Toll-Like Receptor and miRNA-let-7e Expression Alter the Inflammatory Response in Leishmania amazonensis-Infected Macrophages

Parasite recognition by Toll-like receptors (TLRs) contributes to macrophage activation and subsequent control of Leishmania infection through the coordinated production of pro-inflammatory and microbicidal effector molecules. The modulation of microRNA (miRNA) expression by Leishmania infection potentially mediates the post-transcriptional regulation of the expression of genes involved in leishmanicidal activity. Here, the contribution of TLR signaling to the miRNA profile and gene expression was evaluated in Leishmania amazonensis-infected murine macrophages. The infectivity of L. amazonensis was higher in murine bone marrow-derived macrophages from mice knockout for myeloid differentiation factor 88 (MyD88−/−), TLR2 (TLR2−/−), or TLR4 (TLR4−/−) than wild type C57BL/6 (WT). L. amazonensis infection of WT macrophages modulated the expression of 32% of the miRNAs analyzed, while 50% were upregulated. The absence of MyD88, TLR2, and TLR4 altered the percentage of miRNAs modulated during L. amazonensis infection, including the downregulation of let-7e expression. Moreover, the absence of signals mediated by MyD88, TLR2, or TLR4 reduced nitric oxide synthase 2 (Nos2) mRNA expression during infection. Indeed, the inhibition of let-7e increased levels of the Nos2 mRNA and NOS2 (or iNOS) protein and nitric oxide (NO) production in L. amazonensis-infected macrophages (4–24 h). The absence of TLR2 and inhibition of let-7e increased the expression of the arginase 1 (Arg1) mRNA but did not alter the protein level during infection. However, higher levels of the L-arginine transporters Cat2B and Cat1 were detected in the absence of Myd88 signaling during infection but were not altered following let-7e inhibition. The inhibition of let-7e impacted the global expression of genes in the TLR pathway by upregulating the expression of recognition and adaptors molecules, such as Tlr6, Tlr9, Ly96, Tirap, Traf 6, Ticam1, Tollip, Casp8, Map3k1, Mapk8, Nfkbib, Nfkbil1, Ppara, Mapk8ip3, Hspd1, and Ube2n, as well as immunomodulators, such as Ptgs2/Cox2, Csf 2, Csf 3, Ifnb1, Il6ra, and Ilr1, impacting NOS2 expression, NO production and parasite infectiveness. In conclusion, L. amazonensis infection alters the TLR signaling pathways by modulating the expression of miRNAs in macrophages to subvert the host immune responses.


INTRODUCTION
Protozoan parasites of the genus Leishmania are the causative agents of leishmaniases, diseases that affect more than 12 million people worldwide (1). Cutaneous leishmaniasis is widespread in Brazil, the country reporting the greatest number of cases in the Americas, with an estimated incidence of 100,000 cases per year (1). L. amazonensis induces cutaneous and/or diffuse cutaneous manifestations (2,3).
Host-parasite interactions during the innate immune response to Leishmania are mediated by the recognition of pathogenassociated molecules (PAMPs) by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) that are mainly expressed in phagocytes and antigen-presenting cells, as neutrophils, macrophages and dendritic cells (4,5). The surface of Leishmania spp is covered with molecules that are recognized by TLRs (6), which play central roles in macrophage activation, signaling to induce phagocytosis, parasite healing or persistence and in the control of infections by innate and adaptive immunity (7)(8)(9)(10)(11).
The interaction of TLR2 or TLR4 starts the signal transduction cascade through the activation of adaptor molecules, such as MyD88 and Toll-like receptor adapter-inducing interferon β (TRIF/TICAM1) (12), mediating Leishmania recognition and modulation of infectivity in macrophages (13)(14)(15). The activation of the TLR2 or TLR4 signaling cascade by LPG stimulation and L. donovani, L. major or L. amazonensis infections modulates the expression of transcription factors, such as nuclear factor kappa B (NF-κB), interferon regulatory factors (IRFs) and mitogen-activated protein kinase (MAPK) that induce the transcription of pro-inflammatory cytokines, such as tumor necrosis factor (TNF), and NO and superoxide production (14,(16)(17)(18)(19)(20)(21). However, the mechanism regulating the signal required for the activation of TLR signaling is not completely understood.
The NO produced during the initial steps of the inflammatory response may exhibit leishmanicidal activity (14,(22)(23)(24). The outcome of Leishmania infection depends on the balance between the induction of NOS2 expression to produce NO and subsequently kill the parasite (25)(26)(27)(28)(29)(30)(31) and the induction of ARG1 expression to produce polyamines that promote Leishmania survival (28,(32)(33)(34)(35). Interestingly, L-arginine is a substrate for both NOS2 and ARG1. Depending on the environmental conditions and immune response, the prevalence of one activity over the other results in the killing of the parasite or its survival. The expression of cationic amino acid transporters (CATs) and their roles in L-arginine uptake into macrophages determines the fate of the infectiveness (36)(37)(38)(39)(40).
In this study, we analyzed the activation of the TLR signaling cascade and the miRNA-mRNA interactions to understand the outcome of L. amazonensis infection in C57BL/6 macrophages. TLR2 and TLR4 mediated L. amazonensis recognition and infectivity resistance in macrophages. Other MyD88-dependent receptors may participate in macrophage activation in response to L. amazonensis. L. amazonensis infection altered the expression of genes involved in TLR signaling, transcription factors and pro-inflammatory molecules. MyD88, TLR2, and TLR4 regulated miRNA expression, such as let-7e, during the course of infection. In addition, the functional inhibition of let-7e altered the activation of the TLR pathway, Nos2/NOS2 expression and NO production, impacting the infectiveness of the parasite.

