Immune‐Pineal Axis Protects Rat Lungs Exposed to Polluted Air

Abstract

Environmental pollution in the form of particulate matter <2.5 μm (PM2.5) is a major risk factor for diseases such as lung cancer, chronic respiratory infections and major cardiovascular diseases. Our goal was to show that PM2.5, eliciting a proinflammatory response activates the immune‐pineal axis, reducing the pineal synthesis and increasing the extrapineal synthesis of melatonin. Herein, we report that the exposure of rats to polluted air for 6 h reduced nocturnal plasma melatonin levels and increased lung melatonin levels. Melatonin synthesis in the lung reduced lipid peroxidation and increased PM2.5 engulfment and cell viability by activating high‐affinity melatonin receptors. Diesel exhaust particles (DEPs) promoted the synthesis of melatonin in a cultured cell line (RAW 264.7 cells) and rat alveolar macrophages via the expression of the gene encoding for AANAT through a mechanism dependent on activation of the NFκB pathway. Expression of the genes encoding AANAT, MT1 and MT2 was negatively correlated with cellular necroptosis, as disclosed by analysis of Gene Expression Omnibus (GEO) microarray data from the human alveolar macrophages of nonsmoking subjects. The enrichment score for antioxidant genes obtained from lung gene expression data (GTEx) was significantly correlated with the levels of AANAT and MT1 but not the MT2 melatonin receptor. Collectively, these data provide a systemic and mechanistic rationale for coordination of the pineal and extrapineal synthesis of melatonin by a standard damage‐associated stimulus, which activates the immune‐pineal axis, and provides a new framework for understanding the effects of air pollution on lung diseases.

Full text

J Pineal Res. 2020;00:e12636.
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1 of 13
https://doi.org/10.1111/jpi.12636
wileyonlinelibrary.com/journal/jpi
1
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INTRODUCTION
The deterioration of air quality in megacities caused by vehicle
emissions is the fifth highest global mortality risk factor.1,2 The
metropolitan area of São Paulo is characterized by a high level
of air pollution as the result of intense industrial activity and a
fleet of approximately 7 million vehicles.3 Air pollution results
from a complex mixture of thousands of pollutants, including
particulate matter (PM) of variable composition, with aerody-
namic diameters ranging from 5nm to 100μm. Coarse PM,
Received: 28 June 2019
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Revised: 3 February 2020
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Accepted: 4 February 2020
DOI: 10.1111/jpi.12636
ORIGINAL ARTICLE
Immune-pineal axis protects rat lungs exposed to polluted air
Claudia EmanueleCarvalho-Sousa1
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Eliana P.Pereira1
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Gabriela S.Kinker1
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MarianaVeras2
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Zulma S.Ferreira1
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Fernanda P.Barbosa-Nunes3
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Joilson O.Martins3
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Paulo H.N.Saldiva2
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Russel J.Reiter4
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Pedro A.Fernandes1
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Sanserayda Silveira Cruz-Machado1
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Regina P.Markus1
© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
1Laboratory of Chronopharmacology,
Institute of Bioscience – University of São
Paulo, São Paulo, Brazil
2Faculty of Medicine, University of São
Paulo, São Paulo, Brazil
3Faculty of Pharmacy and Biochemistry,
University of São Paulo, São Paulo, Brazil
4Faculty of Medicine, University of Texas
Health Center at San Antonio, San Antonio,
Texas
Correspondence
Regina P. Markus, Institute Bioscience,
University of São Paulo, Rua do Matão, tv
14, CEP 05508-090 São Paulo, Brazil.
Email: rpmarkus@usp.br
Funding information
São Paulo Research Foundation, FAPESP,
Grant/Award Number: 2010/5234-
5, 2011/50198-6, 2012/04508-6 and
2013/13691-1; Brazilian National Council
for Research and Technology, CNPq,
Grant/Award Number: 305378/2009-0;
Coordenaçãode Aperfeiçoamento de
Pessoal de Nível Superior-Brasil (CAPES),
Grant/Award Number: Finance Code 001
Abstract
Environmental pollution in the form of particulate matter <2.5μm (PM2.5) is a major
risk factor for diseases such as lung cancer, chronic respiratory infections, and major
cardiovascular diseases. Our goal was to show that PM2.5 eliciting a proinflammatory
response activates the immune-pineal axis, reducing the pineal synthesis and increas-
ing the extrapineal synthesis of melatonin. Herein, we report that the exposure of rats
to polluted air for 6hours reduced nocturnal plasma melatonin levels and increased
lung melatonin levels. Melatonin synthesis in the lung reduced lipid peroxidation and
increased PM2.5 engulfment and cell viability by activating high-affinity melatonin
receptors. Diesel exhaust particles (DEPs) promoted the synthesis of melatonin in a
cultured cell line (RAW 264.7 cells) and rat alveolar macrophages via the expression
of the gene encoding for AANAT through a mechanism dependent on activation
of the NFκB pathway. Expression of the genes encoding AANAT, MT1, and MT2
was negatively correlated with cellular necroptosis, as disclosed by analysis of Gene
Expression Omnibus (GEO) microarray data from the human alveolar macrophages
of nonsmoking subjects. The enrichment score for antioxidant genes obtained from
lung gene expression data (GTEx) was significantly correlated with the levels of
AANAT and MT1 but not the MT2 melatonin receptor. Collectively, these data pro-
vide a systemic and mechanistic rationale for coordination of the pineal and extrapin-
eal synthesis of melatonin by a standard damage-associated stimulus, which activates
the immune-pineal axis and provides a new framework for understanding the effects
of air pollution on lung diseases.
