miRNA Regulation of NK Cells Antiviral Response in Children With Severe and/or Recurrent Herpes Simplex Virus Infections

Severe and/or recurrent infection with Herpes simplex virus (HSV) is observed in a large group of patients treated in clinical immunology facilities. Atypical and prolonged HSV infection is the most common clinical manifestation of disturbed NK cell development and functions, yet the molecular basis of these disorders is still largely unknown. Since recent findings indicated the importance of miRNA in regulating NK cell development, maturation and functions, the aim of our study was to investigate miRNA expression pattern in NK cells in patients with severe and/or recurrent infections with HSV and analyze the role of these miRNAs in NK cell antiviral response. As a result, miRNA expression pattern analysis of human best known 754 miRNAs revealed that patients with severe and/or recurrent HSV infection had substantially upregulated expression of four miRNAs: miR-27b, miR-199b, miR-369-3p and miR-491-3p, when compared to healthy controls. Selective inhibition of miR-27b, miR-199b, miR-369-3p and miR-491-3p expression in NK-92 cells resulted in profound upregulation of 4 genes (APOBEC3G, MAP2K3, MAVS and TLR7) and downregulation of 36 genes taking part in antiviral response or associated with signaling pathways of Toll-like receptors (TLR), NOD-like receptors, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and type I IFN-related response. Additionally, flow cytometry analysis revealed that miR-369-3p and miR-491-3p inhibitors downregulated NK cell intracellular perforin expression, while the expression of granzyme B and IFNγ remained unchanged. Taken together, our study suggests a novel mechanism which may promote recurrence and severity of HSV infection, based on miRNAs-dependent posttranscriptional regulation of genes taking part in antiviral response of human NK cells.


INTRODUCTION
Severe and/or recurrent infections with HSV are observed in a considerable group of patients treated in clinical immunology facilities. Atypical and prolonged HSV infection is the most common clinical manifestation of disturbed antiviral response caused by primary NK cell deficiencies, that results from either reduced/absent number of NK cells or disturbed function of these cells (1)(2)(3). Nonetheless, in a large group of patients suffering from severe and/or recurrent HSV infection who exhibit normal NK cell numbers and NK cell cytotoxic activity, the molecular basis of their disorder is still largely unknown.
Infections with Herpes viruses are mainly controlled by natural killer (NK) cells, as a part of innate immunity. NK cells are large, granular lymphocytes and their peripheral blood frequency varies between 5 and 20% (4). NK cells have several surface markers/ receptors, such as CD16 (Fc gamma receptor type III), important receptor in antibody-dependent cellular cytotoxicity (ADCC) (5) or CD56, characteristic of NK cells producing large amounts of cytokines (6). NK cells kill infected or neoplastic cells. The lysis of altered cell occurs via perforin (Prf) and granzymes, secreted from cells' lysosomes, or through Fas ligand, tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand (7). NK cells can also secrete a number of cytokines and chemokines, such as interferon g (IFN-g), crucial in antiviral response; TNF, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 5 (IL-5), IL-13, macrophage inflammatory protein (MIP-1) and RANTES (6). The release of these cytokines activates Th1 response, macrophagemediated killing of intracellular pathogens and up-regulates major histocompatibility complex (MHC) class I molecules expression. NK cells can also provide a co-stimulatory signal to T and B lymphocytes, through CD40L and OX40L (8).
NK cells play a vital role in the control of a number of human viruses. Several studies demonstrated NK cell-dependent protective effects during infections with coxsackievirus, human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus and poxvirus, but most importantly, Herpes viruses (7). Furthermore, the most common manifestation of NK cell deficiency, both in number and function, is severe, recurrent or disseminated infection with herpesviruses, including HSV, Varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), etc (2,7).
The discovery of miRNAs has revealed a new mechanism of gene expression regulation that affect many biological systems, including mammalian immune system. miRNA are small, singlestranded non-coding RNA that negatively regulate gene expression by inducing their degradation or repress translation of the target protein-coding mRNA (9). They are encoded by genomic DNA, transcribed by RNA polymerase II and processed from their primary transcript by the enzymes Drosha and DiGeorge syndrome critical region gene 8 (DCGR8). Pre-miRNA are then exported from the nucleus by exportin 5, and after reaching cytoplasm, are further processed by Dicer, resulting in the formation of miRNA duplex. The duplex is packaged into RNA-induced silencing complex (RISC), which comprises several proteins, including Argonaute. The miRNA guides the complex to its 3' UTRs, leading to inhibition of translation or decrease of mRNA stability (9,10). miRNA themselves are coordinately modulated by different effectors when carrying out basic functions. Studies of miRNA show development-and tissue-specific regulation mechanisms, although a considerable amount of known miRNA are expressed universally in various tissues and species (11). miRNA are regulated at the multiple steps of their biogenesis, by sequence-specific RNA binding proteins and double-strand RNA binding domains, as well as by other mechanisms, including RNA editing or methylation (12). miRNA plays a pivotal role in every aspect of cellular activity, such as differentiation, development, metabolism, proliferation and apoptotic cell death. miRNA is also associated with molecular mechanisms of various human diseases, including viral infections and tumorigenesis. Currently, approximately 2,300 human miRNAs have been discovered (13).
Recent advances highlight the importance of miRNA in regulating NK cell development, maturation and functions (14)(15)(16)(17)(18). In mice, Dicer-and Dcgr8-deficient NK cells were significantly impaired in survival and turnover, and had impaired function of ITAM-containing activating cell receptors (17), while Dicer-deficient NK cells exhibited reduced survival and impaired maturation with enhanced degranulation and IFNg production in response to cytokines (19). The role of specific miRNAs in NK cells has also been studied. For example, modulation of miR-150 expression levels has a different effect of NK cells development (20). A number of miRNAs has also been implied to be crucial for NK cell functionality. A number of miRNAs, such as hsa-miR-155 and hsa-miR-181, regulate expression of IFN-g in NK cells (19,21), while other, i.e. miR-223, miR-27a-5p, miR-150, miR-378, and miR-30e, has been reported to suppress the cytotoxic capabilities of NK cells by repressing perforin or granzyme B (22)(23)(24)(25).
Recent studies have also shown that pathogens can influence miRNA regulation of NK cell functions in order to evade immune response. Among herpesviruses, targeted miR-155 deletion in murine NK cells result in dramatically diminished effector activities and reduce memory cell numbers in both lymphoid and non-lymphoid tissues after murine CMV infection (26). However, little is known about a possible association of disturbance of miRNA regulation and severe, prolonged HSV infection.
The aim of the study was to investigate 1) miRNA expression pattern in NK cells in patients with severe and/or recurrent HSV infections, and to describe 2) which miRNAs might be associated with increased susceptibility to prolonged and severe HSV infection, and 3) define the genes associated with antiviral response which expression is altered by studied miRNAs, in order to predict the possible new mechanism of NK cell deficiency leading to propagation of HSV infection.

