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The neonicotinoid insecticide Clothianidin adversely affects immune signaling in a human cell line

  • IPSP-CNR Institute for Sustainable Plant Protection, National Research Council - Piazzale Enrico Fermi 1, 80055 Portici, Naples, Italy

Abstract and Figures

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 effect of Clothianidin on vertebrate defense barriers. In presence of this neonicotinoid insecticide, a pro-inflammatory 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 finding is corroborated by a significant impact of Clothianidin on the transcription levels of different immune genes, characterized by a core disruption of TRAF4 and TRAF6 that negatively influences 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.
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Scientific REPORTs | 7: 13446 | DOI:10.1038/s41598-017-13171-z
The neonicotinoid insecticide
Clothianidin adversely aects
immune signaling in a human cell
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 eect of Clothianidin on vertebrate defense barriers. In presence of this neonicotinoid insecticide, a
pro-inammatory 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 signicant impact of Clothianidin on the transcription levels
of dierent immune genes, characterized by a core disruption of TRAF4 and TRAF6 that negatively
inuences 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 eective at low dosage
and show poor anity 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
eect of these systemic and persistent insecticides on several non-target organisms and ecosystem services3,4. In
particular, pollinators seem to be particularly aected. Indeed, in spite of the fact that acute lethal eects are rarely
observed5, there are a number of reports on sub-lethal eects, such as impaired honeybee learning or homing
behavior68, and a stronger impact on pollinators of various pathogens913. is latter eect 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 barriers1619.
e molecular mechanism underlying the negative eect of the neonicotinoid Clothianidin on insect immune
response has been recently reported14. Basically, this insecticide is able to exert a negative eect 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 inuenced 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 dierent animal groups4,19. It does not require a leap of imagination
to speculate that neonicotinoids may have possible negative eects 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: or R.C. (email:
Received: 6 July 2017
Accepted: 18 September 2017
Published: xx xx xxxx
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Scientific REPORTs | 7: 13446 | DOI:10.1038/s41598-017-13171-z
comprehensive eort 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 eects that this
molecule has on gene expression prole upon immune challenge. is has been done by RNA sequencing in
the monocytic human cell line THP-1, as aected by Clothianidin exposure, and by studying how this latter can
inuence pro-inammatory cytokine release upon immune challenge.
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 eect of Clothianidin on the expression prole of the Tumor Necrosis Factor Alpha (TNF-α), a
pro-inammatory 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 eect on THP1 cells, as demonstrated by lactate dehydrogenase (LDH) release across a range
of dierent 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 signicant (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 eect 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. Aer trimming and quality control of the
obtained sequences, about 97% resulted as high quality reads (Supplementary TableS1), and were mapped against
Homo sapiens reference genome (Ensembl, GRCh38). Principal component analysis (PCA) was applied to the
dataset showing two distinct clusters, conrming replicate uniformity (Supplementary FigureS2). Dierential
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 FigureS3a,b). To
select the most dierentially expressed genes, we applied a more stringent lter for Log2 fold change of >+1
or <−1, which allowed the identication 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), aer 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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modulator of NF-κB activation TRAF4 and the down-regulation ofTRAF6, 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 dierentially expressed and,
then, of particular functional importance to face the stress induced by Clothianidin exposure, we used the String
soware25. is approach allowed the identication 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
signicantly 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 (Students 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 eects.
In this study we demonstrate that human THP-1 cells treated with the neonicotinoid insecticide Clothianidin
react to an inammatory 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
ecacy of antiviral immune barriers controlling DWV replication in honeybees bearing covert infections of this
viral pathogen14. e observed similar eects 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 system2628. 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 anity
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 eect that neonicotinoids may have on vertebrates.
