Neuroimmune remodelling and secondary inflammatory vulnerability after traumatic brain injury

Abstract

Traumatic brain injury (TBI) represents not merely a mechanical insult but a biologically intricate and evolving disorder characterized by neurovascular disruption, sterile inflammation, leukocyte recruitment, and tissue remodelling, all of which collectively influence neurological outcomes. While the early activation of the innate immune system following TBI is well-documented, the progression of this response into T cell engagement and its potential to leave a lasting immune imprint within the injured brain remains less understood. This thesis explores the hypothesis that controlled cortical impact (CCI) induces a temporally ordered neuroimmune response, wherein early innate immune programs influence subsequent T cell immunity, culminating in a persistent tissue-associated immune state that alters susceptibility to subsequent systemic inflammatory challenges. The study particularly emphasizes the interleukin-1β (IL-1β) axis, innate and T cell immune crosstalk, and the emergence of tissue-resident memory T cell (TRM) populations post-injury. To investigate this, adult male C57BL/6J mice were subjected to moderate-severe CCI and examined across acute, subacute, and chronic phases post-injury. Brain mononuclear-cell isolation was optimized for downstream immune profiling, using multiparameter spectral flow cytometry, intracellular cytokine staining, and RT-qPCR to delineate immune composition and effector programs over time. Behavioural outcomes were assessed using beam walk, rotarod, Y-maze, novel object recognition, and SNAP scoring. Mechanistic studies targeted distinct innate pathways through anti-Ly6G mediated neutrophil attenuation, MC-21-mediated CCR2-dependent monocyte depletion, and pharmacological NLRP3 inflammasome inhibition with MCC950. Chronic adaptive remodelling was examined using intravascular anti-CD45 labelling to differentiate tissue-associated from blood-circulating leukocytes, while a secondary systemic inflammatory challenge with LPS, with or without CD49d/VLA-4 blockade, was employed to ascertain whether post-traumatic immune amplification relied on continued leukocyte trafficking or resident tissue-associated compartments. The influence of aging on post-traumatic TRM responses was also investigated. TBI elicited a sequential and compartmentalized neuroimmune response. Early post-injury, inflammatory gene expression was rapidly induced, with Ly6G+ neutrophils and Ly6C+ monocytes emerging as the predominant infiltrating populations and primary cellular sources of IL-1β. As the response progressed, adaptive immune involvement became more pronounced, with γδ+ T cells constituting the main interleukin-17 (IL-17) producing population and interferon-γ (IFN-γ) distributed across multiple T cell subsets. Mechanistic studies revealed that targeting individual innate compartments did not simply suppress downstream adaptive immunity, neutrophil attenuation altered inflammatory composition without clear functional benefit, CCR2-dependent monocyte depletion yielded only modest effects, and NLRP3 inhibition with MCC950 selectively reduced microglial activation while leaving broader leukocyte recruitment and T cell responses largely unaffected. Concurrently, TRM cell populations peaked during the subacute phase, and persisted chronically within the injured hemisphere. Aging amplified this response, and secondary systemic inflammation with LPS triggered robust reactivation of TRM cells. CD49d blockade attenuated T cell trafficking; however it did not eliminate the TRM response. This suggests that both continued trafficking and resident tissue-associated immunity contribute to post-traumatic immune amplification. In conclusion, this thesis elucidates that TBI initiates a prolonged and compartmentalized process of immune remodelling, rather than merely a transient inflammatory response. The findings indicate that post-traumatic pathology is maintained through complex interactions between innate and T cells, persistent tissue-associated immunity, and recruitment-dependent amplification, with aging and secondary challenge further exacerbating vulnerability. These results highlight the IL-1β axis, TRM responses, and trafficking-dependent immune interactions as critical components of post-traumatic neurobiology and as potential therapeutic targets for mitigating chronic inflammation and subsequent vulnerability following TBI.