Ethics Statement
The protocol used for the animal experiments was approved by the Comissão de Ética no Uso de Animais (CEUA) from Instituto de Biociências of the Universidade de São Paulo (the approval number CEUA-IB: 233/2014). This study was performed in strict accordance with the recommendations in the Guide and Policies for the Care and Use of Laboratory Animals of the São Paulo State (Lei Estadual 11.977, de 25/08/2005) and Brazilian government (Lei Federal 11.794, de 08/10/2008).
Approximately 2 × 10 5 BMDMs were plated into 8-well glass chamber slides (Lab-Teck Chamber Slide; Nunc, Naperville, IL, USA) and incubated at 34 • C in 5% CO 2 atmosphere for the infectivity analysis. Approximately 3 × 10 6 BMDMs were plated into 6-well plates (SPL, Lifescience, Pocheon, Korea) and incubated at 34 • C in 5% CO 2 atmosphere for analyses of miRNA and mRNA expression. After 18 h of incubation, non-adherent cells were removed by washes with PBS and the infection was performed with promastigotes in the stationary growth phase (MOI 5:1). After 4 h of incubation , non-phagocytosed promastigotes were removed by washes with fresh medium. Thereafter, the infection course was followed for 4, 24 and 48 h. Non-infected macrophages maintained in culture under the same conditions were used as controls. The infectivity was microscopically analyzed after cells were fixed with acetone/methanol (1:1, v:v, Merck, Darmstadt, Germany) for 20 min at −20 • C, followed by washes with PBS and staining with DAPI (Invitrogen). The infectivity indexes (rate of infected macrophages multiplied by the average number of amastigotes per infected macrophage) were calculated by randomly counting at least 600 macrophages per slide. For the analysis of RNA expression, cells were washed with PBS, resuspended in QIAzol (Qiagen, Germantown, MD, USA) and stored at −20 • C until use.
RNA Extraction, Reverse Transcription, and miRNA PCR Array RNA was extracted using the miRneasy R Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The cDNA templates were produced from mature miRNAs using the miScript II RT kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Briefly, 250 ng of total RNA were added to 2 µL of 5X miScript HiSpec Buffer, 1 µL of 10X Nucleic Acids Mix, 1 µL of miScript Reverse Transcriptase Mix and RNAse-free water to a final volume of 10 µL. The mixture was incubated at 37 • C for 60 min to insert the poly-A+ sequence at the end of the miRNA sequence (downstream) and anneal a T-tail-tag for the elongation of the cDNA. The enzyme was inactivated at 95 • C for 5 min. The reaction was performed in a Thermocycler Mastercycler gradient (Eppendorf, Hamburg, Germany). The cDNAs were stored at −20 • C until use.
The miRNA PCR array was performed with 10-fold diluted cDNAs as the template using Mouse Inflammatory Response and Autoimmunity miRNA PCR Array: MIMM-105Z (Qiagen, Germantown, MD, USA) and the miScript SYBR PCR Kit (Qiagen, Hilden, Germany), as previously described (58). Briefly, the pooled mixture was prepared with 1X QuantiTect SYBR Green PCR Master Mix, 1X miScript Universal Primer, 105 µL of cDNA, and RNAse-free water to a final volume of 2,625 µL, and 25 µL was aliquotted into each well. The specific amplification of let-7e-5p by RT-qPCR was performed with 1X QuantiTect SYBR Green PCR Master Mix, 1X miScript Universal Primer, 1X Specific Primer, 5 µL of cDNAs, and RNAse-free water to a final volume of 25 µL. The reactions consisted of the activation of the HotStart Polymerase for 15 s at 95 • C and 40 cycles of 15 s at 94 • C, followed by 30 s at 55 • C and 30 s at 70 • C, and were performed in the Thermocycler ABI Prism 7300 instrument (Applied Biosystems, Carlsbad, CA, USA). The relative Ct value was analyzed using the miScript miRNA PCR Array Data Analysis software (www.qiagen.com). Triplicate samples were analyzed for each condition. The average Ct value was calculated to represent the gene expression variation with good accuracy. The geometric average Ct values for the miRNAs were normalized based on the average of SNORD95A. The controls for PCR and RT-qPCR were performed according to the manufacturer's instructions and determined based on efficiency reactions. A negative control containing all reaction components except the reverse transcriptase enzyme was included and subjected to RT-qPCR to confirm the absence of DNA contamination in RNA samples. The Fold Regulation (FR) value is defined as the negative inverse of the fold change [function = −1 * (1/fold change value)]. FR levels ≥1.2 were considered to indicate upregulation, and levels ≤1.2 were considered to indicate downregulation. A Venn diagram was designed Venny 2.1 tool (http://bioinfogp.cnb.csic.es/tools/venny/index.html) (59) and modified to include miRNA or mRNA names inside the areas.

