Fluid proteomics of CSF and serum reveal important neuroinflammatory proteins in blood-brain barrier disruption and outcome prediction following severe traumatic brain injury: a prospective, observational study

Abstract

Background: Severe traumatic brain injury (TBI) is associated with blood-brain barrier (BBB) disruption and a subsequent neuroinflammatory process. We aimed to perform a multiplex screening of brain enriched and inflammatory proteins in blood and cerebrospinal fluid (CSF) in order to study their role in BBB disruption, neuroinflammation and long-term functional outcome in TBI patients and healthy controls.

Methods: We conducted a prospective, observational study on 90 severe TBI patients and 15 control subjects. Clinical outcome data, Glasgow Outcome Score, was collected after 6-12 months. We utilized a suspension bead antibody array analyzed on a FlexMap 3D Luminex platform to characterize 177 unique proteins in matched CSF and serum samples. In addition, we assessed BBB disruption using the CSF-serum albumin quotient (Q_A), and performed Apolipoprotein E-genotyping as the latter has been linked to BBB function in the absence of trauma. We employed pathway-, cluster-, and proportional odds regression analyses. Key findings were validated in blood samples from an independent TBI cohort.

Results: TBI patients had an upregulation of structural CNS and neuroinflammatory pathways in both CSF and serum. In total, 114 proteins correlated with Q_A, among which the top-correlated proteins were complement proteins. A cluster analysis revealed protein levels to be strongly associated with BBB integrity, but not carriage of the Apolipoprotein E4-variant. Among cluster-derived proteins, innate immune pathways were upregulated. Forty unique proteins emanated as novel independent predictors of clinical outcome, that individually explained ~ 10% additional model variance. Among proteins significantly different between TBI patients with intact or disrupted BBB, complement C9 in CSF (p = 0.014, ΔR² = 7.4%) and complement factor B in serum (p = 0.003, ΔR² = 9.2%) were independent outcome predictors also following step-down modelling.

Conclusions: This represents the largest concomitant CSF and serum proteomic profiling study so far reported in TBI, providing substantial support to the notion that neuroinflammatory markers, including complement activation, predicts BBB disruption and long-term outcome. Individual proteins identified here could potentially serve to refine current biomarker modelling or represent novel treatment targets in severe TBI.

Keywords: Apolipoprotein E4; Blood–brain barrier; Glasgow Outcome Score; Human; Neuroinflammation; Protein biomarkers; Proteomics; Traumatic brain injury.

Fig. 1

Assessed proteins were predominantly CNS structural proteins. The vast majority of proteins exhibited highest tissue enrichment in the CNS, with the second most frequent category being immune-system organs (a). Notably, numerous proteins were concomitantly expressed in multiple tissues (b). Within the Brain Atlas, the majority were cerebral cortex enriched (c), but few proteins were exclusively expressed within one CNS-niche (d). Protein characterization data was obtained from the Human Protein Atlas. CNS central nervous system

Fig. 2

A severe TBI induces protein alterations in CSF and serum. Individual patient proteomic profiles were different in CSF compared with serum, utilizing tSNE. Following a severe TBI, additional proteomic alterations occur within both of these compartments (a). Individual patient attributes, such as BBB disruption, seemed associated with some of TBI patient heterogeneity, predominantly in CSF (b). At the individual protein level, this was mimicked by altered protein levels in both CSF and serum (c, d). Graphical significance threshold was set to log₂ FC |0.5| and adjusted p value < 0.05, and values not fulfilling these criteria were diminished in size and shaded in light-gray. In CSF, both CNS structural and neuroinflammatory protein levels were increased following a severe TBI (c). This was reflected in pathway upregulations of structural, metabolic, and inflammatory pathways (e). In contrast, fewer protein were altered in serum (d), and upregulated pathways were predominantly neuroinflammatory (f). CSF cerebrospinal fluid, TBI traumatic brain injury, tSNE t-distributed stochastic neighbor embedding. All full protein names are given in Additional file 3: Table S1

Fig. 3

BBB disruption co-occurs with upregulation of innate immune pathways, notably the complement cascade. A severe TBI elicited an acute BBB disruption among a subset of patients, quantified using Q_A (a). Among the n = 114 proteins significantly correlated with Q_A, the majority were nervous system or immune system enriched (b). Using hierarchical clustering on CSF and serum protein measurements respectively, protein levels clearly clustered depending on BBB integrity status in CSF (c), but less so in serum (d). APOE carrier status was not associated with protein levels in either group (c, d). In CSF, this corresponded to pathway upregulation of predominantly innate immune mechanisms (e). Examining proteomic profiles between patients with disrupted and intact BBB, a handful of proteins were significant in CSF (f) and merely one in serum (g). APOE Apolipoprotein E, CSF cerebrospinal fluid, CNS central nervous system, GOS Glasgow Outcome Score, MFI median fluorescence intensity, Q_A albumin quotient, TBI traumatic brain injury. All full protein names are given in Additional file 3: Table S1

Fig. 4

Proteins associated with BBB disruption and TBI-induced protein level alterations were outcome predictors following TBI. Using the hierarchical clustering depicted in Fig. 3d, Q_A associated proteins significantly different between clusters were derived. Of these, n = 90 proteins were found to overlap with proteins altered in CSF following TBI as portrayed in Fig. 2c (a). Similar assessments between CSF clusters and TBI-induced protein alterations in serum yielded an overlap of n = 32 proteins (b). Among these, n = 40 proteins comprised novel outcome predictors following severe TBI, of which an excerpt of proteins with different features are shown (c, d). These analyses were multivariable, meaning that outcome predictors are independently significant even when adjusting for previously known prognostic covariates following a severe TBI. Validation of results were conducted in an independent TBI cohort without CSF samples. Following TBI, many of the matched cohort outcome proteins were upregulated in this validation cohort as well (e). CSF cerebrospinal fluid, MFI median fluorescence intensity, TBI traumatic brain injury. All full protein names are given in Additional file 3: Table S1