DOI: 10.1002/JLB.

3MA0520-113R

ARTICLE

Mycobacterium tuberculosis-infected alveolar epithelial

cells modulate dendritic cell function through the
HIF-1𝜶-NOS2 axis

Tamara Silva Rodrigues1 * Annie Rocio Piñeros Alvarez2 * Ana Flávia Gembre1
Maria Fernanda Pereira de Araújo Demonte Forni4 Bruno Marcel Silva de Melo3 *
José Carlos Farias Alves Filho3 * Niels Olsen Saraiva Câmara4 *
Vânia Luiza Deperon Bonato1 *

1 Department of Biochemistry and Immunology,

Ribeirao Preto Medical School, University of Sao Abstract

Paulo, Ribeirao Preto, Brazil
2 Indiana University – Purdue University
Indianapolis, Indianapolis, Indiana, USA
3 Department of Pharmacology, Ribeirao Preto
Medical School, University of Sao Paulo, Ribeirao
Preto, Brazil
4 Transplantation Immunology Laboratory,
Department of Immunology, Institute of
Biomedical Sciences, University of Sao Paulo,
Sao Paulo, Brazil
Correspondence
Vânia Luiza Deperon Bonato, Department of
Biochemistry and Immunology, University of Sao
Paulo, Ribeirao Preto Medical School, FMRP-
USP. Av. Bandeirantes 3900, Ribeirao Preto, SP
14049–900, Brazil.
Email: vlbonato@fmrp.usp.br
* Basic and Applied Immunology Program –
FMRP-USP.
Tuberculosis kills more than 1 million people every year, and its control depends on the effec-
tive mechanisms of innate immunity, with or without induction of adaptive immune response. We
investigated the interaction of type II alveolar epithelial cells (AEC-II) infected by Mycobacterium
tuberculosis with dendritic cells (DCs). We hypothesized that the microenvironment generated by
this interaction is critical for the early innate response against mycobacteria. We found that AEC-
II infected by M. tuberculosis induced DC maturation, which was negatively regulated by HIF-1𝛼-
inducible NOS2 axis, and switched DC metabolism from an early and short peak of glycolysis to a
low energetic status. However, the infection of DCs by M. tuberculosis up-regulated NOS2 expres-
sion and inhibited AEC-II-induced DC maturation. Our study demonstrated, for the first time, that
HIF-1𝛼-NOS2 axis plays a negative role in the maturation of DCs during M. tuberculosis infection.
Such modulation might be useful for the exploitation of molecular targets to develop new thera-
peutic strategies against tuberculosis.
KEYWORDS
epithelial cells, dendritic cells, mycobacterium tuberculosis, inducible nitric oxide synthase, hypoxia
Inducible factor alpha

1 INTRODUCTION to Mycobacterium tuberculosis are latently infected, innate immunity,

The World Health Organization (WHO) estimates that there are
around 10 million people feeling ill with tuberculosis (TB), and approx-
imately 7 million new cases were notified in 2018. The disease
accounted for 1.5 million of deaths in the same year, making it 1 of the
10 leading causes of death by infectious diseases in the world (World
Health Organization).1 Because the great majority of subjects exposed
Abbreviations: AEC, alveolar epithelial cells; BCG, Bacillus Calmette Guerin; BMDC, bone
marrow-derived dendritic cell; BMDM, bone marrow-derived macrophages; ECAR,
extracellular acidification rate; HIF-1𝛼, hypoxia inducible factor alpha; MMP1,
metalloproteinase 1; NIC, noninfected MLE-15 conditioned medium; OCR, oxygen
consumption rate; OXPHOS, oxidative phosphorylation; TB, tuberculosis.
with or without induction of memory/adaptive immune response, must
play a critical role in the balance of progression of infection and
immunopathology.2 In this context, it is imperative to understand the
crosstalk among the first cells infected by M. tuberculosis, as these cells
drive the molecular mechanisms essential for controlling the dissemi-
nation of mycobacteria.
The alveolar space consists mainly of 2 epithelial cells (ECs): type I
and type II alveolar epithelial cells (AEC-I and AEC-II). AEC-I are squa-
mous cells that constitute ∼95% of alveolar space, being the majority of
ECs in the alveolar space. They are important for gas exchange due to
their large surface area very close to capillaries.3–5 AEC-II are cuboidal
cells and comprises ∼5% of the alveolar space. They protect the lungs

