Chronic complement dysregulation drives neuroinflammation after traumatic brain injury: a transcriptomic study

Abstract

Activation of the complement system propagates neuroinflammation and brain damage early and chronically after traumatic brain injury (TBI). The complement system is complex and comprises more than 50 components, many of which remain to be characterized in the normal and injured brain. Moreover, complement therapeutic studies have focused on a limited number of histopathological outcomes, which while informative, do not assess the effect of complement inhibition on neuroprotection and inflammation in a comprehensive manner. Using high throughput gene expression technology (NanoString), we simultaneously analyzed complement gene expression profiles with other neuroinflammatory pathway genes at different time points after TBI. We additionally assessed the effects of complement inhibition on neuropathological processes. Analyses of neuroinflammatory genes were performed at days 3, 7, and 28 post injury in male C57BL/6 mice following a controlled cortical impact injury. We also characterized the expression of 59 complement genes at similar time points, and also at 1- and 2-years post injury. Overall, TBI upregulated the expression of markers of astrogliosis, immune cell activation, and cellular stress, and downregulated the expression of neuronal and synaptic markers from day 3 through 28 post injury. Moreover, TBI upregulated gene expression across most complement activation and effector pathways, with an early emphasis on classical pathway genes and with continued upregulation of C2, C3 and C4 expression 2 years post injury. Treatment using the targeted complement inhibitor, CR2-Crry, significantly ameliorated TBI-induced transcriptomic changes at all time points. Nevertheless, some immune and synaptic genes remained dysregulated with CR2-Crry treatment, suggesting adjuvant anti-inflammatory and neurotropic therapy may confer additional neuroprotection. In addition to characterizing complement gene expression in the normal and aging brain, our results demonstrate broad and chronic dysregulation of the complement system after TBI, and strengthen the view that the complement system is an attractive target for TBI therapy.

Keywords: Complement inhibition; Complement system; Gene expression; NanoString; Neuroinflammation; Traumatic brain injury.

Fig. 1

Patterns of neuroinflammatory gene expression in TBI. a Number of upregulated and downregulated genes in injured brains at days 3, 7, and 28 after TBI compared to 12-week-old uninjured brains (using NanoString Neuroinflammation panel). b Common changes in gene expression between time points color coded by the time point(s) of differential expression. c Volcano plots of differentially expressed genes per time point, color coded as in panel b, and showing labeled examples of the most dysregulated genes. d Analysis of time points of peak change in expression per gene (color legend) based on statistical difference in fold change (columns) between time points of differential expression (rows). Genes that showed no significant differences were considered to show peak change at all time points of differential expression. The volcano plot on the right shows an example of 29 consistently upregulated genes that had peak expression on days 7 and 28 post injury compared to day 3 post injury. False discovery rate (FDR) was computed for all genes per comparison to determine statistical significance. N = 3 per group

Fig. 2

NanoString pathway analysis highlights involvement of the complement system in TBI. a Bar graphs showing the median fold change of DEGs (column 1, color coded by time point) and the number of DEGs (column 2, color coded by patterns of differential expression) per NanoString pathway. b Volcano plots showing the fold change and FDR of complement genes relative to other genes in the Neuroinflammation panel. FDR was computed for all genes. N = 3 per group

Fig. 3

Complement genes show extensive and chronic dysregulation in TBI. a Quantification of the number of reads of complement genes in normal 12-week-old brain samples (custom-built NanoString panel). Complement genes are organized in order by complement pathway (in text) and class (shape legend). b Quantification of fold change in expression of complement genes in injured brains at 3 days, 7 days, 28 days, 1 year, and 2 years after TBI compared to age-matched uninjured controls. FDR was computed only for genes with p value < 0.05 to increase sensitivity at chronic time points. N = 3 per group. c Combination of IHC for NeuN (blue) and RNAscope in situ hybridization for C1qa gene (red puncta) in TBI brains at days 7 after injury. N = 3 per group. Shown is a representative image of the contralateral and ipsilateral hemisphere in the same animal at 10 × magnification. The boxed areas on both hemispheres are shown at 40 × magnification

Fig. 4

Targeted complement inhibition strongly modulates inflammatory gene expression during TBI. a Number of upregulated and downregulated genes in brains of PBS/vehicle treated (red) and CR2-Crry treated (green) mice at days 3, 7, and 28 after TBI (using NanoString Neuroinflammation panel). b Bar graph showing overall effects of CR2-Crry on gene expression at all time points after TBI. c Principal component analysis of all brain samples. d Volcano plots showing fold change and FDR of genes in PBS group (same as Fig. 1c), color coded by effects of CR2-Crry on gene expression with labeled examples. e Effect of complement inhibition on the pattern of differential expression. Table on the right shows the results of a Reactome pathway analysis of induced and inhibited genes. FDR was computed for all genes in comparisons between sham and injured brains, and for genes with p value < 0.05 in comparisons between injured groups (because of increased variability in gene expression after injury). N = 3 per group

Fig. 5

NanoString pathway analysis of the effect of complement inhibition on gene expression. Bar graphs showing the median fold change of DEGs (column 1, color coded by time point) and the number of DEGs (column 2, color coded by the effect of complement inhibition) per NanoString pathway. The median fold change and number of DEGs in the PBS group are also plotted for comparison (black box with white background). FDR was computed for all genes. N = 3 per group