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R E S E A R C H Open Access
Interleukin-6 actions in the hypothalamus
protects against obesity and is involved in
the regulation of neurogenesis
Vanessa C. Bobbo
1,2
, Daiane F. Engel
2
, Carlos Poblete Jara
1,2
, Natalia F. Mendes
1,2
, Roberta Haddad-Tovolli
2
,
Thais P. Prado
1,2
, Davi Sidarta-Oliveira
2
, Joseane Morari
2
, Licio A. Velloso
2
and Eliana P. Araujo
1,2*
Abstract
Background: Interleukin-6 (IL6) produced in the context of exercise acts in the hypothalamus reducing obesity-
associated inflammation and restoring the control of food intake and energy expenditure. In the hippocampus,
some of the beneficial actions of IL6 are attributed to its neurogenesis-inducing properties. However, in the
hypothalamus, the putative neurogenic actions of IL6 have never been explored, and its potential to balance
energy intake can be an approach to prevent or attenuate obesity.
Methods: Wild-type (WT) and IL6 knockout (KO) mice were employed to study the capacity of IL6 to induce
neurogenesis. We used cell labeling with Bromodeoxyuridine (BrdU), immunofluorescence, and real-time PCR to
determine the expression of markers of neurogenesis and neurotransmitters. We prepared hypothalamic
neuroprogenitor cells from KO that were treated with IL6 in order to provide an ex vivo model to further
characterizing the neurogenic actions of IL6 through differentiation assays. In addition, we analyzed single-cell RNA
sequencing data and determined the expression of IL6 and IL6 receptor in specific cell types of the murine
hypothalamus.
Results: IL6 expression in the hypothalamus is low and restricted to microglia and tanycytes, whereas IL6 receptor
is expressed in microglia, ependymocytes, endothelial cells, and astrocytes. Exogenous IL6 reduces diet-induced
obesity. In outbred mice, obesity-resistance is accompanied by increased expression of IL6 in the hypothalamus. IL6
induces neurogenesis-related gene expression in the hypothalamus and in neuroprogenitor cells, both from WT as
well as from KO mice.
Conclusion: IL6 induces neurogenesis-related gene expression in the hypothalamus of WT mice. In KO mice, the
neurogenic actions of IL6 are preserved; however, the appearance of new fully differentiated proopiomelanocortin
(POMC) and neuropeptide Y (NPY) neurons is either delayed or disturbed.
Keywords: Cytokine, Metabolism, Neuron, Astrocyte, Brain, Diabetes
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data made available in this article, unless otherwise stated in a credit line to the data.
* Correspondence: earaujo@unicamp.br
1
Nursing School, University of Campinas, Campinas, Brazil
2
Laboratory of Cell Signaling, University of Campinas, Rua Cinco de Junho,
350, Cidade Universitária, Campinas, SP 13083-877, Brazil
Bobbo et al. Journal of Neuroinflammation (2021) 18:192
https://doi.org/10.1186/s12974-021-02242-8
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
Interleukin-6 (IL6) is a rather unique cytokine that ex-
erts pleiotropic actions in distinct organs and systems [1,
2]. Factors such as magnitude of production, duration of
response and site of action may have either protective or
damaging impact on organism health [3,4]. For ex-
ample, the rapid activation of IL6 response during early
infection has critical role in host protection [5–7],
whereas in chronic inflammatory diseases and obesity-
associated metabolic inflammation, the enduring actions
of IL6 promote structural and functional losses that may
result in irreversible damage [8–12].
One remarkable advance in the understanding of the
beneficial actions of IL6 was the characterization of its
production by the exercised muscle [13]. Differently of
the pattern of production in infectious and chronic in-
flammatory conditions, during exercise, IL6 is produced
for a short period of time, independently of a previous
stimulation by tumor necrosis factor-alpha (TNFα) and
accompanied by only moderate/low increase of other in-
flammatory substances [14]. This particular mode of IL6
production has been shown to mediate some of the
health promoting actions of exercise, such as increasing
systemic insulin action [15–17], reducing hepatic steato-
sis [18], and reducing hepatic glucose production [17].
The brain is an important site of action of IL6. Studies
have shown that exercise-induced IL6 can attenuate
memory impairment in models of Alzheimer’s disease
[19,20], whereas in the hypothalamus, IL6 produced in
response to exercise can reduce diet-induced inflamma-
tion and correct the abnormal regulation of food intake
[21,22]. The reduction of neuroinflammation is one of
the mechanisms mediating the actions of IL6 in models
of exercise [21]; however, recent studies have shown that
induction of neurogenesis is yet another important
mechanism mediating the actions of IL6 improving cog-
nition in models of traumatic brain injury and Alzhei-
mer’s disease [23,24].
Most studies evaluating adult neurogenesis have fo-
cused on the subventricular and subgranular zones (SVZ
and SGZ, respectively), which provide new neurons for
these specific regions, as well as for neighboring areas,
such as striatum in humans [25,26].
However, evidence suggests that replacement of hypo-
thalamic neurons during life relies on local production,
thus placing the hypothalamus as an autonomous adult
neurogenesis niche [27,28]. In concern with the hypo-
thalamic functions controlling food intake and energy
homeostasis, stimuli such as leptin and insulin, as well
as nutrients, have been shown to regulate hypothalamic
neurogenesis [28–31]. As IL6 produced during exercise
is known to mitigate diet-induced hypothalamic dys-
function [21] and, considering that IL6 neurogenic po-
tential has been shown in other neurogenic niches of the
brain, here, we decided to evaluate the putative neuro-
genic actions of IL6 in the hypothalamus.
Methods
Experimental animals
Six- to 12-week-old male Swiss mice and 8-week-old
male and female C57BL/6J mice (WT; Jackson Labora-
tory stock #000664) were obtained from the University
of Campinas Animal Facility. Male and female, 8-week-
old interleukin-6 knockout (KO; C57BL6/J KO
il6−/−
;
Jackson Laboratory stock # 002254) were obtained from
the Ribeirão Preto Medical School. Male and female,
C57BL/6J and C57BL6/J KO
il6−/−
pups (postnatal day 0–
7) were obtained from the University of Campinas Ani-
mal Facility. All mice were kept in individual cages at 21
± 0.5 °C, in 12/12-h light/dark cycle, with water and
chow available ad libitum. In all experiments, control
and intervention group mice were submitted to the same
experimental settings.
