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The neonicotinoid insecticide
Clothianidin adversely aects
immune signaling in a human cell
line
Gennaro Di Prisco, Marco Iannaccone, Flora Ianniello, Rosalba Ferrara, Emilio Caprio ,
Francesco Pennacchio & Rosanna Capparelli
Clothianidin is a widely used neonicotinoid insecticide, which is a potent agonist of the nicotinic
acetylcholine receptor in insects. This neurotoxic compound has a negative impact on insect immunity,
as it down-regulates the activation of the transcription factor NF-κB. Given the evolutionary conserved
role of NF-κB in the modulation of the immune response in the animal kingdom, here we want to assess
any eect of Clothianidin on vertebrate defense barriers. In presence of this neonicotinoid insecticide, a
pro-inammatory challenge with LPS on the human monocytic cell line THP-1 results both in a reduced
production of the cytokine TNF-α and in a down-regulation of a reporter gene under control of NF-κB
promoter. This nding is corroborated by a signicant impact of Clothianidin on the transcription levels
of dierent immune genes, characterized by a core disruption of TRAF4 and TRAF6 that negatively
inuences NF-κB signaling. Moreover, exposure to Clothianidin concurrently induces a remarkable up-
regulation of NGFR, which supports the occurrence of functional ties between the immune and nervous
systems. These results suggest a potential risk of immunotoxicity that neonicotinoids may have on
vertebrates, which needs to be carefully assessed at the organism level.
Neonicotinoids are among the most widely used insecticides in agriculture, which are eective at low dosage
and show poor anity for the nicotinic acetylcholine receptor of mammalian species1,2. e limited impact on
non-target higher animals is, however, challenged by a growing number of studies, which support a negative
eect of these systemic and persistent insecticides on several non-target organisms and ecosystem services3,4. In
particular, pollinators seem to be particularly aected. Indeed, in spite of the fact that acute lethal eects are rarely
observed5, there are a number of reports on sub-lethal eects, such as impaired honeybee learning or homing
behavior6–8, and a stronger impact on pollinators of various pathogens9–13. is latter eect is in part due to the
immunosuppressive action exerted by neonicotinoids14,15 which further exacerbates the negative impact that viral
pathogens and Varroa destructor have on honeybee defense barriers16–19.
e molecular mechanism underlying the negative eect of the neonicotinoid Clothianidin on insect immune
response has been recently reported14. Basically, this insecticide is able to exert a negative eect on the activation
of the nuclear factor-κB (NF-κB) and of the downstream immune barriers, which promotes uncontrolled viral
replication in honeybees bearing covert infections14. Moreover, other immune responses controlled by this tran-
scription factor, both cellular and humoral, are down-regulated by neonicotinoids15, suggesting the occurrence of
a wider impact of these insecticides on immunity.
NF-κB has a central role in the immune response by animals20, and, therefore, any defense pathway, conserved
across distant evolutionary lineages, under control of this transcription factor could be inuenced by a shared
negative regulation of its activation. is could account for the proposed link between the use of neonicotinoids
and the increasing incidence of pathologies in dierent animal groups4,19. It does not require a leap of imagination
to speculate that neonicotinoids may have possible negative eects on human health, by similarly interfering with
the regulation of the immune system. is is a hypothesis that certainly merits to be investigated, as part of a more
Department of Agricultural Sciences, University of Napoli “Federico II” – Via Università 100, 80055 Portici, Napoli,
Italy. Gennaro Di Prisco and Marco Iannaccone contributed equally to this work. Correspondence and requests for
materials should be addressed to F.P. (email: f.pennacchio@unina.it) or R.C. (email: capparel@unina.it)
Received: 6 July 2017
Accepted: 18 September 2017
Published: xx xx xxxx
OPEN
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Scientific REPORTs | 7: 13446 | DOI:10.1038/s41598-017-13171-z
comprehensive eort towards a thorough characterization of neonicotinoid impact on human health, which,
surprisingly, is still in its infancy21,22.
Here we contribute to ll this gap of knowledge, by focusing our attention on the impact of the neonicotinoid
Clothianidin on the human immune response, using an in vitro model system to characterize the eects that this
molecule has on gene expression prole upon immune challenge. is has been done by RNA sequencing in
the monocytic human cell line THP-1, as aected by Clothianidin exposure, and by studying how this latter can
inuence pro-inammatory cytokine release upon immune challenge.
Results
Clothianidin disrupts NF-κB signaling. Insect immune response is negatively modulated by
Clothianidin, which disrupts NF-κB signaling by up-regulating a negative modulator of this transcription factor14.
