Objective
Fetal alcohol syndrome (FAS) is associated with intellectual disability and neurodevelopmental abnormalities. Neuroprotective peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL) can prevent some of the alcohol-induced teratogenesis including fetal death, growth abnormalities, and learning impairment in part by preventing alcohol-induced alterations in N -methyl- D -aspartate receptor gene expression in a mouse model for FAS. We evaluated a panel of cytokines and chemokines to determine whether NAP plus SAL work through a cytokine/chemokine-mediated pathway in preventing these alterations.
Study Design
Using a well-characterized FAS model, timed, pregnant C57BL6/J mice were treated on gestational day (E) 8 with alcohol (0.03 mL/g), placebo, or alcohol plus peptides. Embryos were evaluated at 2 time points: after 6 hours and 10 days later at E18. A panel of cytokines/chemokines was measured using a microsphere-based multiplex immunoassay (Luminex xMAP; Millipore, Billerica, MA). Statistical analysis included Kruskal-Wallis, with P < .05 considered significant.
Results
Six hours after treatment, interleukin (IL)-6 and keratinocyte chemoattractant cytokine (KC) were not detectable in the control embryos. Alcohol treatment resulted in detectable levels and significant increases in IL-6 (median, 15.7; range, 10.1–45.9 pg/mL) and KC (median, 45.9; range, 32.5–99.1 pg/mL). Embryos exposed to alcohol plus NAP plus SAL had undetectable IL-6 and KC (both P < .003), similar to the controls. Alcohol exposure resulted in a significant increase of granulocyte colony–stimulating factor (G-CSF) ( P < .003) as compared with controls, and treatment with NAP plus SAL prevented the alcohol-induced increase. IL-13 and IL-1β were decreased 6 hours after alcohol exposure, and exposure to alcohol plus NAP plus SAL did not completely ameliorate the decrease. At E18, 10 days after exposure, these alterations were no longer present. Several analytes (regulated upon activation, normal T cell expressed, and secreted, tumor necrosis factor-α, interferon-γ, and IL-4) were not detectable at either time point in any of the groups.
Conclusion
Prenatal alcohol exposure acutely results in a significant elevation of IL-6, G-CSF and the KC, which are known to affect N -methyl- D -aspartate receptors. NAP plus SAL treatment prevented alcohol-induced increases. This provides additional insight into the mechanism of alcohol damage in FAS and NAP plus SAL prevention of neurodevelopmental anomalies.
In the United States, the prevalence of fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorder (FASD) is 1 in 100 births. Most, if not all, children afflicted with FAS or FASD will be susceptible to cognitive learning impairments that are life long. FAS and FASD are the most common, nongenetically inherited, preventable causes of intellectual disability. Neurodevelopmental abnormalities are common in FAS and neurobehavioral deficits in FASD.
To better understand the role of alcohol in the complex mechanisms that render the FAS phenotype, we utilized a well-established animal model (reported by Webster et al ). Induction of the dysfunctional regulatory mechanisms in FAS includes consideration of timing, dose, and duration of embryonic ethanol exposure.
During development, intercellular communication and function facilitated by signal transduction pathways are tightly regulated by growth factors, hormones, and cytokines. These cell-signaling proteins regulate trophoblast growth and cellular differentiation. Specifically the neuropoietic or gp130 cytokines interleukin (IL)-6, IL-13, granulocyte colony–stimulating factor (G-CSF) and keratinocyte chemoattractant cytokine (KC) mediate the tight regulation of the ongoing renewal of neuroepithelia/radial glia cells that are the precursors to neurons, astrocytes, and oligodendrocytes. These developmental processes may be modified by environmental factors such as alcohol and may have a life-long impact on gene regulation and disease susceptibility.
Previously we demonstrated that treatment with the neuroprotective peptides, NAPVSIPQ (NAP) plus SALLRSIPA (SAL) prevents alcohol-induced learning dysfunction as well as the concomitant fetal growth restriction, microcephaly, and oxidative damage. The peptides NAP and SAL are 8 and 9 amnio acids, respectively, and are derived from 2 neuroprotective proteins, activity-dependent neuroprotective protein (ADNP) and activity-dependent neuroprotective factor (ADNF), respectively. These proteins are released and regulated by vasoactive intestinal peptide, an important regulator of embryonic growth. Both ADNP and ADNF have been shown to have numerous neuroprotective mechanisms including enhancing survival in electronically blocked neuronal cells, preventing oxidative damage and altering transcription. NAP and SAL have also been shown to have neuroprotective properties and have been shown to reduce oxidative damage as well as prevent alterations in the N -methyl- D -aspartate and γ-aminobutyric acid receptors induced by alcohol.
