CD4+ T cell-mediated neuroprotection is independent of T cell-derived BDNF in a mouse facial nerve axotomy model.

BACKGROUND: The production of neurotrophic factors, such as BDNF, has generally been considered an important mechanism of immune-mediated neuroprotection. However, the ability of T cells to produce BDNF remains controversial. METHODS: In the present study, we examined mRNA and protein of BDNF using RT-PCR and western blot, respectively, in purified and reactivated CD4(+) T cells. In addition, to determine the role of BDNF derived from CD4(+) T cells, the BDNF gene was specifically deleted in T cells using the Cre-lox mouse model system. RESULTS: Our results indicate that while both mRNA expression and protein secretion of BDNF in reactivated T cells were detected at 24 h, only protein could be detected at 72 h after reactivation. The results suggest a transient up-regulation of BDNF mRNA in reactivated T cells. Furthermore, in contrast to our hypothesis that the BDNF expression is necessary for CD4(+) T cells to mediate neuroprotection, mice with CD4(+) T cells lacking BDNF expression demonstrated a similar level of facial motoneuron survival compared to their littermates that expressed BDNF, and both levels were comparable to wild-type. The results suggest that the deletion of BDNF did not impair CD4(+) T cell-mediated neuroprotection. CONCLUSION: Collectively, while CD4(+) T cells are a potential source of BDNF after nerve injury, production of BDNF is not necessary for CD4(+) T cells to mediate their neuroprotective effects.

IL-10 within the CNS is necessary for CD4+ T cells to mediate neuroprotection.

We have previously shown that immunodeficient mice exhibit significant facial motoneuron (FMN) loss compared to wild-type (WT) mice after a facial nerve axotomy. Interleukin-10 (IL-10) is known as a regulatory cytokine that plays an important role in maintaining the anti-inflammatory environment within the central nervous system (CNS). IL-10 is produced by a number of different cells, including Th2 cells, and may exert an anti-apoptotic action on neurons directly. In the present study, the role of IL-10 in mediating neuroprotection following facial nerve axotomy in Rag-2- and IL-10-deficient mice was investigated. Results indicate that IL-10 is neuroprotective, but CD4+ T cells are not the requisite source of IL-10. In addition, using real-time PCR analysis of laser microdissected brainstem sections, results show that IL-10 mRNA is constitutively expressed in the facial nucleus and that a transient, significant reduction of IL-10 mRNA occurs following axotomy under immunodeficient conditions. Dual labeling immunofluorescence data show, unexpectedly, that the IL-10 receptor (IL-10R) is constitutively expressed by facial motoneurons, but is selectively induced in astrocytes within the facial nucleus after axotomy. Thus, a non-CD4+ T cell source of IL-10 is necessary for modulating both glial and neuronal events that mediate neuroprotection of injured motoneurons, but only with the cooperation of CD4+ T cells, providing an avenue of novel investigation into therapeutic approaches to prevent or reverse motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).

Phenotype of CD4+ T cell subsets that develop following mouse facial nerve axotomy.

We have previously shown that CD4(+) T helper (Th) 2 cells, but not Th1 cells, participate in the rescue of mouse facial motoneurons (FMN) from axotomy-induced cell death. Recently, a number of other CD4(+) T cell subsets have been identified in addition to the Th1 and Th2 effector subsets, including Th17, inducible T regulatory type 1 (Tr1), and naturally thymus-born Foxp3(+) regulatory (Foxp3(+) Treg) cells. These subsets regulate the nature of a T cell-mediated immune response. Th1 and Th17 cells are pro-inflammatory subsets, while Th2, Tr1, and Foxp3(+) Treg cells are anti-inflammatory subsets. Pro-inflammatory responses in the central nervous system are thought to be neurodestructive, while anti-inflammatory responses are considered neuroprotective. However, it remains to be determined if another CD4(+) T cell subset, other than the Th2 cell, develops after peripheral nerve injury and participates in FMN survival. In the present study, we used FACS analysis to determine the temporal frequency of Th1, Th17, Th2, Tr1 and Foxp3(+) Treg CD4(+) T cell subset development in C57BL/6 wild type mice after facial nerve transection at the stylomastoid foramen in the mouse. The results indicate that all of the known CD4(+) T cell subsets develop and expand in number within the draining lymph node, with a peak in number primarily at 7 days postoperative (dpo), followed by a decline at 9 dpo. In addition to the increase in subset frequency over time, FACS analysis of individual cells showed that the level of cytokine expressed per cell also increased for interferon-gamma (IFN-gamma), interleukin (IL)-10 and IL-17, but not IL-4. Additional control double-cytokine labeling experiments were done which indicate that, at 7dpo, the majority of cells indeed have committed to a specific phenotype and express only 1 cytokine. Collectively, our findings indicate for the first time that there is no preferential activation and expansion of any single CD4(+) T cell subset after peripheral nerve injury but, rather, that both pro-inflammatory and anti-inflammatory CD4(+) T cells develop.

