Key role of the CCR2-CCL2 axis in disease modification in a mouse model of tauopathy.

BACKGROUND: For decades, dementia has been characterized by accumulation of waste in the brain and low-grade inflammation. Over the years, emerging studies highlighted the involvement of the immune system in neurodegenerative disease emergence and severity. Numerous studies in animal models of amyloidosis demonstrated the beneficial role of monocyte-derived macrophages in mitigating the disease, though less is known regarding tauopathy. Boosting the immune system in animal models of both amyloidosis and tauopathy, resulted in improved cognitive performance and in a reduction of pathological manifestations. However, a full understanding of the chain of events that is involved, starting from the activation of the immune system, and leading to disease mitigation, remained elusive. Here, we hypothesized that the brain-immune communication pathway that is needed to be activated to combat tauopathy involves monocyte mobilization via the C-C chemokine receptor 2 (CCR2)/CCL2 axis, and additional immune cells, such as CD4+ T cells, including FOXP3+ regulatory CD4+ T cells. METHODS: We used DM-hTAU transgenic mice, a mouse model of tauopathy, and applied an approach that boosts the immune system, via blocking the inhibitory Programmed cell death protein-1 (PD-1)/PD-L1 pathway, a manipulation previously shown to alleviate disease symptoms and pathology. An anti-CCR2 monoclonal antibody (αCCR2), was used to block the CCR2 axis in a protocol that partially eliminates monocytes from the circulation at the time of anti-PD-L1 antibody (αPD-L1) injection, and for the critical period of their recruitment into the brain following treatment. RESULTS: Performance of DM-hTAU mice in short-term and working memory tasks, revealed that the beneficial effect of αPD-L1, assessed 1 month after a single injection, was abrogated following blockade of CCR2. This was accompanied by the loss of the beneficial effect on disease pathology, assessed by measurement of cortical aggregated human tau load using Homogeneous Time Resolved Fluorescence-based immunoassay, and by evaluation of hippocampal neuronal survival. Using both multiparametric flow cytometry, and Cytometry by Time Of Flight, we further demonstrated the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, 12 days following the treatment, which was absent subsequent to CCR2 blockade. In addition, measurement of hippocampal levels of the T-cell chemoattractant, C-X-C motif chemokine ligand 12 (Cxcl12), and of inflammatory cytokines, revealed that αPD-L1 treatment reduced their expression, while blocking CCR2 reversed this effect. CONCLUSIONS: The CCR2/CCL2 axis is required to modify pathology using PD-L1 blockade in a mouse model of tauopathy. This modification involves, in addition to monocytes, the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, and the T-cell chemoattractant, Cxcl12.

A 20-Year Journey from Axonal Injury to Neurodegenerative Diseases and the Prospect of Immunotherapy for Combating Alzheimer's Disease.

The understanding of the dialogue between the brain and the immune system has undergone dramatic changes over the last two decades, with immense impact on the perception of neurodegenerative diseases, mental dysfunction, and many other brain pathologic conditions. Accumulated results have suggested that optimal function of the brain is dependent on support from the immune system, provided that this immune response is tightly controlled. Moreover, in contrast to the previous prevailing dogma, it is now widely accepted that circulating immune cells are needed for coping with brain pathologies and that their optimal effect is dependent on their type, location, and activity. In this perspective, we describe our own scientific journey, reviewing the milestones in attaining this understanding of the brain-immune axis integrated with numerous related studies by others. We then explain their significance in demonstrating the possibility of harnessing the immune system in a well-controlled manner for the treatment of neurodegenerative diseases.

Maternal Type-I interferon signaling adversely affects the microglia and the behavior of the offspring accompanied by increased sensitivity to stress.

