Characterization of serum cytokines and circulating microRNAs that are predicted to regulate inflammasome genes in cutaneous leishmaniasis patients

Journal Pre-proof Characterization of serum cytokines and circulating microRNAs that are predicted to regulate inflammasome genes in cutaneous leishmaniasis patients Lucilla Silva Oliveira Mendonça, Jaqueline Marques Santos, Carla Martins Kaneto, Luciana Debortoli de Carvalho, Jane Lima dos Santos, Danillo Gardenal Augusto, Silvia Maria Santos Carvalho, Jamária Adriana Pinheiro Soares-Martins, Izaltina Silva-Jardim PII:

S0014-4894(18)30568-X

DOI:

https://doi.org/10.1016/j.exppara.2020.107846

Reference:

YEXPR 107846

To appear in:

Experimental Parasitology

Received Date: 21 December 2018 Revised Date:

8 August 2019

Accepted Date: 24 January 2020

Please cite this article as: Mendonça, L.S.O., Santos, J.M., Kaneto, C.M., de Carvalho, L.D., dos Santos, J.L., Augusto, D.G., Carvalho, S.M.S., Soares-Martins, Jamá.Adriana.Pinheiro., Silva-Jardim, I., Characterization of serum cytokines and circulating microRNAs that are predicted to regulate inflammasome genes in cutaneous leishmaniasis patients, Experimental Parasitology (2020), doi: https://doi.org/10.1016/j.exppara.2020.107846. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

Characterization of serum cytokines and circulating microRNAs that are 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 dos Santosc, Danillo Gardenal 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,

CURITIBA – PR – BRAZIL e

DEPARTMENT OF LIBERAL ARTS AND SCIENCES, MILWAUKEE AREA

TECHNICAL COLLEGE (MATC), MILWAUKEE – WI – UNITED STATES

* Corresponding author E-mail: [email protected]

1

Abstract Leishmaniasis is neglected diseases caused by an intracellular protozoan parasite of the genus Leishmania. Infection starts when this protozoan replicates in a phagolysosomal compartment in macrophages, after evading host immune responses. The balance of Th1 and Th2 immune responses is crucial in leishmaniasis because it will determine whether the infection will be under control or if clinical complications will occur. The inflammasome, which is activated during Leishmania infection, involves the action of caspase-1 and release of the proinflammatory 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 leishmaniasis 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 infection.

Keywords: cutaneous leishmaniasis, cytokines, inflammasomes, microRNAs.

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1. Introduction

Leishmaniasis is tropical parasitic diseases 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 cutaneous (CL) and mucosal

leishmaniasis

(ML)

(SisLeish-OPAS/WHO,

2018).

Disseminated

cutaneous leishmaniasis (DL) accounts for approximately 2% of the reported cases of CL in Brazil and difuse cutaneous 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 2,362 new cases are notified annually. The coefficient of detection was found to be at 32.8% risk of transmission in the Bahia state (SINAN and SUVISA, 2016). Depending on the host immune response and the parasite’s entry, tropism, and pathogenicity, the disease can have outcomes ranging from cutaneous lesions at the bite site, mucosal lesions to visceralization, a more severe condition known as visceral leishmaniasis or kala azar (Herwaldt, 1999). The main species of Leishmania found in developed countries cause cutaneous manifestations, which may either heal spontaneously or progress to an intense inflammatory state, making the treatment difficult (Herwaldt, 1999). The ACL may be subdivided into four types: i) localized cutaneous leishmaniasis (LCL), which is characterized as an ulceration with raised edges; ii) mucosal cutaneous leishmaniasis (MCL), which is a progression of the cutaneous form and it is characterized by the destruction of the mucosa tissues, especially in the upper respiratory tract, iii) the rare form DL, which is characterized by the appearance of multiple polymorphic skin lesions in various body regions, and most of the cases documented in the Americas 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 3

