Ethanol Induction of Innate Immune Signals Across BV2 Microglia and SH-SY5Y Neuroblastoma Involves Induction of IL-4 and IL-13
Abstract
:1. Introduction:
2. Methods
2.1. Cell Lines and Treatment
2.2. Transwell Co-Culture Model
2.3. Hippocampal Entorhinal (HEC) Slice Culture
2.4. Real-Time (RT)-PCR
2.5. Enzyme-Linked Immunosorbent Assay (Elisa)
2.6. Statistical Analysis
3. Results
3.1. Co-Culture Modifies bv2 Microglia and SH-sy5y Gene Expression and Ethanol-Induced Cytokines and Tlrs
3.2. Il-4 and IL-13 Expression Is Increased by Ethanol in Co-Cultured BV2 Microglia and SH-SY5Y
3.3. IL-4 and IL-13 Reduce Ethanol-Induced Tnfα and IL-1β in Hippocampal-Entorhinal Slice Culture
3.4. IL-10 and Tgfβ Are Increased by Ethanol in Co-Cultured BV2 Microglia and SH-SY5Y
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene | Species | Forward (5′–3′) | Reverse (5′–3′) |
---|---|---|---|
ADAM10 | Human | ATGGGAGGTCAGTATGGGAATC | ACTGCTCTTTTGGCACGCT |
β-Actin | Human | GAT GCA GAA GGA GAT CAC TGC | ATA CTC CTG CTT GCT GAT CCA |
Caspase 3 | Human | CTCTGGTTTTCGGTGGGTGT | GCTGCATCGACATCTGTACC |
CCR2 | Human | CCACATCTCGTTCTCGGTTTATC | CAGGGAGCACCGTAATCATAATC |
CD200 | Human | ACGTCTGTTACCAGCATCCTC | CTTAAAGTCGGTCACAGTCCC |
ChAT | Human | GGA GGT GGA GGG TTT GTG AC | ATT TCC TTG GCA CCC TGA GG |
CX3CL1 | Human | ACCACGGTGTGACGAAATG | TGTTGATAGTGGATGAGCAAAGC |
CXCL10 | Human | GACTCTGAGTGGAACTCAAGGAAT | GTGGCAATGATCTCAACACG |
DAP12 | Human | ACTGAGACCGAGTCGCCTTAT | ATACGGCCTCTGTGTGTTGAG |
DCX | Human | TTCAAGGGGATTGTGTACGCT | GTCAGACAGAGATCGCGTCAG |
DR3 | Human | AGAGATACTGACTGTGGGACC | CCCAGAACACACCTACTCTGC |
FADD | Human | GAAAACGCGCTCTTGTCGAT | GCCCGAGGCATAGGAACTTG |
Fas | Human | GTCTCCTGCGATGTTTGGC | TTCAAGGAAAGCTGATACCTATTTC |
HDAC1 | Human | CCGCATGACTCATAATTTGCTG | ATTGGCTTTGTGAGGGCGATA |
HDAC2 | Human | ATGGCGTACAGTCAAGGAGG | TGCGGATTCTATGAGGCTTCA |
HMGB1 | Human | GGA GAT CCT AAG AAG CCG AGA | CAT GGT CTT CCA CCT CTC TGA |
HMOX1 | Human | AAGACTGCGTTCCTGCTCAAC | AAAGCCCTACAGCAACTGTCG |
IKKβ | Human | CTGGCCTTTGAGTGCATCAC | CGCTAACAACAATGTCCACCT |
IL-10 | Human | GACTTTAAGGGTTACCTGGGTTG | TCACATGCGCCTTGATGTCTG |
IL-13 | Human | GAGGATGCTGAGCGGATTCTG | CACCTCGATTTTGGTGTCTCG |
IL-13R | Human | ACAACCTGAGCTACATGAAGTG | GGCTTCTGTGCCAATAGTAGAG |
