyordinaryone‐wayANOVAfollowedbyDunne’smultiplecom‐
parisontest.Foreachexperimentalconditionaselectedspecificpaernhasbeenused.
HavingassessedthecytoprotectiveeffectsofT1AM,wemovedontoexaminethe
abilityofT1AMtosuppressβ‐amyloid‐inducedupregulationofproinflammatorycyto‐
kinesproduction.ExposureofHMC3cellsto10µMAβ25–35for24hpromotedasignifi‐
cantincreaseinthesecretionofcommonproinflammatorycytokines(TNF‐αandIL‐6),
andnoeffectonthereleaseofanti‐inflammatorycytokineIL‐10(Figure9A–C).Then,we
0
20
40
60
80
100
120
Cell viability (% vs. CTRL)
A
(µM)
- 1 10 - 1 10
24h 48h
0.007
0.0078
0.0084
0.0069
AB
0
20
40
60
80
100
120
Cell viability (% vs. CTRL)
A
(µM)
- 10 10 10 10
T1AM (µM)
- - 0.1 1 10
0.0025 0.043
0.0089
Figure 8.
MTT assays in HMC3 cells exposed to A
β
. (
A
) Effects on HMC3 cells’ viability of treatment
with
β
–amyloid peptide 25–35 (A
β
) at 1 and 10
µ
M for 24 or 48 h. (
B
) Effects on HMC3 cells’ viability
of pretreatment with T1AM at 0.1, 1, and 10
µ
M, followed by exposure to 10
µ
M A
β
for 24 h. Data
represent means
±
S.E.M. from three independent experiments, performed in triplicate (n= 3).
Statistical analysis was performed by ordinary one-way ANOVA followed by Dunnett’s multiple
comparison test. For each experimental condition a selected specific pattern has been used.
Having assessed the cytoprotective effects of T1AM, we moved on to examine the
ability of T1AM to suppress
β
-amyloid-induced upregulation of proinflammatory cytokines
production. Exposure of HMC3 cells to 10
µ
M A
β
25–35 for 24 h promoted a significant
increase in the secretion of common proinflammatory cytokines (TNF-
α
and IL-6), and no
effect on the release of anti-inflammatory cytokine IL-10 (Figure 9A–C). Then, we repeated
the experiment, exposing HMC3 cells to pretreatment with 1 and 10
µ
M T1AM. Compared
with the A
β
-treated group, the levels of TNF-
α
and IL-6 were found to show a significant
return to normal levels in the group receiving the combined A
β
25–35 and T1AM treatment
Int. J. Mol. Sci. 2023,24, 11569 8 of 15
(Figure 9A,B). In addition, pretreatment with T1AM significantly increased the secretion of
anti-inflammatory cytokine IL-10 (Figure 9C). Taken together, these findings indicated the
potential of T1AM to inhibit A
β
-induced microglia activation. Since TAAR1 has previously
been found to mediate the beneficial effects of T1AM on the inflammatory response of
LPS/TNF
α
-treated HMC3 cells, we subsequently proceeded to examine the role of TAAR1
in mediating the anti-inflammatory effects of T1AM in β-amyloid-induced HMC3 cells.
Int.J.Mol.Sci.2023,24,115699of17
repeatedtheexperiment,exposingHMC3cellstopretreatmentwith1and10µMT1AM.
ComparedwiththeAβ‐treatedgroup,thelevelsofTNF‐αandIL‐6werefoundtoshowa
significantreturntonormallevelsinthegroupreceivingthecombinedAβ25–35and
T1AMtreatment(Figure9A,B).Inaddition,pretreatmentwithT1AMsignificantlyin‐
creasedthesecretionofanti‐inflammatorycytokineIL‐10(Figure9C).Tak e ntogether,
thesefindingsindicatedthepotentialofT1AMtoinhibitAβ‐inducedmicrogliaactivation.
SinceTAAR1 haspreviouslybeenfoundtomediatethebeneficialeffectsofT1AMonthe
inflammatoryresponseofLPS/TNFα‐treatedHMC3cells,wesubsequentlyproceededto
examinetheroleofTAAR 1inmediatingtheanti‐inflammatoryeffectsofT1AMinβ‐am‐
yloid‐inducedHMC3cells.
