Experimental Parasitology 210 (2020) 107846

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Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

Characterization of serum cytokines and circulating microRNAs that are T

predicted to regulate inflammasome genes in cutaneous leishmaniasis
patients
Lucilla Silva Oliveira Mendonçaa, Jaqueline Marques Santosb, Carla Martins Kanetoc,
Luciana Debortoli de Carvalhoc, Jane Lima-Santosc, Danillo G. Augustoc,d,
Silvia Maria Santos Carvalhoc, Jamária Adriana Pinheiro Soares-Martinse, Izaltina Silva-Jardimc,∗
a
Department of Health Sciences, Universidade Estadual de Santa Cruz (UESC), Ilhéus, BA, Brazil
b
Institute of Biomedical Sciences, Universidade de São Paulo (USP), São Paulo, SP, Brazil
c
Department of Biological Sciences, Universidade Estadual de Santa Cruz (UESC), Ilhéus, BA, Brazil
d
Department of Genetics, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil
e
Department of Liberal Arts and Sciences, Milwaukee Area Technical College (MATC), Milwaukee, WI, United States

ARTICLE INFO ABSTRACT

Keywords: Leishmaniasis is a neglected disease caused by an intracellular protozoan parasite of the genus Leishmania.
Cutaneous leishmaniasis Infection starts when this protozoan replicates in a phagolysosomal compartment in macrophages, after evading
Cytokines host immune responses. The balance of Th1 and Th2 immune responses is crucial in leishmaniasis because it will
Inflammasomes determine whether the infection will be under control or if clinical complications will occur. The inflammasome,
microRNAs
which is activated during Leishmania infection, involves the action of caspase-1 and release of the proin-
flammatory cytokines interleukin-1β and interleukin-18. Together, they contribute to the maintenance of an
inflammatory response and pyroptosis. Here, we evaluated the serum levels of cytokines and the expression of
circulating microRNAs related to inflammasome regulation in twenty-seven patients with cutaneous leishma-
niasis in comparison to nine healthy individuals, in the context of the inflammasome activation. Evaluation of
serum cytokines activation (IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-17) was performed by flow cytometry using CBA
kits (cytometric beads array) while the expression of circulating microRNAs (miR-7, miR-133a, miR-146b, miR-
155, miR-223, miR-328, and miR-342) in plasma was measured by quantitative polymerase chain reaction. Our
results showed an increase of the expression of miR-7-5p (p < 10−5), miR-133a (p = 0.034), miR-146b
(p = 0.003), miR-223–3p (p = 10−5), and miR-328–3p (p = 0.002), and cytokine levels for IL-1β (p = 0.0005),
IL-6 (p = 0.001), and IL-17 (p = 0.001) in patients with cutaneous leishmaniasis compared to the controls.
These results suggest that microRNAs and cytokines can play an important role in regulating the human immune
responses to Leishmania infection. Our findings may contribute to the understanding of the mechanisms of the
gene regulation during the cutaneous leishmaniasis and to the identification of possible biomarkers of the in-
fection.

1. Introduction
Leishmaniasis is a tropical parasitic disease caused by different
species of the protozoan Leishmania, which are transmitted to humans
through the bites of the sand fly vector Phlebotomus (WHO, 2018). In
Brazil, American cutaneous leishmaniasis (ACL) is one of the major
concerns regarding dermatological diseases because of its severity and
extensive human deformities (Brasil, 2017).
According to the 2016 epidemiological report, Brazil is among the
most endemic countries in the Americas, with 12,690 cases of cuta-
neous (CL) and mucosal leishmaniasis (ML) (SisLeish-OPAS/WHO,
2018). Disseminated cutaneous leishmaniasis (DL) accounts for ap-
proximately 2% of the reported cases of CL in Brazil and diffuse cuta-
neous leishmaniasis (DCL) another rare manifestation of CL with 1 or 2
cases diagnosed per year (Brasil, 2017). The ACL can be found in all
regions of Bahia state, in which 67.2% of cities have been affected and
an average of 2362 new cases are notified annually. The coefficient of
detection was found to be at 32.8% risk of transmission in the Bahia

∗
Corresponding author.
E-mail address: isjcavalli@uesc.br (I. Silva-Jardim).

https://doi.org/10.1016/j.exppara.2020.107846
Received 21 December 2018; Received in revised form 8 August 2019; Accepted 24 January 2020
Available online 28 January 2020
0014-4894/ © 2020 Elsevier Inc. All rights reserved.
L.S.O. Mendonça, et al. Experimental Parasitology 210 (2020) 107846

