Variants of MIRNA146A rs2910164 and MIRNA499 rs3746444 are associated with the development of cutaneous leishmaniasis caused by Leishmania guyanensis and with plasma chemokine IL-8.

Leishmania are intracellular protozoan parasites that cause a wide spectrum of clinical manifestations in genetically susceptible individuals with an insufficient or balanced Th1 immune response to eliminate the parasite. MiRNAs play important regulatory role in numerous biological processes including essential cellular functions. miR146-a acts as an inhibitor of interleukin 1 receptor associated kinase 1 (IRAK1) and tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) present in the toll-like receptors pathway while miR499a modulates TGF-β and TNF signalling pathways. Here, we investigated whether MIRNA146A rs2910164 and MIRNA499 rs3746444 variants are associated with the development of L. guyanensis (Lg)-cutaneous leishmaniasis (CL). The variants MIR146A rs2910164 and MIR499A rs3746444 were assessed in 850 patients with Lg-CL and 891 healthy controls by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma cytokines were measured using the BioPlex assay. Carriers of rs2910164 CC genotype have 30% higher odds of developing CL (ORadjage/sex = 1.3 [95%CI 0.9-1.8]; Padjage/sex 0.14) compared to individuals with the genotype GG (ORadjage/sex = 0.77 [95%CI 0.56-1.0]; Padjage/sex 0.14) if exposed to Lg-infection. Heterozygous GC individuals also showed lower odds of developing CL (ORadjage/sex = 0.77 [95%CI 0.5-1.1]; Padjage/sex 0.09). Homozygosity for the allele C is suggestive of an association with the development of Lg-CL among exposed individuals to Lg-infection. However, the odds of developing CL associated with the CC genotype was evident only in male individuals (ORadjage = 1.3 [95% CI = 0.9-2.0]; Padjage = 0.06). Individuals homozygous for the G allele tend to have higher plasma IL-8 and CCL5. Similarly, for the MIR499A rs3746444, an association with the G allele was only observed among male individuals (OR = 1.4 [1.0-1.9]; P = 0.009). In a dominant model, individuals with the G allele (GG-GA) when compared to the AA genotype reveals that carriers of the G allele have 40% elevated odds of developing Lg-CL (ORadjage = 1.4 [1.1-1.9]). Individuals with the GG genotype have higher odds of developing Lg-CL (ORadjage/sex = 2.0 [95%CI 0.83-5.0]; Padjage = 0.01. Individuals homozygous for the G allele have higher plasma IL-8. Genetic combinations of both variants revealed that male individuals exposed to Lg bearing three or four susceptible alleles have higher odds of developing Lg-CL (OR = 2.3 [95% CI 1.0-4.7]; p = 0.017). Both MIR146A rs2910164 and MIR499A rs3746444 are associated with the development of Lg-CL and this association is prevalent in male individuals.

Introduction

Leishmaniasis, a vector-borne disease caused by protozoan parasites from the genus Leishmania, is endemic in the tropical and subtropical regions, including more than 98 countries. Nearly, one billion of people are at risk of infection [1]. Leishmania species cause a spectrum of clinical forms of the disease: visceral (VL), cutaneous (CL), diffuse cutaneous, disseminated cutaneous and mucocutaneous leishmaniasis (ML) [2].

CL is considered the most common form of Leishmania-infection. Approximately 0.7 million to 1.2 million human beings are affected by this disease [1,3]. In Brazil, the main species involved in the etiology of CL are L. braziliensis (Lb), L. guyanensis (Lg), L. lainsoni, L. amazonensis, L. shawi, L. naiffi and L. lindbergi [4]. In the Amazonas state of Brazil, Lg is responsible for 95% of CL cases [5].

Resistance to Leishmania-infection or healing is associated with a Th1 cell immune response and production of pro-inflammatory cytokines (IL-12, IFN-γ and TNF-α) [6]. Susceptibility to infection and uncontrolled parasitic replication are associated with a Th2 anti-inflammatory cytokines IL-4, IL-5 and IL-13 [7]. Th17 cells, producing IL-22 and IL-17, and T regulatory (Treg) cells producing IL-10 and TGF-β, also contribute to disease progression [6].

MicroRNAs (miRNAs) are small, single-stranded, untranslated endogenous RNAs, composed of 18–26 nucleotides that regulate gene expression. miRNAs bind to their target messenger RNA (mRNA) through base complementarity mechanism resulting in the regulation or degradation of protein translation [8,9]. More than 30% of human protein-coding genes are under post-transcriptional control of miRNAs [10]. miRNAs can regulate many physiological processes such as cell cycle, metabolism, and apoptosis. miRNAs play a crucial role in haematopoiesis, immune cells development and differentiation, inflammation, and immunomodulation [11,12].

MiRNAs can modulate macrophages polarization during Leishmania-infection, creating mix M1/M2 as shown in murine macrophages infected with L. amazonensis [13,14]. L. donovani glycoprotein can downregulate pre-miRNA-122 affecting post-transcriptional regulation of host mRNA/miRNA interactions leading to accumulation of the parasites in the liver [15]. L. amazonensis upregulates miR-294-30 and miR-721 that bind to Nos2 mRNA leading to low levels of NOS2 and NO and increased infectivity in BALB/c-BMDM [16]. miRNAs, let-7a, miR-25, miR-26a, miR-132, miR-140, miR-146a, and miR-155 are upregulated in L. major-infected human macrophages and negatively correlated with the expression of their corresponding chemokine targets, CCL2, CCL5, CXCL10, CXCL11, and CXCL12 [17]. let-7a, let-7b, and miR-103 are upregulated in L. donovani-infected DCs and macrophages but downregulated in L. major infections [9]. Recently, it was shown that a super-enhancer mediates the transcription of MIR146A and drives the polarization of macrophages into M2 suppressing immune responses in L. donovani-infection [18].

miR146-a is a key modulator of innate immune response and acts as an inhibitor of interleukin 1 receptor associated kinase 1 (IRAK1) and tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) [19]. Both IRAK1 and TRAF6 are signalling transducers of the nuclear factor kappa B (NF-κB) in the Toll-like receptors (TLR) pathways. Inhibitions the NF-κB transcriptional activity leads to the impairment of the biosynthesis of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α [19]. miR499-a modulates different biological functions, including immune cells development and maturation, TGF-β and TNF signalling pathways [20]. miR499-a is correlated to the expression of IL-17RB, IL-23a, IL-2R, IL-6, IL-2, and IL-18R [21].