KEYWORDS
alveolar macrophages, diesel particle, immune-pineal axis, NF-kappa B, phagocytosis

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which has a 2.5-10μm aerodynamic diameter (PM10), is re-
tained in the upper airways (Nemmar et al 2013), while fine and
ultrafine PM, which have aerodynamic diameters from 0.1 to
2.5μm (PM2.5) and smaller than 0.1μm (PM0.1), respectively,
can penetrate the terminal portion of bronchi and the alveolar
portion.4-6 Exposure to PM2.5 is associated with cerebrovascu-
lar and heart diseases, atopic eczema, asthma, rhinitis, chronic
obstructive pulmonary disease, and lung cancer.7-11
Fine PM induces inflammatory responses in the lung and
internal organs by interacting with Toll-like receptors 2 (TLR2)
and TLR4, triggering the nuclear translocation of NFκB (nu-
clear factor kappa-light-chain-enhancer of activated B cells)
in the lung, brain, and other internal organs.12 NFκB dimers
containing the p65 subunit (also known as RelA) trigger a com-
bination of proinflammatory genes, including those that en-
code adhesion molecules, and oxidative and nitrosative stress.
Dimers of the c-Rel subunit are linked to the regulatory phase of
the inflammatory response. The p50/RelA dimer promotes the
transcription of Rel, the gene that encodes c-Rel,13,14 a subunit
associated with the priming of naïve T cells,15 and neuroprotec-
tion.16 Among the genes triggered by p50/c-Rel or RelA/c-Rel is
the Aanat (arylalkylamine N-acetyltransferase) gene, which en-
codes the enzyme that converts serotonin to N-acetylserotonin,
the direct precursor of melatonin.17,18 In contrast, nuclear trans-
location of the p50/p50 dimer in pinealocytes blocks Aanat
transcription, impairing melatonin synthesis.19-21
Activation of the immune-pineal axis, defined as the inhibi-
tion/induction of melatonin synthesis by pinealocytes/activated
macrophages, is mediated by the TLR2/4- or TNFR1-induced
translocation of NFκB dimers.22 Blockade of pineal melatonin
synthesis favors the expression of endothelial adhesion mole-
cules involved in the rolling and adhesion of leukocytes nec-
essary to mount a proper inflammatory response, whereas the
extrapineal synthesis of melatonin by macrophages regulates
this response, potentiating phagocytosis and reducing oxidative
and nitrosative stress.23,24 Many authors have described the pro-
tective effect of melatonin against inflammatory responses due
to its electron-donor property, which requires a concentration
from μm to mmol/L (for review22,25). More recently, exogenous
melatonin was suggested to reduce the expression of proteins
related to inflammation, oxidative stress, apoptosis, and fibrosis
in animals exposed to polluted air and subjected to pulmonary
ischemia-reperfusion injury.26
In contrast, some works suggest that the interaction of
melatonin with high-affinity G protein-coupled receptors
increases the expression or activation of oxygen-detoxifying
enzymes.27-29 Moreover, to the best of our knowledge, no in-
formation regarding the role of lung-synthesized melatonin
in regulating oxidative stress, cell death, and fibrosis induced
by polluted air is available.
Here, we tested whether the inhalation of polluted air ac-
tivates the immune-pineal axis by switching the synthesis of
melatonin from the pineal gland to alveolar macrophages,
enhancing phagocytosis of PM2.5 and reducing necroptosis.
Moreover, using bioinformatics analyses, we evaluated the
association between the expression of genes that encode mel-
atonin biosynthetic enzymes and receptors with necroptosis
and impaired antioxidant enzymatic processes. Our results
confirm activation of the immune-pineal axis under stressful
conditions and provide strong evidence that macrophage-syn-
thesized melatonin retains particulate matter in the lung.
2
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MATERIAL AND METHODS
Drug suppliers and a complete description of the methods are
provided in the Supporting Information.
2.1
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Animals
Adult male Wistar rats (2-3 months old) housed and bred
in the animal facility of the Department of Physiology of
the IBUSP were kept under a 12/12 hour light/dark cycle
(lights on at 06:00 hours) and euthanized by decapitation.
Experiments were conducted in accordance with the ethical
principles and guidelines of the Brazilian National Council
on Experimental Animal Control (CONCEA) and approved
by the Ethics Committee of ICB and IB USP (protocols
057/2012 and 198/2014).
2.2
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Exposure to polluted air
Animals were housed in two isolated compartments of a
Harvard Ambient Particle Concentrator (HAPC) at the
Faculty of Medicine, USP. Ambient particles ranging from
0.1 to 2.5μm in size were concentrated approximately 25-30
times in the experimental chamber,30 or they were filtered
to allowing animals breath clean air (control group). The
concentration of the particles varied according to the daily
air quality and increased linearly with the exposure time
(PM2.5: 700-3300 μg/m3; 1-6 hours). Control and experi-
mental animals were simultaneously exposed to filtered and
concentrated air at the dark phase and light phase of the day.
