There is a robust increase in TNFα expression levels in the CNS during numerous experimental models of both acute injury and chronic neurodegenerative disease, such as AD, suggesting a significant role for this cytokine in the injury or disease process [12, 38]. Neuroinflammation begins early in AD and accompanies Aβ accumulation and neurodegeneration . Still nebulous is whether this AD-related inflammatory response is advantageous or deleterious and what the best approach is to resolving the inflammatory tide while simultaneously allowing beneficial processes to continue. In the current study, we focus on the central role of TNFα and its modulation in inflammatory regulation and cognitive function in the 3 × Tg mouse model of AD.
Although there is ample evidence that TNFα plays a central role in brain development and homeostatic and repair mechanisms , many studies demonstrate a negative role for TNFα in AD pathology. APP/presenilin 1 (PS1) transgenic mice receiving short-term CNS infusion of anti-TNFα monoclonal antibody showed reduced tau pathology and amyloid plaque deposits . Ligation of microglial CD40 with its cognate ligand, CD40 ligand (CD40L, expressed by activated astrocytes associated with beta-amyloid plaques ), synergistically activated microglia to produce TNFα in response to low levels of Aβ peptides. This form of microglial activation was deleterious, as it resulted in TNFα-dependent neuronal injury. Further, when mice deficient in CD40L were crossed with the Tg2576 mouse model of AD, abnormal phosphorylation of tau (an index of neuronal stress) was reduced prior to beta-amyloid deposition, suggesting that the CD40-CD40L interaction is an early event in AD pathogenesis . However, complete abrogation of TNFα is not beneficial in the context of AD. Giuliani and coworkers used the PDAPP mouse model to demonstrate increased amyloid plaque burden and no cognitive improvement following chronic TNFα ablation . The dual mission of TNFα may depend on the timing and progression of damage. In a model of traumatic brain injury, TNFα-null mice exhibited less severe cognitive and motor neuron impairments than wild type (WT) mice in the acute post-traumatic period . While neurological functions recovered by 2 to 3 weeks post-injury in WT mice, TNFα-null animals still demonstrated motor deficits at 4 weeks and brain damage was significantly more extensive in TNFα-deficient mice. What remains unclear after these important studies is which approach to pursue in balancing the dual roles of the inflammatory response in AD. Our data indicate that long-term modulation with the small molecule TNFα inhibitor 3,6′-DT is safe, reduces CNS TNFα levels and improves cognitive function in the early stages of disease in the 3 × Tg mouse. It will be important to assess long-term dosing strategies that encompass later disease stages for safety and impact on the development of the classical neuropathological features of AD, such as tau pathology (not seen at the age of the mice in this study; see [33, 37]) and amyloid accumulation. It is important to note that, at this early phase of the disease, treatment of 3 × Tg mice with either Thal or 3,6′-DT did not increase intraneuronal Aβ or Aβ plaque deposition.
TNFα has already been validated as a drug target with infliximab (Remicade), etanercept (Enbrel) and adalimumab (Humira) in clinical use. Short-term, extrathecal etanercept administration in patients with AD achieved significant cognitive and behavioral improvements [46–48]. As AD treatment necessitates chronic, long-term treatment, perispinal injections are neither practical nor safe in this context and the development of small, drug-like molecules to potently and safely inhibit TNFα is of significant clinical value. Thalidomide, a small molecule glutamic acid derivative demonstrating anti-TNFα actions, enhances the degradation of TNFα mRNA [49–51]. Recent preclinical studies indicate the therapeutic potential of employing thalidomide as an AD treatment. Daily treatment with thalidomide (20 mg/kg) improved recognition memory induced by Aβ(25–35) or Aβ(1–40) in mice . These data suggest the practicability of small molecules that target TNFα as a therapeutic strategy against Aβ-mediated cognitive impairments. However, thalidomide’s use in humans is severely restricted by its well-documented side effects, such as somnolence, deep-vein thrombosis and peripheral neurotoxicity . Thalidomide also has a high IC50 for TNFα inhibition, necessitating chronic high dosing to achieve significant clinical benefit while increasing risk for side effects in patients with AD.