Reverse Transcription and RT-qPCR for Analysis of mRNA Expression
The cDNA templates for the analysis of mRNA expression were synthesized using 2 µg of total RNA, 20 nmol of random primers (Applied Biosystems, Carlsbad, CA, USA) and water to a final volume of 28 µL. The RNA was incubated at 65 • C for 5 min and then cooled to 10 • C. Thereafter, mix 2 containing 8 µL of 5X buffer, 2 µL of 10 mM dNTPs and 2 µL of RevertAid TM Reverse Transcriptase (200 U/µL) (Fermentas Life Sciences, Burlington, Ontario, Canada) were added to the reaction, followed by an incubation at 25 • C for 5 min and 42 • C for 60 min. The enzyme was inactivated at 70 • C for 10 min, and the reaction was stored at −20 • C until further use. A negative control containing all reaction components except the reverse transcriptase enzyme was included and subjected to RT-qPCR to confirm the absence of DNA contamination in the RNA samples.

Reverse Transcription and TLR PCR Array
The cDNA templates were produced from 1 µg of total RNA using RT2 First Strand Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The reactions were performed in a Thermocycler Mastercycler gradient instrument (Eppendorf, Hamburg, Germany) and stored at −20 • C until use.
The TLR PCR array was performed with 10-fold diluted cDNAs as templates using RT² Profiler TM PCR Array Mouse Toll-Like Receptor Signaling Pathway (PAMM-018Z) (Qiagen, Germantown, MD, USA) and RT2 SYBR Green qPCR Mastermix (Qiagen, Hilden, Germany). The reactions were performed with 1X RT2 SYBR Green Master Mix and 100 µL of cDNAs and RNAse-free water to a final volume of 2,700 µL (25 µL/well). The PCR consisted of the activation of the HotStart Polymerase for 10 s at 95 • C and 40 cycles of 15 s at 95 • C, followed by 1 min at 60 • C, and was performed in Thermocycler ABI Prism 7300 (Applied Biosystems, Carlsbad, CA, USA). The relative Ct value was calculated using the RT PCR Array Data Analysis software (www.qiagen.com). Quadruplicate samples were analyzed for each condition. The average Ct value was calculated to represent the gene expression variation with good accuracy. The geometric average Ct values for the mRNAs were normalized based on the average value for the housekeeping gene B2 microglobulin. The PCR and RT-PCR controls were performed according to the manufacturer's instructions and determined using efficiency reactions. A negative control containing all reaction components except the reverse transcriptase enzyme was included and subjected to realtime PCR to confirm the absence of DNA contamination in the RNA samples. The Fold Regulation (FR) value is defined as the negative inverse of the fold change [function = −1 * (1/fold change value)]. FR levels ≥1.2 were considered to indicate upregulation, and levels ≤−1.2 were considered to indicate downregulation.

