Discussion
In this study, we investigated neuroinflammatory markers in former professional and college American football players and the relationship with the neuropsychiatric syndrome, neurobehavioral dysregulation (NBD). Overall, former football players diagnosed with NBD did not have significantly different levels of CSF or plasma inflammatory biomarkers concentrations compared to asymptomatic unexposed individuals. However, CSF IL-6 levels differed between former football players with and without NBD diagnosis and we observed significant associations between levels of CSF IL-6 and elevated scores on specific NBD measures (emotional dyscontrol, affective lability, impulsivity, and overall neurobehavioral dysregulation). This association was not observed for explosivity, and a sensitivity analysis found that the relationship with emotional dyscontrol was dependent of cognitive performance or the use of antidepressants. CSF IL-6 was specifically associated with affective lability, impulsivity, and overall NBD and did not correlate with cognitive performance. Furthermore, in former football players ≥ 60 years, higher concentrations of plasma NfL were associated with more self-reported symptoms of emotional dyscontrol, impulsivity, and overall NBD. Elevated NfL levels were also associated with poorer executive functioning and processing speed. These results collectively show that NBD symptoms among former American football players may be associated with an altered neuroinflammatory response and that, in later life, specific NBD symptoms may be part of a broader clinical syndrome that potentially involves both neuroinflammation and neurodegeneration. The absence of an association between plasma IL-6 and NBD indicates that the inflammatory link is specifically central in nature, rather than systemic.
Our findings suggest that, while multiple biomarkers for neuroinflammation did not show a consistent relationship with NBD, IL-6 demonstrated a more robust and specific association. IL-6 is a pro-inflammatory cytokine that plays a pivotal role in inflammatory responses within the central nervous system. It exhibits pleiotropic features and is produced by various cell types including microglia, astrocytes, and neurons [
24,
25]. The role of IL-6 is multifaceted, involving the regulation of acute-phase inflammatory responses as well as participation in adaptive immune responses through multiple pathways, potentially contributing to chronic ongoing inflammation [
24,
25]. Overall, IL-6 appears to be a more general and sensitive marker capable of detecting subtle changes and correlations [
25]. It should be noted that the studied population is considered relatively "healthy" and not expected to show severe systemic inflammatory responses as seen in primary inflammatory diseases. Consequently, the finding that only IL-6 is associated with NBD symptoms in this population may not be surprising, as this requires a sensitive marker.
The association between IL-6 and neuropsychiatric symptoms has been assessed in a variety of disorders. In the context of AD, studies have demonstrated that increased serum concentrations of IL-6 are associated with overall neuropsychiatric symptoms or higher odds of apathy [
13,
26,
27]. Conversely, other studies did not find significant relationships between CSF or serum IL-6 levels and neuropsychiatric symptoms in AD patients [
27,
28] and a recent study reported that even lower CSF levels of IL-6 were associated with symptoms of depression and anxiety [
29]. In frontotemporal dementia (FTD), patients with disinhibition had higher levels of plasma IL-6 compared to patients without disinhibition [
30]. Elevations in IL-6 have also been linked to various primary psychiatric syndromes and corresponding symptoms. Two meta-analyses have demonstrated that CSF concentrations of IL-6 are elevated in patients with major depressive disorder compared to controls [
31,
32]. In contrast, a recent study that focused on a more homogeneous sample of patients with recent depression did not observe significant elevations in CSF IL-6 levels nor associations with the severity of depressive symptoms [
33]. Elevated levels of IL-6 in blood have been found in patients with bipolar disorder, most strongly during the acute mania phase [
34]. Additionally, higher levels of serum IL-6 have been found in individuals with intermittent explosive disorder and were positively correlated with the level of aggressive symptoms [
35]. Overall, despite conflicting findings, there appears to be an association between elevated levels of central and peripheral IL-6 and the presence of diverse neuropsychiatric symptoms in a variety of neurological and psychiatric conditions. This suggests that elevated IL-6 levels may reflect an altered emotional state commonly observed in these disorders. Consequently, IL-6 has the potential to serve as a sensitive but non-specific marker.