Experimental protocol
In vivo protocol #1
Swiss mice were randomly separated into two groups:
IL6 treatment or vehicle (saline). Thereafter, mice were
fed a HFD for 2 weeks as described at the end of this
paragraph (schematically illustrated in Fig. 1A). To de-
termine caloric intake, the number of daily calories
ingested by each mouse was divided by its weight in
grams (Kcal/g/day); this approach was adopted because
of the considerable variability in body mass among mice.
The macronutrient composition of standard chow was
20 g% protein, 76 g% carbohydrate, and 4 g% fat, result-
ing in energy value of 17.5 kJ/g; the macronutrient com-
position of HFD was 20 g% protein, 45 g% carbohydrate,
and 35 g% fat, resulting in energy value of 24.1 kJ/g.
In vivo protocol #2
Another set of Swiss mice were fed a HFD for 24 h and
then defined as H (25% of animals that consumed high-
est amount of diet) or L (25% of animals that consumed
lowest amount); thereafter, mice were euthanized and
the hypothalami were collected for gene expression ana-
lysis (schematically illustrated in Fig. 3A).
In vivo protocol #3
In order to perform central delivery of palmitate (30
μM, 2 μl; Sigma) or vehicle (saline), Swiss mice were
submitted to a single stereotaxic injection in the third
ventricle under xylazine (10mg/kg, ip) and ketamine
(100 mg/kg, ip) anesthesia. The coordinates from
Bregma were as follows: anteroposterior, 0.0 mm; lateral,
0.0 mm; and depth, 4.5 mm [32,33]. According to a
time-course, mice were euthanized and the hypothalami
were collected for gene expression analysis.
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 2 of 17
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Fig. 1 Effect of exogenous IL6 on caloric intake and body mass. Wild-type mice were fed on chow or high-fat diet (HFD) and treated intraperitoneally (IP) with
exogenous IL6 or saline as depicted in A. Body mass (B,D) and caloric intake (C,E) were determined in mice fed chow (B,C)andHFD(D,E). In all experiments,
n= 5 mice/group. Data was analyzed using two-way ANOVA with repeated measures and Bonferroni post-test. **p< 0.01; ***p< 0.001
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 3 of 17
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In vivo protocol #4
C57BL/6J mice were treated with vehicle (saline) or re-
combinant IL6 (Sigma) (1 ng ip, once a day, for up to 7
days, proliferation assay, or 14 days, survival assay). Bro-
modeoxyuridine (BrdU; Sigma) was used to evaluate cell
proliferation. BrdU is a thymidine analog that is incorpo-
rated into the DNA double-helix during the S-phase of
the cell cycle, and thus labels actively proliferating cells
[34]. Mice received BrdU (0.1 M PBS, pH = 7.2; 50 mg/
Kg 2x day ip for 7 days concomitant with IL6) and were
euthanized 24 h after the last injection (proliferation
assay, schematically illustrated in Fig. 4A) or 14 days
after the last injection (survival assay, schematically illus-
trated in Fig. 4D).
In vivo protocol #5
Swiss mice were treated with a single injection of vehicle
(saline) or recombinant IL6 (Sigma) (1 ng ip) at different
time points (8 h, 6 h, 4 h, 2 h prior to extraction).
Thereafter, mice were euthanized and the hypothalami
collected for gene expression analysis (schematically il-
lustrated in Fig. 5A).
In vivo protocol #6
Adult IL6 knockout (KO) mice fed on chow were com-
pared to the respective WT, C57BL/6J controls. For that,
we determined body mass and blood IL-6 levels at base-
line conditions and 4 h after treatment with recombin-
ant IL6 (Sigma) (1 ng ip) and hypothalamic gene
expression analysis in baseline conditions.
Immunofluorescence staining
Mice were deeply anesthetized with a solution of xyla-
zine (10 mg/kg, ip) and ketamine (100 mg/kg, ip) and
perfused through the left cardiac ventricle with 0.9% sa-
line solution, followed by 4% paraformaldehyde (PFA) in
0.1M PBS (pH 7.4). After perfusion, the brains were re-
moved, post-fixed in the same fixative solution for 24 h
at room temperature (RT), and immersed in a 30% su-
crose solution in PBS at 4 °C. Serial coronal sections (20
μm) of hypothalami were obtained with a cryostat
(LEICA Microsystems, CM1860). In order to determine
cell proliferation and survival in the hypothalamic ven-
tricular zone and parenchyma, a series of one-in-four
free-floating sections were processed for detection of the
BrdU immunoreactivity. Differentiation was assessed by
BrdU/neuronal nuclei (NeuN) double labeling. Briefly,
after DNA denaturation in 2 N HCl at room
temperature (RT) for 1 h and pre-incubation with 10%
blocking solution (0.1M PBS with 10% normal goat or
donkey serum and 0.2% Triton X-100), sections were in-
cubated overnight at 4 °C in rat anti-BrdU (1:200;
Ab6326) and mouse anti-NeuN (1:200; MAB377C3; Cy3
Conjugate) primary antibodies. The sections were then
incubated with secondary antibody goat anti-rat FITC
(1:200; sc2011) for 2 h at RT. All sections were mounted,
cover slipped with Fluor Mount (Sigma), and stored at 4
°C. The morphological analyses were performed on
coded slides, with the executing researcher blinded to
the experimental group. The total numbers of BrdU-
immunopositive cells in the HVZ and PA were esti-
mated by manually counting all positive cells. From all
sections containing the hypothalamus (1.06 to 2.30 mm
posterior to Bregma), one-in-four series of sections were
used for the analysis. The results were expressed as the
counted number of labeled cells multiplied by 4 (the sec-
tion interval), and the resulting number was corrected
using the Abercrombie formula. Newly formed neurons
(BrdU/NeuN-positive cells) were analyzed in one-in-four
series of sections by immunofluorescence double stain-
ing [35]. The results were expressed as the counted
number of labeled cells multiplied by 4 (the section
interval), and the resulting number was corrected using
the Abercrombie formula. The amount of BrdU/NeuN-
positive cells was expressed as percentages. A maximum
of 50 BrdU-labeled cells per mouse were randomly se-
lected for analysis of co-labeling with NeuN. Double la-
beling was confirmed by three-dimensional orthogonal
reconstruction (Imaje J software) of z-series (average of
18 images per series) of confocal microscopy covering
the entire nucleus (or cell) of interest (confocal micro-
scope Upright LSM780-NLO) in sequential scanning
mode to avoid cross-bleeding between channels. Nuclei
diameter was estimated using the Fiji software, as previ-
ously described [36]. Only cells that presented a typical
aspect of NeuN surrounding the nucleus were consid-
ered. The representative images are the stack of the z-
project of all images obtained within the 20 μm section.
