The neuroimmune axis of Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is a genetically complex and heterogeneous disorder with multifaceted neuropathological features, including β-amyloid plaques, neurofibrillary tangles, and neuroinflammation. Over the past decade, emerging evidence has implicated both beneficial and pathological roles for innate immune genes and immune cells, including peripheral immune cells such as T cells, which can infiltrate the brain and either ameliorate or exacerbate AD neuropathogenesis. These findings support a neuroimmune axis of AD, in which the interplay of adaptive and innate immune systems inside and outside the brain critically impacts the etiology and pathogenesis of AD. In this review, we discuss the complexities of AD neuropathology at the levels of genetics and cellular physiology, highlighting immune signaling pathways and genes associated with AD risk and interactions among both innate and adaptive immune cells in the AD brain. We emphasize the role of peripheral immune cells in AD and the mechanisms by which immune cells, such as T cells and monocytes, influence AD neuropathology, including microglial clearance of amyloid-β peptide, the key component of β-amyloid plaque cores, pro-inflammatory and cytotoxic activity of microglia, astrogliosis, and their interactions with the brain vasculature. Finally, we review the challenges and outlook for establishing immune-based therapies for treating and preventing AD.

Background

Alzheimer’s disease (AD) is a neurodegenerative and genetically complex age-related dementia characterized by progressive memory loss. The pathogenesis of AD involves deposition of β-amyloid plaques, and formation of neurotoxic oligomers of the amyloid-β (Aβ) peptide. This results in neurofibrillary tangles (NFTs) made up of the hyperphosphorylated microtubule-associated protein, Tau (p-Tau), neuroinflammation, neuronal and synaptic loss, and, ultimately, onset of dementia [1–4]. Neuroimaging studies have revealed that β-amyloid plaques begin to deposit in the brain a decade or more before the onset of cognitive decline [5]. This indicates that therapeutics aimed at lowering Aβ, the key component of β-amyloid plaques, would be best used pre-symptomatically, preferably a decade or more before the propagation of AD pathologies. This form of prophylactic clinical strategy would be analogous to reducing future risk for heart disease by managing cholesterol levels [6].

β-amyloid deposition and tauopathy, as assessed by levels of Aβ species and p-Tau (p-Tau 181, 217, 231), respectively, in cerebrospinal fluid (CSF) and plasma as well as directly by positron emission tomography (PET), can be used to detect Aβ- and Tau-related neuropathology prior to the onset of cognitive impairment [7]. Recent studies have demonstrated that post-translational alterations of Tau also play a role in the rate of clinical AD progression [8] and variability in tauopathies across brain regions of patients [9]. Collectively, these studies suggest that AD is a heterogeneous neurodegenerative condition with respect to both clinical presentation and progression of AD pathology. Thus, it is important to consider how disease heterogeneity and the temporal order of AD neuropathological features impact target identification, the timing of treatment, and the development of therapeutics to reduce AD pathologies and treat cognitive symptoms.

While aging is the leading risk factor for the onset of AD, family history and genetic risk factors play the second most prominent role. Genetically, AD pathology is a complex and heterogeneous disorder, encompassing a spectrum of genetic effect sizes. They range from fully or highly penetrant mutations causing familial AD (in the AD risk genes, APP, PSEN1, and PSEN2) and the common APOE ε4 allele with the most substantial impact on the risk on sporadic AD to variants with relatively small effects on the risk identified in genome-wide associations studies (GWAS) [1, 2]. GWAS has implicated many genes with potential roles in adaptive and innate immune systems (Table 1), with CD33 as the first of these genes shown to be associated with AD in a family-based GWAS [10]. This is followed by identifying other important AD-associated genes, including TREM2, INPP5D, CLU, CR1, SPI1, ABCA7, EPHA1, and the MS4A cluster [3, 10–14], with related functions to the immune system. CLU and CR1, for example, are well-established AD risk genes and components of the complement cascade. CR1 plays an important role in the activation of the complement system, mediates microglia activity, and promotes the phagocytosis of immune complexes, cellular debris, and Aβ [15–17]. CR1’s role in AD neuropathogenesis is still unknown. However, GWAS variants implicating that the CR1 gene may lead to loss of function (LOF), a reduction of peripheral Aβ clearance by erythrocytes, and a dysregulation of the complement cascade, including effects on inflammation [16, 17]. Moreover, expression of the CR1 gene in several cell types, such as erythrocytes, lymphocytes (T and B cells), and astrocytes, indicates that CR1’s mechanism of action on AD might be mediated through brain-resident cells and/or both peripheral immune cells and brain-specific cell types [18, 19]. Leveraging bispecific antibodies to simultaneously bind soluble Aβ and erythrocyte CR1 is suggested to rapidly decrease circulating Aβ as CR1-associated immune complexes [20]. This strategy might harbor the potential to subsequently prevent β-amyloid deposition in AD brains by an overall reduction of Aβ concentration in the bloodstream and other compartments [20]. Overall, the genetic heterogeneity of AD carries significant implications for drug development, which must be deeply considered in developing effective diagnostics and disease-modifying therapies for AD [21].

Table 1.

Genome-wide significant AD-associated genes with potential roles in innate and adaptive immunity

