Basic science of neuroinflammation and involvement of the inflammatory response in disorders of the nervous system

Introduction

Inflammation plays a key role in highly prevalent diseases, including many cardiovascular, neurological, and rheumatological diseases. While an appropriate immune response can be beneficial and protective, aberrant activation of this response recruits excessive pro-inflammatory cells to cause damage (Figure 1). Neuroinflammation is inflammation within the central nervous system (CNS). Because the CNS is separated from the periphery by the blood-brain barrier that creates an immune-privileged site, it has its own unique immune cells and immune response. Moreover, neuroinflammation can compromise the blood-brain barrier (BBB) causing an influx of peripheral immune cells and factors. This chapter will provide a broad overview of neuroinflammation and describe its unique aspects. Key immune cells and factors in mediating neuroinflammation will be introduced followed by a discussion of neuroinflammation in different neurological diseases.

Figure 1:

Neuroinflammation. When the blood-brain barrier (BBB) is compromised, peripheral immune cells infiltrate into the CNS and interact with microglia and astrocytes. Depending on the stimuli, the resultant response can be either detrimental or protective to the CNS.

I. Key Players in Neuroinflammation

A. Neutrophils

Neutrophils form 50–70% of leukocytes and play a central role in neuroinflammation. As one of the major defenses, neutrophils are quickly activated in the bloodstream and are the first peripheral leukocytes to respond to stimuli. Neutrophils at the injured site will attempt to eliminate pathogens and initiate an inflammatory cascade to repair the damaged tissue. Neutrophils phagocytose pathogens to form a phagosome that undergoes lysosomal degradation to destroy the pathogen’s cell walls and proteins. After ingesting a pathogen neutrophil produces reactive oxygen species (ROS) such as superoxide (O2⁻), hypochlorite (HOCl), hydrogen peroxide (H₂O₂), hydroxyl radicals, and nitric oxide (NO)¹. Neutrophils also secrete myeloperoxidase (MPO), an enzyme that produces HOCl and other reactive species. MPO is also involved in the formation of neutrophil extracellular traps (NETs) which limit pathogens from multiplying by entrapping bacteria, viruses, and fungi². However, NETs can also cause a proinflammatory responses and lead to tissue damage via long-term production of proteolytic enzymes. Formation of NETs can eventually lead to apoptosis, known as NETosis.

The BBB serves as a significant immunological defense from foreign pathogens and toxic molecules. However, with injury and resultant neuroinflammation, the BBB can become compromised allowing peripheral immune cells to infiltrate into the CNS. The infiltration of neutrophils has been related to brain damage due to the appearance of pro-inflammatory cytokines, ROS, and NETs release. However, not all neutrophils are pro-inflammatory. Recently, other neutrophil phenotypes have been described that have roles in tissue repair, tumor killing, and immune regulation³. This has implications in therapy as strategies that indiscriminately deplete or inhibit neutrophils may not always be beneficial.

B. Macrophages

Activated macrophages regulate inflammatory responses and have diverse phenotypes and functions, allowing macrophage to change fluidly into different states depending on its environment⁴. An M1/M2 classification has been described from in vitro observations. Macrophages (M0) can be “classically” stimulated with pro-inflammatory mediators, i.e., LPS and IFN-y, to adopt an aggressive (M1) phenotype. Conversely, cells “alternatively” stimulated with anti-inflammatory cytokines (i.e., IL-4 & IL-13) will adopt an M2 phenotype, thought to have restorative and immunomodulating functions. When inflammation initially occurs, macrophages can polarize into M1-like phenotypes and generate pro-inflammatory cytokines and chemokines like TNF, IL-1β, and IL-12⁵. These macrophages will amplify inflammation by generating large amounts of pro-inflammatory cytokines and ROS to remove pathogens or foreign molecules from the injured site, though prolonged M1 macrophage activity will result in tissue damage and chronic inflammation. M2-like phenotypes are thought to be anti-inflammatory and reduces inflammation to enable tissue repair by secreting anti-inflammatory cytokines, chemokines, and growth factors like IL-10, TGF-β, and CCL18⁶. Underscoring the complexity and diversity of macrophages, the M2 phenotype has been found to have subsets known as M2a, M2b, M2c, and M2d. M2a amplifies cell growth and tissue repair, M2b plays a central role in inflammation, M2c is important for phagocytosis, and M2d stimulates tumor progression⁷. Beyond the M1/M2 phenotypes, other phenotypes are starting to be described, primarily based on advanced techniques such as RNAseq⁸.

