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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Eur J Immunol. 2021 Feb 4;51(3):557–566. doi: 10.1002/eji.201948358

Enriched environment enhances NK cell maturation through hypothalamic BDNF in male mice

Anthony G Mansour 1,2,#, Run Xiao 2,3,#, Stephen M Bergin 2, Wei Huang 2,3, Logan A Chrislip 2,3, Jianying Zhang 4, Seemaab Ali 2,3, Nicholas J Queen 2,3, Michael A Caligiuri 1, Lei Cao 2,3
PMCID: PMC8212871  NIHMSID: NIHMS1708274  PMID: 33169371

Abstract

Macroenvironmental factors, including a patient’s physical and social environment, play a role in cancer risk and progression. Our previous preclinical studies have shown that the enriched environment (EE) confers anti-obesity and anti-cancer phenotypes that are associated with enhanced adaptive immunity and are mediated by brain-derived neurotrophic factor (BDNF). Natural killer (NK) cells have anti-cancer and anti-viral properties, and their absence or depletion is associated with inferior clinical outcomes. In this study, we investigated the effects of EE on NK cell maturation following their depletion. Mice living in EE displayed a higher proportion of NK cells in the spleen, bone marrow, and blood, compared to those living in the standard environment (SE). EE enhanced NK cell maturation in the spleen and was associated with upregulation of BDNF expression in the hypothalamus. Hypothalamic BDNF overexpression reproduced the EE effects on NK cell maturation in secondary lymphoid tissues. Conversely, hypothalamic BDNF knockdown blocked the EE modulation on NK cell maturation. Our results demonstrate that a bio-behavior intervention enhanced NK cell maturation and was mediated at least in part by hypothalamic BDNF.

Keywords: BDNF, environmental enrichment, NK cells, NK maturation, secondary lymphoid tissues

Introduction

Natural Killer (NK) cells are a central component of the innate immune system and constitute the first line of defense against a variety of tumors and microbial pathogens [1, 2]. NK cells were traditionally thought to develop primarily in the bone marrow (BM). However, recent evidence in humans and mice suggests that they can also develop and mature in secondary lymphoid tissues (SLTs) including tonsils, spleen, and lymph nodes (LNs) [3,4]. NK cells are heterogeneous as to their phenotype, function, and anatomic distribution. NK cell precursors circulate in the blood and reside in the spleen, lymph nodes, and other peripheral non-lymphoid organ where they continue to develop and mature [5]. Mouse NK cells undergo a maturation process that is defined by expression of the surface markers CD27 and CD11b and are classified as precursor (stage I, CD11blow CD27low), immature (stage II, CD11blow CD27high), proinflammatory (stage III, CD11bhigh CD27high), or cytotoxic (stage IV, CD11bhigh CD27low) [6].

The regulation of NK cell development is controlled by both genetic and environmental factors, and now represents an ever-expanding field in itself [4, 7]. Our laboratory utilizes the enriched environment (EE) to study the effects of favorable macroenvironmental stimuli on general health and disease progression [8, 9]. In comparison to the standard housing (SE) used in most biomedical research facilities, the EE is a housing environment that provides social, physical, and cognitive stimulation and has a significant influence on the brain function and progression of neurological diseases [10]. We have previously demonstrated that EE leads to anticancer and anti-obesity phenotypes in various mouse models and identifies a specific brain–fat axis as a key underlying mechanism [9, 11–15]. More recently, we have investigated how EE regulates T cell immunity. EE induces phenotypic changes in T cells in secondary lymphoid tissues resulting in an anticancer effect characterized by a decrease in the ratio of CD4 T helper to CD8 cytotoxic T lymphocytes [16]. Moreover, EE regulates thymocyte development and protects against autoimmune challenges [17]. Others and our group have also demonstrated that EE has significant effects on NK cells [9,18] including an increase in the proportion of the mature NK cells with higher natural cytotoxicity against tumor cells [9,19]. However, whether a central mechanism mediates the EE’s peripheral NK modulation is not fully understood. We previously discovered that the hypothalamic brain-derived neurotrophic factor (BDNF) acts along a specific neuroendocrine-immune axis to mediate EE’s favorable effects on the metabolic and cancer phenotypes [9,16,17]. Here we investigated the role of hypothalamic BDNF in the EE-induced NK modulation.