Chr	Gene	Immune-related function	References
1	ADAMTS4	Immunomodulator	Jansen et al., 2019 [22]; Redondo-García et al., 2021 [23]
1	AGRN	Survival and function of monocytes	Mazzon et al., 2012 [24]
1	CR1	Immunity—e.g., microglial phagocytosis and clearance of complement opsonized molecules	Lambert et al., 2009 [25]; Borucki et al., 2020 [26]
1	PSEN2	Innate immune system	Agrawal et al., 2016 [27]; Nam et al., 2022 [28]; Fung et al., 2020 [29]; Mendez et al., 2017 [30]
1	SORT1	Monocytes and T cells	Bellenguez et al., 2022 [31]; Herda et al., 2012 [32]; Mortensen et al., 2014 [33]
2	ADAM17	T cell response	Lambrecht et al., 2018 [34]
2	BIN1	Pro-inflammatory response; endocytosis and phagocytosis	Seshadri et al., 2010 [35], Sudwarts et al., 2022 [36]
2	FHL2	Wound healing and inflammation	Nordhoff et al., 2012 [37]; Wixler et al., 2019 [38]
2	INPP5D	Immunity and microglia function	Efthymiou and Goate, 2017 [19]; Lambert et al., 2013 [39]
2	SPRED2	NK cells; cytokine/chemokine production	Itakura et al., 2017 [40]
2	PRKD3	Thymic selection during T cell development	Ishikawa et al., 2016 [41]
3	IL17RD	Cytokines	Brigas et al., 2021 [42]; Girondel et al., 2021 [43]
3	MME	Neutrophils	Schulte-Schrepping et al., 2020 [44]
4	CLNK	Immune cell-specific adaptors	Utting et al., 2004 [45]
4	RHOH	TCR signaling	Gu et al., 2006 [46]
4	SCARB2	IFN-I production and cholesterol regulation	Guo et al., 2015 [47]; Heybrock et al., 2019 [48]
5	APC	T cell migration	Mastrogiovanni et al., 2022 [49]
5	HAVCR2	Viral receptor; T cells	Zhai et al., 2021 [50]; Wightman et al., 2021 [51]
5	HBEGF	T cells	Macdonald et al., 2021 [52]
5	MEF2C	B cell proliferation, regulate microglia, and antigen presentation	Sao et al., 2018 [53]
5	PFDN1/HBEGF	Macrophage-mediated cellular proliferation	Higashiyama et al., 1991 [54]
5	RASGEF1C	Macrophages; microglia	Srinivasan et al., 2020 [55]
5	TNIP1	NF-κB regulation	G'Sell et al., 2016 [56]; Gurevich et al., 2011 [57]
6	CD2AP	T lymphocyte	Raju et al., 2018 [58]; Tao et al., 2019 [59]
6	HLA-DRB5/ HLA-DRB1	Immunity—e.g., antigen presentation	Lambert et al., 2013 [39]; Lu et al., 2017 [60]
6	TREM2	Phagocytosis, and cellular metabolism in myeloid cells, microglia activation as well as binding to Aβ	Griciuc et al., 2019 [61]; Guerreiro et al., 2013 [13]; Jonsson et al., 2013 [14]; Kunkle et al., 2019 [62]; Bis et al., 2020 [63]; Sims et al., 2017 [64]
7	EPHA1	Immunity, BBB permeability, and immune cell trafficking	Ivanov et al., 2006 [65]; Chen et al., 2018 [66]
7	IKZF1	T/B cell dysregulation	Hoshino et al., 2022 [67]
7	PILRA	Immunoglobulin receptor; viral receptor	Agostini et al., 2019 [68]
7	SEC61G	Antigen presentation	Zehner et al., 2015 [69]
7	TMEM106B	Innate immune system	Rhinn et al., 2017 [70]
8	CLU	Immunity, binding to Aβ, and inhibition of complement system	Tschopp et al., 1993 [71]; Lambert et al., 2009 [25]; Zhao et al., 2015 [72]
8	CTSB	Immune cell infiltration	Ma et al., 2022 [73]; Ha et al., 2008 [74]
8	PTK2B	Spreading, migration, and function of immune cells	Okigaki et al., 2003 [75]; Lambert et al., 2013 [39]
8	SHARPIN	Neuroinflammation and NF-κB activation	Asanomi et al., 2019 [76]
9	ABCA1	Immune modulation; myeloid cells; dendritic cells	Zamanian-Daryoush et al., 2017 [77]; Westerterp et al., 2017 [78]
10	BLNK	B cell linker	Fu et al., 1998 [79]; Han et al., 2016 [80]
10	ECHDC3	NK, monocyte differentiation, cell infiltration	Zhao et al., 2022 [81]
10	TSPAN14	Mast cell function	Orinska et al., 2020 [82]
11	MS4A	Expressed in immune cells, TREM2 regulation, phagocytosis, regulation of complement system	Deming et al., 2019 [83]; Kuek et al., 2016 [84]
11	PICALM	Endocytosis and Aβ clearance	Zhao et al., 2015 [72]; Harold et al., 2009 [85]
11	SORL1	Microglia, monocyte migration, pro-inflammatory cytokines regulation and phagocytosis	Talbot et al., 2018 [86]; Knupp et al., 2020 [87]
11	SPI1	PU.1, altered microglia function	Jones et al., 2021 [88]
12	TPCN1	Antigen-presenting cells	He et al., 2020 [89]
14	FERMT2	Immune cell infiltration	Su et al., 2021 [90]
14	IGHG1	Antigen and immunoglobulin receptor binding activity	Lekhraj et al., 2022 [91]
14	PSEN1	Microglial hyperactivation	Lee et al., 2002 [92]
15	ADAM10	Immune cell function	Lambrecht et al., 2018 [34]
15	CTSH	Autoimmune inflammation, macrophages and phagocytosis, cytokines	Faraco et al., 2013 [93]; Conus et al., 2010 [94]; Zavašnik-Bergant et al., 2004 [95], Li et al., 2010 [96]
15	IGF1R	Stimulates regulatory T cells, autoimmunity	Bilbao et al., 2014 [97]; Andersson et al., 2018 [98]
15	RORA	Inflammatory response; lymphoid cell development; T cell survival; autoimmunity and chronic inflammatory response	Oh et al., 2019 [99]; Lo et al., 2016 [100]; Chi et al., 2021 [101]; Wang et al., 2021 [102]
15	SPPL2A	Catalyzes the intramembrane cleavage of the anchored fragment of shed TNF-α	Fluhrer et al., 2006 [103]
16	IL34	Proliferation, survival and differentiation of monocytes and macrophages	Foucher et al., 2013 [104]; Lin et al., 2008 [105]
16	KAT8	Viral immunity	Huai et al., 2019 [106]
16	MAF	Regulates IL-10	Gabryšová et al., 2018 [107]
16	PLCG2	Involved in multiple pathways in immune cells	Sims et al., 2017 [64]; Magno et al., 2021 [108]
16	ZNF423	B cell differentiation	Harder et al., 2013 [109]
17	ABI3	Immunity, regulation of actin polymerization, and microglia function	Sims et al., 2017 [64]; Satoh et al., 2017 [110]
17	ACE	Innate and adaptive immune system	Bernstein et al., 2018 [111]
17	SCIMP	MHC class II signaling transduction	Draber et al., 2011 [112]
17	TSPOAP1	Microglia-associated gene; IFN signaling	Bhatt et al., 2020 [113]
19	ABCA7	Microglia Aβ clearance and cholesterol metabolism	Hollingworth et al., 2011 [11]; Kim et al., 2006 [114]; Steinberg et al., 2015 [115]
19	APOE	Lipid metabolism; T cell activation	Saunders et al., 1993 [116]; Bonacina et al., 2018 [117]
19	CD33	Immunity, phagocytosis, and transmembrane receptor in myeloid cells	Bertram et al., 2008 [10]; Griciuc et al., 2013 [118]; Griciuc et al., 2019 [61]; Crocker et al., 2007 [119]
19	LILRB2	Immunoglobulin-like receptor; influence immune activation	Deng et al., 2021 [120]
20	RBCK1	Immunodeficiency disorders; LUBAC inhibits TNF-α signaling, dousing inflammation	Boisson et al., 2012 [121]; Bellenguez et al., 2022 [31]
20	CASS4	Eosinophil asthma response	Esnault et al., 2013 [122]
21	ADAMTS1	Immunomodulator	Rodríguez-Baena et al., 2018 [123]; Kunkle et al., 2019 [62]
21	APP	Antimicrobial peptide	Kumar et al., 2016 [124]; Eimer et al., 2018 [125]; Jonsson et al., 2012 [126]

The selected genes were implicated based on independent genetic loci with genome-wide significance in at least one GWAS in AD [12, 22, 25, 31, 39, 51, 85, 127–130] and a potential role in the immune system. IFN-I type I interferon; NK cell natural killer cell; TCR T cell receptor; MHC major histocompatibility complex; NF-κB nuclear factor kappa B; LUBAC linear ubiquitination assembly complex

Neuroimmune interactions introduce further heterogeneity in the etiology and neuropathogenic course of AD. In fact, neuroimmune interactions have increasingly emerged as a major focus in neurodegenerative disease research, including AD, Parkinson’s disease (PD), and multiple sclerosis (MS) [131]. Crosstalk between the brain and the peripheral immune system occurs via either the blood-brain barrier (BBB) [132], choroid plexus (CP) [133, 134], or from the meninges [135–137]. Emerging studies have shown that all these brain interfaces undergo structural and/or biological changes during aging and AD and can act as gateways for infiltrating peripheral immune cells into the brain (Fig. 1). Immunoprofiling studies have demonstrated heterogeneity in microglia [55, 138, 139] and peripheral immune cells, including myeloid cells [140], T cells [141, 142], and B cells [137, 143] in AD. Recent studies have also begun to elucidate the role of peripheral immune cells in brain health and neurodegenerative disease. In addition, genes associated with the immune system have increasingly been associated with AD risk in GWAS, e.g., IGHG1 (Table 1). However, studies to explore the disease-modifying role of peripheral immune cells in human brain are still at a relatively nascent stage. Given the compelling recent evidence for a role of the peripheral immune system in the pathogenesis of AD, future studies are clearly required to fully understand the contribution of peripheral immune cell-related genetics and brain infiltration to brain health and neurodegenerative diseases.

Fig. 1.

Neuroimmune interactions in AD neuropathology. AD is a heterogeneous and multifactorial complex neurodegenerative disease that is characterized by the abnormal aggregation of extracellular β-amyloid plaques and intracellular neurofibrillary tangles. This leads to neuronal cell death, synaptic degradation, and gliosis (microglia and astrocytes), further exacerbating neurodegeneration and ultimately leading to dementia. Under homeostatic conditions, microglia have a predominantly protective role, including phagocytosis and degradation of Aβ, secretion of anti-inflammatory cytokines, and neural network remodeling. However, excessive β-amyloid deposition and neuronal cell death can trigger robust pro-neuroinflammatory activation of microglia, leading to the release of pro-inflammatory cytokines and complement. A vicious cycle of neuropathology, pro-inflammatory glial activation, and excessive neurodegeneration ensues. This pathological cycle affects the BBB integrity and lymphatic drainage, which leads to immune cell infiltration (e.g., T cells) in the brain parenchyma and border zone, immune cell activation, antigen accumulation, and TCR clonal expansion. In this neuroimmune axis model, immunopathogenesis changes can therefore serve as a foundation for designing and developing of disease-modifying therapies for AD. APC, antigen-presenting cells; TCR, T cell receptor

In this review, we highlight immune signaling pathways and genes associated with AD risk, and interactions between innate and adaptive immune cells in AD. We discuss the complexities of AD at the levels of genetics and cellular physiology and their crosstalk with the immune system. We mainly delve into the litany of the role of peripheral immune cells in AD and the mechanisms by which immune cells, such as T cells, influence AD neuropathogenesis (Fig. 1). Lastly, we review the challenges and outlook for developing immune-based therapies for treating and preventing AD.

