5. T cell immunity and neuroplasticity

Abstract

The proneuronal effects of T cells that impact the brain occur from both T cells trafficking into the brain, and from signals in the periphery (e.g., cytokine release and regulation). Recent data indicates that neuroimmunological changes in the brain can modify intrinsic brain processes that are involved in regulating neuroplasticity (e.g., T-cell/microglial interactions, neurotrophins, neurogenesis). We describe: 1) work from our lab and others showing that injury-induced loss of neuronal phenotype and reversal of motor neuron atrophy are associated with normal T cell immunity, and; 2) research indicating that these and other neuroimmunological processes may be generalizable to mechanisms of neuroplasticity involved in cognitive and emotional behavior. These findings are discussed in relation to our lab’s working hypothesis, that T cell immunosenesence may contribute to alterations in brain neuroplasticity related to aging. Greater understanding of the role of adaptive T cell immunity on neuroplasticity could have important clinical implications for developing novel treatment strategies for neurodegenerative diseases (e.g., Alzheimer’s) and brain injury (e.g., stroke, trauma).

The proneuronal actions of T lymphocytes

The literature indicates that T cells have proneuronal effects in the brain, effects that appear to occur from both the periphery (e.g., cytokine release and regulation) and within the brain (Raivich et al., 1998; Ha et al., 2007a,b; Kipnis et al., 2008; Wolf et al., 2009; Meola et al., 2013b; Schwartz et al., 2013). It has long been appreciated that during pathogenic conditions such as multiple sclerosis and brain infection, the presence of T cells in the central nervous system (CNS) were often associated with an increased risk of neuronal damage (Martino and Hartung, 1999; Nau and Brück, 2002). A growing body of research, however, has established that in other contexts T cells act in together with glial cells to promote neuroprotection and survival (Byram et al., 2004; Jones et al., 2005; Ha et al., 2006, 2007a; Kipnis et al., 2008; Huang and Petitto, 2013).

Immune surveillance of the CNS occurs by small numbers T lymphocytes trafficking in and out of the brain, and it is now recognized that under normal physiological conditions T lymphocytes have important effects on neuronal integrity and function (Cose et al., 2006; Hickey et al., 1991). The adaptive arm of the peripheral immune system has been shown to have beneficial effects on neuronal outcomes in various models of trauma (e.g., mechanical, toxic, ischemic, hemorrhagic) (Graber et al., 2009). Perhaps one of the best examples of the neuroprotective role of adaptive immunity is facial nerve axotomy, where T cells have been found to slow the rate of neurodegeneration and neuronal loss after axons are disconnected from their target muscle (Jones et al., 2005; Serpe et al., 2000). Following facial nerve axotomy in mice, T cells cross the blood–brain-barrier (BBB) and traffic to the neuronal cell bodies in the facial motor nucleus (FMN) (Raivich et al., 1988). Immunodeficient mice such as severe combined immunodeficient (SCID) and recombination activating gene knockout (RAG-KO) mice, which both lack functionally mature T and B lymphocytes, exhibit a faster rate of neuronal death than wild-type (WT) mice (Jones et al., 2005; Serpe et al., 2000). The proneuronal or neuroprotective activity resides clearly within the T cell population, as B cells appear to have no effect and are not found within the facial motor nucleus following facial nerve axotomy (Jones et al., 2005; Ha et al., 2006, 2007a,b).

Adapative immunity and neuroplasticity: role in motor neuron atrophy versus death

Identifying the processes by which neurons in the CNS atrophy and lose their phenotype, rather than die, has been of interest since the seminal discovery by Hagg and colleagues (1988, 1989) which showed that axotomized cholinergic septohippocampal projection neurons that shrunk and lost their cholinergic phenotype, could be restimulated to regain their normal size and phenotype following NGF infusion. Understanding these enigmatic processes has important implications for the discovery new treatment strategies (Hagg et al, 1988, 1989; Hottinger et al., 2000; Kwon et al., 2002a,b). McPhail et al. (2004) demonstrated that many adult facial motor neurons undergo a protracted period of atrophy following peripheral resection of the facial nerve in mice, and exist in a lower energy state with decreased ability to uptake dyes such as Nissl. This chronic resection-induced atrophy of facial motor neurons can be reversed by both GDNF delivery (Hottinger et al., 2000) and by reinjuring the same resected facial nerve (McPhail et al., 2004).

