Taenia crassiceps-Excreted/Secreted Products Induce a Defined MicroRNA Profile that Modulates Inflammatory Properties of Macrophages

Helminth parasites modulate immune responses in their host to prevent their elimination and to establish chronic infections. Our previous studies indicate that Taenia crassiceps-excreted/secreted antigens (TcES) downregulate inflammatory responses in rodent models of autoimmune diseases, by promoting the generation of alternatively activated-like macrophages (M2) in vivo. However, the molecular mechanisms triggered by TcES that modulate macrophage polarization and inflammatory response remain unclear. Here, we found that, while TcES reduced the production of inflammatory cytokines (IL-6, IL-12, and TNFα), they increased the release of IL-10 in LPS-induced bone marrow-derived macrophages (BMDM). However, TcES alone or in combination with LPS or IL-4 failed to increase the production of the canonical M1 or M2 markers in BMDM. To further define the anti-inflammatory effect of TcES in the response of LPS-stimulated macrophages, we performed transcriptomic array analyses of mRNA and microRNA to evaluate their levels. Although the addition of TcES to LPS-stimulated BMDM induced modest changes in the inflammatory mRNA profile, it induced the production of mRNAs associated with the activation of different receptors, phagocytosis, and M2-like phenotype. Moreover, we found that TcES induced upregulation of specific microRNAs, including miR-125a-5p, miR-762, and miR-484, which are predicted to target canonical inflammatory molecules and pathways in LPS-induced BMDM. These results suggest that TcES can modulate proinflammatory responses in macrophages by inducing regulatory posttranscriptional mechanisms and hence reduce detrimental outcomes in hosts running with inflammatory diseases.


Introduction
Helminth infections induce polarized T H 2-type biased immune responses that play a role in parasite expulsion, tissue repair, and regulation of unrelated inflammatory and autoimmune responses in the host [1][2][3]. The striking ability of helminth parasites in conferring protection from diseases of immune dysregulation has increased the attention into the immunomodulatory mechanisms evoked by these pathogens. Previous studies in our laboratory, using a murine model of cysticercosis, demonstrated that chronic infection with the helminth Taenia crassiceps or administration of its excreted/secreted products (TcES) ameliorates the development of experimental ulcerative colitis, autoimmune encephalomyelitis (EAE), and type 1 diabetes [4][5][6][7][8]. The ability of T. crassiceps and TcES to counteract these inflammatory responses was demonstrated to be dependent on a population of macrophages that produced markers of alternative activation (M2), such as PD-L2, IL-4Rα, MR, IL-10, ARG1, YM1, and FIZZ1 [9].
Macrophages can be activated towards an M2 phenotype after being stimulated with IL-4 produced by T H 2 lymphocytes during parasitic infections or exposure to allergens [10,11]. In contrast, released IFN-γ and pathogen or danger-associated molecular patterns (PAMPs or DAMPs) during infections or tissue injury, respectively, promote classical (M1) activation in macrophages [1,12]. Although a crucial role for T. crassiceps-induced M2 macrophages in regulating detrimental autoimmune and inflammatory responses has been demonstrated [3], the transcriptional events elicited by TcES that modulate macrophage activation have not been elucidated.
Although we have previously demonstrated a role for the TcES in preventing STAT1 phosphorylation in inflammatory macrophages [17], the influence of TcES in macrophage polarization and the transcriptional pathways regulating this process remain unknown. Here, we determined the effect of TcES alone or in combination with LPS or IL-4, in the regulation of multiple mRNA transcripts and microRNAs induced in macrophages. Our results indicate that TcES decreased the production of inflammatory cytokines (IL-12, TNFα, and IL-6) in LPS-induced macrophages but has a limited role in inducing directly the production of M1-and/or M2-associated molecules. The immune-modulatory ability of TcES was further associated with increased levels of specific microRNAs, which are predicted to target, according to our bioinformatic analysis, numerous inflammatory mRNAs involved in the TNF and NF-κB signaling pathways. These findings suggest a role for TcES in modulating the transcriptional profile of macrophages via altering their microRNA profile and, consequently, the inflammatory properties of these immune cells.