Received: 12 February 2020 Revised: 8 May 2020 Accepted: 17 May 2020

J Leukoc Biol. 2020;1–14. www.jleukbio.org ○
c 2020 Society for Leukocyte Biology 1
2 RODRIGUES ET AL .
from collapse, act on tissue repair after injury, and contribute to the
protection of the alveolar space through the secretion of surfactant
proteins.3,6,7
Human AEC-II (A549 cell line) take up M. tuberculosis through glyco-
proteins described as syndecans8 and they are permissive to M. tuber-
culosis infection.9 AEC-II infected with M. tuberculosis secrete antimi-
crobial peptides,10,11,62 IFN-𝛾,12 CXCL8,13–15 CXCL5,16 NO,17,18
CCL2,13 and metalloproteinase 1 (MMP1).19 However, EC may also
play an anti-inflammatory role as bronchial EC infected with Bacillus
Calmette Guerin (BCG) secreted IL-10 and IL-22.20
The interplay of AEC-II and macrophages has been investigated
in M. tuberculosis infection.21,22 AEC-II stimulated with keratinocyte
growth factor secreted GM-CSF which activates the microbicidal abil-
ity of bone marrow-derived macrophages (BMDM) by mechanisms
dependent on NO production and phagolysosome fusion.21 In a differ-
ent approach, supernatants from THP-1 cells infected with M. tuber-
culosis induced the secretion of S100 proteins by human primary
bronchial ECs.23 Recently, it was shown that the components of the
alveolar lining fluid influence not only M. tuberculosis growth rate inside
AEC-II, but also the ability of those cells to modulate macrophage
inflammatory response.24
Besides AEC and macrophages, lung dendritic cells (DCs) can also
be infected by M. tuberculosis.25,26 M. tuberculosis hampers DC matu-
ration and migration. For instance, the expression of CD80 and CD86
was only significantly enhanced 21 days post infection in the lymph
nodes of infected BALB/c mice.27 It has been shown that M. tubercu-
losis can also prevent DC-mediated antigen presentation by a mech-
anism independent of MHC-II regulation.28 DC activation also relies
on metabolic changes. The activation of immature DCs requires switch
from oxidative phosphorylation (OXPHOS) and oxidation of fatty
acids to higher glycolysis rate.29 Mammalian Target of Rapamycin and
hypoxia inducible factor alpha (HIF-1𝛼) transcription factors have been
shown to be critical for the switch to glycolysis during DC activation.29
However, it has also been shown that HIF-1𝛼 accumulation could
induce prolonged down-regulation of CD80 and CD86 expression by
a mechanism dependent on increased inducible NOS2 expression, an
important HIF-1𝛼 target gene.30
The role of HIF-1𝛼–NOS2 axis and the associated metabolic sta-
tus of DC activation in TB have not been explored. Besides, in the
context of TB, it has not been determined if the interference of
the microenvironment in the alveolar space, such as the infected
ECs, could interfere with DC activation. To address this, we cul-
tured CD11c+ CD11b+ F4/80− bone marrow-derived dendritic cells
(BMDCs) with conditioned media obtained from the supernatants of
M. tuberculosis-infected AEC-II and investigated the maturation and
activation status of DCs. We found that M. tuberculosis-infected AEC-
II induced DC maturation through a pathway partially dependent
on low levels of HIF-1𝛼-NOS2 expression. However, in vivo assays
showed that there was a higher expression of HIF-1𝛼 gene and higher
frequency of NOS2+ CD11b+ DCs in the lungs of M. tuberculosis-
infected mice. Therefore, in DCs infected with M. tuberculosis, infection
increases NOS2 expression such that AEC-II loses their ability to mod-
ulate DC maturation.
2 RESULTS
2.1 AECs are permissive to M. tuberculosis infection,
and they produce proinflammatory mediators
First, we determined if MLE-15, a mouse lung EC line, would be
permissive to M. tuberculosis growth, as previously described for A549
cells, an adenocarcinomic human alveolar basal EC line.9 MLE-15
cells were also permissive to M. tuberculosis infection and allowed a
progressive bacillus growth over 72 h of infection (Figs. 1A and 1B),
indicating that MLE-15 cells are unable to control bacterial growth.
Next, we investigated if M. tuberculosis-infected MLE-15 cells would
produce inflammatory mediators. An increased production of nitrite
(NO2- ), IL-6, CCL5, IFN-𝛾, and S100A9 was observed in the super-
natant of infected cells (Figs. 1C–1G), whereas IL-1𝛽 and IL-10 were
not detected (data not shown).
2.2 AECs infected by M. tuberculosis positively
modulate maturation of DCs
Although AEC-II stimulated with keratinocyte growth factor increased
the ability of BMDM to kill M. tuberculosis,21 it remains unclear if AEC-
II infected by M. tuberculosis affect DC activation. To address this issue,
we used 2 approaches to investigate the direct or indirect effect of
M. tuberculosis-infected AEC-II on BMDC activation. The coculture of
M. tuberculosis-infected MLE-15 and BMDC (Supplemental Fig. 1A)
increased the expression of CD80 (Supplemental Figs. 1B and 1C) and
CD86 (Supplemental Figs. 1D and 1E), as well as increased the produc-
tion of IL-1𝛽 (Supplemental Fig. 1F), IL-6 (Supplemental Fig. 1G), and
IL-10 (Supplemental Fig. 1H).
To assess the indirect effect of MLE-15, BMDCs were cul-
tured/stimulated with supernatants obtained from M. tuberculosis-
infected MLE-15, which were described as M. tuberculosis-infected
MLE-15 conditioned medium (IC). Noninfected MLE-15 conditioned
medium (NIC) was used as a control (Fig. 2A). In addition, BMDCs
were left unstimulated as a negative control or were stimulated
with LPS as a positive control for BMDC activation. BMDCs were
characterized as F4/80− CD11c+ CD11b+ MHC-II+ cells (data not
shown). IC increased the frequency of MHC-II-expressing DC (Fig. 2B),
CCR7 (Fig. 2C), CD80 (Fig. 2D), and CD86 (Fig. 2E) in comparison with
NIC and unstimulated BMDC. As a positive control, LPS increased
the frequency of MHC-II-expressing DC (Fig. 2B), CCR7 (Fig. 2C),
CD80 (Fig. 2D), and CD86 (Fig. 2E) in comparison with unstimulated
BMDC. Besides, IC induced the production of both proinflammatory
and anti-inflammatory cytokines by BMDC compared with NIC. A
significant increase in IL-1𝛽 (Fig. 2F), IL-10 (Fig. 2G), and IL-6 (Fig. 2H)
was observed when BMDCs were cultured with IC. Dotted line in the
Fig. 2H represents the levels of IL-6 quantified in the supernatants of
AEC-II infected by M. tuberculosis (CM-IC). IL-1𝛽 and IL-10 were not
detected in the cultures of infected or uninfected AEC-II. These find-
ings show that indirect contact with AEC-II infected by M. tuberculosis
induced the maturation of DCs and a mixed profile of proinflamma-
tory and anti-inflammatory cytokines. Next, we determined if the
RODRIGUES ET AL . 3

F I G U R E 1 Alveolar epithelial cells are permissive to M. tuberculosis infection and exhibit a proinflammatory profile. Immortalized murine
lung epithelial cells (MLE-15) (1 × 106 cells/well) were infected with M. tuberculosis H37Rv (MOI10). (A) At 4 h after infection, M. tuberculosis was
observed by electronic microscopy. Full black arrows show the bacilli inside the cells. Dotted arrows show the vacuoles formed upon infection.
(B) CFU number was counted at 24, 48, and 72 h of infection. (C) NO2- production, and (D–G) IL-6, CCL5, IFN-𝛾, S100A9 secretion at 48 h of
infection by Griess assay and ELISA, respectively. The results are representative of 3 independent experiments expressed as the mean ± SEM
(n = 4/experimental condition). *P ≤ 0.05. CFU, colony forming units