the disease with infiltrative nodular lesions caused by L. (L.) amazonensis (Barral et al., 1995; Brasil, 2017; David and Craft, 2009). During the course of the infection, the host’s innate immunity plays an important initial role in the immune response by recruiting cells such as neutrophils, macrophages, and dendritic cells. These cells induce the production of reactive oxygen species (ROS) and release of nitric oxide (NO), in addition to pro-inflammatory cytokines that help to fight the pathogen (Kedzierski and Evans, 2014). Cells from the innate immune system detect microorganisms through their pattern recognition receptors (PPRs) which bind to pathogen-associated molecular patterns (PAMP), leading to a more specific immune response (Gurung and Kanneganti, 2016). NOD-like receptors (NLRs), a family of PPRs, were shown to have a crucial role in defending the host against intracellular pathogens. NLRs play a role in forming inflammasome complexes (Martinon and Tschopp, 2005). The term inflammasome was initially described to determine a high molecular weight complex that activates caspase-1 and it is responsible for processing of the proinflammatory cytokines IL-1β and IL-18 (Martinon et al., 2002). Inflammasomes can generate efficient signals and responses against pathogens by triggering the release of cytokines, activation of other immune cells, and programmed cell death (Di Virgilio, 2013). Some molecules can participate in the regulation of immune responses, such as microRNAs (miRNAs) (Sutterwala et al., 2014). MicroRNAs are small endogenous RNAs of approximately 22 non-coding nucleotides, which act as posttranscriptional regulators, cleaving mature mRNAs after binding to their untranslated 3'-end (He and Hannon, 2004). It is known that gene expression regulation by miRNAs also impacts the expression of genes involved in the immune response. Some miRNAs may have their expression increased in certain cell types, such as neutrophils, macrophages, and lymphocytes, ultimately regulating several biological processes such as development, differentiation, and regulation of the immune system (Miska, 2005; Xiao and Rajewsky, 2009). miRNAs play a modulatory role in the assembly of the inflammasome complex (Sutterwala et al., 2014), but the regulation of miRNAs in leishmaniasis is 4

still unclear. Some studies indicate the presence of unregulated miRNAs in human cells and cutaneous lesions, which affects the innate immune response (Diotallevi et al., 2018; Hashemi et al., 2018; Lemaire et al., 2013; Nunes et al., 2018). Up to this date, there are no reports showing the participation of miRNAs in the modulation of inflammasomes in cutaneous leishmaniasis, and this complex might play an essential role in the immune response and tissue damage during the infection. In addition, miRNAs related to inflammasomes could help in the understanding the role of this protein complex during infection. Furthermore, miRNAs could be potential biomarkers for this disease, and detection of their circulating levels could help in the diagnosis, prognosis, and treatment of leishmaniasis.

2. Material and Methods

2.1. Ethical aspects

This study was submitted to evaluation, and it was approved by the Ethics Committee on Human Research at UESC (CAAE 18815013.7.1001.5526). Samples were only collected after patients were informed about the scope of this study and upon signing the informed consent form. The study was conducted according to the Helsinki Declaration and Resolution 510/2016 of the National Health Council of the Ministry of Health that regulates studies involving humans in Brazil.

2.2. Population and sample collection

Our study had 27 patients with active skin lesions and confirmed diagnosis for cutaneous leishmaniasis that were enrolled prior to treatment. These patients were selected at the Health Center CAE III in Ilhéus-BA, and they were diagnosed with CL based on

the clinical characteristics of the disease along

with the

Montenegro test. Often, these patients had one or a few ulcerative cutaneous lesions with raised and delimited borders located in the lower limbs. Patients 5

diagnosed with MCL were not included in this study. The control group was comprised of 9 healthy individuals, in which they were grouped according to their age, gender, and place of residence. The exclusion criteria for the control group included absence of skin lesions or infectious diseases and absence of previous history of leishmaniasis. The information about our study population is presented in table 1.

Table 1. Characteristics of participants at inclusion Groups Variables

Control

Significance (p value)

Patients

N

%

N

%

Male

5

55.6

16

59.3

Female

4

44.4

11

40.7

18-25

2

22.2

5

18.5

26-40

3

33.3

7

25.9

41-50

1

11.1

2

7.41

51-60

2

22.2

5

18.5

61-70

1

11.1

7

25.9

Rural area

3

33.3

16

59.3

Urban area

6

66.7

11

40.7

NA NA

NA NA

10 17

37 63

Sex NS (>0.99)

Age (years) NS (0.91)

Place of residence NS (0.25)

Relapse Yes No Presence of skin lesions

NA Yes

0

0

27

100

No 9 100 0 0 Note: N-number samples. NA- not applicable. NS-not significant (Chi-Square test).

We collected 8 milliliters (ml) of peripheral venous blood from patients with cutaneous leishmaniasis and control group using vacuplast vacuum collection tubes®. We used 4 ml-tubes without additives and containing a clot activator to separate the serum, and 4 ml-tubes with EDTA as an additive to separate the plasma. 6

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, fluorochrome 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 hour at room temperature. After that, 50 µL of mixed detection reagents were added to the samples, followed by incubation in the dark for 2 hours. 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 minutes. After that, tubes were centrifuged at 15000 RPM at 4°C for 15 minutes. 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 minutes. 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. 7

Complementary DNA synthesis (cDNA) was performed from RNA samples extracted from patients’ blood (500ƞg) using miR-specific primers 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 incubated 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 associated 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

Reference

miR-7

- miR-7 as inhibitor of NLRP3 and IL-1β production during

(Zhou et al., 2016).

neuroinflammation in Parkinson's disease. - Suppression of NLRP3 and caspase-1 in neural stem

(Fann et al., 2017).

cells. miR-133

miR-146

Regulation of inflammasome activation via suppression of

(Bandyopadhyay et al.,

UCP2 in THP cells.

