Neuroimmune interactions in Alzheimer’s disease—New frontier with old challenges?

Abstract

The perceived role of the immune system in neurodegenerative diseases has undergone drastic changes over time. Initially considered as a passive bystander, then condemned as a mediator of neurodegeneration and now established as an important player in the pathogenetic cascade, neuroimmune interactions have come a long way to arrive center stage in Alzheimer’s disease research. Despite major breakthroughs in recent years, basic questions remain unanswered as conflicting data describe immune overactivation, inadequate response or exhaustion of the immune system in neurodegenerative diseases. Furthermore, difficulties in translating in vitro and in vivo studies in model systems to the complex human disease condition with multiple overlapping pathologies and the long disease duration in patients suffering from neurodegenerative diseases have hampered progress. Development of novel, advanced model systems, as well as new technologies to interrogate existing disease models and valuable collections of human tissue samples, including brain tissue in parallel with improved imaging and biomarker technologies are guiding the way to better understand the role of the immune system in Alzheimer’s disease with hopes for more effective interventions in the future.

The identification of multiple risk associated variants in genes associated with the immune system has sparked an interest in neuroimmune interactions in neurodegenerative diseases, generated a flurry of new research, and made old challenges facing the field more obvious. For a long time, the immune system has been thought to be a passive bystander in the pathogenic cascade of neurodegenerative diseases, merely responding to damage inflicted by aggregates of pathological proteins. More recent research efforts in animal models and genetic association studies have painted a more diverse picture and assigned a more active role to immune responses in the process of neurodegeneration, ultimately coining the term “cellular phase” of Alzheimer’s disease (AD) pathogenesis, which accounts for the complex interactions between neurons, glial cells and vasculature in modulating the chronic process of neurodegeneration. Despite this significant progress, translational efforts are still hampered by many uncertainties, including: (a) when is the best time to intervene; (b) is inhibition or activation of the immune response the way to go; and (c) what are realistic goals to achieve by manipulating the immune system? Improvements in imaging and biomarker studies have helped elucidate the disease course and the association of inflammation with the emergence of pathological hallmarks, but do not discriminate with high certainty individual cellular immune players and signaling molecules involved. Uncertainty also exists with respect to which type of intervention holds the most promise for success—immunosuppression or immune activation? Given the mostly increased inflammatory activity in the diseased brain, anti-inflammatory intervention seems the obvious choice, but the fact that most of the genetic risk variants associated with AD are full or partial loss of function changes in molecules involved in key immune functions has cast doubt on the merits of anti-inflammatory treatment strategies. Instead, sophisticated intervention strategies may be required aimed at modulating the activity of key signaling pathways, like the Triggering receptor expressed in myeloid cells 2 (TREM2) and downstream molecules. A final area of uncertainty is what to expect from “immune-interventions” in AD, as more and more impressive success stories are published with respect to the use of checkpoint inhibitors or antigen-receptor chimeric T-cells in the cancer field. A big hope lies in the use of antibodies to prevent the accumulation of pathological proteins in neurodegenerative diseases, but there is also a role for manipulation of immune responses to ease the burdens of pathologies thereby improving cognitive decline as well activities of daily living. Progress in these translational efforts is hampered by the difficulty of adapting the findings in commonly used animal models and in vitro systems to the complex situation in the human brain, characterized by high order interactions between multiple cell types, regional differences in neuronal activity and neuro-glial interactions as well as the long period of disease development and clinical symptoms not easily modeled in small rodents and in vitro systems. Advances in experimental technology allow for better studies in human subjects, which will be an important pillar of understanding the complex interactions of the immune system and pathological protein aggregates in neurodegenerative diseases, thereby aiding in the translational efforts for this exciting new field of research.

1. Immune responses in neurodegenerative diseases early days

With the identification of disease causing proteins in major neurodegenerative diseases, including AD^1–4 and Parkinson’s disease (PD),⁵ came the notion that these protein deposits, as well as the neuronal loss with which they are associated, are accompanied by an activation of intrinsic immune cells of the brain, namely microglia and astrocytes. Early studies of brain samples from AD and PD patients demonstrated microgliosis and astrogliosis in regions affected by pathological protein aggregates and neuronal death,^6,7 and this activation of glial cells was suggested as an indicator of disease activity. However, early studies into the interaction of microglia and amyloid β-protein (Aβ) plaques in AD brought a new notion to the table. Microglia processes were found to be in intimate proximity of amyloid fibrils^8,9 and microglia in culture were reported to take up and not degrade, but rather release¹⁰ or even produce¹¹ Aβ, supporting the idea the microglia may be major players in plaque development.¹² Later findings of secretion of “neurotoxic” substances by microglia in contact with Aβ deposits, leading to the death of neurons in culture,^13–15 fueled the concept of “bad” inflammation and suggested innate immune responses to be a driver of disease progression. Early epidemiological studies suggested a possible positive effect of non-steroidal anti-inflammatory drug (NSAID) use on the risk to develop AD,^16–19 but this enthusiasm was dampened by disappointing results from several interventional studies.^20–26 One caveat in interpreting the epidemiological studies is confounding effects of an often-present underlying inflammatory condition in patients taking NSAIDS, which raises an interesting notion, i.e., that possibly pro-inflammatory conditions may be protective.