Ethics Statement.
All experiments in this study were performed according to the guidelines for the Care and Use of Laboratory Animals adopted by the US National Institutes of Health. The Institutional Animal Care and Use Committee (IACUC) at the Research Institute at Nationwide Children's Hospital and the Ohio State University approved all protocols.
2.2. Mice. Adult 6-to 8-week female BALB/c mice were purchased from The Jackson Laboratory. All animals were maintained in a pathogen-free environment and established as breeding colonies in the Transgenic Mouse Facility at the Research Institute at Nationwide Children's Hospital or in specific pathogen-free conditions at the Ohio State University Laboratory Animal Resources. The mice were housed in sterilized polycarbonate cages with basic filter top caging containing pine wood shavings and were offered mouse ration and water ad libitum. The cages were held in Isolation and Containment cubicles (Britz and Co., Wheatland, WY).

Parasites and TcES.
Metacestodes of T. crassiceps (ORF strain) were harvested under sterile conditions from the peritoneal cavity of female BALB/c mice after 8-10 weeks of intraperitoneal (i.p.) infection. The cysticerci were washed four times in physiological saline solution prior to maintaining them in culture with a sterile saline solution at 37°C for 24 h. The supernatant was recovered and centrifuged for 10 min at 1000 g. The heavy fraction of TcES was concentrated using the 50 kDa Amicon Ultra Filter (Millipore), 30 min at 1000 g. Protease inhibitors were added to the >50 kDa fraction, and samples were stored at -70°C until further use.
2.6. Cytokine Assay. Supernatants from cell cultures of stimulated macrophages were recovered at 4 and 24 h poststimulation, and the levels of the cytokines IL-10, IL-6, TNFα, and IL-12 were measured by ELISA according to the manufacturer's instructions (PeproTech).
2.7. RNA Extraction and Arrays. Total RNA was extracted from BMDM stimulated with LPS (M LPS ), TcES (M TcES ), LPS+TcES (M TcES+LPS ), or culture media (M 0 ) using QIAzol reagent (QIAGEN), according to the manufacturer's specifications, and stored at -80°C. Next, RNA was purified following the miRNeasy kit protocol (QIAGEN). RNA concentration and integrity were determined using a Nano-Drop™ spectrophotometer (Thermo Scientific, Wilmington, DE) and Agilent Bioanalyzer 2100, respectively. For transcriptomic analysis, 50 ng/μL of RNA was used for the nCounter Inflammation Panel (NanoString mRNAs) and the nCounter miRNA Assay set (microRNAs). Both mRNA and microRNA arrays were performed following the manufacturer's instructions at the Genomics Shared Resource, OSU. Data analysis for the nCounter Inflammatory Panel (mRNA) and for the nCounter miRNA Assay set was conducted using the nSolver™ Analysis Software according to the manufacturer. For the nCounter Inflammatory Panel (mRNA), we normalized using the normalization factor and subtracted the background (mean of negative controls ± 2 standard deviations). Next, we normalized using the geometric mean of housekeeping genes as reported [20]. Then, using the normalized counts, we calculated the fold change (FC) by comparing M TcES , M TcES+LPS , and M LPS to M 0 . For the nCounter miRNA Assay set, we first normalized using the normalization factor. The background was subtracted from the data using the mean of negative controls ± 2 standard deviations. Finally, we used the top 75 microRNAs [21]. The normalized counts were used to calculate the FC by comparing M TcES , M TcES+LPS , and M LPS to M 0 . Of the 566 total probes measured in the assay, 183 and 236 microRNAs for 4 h and 24 h, respectively, were identified and used for analyzing significant changes in microRNA levels among samples. MultiExperiment Viewer (MeV) was used to generate heat maps, which represent log 2 -transformed data.