Single nucleotide polymorphisms (SNPs) in miRNA precursors may affect the miRNA biogenesis, causing a reduction of mature miRNA expression levels [22]. SNPs in mature miRNA may affect miRNA target specificity and leads to altered cellular protein levels [23,24]. These SNPs or variants in miRNA may by their actions alter the course of various diseases.

MIR499A and MIR146A genes are located on 20q11.22 and 5q33.3 chromosomes, respectively. The variant rs3746444 located in the seed region of mature miR499-a leads to the disruption of miRNA-mRNA interactions and creation of new gene targets [20]. The variant rs2910164 present in the seed sequence of miR146-a precursor results in low production of mature miR146-a and consequently to a decrease inhibition of TRAF6 and IRAK1, leading to a higher production of pro-inflammatory cytokines upon TLRs activation [25]. miRNA146-a and miR499-a have been associated with susceptibility to multiple types of cancer, psoriasis and rheumatoid arthritis [26-29].

Taken into account the potential role of miR146-a and miR499-a in modulating the T helper cell immune response, we investigated whether the variants MIR146A rs2910164 and MIR499A rs3746444 are associated with susceptibility or protection to the development of Lg-CL in the Amazonas. The influence of the MIR146A and MIR499A genotypes on plasma cytokine levels were also investigated.

Methods

Area of study and population

The study was conducted at the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD), the referral regional center for treatment of leishmaniasis. The study population and the endemic area of recruitment of the participant of the study are described elsewhere [30]. Briefly, all the participants are from the perirural areas of Manaus, the capital city of the Amazonas State where L. guyanensis is endemic. Patients with active CL were followed at the FMT-HVD. The healthy controls with no history of CL and devoid of any scar suggestive of CL are from the same endemic area of the patients with CL.

Ethical statement

This study was conducted according to the Declaration of Helsinki and approved by the Research Ethics Committee of the FMT-HVD granted under the file number CAAE:09995212.0.0000.0005 on 31 May 2013. All the participants or their responsible party for individuals less than 18 years of age provided written informed consent for the collection of samples and subsequent analysis.

Sample size calculation

Sample size calculation is described elsewhere [31]. Briefly, using the Genetic Power calculator developed at Harvard University (http://pngu.mgh.harvard.edu/~purcell/gpe), we assumed a minor allele frequency of 5%, disease prevalence of 5%, complete linkage disequilibrium 1 between marker and case-control discrete trait, case-control ratio 1, and 5% type 1 error rates with an odds ratio of 1.5 and 2.0 for heterozygosity and homozygosity, respectively. For 80% power, the genetic allelic model provided a sample size of 789 individuals for cases and 789 for controls.

Leishmania spp identification from biopsy specimens and DNA extraction from whole blood for SNP typing

All the patients with CL provided a biopsy specimen from the cutaneous lesion for the isolation of parasite DNA. For the identification of the Leishmania spp., the discrimination of the Leishmania Viannia subgenus specific PCR was in accordance with established protocols [32,33]. Identification of Leishmania spp. was performed by nucleotide sequencing of a fragment of HSP 70 and miniexon genes [34,35]. Venous blood was drawn from all participants and collected into EDTA-containing Vacutainers (Becton Dickinson, Brazil) to DNA extraction and cytokine assay. Genomic DNA was extracted by the salting out method [36].

SNP genotyping

The SNPs MIR146A rs2910164 and MIR499A rs3746444 were performed by PCR-RFLP with the restriction enzymes HpyCH4 III and HpyCH4 IV (New England Biolabs, Ipswich, MA, United States), respectively. The respective pairs of primers for the amplification the region flanking the SNPs and the fragments generated for alleles discrimination by the restriction enzymes as well as the cycling protocols for PCR are shown in S1 Table.

The pair of primers for MIR499A was designed from the reference sequence NC 000020.11 from NCBI. The underline G nucleotide from the forward primer substitutes A from the reference sequence to eliminate a site of restriction for the HpyCH4IV. Primers for the MIR146A were designed from the reference sequence NC 000005.10 and the underline A nucleotide substitutes C from the reference sequence to create restriction site when the G allele is present.

The PCR mix contains: 0.2 μM of each primer (Thermofisher, MA USA), 40 nM of dNTP (Thermofisher, MA USA), 1.0 mM of MgCl2 (Thermofisher, MA USA), 1 U of Taq DNA polymerase (Thermofisher, MA USA), 1X of 10X Taq polymerase buffer containing 500 mmol/L KCl and 100 mol/L of Tris-HCL (pH 8.3) and 50 ng of DNA in a final volume of 25 μL. A volume of 10 μL of the PCR products was digested with 1 unit of the respective restriction enzyme and buffer in a final volume of 20 uL and size separated in a 3% agarose (Ultrapure Agarose, Thermofisher, MA USA) gel electrophoresis.

Cytokine assay

Cytokine assay of IL-1β, TNF-α, IL-2, IL-6, IL-8, IL-17, IFN-γ, CCL2 and CCL5 in the plasma were measured using the multiplex cytokine commercial kit Human Cytokine Grp I Panel 27-Plex (Bio-Rad, USA) according to the instructions of the manufacturer in the Bio-plex 200 Protein Array System (Luminex Corporation, USA).

Statistical analysis

The genotype and allele frequencies were calculated by direct gene counting. For calculation of Hardy-Weinberg equilibrium (HWE), the website http://ihg.gsf.de/cgibin/hw/hwa1 was used, that also compared cases with the control groups by logistic regressions analysis to determine associations to susceptibility or resistance for the different genotypes and alleles by χ2 test along with OR and 95% confidence interval. The correlation of the different genotypes of MIR146A rs2910164 and MIR499A rs3746444 to the concentration of circulating plasma cytokines was performed by the R software version 4.0.0 of SNPassoc package for quantitative traits analysis and ggplot2 package for visualizing. P values for the correlations of cytokines by genotypes were corrected for false discovery rate (FDR) of Benjamini-Hochberg.

Results

Study population

The study population is the same as described previously [30]. A total of 850 patients with Lg-CL and 891 healthy controls (HC) were included in the study. The HC are from the same endemic area of the patients with Lg-CL. Among the patients with Lg-CL, 639 (75%) patients were male (mean age 34.4 ± SD 13.7 years) and 211 (25%) were females (37.5± SD 15.7 years). In the controls group, 608 (68%) were male (42± SD 17.5 years) and 283 (32%) were female (40± SD 17.4 years). Overall, the mean age among the patients with Lg-CL and controls is 35.17± SD 14.25 and 41.4± SD 17.5 years, respectively. The HC is older than the cases (P<0.0001). Men were older among the HC group compared to group of male patients with Lg-CL (P<0.0001) while there was no age difference among females (P<0.077). All the participants of the study were devoid of HIV and the patients had fewer or equal to six lesions and treatment naïve at the time of enrolment. Pregnant women were excluded from the study. Of note, there are more females in the HC compared to the group of patients with CL (P<0.0013).