Animals exposed during dark phase (ZT12D) were killed at
ZT18, while those exposed during light phase (ZT0L) were
killed at ZT06. The animals stayed awake and were unre-
strained during the exposure.
2.3
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Pineal gland culture
Pineal glands were cultured for 48hours according to a pro-
tocol that promotes complete denervation of the gland31 for
48hours.

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CARVALHO-SOUSA et AL.
2.4
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Cell culture
We used immortalized RAW 264.7 macrophage regular line-
age and a lineage that express the red fluorescent reporter
protein under the control of κB elements in the promoter and
first intron of the Aanat gene (pDsRed2-1-aa-nat-kB-232), and
resident rat alveolar macrophages (see details in Supporting
Information).
2.5
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Cell viability
Cell viability was assessed by MTT colorimetric assay. RAW
264.7 cells were seeded at a density of 2 × 105 cells/well
in 96-well plates and were treated with or without DEP (1-
512μg/mL; 6, 12, and 24hours). Cells incubated with MTT
(0.5mg/mL, 4hours, 37°C) were exposed to dimethyl sul-
foxide (DMSO) (0.5%, 30minutes) under constant stirring.
Absorbance values at 540nm were read on a spectrophotom-
eter. Cell viability was defined according to the percentage of
the optical density of each group compared with that of the
control group.
2.6
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Diesel exhausted particle (DEP)
phagocytosis
Rat alveolar macrophages treated with vehicle or melatonin
(10nmol/L, 30minutes) were incubated with DEPs (128μg/
mL, 2hours) and fixed in acetone/methanol (1:1, 15minutes,
−20°C). Slides were analyzed by confocal microscopy (LSM
510) using a 63× water-immersion objective lens.
2.7
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Dosages—melatonin,
corticosterone, and cytokines
Plasma corticosterone and melatonin levels were assayed
through commercial ELISA kits (IBL International), and
melatonin levels in cultured pineal glands and medium were
determined by high-pressure liquid chromatography (HPLC)
as previously described.33
2.8
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Immunofluorescence analysis and
immunohistochemistry
The activation of microglia in the pineal gland was detected
by immunofluorescence analysis, and the expression of ad-
hesion molecules and iNOS in the lung was detected by im-
munohistochemistry (details in Supporting Information).
The proteins AANAT and PAA-NAT were determined by
immunohistochemistry, immunofluorescence, or Western
blot with Sigma-Aldrich antibodies developed against rabbit.
According to the producer, the anti-AANAT (S0689) recog-
nizes the C-terminal of the phosphorylated and nonphospho-
rylated AA-NAT, whereas the antibody S0814 recognizes
the N-terminal of P-AANAT.
2.9
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Real-time RT-PCR
The relative mRNA expression of the Aanat, Asmt, Sod, Cat,
and Gpx genes normalized to the expression of the Gapdh
(housekeeping) gene was analyzed through real-time RT-
PCR using SYBR Green PCR reagent.34
2.10
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PCR analysis of Toll-like
receptor signaling
The gene expression of TLR signaling machinery compo-
nents in the whole pineal glands of animals killed at ZT18
after 1 or 6hours of exposure to pollution or filtered air was
determined with a commercial PCR kit in real time with a
"Rat Toll-like receptor signaling pathway" array (cat: PARN-
018A; Qiagen) following the manufacturer's instructions.
2.11
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Oxidative stress and
antioxidant activity
The levels of thiobarbituric acid-reactive substances in the
lungs were used as an index of oxidative stress. The activities
of the enzymes superoxide dismutase (SOD; EC1.15.1.1),
catalase (CAT; EC1.11.1.6), and glutathione peroxidase
(GPx, EC1.11.1.9) were determined in portions of the
same lung samples prepared specifically for each enzyme.
Thiobarbituric acid-reactive substances were determined by
the thiobarbituric acid reaction.35
2.12
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Human transcriptome data
Microarray gene expression data from the alveolar mac-
rophages of 15 nonsmokers were obtained from the Gene
Expression Omnibus public repository36 with the accession
number GSE2125.37 GSE2125 data were generated using
Affymetrix Human Genome U133 Plus 2.0 Arrays and
normalized according to the robust multiarray averaging
(RMA) method. Data download was performed using the
GEOquery and Biobase Bioconductor R packages (http://
www.bioco nduct or.org/). GTEx38 RNA-seq gene expres-
sion data from 288 human lung samples were downloaded
on 01/07/2017 from the UCSC Xena browser (http://xena.
ucsc.edu/). GTEx RNA-seq data were generated using the

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Illumina HiSeq 2000 RNA sequencing platform, quanti-
fied using RSEM, normalized by upper quartile, and log2
(x+1) transformed.
2.13
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Gene set enrichment analysis (GSEA)
Genes in both the human alveolar macrophage (GSE2125) and
GTEx lung transcriptome datasets were ranked according to
Spearman's correlation coefficient between their expression and
the expression ofAANAT, MT1, or MT2. GSEA was performed
using KEGG necroptosis pathway39,40 or a curated set of genes
encoding enzymes involved in the cellular antioxidant system
(Table S1). Enrichment scores (ES) were calculated based on
a weighted Kolmogorov-Smirnov–like statistic and normalized
(NES) to account for the size of each gene set. P-values corre-
sponding to each NES were calculated based on 1000 permuta-
tions. The analyses were performed using GSEA v3.0.