Because NF-κB signaling underpins the modulation of several immune reactions in animals, we wanted to assess
if this alteration induced by Clothianidin occurred also in humans. To test this hypothesis, we focused our atten-
tion on the eect of Clothianidin on the expression prole of the Tumor Necrosis Factor Alpha (TNF-α), a
pro-inammatory cytokine regulated by NF-κB via TLR-423, both at transcriptional and translational level, using
an immune cell line (THP-1), which expresses the nicotinic acetylcholine receptor24. Our data clearly indicate that
exposure to Clothianidin disrupts the LPS-mediated induction of TNF-α expression, both in terms of transcript
level (Fig.1a) (One-Way ANOVA: F = 148.09; df = 3; p < 0.001) and protein production (Fig.1b) (One-Way
ANOVA: F = 183.61; df = 3; p < 0.001). e experimental concentration of Clothianidin used (100 ng/ml) did not
have any cytotoxic eect on THP1 cells, as demonstrated by lactate dehydrogenase (LDH) release across a range
of dierent doses of this insecticide (Fig.S1). Collectively, these results demonstrate that Clothianidin inhibits
TNF-α expression, which is under NF-κB control.
To unequivocally demonstrate that Clothianidin exposure interferes with NF-κB activation, we stably trans-
fected the THP-1 cell line with lentiviral particles carrying a NF-κB-responsive luciferase-expressing reporter
gene (CignalLentiReporters, SABiosciences). e cells were incubated overnight, in presence or absence of
Clothianidin, at the same concentration indicated above, and then treated with LPS or le unchallenged. When
LPS challenge was performed in presence of Clothianidin, a signicant (One-Way ANOVA: F = 137.09; df = 3;
p < 0.001) inhibition of LPS-induced enhancement of the reporter gene expression was observed, indicating the
occurrence of a negative eect of this neonicotinoid insecticide on NF-κB signaling (Fig.2).
Clothianidin alters the transcriptome of an immune cell line. In order to identify the molecular
networks underlying the inhibition of NF-κB activation induced by Clothianidin, we performed a transcriptomic
analysis by RNA-Seq of the human THP-1 cell line exposed overnight to this neonicotinoid, at a concentration
of 100 ng/ml, the same used in the experiments described above. Aer trimming and quality control of the
obtained sequences, about 97% resulted as high quality reads (Supplementary TableS1), and were mapped against
Homo sapiens reference genome (Ensembl, GRCh38). Principal component analysis (PCA) was applied to the
dataset showing two distinct clusters, conrming replicate uniformity (Supplementary FigureS2). Dierential
expression analysis by false discovery rate (FDR) (P < 0.05) showed that 2,833 and 2,678 genes were signi-
cantly up- and down-regulated, respectively, in Clothianidin treated cells (Supplementary FigureS3a,b). To
select the most dierentially expressed genes, we applied a more stringent lter for Log2 fold change of >+1
or <−1, which allowed the identication of 36 genes up-regulated and 54 down-regulated; both categories
included immune genes under NF-κB transcriptional control, such as TNF receptor-associated factor 4 (TRAF4),
TNF receptor-associated factor 6 (TRAF6), Fork head box protein O4 (FOXO4), Interleukin-18-binding pro-
tein(IL18BP) and Interleukin-17 receptor (IL17R) (Fig.3). The concurrent up-regulation of the negative
Figure 1. Clothianidin inhibits TNF-α expression induced by LPS treatment. In human THP-1 cells TNF-α
transcription rate was measured by qRT-PCR (a) and TNF-α secreted protein in cell free supernatant by ELISA
(b), aer overnight incubation with Clothianidin (100 ng/ml), followed by LPS stimulation for 1 h (1 µg/ml), and
compared with values obtained in untreated cells or exposed to Clothianindin but le unchallenged. Data are
reported as mean ± SEM and are representative of 3 independent experiments, with 3 replicates each (One-Way
ANOVA, all p < 0.05).
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Scientific REPORTs | 7: 13446 | DOI:10.1038/s41598-017-13171-z
modulator of NF-κB activation TRAF4 and the down-regulation ofTRAF6, exerting an opposite activity, well
account for the reduced expression of genes controlled by this transcription factor. e entire collection of raw
data is available on public database with BioProject Number PRJNA392257 (National Center of Biotechnology
Information, U.S. National Library).
To analyze the putative interactions among proteins encoded by genes found to be dierentially expressed and,
then, of particular functional importance to face the stress induced by Clothianidin exposure, we used the String
soware25. is approach allowed the identication of a network of interactions among nerve growth factor
receptor (NGFR), TRAF4 and TRAF6, which indicate how the core alteration of NF-κB signaling in the immune
cell line THP-1 induced by Clothianidin is associated with a transcriptional change related to neural functions.