CD4(+) T cell-mediated facial motoneuron survival after injury: Distribution pattern of cell death and rescue throughout the extent of the facial motor nucleus.

We have previously demonstrated that CD4(+) T cells transiently rescue facial motoneurons (FMN) from axotomy-induced death in immunodeficient mice. Three subpopulations of motoneurons have been observed within the facial motor nucleus following axotomy: one that always survives axotomy (50%), one that is amenable to rescue from axotomy-induced death through the addition of neurotrophic factors or CD4(+) T cells (30-40%), and one that always dies after axotomy (10-15%). The objective of this study was to anatomically map the extent of axotomy-induced cell death and immune cell rescue in the facial nucleus to study the differential survival capabilities of each subpopulation. Wild-type (WT) mice, recombinase activating gene 2 knockout (RAG-2 KO) mice, and RAG-2 KO mice reconstituted with CD4(+) T cells were subjected to right facial nerve axotomy. At 4 weeks post-axotomy, topographical mapping of axotomy-induced cell death throughout the rostro-caudal extent of the facial nucleus was accomplished in accordance with previously published maps of the subnuclear arrangement of the facial neurons. The results indicate that all 3 subpopulations of FMN can be found in each of the subnuclear groups throughout the entire rostro-caudal extent of the facial nucleus. These data are discussed in context of recent work in amyotrophic lateral sclerosis, a fatal motoneuron disease.

Immune-mediated neuroprotection of axotomized mouse facial motoneurons is dependent on the IL-4/STAT6 signaling pathway in CD4(+) T cells.

The CD4(+) T lymphocyte has recently been found to promote facial motoneuron (FMN) survival after nerve injury. Signal Transducer and Activator of Transcription (STAT)4 and STAT6 are key proteins involved in the CD4(+) T cell differentiation pathways leading to T helper type (Th)1 and Th2 cell development, respectively. To determine which CD4(+) T cell subset mediates FMN survival, the facial nerve axotomy paradigm was applied to STAT4-deficient (-/-) and STAT6-/- mice. A significant decrease in FMN survival 4 weeks after axotomy was observed in STAT6-/- mice compared to wild-type (WT) or STAT4-/- mice. Reconstituting STAT6-/- mice with CD4(+) T cells obtained from WT mice promoted WT levels of FMN survival after injury. Furthermore, rescue of FMN from axotomy-induced cell death in recombination activating gene (RAG)-2-/- mice (lacking T and B cells) could be achieved only by reconstitution with CD4(+) T cells expressing functional STAT6 protein. To determine if either the Th1 cytokine, interferon-gamma (IFN-gamma) or the Th2 cytokine IL-4 is involved in mediating FMN survival, facial nerve axotomy was applied to IFN-gamma-/- and IL-4-/- mice. A significant decrease in FMN survival after axotomy occurred in IL-4-/- but not in IFN-gamma-/- mice compared to WT mice, indicating that IL-4 but not IFN-gamma is important for FMN survival after nerve injury. In WT mice, intracellular IFN-gamma vs. IL-4 expression was examined in CD4(+) T cells from draining cervical lymph nodes 14 days after axotomy, and substantial increase in the production of both CD4(+) effector T cell subsets was found. Collectively, these data suggest that STAT6-mediated CD4(+) T cell differentiation into the Th2 subset is necessary for FMN survival. A hypothesis relevant to motoneuron disease progression is presented.