Patients and Control Group
Thirty-eight patients with severe and/or recurrent HSV-1 infection, including children after HSV-1 infection of central nervous system or ocular infections (3 girls and 2 boys), children with extensive infections of skin and/or mucosa (12 girls and 22 boys), children with accompanying recurrent VZV infection (1 girl and 2 boys) and thirty-nine healthy children (16 girls and 23 boys) were evaluated in this study. There were 2 patients classified to more than one group (1 patient was in group of orolabial herpes and disseminated skin infection, 1 patient was in group of disseminated skin infections and VZV infections) ( Table 1).
All patients were under supervision of the outpatient units of the Department of Clinical Immunology of the University Children's Hospital in Krakow. The study was approved by the Bioethical Committee of the Jagiellonian University (KBET/284/ B/2013).

NK Cell Isolation
NK cells were isolated from blood samples obtained from analyzed patients and healthy controls. In brief, peripheral blood mononuclear cells (PBMC) isolated from EDTA-treated whole peripheral blood by the standard Ficoll/Isopaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. NK cells were isolated from PBMC using MACS technology and a NK cell isolation kit (Miltenyi Biotech), in which NK cells were isolated via negative selection. In particular, non-NK cells were labeled with a cocktail of biotin-conjugated antibodies and antibiotin magnetic beads and were then depleted by passage through a paramagnetic MS Columns (Miltenyi Biotech).
miRNA Expression Analysis miRNA expression analysis was performed in NK cell population isolated from patients and healthy controls. In brief, the total RNA, enriched in small RNAs, was extracted from NK cell samples using High Pure miRNA Isolation Kit (Roche, Germany) according to the manufacturer's protocol. The first strand cDNA was obtained from the total RNA (400 ng) samples with Megaplex ™ RT Primers (pool A and B, Applied Biosystems, Foster City, CA). miRNA expression profile analysis was performed using TaqMan Array Human MicroRNA Card Set v3.0 (Applied Biosystems) according to manufacturer's protocol. Real-time PCR was performed using the QuantStudio 7 System (Applied Biosystems). Data was analyzed using ExpressionSuite version 1 software (Applied Biosystems). The results were shown on volcano plot (p-value vs. fold change), with following settings: fold change boundary 2,0 and p-value boundary 0,05. Volcano plot was performed using GraphPad Prism version 6 software.
The expression of miRNAs that were significantly elevated in patients vs. control group and the differences reached statistical significance were analyzed again, by individual real-time PCRs (TaqMan Gene Expression Assays, Applied Biosystems). Briefly, reverse transcription was performed as described above (Megaplex RT primers pool A or B were used for the selected miRs and U6 as a control) and the PCR reactions were performed in triplicates using the QuantStudio 7 System (Applied Biosystems). The fluorescent signals generated during the informative log-linear phase were used to calculate the relative amount of miRs, while U6 was used as a control for each PCR run. The expression of each miRNA was calculated using 2 −DDCT method.

