Early-life gut fungal dysbiosis in pediatric asthma development.

The microbial community that populates the lower gastrointestinal tract, gut microbiota, is a key player governing human health and disease. Perturbations of the microbiome at the earliest time in life during maximal physiological development have been associated with a risk of non-communicable diseases, such as allergies and asthma. The prevalence of asthma, a chronic disease of the airways, has been steadily increasing in industrialised countries since the 1950s. Factors responsible for the surging asthma rates are not fully understood, but in addition to host genetics, environmental factors appear to play a crucial role. Current research indicates that gut microbes aid in developing and educating the immune system early in life. Disruptions of the gut microbiota caused, for example, by antibiotic use can result in long-term health problems, including asthma. Children are typically at school age when diagnosed with asthma, which has no cure and must be managed through medication. If the disease's origin was recognised as a disruption of the gut microbiota in the earliest stages of life, understanding of the host-microbe interaction in early life could guide the development of preventive asthma therapeutics. We summarised current evidence on host-microbiome intestinal interactions during early life in a recent review (Pettersen VK & Arrieta MC. Current Opinion in Allergy and Clinical Immunology: April 2020 - Volume 20 - Issue 2 - p 138-148). This project was a part of the FRIPRO mobility grant program, and it was an interdisciplinary collaboration between clinical microbiologists, pediatricians, and intestinal microbiome scientists at the Universities of Tromsø (UiT The Arctic University of Norway) and Calgary (UofC, Canada). During the project, we characterised host-fungi interactions relevant to asthma pathogenesis by using fungal species that have been linked to early-life antibiotic use and increased risk of asthma. A critical part of the work was gnotobiotic (i.e., having defined microbiota) animal model of asthma on which we studied the effects of fungal and bacterial gut species on the host immune and physiological functions. Our team completed an animal study that characterised the importance of fungi in the intestine and how they can modulate inflammatory response in the airways (Bernardes, Pettersen, et al. Intestinal fungi are causally implicated in microbiome assembly and immune development in mice. 2020 Nat Commun 11, 2577). In this animal model, we used the so-called "germ-free" mice. The mice were given defined amounts of various bacteria and/or fungi that are common constituents of the human intestinal flora but have previously been shown to be overrepresented in children who have later developed asthma. A group of mice that have been colonised with both bacteria and fungi was also treated with antimicrobial drugs that act against either fungi or bacteria. By modulating the intestinal microbiota composition and then inducing experimental asthma in the mice, we obtained different immunological characteristics in the mice colonised with specific groups of bacteria or fungi. This approach has revealed previously unknown interactions between the fungi and the host that may help understand asthma development. To define mechanisms by which gut fungi might modulate the host immune system, we used high-throughput profiling of small metabolites. By analysing stool samples of the gnotobiotic mice, we identified that gut fungi are unlikely to use secreted metabolites to modulate the host immune system. Therefore we decided to analyse proteins in the stool samples and found that fungi might be using cell-derived vesicles for the host cells modulation ( Pettersen VK, Dufour A, Arrieta MC. Metaproteomic Profiling of Fungal Gut Colonization in Gnotobiotic Mice. Animal Microbiome 2022). Our results suggest that an increased abundance of specific gut fungal species in infancy may impact the developing intracellular balance of epithelial and immune cells. Our work sets the stage for future studies that will explore the details of molecular mechanisms by which gut fungi modulate host physiology. Although the project period has ended, the collaboration between the former project manager Dr Pettersen, now an associate professor at UiT, and Dr Marie-Claire Arrieta from UofC continues. We are currently exploring a hypothesis that fungal extracellular vesicles have immunomodulating properties and might influence the host cells through direct contact.

The results of this project revealed previously unknown interactions between the fungi and the host that may help understand asthma development. Our results also suggest that an increased abundance of specific gut fungal species in infancy may impact the developing intracellular balance of epithelial and immune cells. Our work shows how antibiotic use in infancy, which promotes fungal overgrowth, has unwanted side effects that negatively impact later health.

There is mounting evidence that early-life gut microbiota guides immune development, and that when the balance between gut-residing microbes is disrupted, diseases may follow. One of the diseases associated with antibiotic-driven microbial dysbiosis is asthma. We have provided robust evidence that gastrointestinal bacterial species are causally implicated in early-life immune dysregulation that leads to asthma. Recently, we detected significant alterations in children´s fungal microbiota that were associated with antibiotic use and with asthma risk. We hypothesize that antibiotic-driven dysbiosis of the gut fungal microbiota affects immune development and consequently predisposition to asthma. We will test this hypothesis by analysing changes in infants´ fecal microbiomes that result from common practice antibiotic therapy in the Alberta Children´s Hospital, Canada. After the initial description of the pre- and post-antibiotic microbiomes by amplicon-based sequencing, we will investigate the effects of fungal species that become overrepresented after the antibiotic treatment in a murine model of asthma by immunological and histological techniques. With this translational approach, we aim to describe currently unknown immune interactions between the fungi and the host that might have implications for asthma development. A key challenge is to identify features of the gut fungal microbiota that have the potential to influence the host immune system. We will address it by systematic profiling of the murine host fecal microbiome. Modulation of the fungal species abundance in the mice gut through the use of an antifungal treatment will facilitate detection of quantitatively changed fungal enzymatic pathways and metabolites. By associating this information with the experimental asthma model immunological characterization, we aim to describe metabolic and proteomic traits of the fungal species that can potentially assist in the prediction of asthma development.