IL-1β | Human | ATG ATG GCT TAT TAC AGT GGC AA | GTCGGAGATTCGTAGCTGGA |
IL-4 | Human | CCAACTGCTTCCCCCTCTG | TCTGTTACGGTCAACTCGGTG |
IL-4R | Human | ACACCAATGTCTCCGACACTC | TGTTGACTGCATAGGTGAGATGA |
iNOS | Human | TTCAGTATCACAACCTCAGCAAG | TGGACCTGCAAGTTAAAATCCC |
MCP1 | Human | CTCTCGCCTCCAGCATGAAA | AGGGTGTCTGGGGAAAGCTA |
MD-2 | Human | GAA GCT CAG AAG CAG TAT TGG GTC | GGT TGG TGT AGG ATG ACA AAC TCC |
NGFR | Human | CCTACGGCTACTACCAGGATG | CACACGGTGTTCTGCTTGT |
RAGE | Human | CTA CCG AGT CCG TGT CTA CCA | CAT CCA AGT GCC AGC TAA GAG |
TBK1 | Human | TGGGTGGAATGAATCATCTACGA | GCTGCACCAAAATCTGTGAGT |
TGFβ | Human | CAATTCCTGGCGATACCTCAG | GCACAACTCCGGTGACATCAA |
TH | Human | GGAAGGCCGTGCTAAACCT | GGATTTTGGCTTCAAACGTCTC |
TL1A | Human | TGCAGGACTCACCACATA | CTTGGCTTATCTCCGTCT |
TLR3 | Human | TTGCCTTGTATCTACTTTTGGGG | TCAACACTGTTATGTTTGTGGGT |
TLR4 | Human | CCTGCGTGGAGGTGGTTCC | AGAGGTGGCTTAGGCTCTGATA |
TLR7 | Human | GATAACAATGTCACAGCCGTCC | GTTCCTGGAGTTTGTTGATGTTC |
TNFα | Human | CCC AGG CAG TCA GAT CAT CTT CT | ATG AGG TAC AGG CCC TCT GAT |
TREM1 | Human | GAACTCCGAGCTGCAACTAAA | TCTAGCGTGTAGTCACATTTCAC |
TREM2 | Human | GGTCAGCACGCACAACTTG | CGCAGCGTAATGGTGAGAGT |
Arg1 | Mouse | TTAGGCCAAGGTGCTTGCTGCC | TACCATGGCCCTGAGGAGGTTC |
β-Actin | Mouse | GTA TGA CTC CAC TCA CGG CAA A | GGT CTC GCT CCT GGA AGA TG |
CCR2 | Mouse | ATCCACGGCATACTATCAACATC | CAAGGCTCACCATCATCGTAG |
CD200R | Mouse | TAAGGTGGAGGCATTTCCAGT | GATTCCAATGGCCGACAAAGTA |
CX3CR1 | Mouse | TCTTCACGTTCGGTCTGGTG | TGCACTGTCCGGTTGTTCAT |
DAP12 | Mouse | GAGTGACACTTTCCCAAGATGC | CCTTGACCTCGGGAGACCA |
DR3 | Mouse | CTCACCTTTCTCTTGTGTCCC | GTGCAGTCATTGCCACGTAT |
HMGB1 | Mouse | CGC GGA GGA AAA TCA ACT AA | TCA TAA CGA GCC TTG TCA GC |
IKKβ | Mouse | GGAGCCTGGGAAATGAAAGAA | GCCAGAGCCCTACCTGATTG |
IL-10 | Mouse | GGC AGA GAA GCA TGG CCC AGA A | GGC TGA GGC GCT GTC ATC GAT T |
IL-13 | Mouse | CCTGGCTCTTGCTTGCCTT | GGTCTTGTGTGATGTTGCTCA |
IL-13R | Mouse | TCAGCCACCTGTGACGAATTT | TGAGAGTGCAATTTGGACTGG |
IL-1β | Mouse | CTG GTG TGT GAC GTT CCC ATT A | CCG ACA GCA CGA GGC TTT |
IL-4 | Mouse | TGGGTCTCAACCCCCAGCTAGT | TGCATGGCGTCCCTTCTCCTGT |
IL-4R | Mouse | TCTGCATCCCGTTGTTTTGC | GCACCTGTGCATCCTGAATG |
iNOS | Mouse | GTGGTGACAAGCACATTTGG | CATGGTGAACACGTTCTTGG |
MCP1 | Mouse | ACT GAA GCC AGC TCT CTC TTC CTC | TTC CTT CTT GGG GTC AGC ACA GAC |