In order to avoid overestimation of cell count, we per-
formed Abercrombie Correction according to the for-
mula N=n×(T/(T+ D), where n= cell counting, T=
section thickness (20 μm), and D= estimated diameter
of cell nuclei (approx. 15 μm), as described elsewhere
[37,38].
Serum IL-6 determination
Blood samples were collected immediately after decapi-
tation using a tube with citrate. Plasma was separated by
centrifugation (1100×g) for 15 min at 4 °C and stored at
−80 °C until assay. IL-6 concentrations were determined
using a commercially available Enzyme Linked Immuno-
sorbent Assay kit, according to manufacturer instruc-
tions (Mouse IL-6 DuoSet ELISA, R&D Systems).
Postnatal neurosphere culture
Postnatal day 0–7 pups were euthanized, their brains
immediately removed, and the hypothalamus microdis-
sected. Tissue fragments were repeatedly dissociated
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 4 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
with a Pasteur pipette in PBS with 5.5 mM glucose, 100
U/mL penicillin, and 100 mg/mL streptomycin. Cells
were suspended in 5 mL of proliferation media: Dulbec-
co’s modified Eagle’s medium (DMEM)-F12/Glutamax
(Gibco) supplemented with growth factors (10 ng/mL
human basic fibroblast growth factor, bFGF, and 10 ng/
mL epidermal growth factor, EGF, sigma) 100 U/mL
penicillin, 100 mg/mL streptomycin, and 1% B27 supple-
ment. The floating neurospheres were allowed to grow
in uncoated 25-cm
2
flasks in a humidified incubator with
a5%CO
2
atmosphere. On culture day 7, neurospheres
were collected by centrifugation, mechanically dissoci-
ated using a pipette, and plated using approximately
100,000 cells per well in fresh proliferation medium,
onto Poly-L-Lysine (PLL; Sigma)-coated 12 well culture
plates for RNA extraction or glass coverslips for im-
munocytochemistry. Once the monolayer reached con-
fluence, cell differentiation was induced by switching
proliferation media for differentiation media: neurobasal
medium (Gibco) supplemented with 1% B27 (Invitro-
gen), 500 mM/L-Glutamine (Sigma), and 50 units/mL
penicillin/streptomycin (Life Technologies), without
growth factors. The differentiation protocol was carried
for 5 to 14 days to assess the differences between WT
and KO mice and upon IL-6 (2 pg/ml, 20 pg/ml, 200 pg/
mL, and 2 ng/mL) treatment over the expression of cell
stemness and differentiation markers.
Immunocytochemistry
Cover slips containing differentiated neurospheres were
fixed with 4% PFA in 0.1 M PBS for 10 min at RT. After
washing with PBS, the cells were incubated in blocking
solution (10% normal donkey serum in 0.1 M PBS con-
taining 0.2% Triton X-100) for 1 h at RT. The cells were
then incubated in fresh blocking solution (3% normal
donkey serum) containing rabbit anti-DCX (1:200; Cell
Signaling 4,604), rabbit anti-MAP2 (1:200; ab32454), or
anti-GFAP Cy3 (1:2000; ab49874) overnight at 4 °C. The
cells were washed three times with PBS and incubated in
blocking solution containing donkey anti-rabbit FITC (1:
500; ab6798) for 1 h at RT, followed by DAPI for 10 min
at RT. Following another three PBS washes, the slides
were mounted using fluorescence mounting medium be-
fore image capturing on fluorescence microscopy (Olym-
pus BX41). The results of immunopositive cells
represent the average of 4 cover slips per experimental
replicate, where 4–5 fields were imaged per cover slip
and averaged. The number of immunopositive cells was
quantified per image using the ImageJ software and are
expressed as percentage relative to total nuclei.
RNA extraction and quantitative real-time PCR
RNA samples were prepared using TRIzol (Invitrogen)
according to the manufacturer’s recommendations.
Spectrophotometry was employed for RNA quantifica-
tion. For each sample, 2 μg of RNA was employed for
the synthesis of complementary DNA (cDNA) using the
High Capacity cDNA Reverse Transcription Synthesis
kit (Applied Biosystems). Real-time PCR reactions were
run using the TaqMan system (Applied Biosystems).
Analyses were run using 4 μL (10 ng/μL) cDNA, 0.625
μL primer/probe solution, 1.625 μLH
2
O, and 6.25 μL
2X TaqMan Universal MasterMix. GAPDH
(Mm99999915_g1) was employed as a reference gene.
Gene expression was obtained using the StepOne Plus
System software (Applied Biosystems). As negative con-
trol, no retrotranscriptase was added. All primers used
in the study (Supplementary Table 1) were certified for
efficiency and specificity, as declared by the manufac-
turers. Nevertheless, we further validated the primers by
amplifying the cDNA of each sample in triplicates at six
different concentrations (3-fold serial dilutions). Both
the primers for the target genes and reference gene were
tested. The efficiency of the system was calculated using
the formula: E=10(−1/slope) −1. The quantification
method used for the calculations of the data was the
2
−dct
method. Then, the average data from the control
group was considered as 1, and other groups were rela-
tive to control. This was the fold change calculation
used.
Single-cell RNA sequencing data extraction and
processing
scRNAseq data from the adult mouse arcuate nucleus
was obtained from GEO (GSE93374). Data analysis was
performed with Seurat v3.2.2 [39], a scRNAseq data ana-
lysis toolkit in R. Data was normalized with SCTrans-
form, a negative binomial normalization approach to
scRNAseq data [40]. We then selected top 5000 differen-
tially expressed genes (DEGs) with a dispersion method
and used these in the computation of dbMAP. dbMAP
estimates data structure from a series of random walks
by employing adaptive multiscale diffusion maps for a
first round of dimensional reduction which is followed
by an adapted UMAP implementation on the multiscale
diffusion components and is available in python (https://
doi.org/10.2139/ssrn.3582067). Plots of dbMAP embed-
dings colored by annotated cell type were obtained with
the function DimPlot of Seurat, with cell groupings from
the original data report.