Our lab has shown that the adaptive arm of the immune system appears to be required to reverse the atrophic status of these injured motor neurons (Ha et al., 2007a). Specifically, we used the facial nerve reinjury model to test the hypothesis that the reversal of motor neuron atrophy (i.e., increase in cell number and size) elicited by nerve reinjury would be abnormal in immunodeficient RAG2-KO mice. We found that whereas a substantial portion of chronically resected facial motor neurons reside in an atrophied state that can be reversed at 14 days following reinjury in wild-type (WT) mice, atrophy reversal was abnormal in immunodeficient RAG2-KO mice. It was unclear, however, if the abnormal response at day 14 post-reinjury in immunodeficient mice might be due to differences in the kinetics of the reversal response or an imparted regeneration response. Although our initial study provided the first data in the literature to suggest that a functional adaptive immune systems may be required to regenerate the normal phenotype of atrophied facial motor neurons, it was possible, for example, that RAG2-KO mice have a less sustained reinjury response than WT mice; an attenuated response that could occur earlier, in closer proximity to the time of the reinjury stimulus. Specifically, as it has been shown that a larger response occurs at day 7 than day 14 following reinjury in WT mice, it was possible that the altered reinjury-induced reversal response that we saw in RAG2-KO mice at day 14 post-reinjury might be due to timing.

Microglial proliferation is most pronounced 3 days after facial nerve axotomy. Therefore, in a recent study we sought to extend our initial finding by comparing WT and immunodeficient RAG2-KO mice in the facial nerve reinjury paradigm at day 3, and later in time, at day 28 post-reinjury. We sought to test our working hypothesis that the normal regeneration of atrophied motor neurons is dependent on normal adaptive immunity, and determine if there were differences in kinetics of the reversal response between the groups. Specifically, we sought to determine if the neuronal atrophy reversal response was sustainable out to 1-month duration post-reinjury in immunologically intact WT, and compare immunodeficient RAG2-KO and WT mice across time. To this end, we compared motor neuron survival and size between WT and RAG2-KO mice in the facial nerve reinjury paradigm at 3 and 28 days post-reinjury (Huang and Petitto, 2013). We found that in WT mice, facial motor neurons that were resected for 10 weeks and subsequently reinjured for 3 days were able to regain fully an apparent 40% loss of countable neurons), and nearly 45% of that robust increase in neurons was sustained at 28 days post-reinjury in the WT mice. By contrast, at both 3 and 28 days post-reinjury RAG2-KO mice failed to show any increase in neuronal number. Size measurements showed that the surviving neurons of WT and RAG2-KO mice exhibited substantial motor neuron hypertrophy at 3 days post-reinjury, and similar levels of normal size motor neurons by 28 days post-reinjury. Among the WT mice, small numbers of T lymphocytes where found in the re-injured facial motor nucleus (FMN), and were significantly higher at 3 days, but not 28 days, in the reinjury compared to sham-reinjury groups. No differences were seen between the WT and RAG2-KO mice in overall microglial cell activity using CD11b expression following reinjury. These data suggest that many resected motor neurons did not survive the initial resection in RAG2-KO mice, whereas in WT mice they atrophied and could be restimulated by reinjury to regenerate their phenotype.

We did not expect no response whatsoever in the RAG2-KO. We thought that the response would be impaired or show an altered kinetic profile compared to WT mice. As noted earlier, we speculated that the RAG2-KO mice may have had a less sustained reinjury response than WT mice at 3 days (that was absent by 14 days in our previous study) (Ha et al., 2007a). The fact that there was no atrophy reversal response whatsoever at both day 3 and 28 in the RAG2-KO mice, despite a robust response in WT mice, coupled with the finding that the size of the surviving motor neurons of RAG-2KO and WT were similar in size, indicates that the chronically axotomized motor neurons of RAG2-KO mice likely died from the first resection surgery.

Together, our studies showed that many resected motor neurons did not survive the initial resection in RAG2-KO mice, whereas in WT mice they atrophied and could be restimulated by reinjury to robustly regenerate their phenotype. In fact, the duration of the atrophy reversal response of these resected WT neurons disconnected from their target tissue was sustained at 1 month following reinjury where they surprisingly maintained nearly half the gain in neurons that they exhibited at day 3 post-reinjury. Thus, it appears that normal T cell function is essential for activating regeneration programs of atrophied motor neurons. Moreover, we have not found neurons co-labeled with BrdU and doublecortin in the reinjured FMN model, confirming that the reinjury-induced reappearance neurons are not neurostem cells.

Although the literature has established that the neuroprotective activity of the adaptive arm of the immune system resides within the T cell population in the facial nerve axotomy model, low levels of T cells in the reinjured FMN, the lack of reinjury-induced regeneration suggests that normal T cell function in the CNS and/or the periphery could be essential for activating regeneration programs of atrophied motor neurons (Jones et al., 2005; Ha et al., 2007a). Future studies will need to determine that this effect is generalizable to other types of immunodeficient mice (e.g., SCID), or could be related to some yet unknown function of the RAG2 gene in the brain.

T cells, neuroplasticity and behavior: A role in brain aging?

