Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons (1). Clinical phenotypes of ALS exhibit heterogeneity, including muscle atrophy, weakness, spasticity and pyramidal syndrome, progressive dysphasia, and dysarthria. The average incidence of ALS is estimated to be 1.5 to 2 per 100,000 person­years, with increased incidence observed worldwide over the past decade (2, 3). Several pathological events, including excitotoxicity resulting from excessive glutamate levels, oxidative stress damage, protein misfolding, disruption of the blood-brain barrier (BBB), and reduced energy metabolism, have been hypothesized to contribute to the neurodegenerative process in ALS. TDP-43 is an RNA-binding protein mainly found in the nucleus and plays an important role in RNA cleavage, transport, translation, and stability (4, 5). 95% of ALS patients exhibit abnormal localization of TDP43 in the cytoplasm, which leads to serious consequences such as miscleavage of RNA in the cytoplasm, decreased translation efficiency, and loss of stability, which may be the main cause in ALS (with the exception of SOD1 mutations) (4, 5). However, the exact pathogenesis of ALS remains unclear to date.

Compelling evidence has revealed dysregulated peripheral immunity as a pathological factor of ALS. Whereas the central nervous system (CNS) has traditionally been considered an immune-privileged tissue due to the selective permeability of the blood-brain barrier (BBB), however, a growing number of studies have revealed functional crosstalk between peripheral immune cells and the CNS (6). The peripheral immune system comprises the innate immune system, with neutrophils as its core components, and the adaptive immune system, with lymphocytes as its core components. Different subpopulations of immune cells display different functions in the progression of ALS. For instance, Tregs have shown a protective role (7), while inflammatory monocytes and CD8⁺ T cells may be destructive (8–10). As the key component of innate immunity, neutrophils act as front-line defenders against pathogens by being stimulated and recruited to affected sites through chemotaxis and engaging in degranulation to eliminate pathogens. However, excessive neutrophil activity can result in significant collateral tissue damage. In ALS, researchers mainly focus on monocytes or T cells previously, while potential effects and mechanisms of neutrophils in the pathogenesis of ALS remain neglected. Modulation of peripheral immune cells has been shown to delay the progression of ALS in mouse models (11, 12), offering a feasible and less invasive intervention strategy than modulation of immune cells in CNS and making peripheral immune cells an attractive therapeutic target in ALS treatment (13–15). For example, pharmacological treatment with masitinib delayed peripheral motor pathway degeneration (axonal pathology, secondary demyelination, and the loss of type 2B myofibers) by preventing mast cell infiltration (11). Garofalo et al. found antibody for the α4 integrin, Natalizumab, prolonged the survival time of ALS mice by blocking the extravasation of immune cells in the central nervous system (12). Thereby, searching for precise immunotherapies targeting specific subsets of peripheral immune cells holds great promise in ameliorating immune-mediated pathological deterioration in ALS, and has great benefits in treating ALS. This review aims to summarize the role of neutrophils in ALS in terms of their origin, activation, and functions in ALS, with a specific emphasis on the impacts of the neutrophil granules on ALS, shedding light on the overlooked contributions of neutrophils in ALS and providing a theoretical basis for potential precise immunotherapy.

In recent years, the potential link between circulating neutrophil counts and ALS disease progression has been proposed ( Table 1 ). In 2017, Murdock et al. investigated the longitudinal association between changes in peripheral immune markers and ALS disease progression. They found that an early elevation of neutrophils was associated with accelerated disease progression reflected by the change in the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) (17). However, Cui et al. revealed a different conclusion. They found that neutrophil counts increased gradually over time following ALS diagnosis and were negatively correlated with ALSFRS-R score but didn’t find an association between neutrophil changes and ALS progression (9). In 2020, increased expression of CD16 on the surface of neutrophils was observed, which is considered a marker of oxidative stress and phagocytic activity of neutrophils (25) that is associated with ALS disease progression, indicating a highly-activated state of neutrophils in rapidly progressing ALS (20). In 2021, Murdock et al. conducted a prospective cohort study to explore the association between baseline neutrophil counts and ALS survival. The results showed that higher baseline neutrophil counts are associated with reduced survival in ALS. Furthermore, the effect appeared to be sex-dependent, with a more pronounced hazard ratio in females (19), suggesting a potential role of sex hormones in neutrophil activity. However, evidence from mendelian randomization analysis showed that a genetically determined increased neutrophil count was associated with a reduced risk of ALS (95% CI: 0.858-1.000, P: 0.049) (18). The controversial conclusions between mendelian randomization and observational studies may be attributed to two factors: firstly, the effect of neutrophils on ALS in mendelian randomization is weak and could be influenced by confounding biases; secondly, previous observational studies mainly focused on the link between neutrophils and ALS after diagnosis, while mendelian randomization analysis revealed the causal relationships between neutrophils and ALS incidence. These inconsistent findings also suggest that neutrophils may play both detrimental and protective roles for neurons at different stages of diseases. To date, no positive immunosuppressive drugs for ALS patients have been identified in clinical trials (26). More attention should be paid to applying precise immunotherapy in different disease stages and specific subpopulations.

The neutrophil-derived metric, neutrophil to lymphocyte ratio (NLR), which reflects the balance of peripheral innate and adaptive immunity, has also been indicated as a promising biomarker for ALS disease progression and prognosis. As early as 2009, low-grade systemic inflammation, characterized by an elevated wide-range C-reactive protein (wrCRP), fibrinogen, erythrocyte sedimentation rate (ESR), and NLR, has been proposed as a hallmark of ALS. This correlation was consistently observed in repeated blood draws over time (21). In 2020, research conducted in South Korea divided ALS patients based on their baseline NLR into three groups and revealed that the patients with a high NLR had faster disease progression and shorter survival (22). These findings were subsequently confirmed by a single-center cohort from China and a multicenter cross-sectional cohort study from Italy (23, 24), suggesting that the balance between innate and adaptive immunity plays an important role in ALS progression.