Using STRING software, we highlighted a tight interaction between TRAF4, TRAF6 and the NGFR
(Supplementary FigureS4). 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 inuenced
Figure 2. Clothianidin inhibits the expression of a NF-κB responsive reporter gene. NF-κB induction as
aected by insecticide exposure was measured in human THP-1 cells, using a NF-κB luciferase reporter
aer 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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by the acetylcholine agonist Clothianidin, which enhances the transcription of NGFR. en, the observed tran-
scriptional regulation may potentially inuence cross-communication between the nervous and the immune
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 eects 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 signicantly (FDR <0.05) up- and down-regulated genes with Log2 fold change higher than 1
and lower than 1, respectively.
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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 inuenced 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 inammation35. Recent work has demonstrated
that knockdown of FOXO4 does not aect 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 inuences 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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insects. ese ndings are interesting, but their relevance will have to be assessed at the organism level to see if
they may represent a signicant threat for human health.
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 (ermoScientic), 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
veried 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(ermoScientic), 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 puried 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 (
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-specic 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 dierential expression analysis. Gene Ontology enrichment analysis of the dierentially expressed genes
was performed with the GOStat package.
qRT-PCR. e expression prole 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. Amplications were performed with ABI PRISM 7900HT (Applied Biosystems).
Clothianidin eect 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 manufacturers 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 dierent 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. Dierences 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 dierences 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. Dierences 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 soware, San Diego, CA, USA). All statisti-
cal data are available in the Supplementary Table2.
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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-
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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
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... Oxidative stress is known to modulate the generation of inflammatory cytokines through activation of the NF-κB signaling pathway [15]. Proinflammatory cytokines such as IL-1β and IL-6 play an important role in inflammatory and immunologic responses as a part of the host defense mechanisms. ...
... This demonstrates disruption of the balance between proinflammatory and antiinflammatory immune responses leading to immune system dysregulation. The increased IL-1β and IL-6 levels may result from the oxidative stress and activation of various transcription factors [15,58]. The proinflammatory cytokines may amplify the inflammatory response, which contributes to uncontrolled tissue damage with a massive generation of free radicals [59], and hence there is a need for anti-inflammatory cytokines to alleviate this effect as a protective response. ...
... Previous studies demonstrated that IL-10 overexpression has a pivotal role in protection against inflammation-induced injuries [59][60][61]. In agreement with our results, previous studies on other NNs revealed modulation of inflammatory cytokine production by NNs [15,58]. ...
Full-text available
Thiacloprid (TCP) is a widely used neonicotinoid insecticide with a probable toxic hazard to animals and human beings. This intensified the demand for natural compounds to alleviate the expected toxic insults. This study aimed at determining whether astaxanthin (ASX) could mitigate the hepatotoxic effect of TCP and diminish its suppressive effect on immune responses in rats. Animals received TCP by gavage at 62.1 mg/kg (1/10th LD50) with or without ASX at 40 mg/kg, for 60 days. Intoxicated rats showed modulation of serum transaminases and protein profile. The hemagglutination antibody titer to sheep red blood cells (SRBC) and the number of plaque forming cells in spleen were reduced. The cell-mediated immunity and phagocytosis were suppressed, while serum interleukins IL-1β, IL-6 and IL-10 were elevated. Also, malondialdehyde, nitric oxide and 8-hydroxy-2'-deoxyguanosine levels were increased in the liver, spleen and thymus, with depletion of glutathione and suppression of superoxide dismutase and catalase activities. The expressions of inducible nitric oxide synthase and the high mobility group box protein 1 genes were upregulated with histomorphological alterations in the aforementioned organs. Co-treatment with ASX markedly ameliorated the toxic effects of TCP, and all markers showed a regression trend towards control values. Collectively our data suggest that the protective effects of ASX on the liver and immune system of TCP-treated animals depend upon improving the antioxidant status and relieving the inflammatory response, and thus it may be used as a promising therapeutic agent to provide superior hepato- and immunoprotection.
... For instance, imidacloprid reduces the immune response to viral infections and upregulates the anti-inflammatory cytokine IL-10 in pigs [187]. Acetamiprid reduces B lymphocyte and macrophage activation in rodents [188,189]. Following exposure to another neonicotinoid, clothianidin, and a pro-inflammatory challenge, the human monocytic cell line THP-1 showed down-regulation of NF-κB signaling and of the inflammatory cytokine TNF-α, together with up-regulation of nerve factor growth receptor (NGFR), supporting the functional interface between the immune and nervous systems [188]. ...
