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Role of intestinal fungal communities in arthritic pathology

Periodic Reporting for period 1 - FungArth (Role of intestinal fungal communities in arthritic pathology)

Reporting period: 2017-03-01 to 2019-02-28

The human body is in constant interaction with a complex group of micro-organisms like bacteria, viruses and fungi which populate various body surfaces like our skin, lungs and gut. These organisms generally do not pose a threat to the host but in contrary have many beneficial functions like stimulating the correct development of our immune system. Until now, research has mainly focused on bacteria, while fungi have largely been overlooked. Enteric fungi represent a significant part of the intestinal microbiota, but their role in human health and disease is barely studied. Spondyloarthritis (SpA) is a common immune pathology that causes severe spine and joint inflammation. The lab of Prof. Dirk Elewaut was the first to show a mechanistic link between spondyloarthritis and intestinal inflammation. Interestingly, anti-fungal immune responses require Th17 immune activation, an immune arm commonly hyperactivated in arthritic disease. We, therefore, hypothesize that aberrant antifungal immune responses may be contributing to arthritic pathology. Alternatively, homeostatic fungal-host interactions may be important for both mucosal and systemic immune homeostasis. With the FungArth project, I studied in detail the contribution of naturally occurring intestinal fungi to health and SpA development in a multi-disciplinary manner, using elegant genetic mouse models of disease, state-of-the-art technologies and clinical samples from SpA patients.

The Marie-Curie fellowship is now ended but I am continuing the project in the lab, finishing some experiments and testing new hypotheses. Here are the main findings and the continuation of the project:

I evidenced a role of fungal communities in the SpA disease development. Indeed, antimycotic treatment induced a higher clinical scoring all along the duration of the treatment in mouse models. These results are confirmed at the joint level, where the inflammation is worsened with the antimycotic treatment. While antibiotic treatments have a protective effect in gut inflammation, no effect on arthritis has been observed.
These results suggest a protective effect of fungi, potentially slowing down disease development. The antimycotic treatment targeted specific yeast species, those yeast are currently inoculated in germfree mice and conventional disease models in order to confirm the hypothesis.

I also worked with samples from SpA patients (biopsies and feces): extracting both DNA and RNA in order to profile bacterial and fungal communities but also evaluate the gene expression. The biopsies extractions and sequencing are achieved (385 samples in total) and I am currently analyzing the results.
1. Experiments with mice models:
Several mice experiments have been conducted in order to modulate both bacterial and fungal gut communities and decipher their role in SpA disease development/progression.

Mice models-Experiment 1:
I treated the TNFΔARE mice (a model developing spontaneously arthritis and ileitis) with a large spectrum of antibiotics and an antimycotic molecule.
Results: the antibiotic treatment reduced drastically ileitis but not the arthritis phenotype. The antimycotic treatment increased drastically arthritis but not ileitis phenotype. The profiling of both bacterial and fungal communities from mice feces is ongoing.
Follow-up: This experiment was repeated to validate the results (Experiment-1b).
Because the effect of the antimycotic molecule was specific to few yeast species, we are currently inoculating TNFΔARE mice and germfree mice with these species to test their activity in both disease development and immune system activation.

Mice models-Experiment 2:
TNFΔARE mice have been treated with two specific antibiotics, each targeting few bacterial species in order to modulate more finely the bacterial communities.
Results: One of the treatments increased drastically the arthritis phenotype. The profiling of both bacterial and fungal communities from mice feces is ongoing.
Follow-up: The bacterial species identified in this analysis will be inoculated in TNFΔARE mice and germfree mice to test their activity in both disease development and immune system activation.

Mice models-Experiment 3:
I characterized the phenotype at steady-state of a new mouse model in the lab: SKG mice (a model developing arthritis and psoriasis).

Mice models-Experiment 4:
SKG mice are currently treated with a large spectrum of antibiotics and an antimycotic molecule.


2. Analysis of SpA patient samples:
I am analyzing patient samples from the Be-GIANT cohort (Gent Inflammatory Arthritis and spondylitis cohort). This cohort is composed of new-onset SpA patients receiving an extensive baseline investigation including X-ray analysis, ileocolonoscopy, gut and ilial biopsies, stool and serum isolation. Patients are followed every 6 months systemically for at least 10 years (started in 2010). The objective was to deeply profile both bacterial and fungal communities comparing SpA patients and healthy controls. Both DNA and RNA have been extracted from 385 gut biopsies. The preliminary analysis revealed a strong dysbiosis between SpA patients and healthy controls. The inflammatory status of both colon and ileum influences also the bacterial communities. Regarding the diagnosis, we show for the first time a dysbiosis between patients classified with an Axial (spine involvement) disease compare to Peripheral (joint involvement) disease’s manifestation.

In a previous study, the team analyzed 27 biopsies of SpA patients (only the bacterial communities). They evidenced a correlation between the abundance of the bacterial genus Dialister and the disease activity. The preliminary analysis of the extensive sequencing confirmed a link with the Dialister bacteria (as a marker of the disease or as an actor in the disease progression).

I also performed a genomic analysis which revealed specific pathways enriched in this bacterial genus compare to the other bacteria present in the gut. We are currently conducting inoculation of germfree mice with Dialister bacteria and also testing functionally the involvement of the specific pathways identified in the disease development.
The Marie-Curie fellowship is now ended but I am continuing the project in the lab, finishing some experiments and testing new hypotheses. Here are the main findings and the continuation of the project:

I evidenced a role of fungal communities in the SpA disease development. Indeed, antimycotic treatment induced a higher clinical scoring all along the duration of the treatment in mouse models. These results are confirmed at the joint level, where the inflammation is worsened with the antimycotic treatment. While antibiotic treatments have a protective effect in gut inflammation, no effect on arthritis has been observed.
These results suggest a protective effect of fungi, potentially slowing down disease development. The antimycotic treatment targeted specific yeast species, those yeast are currently inoculated in germfree mice and conventional disease models in order to confirm the hypothesis.

I also worked with samples from SpA patients (biopsies and feces): extracting both DNA and RNA in order to profile bacterial and fungal communities but also evaluate the gene expression. The biopsies extractions and sequencing are achieved (385 samples in total) and I am currently analyzing the results.
Effect of antiobiotic and antimycotic treatment in SpA disease model mice.