Neuroimmune alterations in AD

The healthy brain is an immunologically active organ protected by the resident immune cells and by infiltrating peripheral immune cells [144]. Recent discoveries have highlighted alterations in brain-resident (microglia) and peripheral immune cells (neutrophils, monocytes, T cells, and B cells) as well as crosstalk between innate and adaptive immune cells in the development of AD neuropathology [145–151]. Under homeostatic conditions, it is now clear that the adaptive immune system is present. Though low in levels, adaptive immune cells, including T cells and B cells, can enter the brain meninges, occupy the dura through skull channels—and play crucial roles in brain maintenance, including neuronal function, brain development, and spatial learning [152], mainly due to cytokines released from T cells, including interleukin-4 (IL-4), IL-17, and IFN-γ [153]. It has been shown that a deficiency of IL-4-producing T cells in the meninges [154] or an excess of these cells in choroid plexus during aging [155] negatively impacts the brain, emphasizing the importance of the adaptive immune system contributing to brain function and maintenance during homeostatic conditions. A recent study on the increased clonal expansion and elevated T cell activation in cytotoxic CD8+ T cells in brains of mild cognitive impairment (MCI) and AD highlight the potential disease-modifying roles of T cells in AD [142]. However, it is yet to be determined whether cytotoxic T cells have a pathogenic role in AD patients. Another possibility is that under disease conditions, such as AD, subsequential damage to the BBB due to the pathological changes may lead to gliosis and infiltration of CD8+ T cells from blood and border zones into the brain parenchyma and subsequent T cell receptor (TCR) clonal expansion. However, it is not fully known whether peripheral CD8+ T cells can cross the BBB and infiltrate into the AD brain parenchyma. In addition, reduced levels of circulating IFN-γ-secreting T cells were linked with age-related cognitive decline in mice [156]. Similarly, lower levels of IFN-γ in the plasma of AD patients were associated with progression of cognitive deficiency [157]. While the underlying mechanisms of increased infiltration of adaptive immune cells in neurodegenerative diseases and aging remain to be elucidated, the role of infiltrating T cells on disease progression differs extensively depending on the specific neurodegenerative disease under investigation and their functional programming [153]. In this section, we summarize the current status of research on the role of innate and adaptive immunity and crosstalk between these systems (Fig. 1) in the AD brain.

Microglial activation in AD

Microglia, the primary innate immune cells of the brain are crucial for local immune defense, clearing debris and toxins, as well as for maintaining brain homeostasis [158]. Microglia are also critical during brain development, for maintenance of neuronal networks, and for repair following injury or infection [159, 160]. Like sentinels, ramified microglia constantly survey their environment for detrimental signals and switch rapidly to an activated state upon sensing signals indicative of damage, infection, and other pathological conditions [161]. With regard to AD neuropathogenesis, microglial cells play a variety of roles. Conventionally, microglia, like macrophages, have been oversimplistically divided into M1 type (pro-inflammatory) and M2 type (neuroprotective) [162, 163]. However, single-cell transcriptomic and detailed proteomic studies have revised this simple dichotomy by revealing many functionally distinct microglial cell populations covering a spectrum of activities in the healthy brain and in the progression of AD [164]. This includes disease-related microglial cells such as disease-associated microglia (DAM) [165], microglia neurodegenerative phenotype (MGnD) [166], morphologically activated microglia (PAM) [167], and a multitude of unnamed subsets [164].

DAM are characterized by substantially low expression of homeostatic genes and upregulated expression of genes involved in neurodegenerative diseases, including AD and amyotrophic lateral sclerosis (ALS) [165, 166, 168]. Interestingly, genes upregulated in DAM include several known AD-related genes such as APOE [169], CTSD [170], LPL [171], TYROBP [172], and TREM2 [13, 14, 62, 173]. Major players in converting homeostatic microglia to MGnD are controlled by the APOE/TREM2/CD33 pathways [166]. The molecular DAM signature is defined by upregulated expression of genes related to phagocytosis, chemotaxis, and release of cytokines upon neuronal injury and is accompanied by a suppression of homeostatic genes [166, 174]. Furthermore, the PAM population is associated with Aβ and Tau pathology and the progression of cognitive decline [167, 175]. Nonetheless, microglia functional and phenotypic conversion in AD development and progression has yet to be fully understood with regard to distinguishing between protective and detrimental microglial function.

One of the classic pathological features of AD is nebulously referred to as “microglial activation”, which can be observed over the course of AD neuropathogenesis, possibly even a decade before the onset of clinical symptoms when deposition of β-amyloid plaques first takes place [176]. Increased microglial activation and the transition to DAM can either lead to a neuroprotective effect through Aβ clearance in the early and asymptomatic stages or, as AD pathology progresses, to a persistent inflammatory response leading to neurodegeneration [177]. In mice and AD patients, DAM colocalize with β-amyloid deposits. It has also been shown that DAM can even form a barrier to reduce further deposition of plaques [178, 179] and actively participate in the disassembly and digestion of β-amyloid plaques [165]. However, this process of colocalization can also trigger pro-inflammatory microglial activation and release of pro-inflammatory cytokines. The transition into MGnD is driven by an increase of APOE followed by an upregulation of CLEC7A expression and a further suppression of homeostatic genes [166]. MGnD are induced by phagocytosis of apoptotic neurons and colocalize with neuritic plaques, which are a hallmark of neurodegeneration in AD [166]. Microglia can also exacerbate the propagation of β-amyloid plaques by triggering the NLRP3 inflammasome and release of apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC specks), which quickly bind and cross-seed Aβ peptides extracellularly [180].

Microglia have also been shown to play a role in phagocytosis and propagation, e.g., via exosome secretion of p-Tau [181, 182]. For instance, chronic microglial activation, induced by excessive deposition of β-amyloid or by localized events of neurodegeneration, can lead to both a maladapted inflammatory response and the intraneuronal accumulation of p-Tau [183]. The presence of pathogenic p-Tau aggregates can further exacerbate pro-inflammatory microglial activation, including generation and release of pro-inflammatory cytokines [184, 185]. This can then trigger the activation of neuronal p38 mitogen-activated protein kinase (p38 MAPK), and further stimulation of Tau hyperphosphorylation [186], leading to a vicious cycle of p-Tau formation, neurodegeneration, and pro-inflammatory microglial activation/neuroinflammation.

Recent studies show that the AD-associated gene, CD33 [10] promotes pro-inflammatory activation of microglia while TREM2 promotes phagocytosis and clearance of debris, including Aβ [61, 118]. In addition to this opposing modulation of microglia activation by CD33 and TREM2, the microglial cytosolic protein SHIP1, encoded by the AD-associated gene INPP5D is linked to TREM2-signaling and is more highly expressed in plaque-associated microglia and during progression of AD. Inhibition of Inpp5d in 5XFAD mice revealed that it also regulates Aβ pathology and is associated with plaque density [187]. TREM2-dependent upregulation of genes related to phagocytosis and lipid metabolism results in modulating the neuroprotective function of DAM and therefore represents promising targets for enhancing beneficial microglial function and reducing neuroinflammatory events [3]. Moreover, increased CSF soluble TREM2 (sTREM2) and reduced AD risk and age-at-onset are associated with common variants in the AD-associated MS4A gene cluster [83]. Activating TREM2 or inhibiting CD33 with humanized antibodies are strategies currently underway in AD clinical trials and represent new therapeutic approaches to treating and preventing AD.

In addition to risk gene identification by GWAS, the integration of a multi-omic dataset allows fine mapping of AD risk loci. For instance, examining the population-level variation of gene expression and incorporating chromatin accessibility could verify previously implicated AD risk genes and identify putative AD genes for loci harboring multiple candidate genes (e.g., MS4A4E in the MS4A gene cluster) [188]. Such approaches could provide support for the microglial PU.1 transcription factor that has previously been associated with increased AD risk [189, 190]. The PU.1 downstream target genes have a predominantly immune function, in particular, the contributions of myeloid-/leukocyte-related processes were strongly highlighted [188]. This resource for human microglia-specific regulation of transcription provides further evidence for the critical role of microglia in AD. The functional phenotypes of microglia and their multidimensional roles in the etiological and neuropathogenic processes underlying AD are still not very well understood. However, identification of critical genes and pathways continues to be driven by a growing sample size in genetic studies, advances in multi-omic data integration, and targeted functional follow-up analyses. Eventually, these studies will help improve our understanding of microglial contribution to AD and pave the way for new avenues in developing better therapeutics.