Figure9.T1AMpretreatmentmodulatedthereleaseofproinflammatory(TNF‐α,IL‐6)andanti‐
inflammatory(IL‐10)interleukinsinAβ‐inducedHMC3cells.Barsrepresenttherelease(pg/mL)of
TNF–α(A),IL–6(B),andIL–10(C)inthepresenceofthedrugsattheindicatedconcentrations.Data
representmeans±S.E.M.fromthreeindependentexperiments(n=3),performedinduplicate.Sta‐
tisticalanalysiswasperformedbyordinaryone‐wayANOVAfollowedbyDunne’smultiplecom‐
parisontest.Foreachexperimentalconditionaselectedspecificpaernhasbeenused.
WeobservedthatT1AM’sprotectiveeffectagainstAβ‐inducedmicrogliaactivation
wasabolishedbycoadministrationofTAAR1 selectiveantagonistEPPTB(1µM)(Figure
9A–C).
DysregulationofthetranscriptionfactorNF‐κBhasbeenwidelyassociatedwithAD,
leadingtoglialcellsactivationandneuroinflammation[51].Moreimportantly,theinacti‐
vationofmicroglialNF‐κBhasbeenshowntorestorecognitivedeficitsandhelpto
reestablishahomeostaticphenotypeinmicroglia[52].
Therefore,wedecidedtoevaluatetheinvolvementoftheNF‐κBsignalingpathway
inthebeneficialeffectsobservedwithT1AMinβ‐amyloid‐inducedHMC3cells.Therefore,
werepeatedkeyAβ25–35andT1AMcotreatmentexperimentsandevaluatedNF‐κBac‐
tivity.Thecellswerethencollected,andphospho‐NF‐κBP65(p‐P65)wasdetectedinthe
celllysates(Figure10).TheresultsshowedthatAβ25–35treatmentincreasedthephos‐
phorylationlevelofP65toactivatetheNF‐κBpathway,andT1AMsuppressedthisre‐
sponse(Figure10).Notably,inthepresenceofTA AR1selectiveantagonistEPPTB(1µM),
theeffectofT1AMonNF‐κBactivationwascompletelyabolished(Figure10).
0
200
400
600
800
1000
TNF
[pg/ml]
A
(µM)
- 10 10 10 10
T1AM (µM)
- - 1 10 10
EPPTB (µM) - - - - 1
0.0037 0.0046
0.0041
0
200
400
600
800
IL-6 [pg/ml]
A
(µM)
- 10 10 10 10
T1AM (µM)
- - 1 10 10
EPPTB (µM) - - - - 1
0.0031 0.0042
0.0039
0
100
200
300
400
500
IL-10 [pg/ml]
A
(µM)
- 10 10 10 10
T1AM (µM)
- - 1 10 10
EPPTB (µM) - - - - 1
0.0022
0.0018
AB C
Figure 9.
T1AM pretreatment modulated the release of proinflammatory (TNF-
α
, IL-6) and anti-
inflammatory (IL-10) interleukins in A
β
-induced HMC3 cells. Bars represent the release (pg/mL)
of TNF–
α
(
A
), IL–6 (
B
), and IL–10 (
C
) in the presence of the drugs at the indicated concentrations.
Data represent means
±
S.E.M. from three independent experiments (n= 3), performed in duplicate.
Statistical analysis was performed by ordinary one-way ANOVA followed by Dunnett’s multiple
comparison test. For each experimental condition a selected specific pattern has been used.
We observed that T1AM’s protective effect against A
β
-induced microglia activa-
tion was abolished by co administration of TAAR1 selective antagonist EPPTB (1
µ
M)
(Figure 9A–C).
Dysregulation of the transcription factor NF-
κ
B has been widely associated with
AD, leading to glial cells activation and neuroinflammation [
51
]. More importantly, the
inactivation of microglial NF-
κ
B has been shown to restore cognitive deficits and help to
reestablish a homeostatic phenotype in microglia [52].
