THE EFFECT OF INDOOR TOTAL SUSPENDED PARTICLES (TSP) ON THE HUMAN UPPER RESPIRATORY SYSTEM: FROM THE MICROBIOME TO HERV METHYLATION

Air pollution is well known for its adverse effects on human health. Among all air contaminants, particulate matter is one of the most studied because of its effect on the cardiovascular and respiratory system. Almost the entire global population is exposed daily to an unhealthy level of these contaminants and most of the exposure comes from the indoor setting where we spend a large portion of our daily time. The link between particulate matter exposure and human disease is still not fully understood, although inflammation is commonly observed after exposure to particulate matter. For that reason, investigating how the upper respiratory microbiome responds to particulate matter exposure is critical. These microorganisms that inhabit our upper airways play a crucial role in the homeostasis of the immune system and defend against external environmental stimuli. Furthermore, inflammation is also correlated with the activation of human endogenous retroviral elements integrated in our genome, the HERV genes. These genes regulate essential processes, and their dysregulation is associated with various inflammatory and immune-related diseases. The aim of this project is to investigate the effects of indoor Total Suspended Particles (TSP) on the upper respiratory microbiome and the methylation levels of HERV genes. Additionally, this study aims to examine whether the microbiome can influence the effect that indoor TSP has on HERV methylation. To achieve this aim, we recruited 34 healthy office workers from the University of Milan and the University of Como. We monitored them and their workplaces for six weeks total, three weeks during the winter and three weeks during the summer. At the end of every week, we collected both environmental and biological samples. The TSP samples were collected using an active filter-based technique. The biological samples included anterior nares and nasopharynx swabs to evaluate the upper respiratory microbiome, and buccal brushes to measure the HERV methylation. The first part of the project analyzed the environmental samples collected in the two seasons. The indoor and outdoor TSP concentrations were calculated gravimetrically. Then, we used the DNA extracted from the TSP collected on cellulose ester filters to characterize the TSP microbiome through whole genome shotgun (WGS) sequencing. We observed differences between the outdoor and the indoor TSP in both concentration and microbiome composition. The differences in microbiome composition we observed were probably influenced by the presence of plants, animals, and human activity in the environment. The second part of the project characterized the anterior nares microbiome in healthy subjects and described the seasonal variation in this microbiome using both 16S rRNA and whole genome shotgun (WGS) sequencing. Specifically, we compared the anterior nares microbiome with the nasopharynx microbiome, and we analyzed how the indoor TSP exposure affected them. We observed significant differences between the two seasons in both microbiome diversity and composition in the anterior nares samples. The winter samples were enriched in Moraxella species (Moraxella catarrhalis and Moraxella nonliquefaciens) and the diversity was lower. The microbiome of the anterior nares was similar in taxa composition to the nasopharynx microbiome. They were both mostly dominated by gram-positive bacteria. However, the nasopharynx microbiome reported a higher diversity, and enrichment in Staphylococcus aureus, while the anterior nares microbiome had a high abundance of Corynebacterium, Cutibacterium acnes, and Staphylococcus epidermidis. In both microbiomes, we observed that the indoor TSP exposure affects commensal gram-positive bacteria such as Corynebacterium accolens and Streptococcus pneumoniae. Consistent with in vitro studies, an increase in Staphylococcus aureus relative abundance was identified in response to TSP exposure. Finally, we observed positive correlations in the abundance of some respiratory bacteria found in both TSP and human samples. The third part of the project examined the impact of indoor TSP exposure and the microbiome on HERV methylation, investigating whether the upper respiratory microbiome can modulate the effect of this pollutant on HERV genes. Indoor TSP exposure alone did not affect the DNA methylation of the HERV genes studied. However, the upper respiratory microbiome was associated with changes in methylation of HERV-K. Furthermore, in our analysis of the interaction between indoor TSP exposure and the microbiome on HERV methylation, we identified that at low abundance of Cutibacterium acnes and Actinomyces naeslundii, indoor TSP exposure is associated with increased methylation of HERV-W. This HERV sequence was previously found to be hypermethylated in response to air pollution exposure. In conclusion, this evidence indicates that indoor TSP exposure directly affects the respiratory microbiome, and consequentially the methylation of HERV-K and HERV-W.