TGF-β1 Downregulates the Expression of CX3CR1 by Inducing miR-27a-5p in Primary Human NK Cells

Activity of human natural killer (NK) cells against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX3CR1 that drives these effectors toward peripheral tissues, including tumor sites. To identify possible mechanisms mediating chemokine receptors modulation, we analyzed the microRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX3CR1. We demonstrated the functional interaction of miR-27a-5p with the 3′ untranslated region (3′UTR) of CX3CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX3CR1 3′UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p downregulates the surface expression of CX3CR1. Finally, we showed that neuroblastoma cells induced in resting NK cells a downregulation of the CX3CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX3CR1 expression.

Activity of human natural killer (NK) cells against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX3CR1 that drives these effectors toward peripheral tissues, including tumor sites. To identify possible mechanisms mediating chemokine receptors modulation, we analyzed the microRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX3CR1. We demonstrated the functional interaction of miR-27a-5p with the 3′ untranslated region (3′UTR) of CX3CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX3CR1 3′UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p downregulates the surface expression of CX3CR1. Finally, we showed that neuroblastoma cells induced in resting NK cells a downregulation of the CX3CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX3CR1 expression.
Keywords: TgF-β1, chemokine receptors, micrornas, nK cells, tumor microenvironment inTrODUcTiOn Human natural killer (NK) cells mainly consist of CD56 bright CD16 low/neg KIR neg perforin low and CD56 dim CD16 pos KIR pos perforin high cells, which represent sequential stages of maturation and NK subsets with different function and tissue distribution (1,2). CD56 bright NKs, which release large amount of soluble factors (IFN-γ, GM-CSF, and TNF-α) in response to proinflammatory cytokines, are poorly represented in peripheral blood, while populating most tissues, and, in particular, secondary lymphoid organs (SLOs) (3,4). Conversely, CD56 dim cells, which are highly cytotoxic effectors, represent the largest percentage of circulating NKs, while being detectable only in selected peripheral tissues such as lung and breast (1). Notably, upon appropriate stimulation, CD56 dim cells increase the cytolytic potential, become cytokines producers, and also acquire the capability to migrate to SLO (5,6).
The NK cells distribution in physiological and pathological conditions is dictated by the expression of chemokine receptors that retain or drive the migration toward tissues of one or another NK cell subset. Peripheral blood (resting) CD56 dim and CD56 bright NK cells share the expression of CXCR4 (CXCL12 receptor) (7) and CXCR3 (CXCL4, 9, 10, 11 receptor). CD56 bright NKs selectively express CCR5 (CCL3-5 receptor) and CCR7 that guides their migration toward tissues with high levels of CCL19 and CCL21, such as SLO. Conversely, the expression of CXCR1 (CXCL8 receptor), CXCR2 (CXCL1, 2, 3, 5, 8 receptor), and CX3CR1 (CX3CL1 receptor) is restricted to CD56 dim NKs (4), which are also characterized by the peculiar expression of the ChemR23 specific for chemerin (8).
CX3CL1, also termed fractalkine, is the only known member of the CX3C chemokine family. It is synthesized as a membrane anchored molecule expressed by cells such as endothelial cells, epithelial cells, osteoblasts, and mesenchymal stromal cells. CX3CL1 can be shed by A Disintegrin And Metalloproteinases (ADAM) 10 or 17 in non-inflammatory and inflammatory conditions, respectively (9). Once released, it interacts with the specific receptor (CX3CR1), a molecule belonging to the 7-transmembrane G protein-coupled receptor family, which is expressed by different immune cell types, including monocytes, dendritic cells (DCs), T, and NK lymphocytes.
Tumor tissues set up different strategies to escape immune recognition and in particular to affect chemokine/chemokine receptor axes such as CX3CL1/CX3CR1, which are crucial for the recruitment of cells such as CD56 dim NK cells exerting tumor suppressive properties. A possible strategy is to unbalance in the tumor microenvironment the equilibrium between CD56 dim and CD56 bright attracting chemokines. Immature, poor cytolytic CD56 bright cells represent the major NK subset present in most tumor tissues (4,10), and breast carcinomas have been shown to decrease the expression of CXCL2 and CX3CL1 while increasing that of CCL5 and CCL19 (1). Accordingly, CX3CL1 expression in breast carcinoma specimens was shown to correlate with a good prognosis (11).
Another possible mechanism exerted by tumors for limiting immune surveillance is represented by their capability of modifying the chemokine receptor repertoire of crucial effector cells, thus affecting their responsiveness to given chemoattractant gradients. Among the soluble mediators involved, TGF-β1 shows a pivotal role in chemokine receptors modulation (12). TGF-β1 is pleiotropic cytokine whose major physiologic role is to limit the duration of immune responses and promote tissue repair (13,14). In the tumor microenvironment, its release and activation by cancer cells and immune cells, including tumor-associated macrophages (TAM), is exacerbated (9,15). This results in tumor-promoting side effects including angiogenesis and suppression of anticancer immunity. In NK cells, tumor-derived and recombinant TGF-β1 has been shown to downregulate the expression of NKp30 and NKG2D activating receptors involved in tumor recognition and DC editing (16,17) and to modulate the chemokine receptor repertoire. In particular, TGF-β1 increases the expression of CXCR3 and CXCR4 (12,18), involved in peripheral tissues and bone marrow (BM) recruitment, respectively, and decreases that of CX3CR1, which drives effector cells toward peripheral tissues, including central nervous system (CNS) (19)(20)(21). Interestingly, in glioma cells TGF-β1 has also been shown to downregulate CX3CL1 (22). The presence of CD56 dim NK cells, characterized by low expression of CX3CR1, has been detected in ascitic fluids of ovarian carcinoma patients (10). Moreover, a possible systemic effect has been demonstrated in patients with high-risk (stage 4 or M) neuroblastoma (NB), who were characterized by the presence of unconventional CD56 dim CX3CR1 low NKs, both in metastatic sites, such as BM and peripheral blood (12).
Understanding how TGF-β1 impairs the expression of CX3CR1 in CD56 dim NK has potential clinical implications, since it could improve the current knowledge of the mechanisms that regulate the immune responses and, in cancer, participate in the escape from immune surveillance. MicroRNAs (miRNAs), small non-coding RNA molecules, are key players in the regulation of gene expression and modulate several biological processes, including immune responses. In this study, we performed a profile of NKs to identify miRNA that could play roles in TGF-β1-mediated modulation of chemokine receptors expression. We identified miR-27a-5p as a negative regulator of CX3CR1 expression.

MaTerials anD MeThODs nK cells Purification
NK cells were purified from peripheral blood mononuclear cells of healthy donors using the Human NK Cell Isolation kit (Miltenyi Biotec) (12). A total of 13 unrelated healthy donors were used for all the experiments described. Donors provided an informed consent according to the procedures approved by the Ethics Committee of Ospedale Policlinico San Martino (39/2012).

nK cell Treatments for mirna and mrna expression analysis
To obtain miRNA profiling, NK cells were incubated for 24 h in the presence of complete medium (RPMI 1640 with 10% of fetal bovine serum, 2 mM glutamine, 50 mg/mL penicillin and 50 mg/mL streptomycin) supplemented with TGF-β1 at the final concentration of 40 or 5 ng/mL. Control cells were represented by NK cells cultured in the presence of complete medium alone (12). For miRNA validation and mRNA expression analysis, NK cells were incubated in the presence of 40 or 5 ng/mL of TGF-β1 or complete medium for 12 or 24 h.

rna isolation
RNA containing the small RNA fraction was extracted using the miRCURY RNA Isolation Kit-Cell and Plant (Exiqon) according to the manufacturer guidelines. RNA amount was determined using the Quant-iT RiboGreen RNA Assay Kit (Invitrogen) following to the manufacturer instructions.  (24), and miRmap 5 (25), selected miRNAs were investigated as possible regulators of CXCR4, CXCR3, and CX3CR1 expression.

expression of mirnas
Expression of selected miRNAs was evaluated using TaqMan MicroRNA Assays as described by the manufacturer (Applied Biosystems). Briefly, 10 ng of RNA were reverse transcribed using the TaqMan MicroRNA Reverse Transcription kit primed with the specific RT primers. Real-time PCR was performed in quadruplicate using the specific primers. Expression of each miRNA was normalized to the RNU44 expression.

expression of cX 3 cr1 mrna
To evaluate the expression of CX3CR1 mRNAs, 100 ng of RNA were reverse transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). The cDNA was used for real-time PCR using the specific primers contained in the TaqMan Gene Expression Assay (Applied Biosystems). CX3CR1 expression was normalized to the GAPDH expression. Experiments were performed in quadruplicate.

cX 3 cr1 3′ Untranslated region construct
A 700 bp fragment of the CX3CR1 3′ untraslated region (3′UTR) containing the putative target site for miR-27a-5p was amplified by PCR using primers CX3UTR2F and CX3UTR2R, containing the NheI and XhoI sites in their 5′ ends, respectively. The PCR product was purified using the MinElute PCR Purification Kit (Qiagen), NheI-XhoI digested and ligated to the NheI-XhoI digested pmirGLO vector (Promega) using the Rapid DNA Ligation kit (Roche, Basel, Switzerland). Ligation product was used for transformation of TOP10 Escherichia coli competent cells (Invitrogen). Colonies containing the recombinant plasmid were selected by PCR using primers CX3UTR2F/CX3UTR2R. Plasmid DNA was purified using the Illustra PlasmidPrep Mini Spin Kit (GE Healthcare). The inserted fragment and flanking sequences were sequenced using primers CX3UTR2F, CX3UTR2R, CX3UTR22R2, and CX3UTR22F2. Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) in a 3100 Genetic Analyzer (Applied Biosystems). The selected plasmid is reported as pmirCX-3UTRWT. Primers, when not otherwise indicated, were designed by primerBLAST. 6 Primer sequences are reported in Figure S1 in Supplementary Material.

site-Directed Mutagenesis
A mutated version of pmirCX3UTRWT (pmirCX3UTRMT), containing a C>G mutation in the putative target site for miR-27a-5p, was prepared by site-directed mutagenesis using the Geneart Site-Directed Mutagenesis System (Invitrogen) according to the manufacturer guidelines. Oligonucleotides (CX3MUTF and CX3MUTR) were designed using the QuickChange Primer Design on-line tool (Agilent Technologies 7 ). Presence of the mutation was confirmed by restriction analysis and by sequencing the mutagenized region using primers CX3UTR2FS and CX3UTR22R2.
luciferase reporter assay

cX3cr1-lentiviral Vector generation
Primer CX3ATGKZF, designed to contain a Kozak consensus sequence, and primer CX3UTR2R ( Figure S1 in Supplementary Material) were used for the amplification of a genomic fragment (2,494 bp in length) containing the whole CX3CR1 gene open reading frame (ORF) and a portion of the 3′UTR in which the two putative target sites for miR-27a-5p are included. The product was cloned in a pcDNA 3.1/V5-His-TOPO vector (Invitrogen), excised by XbaI-BamHI digestion, and ligated to an XbaI-BamHI digested pCDH-CMV-MCS-EF1-GFP lentiviral vector (System Biosciences). Ligation product was transformed in Stbl3 E. coli cells (Invitrogen), a positive colony was selected by PCR, endotoxinfree plasmid DNA was extracted using the QIAfilter Plasmid Midi Kit with the EndoFree Plasmid Buffer Set (Qiagen), the whole insert and the flanking vector regions were sequenced using 16 primers (whose sequences are reported in Figure S1 in Supplementary Material).

Virus Packaging
The lentiviral vector containing the CX3CR1 gene was assembled in lentiviral particles using the pPACKH1 HIV Lentivector Packaging Kit (System Biosciences). Briefly, 3 × 10 6 293T cells plated in a 10 cm plate were tranfected with the packaging plasmids and the CX3CR1 lentiviral vector with lipofectamine 2000 (Invitrogen). Medium was changed every 24 h and collected at 48 and 72 h. Virus containing supernatants were pooled and concentrated by the use of a PEG precipitation solution. Titration was performed by flow cytometric detection of GFP fluorescence emitted by infected cells.

heK293T cells Transduction
HEK293T cells (2 × 10 5 ) were plated in six-well plates and infected with viral preparation at a multiplicity of infection of 2 in 500 µL complete medium without antibiotics in the presence of 8 µg/mL of polybrene (Santa Cruz Biotechnology, Dallas, TX, USA). Infected cells were centrifuged for 1 h at room temperature at 2,000 rpm, then incubated at 37°C. 24 h after infection, medium was removed and replaced with 4 mL of fresh medium without polybrene. One week after infection, CX3CR1 positive cells were selected using the anti-CX3CR1 Microbead Kit (Miltenyi Biotec). Clones generated by limited dilution of the selected cells were analyzed for CX3CR1 surface expression by flow cytometry.
Clones showing a good and stable expression of CX3CR1 were expanded and used for transfection with miR-27a-5p and with miR-Neg.

Transfection of cX 3 cr1-expressing heK293T cells
An HEK293T clone stably expressing CX3CR1 was transfected with miR-27a-5p and with miR-Neg to evaluate the modulation of CX3CR1 expression. The 10 5 cells were plated in six-well plates in 2 mL complete medium, transfection was performed using 100 pmol of miRNA mimics and 3 μL of Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer guidelines. Cells were harvested 48 and 72 h after transfection. CX3CR1 expression was analyzed by flow cytometry.

statistical analysis
The statistical level of significance (p) is indicated using Student's t-test, a parametric significance test. Graphic representation and statistical analysis were performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). Pearson's correlation coefficient (two-tailed test) was used to evaluate the correlation between CX3CR1 mRNA and miR-27a-5p expression.

resUlTs mirna Profiling of TgF-β1-Treated nK cells
To identify miRNAs possibly involved in the TGF-β1-mediated modulation of chemokine receptors expression, we investigated the miRNA expression profile variation in TGF-β1-treated NK cells purified from the peripheral blood of three unrelated healthy donors. The analysis, allowing the simultaneous profiling of 754 miRNAs (Figure 1), showed that miRNAs up-or downregulated by the treatment ranged from 127 in donor 2 to 234 in donor 3 ( Figure S2 in Supplementary Material). A total of 14 miRNAs were modulated in NK cells from all donors analyzed upon treatment with either 5 or 40 ng/mL of TGF-β1. Among these 14 miRNAs, 11 were excluded from further analysis being expressed at low levels (threshold cycle <33), while 1 (miR-1201)   1, 2, and 3). Expression relative fold changes are referred to the expression of untreated cells, whose miR-27a-5p expression has been arbitrarily assigned the value 1. **p < 0.01, ***p < 0.001, and ****p < 0.0001. was forsaken due to its sequence overlapping to an annotated small nucleolar RNAs (snoRNA, SNORD126), as reported in miRBase. 8 The remaining two miRNAs (miR-302a and miR-27a-5p) were further investigated as putative regulators of CXCR4, CXCR3, or CX3CR1 expression (Figure 1 and Figure S2 in Supplementary Material).
The differential expression of miR-302a and miR-27a-5p in untreated and TGF-β1-treated NK cells was checked for validation by using specific miRNA assays. The analysis revealed that miR-302a was virtually absent in both untreated and TGF-β1treated NK cells (data not shown). On the contrary, in agreement with data obtained by miRNA profiling, TGF-β1-treated NK cells showed a significant increase of miR-27a-5p expression, which was higher at low TGF-β1 concentration (Figure 2). Thus, miR-27a-5p was further investigated as putative regulator of CXCR4, CXCR3, or CX3CR1 expression using the computational prediction on-line tools TargetScan (see text footnote 3) (23), miRanda (see text footnote 4) (24), and miRmap (see text footnote 5) (25). The analysis indicated miR-27a-5p as a putative regulator of CX3CR1 expression with relatively high scores. The three softwares predicted the same principal site of interaction in the CX3CR1 3′UTR with the miR-27a-5p seed region ( Figure  S3A in Supplementary Material). An additional site in the CX3CR1 3′UTR, with a weaker interaction with miR-27a-5p, was predicted by miRanda and miRmap tools only ( Figure S3B in Supplementary Material).
inverse correlation of mir-27a-5p and cX 3 cr1 mrna expression in TgF-β1-Treated nK cells Since miRNAs properties include the capability of inducing degradation of targeted mRNAs, expression profiles of miR-27a-5p and CX3CR1 mRNAs were simultaneously analyzed over time. As shown in Figure 3A, at both concentrations used, TGF-β1 caused a significant increment of miR-27a-5p and decrease of CX3CR1 mRNA expression. Importantly, expression of miR-27a-5p and CX3CR1 mRNA was inversely correlated (Pearson's correlation coefficient r = 0.766, p < 0.05; Figure 3B). While preliminary data showed that 1 ng/mL of TGF-β1 may be sufficient for induction of miR-27a-5p, concentrations of 5 and 40 ng/mL were chosen for a more systematic investigation. Next, to deepen the molecular mechanism responsible for the TGF-β1-induced increase of miR-27a-5p, we analyzed the expression of the miR-23a-27a-24-2 cluster, precursor of miRNAs, including miR-27a-5p. As shown in Figure 3C, TGF-β1 caused a significant increment of the primary miR-23a-27a-24-2 transcript, thus demonstrating that the increased amount of miR-27a-5p might be due, at least in part, to induction of its gene expression other than, for example, to miR-27a-5p egress from intracellular stores.

mir-27a-5p interacts with the cX 3 cr1 mrna
To unequivocally demonstrate that miR-27a-5p could interact with the 3′UTR of the CX3CR1 mRNA, modulating its expression, we performed a luciferase reporter assay (Figure 4). We cloned a 700 bp fragment of the CX3CR1 gene 3′UTR, containing all the putative sites of interaction with the seed region of miR-27a-5p, into a luciferase reporter vector, downstream of the firefly luciferase gene. Moreover, we also produced a mutated version of the construct, containing a C>G substitution in the main site of the CX3CR1 3′UTR ( Figure S3A in Supplementary Material). The two plasmids were separately cotransfected in HEK293T cells with a miR-27a-5p mimic or with a random sequence miRNA negative control. As shown in Figure 4A, a significant reduction of the normalized luciferase activity was detected in the presence of miR-27a-5p mimic compared to the miRNA negative control. Importantly, this effect was lost when the miR-27a-5p mimic was cotransfected with the construct having the mutated sequence of the CX3CR1 3′UTR, thus confirming the interaction of miR-27a-5p with the CX3CR1 3′UTR. Next, to further confirm miR-27a-5p and CX3CR1 mRNA interaction, before TGF-β1 treatment NK cells were transiently transfected with a specific miR-27a-5p inhibitor or with a scrambled miRNA as negative control ( Figure 4B). As expected, we observed a significantly higher CX3CR1 mRNA expression in TGF-β1-treated NK cells previously transfected with the miR-27a-5p inhibitor when compared with cells transfected with a scrambled miRNA.

mir-27a-5p Downregulates the surface expression of cX 3 cr1
To determine whether miR-27a-5p is able to downregulate the surface expression of CX3CR1, we prepared a lentiviral FigUre 4 | Functional interaction between miR-27a-5p and CX3CR1 mRNA. (a) Luciferase activity in HEK293T cells cotransfected with luciferase reporter construct containing the 3′ untranslated region (3′UTR) CX3CR1 and miR-27a-5p mimics. Firefly luciferase activity was normalized to the renilla luciferase activity, expressed by the same vector. Data in quadruplicate are from one experiment of three performed. Luciferase activities are referred to the activity of cells transfected with the miRNA negative control whose expression has been arbitrarily assigned the value 1. pmirGLO, parental vector; pmirCX3CR1UTRWT, pmirGLO vector containing 700 bp of the CX3CR1 3′UTR downstream of the firefly luciferase gene; pmirCX3CR1UTRMT, mutated version of pmirCX3CR1UTRWT containing the C>G mutation in the CX3CR1 3′UTR target site; miR-Neg, miRNA mimic negative control. **p < 0.01; ns, not significant. (B) CX3CR1 mRNA expression in NK cells transfected with miR-27a-5p inhibitor (Inh) or with a negative control miRNA Inh. Cells were cultured for 72 h in the presence of 5 ng/mL of TGF-β1. Data in triplicate are from one representative healthy donor of three analyzed (donors 8, 9, and 10). GAPDH has been used as reference control. Expression relative fold changes are referred to CX3CR1 mRNA expression in NK cells transfected with the negative control miRNA inhibitor, whom expression has been arbitrarily assigned the value 1. *p < 0.05. vector containing the whole ORF of the CX3CR1 gene and the portion of the 3′UTR region containing the two putative binding sites for miR-27a-5p. HEK293T cells where transduced with the vector and cultured under limiting dilution to obtain CX3CR1 positive clones. A clone (#124) was selected that stably expressed at the cell surface optimal levels of the chemokine receptor. Clone #124 was transfected with a miR-27a-5p mimic or with a miRNA negative control and analyzed by flow cytometry for the chemokine receptor expression. As shown in Figure 5, miR-27a-5p mimic induced a significant downregulation of the percentage of CX3CR1 positive cells. On the contrary, the expression of other molecules such as PVR (CD155) was unaffected ( Figure S4 in Supplementary Material). These data further support the role of miR-27a-5p as modulator of the chemokine receptor.

mir-27a-5p induction in the context of nB-nK cocultures
To analyze the relevance of miR-27a-5p induction in a pathological context, we cocultured under transwell condition resting NK cells and the prototypic SH-SY5Y NB cell line, which we described to induce a TGF-β1-mediated downregulation of CX3CR1 surface expression (12). Accordingly with our previous published data (12), NB conditioning resulted in a significant downregulation of CX3CR1 expression at both protein and mRNA level. Importantly, this modulatory effect matched with a significant increase of miR-27a-5p as compared to unconditioned NK cells (Figure 6).

DiscUssiOn
A previous study by our group reported that TGF-β1 released by NB cells is able to modify the chemokine receptor repertoire of NK cells (12). In particular, it downregulates CX3CR1 expression in resting NK cells. In the present article, we show that TGF-β1-induced miR-27a-5p directly modulates the expression of CX3CR1 mRNA at the post-transcriptional level. Increment of miR-27a-5p expression is due to upregulation of the miR-23a-27a-24-2 cluster, which contains, among others, miR-27a-5p. Importantly, we observed that this regulatory mechanism also occurs in NK cells during NB-conditioning. All our results were obtained using unfractioned blood NK cells from healthy donors, mostly represented (more than 90%) by the CD56 dim population (7,26). Since CD56 bright NK cells virtually do not express CX3CR1, further experiments should clarify whether miR-27a-5p might contribute to their costitutive CX3CR1 low/neg phenotype.
The downregulation of CX3CR1 has important consequences on NK cells function, as CX3CR1, with other chemokine receptors, drives, at steady state, NK cell localization in peripheral tissues as well as their migration under inflammatory conditions (27). In particular, it has been shown that the relative expression of CX3CR1 and CXCR4 regulates NK cell migration within the BM and their egress from it (28,29). Moreover, in a model of experimental autoimmune encephalomyelitis (EAE), CX3CR1/ fractalkine axis specifically recruits NK cells into the brain (30). Indeed in CX3CR1-deficient mice with EAE the amount of NK cells in the inflamed CNS cells was markedly reduced, as compared to wild-type mice, whereas that of other cell types, including T, NKT cells, and monocyte/macrophages, did not show significant variation (30). Importantly, recruitment of CX3CR1+ NK cells toward the inflamed CNS ameliorated the EAE disease severity, since mature NK cells showed a higher cytolytic activity against autoreactive CD4+ T cells (21).
The functional relevance of CX3CR1 in NK cells tropism and activity is further underlined by the identification of viruses producing viral chemokines able to bind CX3CR1: vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, which inhibits naive migration of CD56 dim NK cells blocking the binding of CX3CL1 (33), and vCXCL1, encoded by human cytomegalovirus, which induces NK and neutrophils migration, presumably to facilitate a neutrophil-mediated viral dissemination (34).
CX3CR1 expression also has a pivotal role in tumors, conditioning migration and adhesion of tumor cells, and tumor invasiveness and metastasis (35). In particular, CX3CR1 expression in pancreatic and prostate cancer cells increases invasiveness and metastasis to neuronal tissues (36). Interestingly, however, while CX3CR1 expression in cancer is generally associated with invasiveness and metastasis, the coexpression of CX3CR1 and CX3CL1 by the same cell type, as occurs in human colorectal cancer cells, acts as a retention factor, limiting tumor spreading to metastatic sites (37).
Due to the wide properties of CX3CR1, we can argue that the receptor downregulation caused by TGF-β1 in mature cytolytic NK cells might severely hamper their recruitment and functions at tumor sites. This might occur thanks the TGF-β1-induced upregulation of the miR-23a-27a-24-2 cluster in NK cells, which in turn causes miR-27a-5p upregulation and the consequent CX3CR1 downregulation. It is likely that a tight control of the miR-23a-27a-24-2 cluster expression is necessary, given that its deregulation has been reported in several tumors and other diseases (38). Interestingly, TGF-β1 has been shown to be responsible for upregulation of the miR-23a-27a-24-2 cluster in hepatocellular carcinoma cells (39), in lung adenocarcinoma cells (40) as well as in CD8 T cells (41). In these lymphocytes, additionally, upregulation of the cluster has been associated with inhibition of IFN-γ expression and reduced cytotoxicity (41). miR-27a-5p has been also reported to downregulate perforine 1 (Prf1) and granzyme B (GzmB) expression in resting and IL-15-activated NK cells (42), thus hampering NK cells cytotoxicity. NK92 and primary NK cells overexpressing miR-27a-5p showed a reduced cytotoxicity but unmodified levels of the activating receptors NKG2D, NKp30, NKp44, and NKp46. Moreover, knockdown of miR-27a-5p in NK cells increased in vitro cytotoxicity and decreased tumor growth in a human tumor xenograft model (42). Overall, these data suggest that miR-23a-27a-24-2 cluster could be an important target of TGF-β1, possibly acting as an intermediate inducer of its effects. miR-27a-5p, a product of this cluster, is able to regulate the expression of multiple targets crucial for NK cells function. Thus, it appears as a key node for NK activity control, being able to dampen NK cell recruitment, cytotoxicity, and IFN-γ production, potentially representing, in perspective, an interesting cancer therapeutic target. We apologize the colleagues whose work we could not cite because of space constraints.