Related to this idea are the facts that clustering of activated microglia cells around Aβ plaques has been observed (Fig. 1 and Ref.²⁷) but without obvious evidence of phagocytosis.^28,29 Other studies described senescent or dystrophic microglia with higher frequency in aged and AD brains^30–33 (see also figure 1C in Ref.³⁴), suggesting that impaired or “exhausted” immune responses are important in the pathogenesis of neurodegenerative diseases. These divergent concepts have made clarifying the proper role of microglia in neurodegenerative diseases difficult and increased uncertainty about the importance of immune responses for the pathogenesis and progression of AD and other neurodegenerative diseases, which has reduced the overall enthusiasm for further studies looking into interaction of neuroinflammation and disease processes.

Fig. 1.

Activated microglia (Iba-1, green) surrounding Aβ plaques (NAB228, red) in the brain of a patient suffering from AD. Blue (DAPI). Scale bar=20 μm. Image courtesy of Stefan Prokop, MD.

2. Evidence for immune-overactivation in neurodegenerative diseases

Studies in animal models and in vitro systems, as well as biomarker studies, provide substantial evidence for increased pro-inflammatory immune responses correlating with the progression of pathological protein aggregation and neurodegeneration. A consistent finding is the upregulation of pro-inflammatory cytokines like IL-1,^35,36 TNF-alpha^37,38 and IL-12³⁹ in animal models of AD, as well as AD patient samples. Knockout or inhibitor studies in animal models consistently show that dampening these pathways is associated with reduced Aβ plaque burden and improved cognition,^39–42 suggesting that microglia-associated pro-inflammatory pathways may be viable targets for therapeutic intervention. Studies validating the relevance of these findings demonstrated upregulation of these pathways in human brain tissue,^41,43 CSF³⁹ and peripheral blood.^44,45 Some of the identified pro-inflammatory pathways are also involved in autoimmune conditions, thus drugs already in clinical use that target these molecules could be repurposed for studies in AD patients.⁴⁶ However, very notably, studies demonstrating that over-expression of the pro-inflammatory cytokine IL-6 in the brain can also be beneficial for the clearance of protein pathology⁴⁷ and that over-expression of anti-inflammatory cytokines like IL-4⁴⁸ or IL-10⁴⁹ exacerbates pathology, while genetic knockout of IL-10 improved Aβ-pathology,⁵⁰ are exemplary notes of caution that manipulations of the immune system need to be carefully balanced to be beneficial for successful interventional purposes.

Another important function of microglia is the removal of synapses that are not needed or damaged in a process called synaptic pruning, or simply “pruning”. This process is obligatory for CNS development, but is active throughout life, where it is crucial for synaptic plasticity and removal of dead or damaged synapses in disease.⁵¹ Damaged synapses are tagged for removal by components of the complement system⁵² and multiple studies have demonstrated that this process is more active in brains of AD model mice^53–55 as well as patients suffering from AD.^56–58 Conversely, blocking these pathways has led to reduced pruning and improved cognition, providing another avenue of potential therapeutic interventions^53–55 through dampening unwanted or overactive immune responses.