Therefore, we decided to evaluate the involvement of the NF-
κ
B signaling pathway in
the beneficial effects observed with T1AM in
β
-amyloid-induced HMC3 cells. Therefore,
we repeated key A
β
25–35 and T1AM cotreatment experiments and evaluated NF-
κ
B
activity. The cells were then collected, and phospho-NF-
κ
B P65 (p-P65) was detected
in the cell lysates (Figure 10). The results showed that A
β
25–35 treatment increased the
phosphorylation level of P65 to activate the NF-
κ
B pathway, and T1AM suppressed this
response (Figure 10). Notably, in the presence of TAAR1 selective antagonist EPPTB (1
µ
M),
the effect of T1AM on NF-κB activation was completely abolished (Figure 10).
Int. J. Mol. Sci. 2023,24, 11569 9 of 15
Int.J.Mol.Sci.2023,24,1156910of17
Figure10.TAAR 1activationmodulatedthephosphorylationofNF–kB(NF–kBp65)inAβ–induced
HMC3cells.Datarepresentmeans±S.E.M.fromthreeindependentexperiments(n=3),performed
induplicate.Statisticalanalysiswasperformedbyordinaryone‐wayANOVAfollowedbyDun‐
ne’smultiplecomparisontest.Foreachexperimentalconditionaselectedspecificpaernhasbeen
used.
3.Discussion
IncreasingevidencesupportstheconceptthatT1AM/TAAR1signalingispartofan
endogenoussystemthatcanbemodulatedtopreventneurodegeneration
[16,18,32,41,53,54].ADisthemostfrequentneurodegenerativedisorderintheelderly,
usuallycharacterizedbymemorydeficitsandcognitivedecline.Cognitivefunctionisan
importantdeterminantofqualityoflife,especiallyintheelderly.Theprogressiveincrease
incirculatingproinflammatorycytokinesininflammaginghasbeenassociatedwithage‐
relatedcognitivedecline[55]aswellasenhancedneuroinflammation,neurodegeneration,
andbrainreleaseofcytokinesbythemicroglia,whichactasresidentphagocyticinflam‐
matorycellsinthebrain[56].Microglialcellsarevitalinrecruitingtheseinflammatory
mediators,anddysregulatedmicroglialactivationrepresentsaneuropathologicalhall‐
markinADandmanyotherNDDs[57].MicroglialTA AR1signalinghasnotyetbeen
fullydefined,butconsiderableoverlapexistsbetweentheimmuneactivatedstateand
TAAR1expression[29,58,59].PathologicalchangesinendogenousTAAR1agonists,asob‐
servedinpsychiatricdisorders,couldunderliealterationsinmicroglialfunctions[23,60].
Therefore,ascertainingtheabilityofT1AM,andendogenousagonistforTAAR 1willlead
toabeerunderstandingoftheroleofTAAR1asamodulatorofmicrogliadysregulation,
furtherexpandingthepotentialofdrugsthatinteractwithTAAR1 inthemanagementof
AD.
WeshowedherethatT1AMwasabletoreduceLPS/TNFα‐inducedIL‐6inHMC3
humanmicroglialcells,whileincreasingtheproductionofanti‐inflammatoryinterleukin
IL‐10withoutaffectingthecells’viability.Inaddition,inLPS/TNFα‐inducedHMC3cells,
treatmentwithT1AMwasobservedtodecreasetheexpressionofinflammatoryresponse‐
relatedgenes,includingthemonocytechemoaractprotein‐1(MCP1),themacrophage
inflammatoryprotein‐1(MIP1),andthetranscriptionfactorNF‐kB.
Regardingthemechanismofaction,T1AMdoesnotbindthenuclearthyroidhor‐
monereceptors(TRs)butitstimulateswithnanomolaraffinityTAAR1 ,aGprotein‐cou‐
pledreceptorthatrecentlyemergedtohavearoleinimmunomodulation[53]andneu‐
roinflammation[32].Nevertheless,thereiscurrentlynotmuchinformationregarding
TAAR1expressioninmicroglia[29].Inthepresentstudy,byperformingquantitativereal‐
0.0
0.5
1.0
1.5
2.0
phospho-NF-B p65
(fold increase vs control)
A (µM) - 10 10 10 10
T1AM (µM) - - 1 10 10
EPPTB (µM) - - - - 1
0.0029 0.017
0.0078
Figure 10.
