Differential gene expression in peripheral blood mononuclear cells from children immunized with inactivated influenza vaccine

Figures & data

Table 1. Demographic characteristics of children in the study.

Demographics	Overall N = 16 N (%)	Day 0-Day 3 group N = 10 N (%)	Day 0-Day 7 group N = 6 N (%)	P Value
Age				0.588
3–8 years	5 (31.2)	4 (40.0)	1 (16.7)
9–18 years	11 (68.8)	6 (60.0)	5 (83.3)
Sex, ref. = male				0.604
Female	12 (75.0)	8 (80.0)	4 (66.7)
Race, ref. = non-black				0.500
Black	14 (87.5)	8 (80.0)	6 (100.0)
Parental education level, ref. = some college or higher				0.607
High school or less	10 (62.7)	7 (70.0)	3 (50.0)
Health Insurance, ref. = private
Public insurance	14 (87.5)	8 (80.0)	6 (100.0)	0.500
Parent’s smoking status, ref. = yes				0.600
No	10 (62.5)	6 (60.0)	4 (80.0)

Table 2. HI antibody titer data for children in the study.

Virus	Seroprotection d0	Seroprotection d21	Seroconversion d21
H1N1 CA/07/09	13/13	13/13	5/13
H3N2 HK/4801/14	7/13	12/13	7/13
B Brisbane 60/08	6/13	11/13	9/13
B Phuket 3073/13	7/13	11/13	5/13

Table 3. Common DEGs on day 0 versus day 3 and day 0 versus day 7 post influenza vaccination.

DEGs on day 0 vs. 3 and 0 vs. 7
Increased both	D3 down, D7 up
NCAPG	UBE2O
CHAC2	CKAP4
FANCL	MEIS1
CERS6-AS1	LTF
CCDC73	TTLL7
ENAM	FOXO3
TSIX	SLC35A2
CNTD1	BMP6
NUP62CL	ANKRD9
	MAP1LC3B2
Decreased Both	HSPA5
CHIT1	PROS1
DLG5	GGTA1P
TTC38
MAPK7	D3 Up, D7 Down
AVPI1	WASL
HBEGF
RN7SL600P
ARRDC2
PDZD4
ENC1

Table 4. Enriched pathways from DEGs between day 0 and day 3 post influenza vaccination.

