Gene expression patterns following unilateral traumatic brain injury reveals a local pro-inflammatory and remote anti-inflammatory response

It is estimated that 3.17-3.32 million persons in the United States are living with long-term or lifelong effects of traumatic brain injury (TBI) [1]. Approximately 1.7 million new TBIs are sustained each year [2] resulting in 53,000 deaths [3] and as many as 125,000 additional people that have long-term behavioral deficits [4]. Efforts to implement preventative measures for the leading causes of TBI (motor vehicle-traffic accidents, falls, and assaults) are critical [2, 3] but will not eliminate TBI as a major public health problem. Development of effective clinical treatment protocols post-TBI and potential prophylactic agents for military usage are necessary to address those TBIs that cannot be prevented.

Injury to the brain results in irreversible damage at the site of impact and initiates cellular and molecular processes that lead to delayed or secondary neural injury in the surrounding tissue [5, 6]. Neuroprotective strategies target these processes in an attempt to halt the progression of or prevent the delayed injury [7]. These processes include inflammation, damage to the blood brain barrier, release of excitatory amino acids, oxidative stress, cerebral edema, reduced cerebral blood flow, hypoxia, and ischemia [5, 6]. In the current study, we used gene microarray analysis to determine which genes, pathways and networks were significantly altered after unilateral controlled cortical impact (CCI), an experimental model of TBI.

Microarray technology is a very powerful tool that allows the user to examine expression profiles for thousands of genes at one time. The true power of microarray technology can be maximized when the expression profiles can be attributed to significant alterations in biological functions and molecular pathways. Fortunately, the recent development of advanced bioinformatic analysis tools has made the utilization of microarray data more practical and allows for easier replication. One such tool is the Ingenuity Pathway Analysis (IPA) software program which uses a database built from published scientific literature to draw direct and indirect interactions between genes and to assign genes to specific biological functions and canonical pathways [8]. IPA was used here for functional, canonical pathway, and network analysis of the genes that were altered by TBI. We examined gene expression on both sides of the brain in order to understand the alterations both locally (ipsilateral) and remotely on the opposite side of the brain (contralateral). We observed that the contralateral side of the brain, which has often been used as a control in similar experiments, exhibited significant alterations in gene expression following TBI.

The bioinformatic analysis tools we used also allowed for both identification of key molecules and elucidation of their interactions with each other. We used these interactions to identify molecules and molecular pathways central in the response to TBI that could provide novel targets for therapeutic strategies. Understanding how local and remote gene expression profiles change following TBI, when compared to non-injured brain, will provide valuable insight into delayed neuronal injury mechanisms as well as intrinsic neuroprotective processes.

In this study, we used microarray technology to examine cellular and molecular mechanisms associated with secondary brain injury following TBI. Our findings indicated that the inflammatory response and its associated genes and pathways are significant in the post-TBI molecular response. This is consistent with our published studies indicating that inflammation is involved with delayed, secondary neuronal injury following other acute brain injuries (ABI’s) including stroke and neurotoxin exposure [17–20]. Activated microglia, astrocytes and macrophages have been shown to be the source of several of the inflammatory molecules identified in this study [10, 21–23]. The presence of activated inflammatory cells ipsilaterally and their absence contralaterally was confirmed by immunohistochemistry. This cellular inflammation offers further evidence for pro-inflammatory molecules being produced in the region of tissue damage following TBI.

Despite the absence of activated inflammatory cells on the contralateral side of the brain, our results also showed that TBI resulted in a significant alteration of the inflammatory gene response on the both sides of the brain. The observed IR gene expression pattern suggests that there is a baseline IR throughout the whole brain due to unilateral CCI. This is demonstrated by the nearly 30% of all IR genes that change similarly on both sides of the brain (109 of 372 IR genes). Above this baseline of inflammation, a distinct IR gene expression pattern emerges. The expression level is higher on the ipsilateral side for all the remaining genes expressed on both sides of the brain. 95% of the IR genes expressed only in ipsilateral tissues increase in expression while 74% of the IR genes expressed only in contralateral tissues decrease in expression. Examination of the contralateral gene expression in our GIH showed that 11 genes (3 primary tier, 3 secondary tier, 3 peripheral tier and 2 orphan) decreased in expression contralaterally while either remaining unchanged or increasing in expression ipsilaterally. Only 3 genes (1 secondary and 2 peripheral tier) increased contralaterally and remained unchanged ipsilaterally. This demonstrated that the most significant contralateral IR genes in this analysis show decreased expression.

Taken together with the cellular inflammation data, we can surmise that increased expression of the majority of IR genes ipsilaterally results in the development of functional inflammation that can contribute to secondary neural injury. Similarly, it is likely that the suppression of the majority of IR genes contralaterally prevents this development in brain regions remote to the injury. This summary of the overall IR gene expression does not take into account that some IR genes may be classified as anti-inflammatory in certain contexts. However, any counter effects of anti-inflammatory molecules seems to be negligible as the cellular inflammation pattern follows the IR gene expression pattern assuming the majority of IR genes are pro-inflammatory. The contralateral inflammatory response without detectable cellular inflammation also fits well with the idea that some inflammation may actually be beneficial following TBI [9].