maturation and activation of BMDC by IC would induce the prolifera-
tion of naive CD4+ T cells. To do this, BMDCs were exposed to NIC or
IC, and subsequently cocultured with CD4+ CD62L+ cells stimulated
with mAb anti-CD3. Figure 2I shows that BMDC previously stimulated
with IC significantly increased the proliferation of naive CD4+ T cells
compared with CD4+ T cells stimulated with NIC.
To confirm that the activation of BMDC was indeed associated with
mediators secreted by infected AEC-II and not compounds from the
mycobacterium, BMDCs were stimulated for 48 h with filtered media
from M. tuberculosis culture, which consisted only of mycobacterium
released products. M. tuberculosis-secreted products (Mtb-media)
significantly reduced the expression of MHC-II (Supplemental Figs.
2A and 2B), CCR7 (Supplemental Figs. 2C and 2D), and CD86 (Sup-
plemental Figs. 2E and 2F), and had no effect on NOS2 expression
(Supplemental Figs. 2G and 2H) in BMDC. These findings show that
mediators secreted by infected MLE-15 cells, and not compounds
from the mycobacterium, induce the maturation and activation
of BMDC.
2.3 AECs infected by M. tuberculosis modulate
metabolism of DCs and HIF-1𝜶 accumulation
A metabolic switch to glycolysis has been shown to be involved in DC
activation.29,31 Next, we investigated if the activation induced by MLE-
15 cells infected by M. tuberculosis would induce the same metabolic
pattern in BMDC. BMDCs left unstimulated (media) or stimulated with
LPS as negative and positive controls, respectively, confirmed that the
basal activation of BMDC was characterized by increased levels of
OXPHOS, measured as oxygen consumption rate (OCR) (Figs. 3A and
3C). BMDC activation was characterized by metabolic shift toward
glycolysis, characterized by elevated levels of ECAR and decreased
respiration (Figs. 3E and 3F). BMDC exposed to NIC exhibited the
same pattern described for the cells cultured in media only, with
higher oxidative metabolism (Fig. 3B). However, cells exposed to IC
exhibited decreased ATP generation-dependent oxygen consumption
(Fig. 3B), suggesting an impairment in the oxidative metabolism, and
a prominent metabolic shift toward glycolysis as evident by more
than 4-fold increase in the levels of extracellular acidification rate
(ECAR) (Fig. 3E). After 24 h, the pattern of LPS stimulation remained
consistent with that in the literature, with a significant glycolytic shift
(Figs 3C and 3F). BMDC exposed to NIC presented an even clearer
pattern of solid and strong oxidative metabolism, with increased levels
of basal, maximal, ATP-linked, and spare capacity respiration when
compared with the 2 h stimulation (Figs. 3B and 3D). In addition,
they showed lower levels of ECAR than those stimulated with LPS
(Figs. 3E and 3F). Although ECAR levels were elevated in BMDC
stimulated with IC for 2 h, the levels were consistently lower at 24 h of
IC stimulation compared with LPS and NIC stimulation (Fig. 3F). While
the cells were still able to respond to all the injected drugs, the overall
levels of oxygen consumption were greatly decreased (Fig. 3D), and
they displayed the lowest levels of ECAR after 24 h of IC stimulation
compared with other culture conditions (Fig. 3F).
Next, we assessed the expression of HIF-1𝛼 because of its role in
the maintenance of high glycolytic rates.32–35 HIF-1𝛼 gene and protein
expression had an early significant increase at 12 h and a reduction at
24 h (Figs. 3G and 3H) after IC stimulation. These findings support the
4 RODRIGUES ET AL .

F I G U R E 2 Alveolar epithelial cells infected by M. tuberculosis positively modulate maturation of DCs. (A) BMDC (1 × 106 cells/well) were
cultured for 24 h in different conditions: media (-), LPS 100 ng/ml (LPS), supernatants from noninfected MLE-15 cells (NIC), and M. tuberculosis-free
supernatants from infected MLE-15 cells (IC). (B) MHC-II, (C) CCR7, (D) CD80, (E) CD86 expression on CD11c+ CD11b+ F4/80− cells were analyzed
by flow cytometry. (F) IL-1𝛽, (G) IL-10, and (H) IL-6 secretion were measured at 24 h by ELISA. (I) Flow cytometry analysis of Ki67, a marker of
proliferation of naive CD4+ CD62L+ cells stimulated with mAb anti-CD3 and cocultured for 96 h with nonstimulated BMDC, or BMDC previously
stimulated with NIC or IC for 24 h. The results are representative of 3 independent experiments expressed as the mean ± SEM (n = 4/experimental
condition). *P ≤ 0.05; & P ≤ 0.05 compared with control (-)

early glycolytic peak followed by a lower rate of glycolysis as indicated 2.4 HIF-1𝜶-NOS2 axis plays a negative role in
by the low ECAR at 24 h of stimulation with IC (Fig. 3F). In addition, maturation of DCs
we found an early increase in the gene expression of the glucose trans-
Considering the early peak of glycolysis and the initial accumulation
porter, glut1 (Fig. 3I), and hk2 (hexokinase 2) (Fig. 3J) in IC-stimulated
of HIF-1𝛼, we hypothesized that HIF-1𝛼 would play a role in the
BMDC when compared with NIC-stimulated BMDC. These findings
increased maturation of DCs induced by infected MLE-15 cells. A
indicate an early and nonpersistent increase in both glycolysis and HIF-
prolyl-hydroxylase inhibitor (DMOG) was used to maintain the expres-
1𝛼 expression. To confirm our findings, the expression of NOS2, a HIF-
sion of HIF-1𝛼. DCs treated concomitantly with IC and DMOG lost the
1𝛼 target gene, was also measured by flow cytometry. BMDC exposed
ability to regulate MHC-II (Fig. 4A), CCR7 (Fig. 4B), CD80 (Fig. 4C), and
to IC exhibited no change in NOS2 expression (Figs. 3K and 3L) in com-
CD86 (Fig. 4D), when compared with those stimulated with only IC. As
parison with unstimulated BMDC, BMDC exposed to NIC, and those
expected, the addition of DMOG in BMDC cultures stimulated with IC
stimulated with LPS. NO2- detection in the supernatants of BMDC
induced the expression NOS2 in the CD11b+ DCs population (Fig. 4E).
stimulated with IC are NO2- levels secreted by the single infected MLE-
Interestingly, the majority of NOS2+ cells did not express costimula-
15 cells (CM-IC, dotted line), indicating that the BMDC stimulated with
tory molecules while cells expressing costimulatory molecules barely
IC did not produce extra levels of NO2- (Fig. 3M).
marked positive for NOS2 as shown by the representative analysis
RODRIGUES ET AL . 5

