Neutrophils: underestimated players in the pathogenesis of multiple sclerosis

Abstract

Multiple sclerosis (MS) is a chronic, inflammatory autoimmune disease that is characterized by immunemediated damage to the central nervous system (CNS). In MS, the immune system mistakenly attacks the myelin sheath, the protective covering of nerve fibers. This damage disrupts the transmission of action potentials, leading to a wide range of symptoms, including vision problems, muscle weakness, numbness, and fatigue. Eventually, this ongoing inflammatory response leads to progressive neurological dysfunction. Despite significant research efforts, MS remains an incurable disease, and its exact pathogenesis is not fully understood. Currently available therapies focus primarily on slowing disease progression and managing symptoms rather than halting neurodegeneration or reversing the established damage. Therefore, a constant search is ongoing to identify new biomarkers and possible targets. While it is clear that an interplay between genetic predisposition, environmental factors, and immune dysregulation are involved, the exact mechanisms driving its onset and progression remain elusive. Historically, research has emphasized the roles of T and B cells in MS pathology, but recent studies have highlighted the importance of innate immune cells, including neutrophils, as well. By studying these rather unexpected, possible effector cells and their behavior in MS context, we aim to fill the gaps in our knowledge and gain valuable insights into the disease progression, possibly identifying potential therapeutic targets. Neutrophils, the most abundant leukocytes in the human blood, are often neglected in the context of autoimmunity. These innate immune cells develop in the bone marrow, from which they are released in the circulation where they form the first-line defense mechanism by fighting pathogens. They can be recruited to infected or inflamed tissues, following different stimuli. Once activated in the bloodstream or upon arrival at the site of infection/inflammation, neutrophils can elicit a variety of effector mechanisms. These include phagocytosis of pathogens or particles, degranulation leading to the release of pre-stored antimicrobial peptides and enzymes, generation of reactive oxygen species (ROS), production of cytokines and chemokines, and release of neutrophil extracellular traps (NETs), which are strands of extracellular DNA together with citrullinated histones and enzymes to trap and kill pathogens. When these cells are abnormally activated, these functions can induce tissue damage or contribute to excessive, chronic inflammation, as seen in multiple autoimmune diseases. Based on animal models of MS and human patient samples, it has already been shown that neutrophils might contribute to the onset and inflammatory progression of MS. In this thesis research, we therefore investigated the possible contribution of human neutrophils to MS, by investigating various aspects of the disease. We first explored the possible differences in the phenotype of neutrophils from the circulation of MS patients and healthy donors. Then, we investigated the effect of new MS therapeutics on the function of human neutrophils. Finally, we aimed to clarify the role of neutrophils in the uptake of myelin, and the related mechanisms and consequences. To begin, we explored the phenotype of circulating neutrophils from relapsing-remitting MS (RRMS) patients and healthy donors, using multicolor flow cytometry and single cell multiomics. Here we detected an increased expression of the surface protein CD62L on MS neutrophils compared to healthy controls. This L-selectin is responsible for cell adhesion to the endothelium and an increased expression is associated with newly released neutrophils from the bone marrow. Additionally, we observed multiple small (+/- 3% of all neutrophils) subclusters that were only present in MS neutrophils and were characterized with different combinations of surface proteins. Using an unbiased approach, we investigated these possible differences more in-depth with single cell cellular indexing of transcriptomes and epitopes (CITE) sequencing. Here,we compared peripheral blood neutrophils isolated from untreated RRMS patients (n=3), natalizumab (Tysabri®)-treated patients (n=4) and age- and gender matched healthy donors (n=6). We identified multiple subclusters or phenotypic states within the large, general neutrophil population, all characterized by different transcriptional profiles. Furthermore, we detected some upregulated genes in untreated MS patients, that can potentially serve as possible biomarkers or targets. In the second part, we demonstrated that the use of an appropriate purification method to isolate neutrophils from peripheral blood is crucial for further in vitro experiments and phenotypical studies. We found that conventional density-gradient centrifugation artificially activated neutrophils at baseline, making it impossible to additionally stimulate them in certain assays (ROS and NET production, phagocytosis, polarization and degranulation). This is in contrast to immunomagnetic isolation, which kept neutrophils in a more quiescent state, resembling in vivo neutrophils. After gathering this knowledge, we next evaluated these different effector functions of neutrophils, following treatment with a new, upcoming class of MS drugs, namely the inhibitors of Bruton’s tyrosine kinase (BTK). This intracellular signaling molecule is known for its importance in B cell functioning (e.g., antibody production) but was also proved to be crucial for neutrophil biology. Three BTK inhibitors (evobrutinib, fenebrutinib and tolebrutinib) are currently in phase III clinical trial for the treatment of MS, which we used to treat neutrophils in vitro and investigate how they possibly affect neutrophil functions. We found that pretreating human neutrophils with BTK inhibitors reduced their activation and actin polymerization by the chemokine CXCL8 and the bacterial peptide fMLF. Furthermore, in vitro migration toward CXCL8 and fMLF in a Boyden chamber assay was diminished following BTK inhibitor treatment. Both the formation of ROS and NETs were blocked upon treatment with BTK inhibitors and the production of CXCL8 and IL-1β was decreased as well. In vivo, the recruitment of neutrophils toward intraperitoneallyinjected CXCL1 was not affected, however the locally-attracted neutrophils were less capable to produce ROS. Considering these results, neutrophil functionality should be closely monitored in patients receiving BTK inhibitors, as this might affect their immune responses towards concomitant infections. Furthermore, these findings highlight that, beyond targeting pathogenic B cells, BTK inhibitors can also attenuate specific neutrophil responses. This reveals an additional mechanism of action, further expanding the therapeutic potential of these inhibitors. Finally, while macrophages and microglia are well-known contributors to myelin degradation and myelin debris clearance in CNS diseases, the role of neutrophils in these processes remains unknown. Therefore, we investigated the possibility of human neutrophils to engulf myelin (debris) together with the mechanisms behind this, and the subsequent phenotypic changes. We demonstrated that neutrophils can phagocytose myelin debris, reaching a peak between 3 and 6 hours. This was shown to be primarily dependent on complement opsonization and the activation of complement receptor 3. Extended exposure (12-24 hours) to high myelin concentrations (100 µg/mL) induced a pro-inflammatory phenotype in neutrophils, characterized by elevated ROS production, NET release and formation of inflammatory mediators such as CXCL8 and CCL3. Transcriptomic analysis further revealed a dose-dependent inflammatory signature, characterized by upregulation of CXCL8 alongside a decreased ARG1 expression, indicating a shift toward a pro-inflammatory neutrophil state. These findings uncover novel aspects of neutrophil involvement in myelin degradation and clearance, emphasizing their potential contribution to MS pathology, and other demyelinating diseases. Altogether, this PhD research highlighted the critical involvement of human neutrophils in various aspects of MS pathogenesis, offering valuable insights into their potential contribution to disease progression. This work lays a foundation for further investigations in patient samples or animal models, in an attempt to bridge knowledge gaps and contribute to the development of new therapeutic options for all MS patients.