Through canonical pathway and network analysis combined with identification of the common genes that change differently on each side of the brain, we identified 114 GOI. Many of these genes have been previously associated with the IR following ABI’s (i.e., TBI, stroke, nerve agent exposure) including CCL13/CCL2, CCL4, CCL6, CCL7, CCR5, CD14, CD44, CDKN1A, CEBPB, CEBPD, CXCL13, CXCL9, CSF1, FOS, HBEGF, HSPA1A/HSPA1B, ICAM1, IER3, IL-1β, IRF1, IRF2, JAK2, LCN2, MMP3, MMP9, MYD88, NF-κB, PSMB8, S100A4, SPP1, STAT3, TLR2, TLR4, and TNFRSF1A [10, 13, 21, 24–34]. These results support the utility of our methods for identifying the significant genes related to this biological function. Additionally, several IR genes that appear to be novel in the context of TBI were also identified, providing new targets for future study. These genes included HSP90AA1, ERAP1, PSMB9, CBL, BTK, RORA, THRA, and ITGA5. We wanted to take our analysis one step further and determine which genes were likely the most critical in the observed IR. We accomplished this by creating a network of these genes and determining how many 1^st order connections each gene had with the other genes in the network. A GIH was created based on these numbers and there were some intriguing findings in terms of which tier certain molecule types predominated.

Not surprisingly, a large number of transcription regulators were included in the primary and secondary tiers as these molecules are the point of convergence for many of the inflammatory pathways and the regulatory step in the production of new proteins. Somewhat surprising was the large number of cytokines that fell into the peripheral tier since considerable focus has been placed on cytokines as mediators of inflammation and targets for therapeutic intervention [10, 21, 24]. This result may be due to a near 1-to-1 relationship that these cytokines have with their receptors, limiting the 1^st order connections in the GOI network. While cytokines clearly play an important role in initiating the IR, they may not be the most critical molecules in modulating the IR. Only one cytokine, IL1B, is in the primary tier of our hierarchy while CCL7, CXCL10, and SPP1 are in the secondary tier. This suggests that neuroprotective strategies directed at one of the lower tier cytokines may not be sufficient for limiting the IR [35]. Further, while therapeutic hypothermia after TBI has been shown to improve outcome, inflammatory cytokine levels were unaffected, implying minimal cytokine involvement in the observed neuroprotection [26]. However, targeting the cytokines in the higher tiers may produce effective modification of the IR [36]. Our GIH shows that regulation of transcription, phosphorylation (kinases), extracellular matrix/cell adhesion (FN1, MMPs, ICAM1), and receptors (transmembrane and G-coupled) figure prominently (primary and secondary tiers) in the post-TBI IR. These molecules may, therefore, be more efficient targets for therapeutic strategies to combat post-TBI inflammation because they are activated regardless of the initiating factor.

Two well characterized signaling pathways stood out in our GIH: toll-like receptor/NF-κB signaling and JAK/STAT signaling. There was some concern that our choice of canonical pathway may have skewed our analysis towards these signaling pathways (toll-like receptor/NF-κB and JAK/STAT) because large portions of both of these pathways are included in IL-6 signaling (Figure 4). While 12 GOI are included in that pathway, 8 of these genes were also identified by other analyses used to identify GOI, further supporting their importance to the post-TBI IR. Additionally, interconnection of the genes in IL-6 signaling could not account for the total number of 1^st order connections for these genes in the GOI network used to build the GIH. This was especially true for genes in the primary tier. 19 GOI were either a part of (CEBPB, FOS, NFKB2, EGFR, IKBKB, IL1B, TLR4, MYD88, TLR2, TNFRSF1A, CD14, IL1R2), a product of (IRF1, FN1, CCND1, CASP3), or associated with (IRF7, LYN, HSP90AA1) toll-like receptor/NF-κB signaling [8]. 15 of these genes were in the primary tier of the GIH. Secondarily, 5 GOI (JAK2, STAT3, CDKN1A, IL6R, IL6ST) were a part of JAK/STAT signaling [8], and three of these genes were in the primary tier. Based on our analysis, these signaling pathways are likely candidates for the induction of post-TBI IR. Therefore, therapeutic strategies aimed at the molecules in these pathways may reduce TBI-induced inflammation and, by extension, post-TBI neuronal death.

It should be noted that this study only takes a snapshot at 24 hours post-injury of a dynamic and evolving molecular process initiated by TBI [37]. Therefore, significant molecular events that precede and follow this time point are not reflected in these data. For a more complete examination of the molecular response to TBI, a study including multiple time points before and after 24 hours will be necessary and are underway in our laboratory. Further, confirmation of the biological relevance of any observed gene expression profile following TBI is a critical next step to exploring potential therapies for brain trauma [26, 38].

Microarray analysis is a powerful tool that allows for the analysis of thousands of genes simultaneously. We demonstrated that TBI was associated with a powerful pro-inflammatory response in ipsilateral brain tissues. We also noted the distinct IR gene expression pattern that suggests a remote anti-inflammatory response. The use of multiple network and pathway analyses to identify GOI aided in making our datasets manageable and revealed 2 distinct pathways, toll-like receptor/NF-κB signaling and JAK/STAT signaling, associated with post-TBI secondary neural injury. Our GIH provides a starting point for investigating therapeutic targets in a ranked order that is somewhat different than what has been presented previously in microarray studies. In addition to being a vehicle for identifying potential targets for post-TBI therapeutic strategies, our GIH can also provide a context for evaluating the potential of therapeutic agents currently in development.