... Acetamiprid reduces B lymphocyte and macrophage activation in rodents [188,189]. Following exposure to another neonicotinoid, clothianidin, and a pro-inflammatory challenge, the human monocytic cell line THP-1 showed down-regulation of NF-κB signaling and of the inflammatory cytokine TNF-α, together with up-regulation of nerve factor growth receptor (NGFR), supporting the functional interface between the immune and nervous systems [188]. Overall, while neonicotinoids are plausible immune disruptors, their effects on the developing immune system as well as the impact of the immune effects on human neurodevelopment await elucidation. ...
Diet has long been known to modify physiology during development and adulthood. However, due to a growing number of manufactured contaminants and additives over the last few decades, diet has increasingly become a source of exposure to chemicals that has been associated with adverse health risks. Sources of food contaminants include the environment, crops treated with agrochemicals, inappropriate storage (e.g., mycotoxins) and migration of xenobiotics from food packaging and food production equipment. Hence, consumers are exposed to a mixture of xenobiotics, some of which are endocrine disruptors (EDs). The complex interactions between immune function and brain development and their orchestration by steroid hormones are insufficiently understood in human populations, and little is known about the impact on immune-brain interactions by transplacental fetal exposure to EDs via maternal diet. To help to identify the key data gaps, this paper aims to present (a) how transplacental EDs modify immune system and brain development, and (b) how these mechanisms may correlate with diseases such as autism and disturbances of lateral brain development. Attention is given to disturbances of the subplate, a transient structure of crucial significance in brain development. Additionally, we describe cutting edge approaches to investigate the developmental neurotoxicity of EDs, such as the application of artificial intelligence and comprehensive modelling. In the future, highly complex investigations will be performed using virtual brain models constructed using sophisticated multi-physics/multi-scale modelling strategies based on patient and synthetic data, which will enable a greater understanding of healthy or disturbed brain development.
... Neurotoxicity is not the only possible toxic effect of neonicotinoids (Casida 2011;Casida 2018;Thompson et al. 2020;Mukherjee et al. 2022). Studies indicate that, for vertebrates and also invertebrates, they may be genotoxic (Hong et al. 2018;Senyildiz et al. 2018), immunotoxic (Di Prisco et al. 2017;Hong et al. 2018), hepatotoxic (Wang et al. 2019), and have cytotoxic effects (Senyildiz et al. 2018;Wang et al. 2019). Some studies also (Bal et al. 2012;Lonare et al. 2014;Wessler and Kirkpatrick 2017;Ge et al. 2018;Raby et al. 2018;Picone et al. 2022) point to the possible impairment to the reproductive processes and abilities of vertebrate and invertebrate animals when exposed to neonicotinoid substances. ...
... Down-regulation of a reporter gene under the control of NF-K B promoter was reported along with reduced levels of TNF-α in human monocyte cell line THP1. Transcriptional levels of transcription factors TRAF4 and TRAF6 were also disrupted, which hampered NF-K B signaling 43 . In thiamethoxam-treated mice with concentrations of 43.5 mg/ kg BW and 87.1 mg/kg BW, a dose-dependent decrease was observed in total leukocyte count and an elevated lymphocyte count in both low and high doses. ...