For Bayesian normalization of raw counts data, BA-
SiCS (Bayesian Analysis of Single-Cell Sequencing data)
was employed. Briefly, BASiCS is an integrated Bayesian
hierarchical model that simultaneously performs data
normalization and technical noise quantification to
propagate statistical uncertainty. We used BASiCS new
implementation which does not require the use of Spike-
ins for data normalization and evaluates probabilistic
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 5 of 17
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consistency across batches instead [41]. Briefly, a Mar-
kov Chain Monte Carlo was constructed with the BA-
SiCS_MCMC function of the BASiCS package in R with
default recommended parameters and used for re-
estimation of a denoised counts matrix with the BA-
SiCS_DenoisedCounts function. All processed data and
custom code used for analysis are available upon
request.
Visualization of gene expression
With the exception of plots from Suppl. Fig. 2, BASiCS
probabilistic denoised gene expression was used for
visualization. For dbMAP plots we used the DimPlot and
FeaturePlot functions within Seurat, with the blend par-
ameter set to TRUE for visualization of gene co-
expression. All other visualizations were carried out with
the Scanpy plotting API (https://doi.org/10.1186/s13059-
017-1382-0 ) after conversion to a h5ad AnnData object
via SeuratDisk, an auxiliary Seurat package. Marker
genes for main cell types defined on the original study
were found via the Scanpy default workflow through
Wilcoxon rank-sum tests and visualized in a heatmap.
Statistical analysis
For all experiments, sample size was determined taking
into consideration recommendations published else-
where [42]. Data were analyzed using GraphPad Prism
version 8.0.1. The statistical analyses were carried out
using unpaired two-tailed Student’st-test, one-way ana-
lysis of variance (ANOVA) with Tukey’s multiple com-
parison test, Kruskall-Wallis test with Dunn’s multiple
comparison test, or two-way ANOVA with repeated
measures and Bonferroni post-test when appropriate.
Outliers were identified using Grubbs test. Data are pre-
sented as means ± standard error of the mean (SEM). A
pvalue < 0.05 was considered to be statistically signifi-
cant. Details of statistical analysis of all experiments de-
scribed in this study are presented in Supplementary
Table 2.
Results
Exogenous IL6 protects against diet-induced body mass
gain
The consumption of large portions of dietary fats can in-
duce hypothalamic inflammation, affecting the function
and viability of critical neurons involved in the control
of caloric intake and energy expenditure [43,44]. Here,
we asked if exogenous IL6 could act as a protective fac-
tor against diet-induced obesity. For that, 8-week mice
were randomly divided into four groups as depicted in
Fig. 1A (described in “In vivo protocol #1”). In mice fed
chow, exogenous IL6 resulted neither in body mass (Fig.
1B) nor caloric ingestion (Fig. 1C) changes. However, in
mice fed HFD, IL6 protected against body mass gain
(Fig. 1D), which occurred independently of changes in
caloric intake (Fig. 1E). The combined analysis of all
groups confirmed the effect of IL6 protecting mice from
diet-induced obesity (Suppl. Fig. 1A) without affecting
caloric intake (Suppl. Fig. 1B).
IL6 and its canonical receptor are expressed in the
arcuate nucleus of the hypothalamus of adult mice
Using dbMAP [45] as a dimensional reduction method
for high-resolution visualization, we analyzed 20,921
public single-cell transcriptomes from the arcuate nu-
cleus and median eminence of adult mice [46] into a
comprehensive landscape of cellular heterogeneity. In
this embedding (Fig. 2A), cells are colored by their ori-
ginal main cell type annotations. IL6 (Il6) mRNA was
expressed in only a few cells, classified as microglia and
tanycytes (Suppl. Fig. 2A). To exclude the possibility that
Il6-expressing cells could be a result of random meth-
odological noise, we performed a Bayesian normalization
procedure in order to obtain a denoised, probabilistic-
derived data matrix with BASiCS [47] and show that
these cells indeed express barely detectable Il6,atmuch
lower levels than corresponding marker genes (Suppl.
Fig. 2A). Differently than Il6, its canonical receptor
encoded by Il6ra and its trans-signaling receptor
encoded by Il6st are abundantly expressed across differ-
ent cell types (Fig. 2B, C and Suppl. Fig. 2B-2C). Il6ra is
preferentially expressed in Cx3cr1+ perivascular macro-
phages/microglia (cluster a07), endothelial cells (cluster
a03), and ependymocytes (cluster a09), whereas Il6st is
broadly expressed in tanycytes and arcuate neurons, as
well as neurotrophic receptor Ntrk2 and the neural stem
cell marker Nes (Fig. 2B, C and Suppl. Fig. 2D-2F). Inter-
estingly, Nes (a neural progenitor marker) and Il6st were
co-expressed in tanycytes, which are currently held as
the main hypothalamic neural stem cells [48]. Il6st
mRNA was also detected in vascular leptomeningeal
cells (a08. VLMCs), endothelial cells (a03), some neu-
rons (a13, a16), and the pars tuberalis (a19) (Fig. 2D and
Suppl. Fig. 2G). Il6ra mRNA expression pattern, on the
other hand, was rather restricted to microglia (a07),
ependymocytes (a09) and endothelial cells (a03) (Fig. 2D
and Suppl. Fig. 2H) and so was Il6ra and Il6st co-
expression (Suppl. Fig. 2I). The top five marker genes of
each main cell population are shown on Suppl. Fig. 3.
Hypothalamic IL6 is increased in mice genetically
protected from diet-induced obesity
In outbred mice, body mass gain in response to HFD oc-
curs according to a normal distribution [49]. We have
previously shown that whenever outbred mice are fed on
HFD, there is a direct correlation between caloric intake
during the first 24 h following HFD introduction and
body mass gain overtime [49]. Thus, mice presenting
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Fig. 2 (See legend on next page.)
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 7 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
highest caloric intake are prone to obesity, whereas mice
presenting lowest caloric intake are protected from obes-
ity. Here, we asked if diet-induced obesity predisposition
or protection is accompanied by differences in hypothal-
amic IL6 levels (described in “In vivo protocol #2”).