CNS interfaces and peripheral immune cell infiltration

Crosstalk between the brain and the peripheral immune system occurs via three possible routes: (i) BBB, which provides an interface between the brain and circulation; emerging studies demonstrate the BBB breakdown and dysfunction in AD [132], (ii) choroid plexus (CP), which provides an interface between the blood and the CSF and acts as a gateway for bone-marrow-derived immune cell entry into the brain [133, 134], and (iii) meninges, an immune-blood-brain interface that allows immune cells to bypass the BBB and enter to the brain through specialized skull bone marrow channels [135, 136]. Changes in these CNS borders with advancing age could etiologically initiate disease pathology or exacerbate neuropathogenesis. CP dysfunction exhibits fibrosis, an increase in type I interferons (IFN) and local neuroinflammation, and impaired CP transportation function reduces Aβ clearance in the AD brain [134, 156, 191, 192]. Evidence for T cell infiltration into the brain parenchyma through meningeal lymphatic vessels suggests a broader role for peripheral immune cells in both healthy and diseased brains [193, 194].

Moreover, recent insights into the functions and communications between the glymphatic system and meningeal lymphatics in CNS disorders have recognized new important players in neurophysiology [195, 196]. Based on the lymphatic system, CSF flows directionally within the brain leading to non-selective clearance of metabolic wastes, including Aβ and Tau [197–200]. Disturbances in glymphatic efflux due to, e.g., sleep disorders or chronically impaired glymphatic system have been associated with neurodegenerative diseases such as AD [195, 201–204]. Reduction in meningeal lymphatic drainage has also been linked with aging-associated cognitive decline and an impaired glymphatic system to recirculate CSF through the brain [193, 205]. On the basis of these findings, an aging-related deficit in C-C chemokine receptor type 7 (CCR7) contributes to a reduction in glymphatic influx, cognition, and increased β-amyloid deposits in the brain of 5XFAD mice [206]. It is out of the scope of this review to delve into the immune cell compartmentalization and their changes with brain aging and neurodegenerative diseases, which has been recently reviewed elsewhere [144]. We, therefore, in the following sections, focused on discussing recent findings on infiltrating peripheral immune cells (Table 2), and their beneficial/detrimental implications in AD-related neurodegeneration.

Table 2.

Cellular infiltration in Alzheimer’s disease

Cell type	Model	Age	Disease-modifying role or correlation	Possible recruitment signal	References
Neutrophils	Human	Average age is 74.5 ± 10.4 for AD subjects	Unknown	Unknown	Zenaro et al., 2015 [207]
Neutrophils	5XFAD and 3x-Tg mice	4-month-old (5XFAD), 6-month-old (3xTg mice)	Depletion or inhibition of neutrophils trafficking reduced AD-like pathology and improved cognitive function	LFA-1 integrin	Zenaro et al., 2015 [207]
Monocytes	Appswe/PS1 mice	6–9-month-old	Reduced Aβ burden	Unknown	Malm et al., 2005 [208] Simard et al., 2006 [209]
Monocytes	Tg2576/TGF-β DNR mice	17–18-month-old	Reduced cerebral parenchymal and vascular β-amyloid deposits	TGF-β signaling	Town et al., 2008 [210]
Monocytes	APP/PS1 mice	6–7-month-old	Depletion of monocytes increased β-amyloid deposition	Aβ aggregates on blood vessels	Michaud et al., 2013 [211]
Monocytes	5XFAD mice	10-month-old	PD-1 blockade led to clearance of cerebral β-amyloid plaques and improved cognitive performance	PD-1/IFN-γ pathway	Baruch et al., 2016 [212]
CD8+ T cells	Human	16–81-year-old (Rogers, 1988), 56–96-year-old (Togo, 2002) for AD subjects	Likely have negative consequences for neuronal function and integrity	Unknown	Rogers et al., 1988 [213] Togo et al., 2002 [214]
CD8+ T cells	Human	Average age is 84.1 ± 3.6 for AD subjects	Unknown	Unknown	Merlini et al., 2018 [215]
CD8+ T cells	Human	72–96-year-old for AD subjects	Positive correlation of parenchymal CD8+ T cells with Braak stage	Unknown	Unger et al., 2020 [216]
CD8+ T cells	Human	Average age is 70.74 ± 7.01 for AD subjects	Positive correlation of peripheral TCM and TEM with cognition, and negative correlation of peripheral TEMRA with cognition	Unknown	Gate et al., 2020 [142]
T cells	APP/IFN-γ Tg mice	9-month-old	Clearance of Aβ	IFN-γ cytokine	Monsonego et al., 2006 [217]
CD4+ and CD8+ T cells	ArcAβ mice	12- and 22–24-month-old	No association with β-amyloid deposits	Aβ-induced endothelial cell activation	Ferretti et al., 2016 [218]
CD4+ and CD8+ T cells	APP/PS1 mice	6–7-month-old (Browne, 2013), 10-month-old (McManus, 2017), 18–19-month-old (Unger, 2020), 12–13-month-old (Gate, 2020)	Possible contribution of CD8+ T cells to neuronal dysfunction and modulation of synaptic plasticity	Unknown	Browne et al., 2013 [219] McManus et al., 2017 [220] Unger et al., 2020 [216] Gate et al., 2020 [142]
CD4+ and CD8+ T cells	5XFAD mice	12-month-old	TNF inhibitor treatment reduced CD4+ T cells in the brain, rescued impaired LTP, and decreased β-amyloid plaques	sTNF/TNFR1 signaling	MacPherson et al., 2017 [221] Shukla et al., 2019 [222]
Aβ-specific CD4+ TH1 cells	5XFAD mice	9-month-old	ICV-injected TH1 cells decreased β-amyloid plaques	Unknown	Mittal et al., 2019 [223]
Aβ-specific CD4+ TH1 and TH17 cells	APP/PS1 mice and Aβ42-induced rats	4–5-month-old (Machhi, 2021), 4-month-old (Zhang, 2013)	Teatment with TH1 and TH17 cells in APP/PS1 mice accelerated memory impairment and systemic inflammation, increased amyloid burden, activated microglia, and exacerbated neuroinflammation; TH17 cells mediated neuroinflammation and neurodegeneration in Aβ42-induced rats	Unknown	Machhi et al., 2021 [224] Zhang et al., 2013 [225]
CD4+ T cells	3xTg mice	6–9-month-old	$\alpha$4 blockade reduced Aβ load, Tau hyperphosphorylation and memory decline	$\alpha$4β1 integrin-VCAM-1 signaling	Pietronigro et al., 2019 [226]
Treg cells	5XFAD mice	10-month-old	PD-1 blockade led to clearance of cerebral β-amyloid plaques and improved cognitive performance	PD-1/IFN-γ pathway	Baruch et al., 2016 [227]
Treg cells	APP/PS1 mice	4–7-month-old	Depletion of Treg cells accelerated cognitive deficits and amplification of Treg cells by IL-2 treatment increased plaque-associated microglia, and improved cognitive functions	Unknown	Dansokho et al., 2016 [228]
B cells	3xTg mice	14–16-month-old	Depletion of B cells reduced β-amyloid plaque burden and microglial activation	Unknown	Kim et al., 2021 [229]

T_CM central memory T cells, T_EM effector memory T cells, T_EMRA terminally differentiated memory T cells, sTNF soluble tumor necrosis factor, TNFR1 tumor necrosis factor receptor 1, TGF-β transforming growth factor beta, PD-1 programmed death-1, IFN-γ interferon gamma, LFA-1 lymphocyte function-associated antigen 1, ICV intracerebroventricularly, LTP long-term potentiation