C. Lymphocytes

Lymphocytes are classified into B-cells, T-cells, and natural killer cells (NK). The T- and B-cells participate in acquired or antigen-specific immune responses. B-cells can turn into plasmacytes to produce antibodies. While humoral immunity depends on B-Cells, cell immunity depends on T-cells⁹. T cells play major roles in adaptive immune response to tissue damage and infection. They are able to clone highly polymorphic antigen receptors which allows them to detect protein-based antigens¹⁰. Upon recognition of protein-associated antigens, T cells will undergo clonal amplification and progressively obtain the ability to react to stimuli. Major types of T-cells are CD4+ (helper T-cells) and CD8+ (cytotoxic T-cells)¹¹. T-cell receptors and their related receptors, such as CD3 and CD4, form a complex between the major histocompatibility complex-2 (MHC-II) receptor and the target antigen. CD4+ cells generate cytokines to start an immune reaction and activate T-cell-associated humoral immunity. Once activated, CD8+ cells migrate to its target antigen to eventually kill it. Regulatory T-cells (Tregs) can help manage immune tolerance and suppress extreme inflammation, playing a protective role. Conversely, effector T-cells like Th1 and Th17 cells can play a part in neuroinflammation and tissue damage. NK cells play a versatile role by serving as a rapid defense mechanism against neural damage and helping immune modulation within the CNS.

D. Microglia

Microglia, originating from the yolk sac¹², are the primary immune cells of the CNS, constituting 5–10% of the adult brain cell population. They are the resident macrophages in the CNS and first responders to insults¹³. These cells have highly motile processes allowing them to scan their environment constantly. The three primary functions of microglia are 1) surveying their environments for any changes using their sensomes, which encode various proteins to allow microglia to sense endogenous ligands and microbes¹⁴; 2) physiological housekeeping, which includes migrating to sites of injury¹⁵, synapse pruning and remodeling¹⁶, myelin homeostasis¹⁷, and interacting with astrocytes¹⁸; and 3) protecting the organism against harmful stimuli, which include pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) recognized by the various receptors expressed on microglia, including toll-like receptors, nuclear oligomerization domain-like receptors, and more (Figure 2)¹⁹. As the immune cells of the CNS, microglia protect the brain from harmful antigens, such as apoptotic cells, microbes, protein aggregates, and lipoprotein particles.

Figure 2:

Microglia. Under normal physiological circumstances, “resting” microglia are ramified, consisting of many receptors. Receptor and cytokine interactions maintain the ramified state. Upon activation, microglia can shift toward an amoeboid “M1” phenotype that upregulates a pro-inflammatory response. Activated microglial cells can also shift to the M2-phenotype that is more reparative and anti-inflammatory.

Microglia are also capable of contributing to oxidative stress and have a role in producing ROS that lead to tissue damage when produced in excess. These phagocytes can mediate respiratory burst to produce powerful microbicidal agents downstream of the initial superoxide production²⁰. Activated microglia produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS), and the release of NO leads to neuronal cell death by inhibiting neurons from undergoing cellular respiration²¹. Contributing to oxidative stress is MPO²². Normal brain microglia rarely express MPO^23,24, but activated microglia are positive for MPO²⁵. Elevated MPO activity can be detrimental to tissues and is implicated in many diseases²⁶. In vivo MPO activity can be imaged by molecular imaging agents for MRI^27–31 and PET³².

E. Astrocytes

Astrocytes are the brain’s most common glial cells and help regulate neuronal activity and maintain brain homeostasis. They regulate blood flow, maintain BBB integrity, supply energy metabolites to neurons, modulate synaptic activity, monitor the secretion of neurotrophins, remove dead cells from their surrounding environment, manage the extracellular balance of ions, fluids, and neurotransmitters, and form scars in response to brain injury or damage¹³. Furthermore, astrocytes can phagocytose protein aggregates like amyloid plaques³³. In contrast to microglia, astrocytes tend to engulf distal processes and diffuse neuritic debris³⁴.