state (SINAN and SUVISA, 2016). levels could help in the diagnosis, prognosis, and treatment of leish-
Depending on the host immune response and the parasite's entry, maniasis.
tropism, and pathogenicity, the disease can have outcomes ranging
from cutaneous lesions at the bite site, mucosal lesions to visceraliza-
tion, a more severe condition known as visceral leishmaniasis or kala 2. Material and methods
azar (Herwaldt, 1999). The main species of Leishmania found in de-
veloped countries cause cutaneous manifestations, which may either 2.1. Ethical aspects
heal spontaneously or progress to an intense inflammatory state,
making the treatment difficult (Herwaldt, 1999). The ACL may be This study was submitted to evaluation, and it was approved by the
subdivided into four types: i) localized cutaneous leishmaniasis (LCL), Ethics Committee on Human Research at UESC (CAAE
which is characterized as an ulceration with raised edges; ii) mucosal 18815013.7.1001.5526). Samples were only collected after patients
cutaneous leishmaniasis (MCL), which is a progression of the cutaneous were informed about the scope of this study and upon signing the in-
form and it is characterized by the destruction of the mucosa tissues, formed consent form. The study was conducted according to the
especially in the upper respiratory tract, iii) the rare form DL, which is Helsinki Declaration and Resolution 510/2016 of the National Health
characterized by the appearance of multiple polymorphic skin lesions in Council of the Ministry of Health that regulates studies involving hu-
various body regions, and most of the cases documented in the Amer- mans in Brazil.
icas are caused by Leishmania (V.) braziliensis (Brasil, 2017) and iv)
another atypical form of CL, the DCL, known as anergic-DCL, which is a
very rare and severe form of the disease with infiltrative nodular lesions 2.2. Population and sample collection
caused by L. (L.) amazonensis (Barral et al., 1995; Brasil, 2017; David
and Craft, 2009). Our study had 27 patients with active skin lesions and confirmed
During the course of the infection, the host's innate immunity plays diagnosis for cutaneous leishmaniasis that were enrolled prior to
an important initial role in the immune response by recruiting cells treatment. These patients were selected at the Health Center CAE III in
such as neutrophils, macrophages, and dendritic cells. These cells in- Ilhéus-BA, and they were diagnosed with CL based on the clinical
duce the production of reactive oxygen species (ROS) and release of characteristics of the disease along with the Montenegro test. Often,
nitric oxide (NO), in addition to pro-inflammatory cytokines that help these patients had one or a few ulcerative cutaneous lesions with raised
to fight the pathogen (Kedzierski and Evans, 2014). and delimited borders located in the lower limbs. Patients diagnosed
Cells from the innate immune system detect microorganisms with MCL were not included in this study. The control group was
through their pattern recognition receptors (PPRs) which bind to pa- comprised of 9 healthy individuals, in which they were grouped ac-
thogen-associated molecular patterns (PAMP), leading to a more spe- cording to their age, gender, and place of residence. The exclusion
cific immune response (Gurung and Kanneganti, 2016). NOD-like re- criteria for the control group included absence of skin lesions or in-
ceptors (NLRs), a family of PPRs, were shown to have a crucial role in fectious diseases and absence of previous history of leishmaniasis. The
defending the host against intracellular pathogens. NLRs play a role in information about our study population is presented in Table 1.
forming inflammasome complexes (Martinon and Tschopp, 2005). The We collected 8 mL (ml) of peripheral venous blood from patients
term inflammasome was initially described to determine a high mole- with cutaneous leishmaniasis and control group using vacuplast va-
cular weight complex that activates caspase-1 and it is responsible for cuum collection tubes®. We used 4 ml-tubes without additives and
processing of the proinflammatory cytokines IL-1β and IL-18 (Martinon containing a clot activator to separate the serum, and 4 ml-tubes with
et al., 2002). Inflammasomes can generate efficient signals and re- EDTA as an additive to separate the plasma.
sponses against pathogens by triggering the release of cytokines, acti-
vation of other immune cells, and programmed cell death (Di Virgilio,
2013).
Table 1
Some molecules can participate in the regulation of immune re-
Characteristics of participants at inclusion.
sponses, such as microRNAs (miRNAs) (Sutterwala et al., 2014). Mi-
croRNAs are small endogenous RNAs of approximately 22 non-coding Groups
nucleotides, which act as post-transcriptional regulators, cleaving ma-
Variables Control Patients Significance (p value)
ture mRNAs after binding to their untranslated 3′-end (He and Hannon,
2004). It is known that gene expression regulation by miRNAs also N % N %
impacts the expression of genes involved in the immune response. Some
Sex
miRNAs may have their expression increased in certain cell types, such
Male 5 55.6 16 59.3 NS (> 0.99)
as neutrophils, macrophages, and lymphocytes, ultimately regulating Female 4 44.4 11 40.7
several biological processes such as development, differentiation, and Age (years)
regulation of the immune system (Miska, 2005; Xiao and Rajewsky, 18–25 2 22.2 5 18.5
2009). 26–40 3 33.3 7 25.9 NS (0.91)
41–50 1 11.1 2 7.41
miRNAs play a modulatory role in the assembly of the inflamma- 51–60 2 22.2 5 18.5
some complex (Sutterwala et al., 2014), but the regulation of miRNAs 61–70 1 11.1 7 25.9
in leishmaniasis is still unclear. Some studies indicate the presence of Place of residence
unregulated miRNAs in human cells and cutaneous lesions, which af- Rural area 3 33.3 16 59.3 NS (0.25)
Urban area 6 66.7 11 40.7
fects the innate immune response (Diotallevi et al., 2018; Hashemi
Relapse
et al., 2018; Lemaire et al., 2013; Nunes et al., 2018). Yes NA NA 10 37
Up to this date, there are no reports showing the participation of No NA NA 17 63
miRNAs in the modulation of inflammasomes in cutaneous leishma- Presence of skin lesions
niasis, and this complex might play an essential role in the immune Yes 0 0 27 100 NA