2.14
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Statistical analysis
All the numbers shown refer to individual animals or cultures
and not replicates. The animal experiments required exposure
to air pollution in a special chamber and were always conducted
on at least two different nights. Statistical analysis was per-
formed with GraphPad Prism 8 software. Independent variable
data are presented as box plot whiskers (10%-90%), and depend-
ent variable data (time- or dose-dependent) are presented as the
mean±SEM. Differences between independent variables were
evaluated by ANOVA for parametric or nonparametric values,
followed by the Mann-Whitney or Kruskal-Wallis test. Data on
the dependence on dose or time fitted to the appropriate curve
were tested for regression from zero. Values of P< .05 indi-
cated statistically significant differences. The bioinformatics
statistical analysis was performed as presented above.
3
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RESULTS
3.1
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Systemic and pineal gland
inflammatory responses induced by polluted
air
To determine whether air pollution inhibits pineal melatonin
synthesis and increases the synthesis of melatonin in alveo-
lar macrophages, rats maintained in a 12/12hour light/dark
cycle were placed in Harvard ambient particle concentrator
FIGURE 1 Polluted air triggers systemic and pineal inflammatory responses. A, Plasma cytokine levels of rats exposed to polluted air for
0.5, 1, and 6h; n=4-8 animals per point. B, Heat map plot of genes codifying rat Toll-like receptor signaling pathways expressed in pineal glands
of rats exposed for 1 or 6h to filtered air or polluted air. The data are shown as log2 fold change compared with the vehicle (2 animals per group).
C, AANAT immunoreactivity (green) in the pineal gland of rats killed at ZT6 or ZT18 kept in regular environmental air (naïve) and of rats killed at
ZT18 exposed for 2, 4, and 6h to filtered or polluted air in HAPC. Nuclei were stained with DAPI (blue). In naïve animals, AANAT was expressed
only at nighttime. The expression of AANAT was reduced after 4h in polluted air. Bar=15μm. D, Time course of plasma melatonin (0.5-6h;
upper graph) and corticosterone (5min-6h; lower graph) changes in rats exposed to filtered or polluted air, and those maintained in regular cages
(naïve). All animals were euthanized at ZT18. Data are shown as mean±SEM; n=10-19 rats per point. Different letters indicate significant
differences between the groups (P<.05), *significantly different from naïve and filtered

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chambers before lights off (ZT12L) and sacrificed at ZT18.
Chamber one received filtrated air, and chamber 2 received
concentrated polluted air. The plasma concentrations of IL-
1α, IL-1 β, IL-4, and MCP-1 were transiently increased after
30minutes of exposure and returned to basal values (IL-4)
or significantly lower values (IL-1α and IL-1β) after 1 and
6 hours (Figure 1A). The concentrations of TNF, IL6, and
IL12 were maintained at high levels for 6hours, while the
concentration of IFNγ was reduced after 30minutes. IL-10
and GM-CSF levels were unaffected by polluted air (Figure
1A). The pineal gland also reacted to polluted air and exhib-
ited a rise in the expression of 68 out of 84 proinflammatory
genes (cytokines, Toll-like receptors, adaptor proteins, and
downstream regulatory proteins) (Figure 1B and Table S1).
As described previously, the pineal gland reacts to acute in-
flammatory stimuli by reducing the expression of AANAT22
(Figure 1C). Thus, on the first night of exposure to polluted
air, the pineal gland recognized danger-associated molecu-
lar patterns and mounted an acute inflammatory response.
After exposure to polluted air (4-6hours), plasma melatonin
was significantly reduced to levels below those in animals
exposed to filtered air in chamber 1 of the HAPC or those
maintained outside the apparatus (Figure 1D, upper graph).
To discriminate between stressful effects due to entry into
a new environment (HAPC) from those promoted by air pol-
lution, we simultaneously determined the plasma levels of
corticosterone and melatonin. Plasma corticosterone (5 min-
utes) and melatonin (30minutes) levels were not significantly
different between the three experimental groups. However, at
30minutes, a corticosterone peak was detected, followed by a
melatonin peak at 60minutes. Those peaks were significantly
different from the levels of corticosterone and melatonin in
rats maintained outside the HAPC and euthanized simultane-
ously with polluted air-treated rats (Figure 1D, lower graph).
A similar increase in nocturnal melatonin linked to an increase
in plasma corticosterone was previously observed in restrained
animals41 and after transpineal perfusion with corticosterone at
concentrations mimicking the peak level of corticosterone ob-
served early in the night in rats.42 Thus, these early changes in
corticosterone and melatonin levels reflected stress induced by
a new environment and not stress due to the presence of pollut-
ant material in the air. Furthermore, the late decrease in plasma
FIGURE 2 Polluted air activates rat pineal gland microglia, resulting in the local synthesis of TNF and in the reduction of melatonin
synthesis. A, Inhalation of polluted air (6h) increases the immunoreactivity to the biomarker of activated microglia IBA-1. Nuclei were stained
with DAPI. Data are expressed as mean±SEM and dots show individual data. Bar=100μm. B, Diesel exhaust particles (DEP) inhibit the
noradrenaline (NAd)-induced melatonin synthesis in cultured pineal glands. Superior panel: Time course of induction of TNF in cultured pineal
glands exposed to DEP for 1-6h. The zero-time exposition was obtained from glands exposed to vehicle for 1h. Lower panel shows that the
DEP-inhibition of NAd-induced melatonin synthesis (columns) is blocked in a dose-dependent manner by the antagonists of TLR4 (TAK 242) and
TNFR1 (SPD 304), indicating that pineal gland synthesized TNF inhibits melatonin synthesis. Data are expressed as mean±SEM; n=6 glands
per point

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melatonin levels (4-6hours) was due to an inflammatory re-
sponse in the pineal gland triggered by polluted air.