Impairments in cognitive and emotional behavior may be modulated, in part, by T cell homeostasis (Cushman et al., 2003; Ziv et al, 2006; Kipnis et al., 2008; Wolf et al., 2009; Schwartz et al., 2013). Several lines of evidence suggest that proinflammatory conditions regulated by T cell homeostasis are associated with reduced levels of neurogenesis (Kohman et al., 2013). T cells are pivotal in modulating overall immunological homeostasis, and T lymphocytes and the cytokines that they secrete and/or modulate have been implicated in processes associated with neurogenesis (Mahanthappa and Schwarting, 1993, Farbman and Buchholz, 1996, Satoh and Yoshida, 1997, Newman et al., 2000, Tham et al., 2001, Vallieres et al., 2002, Bauer et al., 2003, Hastings and Gould, 2003; Beck et al., 2005b). Compelling evidence shows that a decline in neurogenesis underlies deterioration in context-dependent learning in aging (Garthe and Kempermann, 2012; Lazarov and Marr, 2013). Moreover, reduced rates of neurogenesis in the dentate gyrus are associated with emotional behavior in animals. Decreased neurogenesis has been associated with increased novelty-induced activity (Lemaire et al., 1999; Kalm et al., 2013) and with deficits in contextual fear discrimination and related forms of fear-based learning (Shors et al., 2002; Nakashiba et al., 2012). Neuroplasticity is altered by the neuroimmunological milieu in the hippocamapus in aging. These changes may be accompanied by alterations in neurobiological processes the underlie brain neuroplasticity including changes in neurotrophins (e.g., NGF and BNDF), glial cell responsiveness and neurogenesis (Beck et al., 2005a,b; Meola et., 2013a).

The actions of T cells in neuronal recovery and function may have important implications for aging. Age-dependent deficits in T cell function in the adaptive arm of the immune system coexist with age-related changes within the innate immune system; however, innate immunity is better preserved, while more severe and often detrimental age-dependent changes occur in the adaptive immune system (Weiskopf et al., 2009). As aging is associated with decreased T cell function (Miller, 1996; Pawelec et al., 1999; Franceschi et al., 2000; Linton and Thoman, 2001; Nikolich-Zugich, 2008), older T cells may be less effective at protecting aging or injured neurons, and be less capable of supporting neurogenesis. Neurogenesis is altered as animals age (Lazarov and Marr, 2013; Kalm et al., 2013). Manipulating T cell age and/or neurogenesis will be important to establish this linkage more directly in future studies to build upon initial observations in the literature (Ziv et al., 2006).

Our lab has found that even as early as late middle-age, for example, that axonal injury induces a marked increase in T cell trafficking to the neuronal cell bodies of origin in the brain (Dauer et al., 2011). As mice age, exaggerated neuroinflammatory responses occur in the hippocampus following infection and LPS (Huang et al., 2008; Richwine et al., 2008; Henry et al., 2009; Jurgens and Johnson, 2012). Aging mice also exhibit higher expression of certain T cell related immune response genes following immune challenge with LPS (Terao et al., 2002). Moreover, in the baseline unchallenged state, aging mice have markedly increased levels of T cells in the hippocampus (Stichel and Luebbert, 2007). Thus, aging alters the neuroimmunological milieu of the brain, and appears to contribute to reduced hippocampal neurogenesis found with brain aging (Lynch and Johnson, 2012; Jurgens and Johnson, 2012; Kohman et at., Lazarov and Marr, 2013).

Neuroimmunology: significance for brain aging and related disorders

Studies in neuroimmunology could enable us to conduct research to disentangle the relative contribution of T cell aging from intrinsic mechanisms of brain aging. Our lab is engaged in approaches to test directly our hypothesis that T cell immunosenescence alters neuroplasticity and neurobehavioral performance by manipulating the immune system of mice. Novel strategies are being employed to examine a unique mechanism whereby the peripheral immune system protects brain neurons and augments brain processes (e.g., glia cells, neurotrophins) that protect neurons from age-related neuroregeneration. New research approaches are continually being sought to impact the progression of neurodegeneration, and to intervene in brain injury and trauma to save or regenerate damaged brain neurons. Novel ways of viewing the biology and the interactions between complex systems, though more risky, are essential to advance the field as well as move through barriers to entertain and devise more effective treatments for clinical conditions affecting the brain. Further research using models such as the facial nerve reinjury model, for example, could identify the molecular signals involved in this powerful and unique form of neuroplasticity (van Kesteren et al., 2011; Yamamoto et al, 2012), and lead new ways to induce or augment neuroregeneration in patients with neurotrauma and other forms of CNS insult and disease. As diminished T cell function associated with normal aging could be involved in brain aging and repair, greater understanding of brain-immune interactions in aging could have important implications for treating neurodegenerative diseases (e.g., Alzheimer’s disease), and improving outcomes for elderly individuals with vascular insults and other forms of CNS trauma.