response and tissue damage during the infection. In addition, miRNAs No 9 100 0 0
related to inflammasomes could help in the understanding the role of
this protein complex during infection. Furthermore, miRNAs could be Note: N-number samples. NA-not applicable. NS-not significant (Chi-Square
potential biomarkers for this disease, and detection of their circulating test).

2
L.S.O. Mendonça, et al.
2.3. Quantification of cytokines
Quantification of the cytokines IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-
17 was performed using the CBA Flex Set System kit (BD®) in patients'
serum samples according to the manufacturer's instructions. Samples
were first diluted at 1:4 ratio and the following assay reagents were
prepared accordingly: standard reagents to calibrate the curve, fluor-
ochrome detection reagent, and mix of the 8 different types of beads.
Briefly, 50 μL of mixed capture beads were added to 50 μL of
standard samples or serum samples and incubated in the dark for 1 h at
room temperature. After that, 50 μL of mixed detection reagents were
added to the samples, followed by incubation in the dark for 2 h.
Samples were then, washed, centrifuged, and resuspended in 300 μL of
washing buffer. Finally, samples were read in a flow cytometer (BD
FACSArray), calibrated with set-up beads. Analysis of results was done
using the FCAP Array software.
2.4. RNA extraction and complementary DNA synthesis (cDNA)
Total RNA was extracted from each plasma sample using TRIzol®
reagent method (Life technologies, USA Molecular Research Center,
Inc).
RNA extractions were performed using 200 μL of plasma and 800 μL
of TRIzol under constant homogenization with micropipette in a tube.
Then, 200 μL of chloroform were added to tubes followed by incubation
at 4 °C for 5 min. After that, tubes were centrifuged at 15000 RPM at
4 °C for 15 min. Supernatants were transferred to a new tube and
subsequently, the RNA was precipitated by adding 800 μL isopropanol
to the aqueous phase. Samples were then placed at −80 °C overnight
and centrifugation at 15000 RPM at 4 °C for 20 min. Supernatants were
discarded, and pellets were washed through centrifugation with 1 mL of
70% ethanol. RNA pellets were resuspended in 30 μL RNAase-free
water. Subsequently, quantification was performed using Nanodrop®
ND-1000 (Thermo Scientific-USA). Only RNA samples with a 260/280
ratio of ≥1.8 were included.
Complementary DNA synthesis (cDNA) was performed from RNA
samples extracted from patients' blood (500ƞg) using miR-specific pri-
mers and Taqman miRNA Reverse Transcription Kit (Applied
Biosystem), in a scaled down volume of 15 μL RT reaction, according to
the manufacturer's instructions. Tubes were homogenized and in-
cubated in the ThermoCycler (Techne Prime, BibbyScientific - UK). The
thermal cycling parameters of reverse transcription were 30 min at
16 °C, 30 min at 42 °C and 5 min at 85 °C x 30 cycles. Subsequently,
samples were diluted at 1:4 ratio and stored at −20 °C, and when
needed, they used as template in quantitative PCR reactions.
2.5. microRNAs and inflammasomes
We did an extensive search in the literature about microRNAs as-
sociated with the modulation of inflammation as shown in Table 2.
Table 2
MicroRNAs associated with inflammasomes and immune response mechanisms found in the in literature.
miR Description
miR-7 - miR-7 as inhibitor of NLRP3 and IL-1β production during neuroinflammation in Parkinson's disease.
- Suppression of NLRP3 and caspase-1 in neural stem cells.
miR-133 Regulation of inflammasome activation via suppression of UCP2 in THP cells.
miR-146 High expression of miR-146 downregulate both the expression of pro-inflammatory cytokines such as IL-1β and IL-18 and the
activation of inflammasomes in diabetic nephropathy.
miR-223 - The expression of miR-223 is downregulated when NLRP3 inflammasome and IL-1β are produced.
- The activation of inflammasomes is negatively regulated by miR-223.
- miR-223 inhibits the production of caspase-1, NLRP3, and IL-1β during intracerebral hemorrhage in animal models.
miR-328 miR-328 is differentially expressed in macrophages infected with Leishmania donovani, suggesting that it may play a role in
phagocytosis.
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Experimental Parasitology 210 (2020) 107846
2.6. Evaluation of the levels of circulating microRNAs in patients and
controls
The following microRNAs were evaluated in our study: hsa-miR-328
(TM 00543), hsa-miR-223 (TM 002098), hsa-miR-7 (TM 000268), hsa-
miR-146b (TM001097), hsa-miR-155 (TM 002287), and hsa-miR-133a
(TM 002246). hsa-miR-342–3p (TM 002260) was used to normalize
miRNA input. microRNA expression was evaluated using quantitative
polymerase chain reaction (qPCR). qPCR reactions contained pre-
viously synthetized cDNA samples as template, specific probes for each
microRNA, and TaqMan® Universal PCR Master Mix, in a final volume
of 20 μL qPCR reactions were performed in duplicate under the fol-
lowing conditions: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C
for 15 s, and 60 °C for 60 s using ABI 7500 Real Time PCR System.
Levels of targeted miRNAs were normalized based on the endogenous
hsa-miR-342–3p, which showed a high expression level, with a low
standard deviation and constant values in both groups, being a suitable
normalizer for our samples according to (Liang et al., 2007). Quanti-
fications of miRNAs levels were calculated using the 2-ΔCT method.
2.7. Prediction of target genes for microRNAs
MicroRNAs found to be differentially expressed were further eval-
