Background
Traumatic brain injury (TBI) is a leading cause of death and disability worldwide and represents a critical public health and socio-economic problem [
1]. TBI can range from mild concussions with short-term and reversible symptoms to severe and permanent brain damage. Current treatment options are restricted to surgical intervention and supportive care. However, secondary pathogenic processes, such as disintegration of the blood-brain barrier (BBB), disturbed autoregulation of cerebral blood flow, impaired tissue oxygenation, and mitochondrial metabolism, which result in the expansion of the damage into the surrounding healthy tissue [
2‐
4], are potential targets of therapeutic approaches in TBI [
5,
6]. In addition, TBI elicits a robust immune response within hours and days [
7]. This immune response is characterized by the infiltration of the damaged tissue by peripheral immune cells and the activation of brain resident astrocytes and microglia, both in patients and in animal models of TBI [
8‐
14]. However, the inflammatory reaction after TBI evokes beneficial and detrimental effects which are still incompletely understood at the mechanistic and molecular level [
15‐
18].
Animal studies using histology, flow cytometry, and/or intravital microscopy provided evidence that neutrophils are the earliest peripheral immune cells entering the injured brain followed by macrophages, dendritic cells, and T cells, eventually after the secondary brain damage occurred [
19‐
22]. T cells infiltrating the damaged tissue were observed at posttraumatic days 3–5 in the controlled cortical impact (CCI) model of TBI [
13,
23]. However, reduced numbers of infiltrating T cells were found at posttraumatic day 7 [
13,
24]. More recently, persistent chronic T cell infiltration was reported 3 months after lateral fluid percussion injury which, however, did not correlate with the severity of chronic inflammation [
25]. Thus, it has been demonstrated that T cells infiltrate the brain parenchyma in TBI during acute and chronic phases. Moreover, some studies indicated that T cells aggravate brain injury [
26,
27]. Accordingly, effector CD4+ T cells adoptively transferred into T cell- and B cell-deficient recombination-activating gene 1 (RAG1) knockout mice exacerbated lesion size and apoptosis after brain injury [
28]. Experimental studies in the early phase of TBI further indicated that the number of circulating T cells correlated with T cell infiltration and inflammatory responses as well as cell death beneath the impact site [
29].
To date, only scarce data exist on the role of regulatory T cells (Tregs) in TBI. This T cell subtype suppresses and thereby controls effector T cells to prevent exaggerated immune responses and in particular autoimmunity [
30‐
32]. Notably, it has been reported that the number of circulating Tregs are positively correlated with a favorable clinical outcome after TBI [
33]. Nevertheless, controversial results on the role of Tregs were reported in different types of CNS injuries [
34], e.g., either removal or addition of Tregs impaired retinal ganglion cell survival following optic nerve injury [
35]. The role of Tregs is also a matter of debate in stroke and its animal models [
36‐
38], which share many pathophysiological features with TBI [
39].
In summary, the inflammatory response in TBI is spatially and temporally dynamic and the role of Tregs therein is largely unexplored. Here, we studied this issue in experimental TBI using diphtheria toxin (DTx)-mediated depletion of Tregs in transgenic DEREG mice. We subjected mice to the CCI model of TBI, examined neurological and motor deficits until 5 days post-injury (dpi) which corresponds to the acute phase of TBI. The consequences of Tregs depletion were evaluated using behavioral, (immuno-) histological, protein, and gene expression analyses.
Discussion
The objective of this study was to investigate the hitherto unresolved role of Tregs in TBI. We subjected DEREG mice to the CCI model of TBI and examined neurological impairment and motor deficits from 1 dpi to 5 dpi, considered as the acute phase of CCI. Our results from histopathological and neurological analyses at 5 dpi do not support a critical role of Tregs for the extent of structural brain damage or the overall neurological outcome. However, Treg-depleted DEREG mice exhibited a trend towards increased NSS and transiently aggravated motor deficits at 1 dpi and increased reactive astrogliosis in the injured brain hemisphere at 5 dpi. Furthermore, we found an increased number of CD3+ T cells infiltrating the perilesional brain parenchyma and elevated IFN-γ gene expression in DEREG mice indicating an immunomodulatory role of Tregs in the brain response to traumatic injury.
The extent of the structural brain damage and the amount of SBDPs as a proxy of Ca
2+-dependent neuronal excitotoxicity [
51] were not different between DEREG and WT mice. These findings indicate that Tregs play a minor role for neuronal survival in the acute phase of TBI. These findings are consistent with results of a genetic approach of Treg depletion after experimental cerebral focal ischemia in mice; Tregs alone, respectively Foxp3 expressing cells, were not capable to limit brain damage or improve functional outcome in mice [
53]. On the other hand, depletion of Tregs with anti-CD25 mAb profoundly increased delayed brain damage and deteriorated functional outcome [
54]. However, anti-CD25 mAb depletes all CD25+ cells, which includes precursor, mature, and activated T and B cells [
55] which may contribute to conflicting results from different approaches of Treg depletion in the MCAO model of stroke.