Congenital or iatrogenic deficiency of NK cell numbers has been associated with an increased incidence of life-threatening infections [20,21]. Multiple studies have shown that rapid and robust NK cell recovery early after hematopoietic cell transplant (HCT) is associated with less relapse, non-relapse mortality, and improved survival [22,23], and abrogation of NK cell maturation can be seen in progression of acute myeloid leukemia (AML) [24,25]. These reports suggest an association of NK cell recovery and NK cell maturation with clinical outcomes [26]. Hence, understanding the factors that control NK cell development can be important to improving clinical outcomes following HSC transplantation and treatment of AML. Thus, we investigated whether EE affected NK cell maturation following their in vivo depletion.

Results

Anti-asialo GM1 antibody rapidly depletes NK cells

Anti-asialo GM1 antibody or rabbit IgG isotype as a control antibody was injected in a single dose i.p. into mice to determine the efficiency of NK cell depletion. We collected the BM and spleen 24 hrs after antibody injection to evaluate the percentage of NK cells present. Anti-asialo GM-1 significantly depleted both splenic and BM NK cells, validating that the dose administered was sufficient to deplete the NK cells with >95% efficiency (Fig. 1A and B). In order to evaluate the effect of EE on the recovery of NK cells and their maturation, we first monitored the relative percentage of NK cells (% of isotype) in the blood of mice treated with anti-asialo GM-1 at different time points following i.p. injection. We noted that 50% recovery of the blood NK cells required approximately 23 days (Fig. 1C). Hence, blood, spleen, and BM were collected 23 days after the injection of anti-asialo GM-1 or a non-reactive rabbit IgG control. The percentage of NK cells in the experimental group was significantly lower in blood, spleen, and BM, and the absolute count of splenic NK cells was significantly lower when compared to mice in the control group (Fig. 1D and E).

Figure 1.

Figure 1.

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Anti-GM1 antibody depletes NK cells in vivo. (A) Relative abundance of splenic and BM NK cells (% of isotype-treated) at day 1 in C57BL/6 mice treated with either anti-GM1 antibody or rabbit IgG isotype antibody on day 0. (B) Representative flow cytometry of splenic NK cells at day 1 in C57BL/6 mice treated with either anti-GM1 antibody or rabbit IgG isotype antibody. (C) Relative abundance (% of isotype) over time of blood NK cells following a single i.p. injection on day 0 with anti-asialo GM1 antibody. (D) The percentage of blood, splenic, and BM NK cells at day 23 after injection of rabbit IgG isotype or anti-asialo GM1 antibody. (E) The absolute count of splenic NK cells at day 23 post depletion of NK cells. Data are mean ± SEM. Data represent a single experiment with n = 3 for IgG group, n = 3 for anti-asialo GM1 antibody group, and measured by flow cytometry. Data were analyzed using the unpaired two-tailed Student’s t-tests to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.

EE enhances NK cell maturation

We tested the influence of EE on NK cell recovery and maturation. Following the depletion of NK cells with anti-asialo GM1 antibody, mice were housed in an EE or SE for 23 days. Our results from day 23 showed that the percentage of splenic NK cells was two-fold higher in the mice housed in the EE compared to those mice housed in the SE (5.06 % vs 2.53 % respectively, p < 0.05; Fig. 2A). There was also a significant increase in the absolute number of splenic NK cells in the EE group compared to the SE group (Fig. 2B).

Figure 2.

Figure 2.

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EE enhances splenic NK cells maturation following anti-asialo GM1 treatment compared to SE. Percentage (A) and absolute number (B) of splenic NK cells (defined as Lin−NK1.1+CD49b+) assessed in mice housed in SE or EE 23 days following treatment with anti-asialo GM1 on day 0. (C) Percentage of splenic NK cells from each of four stages of NK cell maturation assessed in mice housed in SE or EE 23 days following treatment with anti-asialo GM1 on day 0. (D) Absolute numbers of splenic NK cells assessed in mice housed in SE or EE 23 days following treatment with anti-asialo GM1 on day 0. Data shown represent the mean ± SEM and were performed with n = 7 mice in EE and n = 5 mice in SE and represent 1 of 2 experiments. All data were measured by flow cytometry. Data were analyzed using the unpaired two-tailed Student’s t-tests to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001; NS, nonsignificant.