uated using computational prediction on online bioinformatics plat-
forms to identify possible microRNAs-target genes interactions.
Because there are several bioinformatic platform tools and some of
them have different parameters, which can result in different interac-
tions, we selected three online bioinformatics tools widely used in
predictive studies (Targetscan-targetscan.org; Mirdb-mirdb.org and
Mirtarbase-mirtarbase.nbc.nctu.edu.tw), aiming to reduce the chances
of false-positive results when predicting possible miRNAs target genes.
For each online bioinformatic tool, we used the names of the dif-
ferentially expressed microRNAs to determine possible target genes of
each one. To improve the quality of results when predicting target
genes, data were filtered through the intersection of the results of the
three platforms, in which common target genes were found.
After possible target genes were selected by intersecting results,
they were plotted in Cytoscape (available at http://www.cytoscape.
org/) to obtain a graphic design of the interaction between microRNAs
and their possible target genes. By doing so, we were able to have a
better depiction of microRNAs-target genes interactions. Then, using
Cytoscape and BINGO (http://psb.ugente.be/cbd/papers/BINGO), we
characterized the cellular processes that could occur due to the ex-
pression of these genes.
2.8. Data analysis
For statistical analysis of cytokine levels and relative expression of
microRNAs in patients and control group, we applied the non-para-
metric Mann-Whitney test (for two independent samples) using
GraphPad Prism (7.0) software. Results were considered significant
Reference
Zhou et al. (2016).
Fann et al. (2017).
Bandyopadhyay et al. (2013).
Bhatt et al. (2016).
Haneklaus et al. (2012).
Franz Bauernfeind et al. (2012).
Yang et al. (2015b).
Tiwari et al. (2017)
L.S.O. Mendonça, et al.
Fig. 1. Quantification of cytokines in the sera of patients with active lesions of cutaneous leishmaniasis (CT). A – F) Levels of serum cytokines IL-1β, IL-2, IL-4, IL-6,
IL-10, IL-17. (Graphs show the mean of cytokine production using the non-parametric Mann-Whitney t-Test).
when p-value was < 0.05. We used the R software for Spearman cor-
relation analysis tests (for the correlation matrix) and simple x-y plot.
To determine the homogeneity in our group population, we per-
formed a Chi-square statistical test (p > 0.05), which showed that
both groups are similar.
3. Results
3.1. Levels of IL-1β, IL-6, and IL-17 were increased in the serum of patients
with cutaneous leishmaniasis
We quantified the levels of serum cytokines (IL-1β, IL-6, and IL-17)
in patients with cutaneous leishmaniasis (CL) and compared to healthy
controls. Our results showed significant increase in the cytokines levels
for IL-1β (p = 0.0005), IL-6 (p = 0.001), and IL-17 (p = 0.001) in
patients (Fig. 1). Increased cytokines levels in patients with LC may
trigger a Th17 immune response and an increase in IL-1β levels suggests
the activation of inflammasomes.
3.2. Quantification of microRNAs (miR-7-5p, miR-133a, miR-146b, miR-
223, and miR-328–3p) in plasma of individuals with cutaneous
leishmaniasis
Based on the literature, we found five miRNAs that possibly regulate
genes involved in inflammasomes activation. Our results showed in-
creased levels of miR-7-5p, miR-133a, miR-146b, miR-223, and miR-
328–3p in plasma of patients with cutaneous leishmaniasis (p < 0.05;
Fig. 2). The most significant differences were found for miR-7-5p
(p < 10−5) and miR-223 (p = 10−5), in which their relative expres-
sions were 10,000 and 3.8-fold up in patients with cutaneous leish-
maniasis, respectively, whereas miRNAs miR133a, miR 328-3-P and
miR146b presented p-values equal to 0.034, 0.002, and 0.003, respec-
tively.
To understand the role of these miRNAs in gene regulation, we used
in silico prediction to search for their possible targets. After the
4
Experimental Parasitology 210 (2020) 107846
convergence of three different prediction algorithms, our results
showed that these miRNAs possibly regulate several genes related to the
immune response, such as those involved in the regulation of pro-
grammed cell death (DNAJB6, IRS2, DNAJC5, RBPJ, IGF1R, FOXO3,
ECT2, MEF2C, FOXO1, and TGFB2), caspase activity (NLRP3, FOXL2,
F3, SENP1, and SNCA), and response to cytokine stimuli (IRAK1,
TRAF6, IL-6ST, MCL1, BCL2L1) (Fig. 3).
Altogether, our results showed an up-regulation of microRNAs as-
sociated with inflammasomes in plasma of patients with cutaneous
leishmaniasis when compared to healthy controls.
3.3. Correlation between levels of miRNA and cytokines in the
inflammasome activation
To understand the correlation between the levels of miRNAs and
cytokines found in our study in the context of inflammasomes, we did a
correlation matrix (Fig. 4a). We found an inversely proportional cor-
relation between levels of IL-1β and the microRNAs miR-223 and miR-
7, whereas levels of miR-133a, miR-146b, and miR-328 presented po-
sitive values compared to IL-1β levels.
Based on both the literature and computational prediction, our
analyses indicated that miR-7, miR-223, and miR-133a played an im-
portant role in the inflammasome activation. The correlation between
the levels of these miRNAs and IL-1β in our patients’ samples showed
that the higher the levels of IL-1 β, the lower the levels miR-7 and miR-
223, whereas levels of miR-133a correlate positively cytokine levels, as
shown in Fig. 4b–d.
4. Discussion
Inflammation is a crucial factor in the development of lesions during
