The role of neutrophils in autoimmune diseases

preface

Neutrophils are the most abundant immune cells in humans and are the body's first line
of defense against external microorganisms.
In addition, they also contribute to various physiological functions, including angiogenesis, blood clotting, and tissue repair
.
Recent studies have found that neutrophils, especially the extracellular traps released after neutrophil activation, play a central role
in the development and persistence of systemic autoimmune diseases.

However, the study of neutrophils is particularly difficult
due to several technical challenges.
Coupled with the fact that neutrophils have a short half-life (often less than a day), these technical issues have left research in the field of neutrophil biology lagging behind other innate and adaptive immune cell types
.

At present, with the emergence of some new technologies, the complexity of neutrophil biology and its pathology is now gradually beginning to be understood
.
In fact, neutrophils play a central role
in the pathogenesis of several systemic autoimmune diseases.
Therefore, a better understanding of the role of neutrophils in dysregulated chronic inflammatory responses is critical
for the future development of targeted therapies that can limit the clinical manifestations of these diseases.

Biology of neutrophils

In vivo homeostasis

Under healthy, stable conditions, neutrophils are released from the bone marrow into the bloodstream, typically circulating in humans and mice for less than a day
.
Their fate after their release remains a matter of debate and may depend on the inflammatory state
of the host.
During blood circulation, neutrophil surface marker expression and protein load change with lifespan, such as the chemokine receptor CXCR4
.

In fact, under homeostasis conditions, some tissues are infiltrated by neutrophils, especially the spleen, lungs, and liver
.
Neutrophils in these tissues may act as sentinels, ready to respond to invading microbes and be excited
by the local microenvironment.

Upregulation of CXCR4 expression in elderly neutrophils allows them to remain in bone marrow and lymphoid tissues and be phagocytosed by macrophages as a clearance mechanism
.
This cycle is clearly controlled by circadian rhythms, which are regulated by clock genes in an intracellular manner, but also by
external factors such as glucocorticoid signaling and bacterial metabolites.
As cells age, neutrophils produce higher levels of reactive oxygen species (ROS), with an increased
tendency to form extracellular traps (NETs) compared to newly released neutrophils in the bone marrow.
This rhythm is also reflected in disease states, such as rheumatoid arthritis, and the risk of cardiovascular events is often more severe in the morning, which may be driven
, at least in part, by these diurnal changes in neutrophil biology.

heterogeneity

Recently, some subsets of neutrophils have been identified, such as neutrophils
expressing high levels of the type I interferon-stimulating gene (ISG).
Such subsets of interferon reactions are expanded in patients with severe covid, suggesting that it may be related to
the pathology of the disease.
Notably, interferon-induced neutrophils have higher ISG expression levels in women than in men, and this sex difference may be associated with
strong biases in women's risk for many systemic autoimmune diseases.
This observation is supported by a recent study that showed that neutrophils in young adult women are more mature and active than those in young adult men, resulting in a greater response to stimuli from various cytokines and an increased tendency to
form NETs.

In addition, under certain inflammatory conditions, a subset
of pro-inflammatory neutrophils called low-density granulocytes (LDGs) has been identified.
These cells have a pathogenic phenotype and function
that is different from normal neutrophils.
For example, LDG produces higher levels of certain cytokines, including type I interferon, and tends to increase NET formation, with vascular lesions and immunostimulatory features
.

Effector function

When neutrophils encounter a red flag, they respond
differently depending on their surface receptor composition and intracellular protein content.
Neutrophil activation has different functional roles, including migration into tissues, where neutrophils can release granular contents such as neutrophil elastase, collagenase, and lysozyme, disrupting the extracellular matrix and attacking invading pathogens
.

Neutrophils use several strategies to inhibit and kill microbes
.
A central role in killing microorganisms is phagocytosis, where neutrophils phagocytosis fuse microorganisms with intracellular lysosomes to produce phagolysosomes that kill and degrade invading pathogens
under controlled conditions.
The intracellular vesicles of neutrophils store a variety of cytotoxic enzymes, proteins, and peptides that can be released into phagosomes
at any time.
Another component of the neutrophil reaction is the activation of the NADPH oxidase mechanism, which destroys many structures
by producing highly reactive superoxide anions.

Neutrophil extracellular traps

NETs are made up of nuclei and particles that trap and kill fungi, bacteria and viruses
.
Typically, the formation of NET involves programmed cell death of neutrophils, but it can also be done
without affecting cell membrane integrity or immediate neutrophil death.
NET production can be triggered by a variety of stimuli, including microorganisms, cytokines, immune complexes, autoantibodies, crystals, chemicals, and platelets
.

Exposure to these signals ultimately leads to the breakdown of the neutrophil skeleton, the depolymerization and citrullination of histone chromatin, and the assembly of contents such as myeloperoxidase (MPO) and neutrophil elastase
on the DNA scaffolding of genomic and mitochondrial DNA.
This process usually depends on the NAPDH oxidase mechanism and/or mitochondrial ROS production and cyclins such as CDK4 and CDK6, which promote several downstream effects
of NET formation.