NBD is proposed to be a distinct syndrome encompassing neuropsychiatric symptoms reported in athletes exposed to RHI. The specific components comprising NBD may share overlapping features with symptoms observed in other neurological and psychiatric disorders, although there seem to be notable differences. For instance, symptoms similar to NBD can be observed in individuals who have experienced a single TBI, but these symptoms usually remain stable [
36,
37]. Impulsive behavior is also recognized as part of behavioral disinhibition in the behavioral variant of FTD [
38], while other behavioral changes such as stereotypic behavior or hyperorality are not noted in NBD. Furthermore, NBD exhibits overlap with mild behavioral impairment, which refers to the neuropsychiatric symptoms in early AD [
39,
40], but these symptoms seem to develop later in life than NBD in individuals exposed to substantial RHI. Overlap also exists between NBD and corresponding symptoms of primary psychiatric diseases. According to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V), explosivity and impulsive behavior are key features of intermittent explosive disorder, while tearfulness, irritability, and agitation can be present in major depressive disorder or bipolar disorder [
41]. Distinguishing neuropsychiatric syndromes can be difficult due to symptom overlap. This difficulty applies to both primary psychiatric syndromes and differentiating NBD in individuals exposed to RHI. Complexity increases when coexisting or pre-existing psychiatric syndromes are present. Furthermore, rating scales used to assess symptom severity in these syndromes often contain items that share similarities with NBD symptoms. Consequently, we decided not to include psychiatric disease diagnoses or scores from rating scales measuring other neuropsychiatric symptoms, such as depression, hopelessness, apathy, or anxiety in the analyses of our study. Further investigations are necessary to unravel the construct of NBD and its association with other neuropsychiatric syndromes or symptoms.
It remains unclear what the etiology is of the neuroinflammatory association observed in this study in relation to NBD and whether this is directly linked to exposure to RHI. Notably, CSF IL-6 concentrations were not significantly higher in symptomatic former football players compared to asymptomatic unexposed individuals. It may be possible that this is related to limited power due to the small sample size of unexposed individuals with CSF available. This is supported by the finding (see Table
2) that the former players without NBD had almost identical CSF IL-6 means and SDs as the unexposed controls, although there was only a significant difference between the former players with NBD and those without NBD, but not between the former players with NBD and the controls.
Alterations in IL-6 have previously been observed in studies investigating TBI or RHI. Elevated levels of IL-6 were observed following mild TBI and were associated with poorer outcomes [
42‐
45]. Military personnel and veterans who sustained ≥ 3 TBI had higher serum IL-6 concentrations compared to controls, and these elevated IL-6 levels were associated with symptoms of posttraumatic stress disorder (PTSD) [
46]. One recent study found that male individuals with previous RHI exposure through contact or collision sport, of whom the majority met the criteria for TES, had higher levels of plasma IL-6 compared to both healthy controls and AD patients [
47]. Further evidence for chronic microglial activation in RHI-exposed individuals comes from neuropathology studies as well as translocator protein (TSPO)-positron emission tomography (PET) studies [
5,
7,
48,
49]. Neuroinflammation seems to play a substantial role in the complex pathways following RHI. Several experimental mice studies testing anti-inflammatory treatments after RHI demonstrated reduced inflammatory responses and improvement in clinical performance [
50‐
52]. This neuroinflammatory response can be short-term but can also lead to persistent chronic activation, with associated astrogliosis and p-tau pathology [
6,
53], or behavioral changes [
8,
54]. Another study showed evidence of neuroinflammation associated with microvascular injury and p-tau pathology [
4]. The directionality of the relationship between neuroinflammation and p-tau is debatable, because p-tau may also induce neuroinflammation and progressive cell dysfunction. This highlights the complex relationship between neuroinflammation and neurodegenerative diseases, raising questions about whether neuroinflammation after RHI is a causative factor or a consequence of p-tau pathology and neurodegeneration [
55,
56].
Neurodegeneration, reflected by plasma NfL concentrations, also appears to be linked to NBD in former football players. Plasma NfL serves as a biomarker for neuroaxonal damage and is elevated in multiple central nervous system diseases, including several neurodegenerative disorders [
23]. In these disorders, NfL levels demonstrate good discriminative ability in both plasma and CSF, along with correlations to clinical performance [
57‐
59]. The association between plasma NfL and NBD scores solely within the older age group is not unexpected. Plasma NfL levels increase with advancing age, as does the risk of developing neurodegenerative processes [
23,
60], thus implying a higher likelihood of detecting an effect within this older age category. Furthermore, the relationship between plasma NfL levels and NBD scores showed a significant relationship with executive function and processing speed, suggesting the existence of a clinical syndrome characterized by executive and behavioral dysregulation. This syndrome may be related to chronic axonal degeneration later in life. However, further investigation using longitudinal biomarkers and clinical data is warranted to comprehensively examine these associations and clinical trajectories.