To validate the RNA-Seq analysis, the expression of the immune-related genes, found to be markedly
modulated, was further assessed in an independent qRT-PCR experiment, where THP-1 cells were treated
with Clothianidin overnight, at the same concentration used for RNA-Seq analysis. TRAF4 and NGFR were
signicantly up-regulated (Student’s t test: TRAF4, t = −17.064, df = 4, P < 0.001; NGFR, t = −12.275, df = 2,
P = 0.007), while TRAF6, FOXO4, IL18BP and IL17R were down-regulated, as expected on the basis of RNA-Seq
analysis (Student’s t test: TRAF6, t = 5.438, df = 4, P = 0.006; FOXO4,t = 5.444, df = 4, P = 0.006; IL18BPt = 7.995,
df = 4, P = 0.001; IL17R,t = 8.976, df = 4, P = 0.001) (Fig.4).
Collectively, these results allow to conclude that exposure to Clothianidin of the human cell line THP-1
determines a negative modulation of NF-κB signaling, associated with an up-regulation of TRAF4 and a
down-regulation of TRAF6, a negative and a positive modulator, respectively, of NF-κB activation, which partly
account for the observed immunosuppressive eects.
Discussion
In this study we demonstrate that human THP-1 cells treated with the neonicotinoid insecticide Clothianidin
react to an inammatory stimulus by showing a lower expression of the cytokine TNF-α, due to a reduced activa-
tion of NF-κB, which controls its transcription. e negative impact of this neonicotinoid on NF-κB signaling has
been recently reported in insects, and thought to be one of the stress elements that can contribute to the reduced
ecacy of antiviral immune barriers controlling DWV replication in honeybees bearing covert infections of this
viral pathogen14. e observed similar eects of Clothianidin on immune responses by cells of organisms in phy-
logenetically distant lineages indicate the occurrence of conserved mechanisms of cross-modulation between the
nervous and immune system26–28. e nervous and the immune systems are traditionally thought to be separate
functional entities and, as such, are separately studied. However, it is increasingly evident that this is not the case
and their intimate interaction is a fascinating research area that continuously generates novel information on the
subtle mechanisms involved28 and on their wide occurrence in the animal kingdom27. is conceptual framework
nicely accounts for the observed immunomodulation by the acetylcholine agonist Clothianidin, even though the
underlying molecular network that modulates this response remain still largely unexplored14. ese conserved
pathways of neuroimmune regulation and the fact that Clothianidin binds, even though with much lower anity
than in insects, to the human α4β2nicotinic acetylcholine receptor (α4β2AchR)29 were the major elements stim-
ulating the present study, aiming to discover any immunotoxic eect that neonicotinoids may have on vertebrates.
Using STRING software, we highlighted a tight interaction between TRAF4, TRAF6 and the NGFR
(Supplementary FigureS4). A previous study has reported that co-expression of NGFR with TRAF6 enhances
expression of NF-κB, while TRAF4 negatively interferes with this process30. is further reinforces the tight
relation between the nervous and immune systems. Indeed, the NGF in vertebrates can act as a homologue of the
y Toll ligand Spaetzle in eliciting immune reactions31, and its perception by THP-1 cells seems to be inuenced
Figure 2. Clothianidin inhibits the expression of a NF-κB responsive reporter gene. NF-κB induction as
aected by insecticide exposure was measured in human THP-1 cells, using a NF-κB luciferase reporter
aer incubation with Clothianidin overnight (100 ng/ml), followed by LPS stimulation for 1 h (1 µg/ml), and
compared with values obtained in untreated cells or exposed to Clothianidin but le unchallenged. Data are
reported as mean ± SEM and are representative of 3 independent experiments, with 5 replicates each (One-Way
ANOVA, all p < 0.05).
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Scientific REPORTs | 7: 13446 | DOI:10.1038/s41598-017-13171-z
by the acetylcholine agonist Clothianidin, which enhances the transcription of NGFR. en, the observed tran-
scriptional regulation may potentially inuence cross-communication between the nervous and the immune
systems28.
Our experimental data on THP-1 cells clearly indicate that exposure to Clothianidin is detrimental for the
expression of genes under NF-κB control, as similarly observed in insects14. Indeed, both the RNA-Seq and
qRT-PCR data revealed marked eects of Clothianidin exposure on the expression of genes linked to immune
response. In particular, TRAF4 and TRAF6, which are members of the TRAF protein family, largely associated
with the immune response32, resulted up-regulated and down-regulated, respectively. ese proteins are involved
Figure 3. List of signicantly (FDR <0.05) up- and down-regulated genes with Log2 fold change higher than 1
and lower than −1, respectively.