BBB breakdown during AD pathogenesis

As mentioned above, one possible communication route between the CNS and periphery is the BBB, which comprises various cell types, including endothelial cells, astrocytes, pericytes, and smooth muscle cells [230]. The BBB protects the central compartment by selective regulation and transportation of neurotoxins and serum factors via specialized tight junctions and transporters [231]. Vascular contributions to dementia and AD are increasingly being elucidated [132, 232–236]. In experimental imaging and pathological and epidemiological studies, dysfunction in the BBB and each of the cellular components of the neurovasculature have been linked to AD. These observations have led to the “AD neurovascular hypothesis,” which suggests that cerebrovascular impairment contributes to and perhaps even initiates AD pathogenesis and cognitive decline. Vascular dysfunction in AD accelerates BBB breakdown [237–242], degeneration of pericytes [237, 240, 243, 244], and reduction of BBB-associated cells that maintain integrity [239, 243, 245–251]. The presence of vascular Aβ pathology, also called cerebral amyloid angiopathy (CAA), predisposes toward neurovascular impairment and, sometimes, stroke [252]. Recent studies have suggested that BBB dysfunction is correlated with human cognitive impairment [239], including the early clinical stages of AD, and is considered as an early biomarker of the disease [132, 235, 253, 254]. Moreover, the identification of BBB impairment as an aging risk factor has been connected to the presence of peripheral immune cells, e.g., T cells, in the brains of the elderly and patients with age-associated neurodegenerative diseases [132, 255–259]. Pathogenic infiltration of peripheral immune cells into the brain parenchyma may lead to exacerbation of AD pathology [146, 149, 256]. Thus, reducing BBB breakdown could serve as a potentially useful therapeutic approach for limiting the infiltration of detrimental peripheral immune cells into the CNS.

Peripheral innate immunity in AD

Neutrophils are considered the first line of defense in our body during pathological conditions. Infiltrating neutrophils found in the brains of AD patients and transgenic animal models (Table 2) [207, 260] have attracted growing interest in the last few years in MS and AD [261]. In the blood, the neutrophil:lymphocyte ratio has been correlated with cognitive decline in AD [262, 263]. A limited number of studies demonstrated that neutrophils may contribute to the early stages of AD by mediating BBB damage, intravascular adhesion, and invasion of the CNS [207]. Infiltrating neutrophils also induce neurotoxicity by releasing IL-17, a cytotoxic cytokine for neuronal cells and mediating BBB breakdown [264], neutrophils extracellular traps (NETs), and myeloperoxidase (MPO) [207]. Moreover, depletion of infiltrating neutrophils using anti–Ly 6G or anti–Gr-1 antibody in two mouse models of AD (5XFAD and 3xTg mice) has been shown to significantly reduce the amyloid burden and microglial activation and improve performance in the Y-maze spontaneous alternation task and contextual fear-conditioning test [207], suggesting that neutrophils can play an important role in the development of AD.

Although a handful of studies suggest that pro-inflammatory, CNS-infiltrating neutrophils are detrimental in AD, the field would benefit from a deeper understanding of the causal role of neutrophils, their pathological pathways and mechanisms of action in the process of AD-related neuropathogenesis. A recent study identified a unique subset of neutrophils that can promote neuronal survival in the CNS [265]; the salutary effects of this subset of neutrophils could be therapeutically employed in various neurological disorders, including AD. How neutrophils infiltrate the brain and the factors that determine whether they are detrimental or protective are pivotal questions that need to be explored to gain a deeper insight into AD pathogenesis. Moreover, there is a dire need to understand the interactions between infiltrating neutrophils and brain-resident cells, which could offer new avenues for treating and preventing AD.

Another innate immune cell population consists of monocytes, which are found peripherally and less often in the CNS. Circulating monocytes are heterogeneous cells divided into multiple subsets with different surface markers, heterogeneous transcriptional profiles, and different functions. In AD transgenic mouse models (APP/PS1 and 5XFAD), circulating monocytes (CD14⁺/CD16⁻) can infiltrate the brain (Table 2), reduce Aβ burden, and improve cognitive performance [208, 212], suggesting a beneficial disease-modifying role for these cells in AD pathology. The C-C motif chemokine receptor 2 (CCR2) plays a vital role in the recruitment of monocytes into the CNS. CCR2 blockade in transgenic mouse models of AD (App_swe/PS1 and Tg2576) led to detrimental effects—increased β-amyloid pathology and exacerbated memory deficits [266, 267]. Live two-photon imaging studies have shown that patrolling monocytes crawl onto luminal walls of Aβ⁺ veins, internalize Aβ and circulate back into the bloodstream, suggesting that monocytes play a role in the clearance of vascular Aβ in AD [211]. Monocyte-mediated clearance of Aβ may constitute a unique therapeutic approach for reducing AD pathology using circulating monocytes.

Other studies have shown that blocking transforming growth factor-β (TGF-β) signaling on peripheral macrophages results in substantial infiltration and clearance of cerebral Aβ in the Tg2576 mouse model of AD, suggesting another potential anti-amyloid therapeutic approach [210]. It is important to note that while circulating monocytes can infiltrate the brain and eliminate debris such as cerebral deposits of Aβ, these cells are dramatically less effective in clearing pathology in AD with limited phagocytic ability and phenotypes that have been modulated toward pro-inflammatory conditions compared to monocytes of healthy controls [268]. This is in line with human studies, in which peripheral monocytes reveal reduced capacity for Aβ uptake and phagocytosis [268]. Collectively, these results indicate that the impact of chronic neuroinflammatory diseases such as AD on circulating monocytes is still unclear. Additional studies are needed to determine the direct role of circulating monocytes in AD, which may provide a deeper understanding of the underlying mechanisms of AD pathogenesis and lead to novel therapeutic targets for AD and other neurodegenerative diseases.

The contribution of adaptive immunity to AD pathogenesis

T lymphocytes are a pivotal part of the adaptive immune system, and cumulative evidence suggests that adaptive immune cells influence the pathophysiology of neurodegenerative diseases such as AD. Post-mortem brains from AD patients and corresponding AD-like animal models reveal that CD4+ and CD8+ T cells infiltrate the AD brain (Table 2) [142, 213–222]. However, their precise role in AD pathogenesis remains unclear. Although the pathogenic role of infiltrating CD4+ T cells has been controversial in different neurological disorders, there are only a handful of studies that have revealed a beneficial role of infiltrating CD4+ T cells into the brain parenchyma that target β-amyloid plaques, promoting Aβ clearance and neuronal repair [269]. The mechanisms underlying CD4+ T cell infiltration and activation in AD brain are unclear. Additional studies are required to identify underlying mechanisms of infiltrating CD4+ T cells in the course of AD and whether their infiltration has a beneficial impact on the disease pathology. Limited observations revealed that $\alpha$4-integrins on the surface of peripheral CD4+ T cells are highly expressed in the 3xTg mouse model versus wild type, and these cells infiltrate near vascular cell adhesion protein 1 (VCAM-1)+ cerebral vessels [226]. Blockade of $\alpha$4β1 integrin-VCAM-1 signaling reduced leukocyte adhesion to cerebral vessels and activated microglial cells, and improved memory in the 3xTg mouse model, suggesting a detrimental role of CD4+ T cells, in contrast to previous studies [226]. The harmful effect of CD4+ T lymphocytes in the pathogenesis of AD was also demonstrated via infiltration of T helper 17 (T_H17) cells, a subtype of CD4+ T cells into the brain parenchyma, resulting in an increased level of IL-17 and IL-22 cytokines in the CSF, serum, and hippocampus of AD models. Infiltrating T_H17 cells also lead to neuronal apoptosis [225]. Interestingly, serum levels of IL-17 in AD patients have been shown to be elevated [270, 271], and similar observations of infiltrating T_H17 cells into the brain and increased levels of IL-17 in the CSF and blood have been reported in MS patients [272–274].

The impact of CD4+ T cells on neurodegeneration varies depending mostly on their subsets. Regulatory T (T_reg) cells have been associated with diverse neuroinflammatory and neurodegenerative diseases such as AD due to their regulatory characteristics. However, their contribution to AD neuropathogenesis remains controversial. Depletion of T_reg cells in the APP/PS1 mouse model reduced recruitment of β-amyloid plaque-associated microglial cells and accelerated cognitive impairment [228]. By contrast, depletion of T_reg cells in the 5XFAD mouse model has been linked with clearance of β-amyloid plaques and increased recruitment of immune cells through the CP [227]. In this line and to specifically amplify T_reg cell populations, treatment of APP/PS1 AD mice with low-dose peripheral IL-2 administration increased microglia recruitment to β-amyloid plaques and restored memory function [228]. Collectively, these studies suggest that T_reg cells play an important role at the early stages of AD in regulating resident microglial cell-mediated clearance of parenchymal deposits of β-amyloid. However, additional studies are necessary to dissect the precise role of T_reg cells in AD etiology, their underlying mechanisms and whether therapeutic modulation of T_reg cells in AD is beneficial.

