Breathe (in the air) : pulmonary immunology in multiple sclerosis

Abstract

Multiple Sclerosis (MS) is a demyelinating disease of the central nervous system, with etiology still unknown. MS is thought to arise from a complex interplay between genetic and environmental factors. One of the most well established environmental risk factor is smoking, which confers a striking increase in risk of developing MS and especially in interaction with the risk allele HLA-DRB1*15 and absence of the protective allele HLA-A*02.

The major part of this thesis is focused on investigating the involvement of the pulmonary immune system in MS, and further to uncover underlying smoking associated changes that could elucidate on the role of smoking as a risk factor in MS. To characterize the lung immune cells, bronchoalveolar lavage (BAL) cells were obtained by bronchoscopy, from healthy volunteers and MS-patients, smokers and non-smokers. In project I we provide an initial characterization of our study cohort. We could observe that smokers carrying the MS specific risk allele HLA-DRB1*15 did not show a smoking-associated increase in macrophages defined in non-carriers. Smokers showed higher frequency of proliferating T-cells, while non-smoking MS-patients had increased levels of preformed CD40L in CD4+ T-cells. We could further provide a more in-depth characterization of pulmonary T-cells in MS-patients and smokers, in Project III. The majority of CD4+T-cells in both healthy and MS patients showed a tissue resident memory phenotype, characterized by expression of CD69 and CD44, while also expressing both CXCR3 and CCR6. Cells from healthy smokers showed an increased proliferative capacity and we also observed a significantly higher frequency of regulatory T-cells in the lungs of both healthy smokers and MS-patients compared to healthy non-smokers. When investigating the migratory profile of lung T-cells based on integrins VLA-4 and LFA-1, both implicated in MS pathogenesis, we found no upregulation of these in MS patients compared to healthy.

In recent years, it has been suggested that dysbiosis of the commensal microbiome in the gut is involved in the pathogenies of MS. The lungs also host a unique commensal microbiota, which recently was shown to be dysregulated in the autoimmune disease Rheumatoid Arthritis and pulmonary Sarcoidosis. In Project IV we investigated if the microbiota in the lungs of MS patients also show dysbiosis. We found that the microbial composition in the lungs of MS patients differed considerably compared to healthy controls, with increased richness and diversity. We could further report that MS patients also had altered expression and presence of the antimicrobial peptide human beta defensin-1 (hBD1) in the lungs.

In Project II we developed a novel method, called Small-seq, to study small RNAs, such as microRNAs (miRNA) from a scarce source of starting material; a single cell. Previously methods required large quantities of sample material in order to investigate small RNAs, which often can be a limitation to obtain in clinical samples, as well as average out biological variability and heterogeneity within populations. With Small-seq we are were able to capture different types of small RNAs from single cells, such as miRNA, snoRNA and tsRNA. Captured miRNAs revealed cellular heterogeneity in primed hESC, as well as being able to cluster and separate different cell types. The method implemented a masking strategy to efficiently limit capture of the highly abundant 5.8S rRNA, and incorporation of a unique molecular identifier allowed for molecular quantification of the detected small RNAs.

The work provided in this thesis concludes that the pulmonary immune milieu is altered in MS patients, thereby presenting the lungs as an organ of interest for further investigation into the pathology and potential therapeutic opportunities in MS. The described changes in immune cell composition between smokers carrying the MS risk allele HLA-DRB1*15 and noncarriers, could further shed light upon the mechanisms behind the impact of smoking as a risk factor for disease and in exacerbating MS. Herein we further provide the development of a novel technique to capture and investigate small RNA, such as miRNAs in single cells