Therefore, mice were submitted to the protocol depicted
in Fig, 3A and the hypothalami were extracted 24 h after
diet introduction. As shown in Fig, 3B, IL6 expression
was increased in the hypothalamus of obesity-resistant
mice.
Palmitate induces a rapid increase in hypothalamic IL6
expression
Palmitate is the predominant fatty acid in human diet and
also in rodent HFD. It has been shown to cross the blood-
brain barrier [50] and it is increased in the cerebrospinal fluid
of obese humans [51]. Here, we asked if a direct injection of
palmitate in the hypothalamus could increase the expression
of IL6. For that, mice were acutely treated with a single intra-
cerebroventricular (icv) dose of palmitate and the hypothal-
ami were extracted for analysis (Fig. 3C) (described in “In
vivo protocol #3”).AsshowninFig,3D,therewasarapidin-
crease in hypothalamic IL6 transcripts 8 h after treatment.
Palmitate also promoted a trend to increase the transcripts
expression of TNFα(Fig. 3E) and significantly increased the
transcripts expression of IL1βin the hypothalamus (Fig. 3F).
IL6 do not interfere in hypothalamic cell proliferation nor
cell survival
Next, we evaluated the capacity of exogenous IL6 to in-
duce hypothalamic cell proliferation and survival. For
that purpose, mice were submitted to two distinct proto-
cols aimed at evaluating cell proliferation (Fig. 4A–C)
and cell survival (Fig. 4D–F). At the end of the respect-
ive experimental periods, the brains were removed and
used to determine the number of BrdU-positive cells
during the 7- or 28-day time-windows (described in “In
vivo protocol #4”). As depicted in the representative im-
ages of the hypothalamus (Fig. 4B, E) and in the graph-
ical representation of cell counts (Fig. 4C, F), there were
no significant differences between the groups.
IL6 induces hypothalamic neurogenesis, as well as
expression of neurogenesis-related transcripts in the
hypothalamus
Next, we evaluated the effect of IL6 to induce the expression
of neurogenesis-related genes in the hypothalamus. For that,
mice were treated with IL6 according to the protocol as
depicted in Fig. 5A and the hypothalami were extracted for
determination of gene expression (described in “In vivo
protocol #5”).AsshowninFig.5B and C, IL6 promoted in-
creased expressions of Sox6 and Sox2 transcripts that encode
transcription factors involved in neurogenesis, specifically in
neural stem cells, during the proliferative state [52–54]. In
addition, when looking into putative differences in the neuro-
genic response to IL6 comparing obesity-prone (H) and
obesity-resistant (L) mice (described in “In vivo protocol
#2”), we found that ip IL6 promoted a significantly larger ex-
pression of Sox6 transcripts (Fig. 5D) and a trend for increase
of doublecortin transcripts (Fig. 5E) in obesity-resistant mice.
Next, employing the same protocol as depicted in Fig. 4D
(described in “In vivo protocol #4”, survival), we determined
the number of BrdU-positive cells colocalizing with NeuN-
positive cells. As shown in Fig. 5FandG,IL6promotedan
increased neuronal differentiation after 14 days, revealing its
capacity to drive proliferative cells into a neuronal fate (p=
0.008, unpaired T-test, two-tailed). The total number of cells
determined in this experiment are depicted in Suppl. Fig. 4.
IL6 knockout mice are heavier and present a reduced
expression of POMC in the hypothalamus
Next, we evaluated body mass and hypothalamic gene
expression in IL6 knockout (KO) mice (described in “In
vivo protocol #6”). First, we tested if KOs were indeed
completely depleted of circulating IL6, and if exogenous
IL6 could promote a detectable increase of its blood
levels. As shown in Fig. 6A, IL6 KO mice presented no
detectable blood IL6 and the injection of 1 ng of IL6, 4 h
prior to blood collection, resulted in the detection of
blood IL6 in two out of four mice. At age 8 weeks, IL6
KO were heavier than controls (Fig. 6B), and at age 7
days, there were no differences in the expression of
hypothalamic transcripts encoding for peptides related
to energy homeostasis (Fig. 6C). However, at age 8
weeks, the expression of POMC was lower in IL6 KO as
(See figure on previous page.)
Fig. 2 Analysis of single-cell RNA sequencing data reveal the expression pattern of IL6-receptor encoding genes in the arcuate nucleus of the
hypothalamus. ADiffusion-based manifold approximation and projection (dbMAP) embedding of 20,921 single-cell transcriptomes of the arcuate
nucleus of the hypothalamus, colored by their annotated cluster of origin. In this embedding, similar cells are mapped tightly, whereas dissimilar
cells are mapped apart from each other, rendering clusters of cell types. Cells were annotated by their main cell type as defined in the original
study. B(top) Il6ra mRNA expression, key-colored by expression intensity; Il6ra is found mainly in perivascular macrophages/microglia and
ependymocytes and also in astrocytes and endothelial cells. B(bottom) Cx3cr1 (a canonical microglial marker) mRNA expression, key-colored by
expression intensity. C(top) Il6st mRNA expression, key-colored by expression intensity; Il6st is broadly expressed in the arcuate with some
overlapping with the expression pattern of Ntrk2 (bottom), which encodes a neurotrophic receptor. DViolin plot of the mRNA expressions of Il6,
Il6ra, Il6st, Cx3cr1, Vim, Ntrk2, and Gfap in the arcuate hypothalamus, per main cell types. Oligodend, oligodendrocyte; NG2/OPC, oligodendrocyte
progenitor cells; PVMMicro, perivascular macrophages/microglia; VLMCs, vascular and leptomeningeal cells; ParsTuber, pars tuberalis
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 8 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
compared to control (Fig. 6D). Regarding transcripts re-
lated to neurogenesis, at age 7 days, IL6 KO presented
lower levels of hypothalamic Gfap (Fig. 6E), whereas at
age 8 weeks there were no differences between IL6 KO
and control (Fig. 6F).
IL6 increases markers of neurogenesis in hypothalamic
neuroprogenitor cells
Next, we employed neuroprogenitor cells (NPCs) ob-
tained from IL6 KO mice. The cells were expanded
using a method to generate neurospheres. Thereafter,
NPCs were employed to determine the direct effect of
IL6 to promote differentiation towards a neuronal fate.