CD4+CD25+ regulatory T cells and CD1-restricted NKT cells do not mediate facial motoneuron survival after axotomy.

CD4+ T cells rescue facial motoneurons (FMN) from axotomy-induced cell death. The objective of this study is to determine if the CD4+ T regulatory subsets, CD4+CD25+ T or CD1d-restricted NKT cells are critical for FMN survival after facial nerve axotomy. Surviving FMN within facial motor nuclei from axotomized and control sides 4 weeks after axotomy were counted to determine percent FMN survival. Data generated by applying this paradigm to recombination activating gene-2-deficient mice reconstituted with CD4+ T cells depleted of CD4+CD25+ T cells and to CD1-/- mice, deficient in CD1d-restricted NKT cells, suggest that neither regulatory CD4+ T subset is critical for FMN survival.

Brain-derived neurotrophic factor supports facial motoneuron survival after facial nerve transection in immunodeficient mice.

Numerous studies have shown that motoneuron survival can be facilitated by neurotrophic factors (NTF) after injury. However, the ability of specific NTF to rescue facial motoneurons (FMN) from axotomy-induced death in immunodeficient mice has not been tested. Therefore, one goal of this study was to determine if brain-derived neurotrophic factor (BDNF), an NTF with a known ability to rescue FMN from axotomy-induced death, supports FMN from axotomy-induced death in recombinase activating gene-2 knockout (RAG-2 KO) mice that lack functional T and B lymphocytes. Nerve growth factor, which has been shown not to play a role in motoneuron survival, was used as a negative control. Brain derived neurotrophic factor treatment restored FMN survival to wild-type (WT) control levels 4 weeks post-operative (wpo) (80% +/- 1.9, 83% +/- 2.4, respectively). The second goal of this study was to begin to elucidate if CD4+ T cells produce NTF after facial nerve axotomy. Cervical lymph nodes were collected from WT mice 9 days post-operative, re-activated with anti-CD3 and supernatant collected 24 h later. Immediately after injury, the supernatant was administered to RAG-2 KO mice leading to an increase in FMN survival equivalent to WT controls (80% +/- 1.4, 84% +/- 2.1, respectively, 4 wpo). In addition, cervical lymph node supernatant treated with anti-BDNF attenuated FMN rescue in RAG-2 KO mice (62% +/- 3.3) 4 wpo. These data lend support to the hypothesis that CD4+ T cells produce NTF that support motoneuron survival before target reconnection occurs.

Role of the immune system in the maintenance of mouse facial motoneuron viability after nerve injury.

In the field of neuroimmunology, an emerging area of research involves the role that the immune system plays in neural injury and repair. Such immune:neural interactions may involve both neuroprotective and neurodestruction actions. To begin to address the compelling, and clinically relevant, issue of how the immune system impacts neural reparative processes, we combined the well described facial nerve injury paradigm, a simple neural injury model, with various immunodeficient mouse models, in order to delineate the contributing immune cells/factors involved in neural injury and repair. We have discovered a role for the CD4+ T cell in mediating facial motoneuron survival after facial nerve injury in the mouse. In this review, we present an overview of our work to date in this field and discuss future directions relevant to understanding key elements in the crosstalk between the immune:neural systems that develops subsequent to injury and/or trauma.

CD4-positive T cell-mediated neuroprotection requires dual compartment antigen presentation.

Our laboratory discovered that CD4-positive (CD4+) T cells of the immune system convey transitory neuroprotection to injured mouse facial motoneurons (FMNs) (Serpe et al., 1999, 2000, 2003). A fundamental question in the mechanisms responsible for neuroprotection concerns the identity of the cell(s) that serves as the antigen-presenting cell (APC) to activate the CD4+ T cells. Here, we first establish that CD4+ T cells reactive to non-CNS antigen fail to support FMN survival and, second, demonstrate a two-compartment model of CD4+ T cell activation. Mouse bone marrow (BM) chimeras were developed that discriminate between resident antigen-presenting host cell and BM-derived antigen-presenting donor cell expression of major histocompatibility complex II within central and peripheral compartments, respectively. After facial nerve transection, neither compartment alone is sufficient to result in activated CD4+ T cell-mediated FMN survival. Rather, CD4+ T cell-mediated neuroprotection appears to depend on both resident microglial cells in the central compartment and a BM-derived APC in the peripheral compartment. This is the first in vivo report demonstrating a neuroprotective mechanism requiring APC functions by resident (i.e., parenchymal) microglial cells.