In an earlier study, we found that prenatal alcohol exposure increased tumor necrosis factor-α (TNFα) and IL-6 levels in the embryo that was attenuated with treatment with NAP plus SAL. Given the role of cytokines and chemokines in development, we evaluated a panel of cytokines and chemokines in this model of FAS to evaluate their role. We also evaluated whether the neuroprotective properties of NAP plus SAL are mediated at least in part through cytokine release.
Materials and Methods
C57BL6/J female mice (Jackson Laboratories, Bar Harbor, ME) were kept under a 12 hour light, 12 hour dark regimen, with food and water available at all times. The mice received humane animal care in compliance with the National Institutes of Health guidelines for the care and use of experimental animals. The protocol was approved by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Animal Care and Use Committee.
Six-week-old females (21-24 g) were mated with C57BL6/J males for 4 hours. Presentation of the vaginal copulation plug was considered day 0 of pregnancy. Using a well-developed model of fetal alcohol syndrome, pregnant mice were treated on gestational day 8 intraperitoneally with 25% ethyl alcohol in saline (volume/volume) or vehicle alone at 0.03 mL/g body weight. The peptide doses, NAP and SAL (20 µg in 0.2 mL), were administered immediately prior to the alcohol/placebo treatment. NAP was diluted in 50 mL dimethylsulfoxide (99.97%) and diluted in filtered Dulbecco’s phosphate-buffered saline solution. SAL was dissolved and diluted in filtered Dulbecco’s phosphate-buffered saline solution. The peptide doses reflect previously established protective levels in the prevention of alcohol-induced fetal death, growth restriction, and microcephaly. There were 3 treatment groups: alcohol, placebo, and alcohol plus NAP plus SAL.
We chose 2 time points for evaluation, 1 acute and 1 long term to evaluate the panel of cytokines. The acute time point was after 6 hours to assess alcohol-induced alterations. To evaluate longer-term implications, we assessed after 10 days, on gestational day 18, which is 2 days before the typical delivery in a mouse. In 1 set of animals, after 6 hours, embryos were explanted using microdissection and homogenized in a buffer containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% Tween-20, and a cocktail of protease inhibitors (Roche, Indianapolis, IN). For the longer-term evaluation, after 10 days (gestational day [E] 18), the embryos were explanted and brain tissue was isolated and homogenized in a buffer containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% Tween-20, and a cocktail of protease inhibitors (Roche). The homogenates were centrifuged at 10,000 × g for 5 minutes. The supernatant was removed, analyzed for protein concentration and LUMINEX analysis (Millipore, Bedford, MA). Each gestational time point included at least 4 samples with each sample representing 3-7 litters (a gestation typically includes 8-10 embryos).
Luminex analysis was performed for G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IP-10, KC, macrophage chemoattractant protein-1, macrophage inflammatory protein-1α, regulated upon activation, normal T cell expressed, and secreted (RANTES), and TNF-α. Cytokine/chemokine levels were normalized to total protein. Cytokine/chemokine concentrations were determined using antibodies for the analytes covalently immobilized to a set of microspheres according to the protocol developed and validated at LINCO Research, Inc (St. Charles, MO). The analytes on the surface of microspheres were then detected by a cocktail of biotinylated antibodies. Following then binding of streptavidin-phycoerythrin conjugate, the reporter fluorescent signal was measured with a Luminex100 reader (Luminex Corp, Austin, TX).
Data were calculated using a calibration curve obtained in each experiment using the respective recombinant proteins diluted in kit matrix for plasma samples and lysis buffer for tissue samples. Concentrations of cytokines were calculated using StatLIAs software (Brendan Scientific Corp, Carlsbad, CA) with a 5 parameter logistic curve-fitting method and normalized to the amount of protein in each sample. Statistical analysis included Kruskal-Wallis for the comparison of cytokine levels, with P < .05 considered significant. Data are reported as median (range).
Results
Six hours after alcohol exposure, KC and IL-6 levels were not detectable in the control embryos. Alcohol treatment resulted in detectable levels and significant increases in KC (median, 45.9; range, 32.5–99.1 pg/mL) and IL-6 (median, 15.7; range, 10.1–45.9 pg/mL). The peptides NAP plus SAL prevented the alcohol-induced increase in KC and IL-6 with undetectable levels similar to control embryos (both P < .003), similar to control ( Figure ). Alcohol exposure also resulted in elevated levels of G-CSF, and treatment with NAP plus SAL prevented the alcohol-induced increase ( P < .003) ( Figure ). IL-13 and IL-1β were decreased 6 hours after the alcohol exposure, and exposure to alcohol plus NAP plus SAL did not completely ameliorate the decrease.