Viral infection during pregnancy is often associated with neuropsychiatric conditions. In mice, exposure of pregnant dams to the viral mimetic poly(I:C), serves as a model that simulates such pathology in the offspring, through a process known as Maternal Immune Activation (MIA). To investigate the mechanism of such effect, we hypothesized that maternal upregulation of Type-I interferon (IFN-I), as part of the dam's antiviral response, might contribute to the damage imposed on the offspring. Using mRNA sequencing and flow cytometry analyses we found that poly(I:C) treatment during pregnancy caused reduced expression of genes related to proliferation and cell cycle in the offspring's microglia relative to controls. This was found to be associated with an IFN-I signature in the embryonic yolk sac, the origin of microglia in development. Neutralizing IFN-I signaling in dams attenuated the effect of MIA on the newborn's microglia, while systemic maternal administration of IFNß was sufficient to mimic the effect of poly(I:C), and led to increased vulnerability of offspring's microglia to subsequent stress. Furthermore, maternal elevation of IFNß resulted in behavioral manifestations reminiscent of neuropsychiatric disorders. In addition, by adopting a "two-hit" experimental paradigm, we show a higher sensitivity of the offspring to postnatal stress subsequent to the maternal IFNß elevation, demonstrated by behavioral irregularities. Our results suggest that maternal upregulation of IFN-I, in response to MIA, interferes with the offspring's programmed microglial developmental cascade, increases their susceptibility to postnatal stress, and leads to behavioral abnormalities.

Potential immunotherapy for Alzheimer disease and age-related dementia.

Emerging results support the concept that Alzheimer disease (AD) and age-related dementia are affected by the ability of the immune system to contain the brain's pathology. Accordingly, well-controlled boosting, rather than suppression of systemic immunity, has been suggested as a new approach to modify disease pathology without directly targeting any of the brain's disease hallmarks. Here, we provide a short review of the mechanisms orchestrating the cross-talk between the brain and the immune system. We then discuss how immune checkpoint blockade directed against the PD-1/PD-L1 pathways could be developed as an immunotherapeutic approach to combat this disease using a regimen that will address the needs to combat AD.

PD-1/PD-L1 checkpoint blockade harnesses monocyte-derived macrophages to combat cognitive impairment in a tauopathy mouse model.

Alzheimer's disease (AD) is a heterogeneous disorder with multiple etiologies. Harnessing the immune system by blocking the programmed cell death receptor (PD)-1 pathway in an amyloid beta mouse model was shown to evoke a sequence of immune responses that lead to disease modification. Here, blocking PD-L1, a PD-1 ligand, was found to have similar efficacy to that of PD-1 blocking in disease modification, in both animal models of AD and of tauopathy. Targeting PD-L1 in a tau-driven disease model resulted in increased immunomodulatory monocyte-derived macrophages within the brain parenchyma. Single cell RNA-seq revealed that the homing macrophages expressed unique scavenger molecules including macrophage scavenger receptor 1 (MSR1), which was shown here to be required for the effect of PD-L1 blockade in disease modification. Overall, our results demonstrate that immune checkpoint blockade targeting the PD-1/PD-L1 pathway leads to modification of common factors that go awry in AD and dementia, and thus can potentially provide an immunotherapy to help combat these diseases.

Newly Formed Endothelial Cells Regulate Myeloid Cell Activity Following Spinal Cord Injury via Expression of CD200 Ligand.

The central nervous system (CNS) is endowed with several immune-related mechanisms that contribute to its protection and maintenance in homeostasis and under pathology. Here, we discovered an additional mechanism that controls inflammatory responses within the CNS milieu under injurious conditions, involving CD200 ligand (CD200L) expressed by newly formed endothelial cells. We observed that CD200L is constitutively expressed in the mouse healthy CNS by endothelial cells of the blood-cerebrospinal fluid barrier and of the spinal cord meninges, but not by the endothelium of the blood-spinal cord barrier. Following spinal cord injury (SCI), newly formed endothelial cells, located only at the epicenter of the lesion site, expressed CD200L. Moreover, in the absence of CD200L expression by CNS-resident cells, functional recovery of mice following SCI was impaired. High throughput single-cell flow cytometry image analysis following SCI revealed CD200L-dependent direct interaction between endothelial and local CD200R+ myeloid cells, including activated microglia and infiltrating monocyte-derived macrophages (mo-MΦ). Absence of CD200L signaling, both in vitro and in vivo, resulted in a higher inflammatory response of the encountering macrophages, manifested by elevation in mRNA expression of Tnfα and Il1ß, increased intracellular TNFα immunoreactivity, and reduced expression levels of macrophage factors that are associated with resolution of inflammation, Dectin-1, CD206 (mannose receptor), and IL-4R. Collectively, our results highlight the importance of CD200-mediated immune dialogue between endothelial cells and the local resident microglia and infiltrating mo-MΦ within the lesion area, as a mechanism that contributes to regulation of inflammation following acute CNS injury. SIGNIFICANCE STATEMENT: This manuscript focuses on a novel mechanism of inflammation-regulation following spinal cord injury (SCI), orchestrated by CD200-ligand (CD200L) expressed by newly formed endothelial cells within the lesion site. Our study reveals that, in homeostasis, CD200L is expressed by endothelial cells of the mouse blood-cerebrospinal fluid barrier and of the blood-leptomeningeal barrier, but not by endothelial cells of the blood-spinal cord barrier. Following SCI, newly formed endothelial cells located within the epicenter of the lesion site were found to express CD200L at time points that were shown to be critical for repair. Our results reveal a direct interaction between CD200L+ endothelial cells and CD200R+ microglia and macrophages, resulting in attenuated inflammation, biasing macrophage phenotype toward inflammation-resolving cells, and promotion of functional recovery following SCI.