TAAR1 activation modulated the phosphorylation of NF–kB (NF–kB p65) in A
β
–induced
HMC3 cells. Data represent means
±
S.E.M. from three independent experiments (n= 3), performed
in duplicate. Statistical analysis was performed by ordinary one-way ANOVA followed by Dunnett’s
multiple comparison test. For each experimental condition a selected specific pattern has been used.
3. Discussion
Increasing evidence supports the concept that T1AM/TAAR1 signaling is part of an en-
dogenous system that can be modulated to prevent neurodegeneration [
16
,
18
,
32
,
41
,
53
,
54
].
AD is the most frequent neurodegenerative disorder in the elderly, usually characterized
by memory deficits and cognitive decline. Cognitive function is an important determinant
of quality of life, especially in the elderly. The progressive increase in circulating proin-
flammatory cytokines in inflammaging has been associated with age-related cognitive
decline [
55
] as well as enhanced neuroinflammation, neurodegeneration, and brain release
of cytokines by the microglia, which act as resident phagocytic inflammatory cells in the
brain [
56
]. Microglial cells are vital in recruiting these inflammatory mediators, and dys-
regulated microglial activation represents a neuropathological hallmark in AD and many
other NDDs [
57
]. Microglial TAAR1 signaling has not yet been fully defined, but consider-
able overlap exists between the immune activated state and TAAR1 expression [
29
,
58
,
59
].
Pathological changes in endogenous TAAR1 agonists, as observed in psychiatric disorders,
could underlie alterations in microglial functions [
23
,
60
]. Therefore, ascertaining the ability
of T1AM, and endogenous agonist for TAAR1 will lead to a better understanding of the
role of TAAR1 as a modulator of microglia dysregulation, further expanding the potential
of drugs that interact with TAAR1 in the management of AD.
We showed here that T1AM was able to reduce LPS/TNF
α
-induced IL-6 in HMC3
human microglial cells, while increasing the production of anti-inflammatory interleukin
IL-10 without affecting the cells’ viability. In addition, in LPS/TNF
α
-induced HMC3 cells,
treatment with T1AM was observed to decrease the expression of inflammatory response-
related genes, including the monocyte chemoattract protein-1 (MCP1), the macrophage
inflammatory protein-1 (MIP1), and the transcription factor NF-kB.
Regarding the mechanism of action, T1AM does not bind the nuclear thyroid hormone
receptors (TRs) but it stimulates with nanomolar affinity TAAR1, a G protein-coupled
receptor that recently emerged to have a role in immunomodulation [
53
] and neuroin-
flammation [
32
]. Nevertheless, there is currently not much information regarding TAAR1
expression in microglia [
29
]. In the present study, by performing quantitative real-time
PCR analysis (qPCR), we demonstrated that TAAR1 is expressed in the human microglial
HMC3 cell line, and we found that after LPS/TNF
α
stimulation, no changing levels of
TAAR1 expression were observed in HMC3 cells.
Int. J. Mol. Sci. 2023,24, 11569 10 of 15
Using a pharmacological approach, we demonstrated that inhibition of TAAR1 was
sufficient to prevent T1AM’s anti-inflammatory effect on HMC3 cells, and, conversely, the
stimulation of TAAR1 in the absence of T1AM was able to attenuate the LPS/TNF
α
-induced
release of proinflammatory cytokines, as observed in T1AM-treated cells.
In this investigation, the pharmacokinetics results showed that T1AM was rapidly
taken up by HMC3 cells and catabolized by oxidative deamination to TA1, which was
largely released from cells.