The neonicotinoid class of insecticide is nicotine-like neuro-toxicants used to control the pests of agriculture crops and ornamental plants. They act as selective agonists of acetylcholine receptors in the central nervous system of insect pests and work by disrupting their nerve impulses. Some of the properties of this class of insecticides are a long half-life in soil, low volatility, and higher water solubility, leading to their accumulation in soil, underground water, and water bodies due to surface runoff. This, in turn, results in exposure to many beneficial non-target aquatic and soil fauna such as arthropods, fish, birds, mammals, etc. Although it has a selective mode of action for insects, some in vivo and in vitro investigations have also shown toxicity in non-target invertebrates and vertebrates. Initially, neonicotinoid toxicity was observed in honey bees, which are essential pollinators of crops. Later, studies reported the accumulation of neonicotinoid residues leading to the mortality of aquatic fauna, including salt marsh and freshwater mosquitoes, brine shrimp, fleas, and crayfish. Imidacloprid exposure led to disruption of larval development in the Mayfly larvae. Also, earthworms that play a crucial role in enhancing soil fertility were drastically affected by acetamiprid, clothianidin, imidacloprid, nitenpyram, and thiacloprid. Apart from these, toxicological impacts were also observed in vertebrates such as birds, where imidacloprid, clothianidin, acetamiprid, and thiacloprid caused reproductive, metabolic, and morphological alterations. Similarly, imidacloprid and acetamiprid caused gills, brain and liver dysfunction with embryo mortality. Even after the selective action of neonicotinoids, instances of mammalian toxicity were also reported in many in vivo studies. DNA damage and liver dysfunctions due to imidacloprid in rabbits were observed in various studies. In a recent study, imidacloprid exposure led to DNA damage and oxidative stress in bone marrow-derived mesenchymal cells of buffalo. Also, many instances of neurotoxicity, reproductive toxicity, immunotoxicity, genotoxicity and cytotoxicity in mouse and rat models were observed due to different neonicotinoids. Many in vitro studies using mammalian cell lines have also established potential risks of neonicotinoid exposure. This review, therefore, is a compilation of various toxicity studies of different types of neonicotinoid pesticides in both nontarget invertebrates and vertebrates, including several kinds of toxicities caused in mammals with neonicotinoid exposure.
... Unlike the first generation of neonicotinoids, CLOT does not contain a 6-chloro-3pyridylmethyl group in its structure which is replaced by a chlorothiazolyl group typical for the second generation of neonicotinoids. CLOT is toxic to various non-target organisms [41][42][43][44][45][46][47]. ...
Full-text available
We present the utilization of the novel hybrid electrochemical DNA biosensor, as well as the fabrication and testing of novel dGMP (2'-deoxyguanosine 5'-monophosphate) (bio)sensor based on anodically pre-treated "basal-plane" pyrolytic graphite electrode (BPPGE) for the investigation of the interaction between double-stranded DNA (dsDNA) and neonicotinoid insecticide clothianidin (CLOT) via linear-sweep voltammetry (LSV). The results clarify the nature of the interaction as partial-intercalation (marked by high preference towards guanine bases) with binding constant (Kb) of 8.1×10 5 L/mol, free Gibbs energy (∆G 0) of −33.154 kJ/mol, and site-binding size (s) of 0.58 bp. The conclusions obtained by LSV are further supported by molecular docking that confirmed a partial-intercalative mode of interaction with the predominant involvement of the hydrogen bonds (exclusively with the guanine bases) for the stabilization of dsDNA-CLOT complex.
... Little is known on the effect of chronic human low-level exposure to nAChRs' disrupters such as NN, which, especially in human foetuses' and children's developing brains, could potentially lead to later cerebral dysfunctions. In humans, NN have been associated with small-for-gestational-age neonates, congenital malformations, autism spectrum disorder, memory loss and finger tremor [15][16][17][18][19]. NN toxicological studies in rodents or mammals/human cell-lines have been shown to be cytotoxic, genotoxic, hepatotoxic, haematotoxic, nephrotoxic and potentially immunotoxic [20][21][22][23]. Among pesticides, NN definitely represent a potential significant public-health risk. ...
