Profiling Immunological Phenotypes in Individuals During the First Year After Traumatic Spinal Cord Injury: A Longitudinal Analysis

Abstract

Abstract Individuals with SCI are severely affected by immune system changes, resulting in increased risk of infections and persistent systemic inflammation. While recent data support that immunological changes after SCI differ in the acute and chronic phases of living with SCI, only limited immunological phenotyping in humans is available. To characterize dynamic molecular and cellular immune phenotypes over the first year, we assess RNA (bulk-RNA sequencing), protein, and flow cytometry (FACS) profiles of blood samples from 12 individuals with SCI at 0-3 days and at 3, 6, and 12 months post injury (MPI) compared to 23 uninjured individuals (controls). We identified 967 differentially expressed (DE) genes in individuals with SCI (FDR <0.001) compared to controls. Within the first 6 MPI we detected a reduced expression of NK cell genes, consistent with reduced frequencies of CD56^bright, CD56^dim NK cells present at 12 MPI. Over 6MPI, we observed increased and prolonged expression of genes associated with inflammation (e.g. HMGB1, Toll-like receptor signaling) and expanded frequencies of monocytes acutely. Canonical T-cell related DE genes (e.g. FOXP3, TCF7, CD4) were upregulated during the first 6 MPI and increased frequencies of activated T cells at 3-12 MPI. Neurological injury severity was reflected in distinct whole blood gene expression profiles at any time after SCI, verifying a persistent 'neurogenic' imprint. Overall, 2876 DE genes emerge when comparing motor complete to motor incomplete SCI (ANOVA, FDR <0.05), including those related to neutrophils, inflammation, and infection. In summary, we identify a dynamic immunological phenotype in humans, including molecular and cellular changes which may provide potential targets to reduce inflammation, improve immunity, or serve as candidate biomarkers of injury severity.

Keywords: RNA sequencing; functional genomics; human studies; inflammation; spinal cord injury.

FIG. 1.

Longitudinal profiling of whole blood gene expression after SCI. (A) Overview of cohort, including participants without SCI (CTLs), and participants with moderate or severe SCI. Injury severity determined by AIS grade, where A or B indicates motor complete and C or D indicates motor and sensory incomplete injuries. (B) Dendrogram of two-way unsupervised hierarchical clustering shows the first major branch point differentiates between samples collected at the acute visit and all other study visits (n = 967 filtered gene transcripts, 1.5-fold difference, FDR <0.001, Benjamini-Hochberg adjusted). Participant ID numbers are shown above color-coded legend that indicates study visits (red = acute, yellow = 3 MPI, blue = 6 MPI, green = 12 MPI, black = uninjured CTLs). Heat map shows relative gene expression; color corresponds to Z-score (blue: downregulation, red: upregulation). Corresponding gene list with relative expression values is shown in Supplementary Table S2. (C) GOBP enrichment analysis of 756 genes with positive Z-scores (upregulated) and 456 genes with negative Z-scores (downregulated) at the acute time-point. (D) Temporal expression patterns of select genes from upregulated GOBP categories: neutrophil pathways (SerpinB1, ARG1), cytokine stimulation and signaling (ADAM17, IRAK3), and NFκB signaling (TRIM27, NLRC4). AIS, American Spinal Injury Association Impairment Scale; CTL, control; FDR, false discovery rate; GOBP, Gene Ontology biological process; IL, interleukin; MPI, months post-injury; SCI, spinal cord injury; TF, transcription factors; TLR, Toll-like receptor.

FIG. 2.

Pairwise comparison of gene expression profiles during the first year after SCI. Gene expression profiles were compared pairwise between SCI samples at acute, 3 MPI, or 6 MPI study visits and samples from participants without SCI. (A) Venn diagrams show number of DE genes unique to each study visit or shared among visits: top, upregulated (red); bottom, downregulated (blue). (B) GOBP categories that were enriched in DE genes common across acute, 3 MPI, and 6 MPI study visits are shown (top: upregulated, bottom: downregulated). (C) TF identified by TRANSFAC and JASPAR databases accessed in the Enrichr platform that were predicted to bind enriched DE genes common across acute, 3 MPI, and 6 MPI study visits are shown (top: up, bottom: down; *indicates mouse TF; all other TF are human). (D) Temporal expression patterns of genes of interest from enriched GOBP categories highlight the endogenous TLR ligand HMGB1, its receptors, and other TLRs. Boxes indicate range of Q1 to Q3, line indicates median, and whiskers indicate minimum to maximum. Color-coded legend indicates study visits (red = acute, yellow = 3 MPI, blue = 6 MPI, green = 12 MPI, black = uninjured CTLs); *indicates p < 0.02 in pairwise comparison with controls (ANOVA). ANOVA, analysis of variance; CTL, control; DE, differentially expressed; GOBP, Gene Ontology biological process; HMGB1, high mobility group box 1; MPI, months post-injury; SCI, spinal cord injury; TF, transcription factors; TLR, Toll-like receptor.

FIG. 3.