We found no significant associations between CSF IL-6 and cognitive performance and found that the relationship between IL-6 and most NBD factor scores was not influenced by cognitive performance. Previous studies have yielded mixed results regarding the association between IL-6 and cognition. Studies observed altered peripheral IL-6 levels in patients with mild cognitive impairment and AD dementia, but this evidence was less strong for CSF IL-6 [
61]. Other studies found no links between high IL-6 and worse cognitive performance [
62,
63]. Our findings suggest that IL-6 specifically reflects NBD, unlike NfL, which appears to reflect a more general clinical syndrome. Nevertheless, since our data are cross-sectional, these hypotheses should be interpreted with caution and require further investigation in longitudinal studies.
The lack of association between inflammatory markers and explosivity symptoms in this sample suggests that explosivity may be attributed to other factors. Individuals who have already notable levels of aggression and hostility may tend to participate in youth and/or high school football and are more likely to reach college or professional levels later in life. Further research is needed to discover how these symptoms further exacerbate after a football career and what the role is of other factors, such as poor health behaviors and social determinants of health.
Strengths of this study include a substantial sample size of football players and CSF and plasma samples, along with comprehensive clinical and neuropsychiatric data. However, it is important to consider the limitations of this study when interpreting the findings. The study population consisted of male former American football players. As a result, the generalizability of these findings may be limited. Since we did not include symptomatic reference groups, future studies should also focus on non-RHI exposed individuals with symptoms related to NBD, such as those seen in AD patients with neuropsychiatric symptoms, behavioral variant FTD patients, individuals with neuropsychiatric symptoms following single moderate/severe TBI, or patients with primary psychiatric disease. We also did not present results from a medication-free population and did not account for the use of medication other than antidepressants and corticosteroids. For example, we did not account for nonsteroidal anti-inflammatory drug (NSAID) use. Although NSAIDs have been shown to pass the blood–brain barrier, resulting in diluted but detectable levels of NSAIDS in CSF after oral NSAID intake, cytokine levels in CSF did not show significant alterations and we, therefore, expect that NSAID use would not affect our observed CSF results [
64]. This study consisted of cross-sectional data; the neuropsychiatric assessments and CSF included only single measurements while these might be prone to dynamic changes.
Acknowledgements
DIAGNOSE CTE Research Project Current and Former Investigators and Key Personnel: Banner Alzheimer’s Institute—Investigators: Kewei Chen, Ph.D., Hillary Protas, Ph.D., Eric Reiman, M.D. (mPI), Yi Su, Ph.D. Non-Investigators: Connie Boker, M.B.A. (Director, Imaging Center Operations). Boston University Chobanian & Avedisian School of Medicine—Investigators: Michael L. Alosco, Ph.D, Rhoda Au, Ph.D., Robert C. Cantu, Ph.D., Lindsay Farrer, Ph.D., Robert Helm, M.D.*, Douglas I. Katz, M.D., Neil Kowall, M.D.*, Jesse Mez, M.D., Gustavo Mercier, M.D., Ph.D.*, James Otis, M.D.*, Robert A. Stern, Ph.D. (mPI), Jason Weller, M.D. Trainees: Tahlia Bragg, Ph.D. (Postdoctoral Fellow), Irene Simkin, M.S. (Lab Manager, Molecular Genetics Core Facility), Diana Trujillo-Rodriguez, B.Sc., M.Sc. (Ph.D. Student), Suzan van Amerongen, M.D. (Research Fellow). Boston University Project Coordinating Center Staff—Alondra Andino, B.A. (Project Administrative Manager)*, Shannon Conneely, B.A. (Site Coordinator)*, Courtney Diamond, M.B.A. (Project Manager)*, Tessa Fagle, B.A. (Research Assistant), Olivia Haller, B.A. (Recruitment Coordinator)*, Tennyson Hunt, M.B.A. (Project Administrative Manager)*, Nicole Gullotti, M.B.A. (Research Administrator)*, Bailey Kossow, B.S. (Research Assistant), Carrie Kugelmass, B.A. (Research Assistant), Megan Mariani, B.S., B.A. (Project Manager)*, Brian Mayville, B.S. (Site Coordinator)*, Kathleen McLaughlin, B.A. (Research Assistant)*, Mary Nanna, B.A. (Retention Coordinator)*, Marty DiPopolo, B.S. (Retention Coordinator), Taylor Platt, M.P.H. (Recruitment Coordinator)*, Surya Pulukuri, B.A. (Research Assistant), Fiona