Neutrophils constitute 50-70% of total leukocytes, making them the most abundant leukocytes in circulation (27). They are polymorphonuclear leukocytes originating from myeloid precursors in the bone marrow (BM), characterized by lobulated or rod-shaped nuclei and large cytoplasm of neutral granules (28). These granules primarily consist of lysosomes containing rich enzymes, such as myeloperoxidase (MPO) and neutrophil elastase (NE), which play crucial roles in the phagocytic functions of neutrophils.

Under physiological conditions, neutrophils are generated and differentiated from stem cells to neutrophil precursors in the BM. Upon stimulation, neutrophil precursors can be mobilized from BM into the circulation (29), which is regulated by the interplay between the C-X-C chemokine receptors (CXCRs) family and C-X-C chemokine ligands (CXCL) family (29).

Neutrophils remain in the BM when their surface receptor CXCR4 binds to CXCL12, which is produced by hematopoietic stem cells and BM stromal cells (BMSC). Granulocyte colony-stimulating factor (G-CSF) serves as a major regulator of neutrophil homeostasis (30, 31). When external stimulation occurs, G-CSF can shift CXCR4 on neutrophils to CXCR2, thereby inducing neutrophil mobilization and release.

As the principal regulator of neutrophil biology, G-CSF, a 19.6kd hematopoietic cytokine, has garnered significant attention in the field of ALS. However, clinical and pre-clinical studies yielded inconsistent conclusions regarding the effect of neutrophils increment. Clinical observations have suggested that higher neutrophil level is associated with accelerated ALS disease progression and higher mortality rate (9, 17, 19, 22). Assuming that G-CSF-induced neutrophilia is detrimental to ALS patients, increased expression of G-CSF would be expected to accelerate the disease progression. However, the results of pre-clinical studies do not support the assumption. Animal studies have demonstrated that G-CSF has neuroprotective effects by suppressing endoplasmic reticulum stress and inhibiting pro-apoptotic proteins (32–34). In SOD1^G93A transgenic mice, continuous subcutaneous delivery or CNS-targeted overexpression of G-CSF can inhibit the apoptosis of motor neurons (35). The inconsistency may arise from two aspects. First, the effects of G-CSF were complex, with dual roles in both the hematopoietic and nervous systems. Systemic delivery of G-CSF can stimulate the proliferation and differentiation of white blood cells, potentially playing a detrimental role in ALS progression. However, G-CSF receptors are highly expressed in motor neurons (35, 36), and their effect on motor neurons is primarily responsible for their neuroprotective role. As a result, researchers are exploring methods to enhance the central neuroprotective effects of G-CSF while minimizing its peripheral effects. In 2011, Henriques et al. found that intraspinal injection of G-CSF AAV significantly delayed the onset of hindlimb paralysis and prolonged 10% survival in SOD1^G93A mice (37), which is improved compared with systemic injections. Second, G-CSF mobilizes neutrophil production but also modifies their activation. It is possible that the action of G-CSF is due to enhancing beneficial/pro-resolution processes in neutrophils (29). Neutrophil infiltration without degranulation may stimulate efferocytosis and repair processes (29).

Although G-CSF treatment has shown potential neurotrophic effects in both pre-clinical and clinical studies of ALS, it is important to consider its significant systemic effects, particularly its impact on neutrophilia. Researchers are currently exploring strategies to mitigate potential peripheral effects of G-CSF by administering it intrathecally in ALS, however, the harmful effects due to its invasive nature should not be ignored.

As key effector of innate immunity, neutrophils trigger host tissue damages through three major pathways ( Figure 1 ): (1) Phagocytosis: neutrophils can directly phagocytose foreign pathogens and self-components; (2) Degranulation: neutrophils secrete cytotoxic particles during maturation affecting the immune response. The main components of these particles are enzymes, such as MPO, NE, lactoferrin, and metalloproteinases (MMPs) (38); (3) Activated neutrophils can form neutrophil extracellular traps (NETs), which are composed of chromatin components, MPO and NE attached DNA fibers. (4) Neutrophils may crosstalk with other cells, such as microglia and astrocytes. The exact roles of neutrophils playing in ALS pathology and the underlying mechanisms are still unclear. In this section, we describe the three major roles of neutrophils in ALS and propose potential mechanisms by which they contribute to the disease.

Research on the role of neutrophils in ALS is still in its early stages, and there are many intriguing areas yet to be explored. Firstly, in other neurodegenerative disease animal models, such as AD and PD, neutrophils have been shown to infiltrate into the brain due to the disruption of BBB (47, 127, 128). Although the presence of neutrophils in the spinal cord and NMJ of SOD^G93A mice is documented, their ability to cross the BBB at the early stage of ALS remains uninvestigated. Secondly, neutrophil depletion may improve the cognitive function of AD mice (47), while its role in ALS is not yet known. Thirdly, neutrophils exhibit diverse phenotypes, including a subpopulation known as low-density neutrophils (LDN), which has been reported in a variety of disease conditions (129–131). Investigating the potential role of neutrophil subpopulations in ALS is a promising direction. Lastly, ALS is a highly heterogeneous disease, displaying different levels of innate immunity. Establishing an immune stratification based on neutrophils for ALS patients could pave the way for more precise immunotherapy tailored to distinct patient groups. Further research in these areas will advance our understanding of ALS and the contribution of neutrophils to its pathogenesis and progression.

DF and WC contributed to conception and design of the review. WC wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. All authors contributed to the article and approved the submitted version.