Introduction
Neuroinflammation is a major component of secondary injury that occurs after traumatic brain injury (TBI) and contributes to continuous tissue damage long after the initial mechanical insult. As a result, there is great interest in developing therapeutics that target various molecular and cellular drivers of the neuroinflammatory response that occurs after TBI. To date, however, very few anti-inflammatory drugs have been investigated in clinical TBI, with three clinical trials assessing the effects of cyclosporine and minocycline [
1‐
3]. These studies had small sample sizes, and while they reported no life-threatening events requiring termination of the studies, they did show increased incidence of adverse events with cyclosporine treatment and increased levels of injury markers with minocycline treatment, even though biomarker evaluation indicated decreased microglial activation [
3]. While these findings do not rule out the possibility that future larger studies may identify a neuroprotective effect of these drugs, they nevertheless emphasize the need for identifying and assessing novel diagnostic biomarkers and therapeutic targets involved in post-TBI neuroinflammation.
Previous high-throughput studies have analyzed gene and protein expression in various experimental models of TBI, both acutely and chronically after injury [
4‐
7]. Notably, local and systemic changes in the level of complement system components were consistent and of high magnitude across the various injury models at specified time points. The complement system is an integral part of both the innate and adaptive immune systems and consists of more than 50 soluble and membrane-bound proteins. Activation of the complement system triggers an enzymatic cascade that leads to the production of various effector molecules with roles that include anti-microbial defense, clearance of cellular debris and immune complexes, neurodevelopment, tissue regeneration, and modulation of adaptive immune responses [
8]. Using mass spectrometry, Bao et al. [
9] showed that amongst 32 proteins acutely and chronically upregulated in serum of severe TBI patients, 5 proteins were components of the complement system, including the central complement component C3. In rodents, proteomic data showed complement component C3 to be a hub protein in cortical protein–protein interaction networks early after a weight-drop open-head TBI [
10], while gene expression data showed chronic upregulation in the brain at 6 months after injury of the complement genes
C1q,
C1s,
C2, and
C3 in a midline fluid percussion injury [
7]. Together, these data suggest that complement inhibition may be a viable therapeutic target acutely and chronically after TBI.
In accordance with these findings, several animal studies have shown that complement inhibition following open head or closed head brain injury is neuroprotective, and improves behavioral and histological outcomes [
11‐
14]. Notably, recent studies have shown ongoing complement activation up to 6 months after a single controlled cortical impact (CCI), and with an ongoing cognitive decline that was reversed with complement inhibition, even when administered 2 months after injury [
15,
16]. Nevertheless, the complement system comprises more than 50 proteins, and the complexity associated with dissecting the role of complement in pathogenic and protective mechanisms of TBI has limited progress towards clinical application. While complement genes and proteins have been included in various high dimensional analyses, a comprehensive study specifically focused on the complement system in the context of other neuroinflammatory pathways has not been undertaken.
In this study, we investigated the expression of a panel of neuroinflammatory-associated genes, together with an inclusive complement gene panel, at various time points after TBI using a severe murine CCI model. Specifically, we analyzed gene expression at 3 days, 7 days, 28 days, 1 year and 2 years after CCI. We additionally assessed the effects of complement inhibition in different treatment paradigms on gene expression after CCI. Overall, the data highlight an important role for complement and complement gene expression in the progression of neuroinflammatory processes as they occur temporally after TBI, and they strengthen the premise that the complement system represents a promising therapeutic target for treating TBI.
Discussion
In this study, we comprehensively characterized the expression of complement system genes together with an examination of the neuroinflammatory transcriptome after severe brain injury and found that, together with up-regulation of markers of astrocyte activation and inflammatory immune responses, genes belonging to the complement system were among the most highly upregulated. Similar to our approach, one previous study also investigated the brain inflammatory transcriptome chronically after TBI, but used a milder model of injury. By performing microarrays, Boone et al. [
7] quantified gene expression in the cortex and hippocampus of rats after a midline fluid percussion injury and found significant dysregulation of certain complement genes 6 months post injury. Specifically, the authors showed acute upregulation of classical pathway and anaphylatoxin receptor genes, as well as both acute and chronic upregulation of the
C2 and
C3 genes. Additional transcriptomic studies also highlighted activation of the complement system acutely after TBI. Specifically, an analysis of complement genes extracted from an RNAseq dataset acquired from the cortex at 1 day post CCI (GSE79441) [
45], revealed upregulation of
C1qa-c,
C3ar1,
Itgam, Itgax,
Cd44,
CD93,
Ager and
Serping1, and downregulation of regulatory
Csmd1, which is in line with our data. Moreover, a recent qRT-PCR study characterizing the regional expression of classical and lectin initiators up to 5 weeks after CCI reported the upregulation of
C1q,
C1s, and
Fcnb, and the absence of
MBL dysregulation [
4].