F I G U R E 3 Alveolar epithelial cells infected by M. tuberculosis modulate metabolism of DCs and HIF-1𝜶 accumulation. BMDC (1 × 106
cells/well) were cultured in different conditions: media (-), LPS 100 ng/ml (LPS), supernatants from noninfected MLE-15 cells (NIC) and M.
tuberculosis-free supernatants from infected MLE-15 cells (IC). Real time whole-cell respiration was assessed in (A and B) 2 and (C and D) 24 h.
Oligomycin, CCCP and rotenone plus antimycin were added where indicated. Levels of extracellular acidification rate were measured as a proxy for
lactate production during glycolysis at (E) 2 h and (F) 24 h of induction. (G and H) Gene and protein expression of hif-1𝛼/HIF-1𝛼, gene expression of
(I) glut1 and (J) hk2 (hexokinase 2) were evaluated at indicated times. (K) Frequency of CD11c+ CD11b+ MHC-II+ F4/80− NOS2+ and (L) NOS2 MFI
at 24 h of culture. (M) Levels of NO2- . The results are representative of 3 independent experiments expressed as the mean ± SEM (n = 4/experi-
mental condition). *P ≤ 0.05; & P ≤ 0.05 compared with control (-)
6 RODRIGUES ET AL .

F I G U R E 4 HIF-1𝜶-NOS2 axis plays a negative role in maturation of DCs. (A–F) BMDC (1 × 106 cells/well) from WT mice were cultured for 24 h
in different conditions: media (-), LPS 100 ng/ml (LPS), supernatants from noninfected MLE-15 cells (NIC) and supernatants from infected MLE-15
cells (IC), and IC + DMOG (200 𝜇M). (A) MHC-II, (B) CCR7, (C) CD80, (D) CD86, and (F) NOS2 expression were evaluated by flow cytometry. (F)
Representative analysis of costimulatory molecules and NOS2 expression by flow cytometry. (G–J) BMDC (1 × 106 cells/well) from WT and nos2-/-
mice were cultured for 24 h in different conditions: supernatants from noninfected MLE-15 cells (NIC) and supernatants from infected MLE-15
cells (IC), and IC + DMOG (200 𝜇M). (G and H) MHC-II, and (I and J) CD86 were evaluated by flow cytometry. The results are representative of 3
independent experiments expressed as the mean ± SEM (n = 4/experimental condition). *P ≤ 0.05; and P ≤ 0.05 compared with control (-), ns, not
significant

of MHC-II-, CCR7-, CD80-, CD86-, and NOS2-expressing DCs in the
Fig. 4F.
DCs treated with IC plus DMOG produced higher levels of IL-1𝛽
(Supplemental Fig. 3A), IL-6 (Supplemental Fig. 3B), and IL-10 (Supple-
mental Fig. 3C) compared with DCs stimulated with IC. As expected,
NO2- production was dependent on HIF-1𝛼. Dotted line represents
NO2- production by M. tuberculosis-infected MLE-15 cells (Supplemen-
tal Fig. 3D).
Next, we evaluated the expression of MHC-II, CD80, CD86, and
CCR7 on BMDC generated from WT or nos2−/− mice during the con-
comitant treatment with IC and DMOG. As previously observed, IC
stimulation positively regulated MHC-II (Figs 4G and 4H) and CD86
(Figs. 4I and 4J), whereas the addition of DMOG to IC media reduced
the expression of both molecules. The deficiency of NOS2 in DCs
had no effect on the expression of MHC-II (Figs. 4G–4I) and CD86
(Figs. 4G–4I).
RODRIGUES ET AL .
The collected findings of in vitro experiments addressing the inter-
action of AEC-II and DCs show that the maturation of DCs induced
by products secreted after the infection of MLE-15 by M. tuberculo-
sis is associated with a mixed metabolic pattern (low OCR and ECAR),
reduced expression of HIF-1𝛼 and NOS2, and absence or low produc-
tion of NO2- . DC maturation, characterized by positive regulation of
MHC-II, CD80, CD86, and CCR7 expression, is dependent on reduced
expression of HIF-1𝛼 and NOS2.
2.5 M. tuberculosis hampers AEC-II ability to induce
maturation of DCs through HIF-1𝜶–NOS2 axis
We transferred our in vitro findings to an in vivo microenvironment
by evaluating HIF-1𝛼 gene expression in the lungs of animals infected
with M. tuberculosis (Fig. 5A). We found an increased gene expression of
HIF-1𝛼 in the lungs of 30 day-infected animals (Fig. 5B). In addition, we
verified the presence of CD11b+ and NOS2+ in lung CD11c+ MHC-II+
myeloid cells from infected animals, whereas the expression of NOS2
was absent in the cells from noninfected animals (Figs. 5C–5E). Next,
we investigated the interaction of AEC-II with M. tuberculosis-infected
BMDC (Fig. 5F). Corroborating to in vivo results, M. tuberculosis is able
to increase NOS2 expression in infected DCs (Figs. 5G and 5H). Fur-
thermore, there was no significant difference in the NOS2 expres-
sion of infected BMDC and that of infected BMDC stimulated with IC
(Figs. 5G and 5H). Clearly, Mtb-infected cells had a decrease in NOS2
expression when treated with HIF-1𝛼 inhibitor (Figs. 5G and 5H). Fig-
ures 5I–5L show that infected DCs stimulated with IC were not capa-
ble of up-regulating MHC-II or CD86, as previously observed for non-
infected BMDC stimulated with IC. IC plus DMOG were not involved
in the down-regulation of MHC-II and CD86, as previously observed
