Transcriptomic Analysis of Age-Associated Periventricular Lesions Reveals Dysregulation of the Immune Response
Abstract
:1. Introduction
2. Results
2.1. Histological Characterisation of Periventricular White Matter
2.2. Microarray Analysis
2.3. EnrichR
2.4. Computational Deconvolution of Microglial Gene Expression
2.5. Validation of Candidate Gene Expression by NanoString nCounter
2.6. Validation of Candidate Gene Expression by Immunohistochemistry
3. Discussion
4. Materials and Methods
4.1. Human CNS Tissue
4.2. Histological Characterisation
4.3. Laser Capture Microdissection (LCM)
4.4. Microarray
4.5. Transcriptomic Analysis of Microarrays
4.6. Deconvolution of the Microglial Transcriptome
4.7. Validation of Gene Expression Changes—NanoString
4.8. Validation of Gene Expression Changes—Immunohistochemistry
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ABC-HRP | avidin-biotin complex-horse radish peroxidase |
ACTB | actin cytoplasmic 1 |
AKT3 | V-akt murine thymoma viral oncogene homolog 3 |
BBB | blood brain-barrier |
BST2 | CD317, tethrin |
CACNA1E | calcium voltage-gated channel subunit alpha 1E |
CALM | calmodulin |
CAMK2A | calcium/calmodulin-dependent protein kinase II alpha |
CAMK4 | calcium/calmodulin-dependent protein kinase IV |
cAMP | cyclic adenosine monophosphateP |
CCL2 | C-C motif chemokine ligand 2 |
CCL3 | C-C motif chemokine ligand 3 |
CCL4 | C-C motif chemokine ligand 4 |
CCR5 | C-C motif chemokine receptor 5 (gene/pseudogene) |
CD163 | CD163 molecule, Scavenger Receptor Cysteine-Rich Type 1 Protein M130 |
CD274 | cluster of differentiation 274, programmed death-ligand 1 |
CD74 | CD74 molecule, HLA class II histocompatibility antigen gamma chain |
CD80 | co-stimulatory molecule B7-2 |
CD86 | cluster of differentiation 86, B7-2 |
CD8A | CD8a molecule, T-cell surface glycoprotein CD8 alpha chain |
cDNA | complementary DNA |
CFAS | Cognitive Function and Ageing Study |
CNS | central nervous system |
COL6A3 | collagen type VI alpha 3 chain |
CX3CR1 | C-X3-C motif chemokine receptor 1 |
CYBB | cytochrome b-245, beta polypeptide |
DAVID | Database for Annotation, Visualization and Integrated Discovery |
DEPC | diethylpyrocarbonate |
DSCL | deep subcortical lesions |
ENTPD1 | ectonucleoside triphosphate diphosphohydrolase 1 |
EPSTI1 | epithelial stromal interaction protein 1 |
FCER1G | Fc fragment of IgE receptor Ig |
FCGR1A | Fc fragment of immunoglobulin gamma Fc receptor 1A |
FCGR2A | Fc fragment of immunoglobulin gamma Fc receptor 2A |
FGL2 | fibrinogen like 2 |
FPR1 | formyl peptide receptor 1 |
GABA | gamma-aminobutyric acid |
GAPDH | glyceraldehyde-3-phosphate dehydrogenase |
GBP5 | guanylate-binding protein 5 |
GEO | gene expression omnibus |
GRIA | glutamate receptor, ionotropic, AMPA |
GRIN1 | glutamate receptor, ionotropic, N-methyl D-aspartate 1 |
H&E | haematoxylin and eosin |
HAVCR2 | hepatitis A virus cellular receptor 2 |
HLA-DMB | major histocompatibility complex, class II, DM beta |
HLA-DPA1 | major histocompatibility complex, class II, DP alpha 1 |