Even though TA1 is not a ligand of TAAR1 [
61
], previous works have reported that
TA1 may contribute, at least in part, to some effects elicited after the administration of
exogenous T1AM [
41
,
43
–
45
,
62
]. Therefore, we checked whether TA1 administration could
also decrease the inflammatory phenotype of LPS/TNF
α
-stimulated HMC3 cells, but
obtained negative results, confirming the specific anti-inflammatory effect of T1AM.
A great number of studies have documented the ability of A
β
fibrils to directly stim-
ulate microglia
in vitro
to assume a neurotoxic phenotype characterized by secretion of
a plethora of proinflammatory molecules, ultimately leading to neuron loss [
63
,
64
]. We
found that aggregated A
β
25–35 peptide (10
µ
M, 24 h) significantly decreased viability
of HMC3 microglial cells. This was concentration-dependently attenuated by T1AM
(
0.1–10 µM
). Pretreatment with T1AM also suppressed the release of TNF
α
and proinflam-
matory IL-6 while increasing anti-inflammatory IL-10 levels. These effects were almost
completely abolished by treatment with the TAAR1 antagonist EPPTB. In addition, A
β
in-
duced activation of microglial NF-
κ
B by phosphorylation. Application of T1AM attenuated
this effect via TAAR1 activation, suggesting that the T1AM/TAAR1 system may protect
microglial cells against Aβ-induced cell injury by inhibition of inflammation.
In summary, we provided, for the first-time, preliminary evidence that the T1AM/TAAR1
signaling pathway has the potential to normalize factors that regulate microglia-mediated
neuroinflammation, further increasing the relevance of this system in the pathophysiology
of aging-related brain diseases like AD. In animal models, neuroinflammation has been
closely tied to impaired function of the hippocampus, associated with a loss of hippocampal
pyramidal neurons and entorhinal cells, leading to the disruption of long-term potentiation
(LTP) in hippocampal synapses [
65
,
66
]. Notably, the T1AM/TAAR1 system has been previ-
ously shown to restore LTP in the enthorinal cortex (EC) of mouse models of AD, suggesting
that T1AM and TAAR1 are part of an endogenous system that can be modulated to prevent
synaptic and behavioral deficits associated with A
β
-related toxicity [
41
]. The results of the
present study may help to elucidate the mechanisms by which the T1AM/TAAR1 system
exerts a neuroprotective effect, and the investigation of the relationship with neuroinflam-
mation markers could bring the story one step ahead. These preliminary data are strictly
necessary to provide the basis for the ongoing new
in vivo
study, which we are conducting
to clarify the contribution of neuroinflammation to altered hippocampal neuroplasticity, as
observed in mouse models of AD. Furthermore, the identification of cytokines specifically
associated with cognitive decline may provide novel biomarkers for the development of
interventions to slow or reverse AD.
4. Materials and Methods
4.1. Drugs
T1AM and the TAAR1 antagonist N-(3-ethoxyphenyl)-4-(pyrrolidin-1-yl)-3-trifluoromethyl-
benzamide (EPPTB) were purchased from Sigma-Aldrich (Milan, Italy); the TAAR1 agonist
(S)-4-[(ethylphenylamino) methyl]-4,5-dihydrooxazol-2-ylamine (RO5166017) was kindly
provided by Dr. Gainetdinov. T1AM metabolite 3-iodothyroacetic acid (TA1) was kindly
provided by Dr. Scanlan. Aliquots were stored at
−
20
◦
C in DMSO as a 200 mM stock
solution and diluted to the desired final concentration in culture media.
4.2. Analysis of T1AM and TA1
T1AM and its metabolite 3-iodothyroacetic acid (TA1) were assayed in samples by
tandem mass spectrometry coupled to liquid chromatography (LC–MS/MS) by using
Int. J. Mol. Sci. 2023,24, 11569 11 of 15
a previously established procedure that allows the simultaneous detection of T1AM and
TA1 in each sample [
20
,
40
]. T1AM and TA1 intracellular concentrations were also assayed
by exposing cell lysate samples to the same LC–MS/MS procedure.
Briefly, aliquots (0.1 mL) from each sample collection were spiked with 10
µ
L of a suit-
able mixture of internal standards (deuterated T1AM and TA1). After adding methanol
(
0.4 mL
), the samples were shaken for 10 min and centrifuged at 22,780
×
gfor 10 min.