The expressions of Pomc and Npy transcripts were in-
creased in the hypothalamic NPCs obtained from IL6
KO mice (Fig. 7A). When IL6 was added to culture
media, there was an increased expression (mRNA and
protein) of doublecortin and reduced mRNA expressions
of Pomc and Npy (Fig. 7B–D). In addition, IL6 pro-
moted a trend to increase the number of doublecortin
positive cells (Fig. 7C, D) and a significant increase in
the number of MAP2 positive cells (Fig. 7E, F). In NPCs
differentiated for longer time (10–14 days), IL6 pro-
moted increased expressions of doublecortin (Fig. 7G,
H) and NeuroD1 transcripts (Fig. 7I, J), a reduction of
Pomc (Fig. 7K), and no changes in Npy (Fig. 7L) and
Gfap transcripts (Fig. 7M).
Discussion
Adult neurogenesis warrants neuronal renewal throughout
life [25]. It relies on the existence of neural stem/progenitor
cells (NSPCs) that reside in a few brain niches and respond
Fig. 3 Hypothalamic transcript expression of IL6. In order to identify obese-prone (H) and obese-resistant (L) mice, we performed the procedure as depicted in A. The
expression of hypothalamic IL6 was determined using real-time PCR (B). Palmitate was injected in a single intracerebroventricular (ICV) dose and hypothalamus was
collected (C) for determinations of IL6 (D), TNFα(E), and IL1β(F)transcripts.B,n=7–8mice/group;D–F,n=5mice/group;*p< 0.05, **p<0.01,***p<0.001.Inbar
graphs, results are presented as mean ± standard deviation
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 9 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
to distinct endogenous and exogenous stimuli, differentiating
to integrate specific networks [55–59]. The SVZ and the
SGZ are the most important neurogenic niches producing
new neurons involved in a multitude of functions such as
memory, cognition, and olfaction [60–62]. A smaller neuro-
genic niche exists in the hypothalamus and is responsible for
the renewal of neurons involved in whole body energy
homeostasis [27,28].
Neurons of the mediobasal hypothalamus (MBH) con-
trol food intake and energy expenditure in response to
signals delivered by hormones, neural inputs, and nutri-
ents [44,63–67]. In obesity and aging, hypothalamic in-
flammation leads to neuronal loss, which affects the
balance between neuronal subpopulations exerting ana-
bolic and catabolic functions [68–71]. Certain stimuli
can promote a neurogenic response that results in the
Fig. 4 IL6 effect on hypothalamic cell proliferation and survival. Mice were treated with IL6 and BrdU according to the protocols as depicted in Aand D.The
mediobasal hypothalamus was prepared for analysis using immunofluorescence and confocal microscopy; cell counting was performed using 20× and 40×
magnifications. Representative images are presented in B(proliferation assay) and E(survival assay). Total BrdU countings are presented in C(proliferation assay)
and F(survival assay). In both assays, n= 6 mice/group. In bar graphs, results are presented as mean ± standard deviation
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 10 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
generation of neurons that could reestablish homeostasis
[27,28,31]. Thus, it is believed that pharmacological
and nutritional approaches leading to the generation of
new hypothalamic neurons that counterbalance the ef-
fects of obesity and ageing could be useful in therapeu-
tics [63,72–74].
Here, we tested the hypothesis that IL6 could induce
hypothalamic neurogenesis. The hypothesis was based in
two premises: (i) IL6 produced in response to exercise
improves memory and cognition, at least in part, due to
its neurogenic roles in the hippocampus [19,23] and (ii)
IL6 produced in response to exercise reduce obesity-
associated hypothalamic inflammation restoring the con-
trol of food intake and energy expenditure [21].
First, we showed that exogenous IL6 can reduce body
mass gain in mice fed HFD but not in mice fed chow.
These results are in concert with the fact that IL6 KO
mice develop late-onset obesity and the replacement of
exogenous IL6 corrects the phenotype [75]. Most of the
effects of IL6 to reduce body mass depends on its action
in the hypothalamus [75]. It is long known that both IL6
and its receptor are expressed in the hypothalamus [76];
however, with the recent advance in single-cell tran-
scriptome technology, details regarding the expression of
distinct transcripts can be traced to subpopulation levels
[77]. To explore the expression profiles of Il6 and Il6ra
in the hypothalamus cellular landscape, we analyzed
20,921 public single-cell transcriptomes [46] with the
dbMAP dimensionality reduction method [45]. This is
the first single-cell analysis of IL6 and IL6 receptors in
the hypothalamus, providing advance by showing that
Il6 is lowly expressed and restricted to microglia and
Fig. 5 IL6 induces neurogenesis in the hypothalamus. Mice were treated with a single intraperitoneal dose of IL6, according to the protocol as
depicted in A. The hypothalamic expressions of Sox6 (B) and Sox2 (C) were determined using real-time PCR. The transcript expressions of Sox6
(D) and doublecortin (E) were determined in the hypothalami collected from obese-prone (H) and obese-resistant (L) mice and treated with a
single dose of IL6. FMice were treated with IL6 and BrdU according to the protocols as depicted in Fig. 4D. The mediobasal hypothalamus was
prepared for analysis using immunofluorescence and confocal microscopy; cell counting was performed using 20× and 40× magnifications. Total
BrdU/NeuN counting is depicted in G (neurogenesis assay). B,Cn= 5 mice/group; D,En=8–10 mice/group; F,G,n= 5 mice/group. *p< 0.05,
**p< 0.01. In bar graphs, results are presented as mean ± standard deviation
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 11 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
tanycytes, whereas its canonical receptor Il6ra is consist-
ently expressed in ependymocytes, astrocytes, endothe-
lial cells, and particularly in the microglia. On the other
hand, Il6st, which is needed for Il6ra signaling and
might be directed activated by IL6 trans-signaling [78],
is broadly expressed across almost all cell types, and par-
ticularly in neurons and tanycytes, although Il6st and
Il6ra mRNA co-expression was detectable only in micro-
glia and ependymocytes, suggesting classical IL6 signal-
ing through IL6RA is restricted to these cell types in the
arcuate hypothalamus.
In humans, exposure to environmental factors that
predispose to obesity, results in variable body mass gain,
which occurs according to a normal distribution [79].
This reflects the genetic nature of obesity-prone and
obesity-resistant phenotypes [80]. Similar pattern occurs
whenever outbred rodents are fed on HFD [49,81]. Pre-
viously, we showed that rapid regulation of hypothalamic
POMC following the introduction of HFD is an import-
ant factor determining predisposition to obesity [49,70].