Association MIR146A rs2910164 and MIR499A rs3746444 with susceptibility to Cutaneous Leishmaniasis

MIR146A rs2910164 was assessed in 826 patients with Lg-CL and 886 controls. Genotype and allele frequencies for the two variants are demonstrated in Table 1. rs2910164 was in Hardy-Weinberg equilibrium (HWE) among the patients with Lg-CL and HC. The MIR146A rs2910164 CC genotype was prevalent in patients with Lg-CL (12%) compared with HC (9%). Carriers of rs2910164 CC genotype have 30% higher odds of developing CL (ORadjage/sex = 1.3 [95%CI 0.9–1.8]; Padjage/sex = 0.14) compared to individuals with the genotype GG (ORadjage/sex = 0.77 [95%CI 0.56–1.0]; Padjage/sex = 0.14) if exposed to Lg-infection. Heterozygous GC individuals also have lower odds of developing CL compared with homozygous carriers of the C allele (ORadjage/sex = 0.77 [95%CI 0.55–1.1]; Padjage/sex = 0.09). In a recessive model, when homozygous individuals for the C allele are compared with individuals carrying a G allele (CC versus GC—GG), carrier of G allele have 23% lower odds of developing Lg-CL (ORadjage/sex = 0.77 [95%CI 0.56–1.0]; Padjage/sex = 0.098). Homozygous for the C allele have 30% higher odds of developing CL (ORadjage/sex = 1.3 [95%CI 0.9–1.8]; Padjage/sex = 0.098). The C allele is suggestive of an association with the development of Lg-CL.

Table 1

Genotype and allele frequencies for the MIR146A rs2910164 and MIR499A rs3746444 in patients with Leishmania guyanensis-Cutaneous Leishmaniasis (Lg-CL) and healthy controls.

Genotypes and Alleles Frequencies
	Casesa	(%)	HCb	(%)
rs2910164	N = 826		N = 886
GG	375	46	405	46
GC	349	42	398	45
CC	102	12	83	9
G	1099	67	1209	68
C	553	33	563	32
rs3746444	N = 818		N = 851
AA	649	79	706	83
AG	153	19	135	16
GG	16	2	10	1
A	1451	89	1547	91
G	185	11	155	9
Genotypes and alleles comparisons
Comparisons	P valuec	OR [95% CI]d	Padje	ORadj[95%CI]f
rs2910164
GG vs CC	0.084	1.3 (0.96–1.8)	0.14	1.3 (0.9–1.8)
CC vs GC	0.040	1.4 (1.0–1.9)	0.09	1.3 (0.9–1.8)
CC+GC vs GG	0.897	1.0 (0.8–1.2)	0.864	1.0 (0.8–1.2)
GG+GC vs CC	0.047	1.4 (1.0–1.9)	0.098	1.3 (0.9–1.8)
G vs C	0.304	0.9 (0.8–1.0)
rs3746444
AA vs GG	0.167	1.7 (0.8–3.9)	0.15	2.0 (0.83–5.0)
AA vs AG	0.106	1.2 (0.9–1.6)	0.13	1.3 (1.0–1.7)
AA+GA vs GG	0.196	1.7 (0.8–3.7)	0.170	1.8 (0.8–4.0)
AA vs GA+GG	0.058	1.3 (1.0–1.6)	0.067	1.3 (1.0–1.6)
A vs G	0.035	1.3 (1.0–1.6)

aCases: patients with Lg-CL

bHC: healthy controls

cP value: normal p-value

dOR: odds ratio with 95% confidence interval (CI)

ePadj: p adjusted by gender and age

fORadj: odds ratio adjusted by gender and age; P value <0.05 is statistically significant.

As male individuals were prevalent in both patients with Lg-CL and HC, we stratified according to sex as shown in Table 2. The odds of developing CL associated with the CC genotype was evident only in male individuals (ORadjage = 1.3 [95% CI = 0.9–2.0 Padjage = 0.06]) compared with GG genotype. Individuals with GG genotype had lower odds of developing Lg-CL (Radjage = 0.77 [95%CI 0.5–1.0]; p = 0.06). The genotype CC is suggestive of an association with the development of CL compared to GG genotype among Lg-infected individuals.

Table 2

Distribution and Comparison of Genotypes and Alleles frequencies of the MIR146A rs2910164 between Patients with Leishmania guyanensis-Cutaneous Leishmaniasis (Lg-CL) and Controls stratified according to gender.

	Patients with Lg-CL, no. (%)					Controls, no. (%)
Genotypes	Males		Females			Males		Females
	N = 621		N = 205			N = 603		N = 283
G/G	284 (46)		91 (44)			286 (47)		120 (42)
G/C	254 (41)		95 (47)			261 (44)		136 (48)
C/C	83 (13)		19 (9)			56 (9)		27 (10)
Alleles
G	822 (66)		277 (67)			833 (69)		376 (66)
C	420 (34)		133 (33)			373 (31)		190 (34)
Genotypes and alleles comparisons
	Males				Females
	P va	ORb [95%CI]	Padjc	ORadjd [95%CI]	Pva		ORb [95%CI]	Padjc	ORadjd [95%CI]
GG vs CC	0.036	1.5 [1.0–2.2]	0.06	1.3 [0.9–2.0]	0.82		0.9 [0.5–1.8]	0.45	1.0 [0.5–1.8]
CC vs GC	0.03	1.5 [1.0–2.2]	0.04	1.4 [1.0–2.0]	0.98		1.0 [0.5–1.9]	0.45	1.0 [0.5–2.0]
GG+GC vs CC	0.02	1.5 [1.0–2.2]	0.04	1.4 [0.95–2.0]	0.92		1.0 [0.5–1.9]	0.38	1.0 [0.7–1.5]
G vs C	0.12	1.1 [0.96–1.3]			0.71		1.1 [0.8–1.4]

aP v: normal p-value

bOR: odds ratio with 95% confidence interval (CI)

cPadj: p adjusted by age

dORadj: odds ratio adjusted by age. P value < 0.05 is statistically significant.