Longitudinal profiling of immune cell subsets of participants after SCI. Relative expression of genes of interest characteristic of (A) NK cells, (C) monocytes and DCs, and (E) T-cell subsets in participants with SCI compared with CTLs. Boxes indicate range of Q1 to Q3, line indicates median, and whiskers indicate minimum to maximum. Color-coded legend indicates study visits (red = acute, yellow = 3 MPI, blue = 6 MPI, green = 12 MPI, black = uninjured CTLs); *indicates p < 0.02 in pairwise comparison with controls (ANOVA). Flow cytometry was performed to determine relative distribution of immune cell subsets in participants with SCI compared with CTLs: (C) Percentage of CD56+^dim (left) and CD56+^bright (right) NK cells are significantly decreased after SCI. (D) Percentage of classical and intermediate monocytes are increased acutely, whereas plasmacytoid DCs are decreased acutely after SCI compared with uninjured CTLs. (F) Percentage of total CD4+ T cells is significantly decreased at 3 MPI (left upper); percentage of activated CD4+ T cells is increased at 3 MPI, 6 MPI, and 12 MPI after SCI (right upper); percentage of CD8+ T cells is unchanged after SCI (left lower); percentage of activated CD8+ T cells is increased at 3 MPI, 6 MPI, and 12 MPI after SCI (right lower). For flow cytometry, Mann-Whitney U test was performed to determine significance, defined as p < 0.05, with p-values shown: *p = 0.01–0.05, **p = 0.001–0.01, ***p = 0.0001–0.001, and ****p < 0.0001. ANOVA, analysis of variance; CTL, control; DCs, dendritic cells; MPI, months post-injury; NK, natural killer; SCI, spinal cord injury.

FIG. 3.

Longitudinal profiling of immune cell subsets of participants after SCI. Relative expression of genes of interest characteristic of (A) NK cells, (C) monocytes and DCs, and (E) T-cell subsets in participants with SCI compared with CTLs. Boxes indicate range of Q1 to Q3, line indicates median, and whiskers indicate minimum to maximum. Color-coded legend indicates study visits (red = acute, yellow = 3 MPI, blue = 6 MPI, green = 12 MPI, black = uninjured CTLs); *indicates p < 0.02 in pairwise comparison with controls (ANOVA). Flow cytometry was performed to determine relative distribution of immune cell subsets in participants with SCI compared with CTLs: (C) Percentage of CD56+^dim (left) and CD56+^bright (right) NK cells are significantly decreased after SCI. (D) Percentage of classical and intermediate monocytes are increased acutely, whereas plasmacytoid DCs are decreased acutely after SCI compared with uninjured CTLs. (F) Percentage of total CD4+ T cells is significantly decreased at 3 MPI (left upper); percentage of activated CD4+ T cells is increased at 3 MPI, 6 MPI, and 12 MPI after SCI (right upper); percentage of CD8+ T cells is unchanged after SCI (left lower); percentage of activated CD8+ T cells is increased at 3 MPI, 6 MPI, and 12 MPI after SCI (right lower). For flow cytometry, Mann-Whitney U test was performed to determine significance, defined as p < 0.05, with p-values shown: *p = 0.01–0.05, **p = 0.001–0.01, ***p = 0.0001–0.001, and ****p < 0.0001. ANOVA, analysis of variance; CTL, control; DCs, dendritic cells; MPI, months post-injury; NK, natural killer; SCI, spinal cord injury.

FIG. 4.

Injury severity of SCI influences whole blood gene expression profiles. (A) Dendrogram shows unsupervised two-way hierarchical clustering of DE genes according to AIS grade (n = 2876 unique genes, 1.5-fold change in expression, FDR <0.05, Benjamini-Hochberg adjusted). Participant ID numbers are shown above color-coded legend that indicate study visits (red = acute, yellow = 3 MPI, blue = 6 MPI, green = 12 MPI, black = uninjured CTLs) and AIS grade (orange: AIS A or B, purple: AIS C or D). Heat map shows relative gene expression by Z-score (blue: downregulation, red: upregulation). A corresponding gene list with relative expression values is shown in Supplementary Table S2. (B) GOBP categories enriched in genes that are upregulated or downregulated according to AIS grade. (C) Heat map (log₂ fold change) of significantly changed genes related to NF-kB activation of B cells signaling (NFKB), IL-1 signaling (L-1), neutrophil degranulation (neutrophil), viral mRNA translation/influenza infection (viral/flu inf.), mitochondrial translation (Mito), Rho GTPase cycle (RHO GTPase), SUMOylation DNA damage and repair (SUMO), and IL-6 signaling (IL-6) in participants with AIS AB injuries compared with AIS CD injuries. (D) Elevated systemic inflammatory proteins in samples from participants with AIS AB or AIS CD injuries, measured by ELISA. Inflammatory markers, cytokines, and chemokines, plotted as log10 concentrations, are graphed according to injury severity. Boxes indicate a range of Q1 to Q3, line indicates median, and whiskers indicate minimum to maximum. Symbols represent individual participants at any time-point. Mann-Whitney U test was performed to determine significance, defined as p < 0.05, with p-values shown: *p = 0.01–0.05, ** p = 0.001–0.01, ***p = 0.0001–0.001, and ****p < 0.0001. AIS, American Spinal Injury Association Impairment Scale; CTL, control; FDR, false discovery rate; DE, differentially expressed; ELISA, enzyme-linked immunosorbent assay; GOBP, Gene Ontology biological process; IL, interleukin; MPI, months post-injury; Rho GTPase, Rho guanosine triphosphate; SCI, spinal cord injury.