We assessed the changes in stages of NK cell maturation under SE and an EE 23 days after NK cell depletion with anti-asialo GM1 antibody. Mice housed in an EE showed a significantly higher absolute number of stage III and IV splenic NK cells when compared to mice housed in a SE (Stage III NK: 0.568 × 106 ± 0.09 vs. 1.911 × 106 ± 0.263, p < 0.001, Stage IV NK: 0.554 × 106 ± 0.09 vs. 3.144 × 106 ± 0.369, p < 0.001, respectively), while the environment did not have a significant effect on the absolute numbers of stage I or stage II splenic NK cells (Fig. 2C and D). As was observed in the spleen, mice housed in an EE 23 days following NK depletion with anti-asialo GM1 antibody had a significantly greater percentage of NK cells in the BM (1.5-fold; p < 0.05, Fig. 3A) and blood (≈2.5-fold; p < 0.05, Fig. 3B) when compared to mice housed in an SE for 23 days following NK depletion with anti-asialo GM1 antibody. The EE has a significant anti-obesity and anticancer effect that is mediated through hypothalamic BDNF [9, 11]. Therefore, we measured the expression of hypothalamic BDNF by quantitative qRT-PCR and observed a significant upregulation in the mice housed in an EE 23 days following NK depletion with anti-asialo GM1 antibody when compared to mice housed in a SE (Fig. 3C). We observed a significant decrease in the percentage of stage II BM NK cells and a significant increase in the percentage of stage IV BM NK cells in the mice housed in an EE 23 days following NK depletion with anti-asialo GM1 antibody when compared to mice housed in a SE (Fig. 3D). However, we did not see any significant percent changes in the various stages of blood NK cells when comparing the mice housed in an EE 23 days following NK depletion with anti-asialo GM1 antibody to mice housed in a SE (Fig. 3E).

Figure 3.

Figure 3.

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EE enhances maturation of NK cells in the BM. Percentage of BM NK cells (A) and blood NK cells (B) (defined as Lin−NK1.1+CD49b+) assessed in mice housed in SE or EE for 23 days following treatment with anti-asialo GM1 on day 0 and measured by flow cytometry. (C) Relative expression of hypothalamic Bdnf assessed by qRT-PCR in mice housed in SE or EE for 23 days following treatment with anti-asialo GM1 on day 0. The data were normalized to HPRT gene expression. (D) Percent of BM NK cells from each of four stages of NK cell maturation assessed by flow cytometry in mice housed in SE or EE 23 days following treatment with anti-asialo GM1 on day 0. (E) Percent of blood NK cells from each of four stages of NK cell maturation assessed by flow cytometry in mice housed in SE or EE for 23 days following treatment with anti-asialo GM1 on day 0. Data shown represent the mean ± SEM and were performed with n = 7 mice in EE and n = 5 mice in SE and represent one of two experiments. Data were analyzed using the unpaired two-tailed Student’s t-tests to determine statistical significance. *p < 0.05, **p < 0.01; NS, nonsignificant.

Hypothalamic BDNF reproduces the EE effects on NK cell maturation

To determine whether hypothalamic BDNF is responsible for the EE effects on NK cell maturation in the SLTs (LN or spleen), we firstly used recombinant adeno-associated virus (rAAV) vector to overexpress BDNF in the hypothalamus with GFP as a control. Both groups were housed in SE. BDNF overexpression was confirmed with qRT-PCR 3 weeks after injection of the vector [16]. At 5 weeks after rAAV injection there was no significant difference in the percentage of total splenic NK cells (Fig. 4A), yet mice receiving rAAV-BDNF showed a significant decrease in the percentage of stage II splenic NK cells and significant increase in the percentage of stage IV splenic NK cells when compared to mice receiving rAAV-GFP (Fig. 4B and C). Similar effects were observed in LN at 3 weeks after rAAV injection (Fig. 4D–F). Hence, these results suggest that hypothalamic BDNF overexpression reproduces the effects of EE on NK cell maturation in the SLTs characterized as lower stage II NK cells and higher stage IV NK cells (Fig. 2C).

Figure 4.

Figure 4.