The supernatant was dried under a gentle stream of nitrogen, reconstituted with wa-
ter/methanol mixture (70/30 by volume), and injected into the LC–MS/MS system. The
latter included an Agilent 1290 UHPLC system (Santa Clara, CA, USA) coupled to an
AB-Sciex API 4000 triple quadrupole mass spectrometer (Concord, ON, Canada).
4.3. Cell Cultures and Reagents
The human microglial clone 3 cell line (HMC3) (ATCC
®
CRL-3304
™
,
Manassas, VA, USA
)
was cultured in high-glucose DMEM supplemented with 10% FBS, streptomycin (
100 g/mL
),
and penicillin (100 U/mL) (Sigma-Aldrich, Milan, Italy).
LPS (Escherichia coli 0111:B4), TNF
α
, and Amyloid
β
-Peptide 25–35 (A
β
25–35) were
purchased from Sigma-Aldrich (Milan, Italy).
4.4. MTT (Cell Viability Assay)
HMC3 cells were exposed to cell viability assays by using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, Milan, Italy) reagent. Briefly,
HMC3 cells were exposed to increasing concentrations of T1AM (0.1–10
µ
M) and incubated
at 37
◦
C for 24 h. Then, 0.5 mg/mL MTT reagent was added to each well, and the cells
were incubated for 3 h at 37
◦
C. Next, 25
µ
L of the medium was removed from the wells,
and 50
µ
L of DMSO was added. After incubating for 10 min at 37
◦
C, absorbance at 540 nm
was determined with an automated microplate reader (BIO-TEK, Winooski, VT, USA). The
percentage of cell viability was calculated as a percentage of vehicle-treated cells used as
control. The same procedure was also followed to detect the cytotoxic effect produced in
HMC3 cells after incubation with A
β
25–35 (1 and 10
µ
M, for 24 or 48 h) in the absence and
presence of increasing concentrations of T1AM (0.1, 1, and 10 µM).
4.5. Release of Inflammatory Molecules HMC3 Cells Treated with LPS/TNFα
Proinflammatory IL-6 and anti-inflammatory IL-10 levels were evaluated by specific
ELISA assays (RAB0306 (IL-6) and RAB0244 (IL-10), Sigma-Aldrich, Milan, Italy) on col-
lected culture media. Briefly, HMC3 cells were exposed to pretreatment with each of the
compounds under investigation (i.e., T1AM, TA1, or RO51660170) for 1 h followed by
treatment for 24 h with both LPS (10
µ
g/mL) and TNF
α
(50 ng/mL), generally indicated as
LPS/TNF
α
, and used as proinflammatory stimuli. Vehicle-treated cells were used as con-
trol. In competition experiments, the TAAR1 antagonist EPPTB (1
µ
M) was administered
15 min before proceeding with the administration of the selected TAAR1 agonist, namely,
T1AM or RO5166017.
4.6. Release of Inflammatory Molecules from Aβ25–35-Induced HMC3 Cells
The inflammatory response of HMC3 cells to A
β
25–35 (Sigma-Aldrich, Milan, Italy) ex-
posure (10
µ
M, 24 h) was evaluated by performing specific ELISA assays. In addition to IL-6
and IL-10 measurements, performed as described above, the levels of tumor necrosis factor
TNF
α
(RAB1089, Sigma-Aldrich, Milan, Italy) and nuclear factor kB (NF-kB) (
85-86082-11
,
ThermoFisher Scientific, Carlsbad, CA, USA) were also evaluated in collected culture media
and in cell lysates, respectively, following the corresponding manufacturer’s instructions.
Briefly, HMC3 cells were exposed to pretreatment with T1AM (1 or 10
µ
M) for 1 h followed
by A
β
25–35 (10
µ
M for 24 h), used as proinflammatory stimuli. Vehicle-treated cells were
used as control. In competition experiments, the TAAR1 antagonist EPPTB (
1µM
) was
administered 15 min before proceeding with the administration of T1AM (10 µM).