We have previously shown that inhibition of complement with CR2-Crry at 1 h after TBI, as performed here, significantly improved outcome at 4 weeks after injury, including decreased lesion volume and improved cognitive performance on Barnes Maze [
12]. Based on these findings, and in the context of the above findings on complement gene expression, we additionally investigated the transcriptomic changes in the whole hemisphere chronically after CCI, with and without CR2-Crry treatment. We found that complement inhibition exerts a robust inhibitory effect on genes involved in various pathobiological processes in the injured brain, while bolstering expression of genes involved in DNA repair and the slowing of cell death. For instance, complement inhibition reduced the expression of several markers of reactive astrogliosis, including shared and specific markers of neurotoxic A1 astrocytes and A2 astrocytes. Furthermore, complement inhibition decreased the expression of numerous immune cell markers, such as Cd68, Msr1, Itgam and Trem2, suggesting reduced immune cell infiltration and/or activation at the site of brain injury, especially subacutely. Interestingly, complement inhibition also reduced the expression of complement receptor C5ar1 which is known to activate and recruit immune cells upon binding to the anaphylatoxin C5a [
46]. Hence, inhibition of C5a production by CR2-Crry may be a contributing factor to decreased immune cell infiltration/activation, and subsequently C1q expression by immune cells resulting in decreased C1q-mediated polarization of reactive astrocytes to the neurotoxic A1 subtype [
20,
47]. We cannot exclude the possibility that decreased activation of immune and astroglial cells may be at least partially mediated by the inhibitory effect of CR2-Crry on membrane attack complex (MAC) formation, which could otherwise induce cell lysis and the release of pro-inflammatory cytokines, damage-associated molecular patterns, and excitotoxic glutamate [
48]. However, data from our group have shown that specific inhibition of MAC formation in a therapeutic paradigm is not protective in the chronic phase after severe TBI, unlike inhibiting C3 activation [
12].
Our analysis also showed that considerably fewer genes are affected by complement inhibition on day 3 than on day 7 after TBI. This suggests an early and strong response to mechanical injury consisting of hemorrhage, tissue loss, astrogliosis and immune cell infiltration, followed by an increasingly inflammation-driven pathology that is more susceptible to therapeutic modulation. In addition, some pathological processes remain active through day 28 after TBI despite an earlier response to complement inhibition as shown by the chronic upregulation of markers of reactive astrocytes and oligodendrocytes and disease-associated microglia. The continued neuroinflammation after complement inhibition and the diverse response of complement genes to TBI affirms the need for a better understanding of the various roles of complement components in TBI in order to optimize a complement inhibitory strategy.
Several animal studies have investigated the therapeutic effect of complement inhibition on histological and behavioral recovery. Collectively, these studies targeted C1q, C3, C4, factor B, factor H, the terminal pathway, and more recently, components of the lectin pathway in severe open-head and/or closed-head TBI models [
12‐
14,
49]. Interestingly, while inhibition of the alternative pathway, the lectin pathway, or all pathways improved chronic outcome after CCI, inhibition of the classical pathway (C1q knockout) was not protective at 35 days post injury [
12,
13,
50]. Given the high baseline and TBI-induced expression of classical pathway and C1q receptor genes in our study, it is likely that the role of C1q in neuroinflammation and recovery after TBI may involve both complement activation and C1q receptors. For example, C1q has been shown to bind to CD44 to mediate stem cell chemotaxis in spinal cord injury [
51], which if blocked after TBI, could counter the neuroprotective effects of classical pathway inhibition. Moreover, studies using a severe closed-head weight-drop injury model showed that inhibiting or ablating the alternative pathway is neuroprotective [
11,
49,
52], and a side-by-side comparison with a CCI model demonstrated that alternative pathway inhibition was similarly protective to inhibiting all complement pathways at the C3 activation step [
12]. While this was attributed to the alternative pathway functioning as an amplification loop, recent studies showed that Collectin-12, that we show is upregulated at multiple time points after TBI, can act as a pattern recognition molecule for the alternative pathway independently of the classical pathway and lectin pathway [
35,
36]. Thus, the alternative pathway may alone be able to induce and perpetuate neuroinflammation after TBI, and could potentially explain the similar level of neuroprotection conferred by inhibiting the alternative pathway only vs. the additional inhibition of the classical and lectin pathways.