3. Immune exhaustion or inadequate immune responses in AD?

While studies implicating pro-inflammatory mechanisms in neurodegenerative diseases are prominent, a vast amount of data exists demonstrating inadequate or exhausted immune responses in AD and calling for strategies to activate, rather than to inhibit the immune system to intervene with the pathogenic cascade. Microglia surrounding Aβ plaques do not appear to be engaged in phagocytosis and studies demonstrate that certain microglia functions, including phagocytosis or microglial process movement, are inhibited in the Aβ rich environment.^59–62 In line with that, depletion of microglia from the murine CNS did not have a major impact on plaque deposition and maintenance,^63–65 although more recent studies suggest that longer term microglial depletion improves cognitive functions and prevents neuronal loss without major effects on Aβ burden, likely due to blockage of overly active synaptic pruning and pro-inflammatory signaling.^63,65 Evidence for microglia exhaustion also exists in brain tissue from patients suffering from AD. Researchers noticed that a subset of microglia in brain regions affected by pathological protein deposition show a peculiar morphologic phenotype characterized by broken up and vacuolated cellular processes and shrunken cytoplasmic bodies, termed senescent or dystrophic microglia.^{30,32,34,66–68} These forms of microglia are typically observed in chronic, long standing human disease conditions and are difficult to study in animal models.³³ It has been speculated that these exhausted forms of innate immune cells are limited in their ability to respond to pathologic insults and show unregulated secretion of various pro- and anti-inflammatory molecules.⁶⁹ The concept of cellular senescence is well known in the cancer field but recent studies have suggested a role for senescent cells in overall lifespan and brain aging.^70,71 In addition, it has been suggested that removal of senescent cells, including, but not limited to, microglia cells from the CNS environment in a mouse model of tau-pathology prevented tau phosphorylation and neuron loss as well as improving cognitive function.⁷² Better understanding of the process of cellular senescence and the pathways involved in development of senescent microglia may aide our understanding of this unique state of innate immune cells and help to tailor possible therapeutics.

4. Innate or adaptive immunity or both?

Given the clustering of genetic risk variants for sporadic AD in microglia associated genes,⁷³ these main players of the brain’s innate immune system have taken center stage in recent research efforts to understand immune contributions in AD. Mutations in receptors and signaling molecules associated with basic microglial functions are among the prominent genetic variants associated with sporadic AD.^74–76 In fact, the demonstration of mutations in colony stimulating factor receptor 1 (CSF1R) in patients suffering from a white matter dominant neurodegenerative disease termed hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS)⁷⁷ was the first direct link between a microglia-associated gene and causality of a neurodegenerative process. In addition, mutations in key players of other microglial pathways, including TREM2 and downstream signaling molecule DAP12, were associated with familial forms of dementia.^78–81 Variants in TREM2 and “TYRO protein tyrosine kinase binding protein” (TYROBP, also known as DAP12) highlight the close relationship between microglia and peripheral myeloid cells, including monocytes, macrophages and tissue resident histiocyte populations. TYROBP loss of function mutations are associated with Nasu-Hakola disease,⁸² which is characterized by a white matter dominant neurodegenerative phenotype through changes in microglia function as well as bone cysts ascribed to changes in macrophage activation patterns. This close relationship of microglia and peripheral monocytes has muddied the waters for recent research efforts on inflammation. While Microglia are derived from a unique monocyte population residing in the yolk sac,⁸³ which populates the brain during early embryonic development and are sustained by local self-renewal,^84,85 peripheral monocytes are a very mobile population of cells with a relatively short half-life, renewed from bone marrow precursors.⁸⁶ Some of the mutations and genetic variants associated with increased risk of AD affect microglia, as well as monocyte functions, making it hard to ascribe definitive roles for each of the two cell populations. While the contribution of peripheral myeloid cells to the pool of macrophages/microglia cells in the brain appears to be limited,^87,88 several studies suggest that reduced recruitment of peripheral myeloid cells enhances Aβ pathology.^89–91 While an increased influx of (manipulated) peripheral macrophages into the brain was linked to beneficial effects on containing protein pathology,⁹² the mere exchange of myeloid cell populations did not significantly alter AD associated pathology.⁹³ Single cell RNA sequencing technologies reveal a complicated system comprising multiple diverse microglia/myeloid cell populations during development and in the adult CNS in association with specific local functions and implications in disease processes.^94–97

Microglia are highly connected in the complex architecture of the brain and their processes are constantly monitoring the surrounding CNS environment.⁹⁸ Changes in microglial functions and activity are therefore expected to affect other cell types in the CNS. In addition, there is evidence of regional differences in microglia populations,^99–101 which may in return reflect differences in regional vulnerability of certain brain regions for neurodegenerative processes.^99–101 The interaction of microglia and astrocytes has been prominently positioned in recent studies of pathological CNS conditions, where it was demonstrated that microglial-derived cytokines can induce deleterious phenotypes in astrocytes, which serve in return as the actual toxic insult to neurons.^102–105 It is conceivable that the more abundant population of astrocytes is the major effector cell and microglia serve as a mere mediators in the pathological cascade. Oligodendrocytes and myelin also are affected by altered microglia functions.^106–108 This is illustrated by the fact that some of the variants in the TREM2 gene associated with Frontotemporal dementia (FTD) cause dominant degeneration of white matter tracts without major pathological protein deposits⁷⁸ and the progressive leukoencephalopathy in HDLS associated with reduced microglia function.⁷⁷ Recent large scale genomic analyses also suggest a role for myelination defects in AD and other neurodegenerative diseases,⁷⁷ making the interactions of microglia with oligodendrocytes and white matter tracts an even more interesting area for future study.