2.8. Real-Time PCR. TaqMan gene expression assays (Applied Biosystems) were used to quantify and/or validate the levels of mRNAs and microRNA transcripts. cDNA was generated from mRNAs, using a 15 μL RT reaction consisting of 2.0 μL of Buffer (10x), 0.8 μL 100 mM dNTPs (100 mM), 1.0 μL reverse transcriptase, 2.0 μL of mRNA primer, and 1 μg of total RNA. RT reaction was incubated for 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C. For microRNA levels, a 15 μL reaction was prepared with 2.0 μL of buffer (10x), 0.2 μL 100 mM dNTPs (100 mM), 1.0 μL reverse transcriptase, 0.2 μL RNAse inhibitor (20 U/μL), 3.0 μL of microRNA primer, and 100 ng of total RNA. RT reaction was incubated as mentioned before. For both mRNA and microRNAs, quadruplicate real-time PCR reactions were performed in the 7500 Real-Time PCR system. The amplification reaction mix was composed of 10 μL of TaqMan Universal PCR Master Mix (2x), 1 μL of the specific mRNA or microRNA probe, and 1 μL of specific microRNA cDNA. The reactions were preincubated for 10 minutes at 95°C and amplified with 40 cycles consisting of 10 sec at 95°C, 40 sec at 60°C, and 5 sec at 72°C (fluorescence acquisition). To assess possible bias for reference RNA, we used 18S RNA, Actb, and Gapdh mRNAs. Relative quantification was calculated by 2 -ΔΔCt . All mRNA and microRNA assays were tested for reproducibility and linearity (PCR efficiency was between 1.9 and 2.0 for all assays). All primers were purchased from Applied Biosystems. The primer sequences are shown in Table S1.
2.9. mRNA and MicroRNA Target Gene Prediction and Bioinformatics Analysis. Target mRNAs of differentially produced microRNAs were predicted using DIANA-TarBase database v6.0, which includes experimentally validated targets from the literature. To explore the potential biological function of the microRNAs' profile and their targets, DIANA-mirPath v2.0 (http://snf-515788.vm.okeanos.grnet .gr/) was used to perform enrichment analysis of micro-RNA's target mRNAs in the KEGG pathway and in GO terms [22].

Statistical
Analysis. Data analyses were performed using GraphPad Prism 6 software. Statistical comparisons were performed by using Student's t-test. p values less than 0.05 were considered significant. Graphed data are presented as mean ± SD or SEM.

TcES Reduces the Inflammatory Response of LPS-Induced
BMDM. Previously, we demonstrated the ability of TcES in reducing the development of inflammatory and autoimmune diseases in rodent models [4][5][6][7][8]. The effect of TcES in counteracting detrimental inflammatory responses in vivo is associated with the emergence of polarized macrophages towards an M2 phenotype [4,5,11]. Although studies in our laboratory indicate a role for TcES in blocking the IFNγ/STAT1 signaling pathway in macrophages [17], the effect of TcES in inducing directly M2 macrophages remains to be elucidated. To define the macrophage profile elicited by TcES, we first determined the levels of the inflammatory cytokines IL-12, IL-6, TNFα, and IL-10 in cultures from BMDM. The cells were stimulated (Figure 1 Our results suggest that TcES play a role in downregulating the production of proinflammatory cytokines in LPS-induced BMDM, by increasing the production of a regulatory cytokine. To gain insight in the phenotypic profile induced by TcES in macrophages, we used flow cytometry technique to determine the production of intracellular nitric oxide synthase (NOS2), and arginase-1 (ARG1), as the conventional markers for M1 and M2 profiles, respectively, in BMDM. Our results showed that while M LPS and M IL-4 presented increased percentages of NOS2 + and ARG1 + macrophages, respectively, M TcES displayed limited production of these molecules (Figures 2(a) and 2(d)). Additionally, similar percentages of NOS2 + BMDM were