MIR499A rs3746444 was genotyped in 818 patients with Lg-CL and 851 HC. The variant was in HWE in both groups of patients with Lg-CL and HC. The distribution of genotypes among patients with Lg-CL and HC was different as shown in Table 1, revealing a common odds ratio of 1.3 for the G allele compared with the A allele (p = 0.04). The rs3746444 AA genotype was more prevalent among the HC (83%) compared to 79% in the patients with Lg-CL group. Comparison of the genotype AA to GG revealed that individuals with the GG genotype have 100% higher odds of developing Lg-CL, with a 95% CI ranging from a decreased odds of 17% to an elevated odd of 400% (ORadjage/sex = 2.0 [95%CI 0.83–5.0]; Padjage/sex = 0.15). Similarly, the G allele confers 27% elevated odds of developing Lg-CL suggesting that the G allele contribute to susceptibility to the development of Lg-CL (OR = 1.27 (95% CI 1.0–1.6]; p = 0.035). In a dominant model, individuals with the G allele (GG-GA) when compared to the AA genotype reveals that carriers of the G allele have 30% elevated odds of developing Lg-CL (ORadjage/sex = 1.3 [95% CI 1.0–1.6]; Padjage/sex = 0.067).

We stratified into male and female individuals to look for the strength of the association. Like the MIR146A, the association was more evident among the male individuals as shown in Table 3. Male individuals with GG genotype compared with AA genotype revealed an elevated odds of 50% with a 95% confidence interval ranging from a decreased odds of 40% to an increased odds of 310% to the development of Lg-CL (ORadj/age = 1.5 [0.6–4.1]; Padj/age = 0.38). Similarly, female individuals had higher odds of developing Lg-CL (ORadj = 2.3 [95%CI 0.5–10.0]; Padjage/sex = 0.26).

Table 3

Distribution and Comparison of Genotypes and Alleles frequencies of the MIR499A rs3746444 between Patients with Leishmania guyanensis-Cutaneous Leishmaniasis (Lg-CL) and Controls stratified according to gender.

		Patients with Lg-CL, no. (%)					Controls, no. (%)
Genotypes		Males		Females			Males			Females
		N = 614		N = 204			N = 538			N = 268
A/A		478 (78)		171 (84)			488 (84)			218 (81)
A/G		125 (20)		28 (14)			88 (15)			47 (18)
G/G		11 (2)		5 (2)			7 (1)			3 (1)
Alleles
A		1081 (88)		370 (91)			1064 (91)			483 (90)
G		147 (12)		38 (9)			102 (9)			53 (10)
Genotypes and alleles comparisons
	Males					Females
	P va	ORb [95%CI]	Padjc		ORadjd [95%CI]	P va		ORb [95%CI]	Padjc		ORadjd [95%CI]
AA vs. GG	0.32	1.6 [0.6–4.1]	0.38		1.5 [0.6–4.1]	0.29		2.1 [0.5–8.0]	0.26		2.3 [0.5–10.0]
AA vs. AG	0.01	1.4 [1.0–1.9]	0.02		1.4 [1.0–1.9]	0.28		1.3 [0.8–2.2]	0.29		1.3 [0.8–2.2]
AA vs AG+GG	0.01	1.5 [1.0–1.9]	0.01		1.4 [1.1–1.9]	0.48		1.2 [0.7–1.9]	0.51		1.2 [0.7–1.9]
A vs. G	0.009	1.4 [1.0–1.9]				0.76		1.1 [0.7–1.7]

aP value: normal p-value

bOR: odds ratio with 95% confidence interval (CI)

cPadj: p adjusted by age

dORadj: odds ratio adjusted by age. P value < 0.05 is statistically significant.

Genetic combinations of both MIR146A rs2910164 and MIR499A rs3746444 genotypes

The frequencies of the combined genotypes are shown in Supplementary Table 3. Male individuals carrying at least one susceptibility allele (MIR146A rs2910164C or MIR499A rs3746444G) revealed an OR of 1.1 [95%CI 0.8–1.4] in comparison to individuals with two alleles (1.4 [95% CI 1.0–1.9]; p = 0.035) or three or four alleles (2.3 [95% CI 1.0–4.7]; p = 0.017). Notably, these associations were not observed among female individuals strengthening that the associations are mainly among male individuals.

Comparison of circulating plasma cytokines concentrations (pg/mL) with respect to the MIR146A rs2910164 and MIR499A rs3746444 genotypes

miRNA146-a is suggested to suppress IRAK-1 expression leading to a decrease of IL-6 and IL-8 secretions that are key mediators of inflammation [37]. Bone-marrow derived macrophages from knockout MIR146Amice treated with monosodium urate expressed higher levels of IL-1β, TNFα, NLRP3, IRAK-1 and TRAF-6 compared with BMDM from wild type mice [38]. Inhibitors of miR146-a stimulate the expression of IL-8 and CCL5 [39]. We analysed whether the different genotypes of MIR146A may correlate with circulating plasma levels of IL-6, IL-8, IL-1β, TNFα and CCL5 (S1 Fig).

Only IL-8 showed a tendency to correlate with the genotypes of rs2910164 in healthy individuals albeit no statistical significance is reached. In a dominant model, individuals homozygous for the G allele seem to have higher levels of circulating IL-8 compared with individuals bearing a C allele (GG 1.28±0.06 pg/mL vs CC +GC 1.10±0.05 pg/mL; p = 0.02; P corrected for false discovery rates (PFDR) = 0.06) as shown in Fig 1. Similarly, these individuals seem to have increased levels of CCL5 (GG 73.01±30.14 pg/mL compared with individuals with a C allele, CC +GC 31.68±2.68 pg/mL; p = 0.19) (S2 Fig).

Fig 1

MIRNA146A rs2910164 effects on IL-8 plasma levels in healthy controls.

Concentrations of plasma circulating IL-8 were compared between bearers of the AA, AG, and GG genotypes using inheritance models (codominant, dominant, recessive and overdominant). The mean concentrations are displayed in picogram per millilitre (pg/mL) with standard error (SE) of mean. The means are represented by black bars, whereas SE are represented by error bars. *Corrected for Benjamini-Hochberg False Discovery Rate assuming four tests. p < 0.05 is statistically significant.

miRNA499-a modulates many inflammation-related signalling pathways such as TGFβ, TNFα and Toll-like receptors pathways. miR499-a has also been reported to regulate the expression of TNFα, IL-6, IL17RB, IL-18R and IL-23a [21]. We assayed IL-1β, IL-2, IL-6, IL-8, IL-17, IFNγ, CCL2 and TNFα to correlate with the different genotypes of MIR499A rs374644 as shown in S3 Fig. A tendency of plasma circulating levels of IL-8, IL-6 and IL-17 by MIR499A rs3746444 genotypes is observed. Among the HC, the distribution of plasma circulating IL-8 according to the genotypes is statistically different in a codominant model (p = 0.01; PFDR = 0.02) as shown in Fig 2. In a recessive model, individuals homozygous for the G allele seem to have higher levels of IL-8 (2.14±0.64 pg/mL) compared with individuals (AA+GA 1.18±0.04 pg/mL) bearing a A allele (p = 0.003; PFDR = 0.01) (Fig 2).