Besides the impact of the innate immune system, evidence exists that the adaptive arm of the immune system also may play a role in neurodegenerative diseases. Changes in T cell subtypes, as well as B cells, have been reported in peripheral blood of AD patients compared to controls^109–117 and it has been speculated that a general aging of the immune system may predispose to development of neurodegenerative diseases.^118,119 While major lymphocyte infiltrations are not a feature of neurodegenerative diseases, focal infiltrates of lymphocytes have been observed in brains of AD patients and murine model systems.^120–123 Evidence from studies in animal models suggests that manipulating peripheral lymphocyte populations, including lymphocytes residing in periventricular and perivascular locations, will impact microglial and astrocyte functions via secreted mediators.^124–127 Manipulating T cells with checkpoint inhibitors¹²⁸ or using genetically modified T cells to target cancer cells¹²⁹ has shown great promise in cancer therapy, including brain tumors,¹³⁰ but it remains doubtful that these approaches are applicable to neurodegenerative diseases, although some studies report success with checkpoint inhibitors in animal models.^131,132 The brain is regarded as an immune privileged site and therefore infiltrates of lymphocytes are not desirable and potentially harmful, which is illustrated by serious side effects in early trials of Aβ vaccination,¹³³ where the intervention induced a prominent T cell response leading to meningoencephalitis and a detrimental outcome for the patients.¹³⁴ Furthermore, the changes in neurodegenerative diseases are diffuse and consist of extracellular and intracellular protein aggregates as well as damaged cells or structures, which poses a difficult target and may expose self-antigens and trigger unwanted auto-immune responses.

5. Challenges moving forward

In recent years, the notion that the immune system is a component of the pathogenetic cascade in neurodegenerative diseases, sometimes referred to as “cellular phase,”¹³⁵ has been solidified and changes in immune functions have been demonstrated to be the pathogenetic driver in some neurodegenerative disease conditions. Given its central role in the disease course, the immune system is a prime target for therapeutic interventions, but the road to progress is littered with challenges. A major problem is uniqueness of the neurodegenerative process in humans when compared to common animal models used for experimental studies. Pathological protein deposits begin decades before symptom onset and the immune system’s response to these changes also lasts for long periods of time. These processes are hard to mimic in short-lived animal models. Furthermore, the human disease condition is complicated by multiple coexisting protein pathologies and also a multitude of contributing underlying disease conditions.¹³⁶

Recent studies have underscored similarities and differences between murine and human microglia on a single cell transcriptomics level, with the differences being most apparent with respect to aging.^137–141 Thus, translational studies in human tissue specimens, taking advantage of the immense efforts of AD research centers in collecting fluid specimens as well as brain tissue are more important than ever to validate findings from animal model systems and improvements in technology. In particular, possibilities that allow for a detailed, multiplexed protein analysis, as well as spatially resolved protein and gene expression analysis in post mortem brain tissue specimens, will aid these endeavors in the future.

Another important aspect of studying immune cell functions, in particular in the brain, is that context matters. Microglia are a prominent example of isolated cells in culture behaving differently than cells in the context of tissue with major differences in basic cellular functions like migration, process movement, phagocytosis, response to stimuli and cytokine secretion.^139,142 This calls for the development and more frequent use of novel model systems mimicking the cellular context in the brain like organotypic slice cultures^143,144 and induced pluripotent stemcell (iPSC) derived three-dimensional systems.^145,146

It will be important moving forward to think about what to expect from manipulations of the immune system in patients suffering from symptomatic neurodegenerative diseases. In animal models, existing aggregates of misfolded proteins can be cleared and cognitive deficits reversed by manipulations of immune factors, which is unlikely to happen in the complex human disease condition. But it is reasonable to expect symptom improvement and slowing down of the neurodegenerative cascade through targeted interventions in the immune system. These interventions will have to strike a delicate balance between inhibition of toxic or harmful effects and overactivation of desired immune effects, which can then become toxic.¹⁴⁷ Furthermore, when immune manipulations should be initiated needs to be considered when choosing the intervention and therapeutic strategies that may vastly differ between different stages of disease,¹⁴⁸ including preventative efforts in asymptomatic subjects decades before the anticipated onset of disease. These are exciting times for neurodegenerative disease research and studies of the immune system, at a juncture in AD research progress when overcoming some of the hurdles presented to the field will prove important for a deeper understanding of disease mechanisms and guide the way to successful interventions in these devastating conditions.