found between M TcES+LPS and M LPS , and comparable ARG1 + BMDM were observed when analyzing M TcES+IL-4 versus M IL-4 (Figures 2(a) and 2(d)). Levels of mRNA Arg1 by RT-qPCR showed similar trends as the flow cytometric analysis (Figure 2(f)). While the levels of Nos2 mRNA were upregulated in M TcES+LPS compared to M 0 but significantly reduced compared to M LPS (Figure 2(e)). These data suggest that the stimulus with TcES, either alone or in combination with LPS or IL-4, has a limited role in inducing the production of canonical M1 or M2 markers. Nevertheless, these antigens play a role in downregulating the proinflammatory response to LPS in BMDM.  Table S2. As expected, our results indicate increased levels of multiple proinflammatory mRNAs in M LPS with respect to M 0 ( Table 1 and Table S3), including Il1a, Il6, Il12a, Il12b, Tnf, and Nos2, among other mRNAs, at 4 and 24 h poststimulus. These molecules correspond to previously reported markers for LPS-stimulated macrophages [1]. In contrast, M TcES downregulated the levels, with respect to M 0 , of cytokines, chemokines, and transcriptional factors distinctive of M1-activated macrophages, while displaying upregulated levels mainly associated with enzymes, as MAPK pathway, at 4 and 24 h poststimulus ( Table 1 and  Table S4). Noticeably, although M TcES+LPS presented 132 and 96 upregulated mRNAs (Table S5), these macrophages only shared 6 and 3 upregulated mRNAs with M TcES at 4 and 24 h poststimulus, respectively. However, M TcES+LPS shared 89 and 65 upregulated mRNAs with M LPS at 4 and 24 h poststimulus, respectively, including transcripts for cytokines, chemokines, receptors, and transcriptional factors as Il1a, Il1b, Il6, Il12a, Il12b, Ccl3, Ccl5, Ccl2, Ccl7, Cd86, Tlr2, Stat1, Stat3, and Nfkb1 mRNA. The differentially induced mRNAs between M TcES+LPS and M LPS are shown in Table S6. Next, we validated 7 mRNAs associated with M1 (Il1b, Stat1, Cd86, Il6, and Il12b) and M2 (Stat6 and Chi3l3) macrophages by RT-qPCR. The levels of these mRNAs were comparable to those observed in the mRNA array ( Figure 4), which attest for the high quality of our array, supporting that a posttranscriptional mechanism induced by TcES may have a role in macrophage's response to LPS. Interestingly, although the levels of IL-6 and IL-12 in supernatants from M TcES+LPS were significantly reduced respect to M LPS (Figures 1(a) and 1(b)), the levels of their mRNAs of these cytokines were comparable between M TcES+LPS and M LPS . These data suggest that posttranscriptional mechanisms triggered by TcES may have a role in modulating the production of specific inflammatory cytokines.

TcES Modulate the Profile of MicroRNAs in LPS-
Stimulated BMDM. MicroRNAs participate in diverse biological processes at the posttranscriptional regulatory level. The complementary binding of microRNAs to mRNAs reduces either transcription or translation of mRNA transcripts [16]. Recently, a handful of studies indicate a role for helminth parasites and their antigens in inducing micro-RNAs to modulate host immune responses [14,15,23]. To determine whether the ability of TcES in attenuating the inflammatory response of BMDM is associated with the production of specific microRNAs, we performed a microRNA array (see RT-qPCR. M TcES+LPS are shown in Table 2. The complete lists of micro-RNAs are shown in Table S7-S9. Additionally, we found 4 and 2 microRNAs shared among the groups of stimulated BMDM at 4 and 24 h, respectively (Table S10) (Table S11). These data suggest that TcES induce the early production (4 h) of microRNAs, followed by the stimulus with LPS (24 h Table S12 and Table S13. These data suggest a role for TcES in inducing microRNAs that regulate important metabolic, cell signaling, and inflammatory pathways in LPS-stimulated BMDM.