In this report, a novel experimental TNFα synthesis inhibitor, 3,6′-DT [54, 55], a lipophilic analog of the classic orally active thalidomide, was evaluated. 3,6′-DT was shown to be effective in ameliorating the TNFα increase and cognitive impairment resulting from a mild traumatic brain injury in mice . In the current study, 3,6′-DT and thalidomide were equally effective, at a high dose, in preventing CNS TNFα increases resulting from LPS-induced systemic inflammation in WT mice. However, only 3,6′-DT was effective at lowering TNFα in the CNS of 3 × Tg mice and improving cognitive function. The lack of efficacy by thalidomide in the 3 × Tg model may be due to a higher IC50 value than 3,6′-DT or a difference in brain penetrance. 3,6′-DT has an assessed brain to plasma ratio of 1.34 , which is in accord with its partition coefficient (cLogD) value of −0.56 [54, 55], a measure of its balanced aqueous solubility and lipophilicity. Thalidomide has a moderate degree of lipophilicity with a cLogD value of −0.83 . Further, thalidomide has demonstrated poor ability in vitro to reduce LPS-stimulated TNFα while significantly increasing toxic nitrite levels .
Within the CNS, the main executors of innate immunity are perivascular macrophages and parenchymal microglia . Resting microglia are highly ramified with branched processes and have critical physiologic roles, including determination of neuronal fate, migration, axonal growth and synaptic remodeling [58, 59]. In response to pathological conditions, microglia transform into a reactive state [58, 60, 61], losing their ramified morphology and switching from a neurotrophic phenotype to a persistent, reactive phenotype expressing toxic and reparative functions . Inflammation clearly occurs in pathologically vulnerable regions of the AD brain [62, 63], and both animal models and clinical studies strongly suggest that inflammation significantly contributes to AD pathogenesis . 3 × Tg mice showed a large increase in Iba-1 positive microglia at 6.5 months of age compared with Non-Tg mice and the majority of these microglia had an activated morphology. Although both thalidomide and 3,6′-DT reduced the total numbers of Iba-1 positive microglia, only 3,6′-DT improved the ratio of resting to activated microglia and created a microglial morphological profile that was nearly identical to that of the Non-Tg brain. 3 × Tg mice treated with thalidomide had predominately activated Iba-1 positive microglia.
Although considerable focus has been given to the CNS-endogenous innate immune system, the CNS-endogenous and peripheral immune systems do not function in isolation from each other and there is dynamic interplay. In the healthy brain, peripheral immune cells are indispensable for homeostasis whereas specific cell types play different roles in neuroprotection and neurodestruction . This activity must be tightly regulated or the immune system will become hyperactivated, leading to dysregulation and pathological sequelae. One of the many known functions of TNFα is to stimulate the recruitment of innate effectors such as neutrophils/granulocytes and monocytes and to activate these cells in a paracrine manner. After being activated by injury or disease, innate immune system functions focus on the clearance of pathogens and debris, tissue repair and in orchestrating adaptive immune responses [58, 65, 66].
While the inflammation within the AD brain has been assumed to be a locally-mediated inflammatory response by microglia to Aβ , systemic immune responses may also play a role. Prior to entering the CNS, leukocytes encounter formidable barriers to access including the endothelial blood–brain barrier, the epithelial blood-CSF barrier and the tanycyte barrier surrounding the circumventricular organs . The mechanisms by which leukocytes infiltrate the healthy brain during adulthood and aging are not clear , but there is strong evidence that a compromised blood–brain barrier in brain diseases, such as stroke or brain trauma , and AD [70–75] allows leakage of leukocytes into the brain. TNFα can cause vascular endothelial damage and an increase in vascular endothelial permeability . In regards to macrophage infiltration into the CNS, there are no specific immunohistochemical markers to distinguish blood-derived macrophages from brain endogenous microglial cells, making the distinction difficult. Recent human studies demonstrate that recruited monocytic cells express markers for microglia yet are morphologically and functionally separate from the resident microglia in the AD brain [71–75]. Contradicting the importance of leukocytes in AD pathology, histological examination of postmortem AD brains has not demonstrated abundant leukocytic infiltrates. Future studies and more sophisticated methodologies are required to determine if this is a disease stage phenomenon and what contributions infiltrating leukocytes may make to early stage AD and progression of the disease. In this regard, animal models of AD are valuable.