MD-2 | Mouse | CGC TGC TTT CTC CCA TAT TGA | CCT CAG TCT TAT GCA GGG TTC A |
RAGE | Mouse | GAA GGC TCT GTG GGT GAG TC | CCG CTT CCT CTG ACT GAT TC |
TBK1 | Mouse | ACTGGTGATCTCTATGCTGTCA | TTCTGGAAGTCCATACGCATTG |
TGFβ | Mouse | CTCCCGTGGCTTCTAGTGC | GCCTTAGTTTGGACAGGATCTG |
TLR3 | Mouse | GTG AGA TAC AAC GTA GCT GAC TG | TCC TGC ATC CAA GAT AGC AAG T |
TLR4 | Mouse | ATG GCA TGG CTT ACA CCA CC | GAG GCC AAT TTT GTC TCC ACA |
TLR7 | Mouse | ATG TGG ACA CGG AAG AGA CAA | GGT AAG GGT AAG ATT GGT GGT G |
TNFα | Mouse | GAC CCT CAC ACT CAG ATC ATC TTC T | CCT CCA CTT GGT GGT TTG CT |
TREM1 | Mouse | GACTGCTGTGCGTGTTCTTTG | GCCAAGCCTTCTGGCTGTT |
TREM2 | Mouse | CTGGAACCGTCACCATCACTC | CGAAACTCGATGACTCCTCGG |
β-Actin | Rat | CTACAATGAGCTGCGTGT | CAGGTCCAGACGCAGGAT |
IL-1β | Rat | TTGTGCAAGTGTCTGAAGCA | TGTCAGCCTCAAAGAACAGG |
TNFα | Rat | ATGTGGAACTGGCAGAGGAG | ACGAGCAGGAATGAGAAGAGG |
BV2 Microglia | ||||
---|---|---|---|---|
Gene | CON-Alone | EtOH-Alone | CON-CC | EtOH-CC |
NFκB signaling genes | ||||
CCR2 | 100 ± 11 | 185 ± 29 * | 177 ± 23 ns | 294 ± 16@@,## |
HMGB1 | 100 ± 12 | 109 ± 13 | 107 ± 6.9 | 67 ± 8.3 |
IKKβ | 100 ± 29 | 90 ± 21 | 367 ± 11 **** | 500 ± 45@@@@,# |
MCP1 | 100 ± 15 | 62 ± 21 | 58 ± 5.6 | 19 ± 0.7# |
MD-2 | 100 ± 21 | 147 ± 22 ns | 187 ± 5.9 * | 173 ± 22 |
RAGE | 100 ± 8.5 | 117 ± 12 | 342 ± 55 *** | 340 ± 33@@@ |
TBK1 | 100 ± 23 | 106 ± 22 | 631 ± 61 **** | 666 ± 65@@@@ |
Microglial markers | ||||
Arg1 | 100 ± 21 | 73 ± 15 | 160 ± 5.6 * | 137 ± 5.3@ |
CD200R | 100 ± 27 | 217 ± 46 ns | 320 ± 27 * | 658 ± 71@@@@,### |
CX3CR1 | 100 ± 23 | 122 ± 20 | 203 ± 32 * | 234 ± 20@ |
TREM signaling genes | ||||
DAP12 | 100 ± 6.6 | 187 ± 8.2 **** | 75 ± 2.3 ** | 124 ± 4.4@@@@,#### |
TREM1 | 100 ± 11 | 114 ± 18 | 401 ± 34 ns | 1144 ± 199@@@,## |
TREM2 | 100 ± 13 | 138 ± 3 | 86 ± 8.9 | 125 ± 7.1 |
TNF superfamily genes | ||||
DR3 | 100 ± 12 | 199 ± 36 ns | 341 ± 24 ns | 2261 ± 429@@@@,#### |
SH-SY5Y neuroblastoma | ||||
---|---|---|---|---|
Gene | CON-Alone | EtOH-Alone | CON-CC | EtOH-CC |
NFκB signaling genes | ||||
CCR2 | 100 ± 24 | 187 ± 26 | 98 ± 14 | 142 ± 45 |
CXCL10 | 100 ± 32 | 84 ± 12 | 210 ± 32 * | 85 ± 18# |
HMGB1 | 100 ± 5.9 | 71 ± 8.9 | 42 ± 12 | 102 ± 22# |
HMOX1 | 100 ± 8.5 | 580 ± 95 **** | 126 ± 7.5 | 186 ± 25 ns |