Fig 2

MIRNA499A rs3746444 effects on IL-8 plasma levels in healthy controls.

Concentrations of plasma circulating IL-8 were compared between bearers of the AA, AG, and GG genotypes using inheritance models (codominant, dominant, recessive and overdominant). The mean concentrations are displayed in picogram by millilitre (pg/mL) with standard error (SE) of mean. The means are represented by black bars, whereas SE are represented by error bars. *Corrected for Benjamini-Hochberg False Discovery Rate assuming four tests. p < 0.05 is statistically significant.

GG homozygotes have higher levels of IL-8 (GG = 2.14±0.64 pg/mL versus AA = 1.18±0.04pg/mL (p = 0.005; PFDR = 0.01) (Fig 3).

Fig 3

MIRNA499A rs3746444 effects on IL-6, IL-8, and IL-17 plasma levels between AA homozygous versus GG homozygous in healthy controls.

Comparisons of circulating plasma levels of IL-6, IL-8 and IL-17 between the individuals AA and GG homozygous. The mean concentrations are displayed in picogram per millilitre (pg/mL) with standard error (SE) of mean. The means are represented by black bars, whereas SE are represented by error bars. *Corrected for Benjamini-Hochberg False Discovery Rate assuming three tests. p < 0.05 is statistically significant.

In a recessive model, bearers of the A allele among HC (AA+GA; 0.40±0.01 pg/mL) seem to have lower levels of IL-6 (p = 0.09) compared with GG homozygotes (0.64±0.18 pg/mL) (). Individuals homozygous for the G allele have higher levels of IL-6 compared with AA homozygotes individuals (GG = 0.64±0.18 pg/mL versus AA = 0.41±0.01 pg/mL; p = 0.09) (Fig 3).

Similarly, MIR499A rs3746444 have a tendency on influencing the plasma circulating levels of IL-17 among the HC. GG homozygotes (5.48±1.97 pg/mL) have higher levels of IL-17 compared to heterozygotes GA (3.48±0.31 pg/mL) and homozygotes AA (3.98±0.16 pg/mL) (S4 Fig).

Genetic combinations of both variants revealed that individuals bearing three or four G alleles tend to correlate with higher plasma IL-8 (2.24 ± 0.48 pg/mL) and CCL5 (63.4± 29.2 pg/mL) as shown in S5 Fig.

Discussion

The manifestation of clinical symptoms in Leishmania-infected individuals depends on the capacity of the individual to have a fine regulation of the TH1 response to eliminate the parasite and avoid an exacerbation of expression of pro-inflammatory cytokines. Emerging evidence are showing that miRNAs regulate immune response [19,40,41].

In this study, we showed that male individuals homozygous for the C allele of MIR146A rs2910164 have 30% higher odds of developing Lg-CL compared with individuals bearing a G allele. The MIR146A rs2910164 C allele has been shown to be a risk factor for leprosy in Brazil and psoriasis in South African Indian [42,43]. Homozygous individuals for the C allele have increased risk of developing glioma and decreased survival [44]. A meta-analysis study on autoimmune diseases revealed that individuals bearing the C allele have increased risk of developing the disease [45].

Several studies have also reported that MIR146A rs2910164 is not a risk factor in several diseases. A meta-analysis on ischemic stroke showed that MIR146A rs2910164 is not associated with any risk occurrence of ischemic stroke [46]. A lack of association of MIR146A rs2910164 with the development of RA was also reported [47]. Another study cited that susceptibility to pulmonary tuberculosis is not influenced by the MIR146A rs2910164 [48]. However, a meta-analysis approach on psoriasis case-control studies with rs2910164 revealed that the CC genotype is correlated with decreased risk of psoriasis [28].

Conversely, other studies have reported that the G allele is a risk factor in several diseases. The G allele has been associated with pulmonary TB [49] and ankylosing spondylitis [50]. A meta-analysis study on cancer among Asian patients revealed that GG genotypes are associated with increased risk of cancer [51].

We also showed that in male individuals, the GG genotype of the MIR499A rs3746444 is associated with 50% increased odds of developing CL. The G allele confers 27% elevated odds of developing CL suggesting that the G allele may contribute to the development of Lg-CL.

A recent meta-analysis study showed that the MIR499A rs3746444 G allele is associated with high risk of developing breast cancer [52]. The allele G was also associated with hepatocellular carcinoma [53], and adenocarcinoma [54]. Other studies also showed that the G allele is associated with susceptibility to the development of bronchial asthma [20], RA [29,55], Behcet’s disease [56], ankylosing spondylitis [50], myocardial infarction [57] and coronary artery disease [58,59]. Immune response plays critical role in all these diseases. Of note, there is no study of both variants with protozoan infectious diseases to date.

The gender difference observed for both variants in this study may be due to either sexual hormonal interaction with the variant or the small sample size in the female group. Notably, we have 75% and 68% male individuals among patients with CL and HC, respectively. Interestingly, sex-based differences have been shown in L. tropica infected patients. L. tropica-infection manifests commonly in the form of CL. However, females present predominantly CL while male individuals are more incline to develop viscerotropic leishmaniasis [60-62]. Furthermore, peripheral blood from patients with CL caused by L. mexicana stimulated or not with lipophosphoglycan demonstrated higher expression of IFNγ and tumour necrosis factor alpha in females than in males [63]. An increased parasite growth has been observed when L. mexicana promastigotes are treated with physiological doses of male dihydrotestosterone [64,65].

The MIR146A is in the cytokine cluster 5q31 and the MIR146A rs2910164 is situated in the stem region of the pre-miR-146a. The change of guanine:uracil pair (G:U) to cytosine:uracil (C:U) in the stem structure affects the production of mature miR146-a [25,66]. The G-allele is reported to be associated with high expression levels of mature miR146-a while the C-allele with low expression [25, 67]. Other study cited that the C allele is correlated with higher miR146-a and lower TNFα expression from nerve biopsies of leprosy patients [42]. High expression of miR146-a was also observed in specimens of healthy and tumour tissue from patients with gastric cancer bearing genotype CC compared to GG [68]. In Lupus, CC genotypes was correlated with high miR146-a expression [69]. One study observed that the genotype CC is associated with increased expression of IRAK1 and TRAF6 [70]. miR146-a regulates the IL-17 pathway in human keratinocytes, the first line of defence against pathogens in the skin, to restrain IL17-induced inflammation [71]. Overexpression of miR146-a in keratinocytes inhibits the expression of IL-8 and TNFα, suppressing the chemotactic attraction of neutrophils by keratinocytes [72]. Interestingly, we observed in this study that individuals homozygous for the C allele have lower plasma IL-8. Generation of miRNA regulatory network from Cytoscape revealed that miR146-a play key role in the inflammatory response in Leishmania-infection [73].