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Scientific REPORTs | 7: 13446 | DOI:10.1038/s41598-017-13171-z
in a number of transduction pathways of immune signaling molecules, with TRAF6 promoting NF-κB activation,
while TRAF4 is a negative modulator of this transcription factor, as it competes for its binding sites on signal
transduction proteins recruiting TRAF633. e concurrent transcriptional down-regulation of IL17R induced by
Clothianidin, associated with the TRAF6 and TRAF4 changes mentioned above, suggest that IL17 immune sign-
aling is negatively inuenced by neonicotinoids in THP-1 cells. Moreover, we can assume the occurrence of sim-
ilar immune disruption pathways, largely driven by the same mechanism, that can be further aggravated by the
down-regulation of proteins involved in NF-κB activation, such as IL18BP34. In contrast, the down-regulation of
FOXO4 is not easy to interpret. is transcription factor is a member of the FOXO protein family, which is central
in the integration of growth factor signaling, oxidative stress and inammation35. Recent work has demonstrated
that knockdown of FOXO4 does not aect NF-κB activation, suggesting that FOXO4 acts downstream in the
signaling pathway36. en, its down-regulation by Clothianidin treatment could further reinforce the inhibition
of NF-κB activation. is is a likely hypothesis that merits to be investigated. Future studies will have to address
these intriguing neuroimmune pathways, which are poorly known, in particular in non-vertebrate model sys-
tems, in order to fully appreciate the whole impact of neurotoxic molecules on the immune system. is is an
important research area that is currently not adequately considered in toxicological studies.
In conclusion, our data show that the insecticide Clothianidin negatively inuences in a human cell line the
expression of immune related genes, under control of the transcription factorNF-κB, as similarly observed in
Figure 4. Relative gene expression in THP-1 cell treated with Clothianidin: NGFR (a); TRAF4 (b); TRAF6 (c);
FOXO4 (d); IL18BP (e); IL17R (f). Data are reported as a mean ± SEM of 3 independent experiments, with 5
replicates each. (Student’s t test, all p < 0.05).
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Scientific REPORTs | 7: 13446 | DOI:10.1038/s41598-017-13171-z
insects. ese ndings are interesting, but their relevance will have to be assessed at the organism level to see if
they may represent a signicant threat for human health.
Methods
Cell culture. e human monocytic cell line THP-1 (ATCC number: TIB-202; LGC Standards GmbH) was
cultured in RPMI 1640 (Life technologies) medium supplemented with 10% FCS (Life technologies) and 1 mM of
L-glutamine (Life technologies), under 5% CO2 at 37 °C.
Total RNA extraction and cDNA Synthesis. Total RNA extraction from THP- 1 cells was carried out
by using TRIzol (ermoScientic), according to the manufacturer’s instructions. e RNA yield and A260/280
ratio were monitored with a NanoDrop ND 100 spectrometer (NanoDrop Technologies), and RNA integrity was
veried using the 2100 Bioanalyzer (Agilent Technologies). cDNA synthesis was carried out starting from 1 μg
of total RNA and using the High Capacity cDNA Reverse Transcription Kit(ermoScientic), according to the
manufacturer’s protocol.
RNA-Seq. An amount of 106/well cells (control and Clothianidin treated, 3 biological replicates each) were
processed for RNA-Seq analysis. Indexed libraries were prepared using 1 µg of each RNA puried with TruSeq
Stranded mRNA Sample Prep Kit (Illumina), according to the manufacturer’s instructions. Libraries were quanti-
ed using the Agilent 2100 Bioanalyzer (Agilent Technologies) and pooled, so that each index-tagged sample was
present in equimolar amounts, with a nal concentration of the pooled samples of 2 nM. e pooled samples were
subjected to cluster generation and sequencing, using an Illumina HiSeq. 2500 System (Illumina) in a 2 × 100
paired-end format, at a nal concentration of 8 pmol. e raw sequence les generated (.fastq les) underwent
quality control analysis, using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Raw reads were checked for quality with FastQC v0.11.3 and then trimming and removal of adapters were
performed with Trimmomatic v0.33 (minimum quality score 25, minimum length 35). e obtained reads were
then mapped against Homo sapiens reference genome (Ensembl, GRCh38) with STAR (v2.5.0b), providing the
reference gene annotation le with known transcripts. FeatureCounts (v1.4.5-p1) was used to perform read sum-
marization at gene level, with the strand-specic option “reversely stranded”. Statistical analysis of the read counts
was performed with R, using the HTSFilter package, to remove low expressed genes, and the NOIseq package, to
perform dierential expression analysis. Gene Ontology enrichment analysis of the dierentially expressed genes
was performed with the GOStat package.