Full-text available
Background Neonicotinoids (NN) are selective neurotoxic pesticides that bind to insect but also mammal nicotinic acetycholine receptors (nAChRs). As the most widely used class of insecticides worldwide, they are ubiquitously found in the environment, wildlife, and foods, and thus of special concern for their impacts on the environment and human health. nAChRs are vital to proper brain organization during the prenatal period and play important roles in various motor, emotional, and cognitive functions. Little is known on children’s contamination by NN. In a pilot study we tested the hypothesis that children’s cerebro-spinal fluid (CSF) can be contaminated by NN. Methods NN were analysed in leftover CSF, blood, and urine samples from children treated for leukaemias and lymphomas and undergoing therapeutic lumbar punctions. We monitored all neonicotinoids approved on the global market and some of their most common metabolites by ultra-high performance liquid chromatography-tandem mass spectrometry. Results From August to December 2020, 14 children were consecutively included in the study. Median age was 8 years (range 3–18). All CSF and plasma samples were positive for at least one NN. Nine (64%) CSF samples and 13 (93%) plasma samples contained more than one NN. Thirteen (93%) CSF samples had N-desmethyl-acetamiprid (median concentration 0.0123, range 0.0024–0.1068 ng/mL), the major metabolite of acetamiprid. All but one urine samples were positive for ≥ one NN. A statistically significant linear relationship was found between plasma/urine and CSF N-desmethyl-acetamiprid concentrations. Conclusions We have developed a reliable analytical method that revealed multiple NN and/or their metabolites in children’s CSF, plasma, and urine. Our data suggest that contamination by multiple NN is not only an environmental hazard for non-target insects such as bees but also potentially for children.
... Furthermore, exposure to 1 and 500 mg/L of imidacloprid initially reduced mortality from Ranavirus in Xenopus laevis tadpoles (Hrynyk et al., 2018). However, exposure to neonicotinoids has affected other measures of immune function, such as phagocytic activity in rats and cytokine production and expression of immune-related genes in a human cell line (Di Prisco et al., 2017;Mohany et al., 2011). Thus, neonicotinoids (i.e., imidacloprid, clothianidin and thiamethoxam), potentially may impair immune function in some vertebrates (Gibbons et al., 2014). ...
Neonicotinoid pesticide use is widespread and highly debated, as evidenced by recent attention received from the public, academics and pesticide regulatory agencies. However, relatively little is known about the physiological effects of neonicotinoid insecticides on aquatic vertebrates. Amphibians (larval stages in particular) are excellent vertebrate bioindicators in aquatic systems due to their risk of exposure and sensitivity to environmental stressors. Previous work with wood frog (Rana sylvatica) tadpoles exposed to formulated products containing thiamethoxam or clothianidin in outdoor mesocosms found significant shifts in leukocyte profiles, suggesting the tadpoles were physiologically stressed. The main objective of the present study was to characterize this stress response further using complementary measures of stress after exposure to clothianidin on northern leopard frogs (Rana pipiens) during their aquatic larval stages. Laboratory static-renewal exposures were conducted over eight weeks with the technical product clothianidin at 0, 0.23, 1, 10 and 100 μg/L, and diquat dibromide at 532 μg/L was used as a positive control. We assessed tadpole leukocyte profiles and measures of oxidative stress as these sub-lethal alterations could affect amphibian fitness. We found changes in several types of leukocytes at 1 and 10 μg/L, suggesting that these tadpoles exhibited signs of mild physiological stress. Clothianidin also induced an oxidative stress response at 0.23, 1 and 100 μg/L. However, we found no differences in survival, growth, development time or hepatosomatic index in frogs exposed to clothianidin. Our study indicates that tadpoles chronically exposed to clothianidin have increased stress responses, but in the absence of concentration-response relationships and effects on whole-organism endpoints, the implications on the overall health and fitness of these changes are unclear.