3.2
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Inhibition of pineal melatonin
synthesis by polluted air is mediated by
microglia activation
Exposure to polluted air (ZT12L to ZT18) induced a sustained
increase in the pineal gland expression of IBA-1, a marker of
microglial activation (Figure 2A), while the nuclear NFκB
content was transiently increased (Figure S1). To investigate
the mechanisms of these effects, we conducted experiments
in cultured pineal glands stimulated with diesel exhaust par-
ticles (DEPs). DEPs promoted the synthesis of TNF and
blocked noradrenaline (10nmol/L, 5 hours)-induced mela-
tonin synthesis (Figure 2B). This late response was inhibited
by antagonist of TNFR1 (SPD304, 50 mmol/L) or TLR4
(TAK242, 10 mmol/L), indicating that DEP interacts with
TLR4 to activate pineal microglia, which releases TNF, lead-
ing to the inhibition of melatonin synthesis. A similar profile
was previously observed following exposure to LPS.21 Thus,
polluted air and diesel particles activate the pineal gland arm
of the immune-pineal axis.
FIGURE 3 DEP induced the transcription arylalkylamine N-acetyltransferase gene (Aa-nat), the expression of the enzyme that converts
serotonin to N-acetylserotonin in its native (AANAT) and active form (phosphorylated AANAT, P-AANAT), and the synthesis of melatonin
both in RAW 264.7 cells and in alveolar macrophages. A, DEP-induced transcription of a red protein probe (pDsRed2-1) linked to the promoter
of Aanat in RAW 264.7 cells was blocked by inhibiting NFκB binding to DNA with PDTC (25μmol/L). B, DEP induced increase in the
immunoreactivity to antibody that interacts with the phosphorylated and nonphosphorylated form of AANAT (total AANAT) and the synthesis
of melatonin, shown as percentage of basal levels in RAW 264.7 cell line macrophages (melatonin basal levels=5.42±0.22pg/mL; n=4
independent experiments). C, DEP induced the expression of the enzyme P-AANAT and the synthesis of melatonin in rat alveolar macrophages.
Data are shown as mean±SEM for dependent variables and as box plot and whiskers (10%-90%) for independent variables. Immunoreactivity was
determined after 60min and medium melatonin after 6h of DEP incubation. AANAT (S0564) and phospho-AANAT (S0939) antibodies (Sigma-
Aldrich). Bars=20μmol/L

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3.3
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DEP-induced macrophage melatonin
synthesis increased cell viability
We next tested the ability of DEPs to induce melatonin syn-
thesis by macrophages. To that end, we assessed the activa-
tion ofthe Aanatpromoter, the expression of the AANAT
protein and the levels of the active form of the enzyme,
P-AANAT, and melatonin in the medium of RAW 264.7
macrophages or freshly isolated alveolar macrophages.
The exposure of RAW 264.7 macrophages transfected with
pDsRed2-1 associated with the promoter of the Aanat gene to
DEP led to a dose-dependent transcription of the red fluores-
cent protein (Figure 3A). The AANAT promoter constructed
in our laboratory32 contains two kappa B binding sites. Here,
we showed that PDTC (25mmol/L, 30minutes), an inhibitor
of kappa B binding to DNA, reduced the expression of red flu-
orescent protein. We then showed that DEPs induced the ex-
pression of AANAT and its active form (P-AANAT) in RAW
246.7 and alveolar macrophages, respectively, as well as the
synthesis of melatonin in both cultures (Figure 3B,C). These
results clearly show that both RAW 264.7 cells and alveolar
macrophages synthesize melatonin when challenged with die-
sel particles, as previously shown for LPS and zymosan.22,24,32
The incubation of RAW 264.7 cells with DEPs for
24hours promoted the expression of inducible nitric oxide
synthase (iNOS) by a TLR4 activation-dependent mecha-
nism, as it was blocked in a dose-dependent manner by the
TLR4 antagonist TAK-242 (10pmol/L-1 mmol/L) (Figure
4A). Blockade of the binding of NFκB to DNA with PDTC
(25mmol/L, 30minutes) or melatonin receptors with luzin-
dole (10μmol/L, 1 hour) increased necrosis, reducing cell
viability (Figure 4B,C). Upon evaluating the expression of
annexin V, we observed that only PDTC promoted apoptosis,
suggesting that melatonin protected the cells against necrosis
(Figure S2).