The role of neutrophils in autoimmune diseases

For many years, systemic autoimmune diseases have been primarily associated with deficiencies in adaptive immune responses, but research over the past few decades has highlighted the important role
of various innate immune cells and type I interferon pathways in several systemic autoimmune diseases.
Neutrophils have many associated roles
in the pathogenesis of systemic autoimmunity.

Systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by autoreactions to nucleic acids and other nuclear and intracellular components, with an enhanced response to type I interferon, more pronounced in
women.
It is characterized by extensive inflammation of many organs, including the kidneys, synovial joints, skin, lungs, heart, and blood vessels
.
The etiology of this disease is not fully understood, but it is thought to be a multifactorial process
driven by a combination of genetic, epigenetic, environmental, and hormonal factors.

Compared with healthy controls, the number of neutrophils in SLE patients changed significantly, and neutrophil-specific gene expression increased
as the condition worsened.
There is evidence that SLE-associated neutrophils have impaired phagocyte clearance, increased apoptosis, and abnormal
oxidative metabolism.
In particular, there is an increase in circulating LDG in SLE patients, and an enhanced ability of LDG in SLE to form NETs in vitro, which contain higher levels of modified autoantigens and immune-stimulating molecules
than NETs of normal neutrophils.

In addition to the excessive NETs formation observed in SLE, there is evidence that the ability to degrade these structures in the serum of a significant proportion of patients is impaired
.
In general, DNASE1 and DNASE1L3 are involved in the degradation
of DNA in circulation and tissue.
In a significant proportion of SLE patients, different groups of patients exhibit abnormal levels and/or function of these nucleases, resulting in impaired
NET degradation.

Overall, increasing NET formation and decreasing NET degradation can lead to increased levels of these structures, increased exposure to modified autoantigens and promote tissue damage
.
In addition, when NETs accumulate in tissues, they can activate B cells and plasmacyto-like dendritic cells through intracellular toll-like receptors and other intracellular sensors, further promoting inflammatory signaling pathways
.
DNA-bound immunostimulatory molecules in NETs, such as IL-33, HMGB1, and LL37, further enhance this signaling
.

rheumatoid arthritis

Rheumatoid arthritis is the most common systemic autoimmune disease that primarily affects the synovial joints and, if not treated properly, can lead to severe disability, and often also extra-articular tissues such as the lungs and vascular system
.
About 70% of patients with rheumatoid arthritis present anti-citrullinated protein antibodies (ACPA), which is highly specific for rheumatoid arthritis and can form pathogenic immune complexes in joints, promoting inflammation and bone erosion
.

Neutrophils are a major source of citrullinated antigens because they produce enzymes, such as PAD4, that catalyze the modification of arginine to citrulline
.
In inflammatory joints in patients with rheumatoid arthritis, especially in the early stages of the disease, neutrophils are abundant and can be locally released
.

PAD enzymes are present in the active form of NETs and can be detected
in rheumatoid synovial membranes.
Citrullination in rheumatoid joints may also involve neutrophil-derived membrane-lytic proteins such as perforins and membrane attack complexes, which produce pores
.
In the synovium, NET products, including citrullinated proteins, can be endocytosed by fibrous synovial cells, which upregulate MHC class II molecules and deliver NET-derived peptides to CD4+ T cells, thereby establishing an association with
the adaptive immune response of rheumatoid arthritis.
In addition, NET-derived neutrophil elastase can disrupt the structure of cartilage and promote its citrullination, which can lead to synovial inflammation
by increasing its immunogenicity and autoantibody production.

Overall, neutrophils appear to play a central inflammatory role in rheumatoid arthritis, both at the joint level and in other tissues
.
They may play a role
in initiating and maintaining autoantigen exposure through NET formation.

ANCA-associated vasculitis

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a class of autoimmune diseases that cause vascular inflammation
caused by the infiltration and activation of white blood cells in the blood vessel wall.
Unlike other types of vasculitis, ANCA is characterized by systemic inflammation of small blood vessels, usually affecting the lungs and kidneys
.

Similar to SLE, the ability of neutrophils to synthesize NETs is enhanced
in AAV patients.
In addition, as with SLE, the release of NET carrying histones and various matrix metalloproteinases can further damage endothelial cells, leading to a vicious cycle
of inflammation of blood vessels.
In animal studies, disease severity
can be reduced by blocking PAD4-mediated NET formation.
In addition, in clinical trials, LDG levels in peripheral blood correlated
with disease severity.
Overall, there is clear evidence that neutrophils are involved
in the pathogenesis of AAV.

Idiopathic inflammatory myopathy

Idiopathic inflammatory myopathy (IIM) is a heterogeneous autoimmune disease that affects children and adults, usually presenting with muscle weakness but also causing damage
to the skin, joints, lungs, and heart.
Autoantibodies against a variety of molecules, including transporter RNA, melanoma differentiation-associated protein 5 (MDA5), transcriptional mediator 1 (TIF1), or signal recognition particles are commonly found in these patients, resulting in different disease subtypes
.