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Neonicotinoid insecticides contaminate many kinds of foods and drinking water by the agricultural use since 1990’s, the detection in human urine have been reported. They pass through blood brain barrier and placenta, and directly affect developing neurons. To reduce the intake, organic products with Japanese Agricultural Standard (JAS) authentication might be useful because no neonicotinoids are used for the production. Firstly, we recruited sixty-two volunteers lived in Fukushima, Japan, collected urine samples three times a day for three days and analyzed seven neonicotinoids (acetamiprid, imidacloprid, clothianidin, dinotefuran, nitenpyram, thiacloprid, and thiamethoxam) and a metabolite, N-desmethyl-acetamiprid by LC-MS/MS. In thirteen organic farmers, dinotefuran, N-desmethyl-acetamiprid, clothianidin, and thiamethoxam were less quantified (p<0.001, p=0.014, p=0.003, p=0.024, respectively) than others, but not imidacloprid. Secondly, thirty-seven of 62 volunteers, not organic farmers, started to take organic product (rice, vegetable, potatoes, miso-marinaded pork) for five days. We compared the average of urinary neonicotinoids/a metabolite concentration between before intake for three days and the third to fifth day of intake. In twenty-four volunteers no less than 10 years old, concentration of dinotefuran, N-desmethyl-acetamiprid, clothianidin, and thiamethoxam decreased significantly (p<0.001, p=0.041, p<0.001, and p<0.001, respectively), but not imidacloprid. In thirteen children (three to nine years old), clothianidin and thiamethoxam was significantly less quantified after the start of organic foods (p=0.009, p=0.015, respectively), but not dinotefuran, N-desmethyl-acetamiprid nor imidacloprid. Organic products with JAS are effective to reduce the intake of neonicotinoids, and for children, more comprehensive selection of foods and beverages would be necessary.
ネオニコチノイドと有機農産物 ネオニコチノイドは生態系影響および発達神経毒性などの健康影響の懸念がある殺虫剤で、多種類の食品および飲料水に残留しヒトの尿から高頻度で検出される。曝露を減らすため栽培にネオニコチノイドを使わない有機農産物の摂取が有望視されている。福島県の有機栽培農家ではない15家庭の10-49歳の24人(男/女=7/17)に有機農産物(米、卵、野菜、豚肉、麹、味噌)を提供し、摂取開始前3日間と開始3、4、5日目の尿中のネオニコチノイド7種(イミダクロプリド、アセタミプリド、ニテンピラム、チアクロプリド、チアメトキサム、クロチアニジン、ジノテフラン)と代謝物N-デスメチルアセタミプリド (DMAP)の濃度をLC/MS/MSを用いて測定したところ、チアメトキサム、クロチアニジン、ジノテフラン、DMAPの平均濃度が有意に低下した。主な食品を有機農産物に置き換えることで尿中ネオニコチノイド濃度の低減が期待できる。
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Background Widespread environmental contamination can directly interact with human immune system functions. Environmental effects on the immune system may influence human susceptibility to respiratory infections as well as the severity of infectious diseases, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the efficacy of vaccines to respiratory diseases may be impacted by environmental exposures through immune perturbations. Given the quick pace of research about COVID-19 and associated risk factors, it is critical to identify and curate the streams of evidence quickly and effectively. Objective We developed this systematic evidence map protocol to identify and organize existing human and animal literature on high-priority environmental chemical classes (Per- and polyfluoroalkyl substances, pesticides, phthalates, quaternary ammonium compounds, and air pollutants) and their potential to influence three key outcomes: (1) susceptibility to respiratory infection, including SARS-CoV-2 (2) severity of the resultant disease progression, and (3) impact on vaccine efficacy. The result of this project will be an online, interactive database which will show what evidence is currently available between involuntary exposures to select environmental chemicals and immune health effects, data gaps that require further research, and data rich areas that may support further analysis. Search and study eligibility We will search PubMed for epidemiological or toxicological literature on select toxicants from each of the chemical classes and each of the three outcomes listed above. Study appraisal and synthesis of methods For each study, two independent reviewers will conduct title and abstract screening as well as full text review for data extraction of study characteristics. Study quality will not be evaluated in this evidence mapping. The main findings from the systematic evidence map will be visualized using a publicly available and interactive database hosted on Tableau Public.