Rice, M.P.H. (Project Manager)*, Madison Sestak, B.S. (Assistant Recruitment Coordinator) *, Irene Simkin, M.S. (Lab Manager, Molecular Genetics Core Facility). Boston University School of Public Health—Investigators: Michael McClean, Sc.D., Yorghos Tripodis, Ph.D., Data Team Staff: Douglas Annis, M.S. (Systems Analyst)*, Christine Chaisson, M.P.H. (Leader of Data Management Sub-team)*, Diane B. Dixon (Project Manager), Carolyn Finney, B.A. (Data Manager), Kerrin Gallagher, M.P.H. (Statistical Analyst)*, Kaitlin Hartlage, M.P.H. (Statistical Analyst), Jun Lu, M.S. (Data Security and Technology Analyst), Brett Martin, M.S. (Statistical Manager), Emmanuel Ojo, M.P.H. (Statistical Analyst)*, Joseph N. Palmisano, M.A., M.P.H. (Leader of Data Management Sub-team), Brittany Pine, B.A., B.S. (Statistical Analyst), Janani Ramachandran, M.S. (Data Manager)*, Trainees: Zachary Baucom, Ph.D., Fatima Tuz-Zahra, M.S., Eukyung Yhang, B.A. Brigham and Women’s Hospital—Investigators: Sylvain Bouix, Ph.D., Jennifer Fitzsimmons, M.D.*, Alexander P. Lin, Ph.D., Inga K. Koerte, M.D., Ph.D., Ofer Pasternak, Ph.D., Martha E. Shenton, Ph.D. (mPI), Non-Investigators: Hector Arciniega, Ph.D. (Postdoctoral Research Fellow), Tashrif Billah, M.S. (Software Engineer), Elena Bonke, M.S. (Ph.D. Student), Katherine Breedlove, Ph.D. (Postdoctoral Research Fellow), Holly Carrington, B.A. (Research Assistant), Eduardo Coello, Ph.D. (Postdoctoral Research Fellow), Michael J. Coleman, M.A. (Senior Scientist), Omar John, B.S. (Research Assistant), Leonard Jung, (Ph.D. Student), Huijun Liao, B.S. (Study Coordinator), Maria Loy, M.B.A., M.P.H. (Senior Program Coordinator), Elizabeth Rizzoni, B.A. (Research Assistant), Vivian Schultz, M.D. (Postdoctoral Research Fellow), Annelise Silva, B.S. (Research Assistant)*, Brynn Vessey, B.S. (Research Assistant), Tim L.T. Wiegand, (Ph.D. Student). Cleveland Clinic Lou Ruvo Center for Brain Health—Investigators: Sarah Banks, Ph.D. (Now at University of California, San Diego), Charles Bernick, M.D., Jason Miller, Ph.D., Aaron Ritter, M.D., Marwan Sabbagh, M.D.* (Now at Barrow Institute), Non-Investigators: Raelynn de la Cruz, (Psychometrician)*, Jan Durant, (Psychometrician)*, Morgan Golceker (Site Coordinator), Nicolette Harmon, (Site Coordinator)*, Jaeson Kaylegian, (Psychometrician)*, Rachelle Long, (Site Coordinator)*, Christin Nance, (Psychometrician)*, Priscilla Sandoval (Site Coordinator)*, Miranda Staples, Ph.D. (Program Manager). George Washington University School of Medicine and Health Sciences—Investigator: Robert W. Turner, Ph.D., Non-Investigators: Emma F. Clark, B.A. (Research Assistant)*. Invicro (formerly Molecular NeuroImaging)—Investigator: Kenneth L. Marek, M.D. Non-Investigator: Andrew Serrano, M.B.A. Mayo Clinic Arizona—Investigators: Charles H. Adler, M.D., Ph.D., David W. Dodick, M.D. (Now at Atria Academy of Science and Medicine), Yonas Geda, M.D., M.Sc. (Now at Barrow Neurological Institute), Jennifer V. Wethe, Ph.D. Non-Investigators: Amy Duffy, (Site Coordinator)*, Bryce Falk, R.N., Marci Howard, (Psychometrician)*, Michelle Montague, (Psychometrician)*, Thomas Osgood, (Site Coordinator). National Institute of Neurological Disorders and Stroke (NINDS)—Debra Babcock, M.D., Ph.D. (Scientific Program Official), Patrick Bellgowan, Ph.D. (Administrative Program Official)*. New York University—Investigators: Laura Balcer, M.D., M.S.C.E., William Barr, Ph.D., Judith Goldberg, Sc.D., Binu Joseph, M.B.B.S., Ivan Kirov, Ph.D., Yvonne Lui, M.D., Charles Marmar, M.D., Thomas Wisniewski, M.D.*, Non-Investigators: Alhassan Al-Kharafi (Psychometrician)*, Allan George (Psychometrician)*, Lisena Hasanaj (Site Coordinator), Sammie Martin (Psychometrician) , Edward Riley (Psychometrician)*, William Runge (Psychometrician)*, Liliana Serrano. University of Gothenburg, Sweden—Nicholas Ashton, Ph.D., Henrik Zetterberg, M.D., Ph.D., Kaj Blennow, M.D., Ph.D. University of Nevada, Las Vegas—Jeffrey L. Cummings, M.D., ScD (mPI). VA Puget Sound and University of Washington—Investigator: Jeffrey Iliff, Ph.D., Gail Li, M.D., Ph.D., Deidre Janssen, Ph.D., James Meabon, Ph.D., Elaine R. Peskind, M.D., Juan Piantino, M.D., Abigail Schindler, Ph.D., Ronald Thomas, Ph.D, Non-Investigator: Elizabeth Colasurdo (DIAGNOSE CTE Biofluid Bank Manager), Jane Shofer, M.S. Washington University (CNDA)—Investigators: Daniel S. Marcus, Ph.D., Non-Investigator: Jenny Gurney, M.S., Consultants: Richard Greenwald, Ph.D. (Simbex)*, Keith A. Johnson, M.D. (Massachusetts General Hospital), *No longer involved in the project.