for noninfected BMDC stimulated with IC plus DMOG. The inability of
infected DCs to up-regulate MHC-II and CD86 was directly dependent
on NOS2 because M. tuberculosis-infected DCs from nos2-deficient
mice exhibited a significant increase in the expression of MHC-II and
CD86. These data suggest that M. tuberculosis infection induces the
production of NO by DCs. Although M. tuberculosis infection inhibited
the infected AEC-II-induced maturation of DC, it induced the secretion
of high levels of IL-1 𝛽 and IL-6 (Figs. 5M and 5N). Furthermore, soluble
mediators secreted by infected AEC-II had no additional influence on
the production of these cytokines.
Figure 6 outlines our findings. AECs infected by M. tuberculo-
sis induced DC maturation, which was negatively regulated by HIF-
1𝛼-NOS2 axis. However, such modulation was not observed in M.
tuberculosis-infected DCs, which exhibited a significant increase of
NOS2 expression that was not affected by supernatants of infected
AEC-II cells (IC).
3 DISCUSSION
In the present study, we show, for the first time, that murine AEC-II
(MLE-15 cell line) infected by M. tuberculosis induce the maturation of
DCs, characterized by the up-regulation of MHC-II, CD80, CD86, and
7
CCR7 expression, along with the secretion of IL-1𝛽, IL-6, and IL-10.
The maturation of DCs is dependent on soluble mediators, secreted
by infected AEC-II, which switch the metabolism of DCs from an early
and short glycolysis induction to a low as well as a mixed OXPHOS
and glycolysis pattern. Moreover, the outcome of DC activation is
the absence of HIF-1𝛼-induced NOS2 expression, and consequently, a
basal production of NO. However, the maturation of DCs induced by M.
tuberculosis-infected AEC-II is dependent on whether DCs have been
previously infected by the mycobacterium or not. This is a critical issue
to be addressed because the interaction of infected AEC-II might take
place with either noninfected or infected DCs in the airways. Our study
highlights that the interaction of infected AEC-II with noninfected DCs
and their maturation determine the induction of adaptive immune
response, and the subversion of DC maturation by M. tuberculosis infec-
tion cannot be restored by contact with infected AEC-II. In the con-
text of leishmaniasis, noninfected DCs activated by soluble molecules
of the parasite induces the production of TNF and the activation of T
cells.36 Similarly, in TB, we suggest that optimal migration and activa-
tion may also occur through noninfected DCs, which might induce T
cell priming. The activation of noninfected DCs could be dependent
on the microenvironment such as soluble mediators secreted by M.
tuberculosis-infected AEC-II, and on the uptake of the extracellular vesi-
cles, exosomes, apoptotic bodies, and nonvesicular antigens released
by infected cells.37,38
The in vitro infection of DCs with mycobacteria, BCG,39 or M.
tuberculosis40 increased CD80 and CD86 expression. However, in vivo
M. tuberculosis infection down-regulated CD80 and CD86 expression
in lung myeloid cells.27 It has been reported that M. tuberculosis is effi-
cient in reducing the expression of MHC-II, and consequently the func-
tion of DCs.41 Therefore, the down-regulation of MHC and costimu-
latory molecules is 1 of the many strategies used by M. tuberculosis to
disrupt the activation of immune response. Besides, in vivo, we found a
higher gene expression of HIF-1𝛼 in the lungs of mice infected with M.
tuberculosis. A previous study reported that HIF-1𝛼 accumulated in the
lungs of TB patients with chronic infection, and it was associated with
MMP1 expression and lung cavitation.42 In addition, the accumulation
of HIF-1𝛼 in the lungs of infected BALB/c mice indicate that this tran-
scription factor is a negative regulator of apoptosis and an indicator of
the chronic phase of infection.43
Similarly to what was reported by previous studies with A549
human cell line,12–14,44–46 we demonstrated that murine M.
tuberculosis-infected AEC-II also secrete proinflammatory molecules.
We also showed that MLE-15 cells are permissive to M. tuberculosis
infection, and they are unable to control the bacillus growth. An
extensive characterization of soluble mediators secreted by AEC-II
infected by M. tuberculosis is important to seek potential targets that
induce the activation of DCs. In addition to IL-6, IFN-𝛾, S100A9, and
CCL5, infected MLE-15 produce high levels of NO, and we speculate
that some of these molecules modulates the metabolism of DCs, and
consequently their maturation and activation.
Although the interaction of AEC-II and DCs has not been investi-
gated in TB, the interaction of M. tuberculosis-infected macrophages
with noninfected AEC-II has been investigated. It was recently
8 RODRIGUES ET AL .