Transfection of miRNA Inhibitors
BMDMs were collected, plated and incubated as described in a previously study (58). Thereafter, macrophages were incubated with 100 nM of let-7e-5p inhibitor or the negative control (Ambion, Carlsbad, CA, USA) after a previous incubation with 3 µL of the FugeneHD transfection reagent (Roche, Madison, WI, USA) in 500 µL of RPMI 1640 medium (LGC Biotecnologia, São Paulo, SP, Brazil), supplemented as described above, for 20 min at room temperature. After 24 h of transfection, the cells were infected with L. amazonensis promastigotes, as described above.

Flow Cytometry to Detect NOS2 and ARG1 Expression
Infected BMDMs were fixed with 1% paraformaldehyde (1 h at 4 • C) and permeabilized with 0.05% Tween20 for 30 min at 4 • C, followed by blocking with Odyssey blocking buffer (LI-COR, Bad Homburg, Germany) for 1 h at room temperature. Then, samples were incubated with 1:200 dilutions of rabbit anti-NOS2 (sc651) or anti-ARG1 (sc20150) antibodies (Santa Cruz, CA, USA) for 16 h at 4 • C. Samples were then incubated with 1:300 dilution of FITC-conjugated goat anti-rabbit IgG (F7512, Sigma-Aldrich) antibody for 1 h at room temperature. The fluorescence signal from single cells was measured in channel 2 using FlowSight R Amnis (MerckMillipore, Darmstadt, Germany) and analyzed using the Ideas R Software (Amnis Corporation, Seattle, WA, USA).

Quantification of NO Production
BMDMs were seeded in 24-well plates (SPL) (1 × 10 6 cells/well) and incubated for 18 h at 34 • C in 5% CO 2 atmosphere. Then, the macrophages were infected for 4 and 24 h, as described above. Cells were detached with 0.5 M EDTA in PBS for 10 min at 37 • C, scraped and washed with PBS. Cells were incubated with 5 µM DAF-FM (for NO quantification) (Molecular Probes, Life Technologies, Darmstadt, Germany) in PBS for 30 min at 34 • C in 5% CO 2 atmosphere, as described previously (31,58). Fluorescence signals from single cells were acquired in channel 2 using FlowSight R Amnis (MerckMillipore, Darmstadt, Germany) and analyzed using the Ideas R Software (Amnis Corporation, Seattle, WA, USA).

Statistical Analysis
Statistical analyses were performed with Student's t or ANOVA tests using the GraphPad Prism Software 7 (GraphPad Software, Inc., La Jolla, CA, USA). The obtained p-values are indicated throughout the Results section.

RESULTS
Absence of the MyD88, TLR2, or TL4 Gene Increased L. amazonensis Infectivity  (Figure 1A). The number of amastigotes per infected macrophage appeared to increase in all knockout mouse strains compared to WT macrophages during the course of infection ( Figure 1B). Interestingly, the infectivity index appeared to increase in the MyD88 −/− , TLR2 −/− , and TLR4 −/− mice during the course of infection compared to infected WT macrophages (Figure 1C), confirming the involvement of TLR2, TLR4, and MyD88 in the recognition of the parasite and defining the fate of L. amazonensis infection.

