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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 63  |  Issue : 6  |  Page : 263-275

Sympathetic activation of splenic T-lymphocytes in hypertension of adult offspring programmed by maternal high fructose exposure


Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan

Date of Submission21-Oct-2020
Date of Decision20-Nov-2020
Date of Acceptance25-Nov-2020
Date of Web Publication26-Dec-2020

Correspondence Address:
Dr. Julie Y H Chan
Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_85_20

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  Abstract 


Whereas neuroimmune crosstalk between the sympathetic nervous system (SNS) and immune cells in the pathophysiology of hypertension is recognized, the exact effect of SNS on T-lymphocyte in hypertension remains controversial. This study assessed the hypothesis that excitation of the SNS activates splenic T-lymphocytes through redox signaling, leading to the production of pro-inflammatory cytokines and the development of hypertension. Status of T-lymphocyte activation, reactive oxygen species (ROS) production and pro-inflammatory cytokines expression in the spleen were examined in a rodent model of hypertension programmed by maternal high fructose diet (HFD) exposure. Maternal HFD exposure enhanced SNS activity and activated both CD4+ and CD8+ T-lymphocytes in the spleen of young offspring, compared to age-matched offspring exposed to maternal normal diet (ND). Maternal HFD exposure also induced tissue oxidative stress and expression of pro-inflammatory cytokines in the spleen of HFD offspring. All those cellular and molecular events were ameliorated following splenic nerve denervation (SND) by thermoablation. In contrast, activation of splenic sympathetic nerve by nicotine treatment resulted in the enhancement of tissue ROS level and activation of CD4+ and CD8+ T-cells in the spleen of ND offspring; these molecular events were attenuated by treatment with a ROS scavenger, tempol. Finally, the increase in systolic blood pressure (SBP) programmed in adult offspring by maternal HFD exposure was diminished by SND, whereas activation of splenic sympathetic nerve increased basal SBP in young ND offspring. These findings suggest that excitation of the SNS may activate splenic T-lymphocytes, leading to hypertension programming in adult offspring induced by maternal HFD exposure. Moreover, tissue oxidative stress induced by the splenic sympathetic overactivation may serve as a mediator that couples the neuroimmune crosstalk to prime programmed hypertension in HFD offspring.

Keywords: High fructose diet, neuroimmune crosstalk, oxidative stress, programmed hypertension, pro-inflammatory cytokine, spleen, sympathetic nerves, T-lymphocyte


How to cite this article:
Tsai PC, Chao YM, Chan JY. Sympathetic activation of splenic T-lymphocytes in hypertension of adult offspring programmed by maternal high fructose exposure. Chin J Physiol 2020;63:263-75

How to cite this URL:
Tsai PC, Chao YM, Chan JY. Sympathetic activation of splenic T-lymphocytes in hypertension of adult offspring programmed by maternal high fructose exposure. Chin J Physiol [serial online] 2020 [cited 2021 Jan 16];63:263-75. Available from: https://www.cjphysiology.org/text.asp?2020/63/6/263/304861




  Introduction Top


Hypertension is a major risk factor for the development of cardiovascular diseases that becomes an epidemic concern worldwide. Underlying mechanisms of hypertension are multifaceted, but overexcitation of the sympathetic nervous system (SNS) has been recognized as a common feature associated with high blood pressure (BP).[1],[2] In addition to causing direct damages to the cardiovascular organs, including the heart, vasculature, and kidneys,[3],[4] evidence from studies over more than 10 years suggests that SNS overexcitation may lead to hypertension via its regulatory actions to the immune cells.[5],[6],[7] Excitation of SNS in the bone marrow and the spleen, as well as the associated release of hematopoietic cells into the systemic circulation, play pivotal roles in hypertension pathogenesis.[8] In the deoxycorticosterone acetate-salt model of hypertension, an intact sympathetic drive to the spleen is a prerequisite for priming the cell immunity and the consequent rise in BP.[9] In addition, enhanced T-cell infiltration and increased sympathetic discharge have been identified in the carotid body of hypertensive patients.[10] Whether this neuroimmune crosstalk between the SNS and immune cells is unequivocal in hypertension with different underlying etiologies, however, remains to be validated.

The complexity in the pathogenesis of adult hypertension is further accentuated by both human epidemiological observations[11],[12] and animal experimental findings[13],[14],[15] that demonstrate suboptimal environment during fetal and neonatal development primes adult onset of disease; evidence leads to the concept of developmental origins of health and disease (DOHaD) or fetal programming of adult disease.[16] According to the DOHaD theory, we now know that maternal insults, such as malnutrition, program a series of changes in fetal structures and functions that are retained in later life and increase the risks for adult cardiovascular diseases.[11],[12],[13],[14],[15] In a rodent model of programmed hypertension in adult offspring to maternal high fructose diet (HFD) exposure, we reported previously that overexcitation of SNS in the vasculature[17] and the kidneys[18] are engaged in the pathogenesis of programmed hypertension in adult offspring.