At E18, 10 days after exposure, the aforementioned alterations were no longer present. The majority of the analytes were not different between the 3 groups ( Table ). However, alcohol exposure resulted in an increase in GM-CSF and IL-15, and these increases were not prevented by NAP plus SAL. Several analytes (RANTES, IFN-γ, IL4) were not detectable at either time point in any of the groups ( Table ).
| Cytokine | 6 hour alcohol median (range) | 6 hour control median (range) | 6 hour alcohol plus peptide median (range) | 18 hour alcohol median (range) | 18 hour control median (range) | 18 hour alcohol plus peptide median (range) |
|---|---|---|---|---|---|---|
| G-CSF | 35.4 (20.2–44.3) a | 11.1 (9.6–33.4) a | 5.9 (3.6–7.9) a | 0.1 (0.1–4.3) | 0.1 (0.1–9.1) | 0.1 (0.1–0.1) |
| GM-CSF | 0.1 (0.1–0.1) | 0.1 (0.1–20.7) | 0.1 (0.1–0.1) | 20.7 (0.1–47.7) | 0.1 (0.1–31.8) | 20.7 (0.1–35.5) |
| IFN-γ | 0.1 (0.1–0.1) | 0.1 (0.1–4.0) | 0.1 (0.1–0.1) | 0.1 (0.1–7.2) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) |
| IL-10 | 0.1 (0.1–0.1) | 1.8 (0.1–4.5) | 1.6 (0.1–4.3) | 6.5 (3.0–10.5) | 5.2 (3.0–10.5) | 9.49 (6.2–12.9) |
| IL-12 (p70) | 0.1 (0.1–2.2) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) |
| IL-13 | 4.2 (0.1–7.7) a | 35.2 (30.8–49.2) a | 25.6 (3.8–37.7) a | 52.9 (2.9–150.7) | 39.3 (9.7–80.8) | 87.4 (34.7–164.6) |
| IL-15 | 28.3 (14.5–30.4) | 38.5 (0.1–44.4) | 7.0 (0.1–14.5) | 9.3 (0.1–28.3) | 1.1 (0.1–15.8) | 7.0 (0.1–17.0) |
| IL-17 | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) |
| IL-1α | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–16.3) | 0.1 (0.1–4.8) | 0.1 (0.1–0.1) |
| IL-1β | 24.4 (19.8–29.0) a | 48.2 (42.6–83.7) a | 39.9 (24.4–49.5) a | 69.1 (30.1–102.11) | 55.2 (40.0–72.9) | 79.6 (56.7–103.1) |
| IL-2 | 0.1 (0.1–0.1) | 3.3 (0.1–5.6) | 0.1 (0.1–3.4) | 7.6 (0.1–10.49) | 4.3 (0.1–6.3) | 8.9 (5.9–10.5) |
| IL-4 | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) |
| IL-5 | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) |
| IL-6 | 15.7 (10.2–45.7) a | 0.1 (0.1–0.1) a | 0.1 (0.1–0.1) a | 0.1 (0.1–4.8) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) |
| IL-7 | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–4.1) | 0.1 (0.1–0.1) | 0.1 (0.1–3.1) |
| IL-9 | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–109.0) | 0.1 (0.1–0.1) | 0.1 (0.1–102.8) |
| IL-10 | 14.6 (10.4–19.9) | 15.8 (12.2–25.9) | 7.3 (5.0–11.4) | 68.1 (34.74–95.5) | 54.4 (42.4–82.3) | 84.8 (56.8–115.6) |
| KC | 45.9 (32.5–99.1) a | 0.1 (0.1–15.3) a | 0.1 (0.1–0.1) a | 7.1 (0.1–16.7) | 6.9 (0.1–16.7) | 6.4 (0.1–13.8) |
| MCP-1 | 8.7 (6.6–18.1) | 6.6 (6.0–15.4) | 1.6 (0.1–7.2) | 25.4 (10.6–52.1) | 26.7 (18.9–40.7) | 34.7 (15.4–91.0) |
| MIP-1α | 46.8 (42.7–52.8) | 57.8 (51.2–4.8) | 57.8 (34.6–68.4) | 57.8 (28.3–98.2) | 36.2 (0.1–71.9) | 67.3 (52.0–89.7) |
| RANTES | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) |
| TNF-α | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) | 0.1 (0.1–0.1) |
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