Modulation of the Expression of Genes Involved in the NO/Polyamine Pathway Depends on TLR Signaling
We revealed the roles of MyD88, TLR2, and TLR4 in the L. amazonensis infection in vitro. We next analyzed how TLR signaling interferes with L-arginine metabolism to produce polyamines for parasite replication or to produce NO for parasite killing. First, we analyzed the basal levels of the Cat1, Cat2B, Arg1, and Nos2 mRNAs in each mouse strain and compared the values to the WT strain. TLR4 −/− macrophages expressed lower basal levels of the Cat1, Cat2B, Arg1, and Nos2 transcripts (Figures 2A-D). MyD88 −/− macrophages showed lower basal levels of the Arg1 and Nos2 transcripts (Figures 2C,D). TLR2 −/− macrophages did not show differences in the basal levels of any transcript (Figures 2A-D).
We analyzed the levels of those transcripts 4, 24, and 48 h after the L. amazonensis infection. The only transcript whose expression was modulated during the course of infection was Nos2, which was increased at 24 h and decreased at 48 h after infection ( Figure 2D). Compared to WT macrophages, the level of the Cat2B transcript was increased in MyD88 −/− macrophages 48 h after infection and increased levels of the Cat1 transcript were observed in MyD88 −/− macrophages 4, 24, and 48 h after infection (Figures 2A,B, respectively). In contrast, MyD88 −/− macrophages showed decreased levels of Arg1 and Nos2 transcripts in all infection periods analyzed (Figures 2C,D, respectively). The comparison between WT and TLR2 −/− macrophages showed increased levels of the Arg1 transcript 4 h after infection and decreased levels of Nos2 transcript in all infection periods analyzed (Figures 2C,D, respectively). The comparison between WT and TLR4 −/− macrophages revealed decreased levels of the Arg1 and Nos2 transcripts throughout the course of infection (Figures 2C,D, respectively). Based on these data, MyD88, TLR2, and TLR4 signaling altered the expression of genes involved in polyamine/NO production in L. amazonensisinfected macrophages.  represents the mean ± SEM of the values obtained from three independent experiments (n = 6-9). Statistical significance was determined using non-parametric two-way ANOVA. *p < 0.05 compared to WT macrophages. (Figures 3, 4, S1; Table S1) to investigate the importance of miRNA-mediated post-transcriptional regulation of gene expression.
The miRNA profile of infected MyD88 −/− macrophages showed the differential regulation of the expression of 35% (30/84) of analyzed miRNAs: 50% of were upregulated and 50% were downregulated (Figure 3). Unexpectedly, the miRNA profile of infected TLR2 −/− macrophages revealed low levels of miRNA modulation during the course of infection. Only 12% (10/84) of miRNAs were differentially regulated, and 80% were downregulated. No miRNA modulation was observed 48 h after infection. The miRNA profile of infected TLR4 −/− macrophages showed the differential regulation of the expression of 28% (24/84) of the analyzed miRNAs, 92% of these were downregulated (Figure 3).
Altogether, our data revealed the differential regulation of the miRNA profiles in L. amazonensis-infected macrophages via MyD88, TLR2, and TLR4 signaling.

In silico Identification of Let-7e Target mRNAs
Since we observed the modulation of let-7e expression in the MyD88-and TLR2-deficient mice infected with L. amazonensis, we decided to assess the effects of this miRNA on modulating the expression of target mRNAs in the TLR pathway. Based on the results from an in silico analysis using miRecords tool, we identified 3,357 predicted interactions for let-7e (Table S2). On the other hand, based on a search using the Dianna tool for experimentally validated targets, let-7e targeted Tlr4, Tbk1, Map2k4, Map3k1, Chuk, Tnf, Tnfpaip3, Tnfrsf 1a, and Il10 (60).

let-7e Affects L. amazonensis Infectivity by Regulating L-arginine Metabolism
Next, we analyzed the impact of let-7e expression on L. amazonensis-infected macrophages. First, macrophages were transfected with 100 nM let-7e inhibitor or negative-control (NC). Then, macrophages were infected with L. amazonensis for 4 and 24 h. Expression assays were used to validate let-7e inhibition, and the inhibition of this miRNA decreased the expression of the miRNA let-7e 4 and 24 h after infection compared to negative control-infected macrophages ( Figure S2). The impact of let-7e inhibition on L. amazonensis infectivity was also evaluated. As shown in Figures 5A-C, the percentage of infected macrophages, number of amastigotes per infected macrophage and infectivity index at 4 h of infection were reduced by ∼10, 30, and 35%, respectively, compared to NC (Figures 5A-C).
We measured the levels of the Cat2B, Cat1, Arg1, and Nos2 transcripts to determine whether let-7e and the subsequent decrease in infectivity required L-arginine metabolism (Figures 5, S2). We did not observe modulation of the levels of the Cat2 and Cat1 transcripts among the samples (Figure S2). The level of the Arg1 transcript was increased in cells transfected with the let-7e inhibitor 24 h after infection (35%, Figure 5D), but the percentage of cells expressing ARG1 ( Figure 5E) and mean intensity of ARG1 ( Figure 5F) were not noticeably modified. The level of the Nos2 transcript was reduced following let-7e inhibition 4 h after infection, but Nos2 levels were increased 24 h after infection ( Figure 5G). Moreover, the percentage of cells expressing NOS2 (Figure 5H), mean intensity of NOS2 ( Figure 5I) and NO production were increased in let-7e inhibitor-transfected infected macrophages compared to FIGURE 5 | Inhibition of let-7 increases differential gene expression and reduces L. amazonensis infectivity. BMDMs were transiently transfected with 100 nM negative control (NC-filed patterned bar) or let-7e-5p inhibitor (let-7e-blue bar). Twenty-four hours after transfection, the cells were co-cultivated with L. amazonensis (MOI 5:1) for 4 h, and the cultures were then washed. Four and 24 h after infection, the samples were analyzed for infectivity under the microscope by counting the numbers of infected macrophages (A), amastigotes per infected macrophage (B), and infectivity index (C) (n = 1,000 macrophages/treatment). RT-qPCR was used to (Continued) NC-transfected macrophages, as observed by the percentage of DAF-FM+ cells ( Figure 5J) and mean intensity of the DAF-FM signal ( Figure 5K). Thus, let-7e inhibition indirectly impacted the expression of genes involved in L-arginine metabolism, promoting NO production and subsequent infectiveness.