Human studies and AD transgenic animal models have shown that infiltration of cytotoxic CD8+ T cells correlates with a worsening disease, suggesting a role for these cells in AD pathogenesis [213, 214, 216]. Blood immune profiling of AD patients and healthy individuals has revealed a higher percentage of activated HLA-DR+ CD8+ T cells and augmented release of pro-inflammatory cytokines [142, 275], suggesting that circulating cytotoxic T cells are activated in the blood of AD patients. Notably, a recent study powerfully demonstrated the clonal expansion of CD8+ T cells in the brains/CSF of MCI and AD patients, indicating CD8+ T cells may impact neurodegeneration and/or cognitive impairment in AD [142]. Collectively, these findings underscore the critical pathogenic roles of infiltrating T cells in AD. However, to date, these studies have not yet provided direct causative evidence for infiltrating T cells playing an etiological or disease-modifying role in AD. This is partially due to a lack of a comprehensive disease model that recapitulates T cell infiltration and interaction with human brain cells with different human genetic backgrounds.

T cells and microglia crosstalk has been shown to help maintain homeostasis and shape neuropathology during chronic neurodegeneration [276]. Several studies have suggested that crosstalk between microglia and infiltrating CD4+ T cells plays a critical role in orchestrating immunoregulatory mechanisms in AD pathogenesis [223, 224]. For example, Aβ-specific CD4+ T helper 1 (T_H1) cells induce a major histocompatibility complex class II (MHC II)+ population of microglia that abrogate AD-like pathology in the 5XFAD mouse model, likely due to interferon gamma (IFN-$\gamma$) cytokine signaling [223]. In contrast, injecting Aβ-specific CD4+ T_H1 and T_H17 T_eff cells into the brains of APP/PS1 mice has been shown to exacerbate Aβ burden, microgliosis, neuroinflammation, and cognitive impairment [224]. Given the observation of T_reg deficits in AD, this may be partially due to breaking immune tolerance by limiting T_reg cells in the circulation and CNS, thus compromising T_reg immunosuppressive functions [228]. These studies underscore a critical and complex disease-modifying role for CD4+ T cells in AD pathogenesis. Additionally, few other studies revealed the role of microglia as antigen-presenting cells (APCs) to mediate CD8+ T cell infiltration during viral infection [218, 277, 278], which might be relevant to neurodegenerative diseases, including AD and PD. To assess whether age-related T cell infiltration is due to passive extravasation or promoted by microglia as APCs via MHC II receptors, a study using monkeys found that T cell entry into the brain is correlated with activated microglial cells and cognitive impairment [279]. This can go in the other direction in which infiltrating T cells possibly alter microglia phenotypes, neuroinflammation, and neurodegeneration. Thus, it will be important to investigate the impact of the peripheral immune cells, including infiltrating CD8+ T cells and different subsets of CD4+ T cells, as well as other cells, on microglia and their interaction consequences on neuronal cells during AD pathogenesis [280].

In contrast to T cells, the role of B cells and their involvement in AD has been relatively less explored. Mature B cells have been reported in the brains of AD transgenic mice using single-cell RNA sequencing data [173]. Consistent with this observation, recent evidence reveals infiltration of B cells into the brain parenchyma of 3xTg AD mouse model (Table 2), which results in elevated IgG around β-amyloid plaques, activated microglial cells, and has been linked to heightened progression of AD pathology [229]. Depletion or inactivation of B cells at the early stages of AD pathology in transgenic AD mice has demonstrated beneficial therapeutic impact by restoring TGF-β⁺ microglia, which have enhanced ability to clear Aβ oligomers and slow down the progression of AD. The loss of B cells has been shown to significantly reduce β-amyloid plaque burden and reverse behavioral and memory deficits in the 3xTg AD mouse model [229]. This study suggested that while B cells infiltrating the brain parenchyma can produce what may be beneficial IgG around β-amyloid plaques, they also exacerbate the manifestation of AD-like pathology. Although the exact role of B cells in AD neuropathogenesis is still in its infancy, depletion of a specific subset of infiltrating B cells may offer a unique disease-modifying treatment similar to the use of anti-CD20 antibodies in relapsing-remitting and primary progressive MS [281, 282]. The dominant mode of action of most of these antibodies is through selective immunosuppression of pathogenic immune cells (B cells and a small population of T cells), for instance, by blocking α4β1 integrins to halt infiltration of these immune cells to the CNS [283, 284]. Of note that these immunotherapies are effective in controlling inflammation in MS patients with ongoing inflammation, but they fail to halt the disease progression, and their effects are often short-lived [285]. Decelerating the multifaceted vicious cycle of AD neuropathology will be even more challenging.

Role of systemic inflammation in AD

Aging has been linked to alterations in the systemic immune system associated with an increased frequency of inflammation and infection [149]. Emerging evidence suggests that manifestations beyond the brain include systemic inflammatory events (e.g., circulating pro-inflammatory cytokines and chemokines or common cold), early-life or long-life exposure to infection agents (e.g., herpes simplex virus and Chlamydophila pneumonia), or critical disease (e.g., sepsis) have been associated with an increased risk of developing AD and cognitive decline [286]. Investigating this periphery-brain interaction may provide new insights into the understanding of AD pathogenesis. It could offer great promise for novel therapeutic and diagnostic approaches. Studies have suggested that even a single recent infection can modulate peripheral immune-brain communication and accelerate cognitive decline in AD patients and elderly adults [287–289]. For example, increased serum levels of pro-inflammatory cytokines tumor necrosis factor–α (TNFα) and IL-6 are directly linked with neuropsychiatric features in AD [289], suggesting that such systemic inflammatory events may further promote the disease progression. TNFα and vascular endothelial growth factor (VEGF) in combination with Aβ_1-42 were also found to reduce viability of neurons in culture [290]. A study performed in the Han Chinese population found increased serum levels of IL-18, IL-23, and IL-17 in AD patients compared to healthy controls [271]. This observation also confirmed in APP/PS1 mice, where it was found that increased level of IL­12/IL­23 subunit p40 correlates with reduced amyloid burden in APP/PS1 mice lacking IL-12 and IL-23 [291]. Furthermore, a significant linear correlation of cognitive performance and CSF p40 values in AD and control subjects were observed [291], indicating that IL­12/IL­23 signaling plays a crucial role in regulating not only the amount of β-amyloid plaques, but also cognitive impairment.

In contrast to the results from previous observations regarding TNFα, IL-6, and IL-12, we have recently shown that increased plasma levels of the pro-inflammatory cytokines, IL-12, and IFN-γ have been associated with a healthier cognitive trajectory in normal non-demented elderly, particularly in those with β-amyloid-positive brains [157]. Increased levels of these two cytokines would be expected to ramp up T cell-macrophage interactions leading to enhanced defense against infection. Given our recent findings showing that Aβ is an antimicrobial peptide, this is particularly interesting in view of the antimicrobial protection hypothesis of AD [292], which posits Aβ aggregation and subsequent β-amyloid deposition can be triggered by microbial infection in the brain. Perhaps, by affording enhanced protection from peripheral infection, higher levels of plasma IL-12 and IFN-γ may also reduce entry of pathogens into the brain, which would otherwise trigger the antimicrobial response of Aβ to form plaques [157]. Along similar lines, recent studies have also shown that various types of vaccination, from influenza to bacillus Calmette-Guérin (BCG), protect against AD risk [293–298], further suggesting that protection against common infections may help reduce AD neuropathology, along the lines of the antimicrobial protection hypothesis [292].

Recent studies also have attempted to determine whether acute episodes of systemic inflammation influence the risk for AD. In one study, short-term systemic inflammatory attacks were linked with increased serum levels of TNFα cytokine and an enhanced rate of cognitive decline in AD subjects [299]. Another recent study demonstrated that a history of severe infections requiring hospital admission and treatment was associated with an increased long-term risk of vascular dementia and AD [300]. These infections were not limited to CNS infections and covered a variety of hospital-treated viral and bacterial infections, suggesting that exposure to systemic inflammation is sufficient to affect the brain and increase the risk of dementia. These studies collectively underscore the potential roles of infections and systemic inflammatory events in the etiology of dementia and AD. Moreover, they raise the question of whether practices and strategies to improve infection control and general inflammation might mitigate or delay AD risk. There is, of course, a caveat to note that hospitalization per se (and not the infection itself) could also be associated with greater dementia or AD risk [301–303].