The fetal immune system is particularly vulnerable to intrauterine stress. The increase in pro-inflammatory cytokines, such as interleukin 1-β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α), have been observed in the fetal circulation, placenta and amniotic fluid induced by intrauterine growth restriction.[19] Recently, through the whole genome next-generation RNA sequencing to quantify changes in the abundance of RNA transcripts in the aorta of the neonate and 3-week-old HFD offspring, we found that in 29 transcriptomes of which their expressions were altered at both age windows, approximately 10% are related to immune processes.[20] Nonetheless, the functional significance of this change in immune profiling in early life to the development of programmed hypertension in adult HFD offspring is currently unknown.

There appears to be an important role of SNS in the developmental programming of hypertension in adult offspring to maternal HFD exposure.[21] At the same time, our previous findings indicate an intergenerational influence of maternal HFD on transcriptome profiles associated with cell immunity.[20] In the present study, we sought to characterize the coupling between the SNS and immune cells in the spleen to the development of hypertension in adult offspring programmed by maternal HFD exposure. Moreover, we aimed to investigate whether targeting the splenic SNS could inhibit programmed hypertension and, in turn, reveal additional mechanisms underpinning hypertension programming in HFD offspring. We provide evidence that SNS excitation increases cell populations of both CD4+ and CD8+ T-lymphocytes in the spleen and contributes to hypertension in adult offspring programmed by maternal HFD exposure. We further identify tissue oxidative stress may serve as a mediator that couples the SNS with immune cells, as well as expression of pro-inflammatory cytokines in the spleen of HFD offspring.


  Materials and Methods Top


Animals and experimental design

Male and virgin female adult (12–14 weeks of age; n = 12 for male and n = 24 for female) normotensive Sprague-Dawley rats purchased from BioLASCO, Taiwan were used in this study. Animals were housed in an AAALac-International accredited animal facility in our hospital under conditions of controlled temperature (24°C ± 0.5°C) and 12-h light/dark (08:00–20:00) cycle. Standard laboratory rat chow (PMI Nutrition International, St. Louis, MO, USA) and tap water were available ad libitum. All experimental procedures were carried out in accordance with the guidelines for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health and were reviewed and approved by our institutional animal care and use committee (approval no. 2016112503).

After 14 days acclimatization period, one male rat was housed with two females until mating was confirmed by the observation of vaginal plug. Pregnant female rats were randomly assigned to receive regular chow (normal diet [ND], 46% complex carbohydrate, 3.4 kcal/g, Harlan Laboratories, Madison, WI, USA) or HFD (60% fructose, 3.6 kcal/g; TD.89247, Harlan Laboratories) during the entire period of pregnancy and lactation.[13],[15],[17],[20] Given that men are more prone to hypertension at a younger age;[22] only male offspring from litters culled to sizes of eight pups after birth were used in subsequent experiments. After weaning (3 weeks after birth), offspring from mother exposed to ND or HFD returned to ND chow until they were killed at the age of 6, 9, 12, 18, or 24 weeks. At 5 week of age, ND and HFD offsprings were randomly separated into the following groups to receive splenic nerve denervation (SND) by thermoablation, intraperitoneal (i.p.) infusion of nicotine (1 mg/kg/day; Sigma-Aldrich, St. Louis, MO, USA) for 1 week. An additional group of rats received the treatment with a superoxide scavenging antioxidant, tempol (1 mmol/L; Sigma-Aldrich), in the drinking water (ad libitum) for 1 week at the age of 6 weeks. Dosage of the compound was adopted or modified from previous studies.[8],[9],[23],[24]

Preparation of spleen cells

The offspring spleen was harvested at different ages (3, 6, 9, 12, 18, or 24 weeks) and was prepared for single cell (1 × 106) suspension. In brief, ND and HFD offspring were deeply anesthetized with an overdose of pentobarbital sodium (100 mg/kg, i.p.), followed by intracardial infusion with warm normal saline. The spleens from ND and HFD offspring were harvested, dissected, physically disrupted into single cell suspensions in RBC lysis buffer (Biolegend, San Diego, CA, USA) and filtered through Falcon® 70-μm cell strainer (Corning Inc., Phoenix, AZ, USA) to remove large debris. Erythrocyte contamination was removed by the use of red blood cell lysis buffer (15.5 mM NH4 Cl, 1 mM KHCO3, 10 μM EDTA), and cell pellets were collected by centrifugation at 300 g for 5 min at 4°C. The process was repeated once and the suspend cell pellets were dissolved with 1X phosphate-buffered saline (PBS) (pH 7.4). Cells were counted using size threshold of 4.0 μm on a Beckman Coulter counter (Indianapolis, IN, USA), and concentration was adjusted for subsequent staining procedures.