Declarations
Competing interests
SVA: none to report. SVP: none to report. FTZ: none to report. YT: none to report. JDC: none to report. CB: receives research support from the Ultimate Fighting Championship, Top Rank promotions, and Haymon Boxing. YEG: receives funding from the NIH and Roche, and served on Lundbeck Advisory Board. JVW: None to report. DIK: received royalties from Springer/Demos Publishing for a textbook on brain injury; serves as an expert witness in legal cases involving brain injury and concussion; receives a stipend from Encompass Health as program medical director for brain injury and chair of the annual Neurorehabilitation conference; and has received honoraria for a keynote address for the HealthSouth Annual Medical Directors Meeting. MLA: receives royalties from Oxford University Press for a textbook outside the submitted work. CHA: None to report. LJB is Editor-in-Chief of the Journal of Neuro-Ophthalmology. NJA: none to report. KB: has served as a consultant and at advisory boards for Acumen, ALZPath, BioArctic, Biogen, Eisai, Lilly, Moleac Pte. Ltd, Novartis, Ono Pharma, Prothena, Roche Diagnostics, and Siemens Healthineers; has served at data monitoring committees for Julius Clinical and Novartis; has given lectures, produced educational materials and participated in educational programs for AC Immune, Biogen, Celdara Medical, Eisai and Roche Diagnostics; and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program, outside the work presented in this paper. HZ: has served on scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, ALZPath, Annexon, Apellis, Artery Therapeutics, AZTherapies, CogRx, Denali, Eisai, Nervgen, Novo Nordisk, Passage Bio, Pinteon Therapeutics, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures in symposia sponsored by Cellectricon, Fujirebio, Alzecure, Biogen, and Roche. DHD: serves as an expert witness in legal cases involving brain injury and concussion and serves as an advisor and options holder for StataDx. EAC: None to report. JJI: None to report. GL: None to report. ERP: has served as consultant, on scientific advisory boards, or on data monitoring committees for Eli Lilly, Avanir, Acadia, Roche, Regeneron, and ALPHA-Cognition. EMR is a compensated scientific advisor for Alkahest, Alzheon, Aural Analytics, Denali, Green Valley, Retromer Therapeutics, and Vaxxinity, and a co-founder of ALZPath. MES: None to report. JLC has provided consultation to Acadia, Actinogen, Acumen, AlphaCognition, Aprinoia, AriBio, Artery, Biogen, BioVie, Bristol-Myers Squib, Cassava, Cerecin, Diadem, EIP Pharma, Eisai, GemVax, Genentech, GAP Innovations, Janssen, Jocasta, Karuna, Lighthouse, Lilly, Lundbeck, LSP/EQT, Merck, NervGen, Novo Nordisk, Oligomerix, Optoceutics, Ono, Otsuka, PRODEO, Prothena, ReMYND, Roche, Sage Therapeutics, Signant Health, Simcere, Suven, SynapseBio, TrueBinding, Vaxxinity, and Wren pharmaceutical, assessment, and investment companies. He owns the copyright of the Neuropsychiatric Inventory. RAS is a member of the Board of Directors of King-Devick Technologies, Inc. (Chicago, IL, USA), and he receives royalties for published neuropsychological tests from Psychological Assessment Resources, Inc. (Lutz, FL, USA), and consulting fees from Eisai.