In this study, we did not observe any correlation of plasma levels of TNFα, IL-6, IL-1β, IL-2, IL-17, IFNγ and CCL2 by MIR146A rs2910164 genotypes neither among the patients with Lg-CL nor the HC. However, individuals homozygous for the G allele seem to have higher levels of circulating IL-8 and CCL5 compared with individuals bearing a C allele.

Knockout MIR146Amice developed exaggerated pro-inflammatory response upon challenging with lipopolysaccharide due to chronic dysregulation of nuclear factor kappa-light-chain enhancer of activated B cells (NFkB) signalling [74,75]. miR146-a is reported to target IRAK and TRAF-6 in the TLRs downstream pathway to downregulate the upregulation of NFkB leading to a decrease in the transcription of pro-inflammatory cytokines IL-6, IL-8, IL-1β, and TNFα [74,76-80]. A recent study showed that inhibition of miR146-a resulted in a reduced secretion of IL-6 and IL-8 [81], suggesting that miR146-a may downregulate an inflammatory reaction.

miRNA499-a is involved in TLR-signalling [82]. Computational tool analysis suggests that miR499-a may target IL-13 and IL-23 (microRNA.org). MIR499A rs3746444 genotype GG has been suggested to correlate with high expression of miR499-a [83]. Our data showed that individuals homozygous for the G allele have higher plasma IL-8 and a tendency to also have high IL-6, IL-17, and increased risk of developing Lg-CL.

Indeed, in Lb-infected patients with CL, high levels of IL-17 were observed [84] and peripheral blood from these patients released high IL-17 upon stimulation with soluble Leishmania antigen [85]. L. major-infected C57BL/6 mice developed larger lesion size with increased production of IL-17 and neutrophil infiltration at the site of lesion compared with mice treated with anti-IL-17 [85] while L. major-infected BALB/c mice deficient for IL-17 develop smaller lesions [86], suggesting that IL-17 promotes lesions. High levels of IL-6 impaired the cytokine-enhanced antileishmanial activity of human macrophages by inhibiting IFNγ and TNFα [87].

Our study has several limitations. Firstly, we considered that our healthy controls from the same endemic areas as the patients are exposed to the Lg-vector and are infected but did not develop the disease. We did not perform DTH to ensure this despite most of the individuals included in the study are farming or agricultural workers. Secondly, the problem of multiple testing of many associations may result in spurious associations but may also discard a true association after correcting for multiple comparisons. Thirdly, our controls are slightly older than our patients with CL and contain slightly more females. Stratification reduces the sample size.

Considering our data, we may hypothesize that MIR146A rs2910164 CC individuals are prone to develop Lg-CL due to an impairment neutrophil migration at the inoculation of the parasite by the sand fly bite. Additionally, these individuals have lower levels of CCL5. CCL5 has been shown to correlate with resistance to L. major infection in animal model [88]. Furthermore, our MIR499A rs3746444 data suggest that individuals with genotype GG are susceptible to develop Lg-CL by attracting too many neutrophils that may pass the parasite to macrophages leading to parasite persistence as they might be high producers of IL-8, IL-6 and IL-17.

Altogether, this is the first study to date to investigate MIR146A rs2910164 and MIR499A rs3746444 in protozoan infectious diseases. Both variants are associated with the development of Lg-CL male individuals exposed to Lg-infection and correlate with plasma IL-8.

Plasma levels of cytokines and chemokines according to the different genotypes of MIR146A rs2910164 in cases, controls and total individuals.

(TIFF)

Click here for additional data file.

MIR146A rs2910164 effects on circulating plasma CCL5 levels in healthy controls.

(TIFF)

Click here for additional data file.

Plasma levels of cytokines and chemokines according to the different genotypes of MIR499A rs3746444 in cases, controls and total individuals.

(TIFF)

Click here for additional data file.

MIR499A rs3746444 effects on circulating plasma IL-6 and IL-17 levels in healthy controls.

(TIFF)

Click here for additional data file.

Genetics combinations of genotypes by plasma cytokines.

(TIFF)

Click here for additional data file.

Polymerase Chain Reactions protocols for the MIR146A rs2910164 and MIR499A rs3746444.

(DOCX)

Click here for additional data file.

Mean values and standard error (SE) of the mean of plasma cytokines by the MIR146A rs2910164 and MIR499A rs3746444 genotypes according to inheritance models among the total individuals.

(DOCX)

Click here for additional data file.

Genetics combinations of genotypes in patients with Cutaneous Leishmaniasis and Healthy Controls.

(DOCX)

Click here for additional data file.

24 Jul 2021

Dear Dr. Ramasawmy,

Thank you very much for submitting your manuscript "Variants of MIRNA146A rs2910164 and MIRNA499 rs3746444 are associated with the development of cutaneous leishmaniasis caused by Leishmania guyanensis and with plasma chemokine IL-8" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Claudia Ida Brodskyn

Associate Editor

PLOS Neglected Tropical Diseases

Steven Singer

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Please, check the comments of the reviewers. They raised important points that must be addressed to consider the manuscript for publication.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The objective of performing the polymorphism analysis is clear; the cytokine measurements are too indirect. The population is well described, although the sample is highly biased for sex. Some analysis accounted for that but not all. there is no information regarding sample size calculation. No concerns related to ethical requirements.

Reviewer #2: The methods is in consonante with goal proposed in the manuscript, supporting the objetives. The statistical analysis was adequate.

A minor point can be included in the text:

What is the volume of PCR mix and Buffer? And the volume of enzymatic digestion mix?

Reviewer #3: Objectives are clearly stated and the study design and statistical analysis (except correction for multiple testing, see below) in agreement with these objectives. The authors should be recommended for an exceptionally large sample size, which is uncommon in neglected tropical diseases.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Results are clearly presented.

Reviewer #2: Tables and Figures are clearly and showed that an analysis of SNPs MIR146A rs2910164 and MIR499A rs3746444 showed a prevalence of MIR146A rs2910164 CC genotype and MIR499A rs3746444 AA genotype, elevating the risk to develop CL. In MIR146A rs2910164 G allele presented higher levels of IL-8 and RANTES in CL plasma samples. The, MIR499A rs3746444 G allele presented higher levels of IL-8, IL-6 and IL-17.