Next, we selected and validated by RT-qPCR four micro-RNAs (miR-125a-5p, miR-762, miR-155-5p, and miR-484), which are potentially involved in the regulation of inflammatory mRNAs, as indicated by previous studies and our bioinformatics analysis. We found that both M LPS and M TcES showed increased levels of miR-125a-5p (Figure 7(a)), a microRNA reported to reduce the production of inflammatory cytokines (IL-6, IL-12, and TNFα) [24]. The levels of miR-125a-5p were sustained in M LPS and M TcES+LPS until 24 h poststimulus (Figure 7

Discussion
Helminth parasites and their antigens can counteract proinflammatory responses generated during autoimmune diseases [3]. In our laboratory, we have previously demonstrated that infection with the helminth parasite T. crassiceps or the administration of TcES reduced the symptoms of EAE, type I diabetes, and ulcerative colitis, in part due to the polarization of macrophages in vivo towards an M2 phenotype [4][5][6][7][8]26]. However, the functional role of TcES in regulating the activation and inflammatory response of macrophages remains unknown. In this study, we evaluated the effect of TcES on the polarization towards an M2 profile, inflammatory immune response, and transcriptomic profile of macrophages in vitro. . mRNA levels are represented as mean relative (±SD). Data are shown as a representative of two independent experiments. Significance was calculated using t-test. * p < 0 01, * * p < 0 05, and * * * p < 0 001.
We first measure the production of the cytokines IL-6, IL-10, IL-12, and TNFα in BMDM-stimulated with TcES alone or in combination with LPS and observed that TcES increased the levels of the regulatory cytokine IL-10 and reduced the release of the inflammatory cytokines IL-6, IL-12, and TNFα in supernatants from LPS-stimulated BMDM. TcES alone did not increase the production of inflammatory cytokines but induced the release of IL-10 in BMDM. The levels of both mRNAs of Il10 and Tnf measured by RT-qPCR showed similar trends when compared to the levels of cytokines obtained by ELISA assay, suggesting a consistent role for TcES in regulating cytokine production by inhibition of their transcripts.
Here, we evaluated the production of NOS2 and ARG1 in BMDM stimulated with TcES alone or in combination with IL-4 or LPS. M1 macrophages normally produce NOS2, which metabolizes L-arginine to nitric oxide (NO), while M2 macrophages produce ARG1, which metabolizes Larginine to produce prolines and polyamines [2,27]. We found that whereas BMDM stimulated with IL-4 or LPS alone showed increased levels of ARG1 and NOS2, respectively, TcES did not alter the production of both NOS2 and ARG1, after 24 h poststimulation. Our data are in agreement with previous studies using Fasciola hepatica tegumental antigens, which also failed to directly induce the production of molecules associated with M2 macrophages in vitro but not in vivo [28]. The production of M2 canonical molecules such as ARG1 has been reported to be IL-4-dependent, which is produced by T H 2 T cells, natural killer T cells, and basophils but not macrophages [29][30][31]. Therefore, helminth antigen stimulation alone is not enough to induce functional polarization of BMDM towards M2; however, they influence the inflammatory properties of these cells. Therefore, TcES do not induce the production of M2-associated molecules but counteract inflammatory response in macrophages in vitro.
In contrast, M TcES+LPS and M LPS shared more than 60 proinflammatory mRNAs at both 4 and 24 h            at 24 h post stimulus, which could be attributed to TcES's own recognition, as previously have been reported to recognize TcES [58]. These data suggest that posttranscriptional events may be involved in the regulatory mechanism triggered by TcES in regulating macrophage inflammatory responses.
microRNAs, small noncoding RNA molecules, have emerged as a key component of macrophage posttranscriptional regulation [59]. These molecules can silence the translation of mRNAs via base-pairing with complementary sequences within the RNA molecules. Hence, we further analyzed the microRNA profile in BMDM stimulated with  This microRNA has been reported to dampen proinflammatory responses in macrophages through the inhibition of TLRs, NF-κB, and STAT signaling pathways by targeting the mRNAs of Traf6, Irak1, Irak2, Nfκb, Stat1, and Ap1 [60][61][62][63]. This evidence is supported by our KEGG enrichment analysis, which indicates that overproduced microRNAs in M TcES+LPS target mRNAs involved in NF-κB, TNF, and MAPK signaling pathways. Of note, these data also confirm our hypothesis that TcES target proinflammatory pathways and support our previous findings indicating a role for TcES in blocking the IFN-γ/STAT1 signaling pathway in macrophages in vitro [17].