Int. J. Mol. Sci. 2023,24, 11569 12 of 15
4.7. Gene Expression Analysis
Total RNA was extracted from HMC3 cells using the RNeasy Mini kit (74104, Qiagen,
Hilden, Germany) following the manufacturer’s protocol. RNA concentration and purity
were determined by Nanodrop-1000 spectrophotometer and Qubit v.1 fluorometer plus
Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Wilmington, DE, USA).
Total RNA (1
µ
g) was retrotranscribed into first-strand cDNA by using by iScriptTM
gDNA Clear cDNA Synthesis Kit (Bio-Rad, Milan, Italy) following manual protocol indications.
Relative quantity of gene transcripts was measured by real-time PCR on samples’
cDNA using an SYBRGreen chemistry and an CFX Connect Real-Time PCR Detection Sys-
tem (Bio-Rad, Milan, Italy). The PCR cycle program consisted of an initial 30 s denaturation
at 95
◦
C followed by 40 cycles of 5 s denaturation at 95
◦
C and 15 sec annealing/extension
at 60
◦
C. A final melting protocol with ramping from 65
◦
C to 95
◦
C with 0.5
◦
C increments
of 5 s was performed for verification of amplicon specificity and primer dimer formation.
Primers were designed with Beacon Designer Software v.8.0 (Premier Biosoft Inter-
national, Palo Alto, CA, USA) with a junction primer strategy whenever possible. In any
case, negative control of retrotranscription was performed to exclude any interference
from residual genomic DNA contamination. The primer sequences for real-time PCR are
reported in Table 2.
Table 2. Primer sequences of target genes.
Reference Sequence
(RefSeq) RNA Gene Symbol Primer Sequences
NM_002046 GAPDH
(F) 5
0
-CCCTTCATTGACCTCAACTACATG
(R) 50-TGGGATTTCCATTGATGACAAGC
NM_002982.4 MCP1 (F) 50-GAGAGGCTGAGACTAACC
(R) 50-TGATTGCATCTGGCTGAG
NM_002983 MIP1 (F) 50-ACTTTGAGACGAGCAGCCAGTG
(R) 50-TTTCTGGACCCACTCCTCACTG
NM_001404662 NFKB (F) 50-CCTTTCTCATCCCATCTTT
(R) 50-CCTCAATGTCCTCTTTCTG
NM_138327 TAAR1 (F) 50-GAGATCTGCTGAGCACTGTTGG
(R) 5
0
-CAGCATAGTAGCGGTCAATGGAG
All reactions were performed in triplicate and the amount of mRNA was calculated by
the comparative critical threshold (CT) method. To account for possible variations related
to cDNA input or the presence of PCR inhibitors, the endogenous reference gene GAPDH
was simultaneously quantified for each sample, and data were normalized accordingly.
4.8. Statistical Analysis
Statistical analyses were performed using GraphPad Prism version 9.0 for Mac (Graph-
Pad Software, San Diego, CA, USA). Data were subjected to t-tests or one-way ANOVA.
Significant differences among different treatments were calculated according to Dunnett’s
multiple comparisons test. Data are reported as mean
±
SEM. Differences at p< 0.05 were
considered statistically significant.
Author Contributions:
B.P. and G.C. designed the study and wrote the manuscript. C.R. and B.P.
performed ELISA tests and collected biochemical data. B.P. and V.C. conducted gene expression
studies. A.B. and C.R. carried out LC/MS-MS data collection. G.R., F.S. and R.Z. analyzed the data and
discussed the results. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by a grant Pfizer (Project ID: 67562227; G.C.) and a grant from
ETA (Project ID: 549901 to G.R.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Int. J. Mol. Sci. 2023,24, 11569 13 of 15
Acknowledgments:
The authors would like to thank the CISUP Centre for Instrumentation Sharing,
University of Pisa for having placed the Sciex QTrap 6500+ mass spectrometer, which was used for
the T1AM assays, at the disposal of the authors.
Conflicts of Interest:
The authors declare no conflict of interest. The authors declare no competing
financial interest.
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