Natural killer cells do not mediate facial motoneuron survival after facial nerve transection.

The goal of the current study was to determine if natural killer (NK) cells mediate facial motoneuron (FMN) survival following injury. Wild-type (WT), perforin/recombinase activating gene-2 knockout (pfp/RAG-2 KO), and common gamma-chain (gammac)/RAG-2 KO mice received a right facial nerve axotomy. In WT mice, FMN survival was 86+/-1.0% relative to the contralateral control side. In contrast, pfp/RAG-2 and gammac/RAG-2 KO mice exhibited significant decreases in FMN survival ( approximately 20% and approximately 30%, respectively), relative to WT. Reconstitution of pfp/RAG-2 and gammac/RAG-2 KO mice with normal NK cells alone, failed to restore FMN survival levels to those of WT, but did restore functional lytic activity against YAC-1 cells. Reconstitution of pfp/RAG-2 and gammac/RAG-2 KO mice with splenocytes, and pfp/RAG-2 KO mice with CD4+ T-lymphocytes alone or in combination with NK cells, restored FMN survival levels to those of WT. Thus, NK cells appear to not be a component of immune cell-mediated rescue of motoneurons from axotomy induced cell death.

CD4+ T, but not CD8+ or B, lymphocytes mediate facial motoneuron survival after facial nerve transection.

The capacity of facial motor neurons (FMN) to survive injury and successfully regenerate is substantially compromised in immunodeficient mice, which lack T and B lymphocytes (). The goal of the present study was to determine which T cell subset (CD4+ and/or CD8+), and whether the B lymphocyte, is involved in FMN survival after nerve injury. All mice were subjected to a right facial nerve axotomy, with the left (uncut) side serving as an internal control. FMN survival, of the right (cut) side, was measured 4 weeks post-operative, and expressed as a percentage of the left (uncut) control side. FMN survival in wild-type mice was 86%+/-1.5. In contrast, FMN survival in CD4 KO mice was 60%+/-2.0. Reconstitution of either CD4 KO mice, or recombinase activating gene-2 knockout (RAG-2 KO) mice (which lack functional T and B cells) with CD4+ T cells alone restored FMN survival to wild-type levels (85%+/-1.2 and 84%+/-2.5, respectively). There was no difference in FMN survival between wild-type, CD8 KO and MmuMT (B cell deficient) mice. Reconstitution of RAG-2 KO mice with CD8+ T cells alone, or B cells alone, failed to restore FMN survival levels (65%+/-1.5 and 63%+/-1.0, respectively). It is concluded that, of the population of FMN that do not survive injury, CD4+ T lymphocytes, but not CD8+ T lymphocytes or B cells, mediate FMN survival after peripheral nerve injury.

Functional recovery after facial nerve crush is delayed in severe combined immunodeficient mice.

The goal of the current study was to determine if T and B lymphocytes play a role in functional recovery after peripheral nerve injury. The time course of behavioral recovery following facial nerve crush injury at the stylomastoid foramen was established in scid mice which lack functional T and B cells and reconstituted scid mice as compared to wild-type mice. The average time necessary for recovery of full eye blink reflex and vibrissae movements in wild-type mice was 10.3+/-0.2 and 9.9+/-0.34 days, respectively. In contrast, recovery of full eye blink reflex and vibrissae movements took 14.8+/-0.54 and 12.3+/-0.41 days, respectively, in scid mice. Reconstitution of scid mice with whole splenocytes resulted in functional recovery times similar to wild-type, with eye blink reflex recovery and vibrissae movement being 10.5+/-0.3 and 10.0+/-0.0 days, respectively. These results suggest that the delayed behavioral recovery time observed in scid mice may be due to the absence of T and B lymphocytes.