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Hypothalamic BDNF modulates the effects of EE on WT NK cell maturation. (A) Percentage of splenic NK cells (defined as Lin−NK1.1+Nkp46+) 5 weeks after injection of rAAV-BDNF or rAAV-GFP into the hypothalamus of mice and housed in SE for 21 days. (B) Percentage of splenic NK cells from each of four stages of NK cell maturation from mice treated and housed as mice in (A). (C) Representative flow cytometry of splenic NK cell stages of maturation shown in (B). (D) Percentage of LN NK cells (defined as Lin−NK1.1+Nkp46+) from mice treated and housed as mice in (A). (E) Percentage of LN NK cells from each of four stages of NK cell maturation from mice treated and housed as mice in (A). (F) Representative flow cytometry of splenic NK cell stages of maturation shown in (E). (G) Experimental design of hypothalamic Bdnf knockdown experiment on LN NK cells. (H) Percentage of LN NK cells from each of four stages of NK cell maturation from mice having undergone Bdnf knockdown or mock knockdown on day 1 and then housed for 5 weeks in an EE or SE. (I) Representative flow cytometry of splenic NK cell stages of maturation shown in (H). Data shown represent the mean ± SEM and measured by flow cytometry. Data shown in (A–F) are representative of two independent experiments with n = 5 mice in each group. Statistical significance for BDNF overexpression experiment was determined using the unpaired two-tailed Student’s t-tests. Statistical significance comparing the overall maturation of NK cells between environment conditions was determined using two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001; NS, nonsignificant.

To further confirm that hypothalamic BDNF is essential for the effects of the EE on NK cell maturation, we injected mice with either a rAAV vector expressing a microRNA targeting mouse Bdnf or a scrambled sequence (miR-scr) that does not alter any known genes as a control [16]. The miR-Bdnf vector was fully characterized previously and used in published studies [9, 11, 12]. After recovery from stereotaxic surgery, mice receiving each vector were randomized to live either in a SE or an EE for 5 weeks (Fig. 4G). Hypothalamic Bdnf knockdown was confirmed using qRT-PCR at the time of sacrifice [16]. LNs were collected after 5 weeks of exposure to either an EE or a SE, which would allow time for a significant shift in maturation of NK cells. We observed a significant decrease in the percentage of stage II splenic NK cells (p < 0.05) and significant increase in the percentage of Stage IV splenic NK cells (p < 0.01) in mice that received miR-Scr and were placed in the EE, compared to mice that received miR-Scr and were placed in the SE. In contrast, miR-Bdnf blocked this shift of NK cell maturation in mice that were placed in the EE such that there were no differences in NK cell maturation in mice that received miR-Bdnf and were placed in the EE compared to mice that received miR-Bdnf and were placed in the SE (Fig. 4H and I). There was no significant interaction between the housing environment and usage of AAV vector for Bdnf KO except with stage IV NK cells (p < 0.05). These results suggest that the alterations in NK cell maturation seen within SLT when mice are housed in an EE are in fact mediated at least in part by hypothalamic Bdnf.

Discussion

Environmental and psychosocial factors profoundly influence immune homeostasis. Most previous studies have focused on the effect of distress (the negative form of psychological stress) such as repeat social defeat [27] and restraint stress [28]. The impact of benign or positive environmental stimuli has not yet been well investigated. Our recent studies characterize how EE, a complex housing providing social, physical, and cognitive stimuli, regulates T cell immunity in the SLTs [16] and thymus [17]. Here we investigated the effect of EE on NK cell development and maturation. A recent study by Meng et al. has shown that mice exposed to EE exhibit a higher percentage of NK cells without absolute number change in the spleen and a shift toward the mature stage IV, CD11bhighCD27low, and removal of spleen attenuates the EE’s anticancer effects [19]. Our previous study also observed an association between enhanced splenic NK cell cytotoxicity with suppression of tumor progression in EE mice [9].

Anti-asialo GM1 antibody is commonly used as an NK cell-depleting antibody in mice for the study of NK cell functions. We observed that a single injection of anti-asialo GM1 antibody reduced the NK cells to minimal level in BM and spleen requiring approximately 3 weeks to recover to ~50%. This rate of recovery is consistent with NK cell depletion using anti-asialo GM1 antibody from published data [29]. The maturation of splenic NK cells in mice housed in an EE following anti-asialo GM1 antibody-mediated NK cell depletion exhibited decreased stage II NK cells and increased stage IV NK cells, the same maturation pattern as reported by Meng et al. in mice without NK depletion and living in an EE [19]. In addition, BM NK cells exhibited similar changes in stages of maturation as observed in splenic NK cells (Fig. 3D), whereas blood NK cells did not display similar changes in NK cell maturation (Fig. 3E). This suggests that different underlying mechanisms govern different tissues in modulating the maturation or migration of NK cells. Future studies to examine local signaling in respective tissues might help to elucidate the underlying mechanisms.