Staining for T-lymphocyte cell surface markers

Cells isolated from the spleen were resuspended in ice-cold staining solution of PBS buffer containing 1% bovine serum albumin. The following antibodies (1:100) were added and incubated for 30 min at 4°C. They are APC-CD3 (Biolegend clone 1F4) for T-cells, PE-CD4 (Biolegend clone OX-35) for T helper cells and FITC-CD8 (Biolegend clone OX-8) for cytotoxic T cells. Normal rat serum was used as a secondary blocking agent prior to intracellular staining with APC-isotype antibodies (mouse IgM, K; Biolegend), PE-isotype antibodies (mouse IgG2a, K; Biolegend), and FITC-isotype antibodies (mouse Ig1, K; Biolegend). Positive stained cells were washed using the same staining buffer, followed by fixation in 4% paraformaldehyde (Sigma-Aldrich) solution. T-lymphocytes were analyzed by flow cytometry (Gallios Flow Cytometer, Beckman Coulter) and quantified using FlowJo cytometric analysis software (Tree Star, Ashland, OR, USA). Photomultiplier tubes voltage adjustment was performed using nonstained cells, isotype-stained cells and single-stained cells before the acquisition.

Cytokine quantification

Tissue pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, and interferon-γ (INF-γ), were measured with Milliplex® MAP rat cytokine magnetic beads kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer's instructions. Both positive and negative controls were included on each plate. The concentration of cytokines was quantified using the CellQuestPro and CBA software (Becton Dickinson, Franklin Lakes, NJ, USA) on a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA).

Norepinephrine quantification by high-performance liquid chromatography

Tissue norepinephrine (NE) level was measured by the o-phthaldehyde (OPA) method using high-performance liquid chromatography (HPLC) with fluorescence detection.[17] For sample preparation, splenic cells were resuspended and 250 μL samples were mixed with 37.5 μL trichloroacetic acid (Sigma-Aldrich) and vortexed for 20 min. After centrifugation at 10,000 g for 20 min at room temperature, supernatant was collected and passed through 0.22 μm syringe filter (Chroma Technology Corp., Bellows Falls, VT, USA); followed by adding methanol (1:4, v/v) to the supernatant and shaking the mixture gently for 10 min. The sample was centrifugated again at 10,000 g for 15 min, supernatant was collected and passed through 0.22 μm syringe filter. The supernatant was collected and kept at −80°C until analysis.

Derivatization solution was prepared by adding 0.5 mg OPA (Sigma-Aldrich) to 40 mL methanol and passed through 0.22 μm filter. The solution was stored in dark for subsequent use. Prepared sample solution or standard NE solution (at concentrations of 1–50 mM) was mixed with OPA solution in 4:1 (v/v), placed in dark for 15 min and 10 μL of the solution was used for HPLC analysis.

For NE quantification, each 10 μL aliquot was injected into the system using a Rheodyne (model 7125, Merck KGaA, Darmstadt, Germany) injector. The HPLC system (Hitachi CM5000, Hitachi Corp., Tokyo, Japan) is comprised of a 5110 syringe pump system, a 5210 autosampler and a 5440 FL spectrofluorimetric detector. Chromatographic separation was achieved on a ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm; Agilent Technologies, Taiwan). The column temperature was maintained at 30°C and the flow rate was 1 mL/min. Methanol and acetate buffer (20 mM, pH 3.5, with 1 mM Na2 EDTA) with a ratio of 5:4 (v/v) was used as the mobile phase, and the samples were eluted within 20 min. The fluorescence signal was detected with excitation and emission wavelengths of 340 and 450 nm, respectively. The retention time for NE was 2.2–2.6 min. The concentration of NE was computed by comparing the area under the curve of each sample against standard solutions of known NE concentrations. Each sample was analyzed in quadruplicates, and the results are shown as the mean of the four values.

Splenic reactive oxygen species analysis

Splenic cells were resuspended and were incubated with 5 μM dihydroethidium (DHE; Thermo Fisher, Chicago, IL, USA) in dark for 30 min at 37°C. After washing twice with PBS, the percentage of stained cells was analyzed by Flow cytometer (Beckman Coulter) and quantified using FlowJo or ModFit cytometric analysis software. DHE fluorescent intensity reflects intracellular reactive oxygen species (ROS) levels. Cells isolated from the spleen without DHE staining was used as a negative control.