Using GEO microarray data from the human alveolar mac-
rophages of nonsmoking subjects (n=15, GSE 2125), we
also investigated the biological relevance of the expression
ofAANAT,MT1,and MT2 and the main enzymes involved
in cellular necroptosis (RIPK1, RIPK3, and MLKL). The
FIGURE 4 Does macrophage-synthesized melatonin negatively regulates pollution-induced necrosis? A, DEP-induced expression of the
inducible nitric oxide synthase (iNOS) is mediated by Toll-like 4 receptors (TLR4), as it was reduced by the competitive antagonist TAK242. Data
are expressed as percentage of increase related to control group (100%) (n=3 independent experiments). B and C, DEP-induced necrosis and
reduction in cell viability are inhibited by blocking melatonin receptors (luzindole 10μmol/L, 1h) or NFκB binding to DNA (PDTC, 25μmol/L,
30min), (n=3 independent experiments). Inhibitors were added 30min before DEP. D, Activation of the necroptosis signaling pathway in
nonsmokers human macrophages correlates with the expression of genes that codify for AANAT, MT1, and MT2.Necroptosis significantly
correlates with the key enzyme of the synthetic melatonin pathway and the two subtypes of melatonin receptors. Human alveolar macrophage gene
expression data (n=15) were retrieved from the public repository Gene Expression Omnibus (GSE2125).Independent variables are plotted as box
and whiskers (10%-90%), and dependent variables are plotted as mean±SEM

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significant negative correlation between the expression of
AANAT,MT1,andMT2and the genes related to programmed
necrosis (Figure 4E) strongly suggested that melatonin syn-
thesized by human alveolar macrophages also controls cell
viability. Finally, we observed that exogenous melatonin in-
creased the viability of RAW 264.7 cells and the engulfment
of DEPs by alveolar macrophages (Figure 5).
To determine whether the synthesis of melatonin in the
lung was part of the first line of defense against particulate
material present in the polluted air, we used our first experi-
mental model; rats were exposed to concentrated polluted air
and compared with those exposed to filtered air. The inhalation
of polluted air (1hour) at night induced respiratory smooth
muscle thickening, a reduction in the lumen, the invasion of
polymorph and mononuclear leukocytes (Figure 6A, left
panel), and expression of the adhesion molecules PECAM-1
and ICAM-1 and inducible nitric oxide synthase (Figure 6A,
central panel). Regarding the melatonergic system, we first
showed that the animals maintained in the chamber that receive
filtered air presented a robust rise in nocturnal melatonin. After
the first hour of exposure to polluted air, only the nocturnal
level of melatonin increased, reinforcing the conclusion that
this melatonin was produced by the pineal gland. Following
exposure to polluted air (6hours), no change in daytime mel-
atonin was detected, while as shown in Figure 1D, the plasma
melatonin level was reduced to values below that observed in
animals maintained in filtered air (Figure 6B).
In the lung, polluted air abolished the difference between
daytime (ZT6) and nighttime (ZT18) melatonin levels, which
were significantly different when animals were exposed to
filtered air. In addition, after 6hours, there was a signifi-
cant increase in the melatonin content in animals euthanized
during both the light and dark phases of the day (Figure 6B).
This pattern strongly suggests melatonin synthesis by the
lung exposed to polluted air for 6hours. To test this hypoth-
esis, the expression of the gene that encodes AANAT was
determined in the lungs of the same animals. The results con-
firmed the hypothesis, as the expression of Aanat in the lung
was significantly increased in animals exposed to polluted air
compared with those exposed to filtered air or polluted air for
shorter periods of time (Figure 6B). Thus, breathing polluted
air activates the immune-pineal axis, which concomitantly
reduces nocturnal melatonin production by the pineal gland
and increases synthesis of the indoleamine in the lung, inde-
pendent of the time of day.
Based on these in vitro data, the autocrine production of
melatonin should protect the lung against pollution. As mel-
atonin reduced hyperbaric oxygen-induced lung lipid peroxi-
dation43 and that induced by zymosan,44,45, we next evaluated
whether lung-synthesized melatonin regulates the activity of
antioxidant enzymes. Exposure to polluted air enhanced the
gene expression and enzymatic activities of superoxide dis-
mutase (SOD), catalase (CAT), and glutathione peroxidase
(Figure 7A). Although the temporal gene transcription pro-
files differed after exposure to polluted air for 6hours, the
activities of the three enzymes were significantly higher in
animals exposed to polluted air than in animals exposed to
filtered air. The inhibition of melatonin receptors with luzin-
dole reduced the expression of SOD and CAT and increased
lipid peroxidation, indicating that the activation of melatonin
FIGURE 5 Melatonin induces increase in macrophage viability, enhancing its capacity to phagocyte diesel exhausted particles (DEP). A,
Melatonin (0.1-100nmol/L) prevented DEP-induced reduction in cell viability (RAW 264.7 cells; 24h exposition) in a dose-dependent manner;
(n=30-45 cells from 3 independent experiments); B, Representative images of confocal microscopy of rat alveolar macrophages incubated with
vehicle (VEH) or DEP (128μg/mL, 1 or 2h). Melatonin (10nmol/L, 30min) potentiated phagocytosis of DEP by alveolar macrophages. Curves
were fitted to the hyperbolic quadratic equation by the least square regression method and compared by extra-sum-of-squares F test. DEP particles
inside alveolar macrophages were quantified by Image J. Bars—20μm; data shown as mean±SEM, n=3 independent experiments

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CARVALHO-SOUSA et AL.
receptors by lung-synthesized melatonin is protective against
polluted air (Figure 7A). The time course over which the
mRNA levels of SOD changed showed that the effect of air
pollution was transient and occurred after 1hour. Luzindole
treatment after 6hours of exposure to air pollution caused
a dose-dependent reduction in SOD mRNA levels, strongly
suggesting that melatonin plays a role in the upregulation
of SOD Accordingly, in the human lung, the expression
ofAANATandMT1, but notMT2,was positively correlated
with the expression of the following enzymes involved in an-
tioxidant defense: SOD, CAT, peroxiredoxins, thioredoxins,
and GPx (Figure 7B and Table S2).