Our body of work has demonstrated that hypothalamic BDNF is an immediate early gene in the brain mediating several EE-induced phenotypes on systemic metabolism, peripheral cancer, and T cell immunity [9, 11, 12, 16]. Because EE stimulated hypothalamic Bdnf expression in the NK depleted group, we used data sets and samples from previous studies in which hypothalamic Bdnf was genetically manipulated [16]. These data show that overexpression of Bdnf in the hypothalamus largely mimics the effect of an EE on NK maturation as reported by Meng et al. [19] and conversely, knockdown hypothalamic Bdnf prevents the effect of an EE on NK maturation. Thus, this study not only identifies one brain factor that is mediating the effect of EE on NK cell maturation, but also further supports the notion that hypothalamic Bdnf is a molecular hub linking EE stimuli and downstream metabolic and immune phenotypes. These observations indicate that manipulation of a single molecule in the brain could regulate T-cell and NK-cell homeostasis and this mechanism could potentially be utilized to correct cancer-mediated immune deficiencies and to strengthen the efficiency of immunotherapies. Indeed, we have recently shown that there is an abrogation of NK cell maturation in both mice and patients with acute myeloid leukemia (AML) that is mediated at least in part through the release of unknown soluble activators of the aryl hydrocarbon receptor transcription factor, which impedes the differentiation of type 3 innate lymphoid cells to NK cells [24,25,30]. It will be intriguing to investigate if this mechanism of innate immune evasion by AML can be prevented in the setting of EE or enhanced endogenous Bdnf expression, or whether these interventions can enhance NK cell or T cell function following checkpoint inhibition [31].

The downstream factors in the periphery mediating EE modulation of NK cells remain to be identified. The development of NK cells is contingent on the presence of IL-15, which also plays a critical role in NK cell proliferation, activation, and cytotoxicity [32, 33]. In addition, we recently reported that adipocyte overexpression of the IL-15/IL-15Rα complex leads to expansion of the NK cells in the blood and spleen as well as the enhancement of NK cell maturation characterized by a decrease of stage II NK cells and an increase of stage IV NK cells [34]. These results suggest that IL-15 signaling could be a candidate contributing to the enhancement of NK cell maturation in EE mice. Although Meng et al. noted they did not see a change of the level of circulating IL-15 in EE mice [19], we are currently examining whether EE affects different forms of IL-15 in various tissues and organs. Moreover, our previous studies reveal profound remodeling of the adipose tissue upon exposure to EE [9, 11, 12, 14, 15] including decreased leptin levels and increased adiponectin levels. These adipokines play important roles in the communication between the neuroendocrine and immune systems [35, 36]. The role of leptin is worthy of further investigation in the EE-induced immune modulations including NK cell maturation.

In summary, an EE providing physical, social, and cognitive stimuli enhanced NK cell maturation after antibody depletion of NK cells in the spleen and BM. EE led to a shift towards the most mature stage of NK cells in the SLTs. Our findings showed that hypothalamic Bdnf was essential for EE to exert effects on NK maturation in the SLTs, further supporting the notion that hypothalamic Bdnf orchestrating the neuroendocrine and immune systems in response to EE is associated with beneficial health effects.

Materials and methods

Mice

Male C57BL/6 mice (4 weeks old) were purchased from Jackson Laboratories and housed in temperature (22–23°C)- and humidity-controlled rooms with food and water ad libitum. The mice were fed a normal chow diet (NCD, 11% fat, caloric density 3.4 kcal/g, Teklad). All animal experiments were performed in accordance to the guidelines approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee.

In vivo NK cell depletion and recovery

Male C57BL/6 mice, 4 weeks of age were randomized to live in SE in cages of 30.5 cm × 17 cm × 15 cm or EE in a cage of 1.5 m × 1.5 m × 1.0 m supplemented with running wheels, tunnels, igloos, huts, retreats, wood toys, a maze, and nesting material [37]. Young animals were used because of the high brain plasticity, less risk of fighting in EE, and our previous mechanistic studies (genetic and pharmacological) identifying hypothalamic BDNF as a key brain mediator of EE’s anticancer and anti-obesity phenotypes. The mice were housed in their respective environment for one day and then injected intraperitoneally (i.p.) with 20 μL of anti-asialo GM1 antibody (Wako Chemicals, Richmond, VA, USA, Cat. # 986–10001) or IgG rabbit isotype (Southern Biotech, Birmingham, AL, USA, Cat. # 0111-01). Cheek blood was obtained from the mice at day one post injection and stained cells were analyzed using flow cytometry to confirm NK cell depletion. Mice were sacrificed 23 days post-depletion.