Leishmania infection (Kaye and Scott, 2011). Assembly and activation of
the inflammasome complex during Leishmania infection has already
been demonstrated (Gurung et al., 2015; Lima-Junior et al., 2013). It
has been suggested this activation is important for the healing process
L.S.O. Mendonça, et al.
Fig. 2. Serum levels of the microRNAs miR-7, miR-133a, miR-146b, miR-223, and miR-328-3p in patients with American cutaneous leishmaniasis (ACL)
and individuals from the control group using quantitative PCR. A–E: serum levels of miR-7-5p, miR-133a, miR-146b, miR-223, and miR-328–3p, respectively.
Data show means and standard deviations using the non-parametric Man-Whitney U Test.
by stimulating the synthesis of nitric oxide and fighting of parasite
(Lima-Junior et al., 2013). However, this is controversial as other stu-
dies have shown that activated inflammasomes actually contribute to
the severity of this disease (Gurung et al., 2015; Novais et al., 2017).
Depending on the Leishmania species and the host cellular immune
response, different cytokines and chemokines can be produced. In this
study, a significant difference was observed in serum cytokines levels (IL-
1β, IL-6, and IL-17) from patients with CL compared to the control group,
which indicates a similar immune response during leishmaniasis (Espir
et al., 2014; Gomes et al., 2014). From all cytokines, we also found greater
levels for IL-1β, which suggests activation of inflammasomes.
IL-1β is an important mediator of innate and adaptive immune re-
sponses, and its association with inflammasomes activation has been
demonstrated. Exacerbation of IL-1β production and secretion may
trigger the inflammatory process that makes difficult to heal the lesions
in cutaneous leishmaniasis (Novais et al., 2017; Peniche et al., 2017).
However, IL-1β may also be involved in the severity of other forms of
the disease, as in the case of the diffused form caused by Leishmania
mexicana (Fernández-Figueroa et al., 2012).
IL-1β levels may be directly related to both the increase in IL-6 le-
vels and triggering of type Th17 immune response with IL-17 produc-
tion (Dinarello, 2011). IL-6 is a potent inflammatory cytokine that plays
a role in acute immune response. Curing infections or tissue lesions, IL-
6 mediates several physiological functions such as lymphocytes differ-
entiation and cell death mechanisms. IL-17 stimulates the secretion of
IL-6 and IL-8 in human fibroblasts and also participates in several mi-
crobial diseases and autoimmune diseases (Bettelli et al., 2007).
Our data showed a differential expression of the microRNAs miR-7-5p,
miR-133a, miR-146b, miR-223, and miR 328–3p, which may be related to
pathogen-host interaction in cutaneous leishmaniasis. When predicting
possible target genes, we identified several that were related to in-
flammasomes, such as NLRP3 in the miR-223, a protein involved in in-
flammasome assembly, and BCL2L1 and UCP2 in the miR-133a, an anti- or
pro-apoptotic regulator involved in NLR receptor signaling and a protein
that negatively controls inflammasomes activities, respectively (F.
5
Experimental Parasitology 210 (2020) 107846
Bauernfeind et al., 2012; Gupta et al., 2017; Liu et al., 2015a). In addition,
we identified possible target genes in the miR-146b, such as TRAF6 and
IRAK1, which are known to be involved in the immune response mediated
by TLRs (toll like receptors) (Park et al., 2015). It was important to predict
possible genes in the microRNAs miR-7 and miR-328 because they are
involved in processes of cell death, phagocytosis, and autophagy (Tay
et al., 2015; Wang et al., 2017).
Most studies involving miR-146 focused on inflammatory roles, and
this microRNA is extensively described as one of the induced miRNAs in
response to cytokines and pathogenic products in macrophages and
dendritic cells (Marques-Rocha et al., 2015; Park et al., 2015). This
miRNA plays a role as an anti-inflammatory regulator in various types
of immune cells by suppressing NF-κβ signaling and stimulating a ne-
gative feedback. Increased levels of miR-146 lead to the silencing of
IRAK1 and TRAF genes (Echavarria et al., 2015; Park et al., 2015).
miR-328 may have a role in regulating innate immune cells, which
can inhibit cell growth and promote apoptosis through PAK6 due to an
increase in caspases 3 and 9 levels (Liu et al., 2015b). When miR-328
expression is inhibited, phagocytosis increase and chances that patho-
gens will survive are reduced (Tay et al., 2015). microRNAs can
downregulate the PTPRJ gene that encodes for the protein tyrosine
phosphatase (PTP); dysfunction of tyrosine phosphorylation is related
to immune disorders (Paduano et al., 2012). In leishmaniasis, induction
of PTP expression by the parasite can lead to a negative regulation of
host cell functions (Blanchette et al., 1999), in addition to the sup-
pression of matrix metalloproteinase-2 (MMP2) (Yang et al., 2015a).
Increased expression of MMP2 are associated with successful healing
Leishmania lesions (Maretti-Mira et al., 2010).
Studies performed in vivo and in vitro showed that miR-7-5p is a
negative regulator of inflammasomes activation by significantly and
specifically decreasing the levels of the inflammasome protein NLRP3
(Zhou et al., 2016). There are studies that demonstrated the partici-
pation of miR-7-5p in the modulation of IL-1β gene, which suggests that
this microRNA may play a key role in the induction of this cytokine (Liu
et al., 2016).
L.S.O. Mendonça, et al. Experimental Parasitology 210 (2020) 107846