Recent studies in AD animal model pathology have elegantly examined the important role that brain-infiltrating monocytes play in Aβ clearance. Angelucci et al. crossed an AD mouse model with an animal deficient in CC-chemokine receptor 2 . These bigenic mice have markedly diminished recruitment of brain resident microglia and/or peripheral macrophages to sites of beta-amyloid plaques and demonstrate heavier Aβ protein burden than AD model mice alone. While this study does not definitively establish the provenance of the amyloid clearing cells, it demonstrates the importance of brain innate immunity in restricting cerebral amyloidosis. An additional report has shown that depletion of CD11c + cells using a CD11c-diphtheria toxin transgenic mouse bone marrow chimera in an AD mouse model opposes the beneficial effect of T cell-directed immunotherapy, suggesting that peripheral innate immune cells are required for Aβ clearance . These studies, by negatively impacting brain penetration and Aβ homing of these peripheral innate immune cells, lead to the deduction that such cells are critical for reducing amyloid accumulation. However, if these cells are to be targeted as a therapeutic modality, strategies for selectively increasing brain leukocyte infiltration and increasing Aβ clearance potential need to be developed. A recent study demonstrated that blood-borne monocytes can be encouraged to enter the brain and restrict Aβ plaques without producing a potentially damaging neuroinflammatory response .
Previous work  and the current study demonstrate that the 3 × Tg mouse has a robust increase in CNS leukocyte infiltrates early in the disease process, and that intracellular TNFα levels in this population are greatly increased relative to Non-Tg mice. Both thalidomide and 3,6′-DT reduced the total numbers of infiltrating peripheral leukocytes in the CNS of 3 × Tg mice as measured by flow cytometry but 3,6′-DT effected a striking reduction. The similar parallel finding of reduced Iba-1 microglia following thalidomide or 3,6′-DT treatment suggest that reducing the infiltrating leukocyte population contributed to the reduction in Iba-1 positive microglia. Additionally, only 3,6′-DT improved the resting to activated ratio of CNS microglia suggesting that the improved CNS penetrance and lower IC50 of 3,6′-DT compared with thalidomide is necessary for efficacy.
3,6′-DT did not, however, change the percentages of specific cell types within the total leukocytic population or alter TNFα levels in the total CD45hi population. Rather, 3,6′-DT specifically reduced intracellular TNFα levels in the CD45hi/GR1+/Ly6Ghi (granulocytic) subpopulation. Due to a paucity of studies, it is unclear what the role of granulocytes is in the human AD brain, particularly in the early stages of the disease, and further studies are needed to determine if granulocytes migrate through the brain parenchyma or are involved in inflammatory signal transduction from the perivascular regions of the brain. Regardless, these data raise interesting questions about AD immunotherapy and suggests that, in addition to reducing the total number of infiltrating leukocytes, modulation of TNFα by small molecule TNFα inhibitors, in specific subsets of peripheral leukocytes, may be therapeutic.
Chronic neuroinflammation is an important component of AD pathogenesis and undoubtedly contributes to neuronal dysfunction, injury, loss and disease progression. A recent proteomic profiling study examined the CSF of young individuals who will go on to develop familial AD in comparison with age-matched controls not carrying a familial AD mutation . The study noted increases in multiple complement cascade components as much as a decade prior to the onset of overt AD symptomology, indicating that neuroinflammation plays a very early role in the disease process. These and other data underscore the therapeutic potential of targeted anti-inflammatory pharmaceuticals both early and throughout the course of the disease. Unfortunately, our knowledge of CNS-related immune function is currently limited and the study of the interface between the peripheral and CNS-endogenous immune systems is in its infancy. Understanding the molecular manipulations required to produce beneficial changes in leukocyte and microglial activation profiles is necessary to beget more sophisticated immunomodulatory strategies for the treatment of AD.