F I G U R E 5 M. tuberculosis hampers AEC-II ability of inducing maturation of DCs through HIF-1𝜶-NOS2 axis. (A) C57BL/6 mice were infected
with 1 × 105 M. tuberculosis by intratracheal route. (B) HIF-1𝛼 gene expression and (C–E) CD11c+ MHC-II+ CD11b+ NOS2+ cells were evaluated
in the lungs at 30 days of infection. (F) BMDC from WT and/ or nos2−/− mice were infected with M. tuberculosis (MOI2) and cultured in different
in different conditions: media (-), HIF-1𝛼 inhibitor (HIF-1𝛼 i, 100 𝜇M), supernatants from noninfected MLE-15 cells (NIC) and supernatants from
infected MLE-15 cells (IC). (G and H) NOS2, (I and J) MHC, (K and L) CD86, (M) IL-1𝛽, and (N) IL-6 were evaluated at 24 h of infection. The results
are representative of 3 independent experiments expressed as the mean ± SEM (n = 4/experimental condition). *P ≤ 0.05; & P ≤ 0.05 compared with
control (-)
RODRIGUES ET AL .
F I G U R E 6 Interaction of type II alveolar epithelial cells with DCs in the M. tuberculosis infection. Alveolar epithelial cells infected by M. tuber-
culosis secrete mediators that induce the maturation of noninfected DCs by a mechanism dependent on negative modulation of HIF1𝛼-NOS2 axis.
On the contrary, M. tuberculosis infection impairs that maturation of DCs, and alveolar epithelial cells have no capacity to overcome that
reported that macrophages infected by M. tuberculosis positively
modulated MMP1 production by A549 human cell line.42 In addi-
tion, noninfected MLE-15 treated with keratinocyte growth factor
secreted GM-CSF and enhanced the death of M. tuberculosis-infected
macrophages.21 Therefore, it is worth noting that the interaction of
infected macrophages with noninfected AEC-II has been investigated,
but neither the interaction of the contrary nor that of AEC-II with DCs
has been studied.
Although several studies reported that M. tuberculosis or M. tuber-
culosis antigens increase or decrease the response of DCs,25,27,28,47 it
has not been reported that other cells in the microenvironment are
involved in this process. In the present study, M. tuberculosis-infected
MLE-15 cells secreted soluble mediators that induced the maturation
of BMDC. Therefore, M. tuberculosis-infected AEC-II indirectly acti-
vated the maturation of DCs.
Cell metabolism plays an important role in immune activation.48
It has been reported that DC activation is associated with shift in
metabolism from OXPHOS to glycolysis.49 The early increase in the
expression of proteins associated with the glycolytic pathway such
as GLUT1 and HK2, as detected by extracellular acidification on the
Seahorse equipment, suggested the participation of HIF-1𝛼, which was
also accumulated at the early stage of cell culture. The involvement of
HIF-1𝛼 in the metabolic reprogramming of cells for aerobic glycolysis is
well established, even under normal oxygen conditions, a phenomenon
known as Warburg Effect.32,50 This phenomenon is primarily found
in cancer cells, as they have increased expression of HIF-1𝛼 and gly-
9
colytic enzymes, even in the presence of oxygen.51 The energy source
of M. tuberculosis-infected macrophages is dependent on aerobic
glycolysis.52 IFN-𝛾 activation induced the accumulation of HIF-1𝛼 in
infected macrophages, and positively regulated the secretion of IL-1𝛽,
IL-6, NO, and eicosanoids.53 In addition, the deficiency of HIF-1𝛼 in
macrophages impaired mycobacteria death, which inhibits the ability
of IFN-𝛾 to control the pathogen’s growth54 and indicates that IFN-𝛾
downstream signaling needs HIF-1𝛼-dependent pathways. Similarly,
in zebra fish model, HIF-1𝛼 was important in the development of
resistance against M. marinum infection through the up-regulation
of NOS2 expression.55 However, the role of HIF-1𝛼 and cellular
metabolism in DCs during M. tuberculosis infection has not yet been
explored. Our findings show that at the late stage of DC activation by
AEC-II, there was no stabilization of HIF-1𝛼 protein and DCs remained
in a metabolic state of adaptation with low rate of glycolysis and
OXPHOS. Besides, there was a basal expression of NOS2 after 24 h
of stimulation, corroborating the associated lower accumulation of
HIF-1𝛼 at this time-point. DMOG treatment, which stabilizes HIF-1𝛼
expression, induced a significant reduction in the expression of MHC-
II, CD80, and CD86, indicating that prolonged accumulation of HIF-1𝛼
plays a negative role in the maturation of DCs. Our findings confirm
the negative role of HIF-1𝛼 in DC maturation and consequently, its
involvement in impaired DC-induced activation of naive T cells.30 HIF-
1𝛼, via NOS2-NO axis, induced mitochondrial dysfunction, inhibiting
OXPHOS and switching cell metabolism to aerobic glycolysis as the
only energy source.30 In the present study, we expected a high rate of
10
both glycolysis and OXPHOS, but instead, DCs used both glycolysis
and OXPHOS at reduced rates. To address the role of HIF-1𝛼-NOS2
axis in the maturation of DCs induced by M. tuberculosis-infected
AEC-II, we differentiated BMDC from NOS2-deficient mice, and
confirmed that HIF-1𝛼 signaling depends on NOS2 expression and NO
production for the down-regulation of DC maturation.
Prolonged HIF-1𝛼 stabilization also had a negative role in the
expression of CCR7. A recent study demonstrated that early increase
in the rate of glycolysis is essential for the oligomerization of CCR7.56
Therefore, the initial increase in the rate of glycolysis might also be
important for the up-regulation of CCR7. However, energy balance
via the 2 pathways, glycolysis and OXPHOS, may be essential for
the maintenance of the phenotype found in the present study. Low
rates of glycolysis and OXPHOS following indirect contact with M.
tuberculosis-infected AEC-II and the effect of HIF-1𝛼-NOS2 axis in the
maturation of DC is the novelty of this study. In mouse model, NOS2
played a protective role against M. tuberculosis infection,57,58 and the
HIF-1𝛼-NOS2 axis has been studied in the context of macrophage
killing.53,54 However, we show for the first time that HIF-1𝛼-NOS2
axis plays a negative role in the maturation of DCs.
Finally, the crosstalk between nonhematopoietic cells (AEC-II) and
hematopoietic cells (DCs) during TB opens a new perception on the
function of AEC-II in the modulation of a microenvironment that facil-
itates the control of infection. The interaction of AEC-II infected by
M. tuberculosis and DCs generates a microenvironment suitable for
the activation of adaptive immune response. This might represent the