2013).

High expression of miR-146 downregulate both the

(Bhatt et al., 2016).

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

(Haneklaus

NLRP3 inflammasome and IL-1β are produced.

2012).

et

al.,

- The activation of inflammasomes is negatively regulated by miR-223.

(Franz Bauernfeind et

- miR-223 inhibits the production of caspase-1, NLRP3,

al., 2012).

and IL-1β during intracerebral hemorrhage in animal

(Z. Yang et al., 2015).

models. 8

miR-328

miR-328 is differentially expressed in macrophages

(Tiwari et al., 2017)

infected with Leishmania donovani, suggesting that it may play a role in phagocytosis.

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). hsamiR-342-3p (TM 002260) was used to normalize miRNA input. microRNA expression was evaluated using quantitative polymerase chain reaction (qPCR). qPCR reactions contained previously 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 following conditions: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 60 seconds using ABI 7500 Real Time PCR System. Levels of targeted miRNAs were normalized based on the endogenous hsa-miR342-3p, 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). Quantifications of miRNAs levels were calculated using the 2-∆CT method.

2.7. Prediction of target genes for microRNAs

MicroRNAs found to be differentially expressed were further evaluated using computational prediction on online bioinformatics platforms 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 interactions, we selected three online bioinformatics tools widely used in predictive studies (Targetscantargetscan.org;

Mirdb-mirdb.org

and

Mirtarbase-mirtarbase.nbc.nctu.edu.tw), 9

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 differentially 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 expression 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-parametric MannWhitney test (for two independent samples) using GraphPad Prism (7.0) software. Results were considered significant when p-value was < 0.05. We used the R software for Spearman correlation analysis tests (for the correlation matrix) and simple x-y plot. To determine the homogeneity in our group population, we performed 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.

10

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.

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).

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 increased 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 expressions were 10,000 and 3.8-fold up in patients with cutaneous leishmaniasis, 11

respectively, whereas miRNAs miR133a, miR328-3-P and miR146b presented pvalues equal to 0.034, 0.002, and 0.003, respectively.

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.

To understand the role of these miRNAs in gene regulation, we used in silico prediction to search for their possible targets. After the 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 programmed 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).

12

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

13

involved in caspase activity. Pink rectangles represent possible predicted genes involved in response to cytokine stimuli.

Altogether, our results showed an up-regulation of microRNAs associated 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 correlation between levels of IL-1β and the microRNAs miR-223 and miR-7, whereas levels of miR-133a, miR-146b, and miR-328 presented positive 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 important 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 Figure 4b-d.

14

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 .

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 by stimulating the synthesis of nitric oxide and fighting of parasite (Lima-Junior et al., 2013). However, this is controversial as other studies have shown that activated 15

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 IL17) 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 responses, 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 levels and triggering of type Th17 immune response with IL-17 production (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 differentiation and cell death mechanisms. IL-17 stimulates the secretion of IL-6 and IL-8 in human fibroblasts and also participates in several microbial diseases and autoimmune diseases (Bettelli et al., 2007). Our data showed a differential expression of the microRNAs miR-7-5p, miR133a, miR-146b, miR-223, and miR328-3p, which may be related to pathogenhost interaction in cutaneous leishmaniasis. When predicting possible target genes, we identified several that were related to inflammasomes, such as NLRP3 in the miR-223, a protein involved in inflammasome 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. Bauernfeind et al., 2012; Gupta et al., 2017; Y. Liu et al., 2015). In addition, we identified possible target genes in the miR-146b, such as TRAF6 and 16

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 (MarquesRocha et al., 2015; Park et al., 2015). This miRNA plays a role as an antiinflammatory regulator in various types of immune cells by suppressing NF-κβ signaling and stimulating a negative 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 (C. Liu et al., 2015). When miR-328 expression is inhibited, phagocytosis increase and chances that pathogens 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 suppression of matrix metalloproteinase-2 (MMP2) (S.-F. Yang et al., 2015). 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 participation 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).