(caption on next page)

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L.S.O. Mendonça, et al. Experimental Parasitology 210 (2020) 107846

Fig. 3. Computational prediction for miR-7, miR-133a, miR-146b, miR-223, and miR-328-3p. A–E: Representation of the interaction of possible target genes
with miRNAs. Genes were selected based on the intersection of results obtained from three different online bioinformatics platforms (TargetScan, miRDB e
miRTarBase) and then, distributed in the integration network using Cytoscape Computational Program. Yellow ellipse represents miRNAs. Blue rectangles represent
possible target genes predicted in general. Green rectangles represent possible predicted target genes involved in programmed cell death. Red rectangles represent
possible predicted genes involved in caspase activity. Pink rectangles represent possible predicted genes involved in response to cytokine stimuli. (For interpretation
of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

miR-223 is described as a regulator of the inflammasome protein
NLRP3, targeting the protein binding site. When expression levels of
miR-223 are increased, inflammasome activation is compromised be-
cause levels and accumulation of the protein NLRP3 are decreased.
However, other studies suggest that miR-223 expression has an oppo-
site association with NLRP3 protein, and it might be a negative reg-
ulator of inflammasome (F. Bauernfeind et al., 2012; Chen and Sun,
2013; Yang et al., 2015b). Assays performed in vitro and in vivo have
shown that the inflammasome protein NLRP3 is activated in response to
Leishmania infection, and it is important to inhibit parasite's replication
in macrophages.
miR-133a participates in silencing the UCP2 gene, which can reg-
ulate the activation of inflammasomes. In a study with THP1 cells,
where UCP2 gene was silenced, an increase in inflammasome activa-
tion, caspase-1 level, and IL-1β secretion was observed when compared
to cells that exhibited greater expression of this gene. These results
suggest that UCP2 plays a role as a negative regulator of inflamma-
somes activation whereas miR-133a plays a role as a positive regulator
(Bandyopadhyay et al., 2013). Deficiency of UCP2 gene increases the
activation of the inflammasome protein NLRP3 by activating reactive
oxygen species (ROS). Expression of UCP2 gene was found to reduce
NLRP3 gene expression and IL-1β and IL-18 levels, but it did not affect
the expression of the inflammasome protein AIM2 (Moon et al., 2015).
In addition, miR-133a also targets MCL1 and BCLXL (BCL2L1 coding
gene) genes, which are inhibitors of apoptosis, caspase-1, and release of
IL-1β (Liu et al., 2015a). Thus, these results suggest that miR-133a
promotes inflammasomes activation.
Inflammasomes activation is critical during host defense mechan-
isms (Osawa et al., 2011). Still, in leishmaniasis, the role of in-
flammasomes in the host defense mechanism against Leishmania is not
well understood. Recent studies using resistant mice showed that
during L. amazonensis infection inflammasomes are important. How-
ever, during infections caused by L. major, L. mexicana, and L. donovani,
inflammasomes activation might be inhibited by these parasites, con-
tributing to the establishment of a virulent infection (Lima-Junior et al.,
2013; Shio et al., 2015).