Ingenuity canonical pathways	-log p value	Molecules (italics = increased expression on Day 3)
Role of PKR in Interferon Induction and Antiviral Response	5.28	FADD,NFKBID,RELA,CASP9,NFKBIA,CASP3,NFKBIE,NFKB2,NFKBIB,RNASEL,IRF1
TWEAK Signaling	5.12	FADD,NFKBID,RELA,CASP9,NFKBIA,CASP3,NFKBIE,NFKB2,NFKBIB,TNFRSF12A
CD27 Signaling in Lymphocytes	4.15	NFKBID,RELA,SIVA1,MAP3K11,CASP9,NFKBIA,CASP3,NFKBIE,MAP3K8,NFKB2,NFKBIB
CD40 Signaling	3.7	RELA,ICAM1,ATF1,NFKBIE,PIK3R5,TNFAIP3,NFKB2,NFKBID,NFKBIA,IRS2,PTGS2,NFKBIB,MAPKAPK2
TNFR1 Signaling	3.68	FADD,NFKBID,RELA,CASP9,NFKBIA,CASP3,NFKBIE,TNFAIP3,NFKB2,NFKBIB
Role of BRCA1 in DNA Damage Response	3.64	FANCM,GADD45A,MSH2,ATF1,CDKN1A,SMARCB1,FANCF,PLK1,BLM,BRCA1,RBL1,HLTF,FANCL
Induction of Apoptosis by HIV1	3.57	FADD,DIABLO,NFKBID,RELA,CASP9,NFKBIA,CASP3,CXCR4,NFKBIE,NFKB2,NFKBIB
MIF-mediated Glucocorticoid Regulation	3.46	PLA2G4A,NFKBID,RELA,NFKBIA,NFKBIE,PTGS2,NFKB2,NFKBIB
IL-17A Signaling in Fibroblasts	3.46	NFKBID,RELA,NFKBIA,NFKBIE,CEBPB,NFKB2,NFKBIZ,NFKBIB
Angiopoietin Signaling	3.43	NFKBID,RELA,NFKBIA,CASP9,RALA,NFKBIE,SOS1,PIK3R5,IRS2,NFKB2,NCK1,NFKBIB,RASA1
PI3K/AKT Signaling	3.39	RELA,RALA,NFKBIE,PPP2R5B,NFKB2,EIF4E,INPP5D,NFKBID,NFKBIA,FOXO3,CDKN1A,SOS1,MAP3K8, PTGS2,INPP5K,SFN,NFKBIB
p53 Signaling	3.21	PMAIP1,GADD45B,GADD45G,TNFRSF10B,PIK3R5,CSNK1D,HIF1A,TP53BP2,GADD45A,THBS1,CCNK, CDKN1A,IRS2,SFN,BRCA1
Lymphotoxin β Receptor Signaling	3.21	DIABLO,NFKBID,RELA,CASP9,NFKBIA,CASP3,RELB,TRAF4,PIK3R5,IRS2,NFKB2
TNFR2 Signaling	3.15	NFKBID,RELA,NFKBIA,NFKBIE,TNFAIP3,NFKB2,NFKBIB
Role of IL-17A in Arthritis	3.1	NFKBID,RELA,CXCL8,NFKBIA,NFKBIE,PIK3R5,IRS2,NFKB2,PTGS2,NFKBIB,MAPKAPK2
Sirtuin Signaling Pathway	2.87	RELA,TOMM40L,GADD45B,GADD45G,HIF1A,NDUFB3,ATG9B,ATG4C,GADD45A,CRTC2,GABARAPL1,SIRT6,FOXO3,MAP1LC3A,MAP1LC3B,PFKFB3,NDUFAF1,CXCL8,EPAS1,PPIF,TUBA4A,SF3A1,NFKB2,MAP1LC3B2,ATG2A,SIRT2,NR1H2,MAPK7
Small Cell Lung Cancer Signaling	2.85	NFKBID,RELA,CASP9,NFKBIA,NFKBIE,TRAF4,PIK3R5,IRS2,NFKB2,PTGS2,NFKBIB,SKP2
MIF Regulation of Innate Immunity	2.83	PLA2G4A,NFKBID,RELA,NFKBIA,NFKBIE,PTGS2,NFKB2,NFKBIB
IL-6 Signaling	2.8	SOCS3,RELA,CXCL8,RALA,NFKBIE,PIK3R5,CEBPB,IL1R1,NFKB2,VEGFA,NFKBID,NFKBIA,SOS1,IRS2, MAPKAPK2,NFKBIB
Hypoxia Signaling in the Cardiovascular System	2.79	VEGFA,NFKBID,NFKBIA,NFKBIE,CREB3,CSNK1D,HIF1A,UBE2S,UBE2O,NFKBIB,UBE2I
Erythropoietin Signaling	2.76	SOCS3,NFKBID,RELA,RALA,NFKBIA,NFKBIE,SOS1,PIK3R5,PRKCE,IRS2,NFKB2,NFKBIB
iNOS Signaling	2.7	NFKBID,RELA,NFKBIA,NFKBIE,NFKB2,NFKBIB,IRF1,IRAK2
Hereditary Breast Cancer Signaling	2.69	POLR2J2/POLR2J3,FANCM,RALA,GADD45B,GADD45G,FANCF,PIK3R5,FANCL,MSH2,GADD45A,CDKN1A, SMARCB1,IRS2,BRCA1,SFN,BLM,HLTF
GADD45 Signaling	2.63	GADD45B,GADD45A,GADD45G,CDKN1A,BRCA1
Prostate Cancer Signaling	2.59	NFKBID,RELA,NFKBIA,CASP9,RALA,NFKBIE,SOS1,CDKN1A,CREB3,PIK3R5,IRS2,NFKB2,NFKBIB
IL-10 Signaling	2.55	SOCS3,NFKBID,RELA,CCR5,NFKBIA,NFKBIE,IL10RA,NFKB2,IL1R1,NFKBIB
NF-κB Activation by Viruses	2.51	NFKBID,RELA,CCR5,RALA,NFKBIA,NFKBIE,ITGAV,PIK3R5,PRKCE,IRS2,NFKB2,NFKBIB
Death Receptor Signaling	2.51	TNFRSF21,FADD,DIABLO,NFKBID,RELA,CASP9,NFKBIA,CASP3,NFKBIE,TNFRSF10B,NFKB2,NFKBIB

Table 5. Enriched pathways from DEGs between day 0 and day 7 post influenza vaccination.