Astrocytes respond to insults by a process called reactive astrogliosis. Activation of astrocytes changes its molecular expression and morphology such as increasing the number and size of astrocytes expressing glial fibrillary acidic protein (GFAP)³⁵. When astrocytes are activated, they can take one of two phenotypes: the A1-phenotype or A2-phenotype, which are neurotoxic or neuroprotective, respectively¹³. Many intercellular signaling molecules can also trigger reactive astrogliosis, e.g., reactive oxygen species, neurotransmitters, cytokines (FGF2, IL-10, CNTF, IFNγ), and molecules associated with systemic metabolic toxicity³⁶. Activated astrocytes become not only neurotoxic but also lose many of their vital functions pertinent to homeostasis and maintenance of neuronal health. A1-reactive astrocytes negatively affect synaptic activity by disassembling synapses and inducing the formation of fewer synapses. Additionally, A1 astrocytes almost completely lose their ability to phagocytose myelin debris from their environment. Other detrimental functions include production of neurotoxic levels of reactive oxygen species³⁷, releasing potentially excitotoxic glutamate³⁸, and compromising the blood-brain barrier via vascular endothelial growth factor (VEGF) production³⁹.

Not all reactive astrocytes are detrimental. Astrocytes also contribute to normal physiological inflammatory responses to protect the CNS. Some beneficial functions of reactive astrogliosis include uptake of glutamate⁴⁰, protection against oxidative stress by producing glutathione⁴¹, degradation of β-amyloid plaques⁴², facilitating repair of the BBB⁴³, and restricting the spread of inflammatory cells or infectious agents into healthy CNS parenchyma⁴⁴.

F. Identification, Polarization, and Imaging

1. Macrophages and Microglia

To differentiate macrophages from microglia, human and murine microglia specifically express TMEM119, by which they may be identified⁴⁵. Among the putative M1-macrophages/microglia markers are iNOS, CD38, CD80, CD86, and FPPR2 as well as pro-inflammatory cytokines such as TNF, IL-6, IL-12A, IL-23A and IL-27. Putative markers of M2-macrophages/microglia are Arginase-1, CD206, CD163, RELMα, CHI3L3, ALOX15, EGR2, and c-MYC⁴⁶. However, transcriptomic experiments suggest that the M1/M2 nomenclature falls short of the complexity of activation states in vivo. A study comparing homeostatic microglia to those in different diseases found ten transcriptomic patterns during development, which reappeared under disease conditions, and three other distinct clusters associated with neurodegeneration, demyelination, and remyelination, hinting at a highly diverse inflammatory milieu⁴⁷.

Molecular imaging studies aim to unravel some aspects of this convoluted network utilizing established disease models to address different aspects of neuroinflammation. One example is imaging agents targeting MPO. Paramagnetic substrates that are activated and retained at sites of MPO allow for visualization of MPO activity with MRI^27,31,48. Activated innate immune cells phagocytose and store Fe²⁺ which can be visualized by iron oxide nanoparticles⁴⁹. MPO and iron oxide nanoparticle MRI have been used in combination to identify and map M1-dominant and M2-dominant lesions in the CNS (Figure 3)⁵⁰. PET imaging requires significantly less agent to be detectable, which increases sensitivity. Imaging probes targeting the translocator protein (TSPO), which is expressed by activated microglia and to a lesser extent reactive macrophages and astrocytes, are widely used in the research setting⁵¹. A radioligand targeting MPO has been recently developed³². Targets for PET-agents beyond TSPO and MPO include endocannabinoid receptor CB2⁵², cyclooxygenase 1⁵³ and 2⁵⁴, matrix metalloproteinases⁵⁵, and P2RY12⁵⁶.

Figure 3:

Differentiating different macrophage/microglia phenotypes by MRI. MPO activity (MPO-Gd) and iron oxide nanoparticle (CLIO) MR imaging in a murine model of experimental autoimmune encephalomyelitis. In (A), MPO-Gd+ lesions with matched CLIO+ lesions (area 2) represent M1-like cells, corroborated by immunohistochemistry in (B) (red = MPO, yellow = CD206, blue = DAPI). Lesions that were CLIO+ only without corresponding MPO-Gd+ signal (area 1) represent M2-like phagocytes that expressed CD206 but virtually no MPO on immunohistochemistry. Modified from⁵⁰.