AD therapeutics: challenges and opportunities

Providing millions of people living with a debilitating disease like AD with effective treatment is a monumental challenge that clinicians and scientists have faced since the first description of the disease by Alois Alzheimer in 1906. Moreover, preventing AD is of equal importance as our population ages at a dramatic rate thereby increasing the prevalence of AD. Thus far, efforts to treat the disease have been marginally successful in managing symptoms, but with essentially no impact on modification of disease progression. Because of the heterogeneity of AD and complex nature of the brain, effective drug discovery and development will require well-coordinated investigations of the molecular, cellular, and genetic factors involved in AD neuropathogenesis as well as crosstalk between the brain and peripheral immune system.

The development of potential therapeutics to treat and prevent AD has been enormously challenging over the past decades, leading to no disease-modifying drugs. The investigational drugs and proposed targets of AD that have progressed to Phase 2 or 3 clinical trials in the U.S. are summarized in Fig. 2. Although the recent U.S. Food and Drug Administration (FDA) approval of aducanumab and its clinical impact is highly controversial [304, 305], it is a promising sign that we can target specific pathological hallmarks of AD. Aducanumab is an immunotherapy (Biogen) derived from memory B cells developed initially by the Swiss company, Neurimmune. The search for an Aβ immunotherapy at Neurimmune was based on identifying naturally occurring protective human antibodies targeting Aβ oligomers, originally inspired by the findings of Moir et al. [306]. The controversy regarding FDA approval of aducanumab was initially ignited by the Biogen company as they halted two phase III trials of aducanumab as the interim analysis showed that the drug was unlikely to improve cognition of mild AD patients. However, re-evaluation of the data revealed a subset of people that might have benefited, which lead to submission of aducanumab for FDA approval. This is further fueled by the unusual route of an “accelerated approval” pathway of the FDA, which is reserved for treatments that are “reasonably likely,” to help patients, but not certain.

Fig. 2.

Investigational drugs and proposed targets for the treatment of Alzheimer’s disease and related dementia (ADRD), focusing on those that have been approved or progressed to Phase 2/3 or beyond in U.S. clinical trials. This up-to-date dataset was obtained from alzforum.org, a resource provided by FBRI LLC

Several other forms of disease-modifying therapeutics are under development for AD or being clinically tested, e.g., small molecules that target γ-secretase enzyme to reduce Aβ peptide production. γ-secretase modulators selectively decrease the level of Aβ42 over Aβ40 and potentiate formation of nonfibrillar and shorter Aβ peptide species, including Aβ37 and Aβ38 [307]. Since β-amyloid deposition begins one to three decades before symptoms [308], anti-Aβ therapies, e.g., Aβ immunotherapy and γ-secretase modulators [307] would be best used pre-symptomatically as a prophylactic or means of secondary prevention following a positive test for Aβ accumulation in the brain, e.g., by PET or blood test. While most of attempts to improve cognitive symptoms in AD patients by reducing Aβ levels in the brain have largely failed, owing to a number of reasons that have been comprehensively discussed elsewhere [309–311], but new promising results from Biogen and Eisai clinical trial earlier this year bring new hopes to people afflicted with this memory-robbing neurodegenerative disease [312]. This anti-amyloid monoclonal antibody, called lecanemab, showed 27% slower progress in cognitive decline in people with early-stage AD compared to placebo, which might likely be due to its mechanism of action in targeting “protofibrils” strands at earlier stages of the disease before they consolidate into β-amyloid plaques and the length of the trial (i.e., 18 months) that allowed showing a meaningful impact on cognition. Moreover, development of bispecific antibodies by linking them to a BBB transporter moiety may facilitate BBB passing into the brain and improve antibody design, which enables targeting soluble Aβ aggregates with a wide range of sizes in a mouse model [313]. Even though the beneficial effects of Aβ immunotherapy on AD are still uncertain, increasing delivery through the BBB and enhancing antibody binding as well as selectivity to the toxic Aβ aggregates could potentially pave the way to promising therapeutic applications [313].

The identification of AD risk genes by GWAS (Table 1) and whole genome/exome sequencing has progressively expanded our current understanding of AD and emphasized the key role of immune genes involved in AD pathophysiology [3]. New opportunities are emerging for the development of genetic risk scores used to assess the impact of genetic susceptibility factors in risk prediction models. AD and its genetically heterogeneous nature include subtypes that may not homogeneously respond to a specific intervention. Comprehensive risk profiling provides the opportunity to categorize patient subgroups for gene-specific therapeutics, personalized medicine, and translational genomics [31, 314, 315]. The association between Alzheimer’s β-amyloid deposition and sTREM2 [316] points to the notion that one can leverage treatments of microglia-modulating and anti-amyloid therapeutics by targeting, e.g., TREM2 or CD33. Recent studies showed the promising effects of increasing TREM2 in a mouse model using an agonistic antibody design [317]. However, the temporal component is crucial and a beneficial effect of increasing TREM2 is observed, especially in the early stage of AD development, highlighting the dynamic role of TREM2 in modulating β-amyloid deposition and neuritic dystrophy in AD pathogenesis [317, 318]. We previously demonstrated the therapeutic potential of targeting CD33 leveraging an adeno-associated virus vector-based knockdown and observed reduced amyloid accumulation and neuroinflammation in an APP/PS1 mouse model [319].

Additionally, given that the APOE ε2 allele is associated with decreased risk of late-onset AD and has been shown to modulate the immune response of microglia, therapeutic strategies aimed at mimicking the protective effects of the APOE ε2 allele are now being considered as disease-modifying interventions for AD [320]. A viral-mediated overexpression of APOE ε2 in amyloid mouse model brains led to a reduction of the Aβ burden, which might be attributed to an increased Aβ clearance in APOE ε2 expressing animals [321]. Gene delivery of APOE ε2 may halt or lessen Aβ burden in the brain and subsequent neuritic plaques and inflammatory processes. However, long-term APOE ε2 overexpression in human brains should be carefully evaluated and raises concerns since the APOE ε2 allele is associated with a higher risk for other diseases like CAA [322] and stroke [320, 323]. Another limiting factor is that in order to be a successful therapy, APOE ε2 overexpression would need to be established before the onset of β-amyloid deposition long before the onset of symptoms in patients, which poses its own challenges [324].

The heterogeneity of the disease carries significant implications for drug development, which must be deeply considered in developing effective disease-modifying therapies for AD. A great deal of study is still needed to better understand the clinical and neuropathological heterogeneity in AD and its impact on the development of more effective diagnostics and therapies for treatment and prevention [325]. The complexity of AD heterogeneity suggests that individualized treatments or a combination of multiple targets at different stages of the disease progression from pre-symptomatic to prodromal to clinical manifestation of cognitive impairment will be needed. Another important aspect is the heterogeneity of the immune response, both spatially and temporally [326–328]. The immune system’s plasticity, driven by cellular heterogeneity, allows it to adopt various phenotypes and genotypes in response to internal and external signaling, which could be instrumental to the disease onset, and progression. A combination of environment and genetics shape the heterogenous immune system and response [329]. The environment can play an important role in shaping the composition of the immune cells present in individuals, e.g., by exposure to infection and the microbiota. For instance, the microbial status can be transferred from the mother to the baby during birth and fetal development. In this context, children delivered by cesarean section can have significantly lower levels of CXCL10 and CXCL11 chemokines in their blood [330]. Shortly after birth, diet (first by milk components and then solid food) shapes the microbial community and development of the immune system, including effector and T_reg cells, which could have strong impacts later in life [331]. Moreover, genetics is another key factor in determining the level of cytokines produced by the immune cells in response to stimuli.