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Background: Numerous studies have identified detectable levels of neonicotinoids (neonics) in the environment, adverse effects of neonics in many species including mammals, and pathways through which human exposure to neonics could occur, yet little is known about the human health effects of neonic exposure. Objective: This systematic review sought to identify human population studies on the health effects of neonics. Methods: Studies published in English between 2005 and 2015 were searched using PubMed, Scopus, and Web of Science databases. No restrictions were placed on the type of health outcome assessed. Risk of bias was assessed using guidance developed by the National Toxicology Program's Office of Health Assessment and Translation. Results: Eight studies investigating the human health effects of exposure to neonics were identified. Four examined acute exposure: three neonic poisoning studies reported two fatalities (n=1280 cases) and an occupational exposure study of 19 forestry workers reported no adverse effects. Four general population studies reported associations between chronic neonic exposure and adverse developmental or neurological outcomes, including tetralogy of Fallot (AOR 2.4, 95% CI: 1.1-5.4), anencephaly (AOR 2.9, 95% CI: 1.0-8.2), autism spectrum disorder (AOR 1.3, 95% CrI: 0.78-2.2), and a symptom cluster including memory loss and finger tremor (OR 14, 95% CI: 3.5-57). Reported odds ratios were based on exposed compared to unexposed groups. Conclusions: The studies conducted to date were limited in number with suggestive but methodologically weak findings related to chronic exposure. Given the wide-scale use of neonics, more needs to be known about their human health effects.
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Significance The parasitic mite Varroa destructor and the deformed wing virus (DWV) are linked in a mutualistic symbiosis. The mite acts as vector of the viral pathogen, whereas the DWV-induced immunosuppression in honey bees favors mite feeding and reproduction. This functional interaction underpins a rapidly escalating immunosuppression, which can be primed and/or aggravated by a wealth of stress factors that co-trigger colony losses. Our experimental results explain the pivotal role proposed for the Varroa –DWV association in the induction of honey bee colony losses. Here we provide a functional framework for studying the dynamics of this multifactorial syndrome and defining effective strategies to reduce its negative impact on the beekeeping industry.
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Evolution has yielded multiple complex and complementary mechanisms to detect environmental danger and protect tissues from damage. The nervous system rapidly processes information and coordinates complex defense behaviors, and the immune system eliminates diverse threats by virtue of mobile, specialized cell populations. The two systems are tightly integrated, cooperating in local and systemic reflexes that restore homeostasis in response to tissue injury and infection. They further share a broad common language of cytokines, growth factors, and neuropeptides that enables bidirectional communication. However, this reciprocal cross talk permits amplification of maladaptive feedforward inflammatory loops that contribute to the development of allergy, autoimmunity, itch, and pain. Appreciating the immune and nervous systems as a holistic, coordinated defense system provides both new insights into inflammation and exciting opportunities for managing acute and chronic inflammatory diseases. Expected final online publication date for the Annual Review of Immunology Volume 34 is May 20, 2016. Please see for revised estimates.
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The human genome contains a variant form of the α7-nicotinic acetylcholine receptor (α7nAChR) gene that is uniquely human. This CHRFAM7A gene arose during human speciation and recent data suggests that its expression alters ligand tropism of the normally homopentameric human α7-AChR ligand-gated cell surface ion channel that is found on the surface of many different cell types. To understand its possible significance in regulating inflammation in humans, we investigated its expression in normal human leukocytes and leukocyte cell lines, compared CHRFAM7A expression to that of the CHRNA7 gene, mapped its promoter and characterized the effects of stable CHRFAM7A over-expression. We report here that CHRFAM7A is highly expressed in human leukocytes but that the levels of both CHRFAM7A and CHRNA7 mRNAs were independent and varied widely. To this end, mapping of the CHRFAM7A promoter in its 5'-untranslated region (UTR) identified a unique 1kb sequence that independently regulates CHRFAM7A gene expression. Because over-expression of CHRFAM7A in THP1 cells altered the cell phenotype and modified the expression of genes associated with focal adhesion (e.g. FAK, P13K, Akt, rho, GEF, Elk1, CycD), leukocyte trans-epithelial migration (Nox, ITG, MMPs, PKC) and cancer (kit, kitL, ras, cFos cyclinD1, Frizzled and GPCR), we conclude that CHRFAM7A is biologically active. Most surprisingly however, stable CHRFAM7A overexpression in THP1 cells up-regulated CHRNA7, which in turn, led to increased binding of the specific α7nAChR ligand, bungarotoxin on the THP1 cell surface. Taken together, these data confirm the close association between CHRFAM7A and CHRNA7 expression, establish a biological consequence to CHRFAM7A expression in human leukocytes and support the possibility that this human-specific gene might contribute to, and/or gauge, a human-specific response to inflammation.