Here, we showed that obesity-resistant mice express lar-
ger amounts of IL6 than obesity-prone mice. These data
Fig. 6 IL6 deficiency predisposes to obesity. IL6 was determined in the serum of wild-type (WT) and IL6 knockout (KO) mice treated or not with 1.0 ng exogenous
IL6 (A). Body mass was determined at age 8 weeks (B). The expression of transcripts encoding for hypothalamic neurotransmitters (C,D) and neurogenesis-related
genes (E,F) were determined using real-time PCR in hypothalamic samples collected in mice aged 7 days (C,E)or8weeks(D,F). An=3–4mice/group;n=25
mice/group; C,En=5–7 mice/group; D,Fn=9mice/group.*p< 0.05, ***p< 0.001. In bar graphs, results are presented as mean ± standard deviation
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 12 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Fig. 7 (See legend on next page.)
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 13 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
provide additional support for the anti-obesity actions of
IL6 [21,75] and suggests that enhanced capacity to pro-
duce IL6 in the hypothalamus could be a genetically de-
termined mechanism that protects against excessive
body mass gain. This is further supported by human
studies that show both obesity-protection [3] and obesity
predisposition [82] associated with different polymor-
phisms of the IL6 gene.
Next, using a living-mouse model, we showed that ip
IL6 was capable of increasing the number of newly gen-
erated neurons in the arcuate nucleus of the hypothal-
amus. This was accompanied by the increased
expression of neurogenic-related transcripts Sox2 and
Sox6. Moreover, we showed that the neurogenic re-
sponse was more evident in obesity-resistant mice. This
is the first evidence for a pro-neurogenic action of IL6 in
the hypothalamus. There are few studies that have evalu-
ated the potential implication of IL6 in adult neurogen-
esis and all of them were focused on the hippocampus.
Some studies showed that chronically, IL6 could impair
neurogenesis [83,84]; however, two studies have shown
that in the context of exercise, IL6 could promote
neurogenesis impacting positively in memory and cogni-
tion [19,23], whereas another study has shown an in-
crease in hippocampal IL6 occurring in response to a
neurogenic stimulus provided by marrow-derived mes-
enchymal stem cells [85].
Further insight into the roles of IL6 in hypothalamic
neurogenesis was obtained determining the expression of
neurogenic-related genes and neurotransmitters in IL6 KO
mice. First, we reproduced results of previous studies show-
ing that IL6 KO mice are obese-prone [75]. Next, we showed
that in mice aged 7 days there was increased expression of
hypothalamic Gfap, whereas in mice aged 8 weeks, there was
a reduction of POMC. This could indicate an abnormal
neurogenic process, since Gfap is a marker of both astrocytes
and tanycytes, cells that are involved in hypothalamic neuro-
genesis [86,87]. The reduction of POMC could result from
the abnormal neurogenic process and contribute to the
obese-prone phenotype of IL6-deficient mice [31,88,89].
In the last part of the study, we evaluated NPCs pro-
duced from IL6 KO mice hypothalami. Both prolifera-
tion rate and expression of POMC and NPY were
increased at the baseline. Upon treatment with IL6,
NPCs that differentiated for five days presented in-
creased expression of doublecortin and decreased ex-
pression of POMC and NPY. Doublecortin is a
microtubule-associated protein that expresses early dur-
ing the development of neurons [90]; we reasoned that
stimulation with IL6 could be inducing an upsurge of
neurogenesis and only undifferentiated neurons could be
detected at this time. Therefore, we increased differenti-
ation time for 10 and 14 days; NPY expression was nor-
malized, however, increased doublecortin persisted and
was accompanied by similar changes in the expression
of NeuroD1, another important factor associated with
adult neurogenesis [91]. Nevertheless, the IL6-induced
reduction of POMC persisted suggesting that in the IL6
deficient environment the stimulus with exogenous IL6
could generate an abnormal pattern of either survival or
generation of specific neuronal populations.
Because of the functional nature of many neurons in
the MBH, which are involved in the control of caloric
intake and energy expenditure, it was expected that
neurogenic stimuli affecting neurons of this region could
impact on whole body energy homeostasis. In fact, we
showed that exogenous IL6 reduces body mass, whereas
IL6 deficiency results in obesity predisposition. More-
over, in IL6 KO mice, the hypothalamic transcript levels
of POMC are reduced. At least in part, these effects
could be attributed to the hypothalamic neurogenic ac-
tions of IL6. We acknowledge that studding the whole
MBH and not specific cell subpopulations is a limitation
of this study that could be further explored in the future.
Conclusions
In this study, we provide the evidence for a role of IL6
in the regulation of hypothalamic neurogenesis. Both, in
living mice and NPCs, IL6 stimulated cell proliferation
and induced the expression of markers of immature
neurons; however, in IL6 deficiency, exogenous IL6 ad-
ministration seem to modify the pattern of differenti-
ation of neurons. Further studies should define the
mechanism linking IL6-induced neurogenesis with neur-
onal fate determination.
Abbreviations
ASCL1: Achaete-scute homolog 1; BDNF: Brain-derived neurotrophic factor;
BrdU: Bromodeoxyuridine; CEUA: Ethics committee on animal use;
Ct: Control; Cx3Cr1: C-x3-c motif chemokine receptor 1; DCX: Doublecortin;
(See figure on previous page.)