qRT-PCR. e expression prole of the immune genes that showed the most pronounced transcriptional
regulation was also analyzed by TaqManqRT-PCR, using specific primers and probes:TNF-α (AssayID:
Hs00174128_m1), FOXO4 (Assay ID: Hs00172973_m1), NGFR (Assay ID: Hs00609976_m1), IL18BP (Assay ID:
Hs00271720_m1), TRAF4 (Assay ID: Hs01030628_g1), TRAF6 (Assay ID: Hs00939742_g1) and IL17R (Assay
ID: Hs01056316_m1). All probes were normalized to Gapdh (Assay ID: Hs02786624_g1) as internal control
(Applied Biosystems). All fold changes were calculated using the ΔΔCt method (Livack et al., 2001) and com-
pared with untreated cell. Amplications were performed with ABI PRISM 7900HT (Applied Biosystems).
Clothianidin eect on TNF-α expression. Clothianidin was obtained from Sigma (Cat No: 33589) and
used as follows: 106/well cells were seeded in a 24 well plate and were pre-treated with Clothianidin (100 ng/
ml) overnight, then, stimulated for 1 hour with LPS 1 μg/ml (Sigma), and compared with unchallenged cells and
untreated basal controls. Experimental cells were washed in PBS before RNA extraction. A qRT-PCR analysis was
performed to measure the transcription rate of TNF-α gene in THP-1 cell treated as described above. TNF-α pro-
tein secretion was measured in cell free supernatant using TNF-α DuoSet ELISA development kit (R&D system),
following manufacturer’s procedure.
NF-κB reporter gene assay. THP-1 cells were infected with 10 µL lentiviral particles carrying a NF-κB
responsive luciferase-expressing reporter gene (CignalLenti Reporters, SABiosciences), according to the protocol
provided by the manufacturer, followed by selection with puromycin. Once the cell line was established, THP-1
were incubated with Clothianidin (100 ng/ml) overnight and then treated with LPS (1 μg/ml) for 1 hour. NF-κB
activity was measured using Dual Glo Luciferase assay (Promega), according to the manufacturer’s procedure.
Luciferase activity was normalized for all samples with total amount of proteins.
Cytotoxicity assay. THP-1 cell were treated overnight with dierent dose of Clothianidin and cytotoxic-
ity was evaluated by measuring lactate dehydrogenase (LDH) release in the supernatant, using a CytoTox 96®
Non-Radio cytotoxicity assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions.
Statistical analysis. Normality of data was checked with Shapiro-Wilk test, while homoscedasticity was
tested with Levene’s procedure. Dierences in the relative expression of TNF-α, secreted TNF-α protein and
Luciferase activity of the NF-κB responsive reporter gene were analyzed with One-Way ANOVA followed by
Games-Howell post-hoc test (parametric and non-homoscedastic procedure).
Two-tailed parametric non-homoscedastict-test was used to analyze dierences in relative expression of
NGFR, while for TRAF4, TRAF6, FOXO4, IL18BP and IL17R gene expression was analyzed with the two-tailed
parametric homoscedastic t-test. Dierences in the LDH amounts released by cells exposed to Clothianidin were
analysed by One-Way ANOVA, followed by LSD post-hoc test (parametric and homoscedastic procedure). ese
analyses were performed by using Prism v.5 for Mac OSX (GraphPad soware, San Diego, CA, USA). All statisti-
cal data are available in the Supplementary Table2.
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Scientific REPORTs | 7: 13446 | DOI:10.1038/s41598-017-13171-z
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Acknowledgements
e research work reported in this paper was supported by POR Campania FESR 2007–2013, Bio Industrial
Processes-BIP (to FP and RC) and by EU Seventh Framework Program (FP7/2007–2013), under Grant 613960
(SMARTBEES) (to FP). Dr. Marco Iannaccone was supported by research funding from Fondazione con il Sud
(Project no. 2011-PDR-18, ‘Biosensori piezoelettrici a risposta in tempo reale per applicazioni ambientali e agro-
alimentari’).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
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8
Scientific REPORTs | 7: 13446 | DOI:10.1038/s41598-017-13171-z
Author Contributions
R.C. and F.P. conceived the study and designed the research plan; G.D.P., M.I., F.I., R.F. and E.C. performed the
experiments; G.D.P. and M.I. analyzed data; F.P. and R.C. wrote the paper, which was revised and approved by all
co-authors.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-13171-z.
Competing Interests: e authors declare that they have no competing interests.
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