With regards to effector pathways, our data showed upregulation of C3 and several complement anaphylatoxin and phagocytic receptors. Whereas therapeutic studies have implicated C3 opsonization of neurons and synapses in driving secondary injury and cognitive decline in both acute and chronic phases after TBI [
15], the role of complement receptors has not been specifically investigated in TBI. With regard the anaphylatoxin receptors, a recent study showed that C5aR2 knockout aggravated C5aR1-mediated myelin damage and tissue loss in a model of severe spinal cord injury, which was reversed using a selective C5aR1 antagonist [
53]. Moreover, C3aR was shown to be protective after spinal cord injury by antagonizing CXCR2-mediated chemotaxis of neurotoxic neutrophils to the site of injury [
54]. This suggests that unlike C5aR1, C5aR2 and C3aR are neuroprotective in spinal cord injury and may have similar effects in traumatic brain injury. Regarding the terminal pathway, although it is implicated in acute tissue loss, acute inhibition of MAC formation was not protective in the chronic phase after TBI [
12], which aligns with our finding of no chronic dysregulation of terminal pathway genes.
The lack of differential expression for some genes, as in the case of MBL, Masp2, and Cfb, does not exclude a role for the gene products in propagating post-TBI pathology, especially after injuries that incur overt damage to the blood brain barrier and cause hemorrhage, hence allowing entry of peripheral complement proteins to the site of injury. For example, the knockout of lectin pathway initiators, MBL and MASP-2, was recently shown to improve chronic motor outcomes after controlled cortical impact [
13], and the inhibition or knockout of complement factor B improved acute histological outcomes after weight-drop TBI [
49,
52]. Conversely, the local production of complement proteins may play a more prominent role chronically after TBI [
15,
16], or after mild closed head injuries.
To better understand the role of locally expressed inflammatory genes in the brain and their cellular source, Arneson et al. [
47] performed single cell RNAseq on hippocampal cells harvested at 1 day after midline fluid percussion injury. The authors then assessed co-expression of inflammatory genes between astrocytes, neurons, oligodendrocytes, ependymal cells, mural cells, and/or microglia. In particular, correlations between genes encoding secreted proteins in source cells and other genes in target cells was quantified in order to understand potential interactions between the different cell types. Notably, several complement genes were shown to have strong co-expression profiles, such as
C1qa-c,
C3, and the regulatory genes,
Cfh,
C1qbp, and
Clusterin. While
C1qa-c were primarily expressed by microglia (
C1qc was used as a microglial marker), it was also reported that ependymal cells expressing
C3 and
C1q were particularly enriched after TBI. Moreover, microglia expressed the soluble complement inhibitors
Cfh and
C1qbp, and ependymal cells and astrocytes expressed
Clusterin. This study implicated multiple brain cell types in acute complement gene expression and showed that immune cells produce both complement activators and regulatory elements—and hence modulate and not only drive complement activation. In the light of our data showing continuous complement dysregulation at 2 years after TBI (and with aging), it will be important to extend single cell profiling of complement gene expression to chronic time points. Specifically, understanding the cellular source of the upregulated central complement genes C2, C3, and C4a, as well as C1qa-c and C3ar1, will help elucidate the role of complement in delayed-onset neurocognitive deficits after TBI and in TBI-induced tauopathies implicated in Alzheimer-like pathology [
55].
With the advent of antibody-based high-throughput imaging technologies, along with the continuous improvement of single cell mass cytometry and RNAseq workflows, characterizing the spatial and cellular abundance of complement components in various organ systems will become more feasible. Although various complement inhibitors have proven effective in animal models of TBI, such high throughput investigations will potentially allow for design of tailored approaches of complement inhibition that may be needed depending on the type of TBI and the time of treatment after TBI, and where there may be dualling roles of complement in injury and repair.
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