IKKβ | 100 ± 7.5 | 258 ± 57 * | 202 ± 12 | 490 ± 47@@,### |
MCP1 | 100 ± 29 | 42 ± 11 | 180 ± 19 * | 77 ± 7.7## |
MD-2 | n.d. | n.d. | n.d. | n.d. |
RAGE | 100 ± 6.2 | 233 ± 38 ns | 810 ± 63 **** | 796 ± 114@@@ |
TBK1 | 100 ± 33 | 185 ± 47 | 243 ± 24 ns | 642 ± 107@@@,## |
TNF superfamily and death receptor pathway genes | ||||
Caspase 3 | 100 ± 19 | 243 ± 34 ns | 525 ± 78 ** | 636 ± 134@@ |
DR3 | 100 ± 33 | 178 ± 33 | 561 ± 28 **** | 702 ± 70@@@@ |
FADD | 100 ± 7.1 | 124 ± 11.5 | 171 ± 18 | 330 ± 58@@@,# |
Fas | 100 ± 11.8 | 861 ± 352 ns | 1203 ± 357 ns | 2422 ± 238@ |
TL1A | n.d. | n.d. | 100 ± 19 | 43 ± 7.1# |
NGFR | 100 ± 12 | 5127 ± 471**** | 42 ± 1.7 | 331 ± 80@@@@ |
Neuron-specific genes | ||||
CD200 | 100 ± 26 | 183 ± 61 | 290 ± 14 ** | 604 ± 67@@@@,### |
CX3CL1 | 100 ± 6.3 | 123 ± 7.0 | 213 ± 17 **** | 274 ± 12@@@@, ## |
ChAT | 100 ± 16 | 144 ± 43 | 177 ± 29 | 234 ± 38 |
DCX | 100 ± 26 | 108 ± 32 | 729 ± 40 **** | 732 ± 54@@@@ |
TH | 100 ± 15 | 124 ± 40 | 668 ± 113 **** | 110 ± 27#### |
TREM signaling genes | ||||
DAP12 | 100 ± 20 | 70 ± 5.2 | 13701 ± 3140 *** | 11800 ± 1552@@@ |
TREM1 | 100 ± 23 | 177 ± 45 | 485 ± 221 | 1506 ± 343@@@,# |
TREM2 | 100 ± 23 | 140 ± 40 | 100 ± 14 | 172 ± 25 |
Proteases | ||||
ADAM10 | 100 ± 33 | 199 ± 65 | 700 ± 56 ** | 1429 ± 161@@@@,### |
Histone deacetylases | ||||
HDAC1 | 100 ± 11.7 | 131 ± 7.0 | 185 ± 8.8 | 336 ± 64@@ |
HDAC2 | 100 ± 11.7 | 193 ± 23 | 354 ± 72 * | 378 ± 84 |
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Lawrimore, C.J.; Coleman, L.G.; Zou, J.; Crews, F.T. Ethanol Induction of Innate Immune Signals Across BV2 Microglia and SH-SY5Y Neuroblastoma Involves Induction of IL-4 and IL-13. Brain Sci. 2019, 9, 228. https://doi.org/10.3390/brainsci9090228
Lawrimore CJ, Coleman LG, Zou J, Crews FT. Ethanol Induction of Innate Immune Signals Across BV2 Microglia and SH-SY5Y Neuroblastoma Involves Induction of IL-4 and IL-13. Brain Sciences. 2019; 9(9):228. https://doi.org/10.3390/brainsci9090228
Chicago/Turabian StyleLawrimore, Colleen J., Leon G. Coleman, Jian Zou, and Fulton T. Crews. 2019. "Ethanol Induction of Innate Immune Signals Across BV2 Microglia and SH-SY5Y Neuroblastoma Involves Induction of IL-4 and IL-13" Brain Sciences 9, no. 9: 228. https://doi.org/10.3390/brainsci9090228