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Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor β (NGFβ), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFβ or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFβ was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRC4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFβ-TRKA signaling in pathogen-specific host immunity.
A strong immune defense is vital for honey bee health and colony survival. This defense can be weakened by environmental factors that may render honey bees more vulnerable to parasites and pathogens. Honey bees are frequently exposed to neonicotinoid pesticides, which are being discussed as one of the stress factors that may lead to colony failure. We investigated the sublethal effects of the neonicotinoids thiacloprid, imidacloprid, and clothianidin on individual immunity, by studying three major aspects of immunocompetence in worker bees: total hemocyte number, encapsulation response, and antimicrobial activity of the hemolymph. In laboratory experiments, we found a strong impact of all three neonicotinoids. Thiacloprid (24 h oral exposure, 200 lg/l or 2000 lg/l) and imidacloprid (1 lg/l or 10 lg/l) reduced hemocyte density, encapsulation response, and antimicrobial activity even at field realistic concentrations. Clothianidin had an effect on these immune parameters only at higher than field realistic concentrations (50–200 lg/l). These results suggest that neonicotinoids affect the individual immunocompetence of honey bees, possibly leading to an impaired disease resistance capacity. � 2016 Elsevier Ltd. All rights reserved.
Activation of innate immune response, induced after the recognition of double-stranded RNA (dsRNA), formed during replication of most viruses, results in intracellular signaling cascades ultimately culminating in the expression of type I interferon (IFN). In this study, we provide the first evidence that FoxO4 triggers the activation of the innate immune signaling pathway in coupling stimulation of TLR3 and RIG-like receptors by the synthetic dsRNA analog, poly(I:C), to IFN-β and IFN-induced gene induction, whereas knockdown of FoxO4 had opposite effects. Similar effects of FoxO4 were observed during paramyxovirus-mediated IFN-β transcriptional induction. We further found that knockdown of FoxO4 did not affect IRF3 and NF-κB activation by poly(I:C), suggesting that FoxO4 would act downstream in the signaling pathway. In addition, we show that the IFN-induced TRIM22 ubiquitin ligase targets FoxO4 and antagonizes its activity through an unrelated ubiquitin/autophagosomic-lysosomal pathway. Unexpectedly, TRIM22 knockdown strongly sensitizes cells to dsRNA-induced caspase-dependent apoptosis, as early as 2 h after poly(I:C) stimulation, concomitantly to the inhibition of the expression of the antiapoptotic protein, Bcl-2, indicating that TRIM22 might be a key factor for controlling the cell survival after TLR3 stimulation. Taken together, our data demonstrate that the regulation of FoxO4 protein expression and cell survival by TRIM22 controls TLR3-mediated IFN type I gene induction, preventing excessive antiviral response through dsRNA-induced apoptosis.
The widespread losses of honeybee colonies recorded over the past number of years in the northern hemisphere represent a major concern for the beekeeping industry and, more importantly, may have a severe impact on ecological services and biodiversity. There is now a general consensus about the multifactorial origin of colony losses, but the mechanistic basis of this complex phenomenon still remains largely elusive. In this review, we propose a functional framework for interpreting how different stress agents can interact to adversely affect bee immunity and health. This provides a new background rationale in which to develop an integrated approach to bee protection, as part of a more comprehensive strategy for the conservation of insect pollinators. Copyright © 2014 Elsevier Ltd. All rights reserved.