17

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 because levels and accumulation of the protein NLRP3 are decreased. However, other studies suggest that miR-223 expression has an opposite association with NLRP3 protein, and it might be a negative regulator of inflammasome (F. Bauernfeind et al., 2012; Chen and Sun, 2013; Z. Yang et al., 2015). 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 regulate the activation of inflammasomes. In a study with THP1 cells, where UCP2 gene was silenced, an increase in inflammasome activation, 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 inflammasomes 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 (Y. Liu et al., 2015). Thus, these results suggest that miR-133a promotes inflammasomes activation. Inflammasomes activation is critical during host defense mechanisms (Osawa et al., 2011). Still, in leishmaniasis, the role of inflammasomes 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. However, during infections caused by L. major, L. mexicana, and L. donovani, inflammasomes activation might be inhibited by these parasites, contributing to the establishment of a virulent infection (Lima-Junior et al., 2013; Shio et al., 2015). 18

Pathogens evolved to modulate immune responses, such as the activation 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), resulting 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 involved 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 secreted 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 circulating microRNAs, such as miR-7-5p and miR-223, were identified to negatively regulate inflammasomes. In leishmaniasis, the host inflammatory response is important to control the infection and parasite replication. Our innate immune system is able to fight pathogens by assembling complexes such as the inflammasome and it may be that certain miRNAs negatively regulate inflammasomes, which may contribute to 19

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 cytokine also had a proportional increase in miR-133a levels, which may indicate that this microRNA is important in the activation of the inflamasome 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 proinflammatory 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 leishmaniasis, 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.

Conflicts of interest The authors declare that they have no competing interests.

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).

20

Acknowledgments Authors thank patients for participating in this study and SMS-CAEIII employees in Ilhéus-BA for their support during collection of samples.

References Bandyopadhyay, S., Lane, T., Venugopal, R., Parthasarathy, P.T., Cho, Y., Galam, L., Lockey, R., Kolliputi, N., 2013. MicroRNA-133a-1 regulates inflammasome activation through uncoupling protein-2. Biochem. Biophys. Res. Commun. 439, 407–412. https://doi.org/10.1016/j.bbrc.2013.08.056 Barral, A., Costa, J.M.L., Bittencourt, A.L., Barral‐Netto, M., Carvalho, E.M., 1995. Polar and Subpolar Diffuse Cutaneous Leishmaniasis in Brazil: Clinical and Immunopathologic

Aspects.

Int.

J.

Dermatol.

34,

474–479.

https://doi.org/10.1111/j.1365-4362.1995.tb00613.x Bauernfeind, F., Rieger, A., Schildberg, F.A., Knolle, A., Schmid-burgk, J.L., Hornung, V., Schmid-burgk, J.L., Hornung, V., 2012. NLRP3 Inflammasome Activity

Is

Negatively

Controlled

by

miR-223.

https://doi.org/10.4049/jimmunol.1201516 Bettelli, E., Oukka, M., Kuchroo, V.K., 2007. TH-17 cells in the circle of immunity and autoimmunity. Nat. Immunol. 8, 345–350. https://doi.org/10.1038/ni0407345 Bhatt, K., Lanting, L.L., Jia, Y., Yadav, S., Reddy, M.A., Magilnick, N., Boldin, M., Natarajan, R., 2016. Anti-Inflammatory Role of MicroRNA-146a in the Pathogenesis of Diabetic Nephropathy. J. Am. Soc. Nephrol. 27, 2277–2288. https://doi.org/10.1681/ASN.2015010111 Blanchette, J., Racette, N., Faure, R., Siminovitch, K.A., Olivier, M., 1999. Leishmania-induced increases in activation of macrophage SHP-1 tyrosine phosphatase are associated with impaired IFN-γ-triggered JAK2 activation. Eur.

J.

Immunol.

29,

3737–3744.

https://doi.org/10.1002/(SICI)1521-

4141(199911)29:11<3737::AID-IMMU3737>3.0.CO;2-S Brasil, M. da saúde, 2017. Manual De Vigilância Da Leishmaniose Tegumentar, Ministério. ed, Secretaria de Vigilância em Saúde. Departamento de Vigilância das Doenças Transmissíveis. Brasilia. 21

Chen, S., Sun, B., 2013. Negative regulation of NLRP3 inflammasome signaling. Protein Cell 4, 251–258. https://doi.org/10.1007/s13238-013-2128-8 David, C., Craft, N., 2009. Cutaneous and mucocutaneous leishmaniasis. Dermatol.

Ther.

22,

491–502.

https://doi.org/10.1111/j.1529-

8019.2009.01272.x Di Virgilio, F., 2013. The Therapeutic Potential of Modifying Inflammasomes and NOD-Like

Receptors.

Pharmacol.

Rev.

65,

872–905.

https://doi.org/10.1124/pr.112.006171 Dinarello, C.A., 2011. Interleukin-1 in the pathogenesis and treatment of inflammatory diseases. Blood 117, 3720–3732. https://doi.org/10.1182/blood2010-07-273417 Diotallevi, A., Santi, M. De, Buffi, G., Ceccarelli, M., Vitale, F., Galluzzi, L., Magnani, M., 2018. Leishmania Infection Induces MicroRNA hsa-miR-346 in Human

cell

line-derived

macrophages.