Splenic nerve denervation

Animals were anesthetized with isoflurane (5% for induction and 2% for maintenance) via an anesthesia mask for surgical operation. Satisfactory anesthesia was maintained by the absence of withdrawal reflex to hind paw pinch. Animals were allowed to breathe spontaneously with room air, and body temperature was maintained at 37°C by a heating pad. The rat abdominal cavity was opened and the splenic artery was exposed. Thermoablation with a thermal cautery (Bovie Medical Corporation, Clearwater, FL, USA) was used to denervate the splenic nerve. This was performed by gently placing the cautery to the splenic artery for 5–6 s until the splenic artery was dilated.[25] In sham-operated rats, the same experimental procedures were taken, but the thermoablation was not performed. The incision was closed with layered sutures, and animals received intramuscular injection of procaine penicillin (1,000 IU) postoperatively. Only animals that showed progressive weight gain after the operation were used in subsequent experiments.

Immunofluorescence staining

Catecholaminergic fibers in the spleen were visualized using sucrose-potassium phosphate-glyoxylic acid (SPG) method, according to the previously published procedures.[26] In brief, animals were deeply anesthetized with an overdose of pentobarbital sodium (100 mg/kg, i.p.), followed by intracardial infusion with warm normal saline. Fresh spleen tissue was cut into 5–10 mm-thick blocks and immediately placed on a pre-cooled cryostat chunk. Frozen spleen tissue was sectioned into 10 nm slices at −30°C and mounted onto clean slide. The slide was dipped into SPG solution for three times and dried for 15–20 min. Tissue sections were then covered with mineral oil and placed at 95°C hotplate for 2–3 min. Spleen sections were coversliped and viewed under fluorescence microscope (BX-51, Olympus, Tokyo, Japan). The catecholamine signals were observed in bluish-white fluorescence.

Implantation of osmotic minipump

Animals were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) for the implantation of osmotic minipump into the peritoneal cavity. Under satisfactory anesthesia, which was maintained by the absence of withdrawal reflex to hind paw pinch, a midline abdominal incision was made and an osmotic minipump (Alzet 1007D; Durect Co., Cupertino, CA, USA) was placed in the peritoneal cavity.[27] Control animals received identical implantation procedures of saline-filled osmotic minipump. The sham-operated animals were treated identically, except osmotic minipump implanted. Following implantation, the abdominal muscle layers were sutured. Body temperature was maintained at 37°C with heating pads until the animals had recovered from surgery.

Blood pressure measurement

Systolic BP (SBP) was routinely measured at 14:00–16:00 in ND and HFD offspring under conscious condition using the noninvasive tail-cuff method based on electrosphygmomanometry (MK-2000; Momuroki Kikai Co., Tokyo, Japan) reported previously.[17] In brief, animals were handled repeatedly and allowed to adapt to the restraint chamber for at least 3 days before the commencement of actual measurements. SBP determinations were considered valid only when five consecutive readings were recorded from rats under resting condition and the values did not differ by >5 mmHg. The mean of such five readings was recorded as the measured SBP value.

Statistical analysis

Data are expressed as means ± standard error of mean. All analyses were performed in Graphpad Prism 7 analysis software (La Jolla, CA, USA). Comparison between groups was performed with one-way or two-way ANOVA with or without repeated measures, as appropriate, to assess group means; followed by the Tukey's multiple range test for post hoc assessment of individual means. P < 0.05 was considered statistically significant.


  Results Top


Maternal high fructose diet increases sympathetic activity and T-lymphocyte activation in the spleen and programs hypertension development in high fructose diet offspring

To examine the effects of maternal HFD exposure on sympathetic activity and CD4+ and CD8+ cells populations of T-lymphocytes in the spleen, as well as the development of programmed hypertension in offspring, temporal profiles of tissue NE levels and percentage of CD4+ and CD8+ cells in T-lymphocytes of the spleen, as well as SBP in offspring from 3 to 12 weeks of age were evaluated. Compared with age-matched ND offspring, sympathetic activity, estimated by NE levels in spleen tissue, was notably higher in HFD offspring [Figure 1]a. At the same time, splenocytes isolated from the same HFD offspring showed a significant T-cell activation, reflected by the increases in CD4+ and CD8+ cell populations [Figure 1]b and [Figure 1]c. Notably, both events occurred in HFD offspring at age younger than the onset of programmed hypertension that became detectable at age of 9 weeks [Figure 1]a. There is no significant difference in number of total splenic T-lymphocytes (CD3+ cells) between HFD and ND offspring [Figure 1]b and [Figure 1]c.
Figure 1: Maternal HFD exposure programs the increases in sympathetic nerve activity and T-lymphocyte activation in the spleen, as well as the increase in SBP in offspring. (a) Time course of changes in splenic NE level and SBP, (b) gating strategy for isolation of CD3+, CD4+ and CD8+ splenic cells by flow cytometry, (c) representative profiles showing CD4+ and CD8+ cell populations gated on isotype-stained and CD3+ stained cells at age of 6 weeks, and statistical results of changes in CD4+ and CD8+ cell populations of CD3+ cells in the spleen of offspring exposed to maternal HFD or ND at 3, 6, 9, 12, 18 and 24 weeks of age. Data are presented as mean ± SEM, n = 6 per group at each age. *P < 0.05 versus ND group in post hoc Tukey's multiple range analysis. HFD: High fructose diet, SBP: Systolic blood pressure, NE: Norepinephrine, ND: Normal diet, SEM: Standard error of mean.