let-7e Impacts Global Variations in the Expression of TLR Pathway Genes in L. amazonensis-Infected Macrophages
We evaluated and compared the transcript levels of molecules involved in recognition/binding and downstream signaling, such as adapters, transcription factors, cytokines/chemokines and cytokine receptors, in infected WT macrophages or negative control (NC)-and let-7e-inhibited macrophages 4 and 24 h after L. amazonensis infection and compared the results to uninfected macrophages to determine the role of let-7e in regulating the expression of molecules in the TLR pathway involved in pathogen recognition and the establishment of an infection (Figures 6, S3; Table S3). First, we compared the expression of genes in the TLR signaling cascade in infected macrophages 4 and 24 h after infection (Figures 6A,B, S3). A substantial number of genes were upregulated 4 h after infection: Tlr1, Tlr2, C-type lectin domain family 4-member e (Clec4e/Mincle), heat shock protein 1A (Hspa1a/HSP70), interferon regulatory factor 1 (Irf 1), receptorinteracting protein kinase 2 (Ripk2), nuclear factor kappa B subunit 1 (Nfkb1), NF-kB inhibitor alpha (Nfkbia/Ikbalpha), CD86 antigen (Cd86), interleukin 1 alpha (Il1a), prostaglandinendoperoxide synthase 2 (Ptgs2/Cox-2), tumor necrosis factor (Tnf ), and TNF alpha-induced protein 3 (Tnfaip3). Of these genes, only the upregulation of Hspa1a and Il1a was maintained 24 h after infection, and the expression of the heat shock protein 1 (Hspd1) gene was upregulated (Figure 6A).
Additionally, let-7e inhibition increased the levels of predicted target mRNAs, including Traf 6, Ppara, Mapk8ip3/Jip3, Map3k1, and Ube2n, in infected macrophages (Tables S2, S3). Based on these data, the expression of genes involved in TLR pathways is altered during L. amazonensis infection. Indeed, let-7e inhibition during L. amazonensis infection impacts global variations in the expression of genes TLR pathways ( Figure 6) and infectiveness (Figures 5A-C).