Front.

Microbiol.

9,

1–9.

https://doi.org/10.3389/fmicb.2018.01019 Dos Santos, J.C., Damen, M.S.M.A., Oosting, M., De Jong, D.J., Heinhuis, B., Gomes, R.S., Araújo, C.S., Netea, M.G., Ribeiro-Dias, F., Joosten, L.A.B., 2017. The NOD2 receptor is crucial for immune responses towards New World Leishmania species. Sci. Rep. 7, 1–10. https://doi.org/10.1038/s41598017-15412-7 Echavarria, R., Mayaki, D., Neel, J.C., Harel, S., Sanchez, V., Hussain, S.N.A., 2015. Angiopoietin-1 inhibits toll-like receptor 4 signalling in cultured endothelial cells: Role of miR-146b-5p. Cardiovasc. Res. 106, 465–477. https://doi.org/10.1093/cvr/cvv120 Espir, T.T., Figueira, L.D.P., Naiff, M.D.F., Da Costa, A.G., Ramalho-Ortigão, M., Malheiro, A., Franco, A.M.R., 2014. The Role of Inflammatory, AntiInflammatory, and Regulatory Cytokines in Patients Infected with Cutaneous Leishmaniasis in Amazonas State, Brazil. J. Immunol. Res. 2014. https://doi.org/10.1155/2014/481750 Fann, D.Y.-W., Lim, Y.-A., Cheng, Y.-L., Lok, K.-Z., Chunduri, P., Baik, S.-H., Drummond, G.R., Dheen, S.T., Sobey, C.G., Jo, D.-G., Chen, C.L.-H., Arumugam, T. V., 2017. Evidence that NF-κB and MAPK Signaling Promotes 22

NLRP Inflammasome Activation in Neurons Following Ischemic Stroke. Mol. Neurobiol. 1–15. https://doi.org/10.1007/s12035-017-0394-9 Fernández-Figueroa, E.A., Rangel-Escareño, C., Espinosa-Mateos, V., CarrilloSánches, K., Salaiza-Suazo, N., Al., E., 2012. Disease Severity in Patients Infected with Leishmania mexicana Relates to IL-1 b. PLoS Negl Trop Dis 6. https://doi.org/10.1371/journal.pntd.0001533 França-Costa, J., Van Weyenbergh, J., Boaventura, V.S., Luz, N.F., Malta-Santos, H., Oliveira, M.C.S., De Campos, D.C.S., Saldanha, A.C., Dos-Santos, W.L.C., Bozza, P.T., Barral-Netto, M., Barral, A., Costa, J.M., Borges, V.M., 2015. Arginase I, polyamine, and prostaglandin E2pathways suppress the inflammatory response and contribute to diffuse cutaneous leishmaniasis. J. Infect. Dis. 211, 426–435. https://doi.org/10.1093/infdis/jiu455 Gomes, C.., Ávila, L.R., Pinto, S.A., Duarte, F.B., Pereira, L.I.A., Abrahamsohn, I.A., Dorta, M.L., Vieira, L.Q., Ribeiro-Dias, F., Oliveira, M.A.P., 2014. Leishmania braziliensis amastigotes stimulate production of IL-1β, IL-6, IL-10 and TGF-β by PBMC from non-endemic area healthy residents. Parasite Immunol. 36, 225–231. https://doi.org/10.1111/pim.12109 Guimarães, E.T., Santos, L.A., Ribeiro dos Santos, R., Teixeira, M.M., dos Santos, W.L.C., Soares, M.B.P., 2006. Role of interleukin-4 and prostaglandin E2 in Leishmania amazonensis infection of BALB/c mice. Microbes Infect. 8, 1219– 1226. https://doi.org/10.1016/j.micinf.2005.11.011 Gupta, A.K., Ghosh, K., Palit, S., Barua, J., Das, P.K., Ukil, A., 2017. Leishmania donovani

inhibits

inflammasome-dependent

macrophage

activation

by

exploiting the negative regulatory proteins A20 and UCP2. FASEB J. 31, 5087–5101. https://doi.org/10.1096/fj.201700407R Gurung, P., Kanneganti, T.D., 2016. Immune responses against protozoan parasites: a focus on the emerging role of Nod-like receptors. Cell. Mol. Life Sci. 73, 3035–3051. https://doi.org/10.1007/s00018-016-2212-3 Gurung, P., Karki, R., Vogel, P., Watanabe, M., Bix, M., Lamkanfi, M., Kanneganti, T., 2015. An NLRP3 inflammasome – triggered Th2-biased adaptive immune response

promotes

leishmaniasis.

https://doi.org/10.1172/JCI79526DS1 23

J.