HLA-DPB1 | major histocompatibility complex, class II, DP beta 1 |
HLA-DQB1 | major histocompatibility complex, class II, DQ beta 1 |
HLA-DRA | major histocompatibility complex, class II, DR alpha |
IFI35 | interferon induced protein 35 |
IFI44 | interferon induced protein 44 |
IFI44L | interferon induced protein 44-like |
IFIT1 | interferon induced protein with tetratricopeptide repeats 1 |
IFIT3 | interferon induced protein with tetratricopeptide repeats 3 |
IFNAR1 | interferon alpha receptor subunit 1 |
IGSF6 | immunoglobulin superfamily member 6 |
IHC | immunohistochemistry |
IKBKB | inhibitor of nuclear factor kappa B kinase subunit beta |
IL1A | interleukin 1 alpha |
IL1B | interleukin 1 beta |
IRF7 | interferon regulatory factor 7 |
IRF9 | interferon regulatory factor 9 |
ISG15 | interferon-stimulated gene 15 |
ITGAX | integrin, alpha X (complement component 3 receptor 4 subunit), CD11c |
IVT | in vitro transcription |
JUN | jun proto-oncogene, AP-1 transcription factor subunit |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
LCM | laser capture microdissection |
LCP2 | lymphocyte cytosolic protein 2 |
LFB | luxol fast blue |
LY6E | lymphocyte antigen 6E |
MAPK | mitogen-activated protein kinase |
MHC-II | major histocompatibility complex class II |
MRC1 | mannose receptor C-type 1 |
MRI | magnetic resnance imaging |
mRNA | messenger ribonucleic acid |
MS4A6A | membrane spanning 4-domains A6A |
MSR1 | macrophage scavenger receptor 1 |
MX1 | MX dynamin like GTPase 1 |
OAS1 | 2’-5’-oligoadenylate synthetase 1 |
OAS2 | 2’-5’-oligoadenylate synthetase 2 |
OAS3 | 2’-5’-oligoadenylate synthetase 3 |
PCA | principal component analysis |
PHF21A | PHD finger protein 21A |
PI3K-Akt | phosphatidylinositol 3-kinase-protein kinase |
PTGS2 | prostaglandin-endoperoxide synthase 2 |
PVL | periventricular lesions |
QC | quality control |
REC | research ethics committee |
RPLP0 | 60S acidic ribosomal protein P0 |
RSAD2 | radical S-adenosyl methionine domain containing 2 |
RT | room temperature |
SAMSN1 | SAM domain, SH3 domain and nuclear localisation signals 1 |
SLAMF8 | SLAM family member 8 |
TAC | transcriptome analysis console |
TFEC | transcription factor EC |
TREM2 | triggering receptor expressed on myeloid cells 2 |
UBE2L6 | ubiquitin conjugating enzyme E2 L6 |
USP18 | ubiquitin specific peptidase 18 |
WM | white matter |
WML | white matter lesions |
XAF1 | XIAP-associated factor 1 |
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Pathway | Count | p-Value |
---|---|---|
hsa04612: Antigen processing and presentation | 20 | 9.17 × 10−6 |
hsa04145: Phagosome | 24 | 0.003 |
hsa04672: Intestinal immune network for IgA production | 11 | 0.004 |
hsa04662: B cell receptor signalling pathway | 12 | 0.026 |
Pathway | Count | p-Value |
---|---|---|
hsa04020:Calcium signalling pathway | 42 | 3.38 × 10−9 |
hsa04728:Dopaminergic synapse | 34 | 5.29 ×10−9 |
hsa04724:Glutamatergic synapse | 30 | 6.28 × 10−8 |
hsa04725:Cholinergic synapse | 29 | 1.29 × 10−7 |
