Jason G. Weinger

The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating microglial function

Significance Neuroinflammation and activation of innate immunity are pathological hallmarks of Alzheimer’s disease (AD). In contrast, very few studies have examined the impact of the adaptive immune system in AD pathogenesis. Here, we find that genetic ablation of peripheral immune cell populations significantly accelerates amyloid pathogenesis, worsens neuroinflammation, and alters microglial activation state. Critically, it appears that loss of IgG-producing B cells impairs microglial phagocytosis, thereby exacerbating amyloid deposition. Conversely, replacement of IgGs via direct injection or bone marrow transplantation reverses these effects and reduces Aβ pathology. Together, these results highlight the importance of the adaptive immune system and its interactions with microglia in the pathogenesis of AD. The innate immune system is strongly implicated in the pathogenesis of Alzheimer’s disease (AD). In contrast, the role of adaptive immunity in AD remains largely unknown. However, numerous clinical trials are testing vaccination strategies for AD, suggesting that T and B cells play a pivotal role in this disease. To test the hypothesis that adaptive immunity influences AD pathogenesis, we generated an immune-deficient AD mouse model that lacks T, B, and natural killer (NK) cells. The resulting “Rag-5xfAD” mice exhibit a greater than twofold increase in β-amyloid (Aβ) pathology. Gene expression analysis of the brain implicates altered innate and adaptive immune pathways, including changes in cytokine/chemokine signaling and decreased Ig-mediated processes. Neuroinflammation is also greatly exacerbated in Rag-5xfAD mice as indicated by a shift in microglial phenotype, increased cytokine production, and reduced phagocytic capacity. In contrast, immune-intact 5xfAD mice exhibit elevated levels of nonamyloid reactive IgGs in association with microglia, and treatment of Rag-5xfAD mice or microglial cells with preimmune IgG enhances Aβ clearance. Last, we performed bone marrow transplantation studies in Rag-5xfAD mice, revealing that replacement of these missing adaptive immune populations can dramatically reduce AD pathology. Taken together, these data strongly suggest that adaptive immune cell populations play an important role in restraining AD pathology. In contrast, depletion of B cells and their appropriate activation by T cells leads to a loss of adaptive–innate immunity cross talk and accelerated disease progression.

Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer ( approximately 150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.

Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during Experimental Autoimmune Encephalomyelitis

BackgroundAxl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis.MethodsWT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freunds adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays.ResultsAxl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals.ConclusionsThese data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.

In brain, Axl recruits Grb2 and the p85 regulatory subunit of PI3 kinase; in vitro mutagenesis defines the requisite binding sites for downstream Akt activation.

Axl is a receptor tyrosine kinase implicated in cell survival following growth factor withdrawal and other stressors. The binding of Axl’s ligand, growth arrest‐specific protein 6 (Gas6), results in Axl autophosphorylation, recruitment of signaling molecules, and activation of downstream survival pathways. Pull‐down assays and immunoprecipitations using wildtype and mutant Axl transfected cells determined that Axl directly binds growth factor receptor‐bound protein 2 (Grb2) at pYVN and the p85 subunit of phosphatidylinositol‐3 kinase (PI3 kinase) at two pYXXM sites (pY779 and pY821). Also, p85 can indirectly bind to Axl via an interaction between p85’s second proline‐rich region and the N‐terminal SH3 domain of Grb2. Further, Grb2 and p85 can compete for binding at the pY821VNM site. Gas6‐stimulation of Axl‐transfected COS7 cells recruited activated PI3 kinase and phosphorylated Akt. An interaction between Axl, p85 and Grb2 was confirmed in brain homogenates, enriched populations of O4+ oligodendrocytes, and O4− flow‐through prepared from day 10 mouse brain, indicating that cells with active Gas6/Axl signal through Grb2 and the PI3 kinase/Akt pathways.