Recent studies have found that neutrophils are associated
with the pathogenesis of several types of myositis.
Increased levels of various neutrophil-specific molecules, such as neutrophil elastase, PRTN3, and serine proteases
, were detected in the circulating and muscle tissues of IIM patients.
Increased circulating NET levels in patients with specific types of IIM are associated
with specific disease manifestations and severity.
In addition, myositis-specific autoantibodies, including MDA5 antibodies, can directly induce NET formation
in vitro.
Overall, these observations suggest that dysregulated neutrophil pathways may play pathogenic roles in myositis, including induction of autoimmune responses and tissue damage
.

Antiphospholipid antibody syndrome

Antiphospholipid antibody syndrome (APS) is a systemic autoimmune disorder characterized by repeated pregnancy failures, obstetric complications, and a high risk of thrombotic events, associated with the presence of various antiphospholipid antibodies that can affect most organ systems
.
APS can exist alone or overlap significantly with
other autoimmune diseases such as SLE.

Neutrophils in patients with APS have an inflammatory phenotype, ISG upregulation, increased expression of surface adhesion proteins, including P-selectin glycoprotein ligand 1 (PSGL1), which promotes vascular thrombosis by enhancing the binding of neutrophils to endothelial cells of the vascular wall
.
The formation of NET in the vascular system promotes platelet aggregation and the formation of thrombostents, further damaging endothelial cells and promoting upregulation
of adhesion molecules.

Immunotherapy targeting neutrophils

Several therapeutics currently used for a wide range of diseases may directly or indirectly alter neutrophil function
extensively.
These include antibodies that neutralize cytokines such as TNF, IL-17, and G-CSF, as well as other pro-inflammatory molecules that inhibit neutrophil chemotaxis and/or activation (e.
g.
, C5a).

Other drugs that target specific or broader neutrophil function include CXCR2 inhibitors, JAK inhibitors, various cyclooxygenase (COX) inhibitors, including aspirin and nonsteroidal anti-inflammatory drugs, and antimalarial drugs such as hydroxychloroquine
.
These drugs can reduce neutrophil activation, cytokine production, NET formation and migration, and therefore may mediate some of their therapeutic effects
by targeting neutrophils.

Inhibiting the formation of NET is a particularly attractive strategy
.
Histones abundant in NETs have been shown to have a direct pathogenic effect, and blocking H4 could be an attractive new strategy to reduce neutrophil activation and improve cardiovascular status
.
GSDMD is a pore-forming protein and the complex disulfiram targeting GSDMD has shown efficacy in clinical trials targeting neutrophils in patients with novel coronary pneumonia (NCT04485130).

In addition, pan-PAD or PAD4 enzyme inhibitors have successfully improved disease manifestations in some experimental models and have been shown to inhibit the activation
of human neutrophils in vitro.

Reducing the ability of neutrophils to mobilize calcium is another strategy
that may alter PAD activity.
Calcineurin inhibitors, such as cyclosporine A and tacrolimus, have shown beneficial multifactorial effects in SLE, including effects
on neutrophils.
Targeting pathogenic NET products, such as neutrophil elastase, has also shown efficacy in mouse models, but has not been tested
in human trials of autoimmune diseases.
Another strategy for targeting NET products is to use DNases to disrupt and clear DNA structures
.
Lupus mouse models produce fewer autoantibodies and delay mortality
after treatment with DNase.
Unfortunately, however, clinical trials using recombinant DNASE1 have not shown clinical efficacy
in patients with lupus nephritis.
However, different nucleases may be used to further explore this strategy
.

In addition, mitochondrial dysfunction and ROS overproduction are also potential drug targets
.
The use of inhibitors such as N-acetylcysteine to neutralize ROS or inhibit MPO enzyme function can be used to reduce pathogenic NET products
in SLE, AAV, or other autoimmune diseases.
idebenone and mitoQ target mitochondrial dysfunction, reduce NET formation, and are able to mitigate clinical symptoms
in mouse models of lupus.

Overall, there are several strategies that may target the activation and dysregulation of neutrophils and deserve further investigation
.

brief summary

Over the past decade, there has been growing evidence to support the important role of
neutrophils in a variety of inflammatory conditions.
They play a central role
in initiating and maintaining autoimmune diseases by attacking tissues, creating an inflammatory environment, and forming new epitopes.
In addition, it is now increasingly recognized that the function and diversity of neutrophils are more different than previously thought, and the definition of specific neutrophil states in health and disease will be an important area of research
.

In addition, research in neutrophil biology advances our understanding of
how gender, age, and genetic differences affect disease risk and severity.
In addition to providing new tools for diagnosing diseases, interesting biological information
can also be provided.
Future research should focus on further elucidating the role of neutrophils in
specific diseases.
It is believed that future research advances in neutrophil biology will lead to more and more novel therapeutic strategies that will benefit
patients.

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