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Sympathetic innervation in the spleen induces T-lymphocyte activation in high fructose diet offspring

The observations of the SNS activation and the increases in CD4+ and CD8+ cell populations in the spleen of HFD offspring prompted the experiments to determine the contribution of sympathetic innervation to the activation of T-lymphocytes in the spleen of HFD offspring. SNS denervation was performed by thermoablating the splenic nerves in HFD offspring at the age of 5 weeks. One week following the SND, distribution of catecholaminergic containing fibers [Figure 2]a and tissue level of NE [Figure 2]b in the HFD spleen were significantly reduced. The same treatment also significantly attenuated the increases in CD4+ and CD8+ cell populations in the spleen of HFD offspring at the age of 6 weeks [Figure 2]c.
Figure 2: Sympathetic innervation in the spleen induces T-lymphocyte activation in HFD offspring. (a) Representative photomicrographs showing the catecholaminergic containing fibers (arrows), visualized using the sucrose-potassium phosphate-glyoxylic acid method, in the spleen of ND, HFD and HFD offspring subjected to SND performed at 5 weeks of age. (b) Representative HPLC chromatography and group data showing tissue NE levels, and (c) percentage of CD4+ and CD8+ cells within the CD3+ cell population in the spleen of ND, HFD and HFD + SND at 6 weeks of age. Data are presented as mean ± SEM, n = 6 per group in b and c. *P < 0.05 versus ND group and #P < 0.05 versus HFD group in post-hoc Tukey's multiple range analysis. SND: Sympathetic nerve denervation, HFD: High fructose diet, ND: Normal diet, SEM: Standard error of mean, HPLC: High-performance liquid chromatography, NE: Norepinephrine.

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Sympathetic activation induces tissue oxidative stress and cytokine expression in the spleen of high fructose diet offspring

SNS activation has been reported to induce tissue oxidative stress and inflammation,[28],[29] two key factors in the development of hypertension.[6],[27],[30],[31] However, their relationship on programmed hypertension in offspring to maternal HFD exposure has hereinto not been investigated. We, therefore, evaluated the changes in tissue ROS levels and the expressions of pro-inflammatory cytokines in the spleen of offspring exposed to maternal HFD insult. Compared with the age-matched ND group, the increase in SNS activity in the spleen of HFD offspring was accompanied by heightened ROS levels [Figure 3]a and the increase in protein expression of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, and INF-γ [Figure 3]b, but a decrease in IL-10 expression [Figure 3]c in splenocytes isolated from HFD offspring. In addition, SND at the age of 5 weeks profoundly reversed these molecular events detected at 6 weeks of age [Figure 3]a, [Figure 3]b, [Figure 3]c.
Figure 3: Sympathetic innervation primes tissue oxidative stress and expression of pro-inflammatory cytokines in the spleen of HFD offspring. (a) Time course of changes in splenic ROS level and (b) expression of IL-1β, IL-6, TNF-α, INF-γ and (c) expression of IL-10 in the spleen of ND and HFD offspring at 3, 6, 9, 12 and 18 weeks of age, as well as their modulations by SND (HFD + SND) at 6 weeks of age (insets on the right-hand side in figures). Data are presented as mean ± SEM, n = 5–6 per group at each age. *P < 0.05 versus ND group and #P < 0.05 versus HFD group in post hoc Tukey's multiple range analysis. ROS: Reactive oxygen species, IL: Interleukin, TNF-α: Tumor necrosis factor-α, INF-γ: Interferon-γ, HFD: High fructose diet, ND: Normal diet, SEM: Standard error of the mean, SND: Splenic nerve denervation.

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The potential role of sympathetic innervation to the production of ROS and expression of pro-inflammatory cytokines in the spleen was further investigated by i. p. infusion of nicotine (1 mg/kg/day, for 7 days at age of 5 weeks) into ND offspring. This treatment scheme has been reported to depolarize the sympathetic nerve terminals to promote NE release in the spleen.[23] Both ROS and NE levels in the spleen of the treated ND offspring were increased [Figure 4]a, accompanied by the increases in protein expression of IL-1β, IL-6, TNF-α and INF-γ [Figure 4]b, but a decrease in IL-10 [Figure 4]c in splenocytes isolated from ND offspring at age of 6 weeks.
Figure 4: Sympathetic activation induces tissue oxidative stress and pro-inflammatory cytokine expression in the spleen of ND offspring. (a) Tissue NE and ROS levels and (b) expression of IL-1β, IL-6, TNF-α, INF-γ and (c) expression of IL-10 in the spleen of ND offspring at 6 weeks of age that received saline (ND + saline) or nicotine (ND + nicotine) infusion (1 mg/kg/day, for 7 days at the age of 5 weeks). Data are presented as mean ± SEM, n = 4–6 per group at each age. *P < 0.05 versus ND group in post hoc Tukey's multiple range analysis. ROS: Reactive oxygen species, IL: Interleukin, TNF-α: Tumor necrosis factor-α, INF-γ: Interferon-γ, ND: Normal diet, SEM: Standard error of the mean, NE: Norepinephrine.