In addition, given the extreme complexity and heterogeneity of AD, it will be necessary to stratify AD patient groups through deep phenotyping and genotyping along with the application of algorithms that incorporate comprehensive clinical, imaging, biomarker, and pathology data to limit misclassification bias and enable more precise and predictive models for drug discovery and personalized treatment. Successful therapeutics aimed at prevention of AD would require targeting the earliest signs of pathology in the earliest stages of the disease, e.g., early detection of β-amyloid deposition, NFT formation, and neuroinflammation. Recently, substantial advances have been made in efforts to identify the pre-symptomatic stages of AD using CSF and blood-based biomarkers, including Aβ42/40 ratio, p-Tau phosphorylated at threonine-181, 217, or 231, and neurofilament light (NfL) [308]. These early events of abnormal proteostasis and glial activation initiate the disease process pre-symptomatically and drive widespread neuroinflammation, further modified by peripheral immune cell infiltration, which can either ameliorate or exacerbate neuronal cell death leading to dementia. Detecting and targeting peripheral immunity could initiate an exciting new era in the discovery and development of neuroimmune treatments to treat and prevent neurodegenerative disorders, such as AD [332, 333]. Therapeutic strategies focusing on pathogenic or protective peripheral immune cells could enable new immune-based therapeutic opportunities for neurodegenerative diseases, including immunosuppressive drugs that directly target specific populations of brain-infiltrating T cells and other immune cells, such as anti-CD3 antibodies, TNF antagonists, or calcineurin inhibitors [334]. Moreover, mammalian target of rapamycin (mTOR) inhibitors such as rapamycin may help promote beneficial T_reg cells while inhibiting detrimental T_H17 cells in AD [334].

The role of specific T cell subsets in AD pathophysiology remains to be fully elucidated. Other new immune-based therapeutic approaches, including depletion of B cells at the early stages of the disease, have already shown promise in delaying AD progression in animal models [229]. Future studies are now necessary to explore whether therapeutics targeting beneficial peripheral immune cells, e.g., monocytes, T_reg cells, or detrimental cells such as T_H17 cells, by cell-specific immunotherapies or other strategies will be helpful in treating and preventing AD. For instance, IL-17-producing T cells have been shown as crucial players in promoting BBB disruption and disease progression in multiple neurodegenerative diseases, including AD, PD, and MS [42, 264, 335]. Most importantly, neutralization of IL-17 cytokine was shown to prevent cognitive impairments, synaptic dysfunction, and rescue neuroinflammation in AD animal models [42, 336]. These findings indicate that tuning the exacerbated levels of IL-17 cytokine in AD might be an important therapeutic target to prevent its deleterious effect on disease progression. Conversely, it is known from several investigations that depletion of T_reg cells in AD mouse models exacerbates disease progression. In this line, treatment of AD mice with low-dose IL-2 cytokine to specifically amplify T_reg cell populations rescued cognitive function [337]. Moreover, the recent CNS-specific gene delivery of IL-2 provides a critical and important therapeutic method for an effective IL-2 delivery system in preclinical models [338]. Although presence of T_reg cells in the brain parenchyma is mostly beneficial, a higher level of T_reg cells in peripheral blood is associated with immune aging and chronic systemic inflammation [339]. This amplifies the complexity of the immune system and sheds light on the importance of not only the immune cells’ function but also their location, which can dictate different outcomes and inform future studies for designing more efficient therapeutics, e.g., amplification of T_reg cells in individuals without inducing systemic immune inflammation.

While immune system heterogeneity has long been acknowledged, limitations of conventional experimental models have constrained our ability to systematically dissect the underlying mechanisms and causes. Recent advances in microfluidic technology, single-cell omics, molecular biology, and imaging now enable the profiling and tracking of immune cells at a single-cell resolution [327]. Single-cell RNA sequencing technologies have had a substantial impact and allow for a better understanding of the immune system heterogeneity and immune function [329]. Also, simultaneous readouts using multi-omic profiling, including cell surface proteins, gene expression, and receptor sequences, provided new insights into a highly heterogeneous immune system. Moreover, more physiologically relevant models (mouse and multicellular in vitro) are needed to accurately predict immune responses to drug treatments in preclinical models of disease. These new advancements will undoubtedly refine our understanding of the highly heterogeneous immune cells and expedite the search for better therapies. Along these lines, patterns of immune response change during the (long) course of progression in neurodegenerative diseases. Interventions that might be beneficial in the early stages of disease could be detrimental in the late stages. Another level of complexity to consider is that this temporal sequence might not be only observed longitudinally but also at a single time point in a patient’s brain since pathology possibly affects the brain in a stage-wise fashion leading to the coexistence of early and late stages of the inflammatory response in different brain regions at a given point in time. New knowledge derived from the heterogeneity of the immune system will accelerate the development of novel and effective immune-based treatments that skew the balance between detrimental and reparative effects to beneficial for AD patients.

Additionally, with the high complexity and phenotypic variability, as well as a high rate of failures from clinical trials of AD, precision medicine has the potential to not only improve the success of the clinical trials but also reduce financial costs and sample sizes [340]. In this line, studying the disease systematically by considering sex differences, risk factors, blood-based biomarkers, disease progression, and responses to therapeutic treatments are a few pillars that are critical for the implementation of precision medicine in finding a cure and increasing diagnostic accuracy for AD.

Concluding remarks

AD pathology begins a decade or more prior to the onset of cognitive decline. However, existing therapeutics targeting pre-symptomatic “initiating” pathologies of abnormal proteostasis—β-amyloid aggregation and deposition and induction of p-Tau by Aβ oligomers—are most often applied at the onset of clinical symptoms when neuroinflammation has already inundated affected brain regions. As such therapeutics targeting AD-related proteinopathy have largely failed to improve cognitive symptoms and would best be applied when β-amyloid plaque and tangle pathology and glial activation first begin, usually a decade or more before symptoms, in a form of prophylaxis or secondary prevention. Human resilient brains, those revealing abundant levels of plaques and/or tangles, in the absence of cognitive deficits at death, have revealed that excessive neurodegeneration leading to clinical dementia requires robust events of neuroinflammation, e.g., microglial activation, pro-inflammatory cytokine release, and reactive astrocytes [341]. With the advent of dozens of AD genes emerging from GWAS that implicate immune cells, e.g., microglia and innate immune mechanisms, novel therapeutics aimed at attenuating neuroinflammation have entered into clinical trials, e.g., targeting CD33 and TREM2 [3]. The major question now remaining to be answered is whether therapies aimed at abating neuroinflammation and neurodegeneration owing to neuroinflammation will be more successful at effectively treating the symptoms of AD than those targeting abnormal proteostasis—plaques and tangles—which may be better suited for prevention.

The precise mechanisms by which infiltration of peripheral immune cells such as T cells is mediated during age-associated neurodegenerative diseases remain to be elucidated. Knowledge gained from studies of other neuroinflammatory conditions may be useful in this regard. For example, in patients with MS, T_H17 cells disrupt the BBB, infiltrate the brain parenchyma, and promote neuroinflammation through IL-17 and IL-22 [264]. Understanding the causative roles (protective or detrimental) of resident immune cells, particularly microglia, and infiltrating peripheral immune cells such as T cells in AD will hopefully guide therapeutic avenues to target the immune system at different stages of the disease from pre-symptomatic onset of pathology to clinical symptoms.

Dissecting the roles of immune cells in AD pathogenesis has been challenging, and much of the work discussed in this review have been conducted using mouse models. This field would benefit from model systems that recapitulate the roles of peripheral immune cells and vascularization, e.g., in vitro three-dimensional models with increased cellular complexity, incorporating peripheral immune cells and vascular elements, single-cell level imaging and interactions, and patient-derived cells. Advances in microfluidics, multicellular human models, and generating iPSC-derived microglia (microglia-like cells) offer a new toolset to dissect immune signaling and complex cell interactions that go awry in neurodegeneration. The elaboration of such models will be essential for designing future therapeutic strategies targeting immune pathways.

Taken together, AD progression is an outcome of a complex interplay of several key players, from dysfunctional neurons to resident immune cells, microglia to the peripheral immune system, and dissecting this entangled and highly heterogenous circuit will take time. The emerging neuroimmune axis of AD emphasizes the need to, someday, additionally classify patients according to their AD-related immunogenetic status (Table 1) together with deep genotyping/phenotyping of innate and adaptive immune cells (Table 2), both inside and outside of the brain, to assess effects on AD risk and pathogenesis and to guide the most effective therapies for treatment and prevention. Ultimately, the successful implementation of multidisciplinary studies among experts with disparate and complementary areas of expertise across neuroscience (including computational neuroscience), genetics, immunology, neurology, and bioengineering will be necessary to engender the paradigm shift needed to successfully develop effective treatments aimed at modifying, halting, or reversing AD neuropathogenesis.

Authors’ contributions

MJ, AM-H, and RET wrote the paper. The authors read and approved the final manuscript.

Funding

This work was supported by the Cure Alzheimer’s Fund and the JPB Foundation.

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Competing interests

The authors declare that they have no competing interests.