hsa04727:GABAergic synapse | 23 | 1.82 × 10−6 |
hsa04612:Antigen processing and presentation | 20 | 1.58 × 10−5 |
hsa04024:cAMP signalling pathway | 34 | 1.54 × 10−4 |
hsa04010:MAPK signalling pathway | 40 | 2.23 × 10−4 |
hsa04145:Phagosome | 25 | 0.002 |
hsa04062:Chemokine signalling pathway | 28 | 0.005 |
hsa04151:PI3K-Akt signalling pathway | 45 | 0.005 |
hsa04660:T cell receptor signalling pathway | 17 | 0.011 |
hsa04662:B cell receptor signalling pathway | 13 | 0.014 |
hsa04068:FoxO signalling pathway | 19 | 0.038 |
Pathway | Count | p-Value |
---|---|---|
hsa04020:Calcium signalling pathway | 46 | 4.71 × 10−11 |
hsa04724:Glutamatergic synapse | 29 | 3.97 ×10−7 |
hsa04727:GABAergic synapse | 21 | 3.58 × 10−5 |
hsa04725:Cholinergic synapse | 23 | 2.30 × 10−4 |
hsa04310:Wnt signalling pathway | 26 | 3.83 × 10−4 |
hsa04024:cAMP signalling pathway | 33 | 5.36 × 10−4 |
hsa04022:cGMP-PKG signalling pathway | 28 | 5.96 × 10−4 |
hsa04728:Dopaminergic synapse | 22 | 0.0039 |
hsa04010:MAPK signalling pathway | 34 | 0.0143 |
Pathway/Functional Group | p-Value |
---|---|
KEGG pathway | |
hsa04612: Antigen processing and presentation | 6.77 × 10−6 |
hsa05169: Epstein-Barr virus infection | 1.06 × 10−4 |
hsa05215: Prostate cancer | 1.49 × 10−4 |
hsa05140: Leishmaniasis | 4.58 × 10−4 |
hsa05168: Herpes simplex infection | 8.04 × 10−4 |
hsa05332: Graft-versus-host disease | 8.61 × 10−4 |
hsa04940: Type I diabetes mellitus | 0.0012 |
hsa04915: Estrogen signalling pathway | 0.0015 |
hsa05330: Allograft rejection | 0.003 |
hsa05164: Influenza A | 0.0031 |
Panther pathway | |
P00031: Inflammation mediated by chemokine and cytokine signalling | 1.20 × 10−4 |
P00053: T cell activation | 0.0068 |
P02756: N-acetylglucosamine metabolism | 0.0199 |
P02776: Serine glycine biosynthesis | 0.0199 |
P00010: B cell activation | 0.0217 |
P00007: Axon guidance mediated by semaphorins | 0.0257 |
P00049: Parkinson disease | 0.0359 |
P00054: Toll receptor signalling pathway | 0.0391 |
P00006: Apoptosis signalling pathway | 0.0426 |
P04386: Histamine H2 receptor mediated signalling pathway | 0.0443 |
Gene Ontology (cellular component) | |
GO:0042613: MHC class II protein complex | 1.24 × 10−4 |
GO:0016607: nuclear speck | 3.02 × 10−4 |
GO:0045121: membrane raft | 4.73 × 10−4 |
GO:0012507: ER to Golgi transport vesicle membrane | 5.67 × 10−4 |
GO:0030671: clathrin-coated phagocytic vesicle membrane | 9.54 × 10−4 |
GO:0016606: LYSP100-associated nuclear domain | 0.0022 |
GO:0010445: nuclear dicing body | 0.0022 |
GO:0071601: sphere organelle | 0.0022 |
GO:0016604: nuclear body | 0.0022 |
GO:0035363: histone locus body | 0.0023 |
Group A (Inactivated Microglia) | Group B (Activated Microglia) |
---|---|
ISG15 | HLA-DPB1 |
MX1 | HLA-DQB1 |
IFI44L | HLA-DMB |
OAS2 | HLA-DPA1 |
IFI35 | LCP2 |
OAS1 | FCER1G |
LY6E | GBP5 |
IFIT1 | CYBB |
IFI44 | SAMSN1 |
UBE2L6 | SLAMF8 |
IFIT3 | MS4A6A |
IRF7 | IGSF6 |
XAF1 | HAVCR2 |
RSAD2 | TFEC |
OAS3 | CD163 |
IRF9 | FCGR2A |
USP18 | MSR1 |
Microarray | NanoString | |||
---|---|---|---|---|