Cell Sorting
The NK-92 cells transfected with miRNA inhibitors were suspended in phosphate-buffered saline buffer (PBS) and sorted using the FACSAria II cell sorter (BD Biosciences, San Jose, CA, USA). The cells were sorted to obtain FAM-miRNA inhibitorstransfected cells. Sorter was equipped with 488 nm laser for excitation of FITC and PE. The following band-pass filters were used for the measurement of fluorescence (wavelength in nm/ power in mW): 530/30 for FITC and 582/15 for PE. Data was acquired and analyzed using BD FACSDiva ™ Software (Becton Dickinson). Sorting was performed using 85 µm nozzle with the sheath pressure set at 45 PSI and a flow rate dependent on the concentration of cells, at the sort rate of less than 3600 events/s, using the sort precision mode set for "Good Purity" to obtain pure (usually 97-98%) subpopulations. Sorted cells were collected into FBS-coated polypropylene tubes (BD Biosciences).

Antiviral Response Gene Expression Analysis
Total RNA was extracted from cell suspension using GenElute ™ Total RNA Purification Kit (Sigma Aldrich), then cDNA was generated with the iScript Advanced cDNA synthesis kit (Biorad, Carlsbad CA). For antiviral response gene expression analysis, the PrimePCR predesigned plates (384-well format, Bio-Rad) were used, altogether with SsoAdvanced SYBR Green supermix (Bio-Rad), according to manufacturer's protocol. Real-time PCR reaction was performed on QuantStudio 7 System. Data analysis was performed with PrimePCR Analysis Software V1.0 (Bio-Rad). The expression of each gene was presented as relative expression normalized to GAPDH, which presented the lowest expression variability.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 6 (GraphPad Software Inc., San Diego, CA). The normal distribution of values was verified using Shapiro-Wilk test. A non-parametric Kruskal-Wallis test with Dunn's post-hoc test was used and median ± interquartile range (IQR: Q1-25%, Q3-75%) was shown. The P values <0.05 were considered significant.

RESULTS miRNA Expression Pattern Analysis in NK Cells
To evaluate the miRNAs expression pattern in NK cells of patients with severe and/or recurrent HSV infection and healthy control subjects, we performed real-time PCR quantification of the best known 754 human miRNAs in NK cells isolated from peripheral blood of all analyzed individuals. As a result, we noticed nine miRNAs that differed between patient and control group (fold change of DCt values of the miRNAs was higher than 2) and the difference was statistically significant (Figure 1), where the expression of four miRNA was significantly upregulated in patients group and five were significantly downregulated in patient group (miR-99b, miR-181c, miR-519e, miR-500, miR-552), when compared to healthy controls ( Figure 1A). Since here we decided to focus on miRNAs which expression was significantly elevated in patient group, we performed individual real-time PCR reactions for these four miRNAs (miR-27b, miR-199b, miR-369-3p, and miR-491-3p) in all patients and healthy subjects, in order to confirm primary results. Again, the differences of analyzed miRNAs expression between patients and control group reached statistical significance ( Figure 1B). These miRNAs were thus chosen for further analysis.
The Effect of miRNA Inhibition on Prf, GrzmB, and IFNg Expression in NK-92 Cells At last, we additionally analyzed the expression of Prf, GrzmB and IFNg intracellular expression in NK-92 cells transfected with miR-27b, miR-199b, miR-369-3p and miR-491-3p inhibitors by flow cytometry (Figure 3). The results showed that only perforin expression was significantly upregulated in NK-92 cells transfected with miR-369-3p or miR-491-3p inhibitors, when compared to negative control ( Figures 3A, D). In the case of miR-369-3p inhibitor, both the percentage of cells expressing perforin, and perforin MFI was markedly elevated, while in the case of miR-491-3p inhibitor we only noted upregulation of the percentage of Prf-positive cells. There were no differences in the percentage of cells or MFI when GrzmB (Figures 3B, E) and IFNg ( Figures 3C, F) expression was analyzed.