Suppression of Inflammatory Responses during Myelin Oligodendrocyte Glycoprotein–Induced Experimental Autoimmune Encephalomyelitis Is Regulated by AKT3 Signaling

AKT3, a member of the serine/threonine kinase AKT family, is involved in a variety of biologic processes. AKT3 is expressed in immune cells and is the major AKT isoform in the CNS representing 30% of the total AKT expressed in spinal cord, and 50% in the brain. Myelin-oligodendrocyte glycoprotein–induced experimental autoimmune encephalomyelitis (EAE) is a mouse model in which lymphocytes and monocytes enter the CNS, resulting in inflammation, demyelination, and axonal injury. We hypothesized that during EAE, deletion of AKT3 would negatively affect the CNS of AKT3−/− mice, making them more susceptible to CNS damage. During acute EAE, AKT3−/−mice were more severely affected than wild type (WT) mice. Evaluation of spinal cords showed that during acute and chronic disease, AKT3−/− spinal cords had more demyelination compared with WT spinal cords. Quantitative RT-PCR determined higher levels of IL-2, IL-17, and IFN-γ mRNA in spinal cords from AKT3−/− mice than WT. Experiments using bone marrow chimeras demonstrated that AKT3−/− mice receiving AKT3-deficient bone marrow cells had elevated clinical scores relative to control WT mice reconstituted with WT cells, indicating that altered function of both CNS cells and bone marrow–derived immune cells contributed to the phenotype. Immunohistochemical analysis revealed decreased numbers of Foxp3+ regulatory T cells in the spinal cord of AKT3−/− mice compared with WT mice, whereas in vitro suppression assays showed that AKT3-deficient Th cells were less susceptible to regulatory T cell–mediated suppression than their WT counterparts. These results indicate that AKT3 signaling contributes to the protection of mice against EAE.

MHC Mismatch Results in Neural Progenitor Cell Rejection Following Spinal Cord Transplantation in a Model of Viral‐Induced Demyelination

Transplantation of syngeneic neural progenitor cells (NPCs) into mice persistently infected with the JHM strain of mouse hepatitis virus (JHMV) results in enhanced differentiation into oligodendrocyte progenitor cells that is associated with remyelination, axonal sparing, and clinical improvement. Whether allogeneic NPCs are tolerated or induce immune‐mediated rejection is controversial and poorly defined under neuroinflammatory demyelinating conditions. We have used the JHMV‐induced demyelination model to evaluate the antigenicity of transplanted allogeneic NPCs within the central nervous system (CNS) of mice with established immune‐mediated demyelination. Cultured NPCs constitutively expressed the costimulatory molecules CD80/CD86, and IFN‐γ treatment induced expression of MHC class I and II antigens. Injection of allogeneic C57BL/6 NPCs (H‐2b background) led to a delayed type hypersensitivity response in BALB/c (H‐2d background) mice associated with T‐cell proliferation and IFN‐γ secretion following coculture with allogeneic NPCs. Transplantation of MHC‐mismatched NPCs into JHMV‐infected mice resulted in increased transcripts encoding the T‐cell chemoattractant chemokines CXCL9 and CXCL10 that correlated with increased T‐cell infiltration that was associated with NPC rejection. Treatment of MHC‐mismatched mice with T‐cell subset‐specific depleting antibodies increased survival of allogeneic NPCs without affecting commitment to an oligodendrocyte lineage. Collectively, these results show that allogeneic NPCs are antigenic, and T‐cells contribute to rejection following transplantation into an inflamed CNS suggesting that immunomodulatory treatments may be necessary to prolong survival of allogeneic cells. STEM CELLS2012;30:2584–2595

Mice Devoid of Tau Have Increased Susceptibility to Neuronal Damage in Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis

Abstract The abundant axonal microtubule-associated protein tau regulates microtubule and actin dynamics, thereby contributing to normal neuronal function. We examined whether mice deficient in tau (Tau−/−) or with high levels of human tau differ from wild-type (WT) mice in their susceptibility to neuroaxonal injury in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. After sensitization with MOG35–55, there was no difference in clinical disease course between human tau and WT mice, but Tau−/− mice had more severe clinical disease and significantly more axonal damage in spinal cord white matter than those in WT mice. Axonal damage in gray matter correlated with clinical severity in individual mice. By immunoblot analysis, the early microtubule-associated protein-1b was increased 2-fold in the spinal cords of Tau−/− mice with chronic experimental autoimmune encephalomyelitis versus naive Tau−/− mice. This difference was not detected in comparable WT animals, which suggests that there was compensation for the loss of tau in the deficient mice. In addition, levels of the growth arrest–specific protein 7b, a tau-binding protein that is stabilized when bound to tau, were higher in WT than those in Tau−/−spinal cord samples. These data indicate that loss of tau exacerbates experimental autoimmune encephalomyelitis and suggest that maintaining tau integrity might reduce the axonal damage that occurs in inflammatory neurodegenerative diseases such as multiple sclerosis.

Two-photon imaging of remyelination of spinal cord axons by engrafted neural precursor cells in a viral model of multiple sclerosis

Significance Stem cell transplantation has emerged as a promising cell-based therapy for the treatment of demyelinating diseases such as multiple sclerosis (MS). This study provides the first real-time imaging of transplanted stem cell-mediated remyelination in a mouse model of MS. Whereas current treatments solely delay disease progression, transplanted stem cells actively reverse clinical disease in animal models. Using two-photon microscopy and viral-induced demyelination, we describe a technique to visualize cellular migration and remyelination in the mouse spinal cord. Transplanted neural precursor cells physically wrap damaged axons with newly formed myelin, preserving axonal health. Neural precursor cells (NPCs) offer a promising approach for treating demyelinating diseases. However, the cellular dynamics that underlie transplanted NPC-mediated remyelination have not been described. Using two-photon imaging of a newly developed ventral spinal cord preparation and a viral model of demyelination, we describe the motility and intercellular interactions of transplanted mouse NPCs expressing green fluorescent protein (GFP) with damaged axons expressing yellow fluorescent protein (YFP). Our findings reveal focal axonal degeneration that occurs in the ventral side of the spinal cord within 1 wk following intracranial instillation with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Axonal damage precedes extensive demyelination and is characterized by swelling along the length of the axon, loss of YFP signal, and transected appearance. NPCs engrafted into spinal cords of JHMV-infected mice exhibited diminished migration velocities and increased proliferation compared with transplanted cells in noninfected mice. NPCs preferentially accumulated within areas of axonal damage, initiated direct contact with axons, and subsequently expressed the myelin proteolipid protein gene, initiating remyelination. These findings indicate that NPCs transplanted into an inflammatory demyelinating microenvironment participate directly in therapeutic outcome through the wrapping of myelin around damaged neurons.

Surgical transplantation of mouse neural stem cells into the spinal cords of mice infected with neurotropic mouse hepatitis virus.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

T cell mediated suppression of neurotropic coronavirus replication in neural precursor cells

Abstract Neural precursor cells (NPCs) are the subject of intense investigation for their potential to treat neurodegenerative disorders, yet the consequences of neuroinvasive virus infection of NPCs remain unclear. This study demonstrates that NPCs support replication following infection by the neurotropic JHM strain of mouse hepatitis virus (JHMV). JHMV infection leads to increased cell death and dampens IFN-γ-induced MHC class II expression. Importantly, cytokines secreted by CD4+ T cells inhibit JHMV replication in NPCs, and CD8+ T cells specifically target viral peptide-pulsed NPCs for lysis. Furthermore, treatment with IFN-γ inhibits JHMV replication in a dose-dependent manner. Together, these findings suggest that T cells play a critical role in controlling replication of a neurotropic virus in NPCs, a finding which has important implications when considering immune modulation for NPC-based therapies for treatment of human neurologic diseases.