Fig. 4. Correlation between levels of cytokines and microRNAs. A – Correlation matrix between levels of all cytokines and miRNAs. Circles and corresponding
sizes represent the significance level. Colors represent the directionality of the correlation (blue - positive correlations; red - negative correlations) B – The correlation
between levels of IL-1β and miR-133a indicates a positive correlation. C- The correlation between levels of IL-1β and miR-7, indicates a negative correlation. D - IL-1β
and miR-223 levels indicate a negative correlation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this
article.)

7
L.S.O. Mendonça, et al.
Pathogens evolved to modulate immune responses, such as the ac-
tivation of inflammasomes by preventing its assembly (Lamkanfi and
Dixit, 2012). One of the proposed mechanisms that leads to inhibition
of the inflammasome protein NLRP3 involves the prostaglandins (PG)
signaling pathway. Specifically, PGE2 suppresses the activation of the
inflammasome protein NLRP3 via induction of cyclic adenosine
monophosphate (cAMP), which binds to protein kinase A (PKA), re-
sulting in NLRP3 phosphorylation and inactivation of inflammasomes
(Mortimer et al., 2016). Leishmania donovani is able to induce PGE2 in
macrophages by stimulating cyclooxygenase 2 (COX2), an enzyme in-
volved in inflammation and production of prostaglandins. The later can
act as immunosuppressants during infection (Matte et al., 2001).
Studies have shown induction of PGE2 by Leishmania species that
cause the cutaneous disease form, in which animals infected with
Leishmania amazonensis were treated with prostaglandin antagonists
and parasitemia decreased (Guimarães et al., 2006). Furthermore, it has
been shown that patients with cutaneous leishmaniasis have higher
levels of plasma PGE2 compared to healthy individuals, suggesting that
prostaglandins are involved in the course of infection and they have an
important role in the persistence of the pathogen and modulation of the
host immune response (França-Costa et al., 2015).
Our results showed an increase in cytokines levels in the sera of
patients with American cutaneous leishmaniasis (ACL), especially for
IL-1β, which is an important cytokine that can be produced and se-
creted upon inflammasomes activation. Furthermore, increased IL-6
and IL-17 levels may be induced via NOD2 receptor by Leishmania
species that cause the cutaneous form of the disease (Dos Santos et al.,
2017). The differential expression of miR-133a has been described as a
positive regulator of inflammasomes activation. However, other circu-
lating microRNAs, such as miR-7-5p and miR-223, were identified to
negatively regulate inflammasomes.
In leishmaniasis, the host inflammatory response is important to con-
trol the infection and parasite replication. Our innate immune system is
able to fight pathogens by assembling complexes such as the inflamma-
some and it may be that certain miRNAs negatively regulate inflamma-
somes, which may contribute to pathogens’ immune evasion mechanisms,
ultimately inactivating inflammasomes. Our study showed the role of
microRNAs target genes in inflamasomes activation, such as miR-7 and
miR-223, which may inhibit the complex and facilitate the spread of the
parasite. These miRNAs were detected at higher levels in patients with
lower levels of IL-1 β, whereas patients with increased levels of this cy-
tokine also had a proportional increase in miR-133a levels, which may
indicate that this microRNA is important in the activation of the in-
flamasome and may contribute to resolution of this disease.
In sum, our results showed that certain microRNAs are differentially
expressed during cutaneous leishmaniasis, suggesting that they can be
used as possible biomarkers of the disease, especially because studies
involving miRNAs and leishmaniasis are still scarce. Our data also
showed an increase in pro-inflammatory cytokines levels (IL-1β, IL-6,
and IL-17), suggesting that these cytokines and microRNAs participate
in the regulation of the host immune response during cutaneous leish-
maniasis, and they might play a role in modulating the response to
inflammasomes activation.
Authors' contributions
LSOM, ISJ and JLS participated in the study design; LSOM, JMS, and
SMSC performed the selection of patients and collection of samples; LSOM,
JMS, CMK, and LDC performed the experiments; LSOM and ISJ interpreted
the results; LSOM, ISJ, LDC, DGA, and JAPSM prepared this manuscript.
Funding
This work was supported by both the Fundação de Amparo à
Pesquisa do Estado da Bahia (FABESB Grant term PET0033/2013) and
the Universidade Estadual de Santa Cruz (UESC).
8
Experimental Parasitology 210 (2020) 107846
Declaration of competing interest
The authors declare that they have no competing interests.
Acknowledgments
Authors thank patients for participating in this study and SMS-
CAEIII employees in Ilhéus-BA for their support during collection of
samples.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.exppara.2020.107846.
