Immunity and inflammation in amyotrophic lateral sclerosis : an epidemiological approach

Abstract

Amyotrophic lateral sclerosis (ALS) is a rare but lethal neurodegenerative disease, characterized by degeneration of both upper and lower motor neurons in the brain and spinal cord. The pathogenesis of ALS is, however, not completely clear. One of the hypothesized mechanisms, neuroinflammation, is considered a pathological hallmark in ALS, as in other neurodegenerative diseases. A crosstalk between neuro- and systemic inflammation has been demonstrated repeatedly in ALS, which provides a foundation to better understand the disease mechanism through studying systemic immune responses. In this thesis, we demonstrated the relationships of previous autoimmune diseases (Study I) and peripheral immune biomarkers (Study II) with the risk of ALS diagnosis, as well as the associations of immune cells measured in blood and cerebrospinal fluid (CSF) with the prognosis of ALS (Study III and Study IV).

In Study I, we comprehensively assessed the associations of 43 autoimmune diseases with the subsequent risk of ALS and further evaluated if familial confounding contributed to these associations. Based on the Swedish National Patient Register (NPR), we conducted a nationwide study including 1) comparison between 3,561 ALS patients diagnosed during 1990-2013 in Sweden and 35,610 population-controls individually matched to the cases by age, sex, and county of birth, and 2) comparison between first-degree relatives of the ALS patients and their controls. We found an overall 47% higher risk of previous diagnosis of any autoimmune disease among ALS patients, compared with controls. An increased risk was also noted for myasthenia gravis (MG), polymyositis or dermatomyositis, Guillain-Barre syndrome (GBS), type 1 diabetes diagnosed younger than 30 years, multiple sclerosis (MS), and hypothyreosis. However, there was no increased risk of autoimmune disease among first-degree relatives of ALS patients, compared with the first-degree relatives of the controls.

In Study II, we described the temporal patterns of serum creatinine and C-reactive protein (CRP) before and after the diagnosis of ALS, MS, and Parkinson’s disease (PD). Through cross-linking the Stockholm CREAtinine Measurements (SCREAM) project to the Swedish regional and national health registers, we performed a population-based case-control study including all newly diagnosed patients with ALS (N=525), MS (N=1,815), and PD (N=3,797) during 2006-2013, as well as their individually matched controls (N=2625 for ALS, N=9,063 for MS, and 18,960 for PD). We observed consistently lower levels of creatinine from two years before diagnosis onward, whereas lower levels of CRP before diagnosis and higher levels after diagnosis, in ALS patients, compared with controls. Among patients with ALS, the creatinine level decreased continuously from one year before diagnosis until two years after diagnosis, whereas CRP level increased from diagnosis until two years after diagnosis. There were, however, no similar patterns noted in MS or PD.

In Study III, we evaluated the correlation between leukocyte subpopulations in blood and the prognosis of ALS. Through the Swedish Motor Neuron Disease (MND) Quality Registry, we conducted a longitudinal cohort study of 288 ALS patients with up to 5 years of follow-up during 2015-2020 in Stockholm, Sweden. The results showed that the counts of peripheral leukocytes, neutrophils, and monocytes increased gradually over time since ALS diagnosis and were positively correlated with disease severity, but not associated with risk of death or disease progression rate. Focusing on the lymphocyte subpopulations, inverse associations were found between the counts of natural killer (NK) cells and proportions of Th2-differentiated CD4+ central memory (CM) T cells with the risk of death, while positive associations were observed between proportions of CD4+ effector memory cells re-expressing CD45RA (EMRA) T cells and CD8+ T cells with the risk of death. None of the lymphocyte subpopulations was correlated with disease severity or progression rate.

In Study IV, we further estimated the association of T cell responses in both the periphery and the intracranial compartment with the prognosis of ALS. We performed a cohort study including 89 newly diagnosed ALS patients in Stockholm, Sweden. Flow cytometry was used to collect information on T cells from blood and CSF of these ALS patients. The results showed that levels of CD4+FOXP3- effector T (Teff) cells were negatively associated with survival, whereas levels of activated regulatory T (aTreg) cells were positively associated with survival. Furthermore, the levels of peripheral Treg cells and composite T cell profile were also found to be associated with disease progression rate. Although these results were comparable between blood and CSF, the markers measured in blood and CSF demonstrated only a moderate correlation with each other. Finally, we performed single-cell RNA sequencing analysis on additional CSF samples collected from five ALS patients and four ALS-free controls and found that the cytotoxic effect of Teff cells noted in the flow cytometric analysis might be due to the elevated cytotoxic gene expressions and clonal expansions.

In conclusion, in Study I, we found a positive association between several autoimmune diseases and ALS, suggesting that chronic inflammation might contribute to the subsequent development of some ALS cases. In Study II, we found that serum creatinine and CRP showed distinct temporal patterns before and after diagnosis of ALS, compared with other neurodegenerative diseases, indicating that there might be specific features of inflammation in ALS compared to other neurodegenerative diseases. In Studies III and IV, our findings suggest that leukocyte populations, primarily lymphocytes, play an important role in ALS prognosis, whereas neutrophils and monocytes primarily reflect functional status. Taken together, these studies add new knowledge to the current understanding of immune responses in the risk and prognosis of ALS and shed light on potential new intervention strategies in the prevention and treatment of ALS.