DISCUSSION
The importance of Leishmania-TLR interactions in enabling the activation of macrophage during healing and protection against leishmaniases has been extensively studied; however, the findings are very controversial. Macrophages have been reported to recognize the parasite, inducing a pro-inflammatory response and microbicidal mechanisms. TLR4 induces TIRAP-MyD88 signaling and then endocytosis, whereas TRIF-TRAM is activated in early endosomes (12). The trafficking of TLRs 3, 7, 8, and 9 from the endoplasmic reticulum to the endolysosomal compartment collaborates in the recognition of PAMPS and DAMPs (12). Here, we reported the roles of TLR2, TLR4, and the adaptor molecule MyD88 in mediating the phagocytosis of L. amazonensis and conferring resistance to infection, in contrast to the higher infectivity index observed in the absence of those molecules.
Several studies have described the participation of transcription factors and miRNAs in the transcriptional and post-transcriptional regulation of gene expression, respectively (64). L. major (65), and L. donovani (41,42) have been reported to modulate miRNA expression in infected human macrophages. In BALB/c macrophages infected with L. amazonensis, the miRNA profile was altered and the involvement of miR-294, miR-497a and miR-721 in infection was confirmed (31). The results presented in this study corroborate these data, showing that L. amazonensis infection regulated the miRNA profiles and let-7e expression in infected C57BL/6 WT, MyD88 −/− , TLR2 −/− , and TLR4 −/− macrophages. The miRNAs let-7e, let-7f, let-7g miR-30b, miR-369, miR-495, and miR-712 were only upregulated in infected C57BL/6 WT macrophages, while TLR2-, TLR4-, or MyD88-deficient mice exhibited alterations in the expression of other miRNAs during L. amazonensis infection. Based on this evidence, the expression of let-7e, let-7f, and let-7g require MyD88, TLR2, and TLR4 signaling during L. amazonensis infection, and our findings reveal the role of the TLR pathway in the transcriptional and post-transcriptional regulation of gene expression during Leishmania infection.
The roles of TLR/IL-1R signaling pathways in the ubiquitindependent NF-κB activation and subcellular trafficking have been extensively studied. The ubiquitination system is based on the ubiquitin-activating E1 enzyme, ubiquitin-conjugating E2 enzymes, and ubiquitin E3 ligases. The E2 UBE2n dimerizes with UBE2v, forming K63-linked ubiquitin chains to polyubiquitinate adaptor molecules, such as TRAF6, which is recruited to the TAB2/TAK1 complex and activates NF-κB, as shown in Figure 7 (70)(71)(72)(73). However, the role of ubiquitination in the immune response to Leishmania has not been studied. Another molecule, PPARα was not previously reported to be involved in Leishmania infection. However, TLR4 transcription and signaling are negatively regulated via PPARα, which inhibits the NF-κB signaling pathway (74). PPARα also regulates the expression of genes involved in free fatty acid metabolism, could bind to TLR4 and interfere with the inflammatory response. In addition, Tollip is a negative regulator of the TLR signaling pathway, regulates the production of the pro-inflammatory cytokines IL-6 and TNF (75) and potentially influences the response to cutaneous L. guyanensis infection (76). Mapk8ip3/JIP3 helps the TLR4 signaling cascade enhance the activation of the JNK and MEK1/2 pathways (77,78).
Indeed, an early step in the infection of human macrophages with L. amazonensis and L. major occurs the upregulation of proinflammatory cytokine genes, such as Il1b, Tnf, and Il6, as well as other molecules involved in tissue growth and repair, such as Csf 2 and Ptgs2/Cox2 (79). Similarly, L. major infection of murine macrophages increases the levels of Tnf, Il1, Il6, Nos2, I1rap, and Csf 3, as well as molecules in the NF-κB and MAPK signaling pathways (21).
Here, TLR2 and TLR4 induced the expression of the Cat2B and Cat1 mRNAs in L. amazonensis-infected macrophages, while a MyD88 deficiency induced their expression, helping control L-arginine uptake. L-arginine uptake mediated by Cat2B helps control L. amazonensis infectiveness (40). The regulation of polyamine production by Arg1 mRNA expression depended on TLR4 and MyD88. Thus, Nos2 expression depends on TLR2, TLR4, and MyD88 molecules during infection and its activation in C57BL/6 mouse macrophages may enhance the resistance to infection, as previously shown in murine macrophages lacking Nos2 (80). Notably, let-7e inhibition regulated Arg1 and Nos2 expression during infection, corroborating the importance of the TLR signaling cascade in the mechanism regulating Arg1 and Nos2 expression at the transcriptional level. The NF-κB pathway regulates Nos2 expression at the transcriptional level (81). In cells infected with L. major and L. amazonensis increasing in the nuclear translocation of the NF-κB p50/p50 dimer correlates with the decrease in the levels of the Nos2 and Cat2B transcripts (16). In response to an L. amazonensis infection, miR-294-3p and miR-721 post-transcriptionally degrade the Nos2 mRNA in BALB/c macrophages (31). Leishmania also induces L-arginine uptake mediated by amino acid permease 3 (82-84), amino acid metabolism via arginase activity (85,86), and NOS-like activity (87). L-arginine is essential for parasite survival and interferes with macrophage activation during infection (88). Indeed, parasite arginase competes with L-arginine and reduces Nos2 expression and NO production in L. amazonensis-infected hosts (31).