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Lucilla Silva Oliveira Mendonça. Master's Degree student
at the Universidade Estadual de Santa Cruz (Ilhéus-BA-
Brazil) from 2016 to 2018. She was part of the “Role of
Inflammasomes in Cutaneous Leishmaniasis” research
group and currently is PhD student in the same institution
from 2018 to 2022, acting on a project related to im-
munomodulation of human phagocytes.
L.S.O. Mendonça, et al.
Jaqueline Marques dos Santos. Graduated in Nursing at
the State University of Santa Cruz in the year 2013, com-
pleting it in 2018. During this period, she was a student of
Scientific Initiation at the Laboratory of Immunobiology,
where she developed the project entitled: Role of
Inflamamosomes and Polymorphisms Genetics in
Resistance and Susceptibility to American Cutaneous
Leishmaniasis. He is currently a student of the Graduate
Program in Immunology of the University of São Paulo,
developing his research project in the Immunology
Laboratory of Mucosa, studying more specifically the mu-
cosa of the gastrointestinal tract.
Dr. Carla M. Kaneto is a professor of Human Genetics at
Santa Cruz State University. She has coauthored publica-
tions including stem cell differentiation and gene expres-
sion in Cancer, Mesenchymal Stem Cells and Cardiovascular
Diseases. The current research interests in Professor
Kaneto's group include MicroRNAs expression in cardio-
vascular diseases, diabetes and leukemia.
Dr Luciana Debortoli de Carvalho is a visiting professor
in the area of microbiology and immunology at the Santa
Cruz State University. I have experience in Microbiology
working mainly in the following subjects: Immunology,
Virology, Bacteriology, General and Medical Mycology,
Clinical Analyzes, Molecular biology and bioprospecting of
plants with biological activity against microorganisms.
WCTC) in Milwaukee area.
Jane Lima dos Santos. PhD in Biochemistry and
Immunology, MA in Microbiology, and BSc in Biological
Science. Currently working as a research/professor at
Universidade Estadual de Santa Cruz (UESC), Ilhéus/BA.
Some areas of research include cellular immunology, im-
munity to microorganisms with emphasis on host-micro-
organism interaction In vitro and In vivo, and the impact of
biofungicide and biofertilizers in this interaction. Advisor
to master degree and PhD students connected to UESC
Biology and Biotechnology of Microorganism
Postgraduation program. Engaged in university extension
activities, such as projects to disseminate and popularize
Science in the area of health knowledge.
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Experimental Parasitology 210 (2020) 107846
Danillo Gardenal Augusto. Bachelor in Biological Sciences
(2006), Master in Genetics (2008) and PhD in Genetics
awarded by Federal University of Paraná. Brazil. (2012).
Postdoctoral experience in prestigious institutions such as
Harvard Medical School (USA) and the National Institutes
of Health (USA). Experienced in Genetics and Immunology,
with emphasis on human molecular genetics, especially in
immunogenetics, population genetics, KIR and HLA genes,
genetic susceptibility to complex and infectious diseases,
molecular biology, gene expression and next generation
sequencing. Currently holds a position of Full Specialist at
the University of California, San Francisco.
Silvia Maria de Carvalho. Bachelor in Biological Sciences
at the Catholic University of Salvador; Master in Genetics at
the Federal University of Pernambuco; PhD in Public Health
at the Ageu Magalhães Research Center, FIOCRUZ-PE.
Currently working as a Professor of Human Parasitology,
Medical Parasitology and Medical Entomology at State
University of Santa Cruz. Experienced in Leishmaniasis and
enteric parasites.
Jamária A. P. Soares Martins. Dr. Martins graduated in
Biological Sciences at Universidade Federal de Viçosa in
2002, where she started working with microbiology. She
earned her Master (2004) and Ph.D. (2007) both in
Microbiology at Universidade Federal de Minas Gerais, still
in Brazil. While working there, Dr. Martins' research helped
to elucidate critical differences in the cellular signaling
pathways induced by genetically related poxviruses,
leading to viral tropism. Dr. Martins completed her
Postdoctoral Fellowship at The Medical College of
Wisconsin in Milwaukee, USA (2013). She currently teaches
science classes at the technical colleges (both MATC and
Izaltina Silva-Jardim. Bachelor in Biological Sciences at
the Federal University of Minas Gerais, Master in Basic and
Applied Immunology and PhD degree in Biochemistry
awarded by Ribeirão Preto Medical School, University of
São Paulo. From 2005 to 2010, she was a researcher at
Institute of Research in Tropical Disease – IPEPATRO
(current FIOCRUZ – Rondônia) and a visitant professor at
Federal University of Rondônia. Postdoctoral experience at
São Carlos Institute of Physics, University of São Paulo.
Currently working as a professor/researcher at State
University of Santa Cruz –UESC. Her research focuses in
chemotherapy for leishmaniasis and host-parasite interac-
tion.