This information open some questions:

- Did you analyze the prevalence of both MIR146A rs2910164 CC and MIR499A rs3746444 AA genotypes in the same CL patients? These correlates?

- In MIR146A rs2910164 G allele presented higher levels of IL-8 and RANTES in CL plasma samples. The, MIR499A rs3746444 G allele presented higher levels of IL-8, IL-6 and IL-17. Have some correlation of presence of both rs2910164 G allele and rs3746444 G allele and cytokines in the same CL patients?

Reviewer #3: Major comments:

1. Correction for multiple testing is strictly necessary for the cytokine analysis, since the Methods sections mentions 8 different cytokines/chemokines were measured: “Cytokine assay of IL-1β, TNF-α, IL-2, IL-6, IL-8, IL-17, IFN-γ, MCP-1 and RANTES in the plasma were measured using the multiplex cytokine commercial kit Bio-PlexProTM Human Cytokine Grp I Panel 27-Plex (Bio-Rad, USA).” Bonferroni correction (all p-values multiplied by 8) might be too harsh, but at least FDR correction (Benjamini-Hochberg or similar) should be applied to all comparisons. In fact, this is a minimal approach, since for each association 4 genetic models were tested in 3 situations (cases, controls and total), correcting for all these would result in no significant associations in the entire study.

However, it seems 27 different chemokines/cytokines were actually quantified with the Bioplex kit, according to the manufacturer:

• FGF basic

• Eotaxin

• G-CSF

• GM-CSF

• IFN-γ

• IL-1β

• IL-1ra • IL-2

• IL-4

• IL-5

• IL-6

• IL-7

• IL-8

• IL-9 • IL-10

• IL-12 (p70)

• IL-13

• IL-15

• IL-17A

• IP-10

• MCP-1 (MCAF) • MIP-1α

• MIP-1β

• PDGF-BB

• RANTES

• TNF-α

• VEGF

Please mention if the cytokines/chemokines not appearing in the manuscript were undetectable (not very likely for e.g. MIP-1alpha/beta and CXCL10), or why they were not selected for analysis. I assume some of these quantifications were previously published (IFN-gamma, IL1beta, IL1RA,…), so it would be correct to state this as a reanalysis.

2. Statistical reporting of the results is incorrect and often misleading:

2.1. Odds ratios are interpreted as if they were relative risk (RR): RR = 1.74 means exposed individuals are 74% more likely to be diseased, OR = 1.74 means that the odds of disease is 74% higher in exposed individuals, which is intrinsically different. For instance, in line 412: “Here, we showed that the GG genotype of the MIR499A rs3746444 is associated with a 74% increased risk of developing CL. Similarly, the G allele confers a 27% elevated risk of developing CL suggesting that the G allele may contribute to the development of Lg-CL.”

2.2. Large CI including 1 are often arguing against the significance claimed by the authors, e.g. “Carriers of rs2910164 CC genotype have a 33% elevated risk with a 95% confidence limits ranging from less 4% to an increase of 83%” should be reported as “no significantly increased odds of disease for the CC genotype.

2.3. Raw p-values, uncorrected for age and gender are mentioned in the abstract, while there is a bias between cases and controls and none of the associations remain. As discussed below, the significant findings in males only also make more sense if occupational exposure is considered, this should be emphasized instead of the many weak associations.

Please correct the entire results section in the abstract accordingly: “Carriers of rs2910164 CC

60 genotype have a 33% elevated risk while individuals with the genotype GG have lower risk

61 of developing CL (OR=0.75 95%CI 0.55 - 1.0). Heterozygous GC individuals also have

62 lower risk of developing CL compared with homozygous carriers of the C allele (OR= 0.71

63 95%CI 0.52 - 0.99 p=0.04). In addition, individuals homozygous for the G allele seem to

64 have higher plasma IL-8 and RANTES. For the MIR499A rs3746444, individuals with the

65 GG genotype have a 74% increased risk of developing CL when compared to AA genotype.

66 The G allele confers a 27% elevated risk of developing CL suggesting that the G allele may

67 contribute to susceptibility to the development of Lg-CL (OR=1.27 (95% CI 1.0 – 1.6] p=

68 0.035). Individuals homozygous for the G allele have higher plasma IL-8 with a tendency to

69 also have high IL-6 and IL-17. Both MIR146A rs2910164 and MIR499A rs3746444 are

70 associated with the development of Lg-CL and this association is more prevalent in male

71 individuals.”

3. The paper does not have a “Limitations of the study” section. This should be inserted and at least include 1) the problem of multiple testing of a large number of associations, 2) the significant bias in both age and gender in cases vs controls, and 3) the assumption that all controls were exposed by merely living in the same endemic area might not hold, thus precluding a correct interpretation of the genetic associations. Other genetic studies have included DTH+ individuals which have been exposed to Leishmania In fact, the significant associations observed in males but not females might hint at different exposures by gender (mostly agricultural workers.

Minor comments:

1. Several typos and spelling/grammatical errors are found throughout the manuscript.

2. A (post-hoc) power calculation would be useful for the correct interpretation of the results, e.g. “with the available number of xx cases and xx controls and MAF of >0.15, an OR of xx (or larger) could be detected with 80% power.”

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Conclusions are partially supported by the data, as the authors presented a lengthy and highly speculative discussion, which should be modified. Some limitations are presented in the discussion, but it would be beneficial to list them separately, rather than embedded in the discussion.

Reviewer #2: Authors studied the SNPs in miR1461 and miR499 in Leishmania guyanensis CL patients. The manuscript Bring a new View point to understand the function of the SNP in the miRNAs, a molecules that was found impacting in the host-parasite interaction, correlating with suceptibility or resistance to infection.

Reviewer #3: Conclusions, as presented in the abstract, are not supported by the data (as detailed above) and should be rephrased, with an emphasis on the findings in males. A "limitations of the study" section is lacking. Public health relevance has been thoroughly addressed for cutaneous leishmaniasis.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: - Please see nomenclature for chemokines (RANTES and MCP1 no longer used).

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The manuscript by Mesquita et al. evaluated the association between genetic variants of miR146-a and miR499a and cutaneous leishmaniasis (CL) caused by L. guyanensis. They evaluated the frequency of the variants rs2910164 and rs3746444 in 850 cases and 891 controls by RFLP. In addition, the authors evaluated the expression of selected cytokines using Bioplex. The data showed that CC genotype for the rs2910164 is slightly associated with CL, while the GG genotype for rs3746444 is associated with a 75% higher risk of developing CL. The paper also shows the expression of selected inflammatory cytokines and chemokines, attempting to correlate miRNA variants and cytokine levels.