Gene | Fold Change | p-Value | Fold Change | p-Value |
AKT3 | 1.33 | 0.002 ** | 1.52 | 0.127 |
CCL2 | −1.7 | 0.05 * | −1.88 | 0.290 |
CCL3 | −1.31 | 0.05 * | −16.11 | 0.008 ** |
CCL4 | −1.16 | 0.060 | −5.14 | 0.098 |
CCR5 | −1.49 | 0.01 * | −2.17 | 0.0003 *** |
CD163 | −3.27 | 0.315 | −1.07 | 0.928 |
CD74 | −2.67 | 0.006 ** | −1.54 | 0.013 * |
CD8A | −1.28 | 0.016 * | −1.23 | 0.567 |
COL6A3 | −1.62 | 0.01 * | −1.72 | 0.074 |
CX3CR1 | −1.5 | 0.005 ** | −1.64 | 0.01 * |
CYBB | −1.62 | 0.013 * | −1.17 | 0.577 |
EPSTI1 | −1.31 | 0.839 | −1.39 | 0.101 |
FCER1G | −1.52 | 0.076 | −1.28 | 0.265 |
FGL2 | −1.77 | 0.012 * | −1.29 | 0.190 |
FPR1 | −2.39 | 0.017 * | −2.82 | 0.066 |
HLA-DRA | −1.71 | 0.004 ** | −1.51 | 0.071 |
IFNAR1 | 1.79 | 0.004 ** | 1.25 | 0.190 |
IGSF6 | −1.27 | 0.257 | −1.12 | 0.585 |
IKBKB | 1.38 | 0.839 | 1.88 | 0.097 |
IL1A | −1.06 | 0.206 | −1.71 | 0.061 |
IL1B | −1.34 | 0.01 * | −4.72 | 0.007 ** |
IRF7 | 1.04 | 0.673 | −1.04 | 0.846 |
JUN | 1.33 | 0.008 ** | 1.98 | 0.008 ** |
MSR1 | −1.95 | 0.023 * | −1.06 | 0.877 |
OAS1 | −1.12 | 0.83 | 1.14 | 0.644 |
PHF21A | 1.36 | 0.008 ** | 1.5 | 0.121 |
PTGS2 | −1.3 | 0.011 * | −2.59 | 0.007 ** |
RSAD2 | −1.2 | 0.065 | 1.09 | 0.757 |
SLAMF8 | −1.03 | 0.679 | −1.48 | 0.327 |
WM Group | Control | Pre-Lesional | PVL |
---|---|---|---|
CD74 | 3.47 (3.08–4.15) | 1.66 (0.9–2.14) | 1.3 (1.13–1.66) |
IL-1β | 3.87 (1.91–4.86) | 2.76 (1.91–4.07) | 3.43 (2.48–4.22) |
CD163 | 0.93 (0.75–1.21) | 0.39 (0.29–1.08) | 0.95 (0.59–1.32) |
CD86 | 4.55 (4.52–5.66) | 5.34 (3.98–6.23) | 5.04 (3.35–6.03) |
Experimental Approach | Classification | Median Age (y) (Range) | Gender (M/F) | PMD (h) (Range) |
---|---|---|---|---|
Microarray | Control (n = 11) | 84 (70–89) | 4/7 | 11 (6–42) |
PVL (n = 7) | 89 (85–95) | 3/4 | 28 (7–72) | |
NanoString | Control (n = 7) | 84 (70–89) | 2/5 | 14 (6–42) |
PVL (n = 5) | 88 (85–95) | 2/3 | 28 (18–72) |
Antibody | Isotype | Dilution (Time, Temperature) | Supplier |
---|---|---|---|
CD163 | Monoclonal Mouse IgG | 1:100 (1 h RT) | Bio-Rad, UK |
CD68 (PG-M1) | Mouse, IgG3κ | 1:100 (1 h RT) | Dako, UK |
CD74 | Polyclonal Rabbit IgG | 1:200 (1 h RT) | Sigma, UK |
CD80/B7-1 | Monoclonal Mouse IgG1 | 1:20 (overnight 4 °C) | R&D Systems, UK |
CD86/B7-2 | Polyclonal goat IgG | 1:20 (overnight 4 °C) | R&D Systems, UK |
CX3CR1 | Monoclonal Mouse IgG1 | 1:25 (overnight 4 °C) | Biolegend, UK |
IL-1β | Polyclonal Rabbit IgG | 1:200 (1 h RT) | Proteintech, UK |
MHC-II (HLA-DR) | Mouse Monoclonal IgG | 1:20 (1 h RT) | Dako, UK |
Gene Symbol | Gene Name | |
---|---|---|
Group A | CD317 (BST2) | Bone marrow stromal cell antigen 2 |
Group B | CD39 (ENTPD1) | Ectonucleoside Triphosphate Diphosphohydrolase 1 |
HLA-DRA | Major Histocompatibility Complex, Class II, DR Alpha | |
CD86 | Cluster of differentiation 86 | |
CD80 | Cluster of differentiation 80 | |
CD274 | Cluster of differentiation 274 | |
TIMD4 | T Cell Immunoglobulin and Mucin Domain Containing 4 | |
CD11C (ITGAX) | Integrin Subunit Alpha X | |
TREM2 | Triggering Receptor Expressed on Myeloid Cells 2 | |