mechanism associated with latent infection, as well as the opportunity
to exploit molecular targets to develop new therapeutic tools.
4 METHODS
4.1 Animals
C57BL/6 mice and nos2−/− mice (6–8 weeks) were obtained from the
breeding facility of the Ribeirao Preto Medical School at University
Sao Paulo (FMRP-USP). All animals were housed in Animal Biological
Safety Level 3 (ABSL3) with sterile water and food. Mice experiments
were conducted in accordance with the ethical guide on the use of ani-
mals of FMRP-USP, and were approved by the Ethics Committee on
Animal Use of FMRP-USP (protocol number 198/2017).
4.2 Bacteria cultivation
M. tuberculosis H37Rv strain (American Type Culture Collection 27294)
was grown in 7H9 Middlebrook Broth (DIFCO Laboratories, Detroit,
MI, EUA) for 10–11 days at 37◦ C. The culture was collected as previ-
ously described.59
4.3 Epithelial and DCs
Immortalized murine lung epithelial cells (MLE-15) were used. MLE-
15 cells were cultured in RPMI medium (Sigma–Aldrich, St Louis, MO,
RODRIGUES ET AL .
USA) supplemented with 2% FBS (Thermo Fisher Scientific, Waltham,
MA, USA), 5 mg/L insulin, 10 𝜇g/ml transferrin, 3 ng/ml selenite,
sodium, 10 nM hydrocortisone, 10 nM 𝛽-estradiol, and 1% peni-
cillin/streptomycin (complete RPMI).
Dendritic cells were differentiated from bone marrow cells from
C57BL/6 mice. After removing the femurs and performing the bone
marrow flush, cells were incubated in RPMI media supplemented
with 20 ng/ml GM-CSF, 10% FBS, 1% glutamine, and 0.1% 𝛽-
mercaptoethanol. In the third day, GM-CSF was added maintaining the
20 ng/ml concentration. After 7 days of culture, the cells are essentially
CD11c+ CD11b+ DCs. Atmosphere for both cells: 95% air; 5% CO2 .
Temperature: 37◦ C.
4.4 In vitro infection
MLE-15 murine immortalized cells (1 × 106 cells) or BMDCs (previ-
ously differentiated from bone marrow cells in RPMI media supple-
mented with 20 ng/ml GM-CSF, 10% FBS 1% glutamine, and 0,1%𝛽-
mercaptoethanol for 7 days) were placed in 24 wells plaques (Corn-
ing Incorporated, New York, USA). After 24 h, MLE-15 or BMDCs were
infected with M. tuberculosis H37Rv for 4 h. MLE-15 was infected with
MOI10 and BMDC with MOI2. Bacilli growth by CFU (Counting Form-
ing Units) counts was evaluated at times 4, 24, 48, and 72 h post infec-
tion. After washing, cells were lysed with 0.05% saponin. Serial dilu-
tions were plated on 7H11 media (Difco Laboratories, Detroit, MI,
EUA), and number of CFU was counted after incubation for 28–30 days
at 37◦ C and 5% CO2 . In order to inhibit HIF-1𝛼, a HIF-1𝛼 inhibitor (Cal-
biochem, San Diego, CA, USA) 100 𝜇M was used in infected cells.
4.5 In vivo infection
C57BL/6 mice were randomly divided in noninfected (control) and
infected groups. Infection was performed by intratracheal administra-
tion of 1 × 105 bacilli.60 The lower and middle right lobes of the lungs
were collected and processed as previously reported.61 Part of the
cells (1 × 106 were used for DC characterization by flow cytometry
analysis. For the CFU assay, serial dilutions (100, 1000, 10,000, and
100,000) of digested lungs were plated on supplemented 7H11 agar
medium. CFU number was counted after 28 days of incubation at 37◦ C,
and the results were expressed as the log10 of CFU/lung. Lung cells
were counted in a CountessTM automated cell counter (Thermo Fisher
Scientific, Waltham, MA, USA). No blinding was used in animal experi-
ments.
4.6 In vitro experimental models
4.6.1 DC and MLE-15 indirect contact assay
1 × 106 immortalized mouse lung epithelial cells (MLE-15) were plated
in 24 wells plate overnight for adhesion. Next, the cells were incubated
for 4 h with M. tuberculosis H37Rv (MOI 10). The supernatants or
conditioned medium (CM) of noninfected (NIC) or infected (IC) cells
was collected after 48 h of incubation. CM was filtered with 0.22 𝜇M
filter (Corning Incorporated, New York, USA) to confirm the absence
RODRIGUES ET AL .
of bacilli. Concurrently, BMDCs, predominantly immature CD11c+
CD11b+ , were incubated for 24 h with IC or NIC as well as media (neg-
ative control), LPS 100 ng/ml (positive control). In order to stabilize
HIF-1𝛼, a prolylhydroxylase inhibitor, DMOG, dimetiloxaloilglicine,
(Calbiochem, San Diego, CA, USA) 200 𝜇M was used in addition to IC.
Cells were analyzed for expression of surface markers, cytokines, HIF-
1𝛼, and target genes. To exclude the role of M. tuberculosis products in
our results from IC group, we used as a control, M. tuberculosis-media,
which consisted in RPMI media incubated with M. tuberculosis (1 × 107
bacilli/well) for 48 h at the same conditions as infected MLE-15 cells.
After 48 h, the media was collected and filtered with 0.22 𝜇M filter
(Corning Incorporated, New York, USA).
4.6.2 Coculture of MLE-15 cells and BMDCs
1× 106 MLE-15 cells were incubated for 4 h with H37Rv (MOI 1:10).
The wells were then washed twice with 1× PBS, and 200 𝜇g/ml gen-
tamicin was added to ensure the absence of extracellular bacilli. Then,
0.5 × 106 BMDC, predominantly immature CD11c+ CD11b+ , were
cocultured for 48 h with infected or uninfected MLE-15 cells. BMDCs
were analyzed for expression of surface activation markers (MHC-II,
CD80, CD86) and cytokines (IL-1𝛽, IL-6, and IL-10).
4.6.3 Coculture of BMDC with naive T cells
BMDCs (0.5 × 105 per well) were stimulated with NIC or IC for 24 h,
and cocultured for 96 h with CD4+ CD62L+ cells (1 × 105 per well)
stimulated with mAb anti-CD3 (1 𝜇g/ml; BD Biosciences, Franklin
Lakes, NJ, USA) in U-bottom 96-well plates (BD Biosciences, Franklin
Lakes, NJ, USA). Naive CD4+ T cells were purified from spleen and
lymph nodes of naive C57BL/6 mice following the instructions of the
CD4+ CD62L+ T Cell isolation kit (Miltenyi Biotec, San Diego, CA, USA).
Proliferation rate was evaluated in CD4+ Ki67+ cells.
4.7 Methods details
4.7.1 Cytokines and Griess assay
The levels of cytokines in the supernatants of cell cultures were mea-
sured using ELISA kits obtained from (BD Biosciences, Franklin Lakes,
NJ, USA), BD OptEIATM Set (IL-10) and R&D Systems, Minneapolis, MN,
USA (IL-6, IL-1𝛽), according to the manufacturer’s instructions. Griess
assay was performed to analyze nitrite production in the supernatants
as previously described.17
4.7.2 RNA extraction, cDNA, and q-PCR
RNA was extracted from 1 × 106 cells using illustra RNAspin Mini RNA
isolation kit (GE healthcare, Chicago IL, USA), and c-DNA was made
using High Capacity cDNA Reverse Transcription kit (Thermo Fisher