4
|
DISCUSSION
Collectively, these data provide a systemic and mechanis-
tic rationale for coordination of the pineal and extrapineal
synthesis of melatonin by a common damage-associated
stimulus in a megalopolis. It is well known that air pollution
induces lung and systemic inflammatory responses.5,46,47 We
confirm that acute exposure to particulate matter (2-6hours)
induces an increase in circulating proinflammatory cy-
tokines12 and show a transient increase in the transcription of
proinflammatory genes and production of proinflammatory
proteins, including TNF, in the pineal gland for the first time.
Because the environment in the fine particle concentra-
tor chamber could stress the animals and because adrenal
corticosterone potentiates the synthesis of melatonin,22,38,42
we measured the blood concentrations of corticosterone and
melatonin over 6hours after the animals were introduced to
the exposure chambers filled with filtered or polluted air to
validate the present model. This new environment, indepen-
dent of the presence of polluted air, induced transient corti-
costerone (30minutes) and melatonin (1hour) plasma peaks,
reinforcing the idea that corticosterone at the concentration
FIGURE 6 Lung damage induced by particulate matter 2.5μm (PM2.5) is attenuated by melatonin synthesized in the lung. (A, left panel)
Inhalation of particulate material (1h) induced an inflammatory process in the lung. Hematoxylin-eosin photomicrograph showing a reduction
in the lumen of a bronchiole. Bars=20μm. (A, central & right panels) Immunohistochemistry of pulmonary veins showing the expression
of the adhesion molecules PECAM-1 and ICAM-1 and the enzyme, induced nitric oxide synthase (iNOS). Data were quantified by ImageJ.
Bar=20μm; n=5 lungs; P<.05 as compared to filtered air. B, Plasma and lung melatonin content and transcription of the Aanat gene from
lungs of animals exposed to polluted air for 1-6h. Light (L)/Dark (D) melatonin content was determined in animals exposed to filtered or polluted
air at ZT6 and ZT18, respectively. Melatonin was detected by commercial ELISA assay and Aanat gene by qPCR. n=10-19 animals from two
different experiments; Bar=20μm; *blue and red brackets indicates significant difference between light/dark phase or filtered and polluted air,
respectively—P<.05)

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produced under mild stress potentiates pineal melatonin syn-
thesis.41,42,48 This transient melatonin response to stress was
not observed in animals kept in their cages outside the ap-
paratus or exposed to pollution during the light phase of the
day. Thus, this effect was not induced by air pollution. When
animals that were exposed to polluted versus filtered air were
compared, a significant reduction in plasma melatonin was
detected after 4hours in a polluted environment. Other au-
thors that maintained animals under a polluted atmosphere
for ten months confirmed a reduction in circulating mela-
tonin after this long-term exposure.47 Thus, as expected for
an acute inflammatory response,21 a reduction in pineal mel-
atonin synthesis was observed already during the first night.
Here, we confirmed that DEPs triggers TLR4,43,45,49,50
and the NFκB pathway in both pineal microglia and alve-
olar macrophages. As previously observed following LPS
exposure,20 the DEP-induced blockade of microglial TNF
production triggered TNFR1 in pinealocytes, blocking pi-
neal melatonin synthesis. Moreover, consistent with a pattern
already detected in bacteria, fungi, and parasites,24,51 DEP-
mediated activation of the TLR4/ NFκB pathway led to the
synthesis of melatonin in alveolar macrophages, which were
shown to develop an inflammatory response.43,50 The activa-
tion of NFκB is a common step for alteration of melatonin
synthesis in pinealocytes and macrophages.18 Here, polluted
air-induced NFκB nuclear translocation decreased the ex-
pression of Aanat in pinealocytes, while transcription of the
same gene was induced in macrophages and the lung.