Ingenuity canonical pathways	-log p value	Molecules (italics = decreased expression on Day 7)
Actin Cytoskeleton Signaling	4.32	MYL9,ARHGEF12,ACTA2,PDGFA,DIAPH3,WASL,EGF,TLN1,MYLK,VCL,GSN,IQGAP1,PDGFC, ACTN1,MATK
Arginine Degradation VI (Arginase 2 Pathway)	3.87	ARG2,PYCR1,ARG1
Extrinsic Prothrombin Activation Pathway	3.69	PROS1,F5,F13A1,TFPI
Integrin Signaling	3.48	MYL9,ITGA2B,ACTA2,LIMS1,WASL,TLN1,MYLK,VCL,GSN,CTTN,ITGB5,ACTN1,ITGB3
Primary Immunodeficiency Signaling	3.43	IGHG3,IGLL1/IGLL5,IGHG4,TNFRSF13B,IGHG1,IGHG2
Coagulation System	3.29	PROS1,F5,F13A1,VWF,TFPI
Epithelial Adherens Junction Signaling	3.18	MYL9,TUBB1,ACTA2,TGFBR3,WASL,EGF,VCL,IQGAP1,PARD3,ACTN1
Sertoli Cell-Sertoli Cell Junction Signaling	3.05	EPB41,TUBB1,PRKAR2B,CLDN5,TJP2,ACTA2,JAM3,TGFBR3,VCL,GUCY1B1,ACTN1
Oxidative Phosphorylation	2.92	MT-CO1,MT-ATP6,MT-ND1,MT-CYB,MT-CO3,MT-ND4,MT-CO2,MT-ND3
Tight Junction Signaling	2.83	MYL9,FOS,EPB41,PRKAR2B,CLDN5,TJP2,ACTA2,JAM3,MYLK,VCL
Remodeling of Epithelial Adherens Junctions	2.68	TUBB1,ACTA2,DNM3,VCL,IQGAP1,ACTN1
Arginine Degradation I (Arginase Pathway)	2.67	ARG2,ARG1
Clathrin-mediated Endocytosis Signaling	2.64	STON2,ACTA2,PDGFA,WASL,EGF,DNM3,CTTN,PDGFC,ITGB5,CLU,ITGB3
ERK5 Signaling	2.62	SH2D2A,FOS,FOXO3,EGF,MAPK7,WNK1
Autoimmune Thyroid Disease Signaling	2.62	IGHG3,GZMB,IGHG4,IGHG1,IGHG2
Hematopoiesis from Pluripotent Stem Cells	2.62	CD247,IGHG3,IGHG4,IGHG1,IGHG2

Table 6. Enriched pathways from DEGs between younger and older children post influenza vaccination.

Ingenuity canonical pathways	-log p value	Molecules (italics = decreased expression in Older)
IL-23 Signaling Pathway	5.27	IL12RB2,IL1B,IL23R,NFKBIA,RORC,TNF
Differential Regulation of Cytokine Production in Macrophages and T Helper Cells by IL-17A and IL-17F	4.54	CCL3,CCL4,IL1B,TNF
Communication between Innate and Adaptive Immune Cells	4.24	CCL3,CCL3L3,CCL4,CD83,IL1B,TNF,TNFRSF17
Differential Regulation of Cytokine Production in Intestinal Epithelial Cells by IL-17A and IL-17F	4.1	CCL3,CCL4,IL1B,TNF
Granulocyte Adhesion and Diapedesis	4.02	CCL3,CCL3L3,CCL4,CCL4L1/CCL4L2,IL1B,ITGB2,PPBP,TNF,XCL1
TREM1 Signaling	3.93	CCL3,CD83,IL1B,NLRP3,NOD2,TNF
Agranulocyte Adhesion and Diapedesis	3.79	CCL3,CCL3L3,CCL4,CCL4L1/CCL4L2,IL1B,ITGB2,PPBP,TNF,XCL1
TNFR2 Signaling	3.63	NFKBIA,NFKBIE,TNF,TNFAIP3
PPAR Signaling	3.16	IL1B,INSR,NFKBIA,NFKBIE,PDGFD,TNF
Role of Hypercytokinemia/ hyperchemokinemia in the Pathogenesis of Influenza	3.02	CCL3,CCL4,IL1B,TNF
Inflammasome pathway	2.98	IL1B,NLRP3,NOD2
TNFR1 Signaling	2.77	NFKBIA,NFKBIE,TNF,TNFAIP3
IL-6 Signaling	2.74	ABCB1,IL1B,NFKBIA,NFKBIE,TNF,VEGFA
NF-κB Signaling	2.61	IL1B,INSR,NFKBIA,NFKBIE,TNF,TNFAIP3,TNFRSF17
Dendritic Cell Maturation	2.55	CD1D,CD83,IL1B,IL32,NFKBIA,NFKBIE,TNF
Th2 Pathway	2.54	BHLHE41,CXCR6,IL12RB2,IL2RB,ITGB2,NOTCH4