2. Astrocytes

Even though GFAP has been commonly used to characterize reactive astrogliosis, many caveats exist. One of the main limitations includes regional differences within the brain regarding GFAP expression. Most importantly, GFAP does not allow for the differentiation between the A1- and A2-phenotypes. Transcriptomic studies found 57 genes, such as C3 and guanine nucleotide-binding protein 2 (GBP2), that exhibited preferential expression in A1-astrocytes, as well as 150 genes, such as the S100 calcium-binding protein A10 (S100a10), that were preferentially expressed in A2-astrocytes⁵⁷. Of these genes, the most used marker for A1-astrocytes is C3, while the most common markers for distinguishing A2-astrocytes include S100a10 and pentraxin-3 (PTX3)⁵⁸. Imaging astrocytes includes PET agents targeting monoamine oxidase B, acetate metabolism, and imidazoline₂ binding sites⁵⁹, although none differentiate between the different phenotypes.

II. Neuroinflammation in Diseases

A. Multiple Sclerosis

People living with MS (PwMS) develop neurodegeneration due to neuroinflammation throughout the brain and spinal cord, leading to accelerated atrophy. Neuroinflammation in MS results from a complex interplay of adaptive and innate immune cells⁶⁰. Traditionally, MS has been viewed as a T-cell-mediated disease. Autoreactive CD4⁺ and CD8⁺ T-cells migrate across the broken-down blood-brain barrier and are present in active demyelinating lesions⁶¹. Dysregulated regulatory T-cells may also contribute to the development of autoreactivity in MS⁶².

While T-cells play a central role in MS neuroinflammation, the critical importance of B-cells has recently been recognized. B-cells act through interaction and antigen presentation to T-cells, the release of inflammatory cytokines and chemokines, and the production of pathogenic antibodies⁶³. PwMS have immune cell infiltrates and ectopic lymphoid follicles in the meninges⁶⁴. Innate immune cells are also important in MS. Microglia and macrophages are abundant in active demyelinating lesions⁶⁵. Active demyelinating lesions predominate early in the disease course but become less frequent later in the disease⁶⁶. These later lesions slowly expand, called smoldering plaques, and have iron-containing microglia and macrophages at the periphery⁶⁷ that may play an important role in the gradual progression of disability in PwMS⁶⁸.

B. Neurodegenerative diseases

Alzheimer’s disease (AD) is the most frequent cause of dementia. A substantial unmet need exists to elucidate the mechanisms underlying AD and find robust non-invasive biomarkers to aid the diagnosis and earlier detection of AD, but also the development and assessment of new therapeutic strategies. In AD, there are multiple potential triggers of inflammation, among which protein aggregates seem to be playing a pivotal role⁶⁹, and crosstalk of protein misfolding and inflammation are a constant part of the neurodegenerative processes⁷⁰. Persistent activation of glial cells that compromises neuronal functionality can result from continuous exposure to an inflammatory stimulus or impairment of resolution⁷¹. Recent studies reveal a significant number of novel AD-risk single nucleotide polymorphisms are exclusively or dominantly expressed in microglia, emphasizing the critical roles these cells play in AD⁷². Different phenotypes of microglia have been found in AD, including activated response microglia (ARMs), interferon-responsive microglia (IRMs), human AD microglia (HAMs)⁷³, disease-associated microglia (DAM)⁷⁴, microglial neurodegenerative (MGnD)⁷⁵, and dark microglia⁷⁶. Research is ongoing to elucidate the roles these different types of microglia play in AD and other neurodegenerative diseases.