Scientific Baltics, UAB, Vilnius, Lithuania), following the manufacture’s
instructions. PCR was performed using Maxima SYBR Green/ROX
qPCR Master Mix (2×) (Thermo Fisher Scientific, Waltham, MA, USA)
in the StepOnePlusTM Real-Time PCR System (Applied Biosystems,
Foster City, CA, USA). The following conditions were used for amplifi-
cation: initial denaturation at 95◦ C for 10 min, proceeded by 40 cycles
11
of 95◦ C for 15 s, an annealing phase at 58◦ C for 30 s and an exten-
sion phase at 72◦ C for 30 s. The threshold cycle (Ct) was used to ana-
lyze samples. Gene expression was calculated as 2−(ΔΔCt), where ΔΔ
Ct = Δ Ct (sample) − Δ Ct (calibrator) and where Δ Ct (sample) = Ct
(target gene) − Ct (normalizer = 𝛽-Actin). The primer sequences used
for detection are the following: 𝛽-Actin (forward, 5′ -CCC TAG GCA
CCA GGG TGT GA-3′ ; reverse, 5′ -GCC ATG TTC AAT GGG GTA CTT
C-3′ ), hif-1𝛼 (forward: 5′ -ACC TTC ATC GGA AAC TCC AAA G-3′ ;
reverse: 5′ -CTG TTA GGC TGG GAA AAG TTA GG-3′ ), hk2 (forward,
5′ -CGG AAT GGG GAG CCT TTG G-3′ ; reverse: 5′ -GCC TTC CTT ATC
CGT TTC AAT GG-3′ ). Glut1 (forward, 5′ - ATG GAT CCC AGC AGC
AAG-3′ ; reverse: 5′ - CCA GTG TTA TAG CCG AAC TGC-3′ )
4.7.3 Western blotting
HIF-1𝛼 expression in BMDC was determined by Western blotting
according to manufacturer’s instructions. Briefly, after transference,
the nitrocellulose membrane was incubated with monoclonal anti-HIF-
1𝛼 (Sigma–Aldrich, St Louis, MO, USA) at 4◦ C overnight, followed by
incubation with anti-rabbit horseradish peroxidase-linked secondary
antibody (Sigma–Aldrich, St Louis, MO, USA) and developed using
enhanced chemiluminescence technique (ChemiDoc XRS System; Bio-
Rad Laboratories, Hercules, CA, USA). The membrane was stripped and
reprobed with Gapdh as a loading control.
4.7.4 Flow cytometry
Cell suspensions (1 × 106 cells) obtained from lungs or in vitro cul-
tures were incubated with supernatant of 2.4G2 cell lineage (con-
taining antibodies anti-Fc𝛾 RII/III) for 20 min at 4◦ C. Then, the cells
were incubated for 30 min at 4◦ C with monoclonal antibodies: F4/80
PercP Cy5.5 (Clone BM8- Cat No.:123127; Biolegend, San Diego,
CA, USA), CD11c-PECy7 (Clone N418 - Cat No.: 25-0114-82; eBio-
science, San Diego, CA, USA), CD11b BV711 (Clone M1/70 – Cat no.
563168; BD Biosciences, Franklin Lakes, NJ, USA), CD11b APCcy7
(Clone M1/70 - Cat No.:557657; BD Biosciences, Franklin Lakes, NJ,
USA), MHC-II Alexa 488 (Clone M5/114.15.2 - Cat No.:562352; BD
Biosciences, Franklin Lakes, NJ, USA), CD80 PE (Clone L307.4; Cat
No.:557227; BD Biosciences, Franklin Lakes, NJ, USA), CD86 PE (Clone
GL-1; Cat No.:553692; BD Biosciences, Franklin Lakes, NJ, USA), CCR7
PE (Clone 4B12; Cat No.:560682; BD Biosciences, Franklin Lakes, NJ,
USA), CD4 PEcy7 (Clone RM4-5; Cat No.:561099; BD Biosciences,
Franklin Lakes, NJ, USA), Nos2 APC (Clone CXNFT; Cat No.:17-5920-
82; eBioscience, San Diego, CA, USA), Ki67 FITC (Clone SolA15; Cat
No.:11-5698-82; eBioscience, San Diego, CA, USA). For intracellular
staining, cells were previously permeabilized with 2% saponin. Stained
cells were washed with PBS with 2% FBS (Thermo Fisher Scientific,
Waltham, MA, USA) and fixed with PBS containing 1% formaldehyde
(J.T. Baker Phillipsburg, NJ, USA). The samples (100,000 events) were
assessed using a FACSCantoTM II cytometer (BD Biosciences, Franklin
Lakes, NJ, USA) and CellQuest software (BD Biosciences, Franklin
Lakes, NJ, USA). FlowJo software (v 7.6.5; Tree Star, Ashland, OR,
USA) was used for cell analysis. First, the cells were gated on FSC-A
and FSC-H for exclusion of doublets, and then gated on FSC-A (size)
12
and SSC-A (granularity) to select DC, which were characterized as
F4/80− CD11c+ CD11b+ /MHC-II+ .
4.7.5 Mitochondrial respiration and extracellular
acidification assays
Mitochondrial respiration and ECARs were assessed using Seahorse
Extracellular Flux XFe96 Analyzer (Seahorse Bioscience, Agilent
Technologies, Santa Clara, CA, USA). Before plating, the 96-well
Seahorse plate was coated for 2 h with poly-D-lysine solution. Initially,
100,000 DC were plated in each well with media. Twenty-four hours
later, media was replaced by different conditioned or control media
and maintained for 2 or 24 h, when the assay was performed. On the
day of the assay, media was changed to the XF Seahorse assay media
and cells were maintained at 37◦ C on an incubator without CO2 for
1 h before being placed in the equipment. Four cycles were used to
measure basal OCR, followed by automatic drug injections delivered
in each well. First, oligomycin (1 𝜇g/ml; Sigma–Aldrich, St Louis, MO,
USA) was employed to obtain respiration-driven ATP synthesis and
proton leak-driven respiration. After 3 cycles, Carbonyl cyanide
chlorophenyl hydrazine (CCCP, 5 𝜇M; Sigma–Aldrich, St Louis, MO,
USA) was delivered to analyze maximal respiratory capacity. At final,
rotenone (1 𝜇M; Sigma–Aldrich, St Louis, MO, USA) and antimycin
(1 𝜇M; Sigma–Aldrich, St Louis, MO, USA) were added to inhibit
complexes I and III and thus ablate mitochondrial oxygen consumption
in order to determine nonmitochondrial respiration. The levels of
extracellular acidification were measured simultaneously in the same
wells to account for the acidification produced by lactate production
and secretion as a readout of glycolysis.
4.7.6 Statistical analysis
Data were presented as the mean ± SEM and analyzed by Prism 6.0
(GraphPad Software, San Diego, CA). For the majority of in vitro assays,
we used n = 4/group, and for in vivo assays we used n = 6/group. For
comparisons between experimental groups, 1-way ANOVA was per-
formed followed by Newman–Keuls multiple comparisons post-tests.
Individual groups were compared using the Mann–Whitney for non-
parametric data or 2-tailed Student’s t-test for parametric data. Statis-
tically significant differences were indicated by P values ≤ 0.05. For in
vivo studies, the was no exclusion from analysis.
ACKNOWLEDGMENTS
We thank Denise Ferraz, Izaira Brandão, and Wendy Martin Rios
for technical support. We are also grateful to professor Niels Olsen
Saraiva Câmara (ICB- USP) for allowing us to use the Seahorse equip-
ment (grant 2018/08479-7). This work was supported by grants from
FAPESP (grants 2017/16538-0, and 2017/21629-5).
DISCLOSURES
The authors declare no competing interests.
RODRIGUES ET AL .
ORCID
Vânia Luiza Deperon Bonato
https://orcid.org/0000-0003-4189-2685
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