Clin.

Invest.

125,

1329–1338.

Haneklaus, M., Gerlic, M., Kurowska-Stolarska, M., Rainey, A.-A., Pich, D., McInnes, I.B., Hammerschmidt, W., O’Neill, L.A.J., Masters, S.L., 2012. Cutting Edge: miR-223 and EBV miR-BART15 Regulate the NLRP3 Inflammasome

and

IL-1

Production.

J.

Immunol.

189,

3795–3799.

https://doi.org/10.4049/jimmunol.1200312 Hashemi, N., Sharifi, M., Masjedi, M., Tolouei, S., Hejazi, S.H., 2018. Locked nucleic acid -anti- let-7a induces apoptosis and necrosis in macrophages infected

with

Leishmania

major.

Microb.

Pathog.

https://doi.org/10.1016/j.micpath.2018.03.057 He, L., Hannon, G.J., 2004. MicroRNAs: Small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522–531. https://doi.org/10.1038/nrg1379 Herwaldt,

B.L.,

1999.

Leishmaniasis.

Lancet

354,

1191–1199.

https://doi.org/10.1016/S0140-6736(98)10178-2 Kaye, P., Scott, P., 2011. Leishmaniasis : complexity at the host – pathogen interface. Nat. Publ. Gr. 9, 604–615. https://doi.org/10.1038/nrmicro2608 Kedzierski, L., Evans, K.J., 2014. Immune responses during cutaneous and visceral

leishmaniasis.

Parasitology

141,

1544–1562.

https://doi.org/10.1017/S003118201400095X Lamkanfi, M., Dixit, V.M., 2012. Inflammasomes and Their Roles in Health and Disease.

Annu.

Rev.

Cell

Dev.

Biol.

28,

137–161.

https://doi.org/10.1146/annurev-cellbio-101011-155745 Lemaire, J., Mkannez, G., Guerfali, F., Gustin, C., Attia, H., Sysco-Consortium, Laouini, D., Sghaier, R.M., Dellagi, K., Renard, P., 2013. MicroRNA Expression Profile in Human Macrophages in Response to Leishmania major Infection.

PLoS

Negl.

Trop.

Dis.

7.

https://doi.org/10.1371/journal.pntd.0002478 Liang, Y., Ridzon, D., Wong, L., Chen, C., 2007. Characterization of microRNA expression profiles in normal human tissues. BMC Genomics 8, 1–20. https://doi.org/10.1186/1471-2164-8-166 Lima-Junior, D.S., Costa, D.L., Carregaro, V., Cunha, L.D., Silva, A.L.N., Mineo, T.W.P., Gutierrez, F.R.S., Bellio, M., Bortoluci, K.R., Flavell, R. a, Bozza, M.T., Silva, J.S., Zamboni, D.S., 2013. Inflammasome-derived IL-1β 24

production induces nitric oxide-mediated resistance to Leishmania. Nat. Med. 19, 909–15. https://doi.org/10.1038/nm.3221 Liu, C., Zhang, L.E.I., Huang, Y., Lu, K.A.I., Tao, T.A.O., Chen, S., Zhang, X., Guan, H.A.N., Chen, M., Xu, B.I.N., 2015. MicroRNA ‑ 328 directly targets p21 ‑ activated protein kinase 6 inhibiting prostate cancer proliferation and enhancing

docetaxel

sensitivity

7389–7395.

https://doi.org/10.3892/mmr.2015.4390 Liu, W., Zhang, Y., Xia, P., Li, S., Feng, X., Gao, Y., Wang, K., Song, Y., Duan, Z., Yang, S., Shao, Z., Yang, C., 2016. MicroRNA-7 regulates IL-1β-induced extracellular matrix degeneration by targeting GDF5 in human nucleus pulposus

cells.

Biomed.

Pharmacother.

83,

1414–1421.