M TcES+LPS also overproduced microRNAs previously reported to target inflammatory mRNAs; for instance, let-7i and let-7e target Tlr4 mRNA, which causes a drop in the recognition of proinflammatory antigens [64][65][66]. Moreover, miR-24-3p production in macrophages has been reported to significantly decrease the production of IL-6 and TNFα [67]. Furthermore, M TcES+LPS and M TcES shared upregulated microRNAs previously reported to be elicited in macrophages exposed to E. multilocularis antigens (e.g., miR-146a-5p) and S. japonicum (miR-365 and miR-24) [14,68]. These data suggest the presence of conserved antigens among helminths that could trigger similar posttranscriptional mechanisms to modulate immune responses in the host.
Finally, we selected four upregulated microRNAs to validate their levels by RT-qPCR and confirm the high quality of our array. We observed increased levels of miR-125a-5p in M TcES and M LPS , as early as 4 h poststimulus. The combined stimulus with TcES and LPS induced an additive effect in the levels of this microRNA. miR-125a-5p has been reported to increase after TLR2/4 signaling and has a key role in reducing the production of inflammatory cytokines (IL-6, IL-12, and TNFα) by targeting NF-κB and KFL4 signaling pathways [24,[69][70][71]. These data are associated with our previous studies suggesting that TcES is a ligand of TLR2 in phagocytic cells [58,72]. In addition, miR-762 was selectively induced in M TcES and M TcES+LPS at 4 h poststimulus. miR-762 has been demonstrated to increase in ovarian and breast cancer and ocular tissue [73][74][75] where macrophages normally acquire an M2-like phenotype [76,77]. Furthermore, by using bioinformatic tools, we found Il12b, Il6, Tnf, Nfkb, and Cd86 mRNAs as possible targets of miR-762 in M TcES and M TcES+LPS . The microRNA miR-484 was found to be upregulated in all the groups of stimulated BMDM at 4 h; however, its levels were only sustained in M TcES at 24 h poststimulation. miR-484 has been previously identified in multiple types of cancers [78][79][80][81][82] and the cerebral cortex [83]; such microenvironments are known to promote an antiinflammatory phenotype in macrophages. Our bioinformatic analysis shows that Il1b, Nfkb, Stat5a, Irf1, Myd88, Stat1, and IL-12a mRNAs are possible targets for miR-484, which suggest a possible role for miR-484 in immune tolerance.
In summary, our study demonstrates a role for TcES in regulating the production of key inflammatory cytokines, possibly by inducing microRNAs that target inflammatory transcripts and promoting the release of IL-10 in macrophages. This phenomenon shapes the transcriptomic profile of macrophages and consequently the outcome of the immune response. Although we found clear associations between TcES-induced microRNAs and mRNAs involved in multiple inflammatory pathways as their targets, our study has the limitation that we did not prove a direct interaction between microRNAs and mRNAs. Therefore, future studies in our laboratory will focus on elucidating the functional roles and significance of the different microRNAs described here. These findings increase our understanding of how released molecules from helminths regulate inflammation and may offer new approaches for the treatment of autoimmune and inflammatory diseases.

Data Availability
The array data used to support the findings of this study have been deposited in the GEO (Gene Expression Omnibus) database of the NCBI with the accession numbers GSE125170 for RNAm and GSE125171 for microRNA as part of the SuperSerie GSE125172 which are public once this article is published.

Conflicts of Interest
The authors have no financial or other conflicts to declare. Table S1: sequence of primers used in RT-qPCRs to amplify the specified mRNAs (A) or microRNAs (B).