For the first time since we proposed the theory of the im-
mune-pineal axis,52 we had the opportunity to explore both
arms of the immune-pineal axis simultaneously. Interestingly,
the reduction in plasma melatonin and increase in lung mel-
atonin followed a similar time course, reinforcing the idea
that the source of melatonin and its function alternate from
pineal gland/ darkness hormone to macrophages/cell protec-
tion. This effect is mediated by increasing the transcription
of genes and the expression of proteins involved in regulating
oxidative stress (superoxide dismutase, catalase, and gluta-
thione peroxidase). The synthesis of melatonin by the lungs
probably facilitates the clearance of particulate material by
alveolar macrophages, which is expelled by coughing, and
a reduction in pulmonary lipid peroxidation to avoid the es-
tablishment of a proinflammatory response. Interestingly,
the short-term exposure of children to polluted air in China
FIGURE 7 Melatonin receptors mediate the protective effect of endogenous melatonin against polluted air. A, Effect of air pollution on the
expression and activity of enzymes involved in reducing oxygen free radicals in the lung—superoxide dismutase (SOD; red), catalase (CAT; pink),
and glutathione peroxidase (GPx; green); (n=4-7 lungs, *P<.005). Effect of luzindole (ip) on the expression of the enzymes SOD (red), CAT
(pink), GPx (green), and lipid peroxidation (gray) in the lungs from animals exposed to polluted air. Luzindole was injected at ZT 11, and animals
were killed at ZT18. (n=5-6 lungs; *P<.001). B, Relationship between the activation of the cellular antioxidant system and the expression of
AANAT, MT1, or MT2 in human lung samples. Enrichment score for antioxidant genes significantly correlated with AANAT and MT1, but not
with MT2 melatonin receptor, suggesting that lung-synthesized melatonin increased the activity of antioxidant pathways via activation of MT1
receptors. Lung gene expression data (n=288) were obtained from the GTEx database. Genes were ranked according to the Spearman's correlation
coefficient between their expression and the expression of AANAT, MT1, or MT2. Normalized enrichment scores and P-values were calculated
using GSEA v3.0 and the curated gene set of enzymes involved in the cellular antioxidant system

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CARVALHO-SOUSA et AL.
was shown to confer protection against allergy and rhinitis,53
strongly suggesting that the lungs are protected against proin-
flammatory responses. Notably, other tissues considered to
be barriers to environmental dangers, such as the skin25 and
gut,54 also synthesize their melatonin in a both constitutive
and inductive manner.
Finally, we evaluated whether similar melatonin synthesis
activation could occur in the human lung. By using a bio-
informatic approach, we show the enrichment of alveolar
macrophages and lung tissues for AANAT, MT1, and MT2
gene expression, which differed according to the grade of
necroptosis in macrophages (GSE2125) or the transcription
of genes linked to oxidative stress in the whole lung (GTEx).
The process of necroptosis was negatively correlated with
enrichment in these three genes, while the transcription of
genes that encode proteins involved in antioxidant processes
was positively correlated with the AANAT and MT1 genes,
but not the MT2 gene. Therefore, we infer that the activity of
melatonin at both melatonin receptor subtypes would reduce
alveolar macrophage death, increasing the probability that
particulate matter is retained in the lung or even expelled by
coughing. In contrast, protection of the lung against oxidative
stress was positively correlated with only the MT1 receptors.
As mentioned above, melatonin synthesis by lungs exposed
to air pollution may be a protective factor against not only a
polluted environment but also other lung pathologies based
on the reduction in defense cells or oxidative stress.55
In summary, we explored both arms of the immune-pi-
neal axis in the same animals and have provided experimental
support to understand the relevance of the coordinated switch
of the melatonin function from a timing-related hormone to
a local anti-inflammatory mediator, which accelerates the in-
flammatory response toward a regulatory phase.
AUTHOR CONTRIBUTIONS
CEC-S and EPP equally contributed to the manuscript that
is a result of their PhD thesis. They contributed to the design
and implementation of the research, executed and analyzed
experiments CEC-S showed that the immune-pineal axis
is activated by air-pollution and diesel particles (in vitro),
and EPP determined the mechanism of action of lung-syn-
thesized melatonin; GSK contributed to the design and the
analysis of the data with human data banks; MMV contrib-
uted to acquisition and analyses of the data regarding lung
inflammatory response; ZSF contributed to hormonal dos-
age and manuscript; PHNS expert in air pollution, helped
to design the experiments,allowed the use of the Harvard
Ambient Fine Particle Concentrator, and participated in the
final discussion of the manuscript; RJR discussed the data
and reviewed the manuscript; PAF contributed to the imple-
mentation of the research, following each of the steps, exe-
cuted experiments, performed analysis, and wrote the paper;
SSCM contributed to collection of data, both “in vivo” and
“in vitro,” coordinated the final form of the figures, and col-
laborated in the writing of the manuscript; and RPM de-
vised the project, the main conceptual ideas, and provided
the grants; designed the Experiments and analyzed the data;
and wrote the manuscript.
ORCID
Gabriela S. Kinker https://orcid.
org/0000-0003-1676-442X
Mariana Veras https://orcid.org/0000-0002-8363-4329
Zulma S. Ferreira https://orcid.
org/0000-0001-6571-837X
Joilson O. Martins https://orcid.
org/0000-0003-2630-7038
Paulo H.N. Saldiva https://orcid.
org/0000-0003-2005-8253
Russel J. Reiter https://orcid.org/0000-0001-6763-4225
Pedro A. Fernandes https://orcid.
org/0000-0002-4871-8201
Sanseray da Silveira Cruz-Machado https://orcid.
org/0000-0002-6937-7207
Regina P. Markus https://orcid.
org/0000-0003-4606-6120
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SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section.
How to cite this article: Carvalho-Sousa CE, Pereira
EP, Kinker GS, et al. Immune-pineal axis protects rat
lungs exposed to polluted air. J Pineal Res.
2020;00:e12636. https://doi.org/10.1111/jpi.12636