https://doi.org/10.1016/j.biopha.2016.08.062 Liu, Y., Zhang, X., Zhang, Y., Hu, Z., Yang, D., Wang, C., Guo, M., Cai, Q., 2015. Identification of miRNomes in human stomach and gastric carcinoma reveals miR-133b/a-3p as therapeutic target for gastric cancer. Cancer Lett. 369, 58– 66. https://doi.org/10.1016/j.canlet.2015.06.028 Maretti-Mira, A.., Oliveira-Neto, M.P., Da-Cruz, A.M., Craft, N., Pirmez, C., 2010. Therapeutic failure in American cutaneous leishmaniasis is associated with gelatinase activity and cytokine expression. Clin. Exp. Immunol. 163, 207– 214. https://doi.org/10.1111/j.1365-2249.2010.04285.x Marques-Rocha, J.L., Samblas, M., Milagro, F.I., Bressan, J., Martínez, J.A., Marti, A., 2015. Noncoding RNAs, cytokines, and inflammation-related diseases. FASEB J. 29, 3595–3611. https://doi.org/10.1096/fj.14-260323 Martinon, F., Burns, K., Tschopp, J., 2002. The Inflammasome: A molecular platform triggering activation of inflammatory caspases and processing of proIL-β. Mol. Cell 10, 417–426. https://doi.org/10.1016/S1097-2765(02)005993 Martinon, F., Tschopp, J., 2005. NLRs join TLRs as innate sensors of pathogens. Trends Immunol. 26, 447–454. https://doi.org/10.1016/j.it.2005.06.004 Matte, C., Maion, G., Mourad, W., Olivier, M., 2001. Leishmania donovani induced macrophages cyclooxygenase-2 and prostaglandin E 2 synthesis. Parasite

Immunol.

23,

177–184. 25

https://doi.org/10.1046/j.1365-

3024.2001.00372.x Miska, E.A., 2005. How microRNAs control cell division, differentiation and death. Curr.

Opin.

Genet.

Dev.

15,

563–568.

https://doi.org/10.1016/j.gde.2005.08.005 Moon, J.S., Lee, S., Park, M.A., Siempos, I.I., Haslip, M., Lee, P.J., Yun, M., Kim, C.K., Howrylak, J., Ryter, S.W., Nakahira, K., Choi, A.M.K., 2015. UCP2induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis. J. Clin. Invest. 125, 665–680. https://doi.org/10.1172/JCI78253 Mortimer, L., Moreau, F., MacDonald, J.A., Chadee, K., 2016. NLRP3 inflammasome inhibition is disrupted in a group of auto-inflammatory disease CAPS

mutations.

Nat.

Immunol.

17,

1176–1186.

https://doi.org/10.1038/ni.3538 Novais, F.O., Carvalho, A.M., Clark, M.L., Carvalho, L.P., Beiting, D.P., Brodsky, I.E., Carvalho, E.M., Scott, P., 2017. CD8+ T cell cytotoxicity mediates pathology in the skin by inflammasome activation and IL-1β production. PLOS Pathog. 13, e1006196. https://doi.org/10.1371/journal.ppat.1006196 Nunes, S., Silva, I.B., Ampuero, M.R., Lopes, A.L., 2018. Integrated Analysis Reveals That miR-193b, miR-671, and TREM-1 Correlate With a Good Response to Treatment of Human Localized Cutaneous Leishmaniasis Caused

by

Leishmania

braziliensis

9,

1–13.

https://doi.org/10.3389/fimmu.2018.00640 Osawa, R., Williams, K.L., Singh, N., 2011. The inflammasome regulatory pathway and infections: Role in pathophysiology and clinical implications. J. Infect. 62, 119–129. https://doi.org/10.1016/j.jinf.2010.10.002 Paduano, F., Dattilo, V., Narciso, D., Bilotta, A., Gaudio, E., Menniti, M., Agosti, V., Palmieri, C., Perrotti, N., Fusco, A., Iuliano, R., 2012. Protein tyrosine phosphatase PTPRJ is negatively regulated by microRNA-328. FESB J. 280, 401–412. https://doi.org/10.1111/j.1742-4658.2012.08624.x Park, H., Huang, X., Lu, C., Cairo, M.S., Zhou, X., 2015. MicroRNA-146a and microRNA-146b regulate human dendritic cell apoptosis and cytokine production by targeting TRAF6 and IRAK1 proteins. J. Biol. Chem. 290, 2831–2841. https://doi.org/10.1074/jbc.M114.591420 26

Peniche, A.G., Bonilla, D.L., Palma, G.I., Melby, P.C., Travi, L., Osorio, E.Y., 2017. A secondary wave of neutrophil infiltration causes necrosis and ulceration in lesions of experimental American cutaneous leishmaniasis. PLoS One 12, 1– 20. https://doi.org/https://doi.org/10.1371/journal. pone.0179084 Schriefer, A., Guimarães, L.H., Machado, P.R.L., Lessa, M., Lessa, H.A., Lago, E., Ritt, G., Góes-neto, A., Schriefer, A.L.F., Riley, L.W., Carvalho, E.M., 2009. Geographic Clustering of Leishmaniasis in Northeastern Brazil. Emerg. Infect. Dis. 15, 871–876. https://doi.org/10.3201/eid1506.080406 Shio, M.T., Christian, J.G., Jung, J.Y., Chang, K., 2015. PKC / ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania ZincMetalloprotease

during

Infection

1–21.