In the present study, we found overall increased GFAP protein levels in lysates from injured brain tissue of DEREG mice and an increased number of GFAP+ astrocytes indicating exaggerated reactive astrogliosis. Reactive astrogliosis is a general response after brain lesion, which has been linked both to beneficial and detrimental processes [
56]. Normally, astrogliosis and scar formation improve the outcome and provide neuronal protection, BBB repair, and restriction of CNS inflammation [
57], but astrocytes can also adopt a neurotoxic phenotype at injury sites [
58]. Furthermore, astrocytes become reactive in response to neuroinflammatory stimuli and peripheral immune cells can communicate with astrocytes and other brain resident cells to create a highly inflammatory microenvironment [
18]. However, these processes depend on different contexts, e.g., astrocytes may recruit T cells into the brain parenchyma at early stages while helping to clear T cells from the brain in later inflammation resolution [
59]. On the other side, T cells (i.e., Th17 effectors) were proposed to preferentially target astrocytes to promote neuroinflammation [
60]. Together with our results showing that DEREG mice are harboring more CD3+ T cells and GFAP+ astrocytes at perilesional sites, these findings suggest a T cell-dependent mechanism leading to preferential activation of astrocytes and reactive astrogliosis. In support of this hypothesis, gene expression of IFN-γ, a major T cell-released factor in the immunological activation of astrocytes [
61,
62], was significantly increased in DEREG mice after CCI.
Intriguingly, IFN-γ is associated with greater disability in the post-acute phase after severe TBI in humans [
63] and can modulate neuronal networks [
64]. Other studies associated impaired motor function with increased expression of inflammation-related genes either in response to environmental factors [
65] or to genetic factors [
66]. Along with the observation of increased IFN-γ expression, our results from the rotarod performance test indicate that the depletion of Tregs causes transient aggravation of motor deficits at 1 dpi. In addition, a trend towards increased neurological deficits was observed at 1 dpi in DEREG mice. These effects are likely due to the DTx-mediated depletion of Tregs, as previous investigations confirmed that untreated DEREG do not show phenotypic abnormalities compared to C57Bl6 mice [
67]. Possibly, the rotarod task may represent a more sensitive task to assess motor impairment in the context of murine TBI. It has been reported that rotarod performance is a very sensitive and efficient index for assessing motor impairment compared to the beam balance and beam walking tasks [
68], which are parts of the composite NSS used in the present study [
44]. However, neither the rotarod task nor the NSS revealed differences between DEREG and WT mice at 5 dpi, the endpoint of our study. Thus, our findings point to a (immuno-) modulatory role of Tregs without major impact on brain tissue loss and persistent neurological impairment in TBI. A major limitation of the present study is that only a single posttraumatic time point has been investigated. To better understand the immunomodulatory role of Tregs in TBI, additional studies are required including long-term studies to examine their contribution to chronic brain inflammation and associated co-morbidities such as epilepsy and neuropsychiatric disorders. Along this line, a recent study demonstrated that Tregs during the chronic phase of experimental stroke suppress reactive astrogliosis and enhance neurological recovery [
69]. Also, molecular characterization of immune cell infiltrations and reactive astrocytes in the injured brain using cell sorting and single-cell RNA sequencing applications in combination with appropriate animal models might be instrumental to reveal molecular pathways and targets for preclinical studies. Furthermore, adoptive transfer of Tregs after TBI may exert neuroprotective and anti-inflammatory actions similar to previous findings in animal models of post-stroke hemorrhage [
70] or intracerebral hemorrhage using autologous blood infusion [
71]. It will be further important to investigate whether adoptive transfer of Tregs may slow or prevent effector T cell brain infiltration and their potentially detrimental actions in animal models of stroke and TBI.
Up to date, inflammation targeted therapy has not been translated into the clinical setting of TBI, despite promising results in animals studies. Previous attempts to directly influence T cells in humans resulted in a life-threatening systemic inflammatory response syndrome, leading to multiorgan failure after administration of an anti-CD28 monoclonal antibody [
72]. In clinical practice, self-tolerance is especially important in patients with autoimmune diseases or after organ transplantation and immunosuppressive drugs such as rapamycin are widely used. Experimental research provided evidence that rapamycin decreases the number of CD4+ T cells and simultaneously increases CD4+/CD25+ Tregs [
73,
74]. Also in murine cerebral ischemia, Treg amplification with a CD28 superagonistic monoclonal antibody [
75] or rapamycin treatment [
76] attenuated the neurological outcome after stroke induction. These effects are to some extent in line with our conclusions and are most probably caused by the modulation of Tregs. In support of this hypothesis, an increased number of Tregs in the circulation appears to be associated with a better outcome after TBI in humans [
33]. However, recent work suggests that conclusions on the numerical increase of Tregs are complicated due to the fluctuating expression of Foxp3 and CD25 and therefore may not be decisive for the therapeutic success by modulation of Tregs [
77].
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