Table 7. Enriched pathways from DEGs post influenza vaccination between those with or without prior year vaccination.

Ingenuity canonical pathways	-log p value	Molecules (italics = increased expression in prior Vax group)
Inflammasome pathway	9.18	CASP1,CASP5,CTSB,IL18,IL1B,MYD88,NLRC4,NLRP3,PYCARD,TLR4
TREM1 Signaling	8.74	CASP1,CASP5,CD86,CXCL8,ICAM1,IL18,IL1B,MPO,MYD88,NLRC4,NLRP3,NLRP6,TLR4,TLR5,TLR8, TREM1,TYROBP
IL-10 Signaling	8.42	BLVRA,BLVRB,CCR1,CD14,FCGR2A,FOS,HMOX1,IL10RB,IL18,IL1B,IL1R2,IL1RAP,IL1RN,NFKBID, NFKBIE,SOCS3
Communication between Innate and Adaptive Immune Cells	7.82	B2M,CCL4,CCR7,CD86,CXCL10,CXCL8,FCER1G,HLA-B, HLA-F,IGHD,IL15,IL18,IL1B,IL1RN,TLR4,TLR5,TLR8,TNFSF13B
Phagosome Maturation	7.36	ATP6AP1L,ATP6V0B,ATP6V0D1,B2M,CTSB,CTSC,CTSH,CTSS,CTSW,CTSZ,DYNLT1,HLA-B, HLA-F,MPO,NAPA,NCF2,PRDX5,PRDX6,RILP,TUBA1A,TUBB4B,VAMP3
Dendritic Cell Maturation	6.42	B2M,CCR7,CD58,CD86,FCER1G,FCGR1A,FCGR1B,FCGR2A,FCGR3A/FCGR3B,HLA-B, HLA-F,ICAM1,IL15,IL18,IL1B,IL1RN,LTBR,MYD88,NFKBID,NFKBIE,TLR4,TNFRSF1B,TYROBP
Role of Macrophages, Fibroblasts and Endothelial Cells in Rheumatoid Arthritis	6.08	C5AR1,CEBPB,CEBPD,CXCL8,F2RL1,FCGR1A,FCGR3A/FCGR3B,FOS,ICAM1,IL15,IL17RC,IL18,IL1B, IL1R2,IL1RAP,IL1RN,LTBR,MYD88,NFKBID,NFKBIE,OSM,RALB,RRAS,SOCS1,SOCS3,TCF7L2,TLR4, TLR5,TLR8,TNFRSF1B,TNFSF13B
Neuroinflammation Signaling Pathway	5.49	B2M,CASP1,CD86,CSF1R,CXCL10,CXCL8,FAS,FOS,GLUL,GRINA,HLA-B, HLA-F,HMOX1,ICAM1,IFNGR2,IL18,IL1B,MYD88,NCF2,NLRP3,PTGS2,PYCARD,SLC1A3,SNCA,SOD2, TLR4,TLR5,TLR8,TYROBP
Acute Phase Response Signaling	5.43	CEBPB,FOS,FTL,HMOX1,IL18,IL1B,IL1RAP,IL1RN,MYD88,NFKBID,NFKBIE,OSM,RALB,RBP7,RRAS, SERPINA1,SERPING1,SOCS1,SOCS3,SOD2,TNFRSF1B
Type I Diabetes Mellitus Signaling	5.43	BID,CD86,FAS,FCER1G,HLA-B, HLA-F,IFNGR2,IL1B,IL1RAP,IRF1,MYD88,NFKBID,NFKBIE,SOCS1,SOCS3,TNFRSF1B
IL-6 Signaling	5.37	CD14,CEBPB,CXCL8,FOS,IL18,IL1B,IL1R2,IL1RAP,IL1RN,NFKBID,NFKBIE,RALB,RRAS,SOCS1,SOCS3,TNFAIP6,TNFRSF1B
Phagosome Formation	5.37	C5AR1,FCAR,FCER1G,FCER2,FCGR1A,FCGR1B,FCGR2A,FCGR3A/FCGR3B,MARCKS,MARCO,MRC2,MSR1,RHOG,RHOT1,TLR4,TLR5,TLR8