https://doi.org/10.1371/journal.pntd.0003868 SINAN, SUVISA, 2016. SITUAÇÃO EPIDEMIOLÓGICA DA LEISHMANIOSE T E G U M E N TA R A M E R I C A N A ( LTA ) . B A H I A , 2 0 1 6 . Secr. Saúde do Estado da Bahia. SisLeish-OPAS/WHO, 2018. LEISHMANIASIS: Epidemiological Report of the Americas. Sutterwala, F., Haasken, S., Cassel, S., 2014. Mechanism of NLRP3 inflammasome activation Fayyaz. Ann. N. Y. Acad. Sci. 1319, 82–95. https://doi.org/10.1111/nyas.12458.Mechanism Tay, H.L., Kaiko, G.E., Plank, M., Li, J., Maltby, S., 2015. Antagonism of miR-328 Increases the Antimicrobial Function of Macrophages and Neutrophils and Rapid Clearance of Non- typeable Haemophilus Influenzae ( NTHi ) from Infected

Lung.

Plos

Pathog.

1–21.

https://doi.org/10.1371/journal.ppat.1004549 Tiwari, N., Kumar, V., Gedda, M.R., Singh, A.K., Singh, V.K., Singh, S.P., Singh, R.K., 2017. Identification and characterization of miRNAs in response to leishmania donovani infection: Delineation of their roles in macrophage dysfunction.

Front.

Microbiol.

8,

1–11.

https://doi.org/10.3389/fmicb.2017.01190 Wang, Y., Wang, Q., Song, J., 2017. Inhibition of autophagy potentiates the proliferation inhibition activity of microRNA ‑ 7 in human hepatocellular 27

carcinoma cells 3566–3572. https://doi.org/10.3892/ol.2017.6573 WHO, 2018. Media Centre Leishmaniases [WWW Document]. Fact sheets. URL http://www.who.int/mediacentre/factsheets/fs375/en/ (accessed 8.20.03). Xiao, C., Rajewsky, K., 2009. MicroRNA Control in the Immune System: Basic Principles. Cell 136, 26–36. https://doi.org/10.1016/j.cell.2008.12.027 Yang, S.-F., Lee, W.-J., Tan, P., Tang, C.-H., Hsiao, M., Hsieh, F.-K., Chien, M.H., 2015. Upregulation of miR-328 and inhibition of CREB-DNA-binding activity are

critical

metalloproteinase-2 osteosarcomas.

for resveratrol-mediated and

subsequent

suppression of

metastatic

Oncotarget

ability

in

6,

matrix human

2736–2753.

https://doi.org/10.18632/oncotarget.3088 Yang, Z., Zhong, L., Xian, R., Yuan, B., 2015. MicroRNA-223 regulates inflammation and brain injury via feedback to NLRP3 inflammasome after intracerebral

hemorrhage.

Mol.

Immunol.

65,

267–276.

https://doi.org/10.1016/j.molimm.2014.12.018 Zhou, Y., Lu, M., Du, R., Qiao, C., Jiang, C., Zhang, K., Ding, J., 2016. MicroRNA7

targets

Nod-like

receptor

protein

3

inflammasome

to

modulate

neuroinflammation in the pathogenesis of Parkinson ’ s disease. Mol. Neurodegener. 1–15. https://doi.org/10.1186/s13024-016-0094-3

28

Short biography – Vitae no photo

Izaltina Silva-Jardim. Bachelor in Biological Sciences at the Federal University of Minas Gerais, Master in Basic and Applied Immunolgy 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 interaction.

29

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 immunomodulation of human phagocytes.

/ Jaqueline Marques dos Santos. Graduated in Nursing at the State University of Santa Cruz in the year 2013, completing 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 30

of São Paulo, developing his research project in the Immunology Laboratory of Mucosa, studying more specifically the mucosa of the gastrointestinal tract.

Dr. Carla M. Kaneto is a professor of Human Genetics at Santa Cruz State University. She has coauthored publications including stem cell differentiation and gene expression in Cancer, Mesenchymal Stem Cells and Cardiovascular Diseases. The current research interests in Professor Kaneto’s group include MicroRNAs expression in cardiovascular diseases, diabetes and leukemia.

31

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.

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, immunity to microorganisms with emphasis on host-microorganism 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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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 33

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 WCTC) in Milwaukee area.

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Highlights •

The role of inflammasomes during human leishmaniasis has not been elucidated yet.

•

MicroRNAs may contribute to the understanding of leishmaniasis immunopathogenesis.

• This work found serum cytokines and circulating microRNAs in patients with cutaneous leishmaniasis. •

IL-1β, miR-7, miR-133a and miR-223 may have an important role during infection.