DISCUSSION
In this report, we show that NK cells isolated from patients suffering from severe and/or recurrent HSV infection had dysregulated miRNA expression, what may alter antiviral response of these cells and, as a consequence, escalate HSV infection. First of all, the results of miRNA expression profiling showed that patients suffering from severe and/or recurrent HSV infection had significantly upregulated expression of four miRNAs: hsa-miR-27b, hsa-miR-199b, hsa-miR-369-3p and hsa-miR-491-3p, when compared to healthy control subjects. None of these miRNAs has been previously reported to play an important function in NK cells, although miR-27a, a close homolog to miR-27b (20/21 nucleotide identity), was shown to repress the cytotoxic capacity of NK cells (24). However, miR-27b, miR-199b, and miR-491-3p had been previously associated with viral infections, also caused by Herpesviruses. miR-27b was up-regulated in human CMV-infected glioma cells (27), and both miR-27a and miR-27b have antiviral activity against murine CMV (28). miR-199b was significantly downregulated in Kaposi's sarcoma specimens (29), while miR-491 was one of the several miRNAs which expression levels were altered by HCV infection in hepatoma cells (30). There is limited data about the role of miR-369-3p in immune response with most of the research concerning its impact in different types of cancer (31)(32)(33).
Selective inhibition of miR-27b, miR-199b, miR-369-3p and miR-491-3p expression in NK-92 cells resulted in down-or upregulation of a number of genes associated with antiviral response. These genes might be involved in the modulation of 4 signaling pathways: Toll-like receptor (TLR) signaling (34)  NOD-like receptor signaling (35), the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling (36) and type I IFN signaling and response (37). To our surprise, among analyzed miRNAs, miR-27b, miR-199b and miR-491-3p inhibition resulted in upregulation of only four genes in total, while the use of all four inhibitors resulted in downregulation of 36 genes altogether. Both miR-27b and miR-199b inhibition resulted in upregulation of the mitochondrial innate immune adaptor (MAVS) gene, while miR-27b inhibitor caused upregulation of two more genes: MAP2K3 and TLR7. MAVS (also known as IPS-1/VISA/Cardif) interacts with the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) such as RIG-I and melanoma differentiation-associated gene 5 (MDA-5), which recognize viral RNA, and activate the expression of type I interferons and IFN-stimulated genes (ISGs) (38,39). Although, HSV is a double-strand DNA virus, it has evolved multiple mechanisms to evade the host innate immune response, including MAVS-mediated response and early ISGs production (40,41). Moreover, the absence of MAVS alters normal NK cell development and maturation (42). Thus, we believe that MAVS might be a key protein associated with alteration of NK immune response against HSV caused by upregulation of miR-27b and miR-199b in our patients suffering from severe and/or recurrent HSV infection. However, it should be also pointed out that although we did not noticed upregulation of type I interferon signaling and response genes following MAVS upregulation, it is possible the time point of harvesting NK-92 cells after stimulation was too short to observe MAVS-mediated gene expression stimulation. miR-27b inhibitor significantly upregulated TLR7 gene and MAP2K3 gene, which mediate downstream signaling of TLR receptors. TLR7 and TLR8 recognize the single-stranded RNAs (ssRNAs) found in many viruses, leading to IFN-a production in virus-infected macrophages and dendritic cells (43,44). It has been shown, though, that infection of human corneal epithelial cell (HCEC) line by HSV-1, which is a dsDNA virus, can lead to upregulation of TLR7 expression level and proinflammatory cytokine and IFNs production (45). On the other hand, MAP2K3 engagement in the activation of p38-STAT1 pathway and activation of a subset of ISGs in antigen-presenting cells during HIV-1 infection was previously reported to facilitates expansion of the infection (46). MAP2K3/P38 MAPK signaling pathway plays also essential role in coxsackievirus B3 infection, favoring viral progeny release from infected cells (47). MAP2K3 has been also shown to be inhibited by miR-21 (47), which promotes NK cell proliferation (48). Taken together, it is possible that upregulation of miR-27b expression in NK cells of our patients promotes HSV infection. Moreover, MAP2K3/P38 MAPK signaling pathway involvement in HSV infection needs further investigation. miR-491-3p inhibition resulted in upregulation of APOBEC3G (A3G) gene expression, which is a type I IFNresponsive gene. A3G is a cytidine deaminase, an enzyme that belongs to a larger group that can edit nucleic acids. A3G is as a well-known interaction partner of the HIV-encoded Viral Infectivity Factor (Vif) protein, which counteract the antiviral activity of A3G proteins (49). However, increased expression of A3G in NK cell lines profoundly alters the coding transcriptome of NK cells observed under hypoxic stress (50). Since both hypoxia and chronic infections are stress factors for NK cells, we might assume that A3G could play an important role in NK cell functions during prolonged HSV infection. Latent HCMV infection has already been shown to downregulate APOBEC3G in CD34 + progenitor cells (51).
In conclusion, our study suggests a novel mechanism which may promote recurrence and severity of HSV infection based on the miRNAs-dependent posttranscriptional regulation of genes taking part in antiviral response of human NK cells.