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Tissue oxidative stress-dependent activation of T-lymphocytes in the spleen of high fructose diet offspring

To identify a potential mechanistic role for tissue oxidative stress in the activation of T-lymphocytes in the spleen of HFD offspring, animals were treated with a superoxide scavenging antioxidant, tempol (1 mmol/L), in the drinking water for 1 week commenced at the age of 6 weeks. Oral intake of tempol significantly attenuated the augmented tissue ROS levels [Figure 5]a, the increases in the cell populations of CD4+ and CD8+ cells in the spleen [Figure 5]b, and programmed hypertension [Figure 5]c at the age of 9 weeks. The same treatment, on the other hand, had no appreciable effect on NE levels in the spleen of HFD offspring [Figure 5]a.
Figure 5: Redox-dependent activation of T-lymphocytes in the spleen of HFD offspring. (a) Tissue ROS and NE levels, (b) percentage of CD4+ and CD8+ cells within the CD3+ cell population in the spleen, and (c) SBP of ND or HFD offspring at 9 weeks of age that received tempol treatment in the drinking water (1 mmol/L) for 1 week at 6 weeks of age. Data are presented as mean ± SEM, n = 5–6 per group at each age. *P < 0.05 versus ND group and #P < 0.05 versus HFD group in post hoc Tukey's multiple range analysis. ROS: Reactive oxygen species, ND: Normal diet, SEM: Standard error of the mean, NE: Norepinephrine, HFD: High fructose diet, SBP: Systolic blood pressure.

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Neuroimmune crosstalk in the spleen contributes to programmed hypertension in high fructose diet offspring

Both loss-of-function and gain-of-function approaches were employed to verify the functional significance of the above-identified cellular and molecular events on hypertension programmed by maternal HFD exposure. The former was performed by thermoablating the SNS in the spleen of HFD offspring, and the latter was carried out in ND offspring subjected to nicotine infusion (1 mg/kg/day, for 7 days). Compared to the sham-operated control, ablation of splenic nerves lessened the increase in SBP of HFD offspring [Figure 6]a, whereas, in comparison to saline infusion, peripheral nicotine treatment significantly increased basal SBP in young ND offspring [Figure 6]b.
Figure 6: Neuroimmune crosstalk in the spleen contributes to the programmed hypertension in HFD offspring. Time course of changes in SBP in HFD or ND offspring subjected to (a) SND or (b) treatment with nicotine infusion (1 mg/kg/day, for 7 days) at 5 weeks of age. Data are presented as mean ± SEM, n = 4–6 per group at each age. *P < 0.05 versus ND group and #P < 0.05 versus HFD group in post hoc Tukey's multiple range analysis. ND: Normal diet, SEM: Standard error of mean, HFD: High fructose diet, SBP: Systolic blood pressure, SND: Splenic nerve denervation.

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  Discussion Top


The most salient findings of the current study are: (1) maternal HFD exposure during gestation and lactation enhances SNS activity and activates both CD4+ and CD8+ T-lymphocytes in the spleen of young offspring, (2) the same exposure also induces tissue oxidative stress and expression of pro-inflammatory cytokines in the spleen of young HFD offspring, (3) SND prevents T-lymphocyte activation, tissue oxidative stress and pro-inflammatory cytokine production in HFD spleen, (4) in contrast, activation of splenic sympathetic fibers results in tissue oxidative stress and activation of CD4+ and CD8+ T cells in the spleen of ND offspring; these molecular events are attenuated by the treatment with tempol, (5) finally, hypertension programmed in adult offspring by maternal HFD exposure is diminished by SND and tempol, whereas activation of splenic sympathetic fibers increases basal SBP in young ND offspring. These findings are interpreted to suggest that the sympathetic innervation in the spleen may activate T-lymphocytes, leading to hypertension programming in adult offspring induced by maternal HFD exposure. Moreover, tissue oxidative stress induced by this sympathetic overactivation may serve as a mediator that couples the neuroimmune crosstalk to prime hypertension programming in HFD offspring.