Microglia development follows a stepwise program to regulate brain homeostasis.

Microglia, the resident myeloid cells of the central nervous system, play important roles in life-long brain maintenance and in pathology. Despite their importance, their regulatory dynamics during brain development have not been fully elucidated. Using genome-wide chromatin and expression profiling coupled with single-cell transcriptomic analysis throughout development, we found that microglia undergo three temporal stages of development in synchrony with the brain--early, pre-, and adult microglia--which are under distinct regulatory circuits. Knockout of the gene encoding the adult microglia transcription factor MAFB and environmental perturbations, such as those affecting the microbiome or prenatal immune activation, led to disruption of developmental genes and immune response pathways. Together, our work identifies a stepwise microglia developmental program integrating immune response pathways that may be associated with several neurodevelopmental disorders.

Thioredoxin-mimetic peptide CB3 lowers MAPKinase activity in the Zucker rat brain.

Diabetes is a high risk factor for dementia. High glucose may be a risk factor for dementia even among persons without diabetes, and in transgenic animals it has been shown to cause a potentiation of indices that are pre-symptomatic of Alzheimer's disease. To further elucidate the underlying mechanisms linking inflammatory events elicited in the brain during oxidative stress and diabetes, we monitored the activation of mitogen-activated kinsase (MAPKs), c-jun NH2-terminal kinase (JNK), p38 MAP kinases (p38(MAPK)), and extracellular activating kinsae1/2 (ERK1/2) and the anti-inflammatory effects of the thioredoxin mimetic (TxM) peptides, Ac-Cys-Pro-Cys-amide (CB3) and Ac-Cys-Gly-Pro-Cys-amide (CB4) in the brain of male leptin-receptor-deficient Zucker diabetic fatty (ZDF) rats and human neuroblastoma SH-SY5Y cells. Daily i.p. injection of CB3 to ZDF rats inhibited the phosphorylation of JNK and p38(MAPK), and prevented the expression of thioredoxin-interacting-protein (TXNIP/TBP-2) in ZDF rat brain. Although plasma glucose/insulin remained high, CB3 also increased the phosphorylation of AMP-ribose activating kinase (AMPK) and inhibited p70(S6K) kinase in the brain. Both CB3 and CB4 reversed apoptosis induced by inhibiting thioredoxin reductase as monitored by decreasing caspase 3 cleavage and PARP dissociation in SH-SY5Y cells. The decrease in JNK and p38(MAPK) activity in the absence of a change in plasma glucose implies a decrease in oxidative or neuroinflammatory stress in the ZDF rat brain. CB3 not only attenuated MAPK phosphorylation and activated AMPK in the brain, but it also diminished apoptotic markers, most likely acting via the MAPK-AMPK-mTOR pathway. These results were correlated with CB3 and CB4 inhibiting inflammation progression and protection from oxidative stress induced apoptosis in human neuronal cells. We suggest that by attenuating neuro-inflammatory processes in the brain Trx1 mimetic peptides could become beneficial for preventing neurological disorders associated with diabetes.