NOS2 activity induces NO production to trigger the macrophage-mediated inflammatory response and kill the parasites (39,(89)(90)(91). The balance between polyamine FIGURE 7 | Schematic of the TLR pathway and let-7e target mRNAs. TLR activation is regulated by let-7e-mediated post-transcriptional mechanisms acting directly (predicted targets in red) or indirectly (molecules in orange) on mRNA/protein expression by modulating the expression of molecules involved in recognition, adaptors and effector molecules, as well as transcription factors (green) that regulate the expression of cytokines, chemokines and other immunomodulators.
production via ARG1 and NO production via NOS2 influences the fate of infectivity in different mouse models. IL-12 production and NOS2 expression reduce the infectivity in C57BL/6 mice. In contrast, IL-4 production and Arg1 expression increase the infectivity in BALB/c mice (37,38,88). The cytokines IL-12 and IL-4 induce CAT2B expression and the uptake of L-arginine, a substrate of ARG1 and NOS2, altering outcome of infection, while the negative regulation of CAT2B expression also reduces the infectivity of L. amazonensis (40).
Interestingly, TLR4 contributes to the control of parasite growth in the initial and later steps of L. major infection (13). In addition, L. major infection induces NOS2 expression by activating TLR4, although ARG1 expression is independent of TLR4 during infection in vivo and in vitro (13). TLR9 induces NOS2, IL-12 and IFN-gamma expression and helps control the parasite burden in L. major-infected BALB/c and C57BL/6 mice (92). However, MyD88 signaling increases the ratio of IL-12/IL-10 in L. amazonensis-and L. braziliensisinfected dendritic cells (15). MyD88 mediates the resistance to L. braziliensis infection in C57BL/6 mice, but TLR2 enhances regulation of the immune response by reducing the ratio of IL-12/IL-10 (15).
L. amazonensis infection has been reported to regulate the activation of MAPK in BALB/c macrophages by regulating IL-10 production (28). However, p38-MAPK activation reduces the infectivity of L. donovani (93). On the other hand, L. mexicana glycosylphosphatidylinositol-anchored lipophosphoglycan (LPG)/TLR stimulation regulates the activity of the MAPK and NF-κB pathways to alter IL-12 production (94,95). The chaperone HSP60 serves as danger signal for stressed or damaged cells and its effects are potentially mediated by its anti-apoptotic activity induced by the formation of the HSP60/pC3 complex (96). TNF or TLR signaling leads to the formation of a Fasassociated protein with death domain (FADD)/pro-caspase 8 complex and activation of caspase 8, which induces apoptosis via direct cleavage of downstream caspases, caspase-3/7 (97). In response to an L. major infection, the inhibition of caspase 8 increases the production of Th1 and Th2 cytokines and NO (98), in contrast to the antiapoptotic signaling mediated by caspase 8 during L. infantum infection (99). The modulation of the expression of mRNAs involved in the TLR pathway impacted the infectivity of L. amazonensis.
Our data corroborated the idea that Leishmania infection may regulate the TLR pathway and subsequent cytokine and chemokine production, as well as NO/polyamine production to control macrophage activation and infectiveness. We emphasize the importance of TLR signaling and miRNA expression during infection and the integration of these molecules in the mechanism regulating gene expression required to determine the fate Leishmania infection, as well as the identification of potentially new targets to control infectivity and pathogenesis.   Figure S2 | Inhibition of let-7 functions. BMDMs were transiently transfected with 100 nM negative control (NC) or let-7e-5p inhibitor. Twenty-four hours after transfection incubation, cells were co-cultivated with L. amazonensis (MOI 5:1) for 4 h, and the cultures were then washed. After 4 and 24 h of infection, the samples were analyzed for let-7e levels normalized to SNORD95 (A), and levels of the Cat2B (B), and Cat1 (C) mRNAs using RT-qPCR. The values were normalized to the average values of NC-transfected, infected macrophages (100%). Each bar represents the average ± SEM of the values obtained from three independent experiments (n = 3-6). Statistical significance was determined using two-tailed Student's t-tests. * p < 0.05 compared to negative control-transfected, infected macrophages.  Table S2 | Predicted target mRNAs of the let-7e miRNA. The miRNA-mRNA interactions predicted using miRecord tools (http://c1.accurascience.com/ miRecords/) were based on the integration of the predicted targets obtained from at least three of the following prediction tools: DIANA-microT, MicroInspector, miRanda, MirTarget2, miTarget, NBmiRTar, PicTar, PITA, RNA22, RNAhybrid, and TargetScan/TargertScanS.