Evidence from studies over the past two decades argues for the interplay between the autonomic nervous system, particularly the SNS, and immune cells in the pathophysiology of hypertension,[5],[6],[7],[8],[9],[10] albeit the exact effect of SNS on T-lymphocyte activation and function in hypertension remains controversial. It was reported that sympathetic stimulation induces NE-mediated T cell activation and vascular inflammation, contributing to hypertension induced by angiotensin II (Ang II)[26] or in Dahl salt-sensitive model.[32] In contrast, suppression of splenic T-lymphocyte activation occurs in a NE-driven model of hypertension.[33] Herein, in a rodent model of adult hypertension of developmental origin, we found that maternal HFD exposure stimulated the SNS activation, reflected by the increase in tissue NE levels in the spleen, and led to activation of splenic T-lymphocytes, indicated by the increases in cell populations of CD4+ and CD8+ T cells. Our observations that splenic NE depletion following thermoablation of splenic nerve resulted in the attenuation of both CD4+ and CD8+ cell activations in HFD offspring provide evidence to suggest a permissive role of the sympathetic innervation in the spleen to T-lymphocyte activation programmed by maternal HFD. These observations provide new evidence not only to support the stimulatory effect of the SNS on T-lymphocyte activation, but also to suggest the modulation of neuroimmune crosstalk identified in adult may have its origin in fetal life.

Pro-inflammatory cytokines generated by immune cells are well recognized for their roles in hypertension.[34] We found in the present study that maternal HFD exposure that stimulated T-lymphocyte activation also caused the increase in pro-inflammatory cytokine production in the spleen of offspring. Conversely, the primed expression of pro-inflammatory cytokines in HFD offspring was appreciably diminished under the scenario in which splenic T-lymphocyte activation was attenuated by SND, suggesting an association of the two events in the spleen primed by maternal HFD exposure. Both CD4+ and CD8+ T cells produce INFγ in hypertensive animals[35] and humans.[36] In addition, pro-inflammatory cytokines, such as TNF-α, IL-1, and INF-γ, produced by T-lymphocytes have been demonstrated to amplify BP elevation and/or renal injury.[37] Adoptive transfer of CD4+ T cells from placental ischemic rats into normal pregnant rats increases BP by the increase in circulating levels of pro-inflammatory TNF-α and IL-6.[38] We also found in the present study a significant decrease in IL-10 expression in the spleen of HFD offspring. IL-10 is an anti-inflammatory cytokine that protects renal and vascular damages in hypertension.[36],[37],[39] The CD4+ T cell populations are composed of helper T cells, including type 1 (Th1), type 2 (Th2), and Th17 cells, as well as immuno-modulatory regulatory T (Treg) cells. Pro-inflammatory cytokines, such as TNF-α, IL-1, and INF-γ, are produced primarily by Th1 cells, whereas the anti-inflammatory cytokine IL-10 is produced by Th2 and Treg cells.[37] In general, the Th1 cells are pathogenic, whereas Th2 and Treg cells are protective in the diseases. The observed increases in TNF-α, INF-γ and IL-1β expressions, together with a decreased IL-10 expression, in this study, are interpreted to imply that a shift in the cell population of Th1 versus Th2 cells in the spleen may underpin the pro-inflammatory status and hypertension programming in HFD offspring. Additional experiments, nonetheless, are required to verify this postulation.

The mediators coupling the neuroimmune communication in the spleen is not well defined. As such, another important contribution of the current study is to identify ROS, particularly the superoxide anion, as a molecule that couples the SNS activation and T-lymphocyte activation in the spleen of HFD offspring. Superoxide anion has been demonstrated in previous studies as a key signal that mediates an array of cellular responses following sympathetic activation and/or NE stimulation.[23],[31] The programmed hypertension of developmental origin offers a unique model to allow for temporal separations on SNS activation, ROS production, T-lymphocyte activation in the spleen to the manifestation of BP phenotype; at the same time, allude to possible cause and effect relationships among them. First, the primed augmentation of tissue ROS levels is correlated positively, at a temporal manner, with the activation of splenic SNS and CD4+ and CD8+ immune T cells in young HFD offspring. Second, all these events in the spleen occurred at age (6 weeks) that precede the increase in BP phenotype (9 weeks of age) in HFD offspring. This time lag is interpreted to suggest egression of activated T cells from the spleen and consequent infiltration to target organs engaged in BP regulation. In support of this speculation, significant immune infiltration to the kidney and perivascular tissues precedes hypertension.[29],[40],[41] Third, SND that attenuated T-lymphocyte activation also ameliorated ROS production in the HFD spleen. A causal role of ROS in this neuroimmune communication was further consolidated from the observations that treatment with tempol, a superoxide scavenging antioxidant, significantly attenuated T-lymphocyte activations in response to splenic SNS activation induced by nicotine infusion. There are other mediators serving as neuroimmune links in the spleen. It has been reported that Ang II infusion leads to an increase in sympathetic activity that induces the production of placental growth factor in the spleen.[9],[42] Since the placental growth factor is required for T cell mobilization into target tissues, this factor has been postulated to serve as a neuroimmune link that connects the SNS with the splenic immune activation.