CCR5 | C-C chemokine receptor type 5 | |
MRC1 | Mannose Receptor C-Type 1 |
Function | Gene Symbol | Gene Name |
---|---|---|
Inflammation | AKT3 | V-akt murine thymoma viral oncogene homolog 3/RAC-gamma serine/threonine-protein kinase |
CCL2 | C-C motif chemokine ligand 2 | |
CCL3 | C-C motif chemokine ligand 3 | |
CCL4 | C-C motif chemokine ligand 4 | |
CCR5 | C-C motif chemokine receptor 5 (gene/pseudogene) | |
COL6A3 | Collagen type VI alpha 3 chain | |
CX3CR1 | C-X3-C motif chemokine receptor 1 | |
IFNAR1 | Interferon alpha and beta receptor subunit 1 | |
IKBKB | Inhibitor of nuclear factor kappa B kinase subunit beta | |
IL1A | Interleukin 1 alpha | |
IL1B | Interleukin 1 beta | |
JUN | Jun proto-oncogene, AP-1 transcription factor subunit | |
PTGS2 | Prostaglandin-endoperoxide synthase 2 | |
Antigen processing and presentation | CD74 | CD74 molecule, HLA class II histocompatibility antigen gamma chain |
HLA-DRA | Major histocompatibility complex, class II, DR alpha | |
CD8A | CD8a molecule, T-cell surface glycoprotein CD8 alpha chain | |
CD163 | CD163 molecule, Scavenger Receptor Cysteine-Rich Type 1 Protein M130 | |
CD14 Monocytes | EPSTI1 | Epithelial stromal interaction protein 1 |
FGL2 | Fibrinogen like 2 | |
FPR1 | Formyl peptide receptor 1 | |
IGSF6 | Immunoglobulin superfamily member 6 | |
PHF21A | PHD finger protein 21A | |
Inactivated Microglia | IRF7 | Interferon regulatory factor 7 |
OAS1 | 2’-5’-oligoadenylate synthetase 1 | |
RSAD2 | Radical S-adenosyl methionine domain containing 2 | |
Activated Microglia | FCER1G | Fc fragment of IgE receptor Ig |
MSR1 | Macrophage scavenger receptor 1 | |
SLAMF8 | SLAM family member 8 | |
CYBB | Cytochrome b-245, beta polypeptide | |
Housekeeping Genes | ACTB | Actin cytoplasmic 1 |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase | |
RPLP0 | 60S acidic ribosomal protein P0 |
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Fadul, M.M.; Heath, P.R.; Cooper-Knock, J.; Kurz, J.M.; Al-Azzawi, H.A.; Ali, Z.; Smith, T.; Matthews, F.E.; Brayne, C.; Wharton, S.B.; et al. Transcriptomic Analysis of Age-Associated Periventricular Lesions Reveals Dysregulation of the Immune Response. Int. J. Mol. Sci. 2020, 21, 7924. https://doi.org/10.3390/ijms21217924
Fadul MM, Heath PR, Cooper-Knock J, Kurz JM, Al-Azzawi HA, Ali Z, Smith T, Matthews FE, Brayne C, Wharton SB, et al. Transcriptomic Analysis of Age-Associated Periventricular Lesions Reveals Dysregulation of the Immune Response. International Journal of Molecular Sciences. 2020; 21(21):7924. https://doi.org/10.3390/ijms21217924
Chicago/Turabian StyleFadul, Motaz M., Paul R. Heath, Johnathan Cooper-Knock, Julian M. Kurz, Hayder A. Al-Azzawi, Zarki Ali, Taylor Smith, Fiona E. Matthews, Carol Brayne, Stephen B. Wharton, and et al. 2020. "Transcriptomic Analysis of Age-Associated Periventricular Lesions Reveals Dysregulation of the Immune Response" International Journal of Molecular Sciences 21, no. 21: 7924. https://doi.org/10.3390/ijms21217924