Toll-like Receptor Signaling	5.36	CD14,FOS,IL18,IL1B,IL1RN,LY96,MYD88,TLR4,TLR5,TLR8,TNFAIP3,UBB,UBC
Crosstalk between Dendritic Cells and Natural Killer Cells	5.28	CCR7,CD69,CD86,CSF2RB,FAS,HLA-B,HLA-F,IL15,IL18,LTBR,TLR4,TNFRSF1B,TNFSF10,TYROBP
Granulocyte Adhesion and Diapedesis	4.88	C5AR1,CCL4,CXCL10,CXCL16,CXCL8,FPR1,FPR2,ICAM1,IL18,IL1B,IL1R2,IL1RAP,IL1RN,MMP28, MMP8,PF4,PPBP,SDC4,TNFRSF1B,XCL1
Systemic Lupus Erythematosus In B Cell Signaling Pathway	4.82	BCL2L1,BLNK,CD72,CXCL8,FCGR2A,FGR,FOS,FOXO4,HCK,IFIT2,IFNGR2,IGHD,IL15,IL18,IL1B,ISG15,LILRA6,LYN,MYD88,OSM,PIK3AP1,RALB,RRAS,TLR8,TNFSF10,TNFSF13B
Role of NFAT in Regulation of the Immune Response	4.81	BLNK,CD86,FCER1G,FCGR1A,FCGR1B,FCGR2A,FCGR3A/FCGR3B,FOS,GNA15,GNAZ,GNB2,GNB4, GNG5,HLA-B,LAT,LYN,NFKBID,NFKBIE,RALB,RRAS
IL-17A Signaling in Fibroblasts	4.45	CEBPB,CEBPD,FOS,IL17RC,LCN2,NFKBID,NFKBIE,NFKBIZ
iNOS Signaling	4.44	CD14,FOS,IFNGR2,IRF1,LY96,MYD88,NFKBID,NFKBIE,TLR4
Role of Tissue Factor in Cancer	4.37	BCL2L1,BLK,CXCL8,EGR1,F2RL1,FGR,GNA15,HBEGF,HCK,IL1B,LIMK2,LYN,PLAUR,RALB,RRAS
NF-κB Signaling	4.36	FCER1G,IL18,IL1B,IL1R2,IL1RN,LTBR,MAP3K8,MYD88,NFKBID,NFKBIE,PELI1,RALB,RRAS,TLR4,TLR5,TLR8,TNFAIP3,TNFRSF1B,TNFSF13B
Interferon Signaling	4.36	IFI6,IFITM1,IFITM2,IFITM3,IFNGR2,IRF1,ISG15,SOCS1
LXR/RXR Activation	4.33	ABCG1,CD14,IL18,IL1B,IL1R2,IL1RAP,IL1RN,LDLR,LY96,MSR1,PTGS2,S100A8,SERPINA1,TLR4, TNFRSF1B
Osteoarthritis Pathway	4.27	ALPL,ANXA2,CASP1,CASP4,CASP5,CEBPB,CXCL8,DDIT4,HIF1A,IL1B,IL1R2,IL1RAP,NAMPT,PTGS2, S100A8,S100A9,SDC4,SPHK1,TCF7L2,TLR4,TNFRSF1B

Figure 1. Prior year vaccination status effects PBMC response to subsequent vaccination.

PBMC gene expression on Day 3 postvaccination was compared between children who did not receive a vaccine in the prior year (N = 4) and those who had (N = 6). 45 DEGs (42 identified) were found between groups after FDR. Higher overall gene expression was seen in children who do not have a prior year vaccine (No) versus those who did (Yes). Heat map cluster analysis was performed using gplots R package. The FDR for each gene was calculated using the Benjamini–Hochberg method within the edgeR R package.