Another important finding of the current study is the illustration of the functional significance of the identified neuroimmune crosstalk in the pathogenesis of programmed hypertension in HFD offspring. We employed both gain- and loss-of-function approaches in this study to demonstrate that SBP in young ND offspring was notably increased on nicotine treatment that activated sympathetic activity and increased the production of pro-inflammatory cytokines in the spleen; whereas SND in the spleen attenuated T-lymphocyte activation and the programmed hypertension in HFD offspring. Both CD4+ and CD8+ T cells are engaged in the development of hypertension. In spontaneously hypertensive rats, the content of Th1 cells is increased and CD4+/CD8+ ratio is higher.[43] In a humanized mouse model in which the murine immune system is replaced by the human immune cells, CD4+ T cells in the thoracic lymph nodes, thoracic aorta, and kidney are increased in response to Ang II infusion.[36] In CD8+ knockout mice, the cardiovascular sensitivity to Ang II is significantly blunted and is restored by systemic adoptive transfer with CD8+ T cells.[44] It is also interesting to note that the induction of CD4+ T cells in the spleen due to reduced uterine perfusion pressure plays an important role in the pathophysiology of hypertension during pregnancy.[38] In addition, the increased cell populations of CD4+ Th1 cells and CD8+ cytotoxic T cells in the spleen and their infiltrations into the kidney are closely correlated with renal damage in several experimental models of hypertension[40],[41] and that observed in patients with hypertension.[36] Renal denervation, on the other hand, reduces the sympathetic innervation in the kidney and prevents immune cell activation and renal inflammation in Ang II-induced hypertension.[45] At the same time, human immunodeficiency virus-positive patients with low CD4+ T cell counts are less prone to hypertension than those on antiretroviral therapy with normal CD4+ T cell counts, who developed hypertension to a similar degree as the normal population.[46] It is noteworthy that our findings extend previous findings and provide new insights on the roles of splenic CD4+ and CD8+ T cells in the pathogenesis of adult hypertension of development origin. Together with the existing evidence, our findings highlight the functional significance of the neuroimmune crosstalk in the pathogenesis of hypertension with different underlying etiologies.

There are several limitations of the current study. First, although we demonstrated the contributing roles of CD4+ and CD8+ T cells in the pathogenesis of adult hypertension of development origin, the influence of maternal HFD on immune responses in different T cell subsets leading to pro-inflammatory condition in young offspring awaits further elucidation. At the same time, ROS production in different T cell subsets also warrants further investigation. In addition, it is unclear from the current study how SNS activation in the spleen stimulates ROS production in T-lymphocytes of HFD offspring. To this end, immune cells express primarily β adrenoceptors,[47] which bind the sympathetic neurotransmitter NE to initiate immuno-modulatory responses. Given that upregulation of β adrenoceptor mRNA is responsible for immune dysfunction in the spleen under disease conditions[48] and that ROS production can be induced by adrenoceptor activation,[33] additional experiments are required to dissect on the complexity of redox-dependent activation of T-lymphocyte subsets to better comprehend the neuroimmune crosstalk in the pathogenesis of programmed hypertension in adult offspring to maternal HFD insults during pregnancy and lactation. We also recognize that ROS levels, T-lymphocyte activation, and cytokine expressions were evaluated in the spleen of ND and HFD offspring only at a single time point (6 weeks of age) after splenic SND or the treatment with nicotine. Although this age in offspring is the time when the maximal changes in those parameters occurred, actions of these interventions on the same parameters at different ages of offspring remain to be evidenced. The causal role of splenic T-lymphocyte activation in hypertension programming in adult offspring to maternal HFD exposure also requires further investigation.


  Conclusion Top


In conclusion, the current study provides novel findings to suggest the sympathetic overexcitation in the spleen may prime hypertension programming in adult offspring via activation of T-lymphocytes induced by maternal HFD exposure. Moreover, tissue oxidative stress induced by the overexcited sympathetic innervation in the spleen could be a mediator that couples the neuroimmune crosstalk and primes hypertension programming in HFD offspring. Therefore, targeting the neuroimmune crosstalk in the spleen might be considered in the future as a new intervention for the prevention of adult hypertension of developmental origin.

Financial support and sponsorship

Research grants from the Ministry of Science and Technology, Taiwan (MOST106-2320-B-182A-005-MY3), and Chang Gung Medical Foundation (CMRPG8J0261) to J.Y.H. Chan. Y.M. Chao was supported by a Postdoctoral Fellowship (MOST108-2811-B-182A-511 and MOST109-2811-B-182A-524) from the Ministry of Science and Technology, Taiwan.

Conflicts of interest

There are no conflicts of interest.



 
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