C. Stroke

Stroke induces a complex cascade of changes dominated by the innate immune response, with activation of local microglia, influx of leukocytes into the brain, and production of ROS that elevate oxidative stress and contribute to stroke progression⁷⁷. Whereas neutrophils become scarce after 3 days, activated microglia and macrophages are elevated within hours and can last for weeks to months after stroke^29,78. Neutrophils arrive in the ischemic brain within 24 hours after stroke, with macrophages peaking about 3 days after⁷⁹. Pro-inflammatory molecules and cytokines such as TNF and IL-1β are elevated in the blood of patients. The levels of pro-inflammatory molecules and leukocytes correlate with outcomes after stroke⁸⁰. Similarly, decreasing leukocyte recruitment and pro-inflammatory and oxidizing molecules, such as MPO, have decreased infarct size by 30–60%⁸¹, demonstrating that inflammation extends ischemic injury. Another example is Rho-associated kinase (ROCK), which is elevated in patients after stroke and is an independent predictor for recurrent stroke.^27,28 ROCK inhibition reduced neutrophil accumulation in the ischemic region and resulted in reduction of infarction volume.^30,31

D. Glioma

The tumor microenvironment (TME) in glioma is characterized by a complex interplay of various cell types, including tumor cells, immune cells, and endothelial cells, along with the secretion of various pro and anti-inflammatory cytokines⁸². Glioma-associated macrophages/microglia (GAMs) can manipulate immune response and tumor development. M1-like GAMs exhibit an anti-tumor effect, while M2-like GAMs have an immunosuppressive impact. The presence of macrophages in low-grade gliomas decrease survival and they are more abundant in higher-grade gliomas compared to microglia⁸³. Similarly, tumor-infiltrated neutrophils (TINs) can modulate other immune cells and promote tumor infiltration. T-lymphocytes also have a distinct role in glioma progression and suppression but at lower fractions than in brain metastasis⁸⁴. T-cells constitute 1–5% of total glioma cellular content. The percentages of both CD4⁺ and CD8⁺ tumor-infiltrating T-cells increase with tumor grade⁸⁵.

Conclusion

Neuroinflammation is a complex interplay between different peripheral and central immune cells that can have damaging and beneficial effects depending on the context. Imaging of these cells and their functions is still at a relatively early stage and represents opportunities in imaging and clinical research to advance our knowledge and improve patient care.

Key Points.

1. Neuroinflammation is a key immune response in the central nervous system (CNS) observed in many neurological diseases.

2. The CNS is separated from the periphery by the blood-brain barrier that creates an immune-privileged site with its own unique immune cells and immune response.

3. BBB compromise causes an influx of peripheral immune cells and factors that interact with resident immune cells in the CNS.

4. Neuroinflammation can exert damaging as well as beneficial effects depending on the context.

Synopsis:

Neuroinflammation is a key immune response observed in many neurological diseases. While an appropriate immune response can be beneficial, aberrant activation of this response recruits excessive proinflammatory cells to cause damage. Because the central nervous system is separated from the periphery by the blood-brain barrier that creates an immune-privileged site, it has its own unique immune cells and immune response. Moreover, neuroinflammation can compromise the blood-brain barrier causing an influx of peripheral immune cells and factors. Recent advances have brought a deeper understanding of neuroinflammation that can be leveraged to develop more potent therapies and improve patient selection.

Abbreviation:

CNS

central nervous system

BBB

blood-brain barrier

IL

interleukin

ROS

reactive oxygen species

MPO

myeloperoxidase

NET

neutrophil extracellular trap

LPS

lipopolysaccharide

IFN

interferon

TNF

tumor necrosis factor

TGF

transforming growth factor

CCL

chemokine ligand

NK

natural killer cell

CD

cluster of differentiation

MHC

major histocompatibility complex

PAMP

pathogen-associated molecular patterns

DAMP

damage-associated molecular patterns

GFAP

glial fibrillary acidic protein

FGF2

fibroblast growth factor 2

CNTF

ciliary neutrotrophic factor

FPPR2

formyl peptide receptor 2

RELMα

Retnla resistin like alpha

CHI3L3

Chitinase-3-like-3

ALOX15

arachidonate 15-lipoxygenase

EGR2

early growth response 2

TSPO

translocator protein

P2RY12

purinergic receptor P2Y₁₂

ROCK

Rho-associated kinase

GAM

glioma-associated microglia/macrophage

TIN

tumor infiltrated-neutrophil

iNOS

inducible nitric oxide synthase