Fig. 7 IL6 induce neurogenic factors expression in hypothalamic neuroprogenitor cells (NPCs). Transcripts were determined using real-time PCR in the NPCsof
WT and IL6 KO mice after a 5-day differentiation period: first, WT and IL6 KO hypothalamic cells were analyzed in basal conditions (A), and then, IL6 KO NPCs
were treated with exogenous IL6 in doses depicted (B). Representative immunocytochemistry images of 7-day differentiated cells (green, DCX; red, GFAP; blue,
DAPI) (C) and DCX-positive cell counting (D), and representative immunocytochemistry images of 14-day differentiated cells (green, MAP2; red, GFAP; blue,
DAPI) (E) and MAP2-positive cell counting (F). After 10- (G,I,K–M) or 14-day (Hand J) differentiation period, transcripts were determined using real-time PCR in
the NPCs of WT and IL6 KO mice treated with exogenous IL6 in doses depicted in the panels (G-M). A,Bn=12(3–4 well/group, 2 independent experiments);
C–Fn= 24 (4 well/group, 2 independent experiments); G–Mn=48(6–9 well/group, 3 independent experiments). *p< 0.05, **p< 0.01. In bar graphs, results
are presented as mean ± standard deviation
Bobbo et al. Journal of Neuroinflammation (2021) 18:192 Page 14 of 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
DMEM: Dulbecco’s modified eagle medium; DNA: Deoxyribonucleic acid;
EGF: Endotelial growth factor; FGF: Fibroblast growth factor; GAPD
H: Glyceraldehyde 3-phosphate dehydrogenase; GFAP: Glial fibrillary acidic
protein; HFD: High-fat diet; ICV: Intracerebroventricular; IL-1β: Interleukin-1β;
IL-6: Interleukin-6; IL6ra: Interleukin-6 receptor subunit alpha;
IL6st: Interleukin-6 cytokine family signal transducer; IP: Intraperitoneal;
KO: Knockout; L: Low intake of high-fat diet; H: High intake of high-fat diet;
HCl: Hydrochloric acid; MAP 2: Microtubule-associated protein 2;
MBH: Mediobasal hypothalamus; mRNA: Messenger ribonucleic acid;
NeuroD1: Neuronal differentiation; NeuN: Nuclear neuronal antigen;
NPC: Neural progenitor cells; NSC: Neural stem cells; Ntrk2: Neurotrophic
receptor tyrosine kinase 2; NPY: Neuropeptide Y; PBS: Phosphate saline
buffer; PCR: Polymerase chain reaction; POMC: Proopiomelanocortin;
RNA: Ribonucleic acid; SGZ: Subgranular zone; SOX: Sex determining region
Y box; SVZ: Subventricular zone; TNF-α: Tumor necrosis factor α; WT: Wild-
type
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12974-021-02242-8.
Additional file 1: Supplementary Figure 1. Effect of exogenous IL6
on caloric intake and body mass. Wild-type mice were fed on chow or
high-fat diet (HFD) and treated intraperitoneally (IP) with exogenous IL6
as depicted in Figure 1A. Body mass (A) and caloric intake (B) were deter-
mined in mice fed chow and HFD. In all experiments, n=5 mice/group.
Two-way ANOVA with repeated measures and Bonferroni post-test was
applied for the statistics of all data. *p<0.05, IL6 HFD vs. saline HFD (day 7
and day 14). *p<0.05, saline HFD vs. saline chow (day 14). *p<0.05, saline
HFD vs. IL6 chow (day 14). Supplementary Figure 2. Bayesian analysis
of single-cell RNA sequencing data and key genes coexpression patterns.
A-F. dbMAP embeddings of the arcuate nucleus and median eminence
scRNAseq data, colored by raw mRNA expression levels (left) or corre-
sponding Bayesian probabilistic-denoised counts levels (right). Bayesian
analysis was performed with BASiCS (Bayesian Analysis for Single-Cell Se-
quencing). G. dbMAP embedding key-colored by Nes (red) and Il6st
(green) mRNA co-expression levels. H. dbMAP embedding key-colored by
Cx3cr1 (red) and Il6ra (green) mRNA co-expression levels. I. dbMAP em-
bedding key-colored by Il6ra (red) and Il6st (green) mRNA co-expression
levels. Supplementary Figure 3. Heatmap of top five marker genes for
the arcuate nucleus and median eminence main cell types. Heatmap
showing mRNA expression of top five marker genes from the main cell
types of the arcuate nucleus and median eminence. Marker genes were
found with a Wilcoxon rank-sum test and then scaled. Z-scores are then
used for coloring. Supplementary Figure 4. IL6 effect on hypothalamic cell
proliferation and survival. Mice were treated with IL6 and BrdU according
to the protocols as depicted in Fig. 4A and 4D. The mediobasal hypothal-
amus was prepared for analysis using immunofluorescence and confocal
microscopy; cell counting was performed using 20X and 40X magnifica-
tions. Abercrombie corrected total BrdU countings are presented in A
(proliferation assay) and B (survival assay). In both assays, n=6 mice/
group). In bar-graphs results are presented as mean ± standard deviation.
Supplementary Figure 5. Total number of cells determined in the experi-
ment depicted in Figure 5G. Mice were treated with IL6 and BrdU accord-
ing to the protocols as depicted in Figure 4D. The mediobasal
hypothalamus was prepared for analysis using immunofluorescence and
confocal microscopy; cell counting was performed using 20X and 40X
magnifications. N=5 mice/group. Supplementary Table 1.. Detailed in-
formation of primers used in the study. All primers were purchased from
Thermo Fischer Scientific. Supplementary Table 2. Raw data for statis-
tical analysis.
Acknowledgements
Vanessa Bobbo received financial support from the São Paulo Research
Foundation. The authors thank Erika Roman, Marcio Cruz, and Gerson Ferraz
for laboratory management. The study was supported by grants from the
São Paulo Research Foundation (2013/07607-8) and Conselho Nacional de
Pesquisa e Desenvolvimento Científico.
Authors’contributions
Conceptualization, VCB, JM, LAV, and EPA. Methodology, VCB, DFE, and JM.
In vivo experiments, VCB, DFE, CPJ, NFM, RH-T, TPP, and JM. Ex vivo experi-
ments, VCB and DFE. Single-cell RNA data analysis, DS-O. Resources, LAV and
EPA. Analyses, VCB, JM, LAV, and EPA. Writing, VCB, DFE, LAV, and EPA. The
authors read and approved the final manuscript.
Funding
The study was supported by grants from the São Paulo Research Foundation
(2013/07607-8) and Conselho Nacional de Pesquisa e Desenvolvimento
Científico.
Availability of data and materials
Data and materials are available upon request.
Declarations
Ethics approval and consent to participate
All experiments were conducted according to the “Guide for the Care and
Use of Laboratory Animals of the Institute of Laboratory Animal Resources,
US National Academy of Sciences”and were approved by the Ethics
Committee (CEUA IB/UNICAMP #3987-1, #4361-1, #4775-1). All animal
experiments were performed in a biosafe facility and were approved by the
University of Campinas Biosafety Committee.
Consent for publication
All authors have provided informed consent for publication of this study.
Competing interests
The authors declare that they have no competing interests.
Received: 22 April 2021 Accepted: 18 August 2021
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