The paper deals with an important subject, which is to find markers of susceptibility to Leishmania infection, a devastating disease. I feel that the manuscript should focus on the association of the variants with disease, as the experiments with cytokine levels are too indirect. The results can be combined as a short report. Please see some specific comments below:

- The authors adjusted the statistical analysis for sex and age in table 1 (Padj column). This correction did not return statistically significant differences. However, data presented in table 2, where they segregated by sex (not considering age) showed statistically significant associations.

Does this suggest that age, in association with sex, is influencing the results? How do the authors explain that?

- Does the combined genotype analysis (for example: occurrence of CC genotype for rs2910164 together with GG genotype for rs3746444) show a higher risk of disease development?

- Cytokine expression is influenced by several parameters (such as immunological, environmental, genetic, and epigenetic). However, while these miRNAs have been associated with consequences in cytokine expression due to their action in signaling molecules, it is not clear that the variants for these miRNA are functional regarding the expression of the miRNA itself.

Have the authors measured the levels of miR146-a and miR499a in individuals with distinct genotypes? This would help make the argument of the functional activity of the variants and would help associate with cytokine expression.

- The fact that the variants are associated with expression of some but not all cytokines that are related to the molecules inhibited by the miRNA suggests that factors other than the variants influence cytokine expression. In addition, as mentioned above, the authors do not show an association between the variant and the expression of the miRNA. Thus, the cytokine data represents too far of an extrapolation of between the variant for the miRNA and its functional activity and because of that it should be removed.

- Discussion is too long and speculative.

Reviewer #2: I consider the work address a higher relevance to understand how the host-genetic interfere in the post-transcriptional regulation of gene expression and consequences to inflammation. I recommend a minor revision.

Minor points:

Abstract: include the main conclusion in the final of abstract.

In the introduction section:

author can cite the original works that describe the miRNA function, as Refs: Baltimore et al., 2008; Bartel, 2004; Bartel, 2009.

The work from Geraci and others can be used to explain the impact of miRNAs in host susceptibility to infection, as REF: Lemaire et al., 2013; Geraci et al., 2015; Muxel et al., 2017,2018; Acuna et al., 2020; Tiwari et al., 2017, Nunes et al., 2018,

Kumar et al, 2018; Das et al., 2020; Souza et al.,2021 and others

-

Are the miR-146a and miR-499 upregulated in Leishmania-infected human/mouse host to support the evaluation of miRNA polymorphisms? Inlcude some information in the introduction and discussion sections.As REFs:

- Kumar V, Kumar A, Das S, et al. Leishmania donovani Activates Hypoxia Inducible Factor-1α and miR-210 for Survival in Macrophages by Downregulation of NF-κB Mediated Pro-inflammatory Immune Response. Front Microbiol. 2018;9:385. Published 2018 Mar 8. doi:10.3389/fmicb.2018.00385 ;

- Das S, Mukherjee S, Ali N (2021) Super enhancer-mediated transcription of miR146a-5p drives M2 polarization during Leishmania donovani infection. PLoS Pathog 17(2): e1009343. https://doi.org/10.1371/journal.ppat.1009343

-Diotallevi A, De Santi M, Buffi G, Ceccarelli M, Vitale F, Galluzzi L and Magnani M (2018) Leishmania Infection Induces MicroRNA hsa-miR-346 in Human Cell Line-Derived Macrophages. Front. Microbiol. 9:1019. doi: 10.3389/fmicb.2018.01019

Reviewer #3: A large sample size and correct design are the strengths of this study, while overselling and not correcting statistical findings are the main limitations.

--------------------

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Reviewer #1: No

Reviewer #2: Yes: Sandra Marcia Muxel

Reviewer #3: No

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13 Aug 2021

Submitted filename: Response to reviewers.docx

Click here for additional data file.

7 Sep 2021

Dear Dr. Ramasawmy,

We are pleased to inform you that your manuscript 'Variants of MIRNA146A rs2910164 and MIRNA499 rs3746444 are associated with the development of cutaneous leishmaniasis caused by Leishmania guyanensis and with plasma chemokine IL-8' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Claudia Ida Brodskyn

Associate Editor

PLOS Neglected Tropical Diseases

Steven Singer

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

The authors answered all the questions raised by the referees. and the manuscript deserves to be published. Please, note that there is a minor correction raised by the reviewer 3 the needs to be observed.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: YES

Reviewer #2: The objetives are clearly delivered in the study, presentign a adequate sampleou size and statistical analysis.

Reviewer #3: Minor correction needed: please specify in the Methods section which FDR correction was used (Benjamini-Hochberg, Benjamini-Yekutieli, or another?).

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: YES

Reviewer #2: The manuscript present an adequate analysis of polymorphism in MIR146A rs2910164 and miR499A rs3746444 in Lg-CL patients. The data analysis of polymorphism in MIR146A rs2910164 in Lg-CL patients correlates the allele C with higher risk to develop leishmaniasis in male, previously implicated in psoriasis. Also, authors identified a lower of plasma levels of IL-8 in Lg-CL with homozygous allele C, which can be Target by miR-146.

Reviewer #3: Yes

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: YES

Reviewer #2: The comparative analysis of distributiion of genotypes and allelesin MIR146A rs2910164 and miR499A rs3746444 in Lg-CL patients support the conclusions presented in the manuscript.

Reviewer #3: Yes

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: N/A

Reviewer #2: (No Response)

Reviewer #3: Minor correction needed: please specify in the Methods section which FDR correction was used (Benjamini-Hochberg, Benjamini-Yekutieli, or another?).

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have responded satisfactorily to the comments I presented.

Reviewer #2: The manuscript bring a new approach for understand the funcional role of miRNAs during Leishmania infection by focus in the polymorphism of MIR146A rs2910164 and miR499A rs3746444 in Lg-CL patients. The allele frequências and genotypes in these genes can interfere in resistance or suceptibility to leishmaniasis, including the control of proinflammatory response to parasite.

Reviewer #3: I agree with all changes to the manuscript and endorse its publication in the present form, with the minor correction detailed above.

**********

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Reviewer #1: No

Reviewer #2: Yes: Sandra Marcia Muxel

Reviewer #3: No

15 Sep 2021

Dear Dr. Ramasawmy,

We are delighted to inform you that your manuscript, "Variants of MIRNA146A rs2910164 and MIRNA499 rs3746444 are associated with the development of cutaneous leishmaniasis caused by Leishmania guyanensis and with plasma chemokine IL-8," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases