Declined miR‐181a‐5p expression is associated with impaired natural killer cell development and function with aging

Abstract MicroRNAs (miRNAs) regulate gene expression and thereby influence cell development and function. Numerous studies have shown the significant roles of miRNAs in regulating immune cells including natural killer (NK) cells. However, little is known about the role of miRNAs in NK cells with aging. We previously demonstrated that the aged C57BL/6 mice have significantly decreased proportion of mature (CD27−CD11b+) NK cells compared with young mice, indicating impaired maturation of NK cells with aging. Here, we performed deep sequencing of CD27+ NK cells from young and aged mice. Profiling of the miRNome (global miRNA expression levels) revealed that 49 miRNAs displayed a twofold or greater difference in expression between young and aged NK cells. Among these, 30 miRNAs were upregulated and 19 miRNAs were downregulated in the aged NK cells. We found that the expression level of miR‐l8la‐5p was increased with the maturation of NK cells, and significantly decreased in NK cells from the aged mice. Knockdown of miR‐181a‐5p inhibited NK cell development in vitro and in vivo. Furthermore, miR‐181a‐5p is highly conserved in mice and human. MiR‐181a‐5p promoted the production of IFN‐γ and cytotoxicity in stimulated NK cells from both mice and human. Importantly, miR‐181a‐5p level markedly decreased in NK cells from PBMC of elderly people. Thus, our results demonstrated that the miRNAs profiles in NK cells change with aging, the decreased level of miR‐181a‐5p contributes to the defective NK cell development and function with aging. This opens new strategies to preserve or restore NK cell function in the elderly.


| INTRODUC TI ON
MicroRNAs are a class of short (~22nt), endogenously initiated noncoding RNAs, which post-transcriptionally control gene expression via either translational repression or mRNA degradation (Cai et al., 2009). Each miRNA may suppress multiple mRNA targets, while one mRNA can be targeted by several miRNAs for precise control of multiple cellular processes, such as cell differentiation, proliferation, apoptosis, tumorigenesis, and host-pathogen interactions (Huang et al., 2011). MicroRNAs are crucial post-transcriptional regulators of the immune system (Contreras & Rao, 2012), by negatively regulating the expression levels of important genes to significantly influence the development and function of the immune cells (Mehta & Baltimore, 2016).
Natural killer (NK) cells are crucial innate effector cells serving as the first line of defense against certain infectious pathogens and tumors due to their ability to rapidly release inflammatory cytokines and kill infected or transformed cells (Vivier et al., 2011).
Our previous studies demonstrated that the proportion of R3 NK cells are significantly decreased in aged mice, indicating defective NK cell development with aging (Fang et al., 2010). The impaired NK cell development results in reduced effector functions of NK cells and susceptibility to poxvirus or influenza A virus infection in the aged mice (Beli et al., 2011;Fang et al., 2010). Previous studies have demonstrated that aging differentially affects NK cell subsets and NK cell function (Manser & Uhrberg, 2016). However, whether miR-NAs are involved in this process is currently unknown.
In this study, we investigated the miRNA profiles of R1 and R2 NK cells from young and aged mice and identified differentially expressed miRNAs between young and aged NK cells.

| Differential miRNA expression profiles of R1 and R2 NK cells between young and aged mice
Our previous studies showed impaired NK cell development in mice with aging (Fang et al., 2010). We reproduced this results in the current study. As shown in Figure S1, compared with the young mice, the percent of R2 and R3 NK cells were dramatically decreased in the spleen, LN and BM of aged mice. On the contrary, the percent of R1 NK cells was increased in the spleen, LN, and BM of aged mice, while there was no significant difference in the percent of R0 NK cells between the young and aged mice. Thus, compared with the young mice, the maturation of NK cells in the aged mice was severely impaired.
To study the mechanisms of the impaired NK cell maturation in the aged mice, we sorted the R1 and R2 NK cells (CD27 + NK cells) from the BM of young and aged mice. Deep sequencing revealed the miRNA profiling of CD27 + NK cells in the young and aged mice. Differential expression analysis was performed to identify differentially expressed miRNAs between young and aged mice. MicroRNAs that displayed a twofold or greater difference in expression level between young and aged mice were considered as differentially expressed, and lower values were not considered. A total of 49 miR-NAs (from known miRNAs) displayed a twofold or greater difference in expression level. Among these, 30 miRNAs were upregulated and 19 miRNAs were downregulated in the aged NK cells compared with the young NK cells (Figure 1a,b). Those 49 miRNAs were further analyzed in heat map, 10 miRNAs were found to be highly consistent in three individual repeats and significantly different between young and aged NK cells (Figure 1b). Thus, we chose the 10 miRNAs as the candidate miRNAs.
To further confirm the functions of potential targets regulated by the miRNAs, we performed mRNA profile analysis of the CD27 + NK cells from the young and aged mice. mRNAs that displayed a twofold or greater difference in expression level between young and aged NK cells were considered as differentially expressed. We F I G U R E 1 Differentially expressed miRNAs in CD27 + NK cells from young and aged mice. (a) The MA plot of differentially expressed miRNAs in CD27 + NK cells from young mice and aged mice. Each dot represents a differentially expressed miRNA in aged mice. The log2 fold change of each miRNA was calculated by the mean of normalized counts. P: p-value; FC: fold change. (b) The heatmap of differentially expressed miRNAs profiled in CD27 + NK cells from the young mice (Y1-Y3 represent three individual repeats), and the aged mice (A1-A3 represent three individual repeats). The color scale shown on the left illustrates the relative expression levels of the miRNAs. The upregulated miRNAs are depicted in red color whereas the downregulated miRNAs are depicted in blue color conducted the miRNA-target network analysis by combining the predicated target mRNAs and the differentially expressed mRNAs.

| MiR-181a-5p and miR-223-3p both decline in the aged NK cells
Previous studies have shown that miR-181 and miR-223 are involved in regulating the development and function of NK cells (Leong et al., 2014). miR-181a-5p is one of the most well-studied members of the miR-181 family, which include four highly conserved mature miR-NAs: miR-181a, b, c and d (Cichocki et al., 2011;Li et al., 2015). In this study, we used qRT-PCR to verify the expression of miR-181a-5p and miR-223-3p in CD27 + NK cells sorted from the BM of the young and aged mice. Significantly decreased expressions of miR-181a-5p and miR-223-3p were found in the aged NK cells (Figure 3a), which were consistent with the results of RNA sequencing. To further determine the miRNAs levels during the maturation of NK cells from R0 to R3, qRT-PCR was performed in the sorted R0, R1, R2, and R3 NK cells from mouse spleens. As shown in Figure 3b, with the maturation of NK cells, the transcript of miR-l8la-5p increased, with the highest expression level in R3 NK cells. The highest expression level of miR-223-5p was also in the R3 NK cells. Our study demonstrated that the expression levels of mi-181-5p and miR-223-5p in NK cells both declined in the aged mice, and the highest expression level of miR-l8la-5p and miR-223-5p were both in the terminally matured R3 NK cells.

| miR-181a-5p promotes the maturation of NK cells in vitro and in vivo
To study whether the above changed miRNAs affect the development and maturation of NK cells, we chose miR-181a-5p for further research. We established a system to imitate the in vitro development of NK cells on the basis of the study by Yin et al. (2015). We used FAM labeled-miR-181a-5p antagomir to knock down miR-181a-5p, which can easily transfect cells. An antagomir from Caenorhabditis elegans (cel-miR-67-3p) was used as a negative control (NC). The hemopoietic stem cells (HSC) isolated from BM of young mice were treated with miR-181a-5p antagomir or NC antagomir and cultured for 12 days as schematized in Figure 3c. Then, we tested the development and maturation of NK cells. We found that transfection of NC antagomir had no effect for the maturation of NK cells. However, the R1 NK cells were markedly increased, while R3 NK cells were significantly decreased in the miR-181a-5p antagomir treated group (FAM + ) compared with the NC antagomir treated group (FAM + ), respectively ( Figure 3d). Thus, NK cell development in vitro was impaired with knockdown of miR-181a-5p.
To further confirm the role of miR-181a-5p in the maturation of NK cells, we set up the in vivo system using the RAG1 −/− γc − mice.
HSCs isolated from BM of young mice were treated with antagomir for 48 h, then transferred into the RAG1 −/− γc − mice to allow further development in vivo for another 7 days ( Figure 4a). We analyzed the percentages of R0, R1, R2, and R3 NK cells in lung, liver, spleen, and BM of the recipient mice. Compared with the control, miR-181a-5p antagomir treated group exhibited a significant reduction of R3 NK cells in the lung, spleen, liver, and bone marrow. Meanwhile, the proportion of R1 NK cells in lung, liver, spleen, and BM were increased accordingly ( Figure 4b). These results were resembled with the NK cell development in the aged mice ( Figure S1), in which the miR-181a-5p level was declined. These results further demonstrated that the reduced miR-181a-5p inhibited the NK cell development and maturation in vivo, and the critical requirement for miR-181a-5p in NK cell development was cell intrinsic.

| NLK and BCL2 are targets of miR-181a-5p in NK cells
The miRNAs control gene expression via either translational repression or mRNA degradation. To study the molecular mechanisms, we further investigated the target genes of miR-181a-5p in regulating the maturation of NK cells in aged mice. We used Targetscan database and miRDB database to predict potential targets for miR-181a-5p.
674 potential targets were found for miR-181a-5p. We searched this list for genes with potential relevance to NK cell development and identified three target genes (BCL2, NLK, and MAPK1) that had been reported to be involved in NK cells development and function (Table 1). We verified the mRNA levels of these three genes in R1 and R2 NK cells of BM from both young and aged mice. As shown F I G U R E 2 Potential targets and function analysis of the 10 candidate miRNAs. (a) The function of miRNA was analyzed based on the target gene information by KEGG pathway enrichment analysis. The abscissa represents miRNA, and the ordinate represents enriched function. Nodes represent enrichment extent. (b) Interactive network diagram of the four upregulated miRNAs in CD27 + NK cells from aged mice and their downregulated potential target genes. (c) Interactive network diagram of the five downregulated miRNAs in CD27 + NK from aged mice and their upregulated potential target genes. Rhombic dots represent the differentially expressed miRNAs. Round dots represent the differentially expressed target genes  in Figure 5a, the transcripts of all the three target genes were significantly increased in aged mice (Figure 5a), which were concordant with the reduced expression of miR-181a-5p in aged mice (Figure 3a).
To further confirm that BCL2, NLK, and MAPK1 are the target genes of miR-181a-5p, we knocked down miR-181a-5p in 293 T cells  Figure 5f, the expression level of both NLK and BCL2 were significantly higher in the R1 and R2 NK cells from aged mice than that of young mice. Therefore, both the mRNA and protein levels of NLK and BCL2 were inversely related to the miR-181a-5p level in R1 and R2 NK cells. Our data supported a role for miR-181a-5p in the negative regulation of NLK and BCL2 expression in NK cells.

| miR-181a-5p regulates the function of NK cells
Previous studies have shown that miR-181 regulates the secretion of IFNγ in NK cells (Leong et al., 2014). To further study the effect of miR181a-5p on NK cell function, we treated NK cells from mouse spleen with miR-181a-5p antagomir or the NC antagomir, YAC-1 cells were then added to the cell culture to stimulate NK cells. As shown in  (Figure 3a). In summary, our data indicated that miR-181a-5p was also important for the function of NK cells in mice and human.

NK cells from elderly people
So far, our data have demonstrated that miR-181a-5p is important for the development and function of NK cells, and the expression level F I G U R E 3 The declined expression of miR-181a-5p influences the maturation of NK cells in aged mice. (a) The expression levels of miR-181a-5p (left) and miR-223-3p (right) in CD 27 + NK cells of young or aged mice were detected using qRT-PCR. The cells were sorted from bone marrow of the young and aged mice by using a FACSAria II (BD Biosciences) with purities >98%. (b) The expression levels of miR-181a-5p (left) and miR-223-3p (right) in different NK cell subsets of young mice. The cells were sorted from spleen, peripheral lymph nodes and bone marrow of the young mice. (c) Schematic of in vitro mouse NK cell differentiation using a two-step cell culture system as described in Materials and Methods. HSCs purified from bone marrow of young CD45.1 mice were transfected with FAM-labeled miR-181a-5p antagomir or FAM-labeled NC antagomir. (d) Flow cytometry (above) and statistical results (below) of R0, R1, R2 and R3 NK cells in (c). Data were from three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001 BM of miR-181a-5p decreases in mice NK cells with aging. To further test whether the expression level of miR-181a-5p also declines in human NK cells with aging, we isolated NK cells from PBMC of young and elderly people. Consistent with the observation in mouse, we observed a 75% reduction in the transcript levels of miR-181a-5p in NK cells from the elderly people compared with the young (Figure 6f).
Thus, our results indicate that the declined miR-181a-5p expression is associated with impaired NK cell function with aging, both in mice and human.
Among the 10 candidate miRNAs in Figure 1b, four miRNAs (miR-223-3p, miR-126a-5p, miR-151-3p, and miR-27b-5p) were also highly conserved between human and mouse. Among them, we chose miR-223-3p and miR-126a-5p to investigate the levels of those miRNAs in human NK cells with aging. As shown in Figure 6f, the expression of miR-223-3p and miR-126a-5p were all decreased in the NK cells from the elderly people compared with the young, which was consistent with the results from mice. Thus, multiple miRNAs levels change in NK cells with aging, both in mice and human. However, whether each individual miRNA plays any role in the defective development and function of aged NK cells awaits further investigations.

| DISCUSS ION
NK cells are important immune cells, playing an important role in defense against infections and tumors. Thus, altered NK cell functionality with physiological aging has critical impact on overall immunity (Camous et al., 2012). This is of particular relevance because the elderly is more susceptible to infection, cancer, and other diseases.
The contribution of the immune system to healthy aging and longevity remains largely unknown. A study of 108 immunologically normal elderly subjects indicated that the low NK cell activity is associated with the development of infections and death in the elderly people with impaired performance status (Ogata et al., 2001). Another study about lymphocyte activity in people of different ages found that the cytotoxic activities of NK cells are well preserved in the centenarians, suggesting that the well-preserved NK cell activities likely contribute to their successful aging in centenarians (Sansoni et al., 1993). Therefore, keeping high performances of NK cells might be helpful for achieving healthy longevity in the elderly.
A broad range of miRNAs are known to regulate the development and function of NK cells. However, little is known about the association between miRNAs and the altered NK cell functionality with physiological aging. In this study, based on analysis of miRNomes, we found that the profiles of miRNAs in NK cells changed profoundly in mice with aging. MiR-181a-5p was one of those miRNAs, and was reduced in the aged CD27 + NK cells. By in vitro and in vivo experiments, we demonstrated that knockdown of miR-181a-5p intrinsically impaired the terminal maturation of NK cells. Therefore, the reduced level of miR-181a-5p may play a role in the defective maturation of NK cells with aging.
NK cells develop from BM-derived HSCs and undergo terminal differentiation in both the bone marrow and various tissues, such as thymus, lymph node, and liver, as a means of generating functionally distinct and tissue-specific NK cells. As shown in Figure S1 Combined with mRNA sequencing, we identified that BCL2, NLK, and MAPK1 might be the targets of miR-181a-5p. The transcripts of all the three target genes were significantly increased in aged CD27 + NK cells, which were concordant with the reduced expression of miR-181a-5p. Furthermore, the protein level of BCL2 and NLK significantly increased in both mice spleen NK cells and BM CD27 + NK cells from the aged mice. Thus, our data suggested that reduced expression of miR-181 influenced the terminal maturation of NK cells in the aged mice, partly through upregulation of NLK and BCL2. NLK is an inhibitor of Notch signaling and Notch signaling is important in NK cell development (Bachanova et al., 2009;Cichocki et al., 2011). MiR-181 targets multiple Bcl-2 family members (Ouyang et al., 2012), and BCL2 is required for the survival of NK cells (Viant et al., 2017). How miR-181a-5p influences the maturation of NK cells through BCL2 with the aged NK cells still needs more in-depth research. Meanwhile, miR-181a-5p has diverse functions via distinct target genes in different cell types (Liang & Xu, 2020;Wen et al., 2020;Zhao et al., 2020). In our research, the putative target genes of miR-181a-5p antagomir were also varied in different cell types (293 T cells, NKL cells, or NK cells from spleen of mice).
MiR-181a-5p antagomir treatment dramatically reduced the production of IFNγ in NK cells, indicating that the cellular level of miR-181a-5p has a significant impact in NK cell secretion of IFNγ, which is consistent with previous report (Leong et al., 2014). In addition, miR-181a-5p antagomir treatment also resulted in a slight but statistically significant decrease in NK cell cytotoxic function.
Furthermore, the cytotoxic function of NK cells was also decreased in the aged mice, which resembled the function of miR-181a-5p antagomir ex vivo. Thus, our data suggested that miR-181a-5p also regulates NK cell function, mainly on the production of IFNγ and F I G U R E 4 MiR-181a-5p is important for the maturation of NK cells in vivo. (a) Schematic of in vivo mouse NK cell differentiation using an adoptive transfer system as described in Materials and Methods. (b) Flow cytometry (above) and statistical results (below) of R0, R1, R2, and R3 NK cells in the lung, liver, spleen, and BM of the recipient mice. Data represented three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001 also on the cytotoxicity. However, whether miR-181a-5p regulation of NK cell cytotoxicity has physiological significance awaits further investigations.
In human, NK cells can be divided into two subsets based on the expression level of CD56 and CD16. CD56 dim CD16 + NK cells have high cytotoxic function, and CD56 bri CD16 − NK cells have immune regulation function by producing cytokines (Melsen et al., 2016).
During the development of human NK cells, CD56 bri CD16 − NK cells subsequently differentiate into CD56 dim CD16 + NK cells (Cichocki et al., 2019). A previous study reported that from CD56 bri CD16 − NK cells to CD56 dim CD16 + NK cells, the expression of miR-181a-5p was increased (Pesce et al., 2018), which was in accordance with our data that the terminally matured R3 NK cells in mice had the highest expression level of miR-181a-5p. Many studies reported that the function of NK cells was impaired in aged people (Witkowski et al., 2020).
Our data showed that the expression of miR-181a-5p in NK cells from human PBMC declined with aging, indicating miR-181a-5p may be associated with the function changes of NK cells in the elderly. To our knowledge, this is the first report demonstrating the connection of changes of miRNAs to the developmental and functional defect of NK cells with aging. Our studies suggest that miRNAs might be used as potential treatment targets for regulating the development and function of NK cells with aging, which opens a new avenue for the mechanisms in immunosenescence and treatment for aging-related disease. Furthermore, our researches provide ideas for improving the development and function of NK cells through miRNA regulation to improve healthy longevity for the elderly.

| Ethics statement
The animal protocol used in this study was approved by the

| Cells culture and transfection
As standard tissue culture medium, we used complete RPMI or DMEM which consisted of the indicated tissue culture medium supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/ml strepto-

| Bone marrow NK cell purification and deep sequencing
For RNA sequencing, R1 and R2 NK cells (CD3 − NK1.1 + CD27 + ) in bone marrow (BM) of young B6 mice (six mice per group) or aged B6 mice (six mice per group) were sorted by using a FACSAria II (BD Biosciences) with purities >98%. Three individual experiments were performed to sort CD27 + NK cells. Then, the total RNA was isolated and deep sequencing was performed in the sequencing facility at Beijing Institute of Genomics.

| Human PBMC isolation and NK cells purification
The human peripheral blood mononuclear cells (PBMC) from young (25-35 aged) and elderly people (>75 aged) were isolated with PBMC separation medium (TBD science, LTS1007) according to the manufacturer's instruction. The cells were washed twice with complete RPMI-1640 medium. Then, the CD3 − CD56 + NK cells were sorted using a FACSAria II (BD Biosciences) with purities >98%.

| Quantitative real-time PCR
Total RNA was extracted from sorted NK cells, 293 T cells, and NKL cells with TRIzol (Invitrogen). The miRNA was quantified using a TaqMan miRNA kit (Applied Biosystems) according to the manufacturer's protocols. The endogenous control U6 was used for normalization. The primers used were get from Ruibo. For mRNA, Universal Probe Library probes were purchased from Roche. Primers for mRNA were synthesized at Sangon Biotech. The primers used for mRNA were listed in Table 2. First-strand cDNA was synthesized using oligo-dT primers. qRT-PCR was performed using a LightCycler 480 (Roche). The cycling conditions for RT-PCR were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s. The fold increase in mRNA expression was determined using the ΔΔCt method relative to the values for the mocktreated samples after normalization to glyceraldehyde 3-phosphate dehydrogenase gene expression. (100 ng/ml; R&D systems), and IL-2 (500 U/ml; R&D systems).

| Hematopoietic stem cells isolation, and in vitro culture
After 24 h of culture, HSCs were transfected with 200 nM miR-181a-5p antagomir or NC antagomir in 96-well plate. Three days later, fresh medium containing the above indicated cytokines was used to replace three quarters of the old medium, and continuing culture for another 3 days. Then, the culture medium was switched to complete 1640 medium supplemented with IL-15 (30 ng/ml; R&D systems) and IL-15 receptor α (120 ng/ml; R&D systems) and cells were further cultured for 6 days. Using this two-step stromal cellfree cell culture system, HSC cells developed into mature NK cells ( Figure 4a).

| Isolation of lymphocytes
Lymphocytes from different organs were collected and processed individually. For spleen and BM, single-cell suspensions were obtained by gentle mechanical dissociation in PBS containing 2% FBS.
After hemolysis with 0.84% NH4Cl solution, cells were washed and resuspended in complete RPMI 1640 medium. The isolation of lung and liver lymphocytes was adapted as described previously (Lu et al., 2018). Briefly, the lung and liver were removed and passed through a cell strainer (BD Falcon) to obtain a single-cell suspension. The cells were resuspended in 35% Percoll solution (in D-Hank's buffer) and centrifuged at 830 g for 20 min at room temperature. The upper liquid phase was removed from the tube; the lymphocyte pellet was resuspended in 0.84% NH 4 Cl solution to lyse the red blood cells (RBCs) and then washed twice with PBS containing 2% FBS. Cells were washed and resuspended in complete RPMI medium.

| Flow cytometric analysis
To determine NK cell maturation, up to 2 × 10 6 lymphocytes were stained with surface antibodies at 4°C for 30 min. Detection of NK cell responses was performed as previously described (Lu et al., 2018). Briefly, 2 × 10 6 splenocytes from mice (or NKL cells) were transfected with 200 nM miR-181a-5p antagomir or NC antagomir for 24 h and cultured at 37°C for 48 h in 96-well plates. Then, 4 × 10 5 YAC-1 cells (or K562 cells when indicated) and anti-CD107a antibody were added. After 3 h, brefeldin A was added to block the secretory pathway for the accumulation of IFNγ inside the cells.
After incubation, cells were washed with pre-cold PBS, stained with Annexin V-APC and 7AAD Apoptosis Detection Kit (sungene biotech, ao2001-11a-ha-h) and immediately analyzed by flow cytometer.

| Western blot analysis
Detection of NLK, BCL2, or ERK2 (MAPK1) protein level was performed with rabbit anti-NLK polyclonal antibody, rabbit anti-BCL2 polyclonal antibody or rabbit anti-ERK2 polyclonal antibody (all from Bioworld), respectively. Then, the bound Abs were detected using an HRP-conjugated goat anti-rabbit IgG secondary Ab (zhong shan jin qiao). Densitometric analysis was performed using an ECL Documentation system.

| Statistical analysis
Statistical analysis was performed using Prism software (GraphPad).
All statistical analyses were performed using an unpaired two-tailed Student's t test or two-way ANOVA test as applicable. When applicable, data were displayed as mean ± SEM.

ACK N OWLED G M ENTS
This work was supported by National Natural Science Foundation of China (91749112, 31970164).