rAAV Vector Construction and Packaging

The rAAV plasmid contains a vector expression cassette consisting of the CMV enhancer and chicken β-actin (CBA) promoter, WPRE, and bovine growth hormone poly-A flanked by AAV inverted terminal repeats. Human BDNF cDNA was inserted into the multiple cloning sites between the CBA promoter and WPRE sequence. EGFP was cloned into the rAAV plasmid as a control. rAAV serotype 1 vectors were packaged, purified and the vectors were adjusted to 2 × 1013 vg/ml in PBS for injection.

rAAV-mediated BDNF overexpression in the hypothalamus

Five-week-old male C57BL/6 mice were randomly assigned to receive AAV-BDNF or AAV-GFP. Mice were anesthetized with ketamine/xylazine and secured via ear bars and incisor bar on a Kopf stereotaxic frame. A midline incision was made through the scalp to reveal the skull, and two small holes were drilled into the skull with a dental drill above the injection sites (−1.2 AP, ± 0.5 ML, −6.2 DV, mm from bregma). rAAV vectors were injected bilaterally into the hypothalamus (0.5 μL per site) at a rate of 0.1 μL per minute using a 10-mL Hamilton syringe attached to Micro4 Micro Syringe Pump Controller (World Precision Instruments)[16]. After infusion, the syringe was slowly removed from the brain, and the scalp was sutured. Animals were carefully monitored post-surgery until fully recovered from anesthesia. The mice were then housed in SE and sacrificed after 3 or 5 weeks.

AAV-microRNA experiment

AAV vectors containing microRNA targeting Bdnf (miR-Bdnf) and the scramble control (miR-scr) were described previously [9]. Six-week-old male C57BL/6 mice were randomized to receive AAV-miR-Bdnf or AAV-miR-scr. Then 0.7 μL of AAV vectors (1.4 × 1010 per site) was injected bilaterally into the hypothalamus at the stereotaxic coordinates described above. One week after surgery each vector-injected group was split to live in EE or SE housing. After 5 weeks the mice were sacrificed.

Isolation of leukocytes and flow cytometry

Mice were anesthetized with 2.5% isoflurane followed by decapitation then truncal blood was collected and the spleen, lymph nodes (LN), and BM were dissected. The blood sample was treated twice with Ammonium Chloride Solution (Stem Cell Technologies) to lyse the red blood cells (RBCs), washed, and then suspended in FACS buffer (1% FBS in PBS). Spleens or LNs were mechanically dissociated through a 70-μm strainer to obtain single-cell suspension. RBCs were lysed with ammonium chloride solution then washed and re-suspended in FACS buffer. For the BM cells, the tibia and femur were isolated and crushed using a mortar and pestle. The cell suspension was filtered using a 70-μm strainer and the pellet was treated with ammonium chloride solution to lyse the RBCs, washed, and re-suspended in FACS buffer. Cells were counted using the Cellometer Auto 2000 (Nexcelom Bioscience). For surface staining, cells were stained with a fluorescent dye conjugated antibody with the appropriate surface markers for 20 min. The antibodies used for flow cytometry immunophenotyping are listed in Supporting Information Table 1. Cell events were acquired using LSRII flow cytometry (BD Biosciences) and the results were analyzed using FlowJo v10 software (Tree Star). The flow cytometry experiments were designed following the recent guidelines published for flow cytometry [38].

Statistical analysis

Continuous and normally distributed measurements values were summarized as mean ± SEM and were analyzed using GraphPad Prism 7 (GraphPad Software) or SAS 9.4 (SAS Institute Inc, 2012). Student’s t test was utilized to compare two independent groups. Two-way ANOVA models were used to assess the 2 × 2 interactions between two factors such as environment (SE vs. EE) and depletion (miR-Scr vs. miR-BDNF). p-Values were adjusted for multiple comparisons and a p-value of 0.05 or less was considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001.

Supplementary Material

S1

Acknowledgements:

We thank Dr. Bethany Mundy-Bosse and Dr. Aharon Freud from The Ohio State University Comprehensive Cancer Center for helpful comments and discussion on the manuscript. We would also like to thank Quais N. Hassan II and Jacqueline M. Anderson for technical assistance with experiments. This work was supported by NIH grants CA163640, CA166590, CA178227, and AG041250 to L. Cao and CA163205, CA068458, CA185301, and CA210087 to M.A. Caligiuri.

Footnotes

Conflict of Interest: M.A.C. is co-founder of CytoImmune Therapeutics Corp. The other authors declare no commercial or financial conflict of interest.

Peer review: The peer review history for this article is available at https://publons.com/publon/10.1002/eji.201948358.

Data availability statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

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