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Mucosal Tolerance and Allergic Predisposition: Does it all start in the gut?

Periodic Reporting for period 2 - ALLERGUT (Mucosal Tolerance and Allergic Predisposition: Does it all start in the gut?)

Reporting period: 2019-01-01 to 2020-06-30

Worldwide, a steady increase in allergic diseases can be observed within the last decades. This rapid increase cannot be explained by genetic reasons as much more time would be required when considering time span of human generations. Thus, alterations of our environment induced by changes in life style conditions, mode of birth, food intake, use of medication such as antibiotics, etc. is likely a contributing factor to the recent rise in allergic diseases. Interestingly, most of the currently discussed changes in life style factors affect directly or indirectly the composition of our microbiome (including bacteria, fungi and viruses). Our microbiome virtually colonizes all our body surfaces and is part of any healthy individual. Currently it is believed that alterations of this microbiome is one of the most likeliest factors leading to a raise in allergic diseases. This concept is supported by epidemiological studies demonstrating that growing up in a rural area or farm environment associated with traditional farming (in contrast to industrial farming) is indeed a major protective factor to prevent e.g. allergic asthma. Thus, the microbiome is very likely one of the key determinants affecting susceptibility to allergic diseases but underlying mechanisms remain poorly understood.
In this project, we hypothesize that alterations in the immune system due to differences in microbial compositions is a common and underlying cause for the above described observations. In particular, the immune system of the gastrointestinal tract where microbial burden is highest and were a big surface area allows for various interactions between the host and its microbial symbionts is of particular importance. Based on our previous results based on in vivo animal models we hypothesize that a specialized population of regulatory T cells (so-called type 3 Tregs) co-expressing the transcription factors Foxp3 and RORgt is a central player in this process for the following reasons: a) Type 3 Tregs develop after weaning and reside within the intestinal tract b) Type 3 Tregs have the highest frequencies where microbial exposure is highest (colon>small intestine>secondary lymphoid tissues associated to the intestinal tract>spleen/distal lymph nodes>thymus) c) Type 3 Tregs are absent in germfree or broad spectrum antibiotic-treated animals and d) genetic ablation of Type 3 Tregs leads to exaggerated type 2 immune responses also found in germfree conditions. Assessing the microbial habitat within the intestinal tract is still a very difficult task because interindividual variety is high and complex interdependent interactions between hundreds of microbial species cannot be easily studied in vitro because many of these species are not yet cultivable. Therefore, type 3 Tregs may serve as a faithful readout for inefficient induction of intestinal homeostasis which can affect both local and systemic diseases associated to a loss of immunological tolerance.
In this project we aim to identify thus both microbial and host-intrinsic pathways that positively or negatively regulate the induction and maintenance of type 3 Tregs. We anticipate that this knowledge can serve as a useful tool to diagnose and in the future possibly also prevent allergic and other diseases associated to a loss of immunological tolerance to foreign antigens.
Since the beginning of the project a number of projects have been started to study the above-mentioned hypothesis. A number of recent high-impact publications from other research groups worldwide support the core concept of the ALLERGUT project making it highly competitive.
So far, we have been able to reveal a key role for dendritic cells in regulating the fate decisions for the differentiation of Type 3 Tregs from naïve T cells. Notably, dendritic cells with constitutively activated CD40 signaling fail to induced type 3 Tregs and induced efficient oral tolerance (assessed in a collaboration). In line with this result, we found recently that genetic ablation of alternative NF-kB signaling in dendritic cells leads to an inefficient induction of type 3 Tregs in vivo suggesting again impaired oral tolerance. Interestingly, we observed a systemic accumulation of ‘conventional’ Tregs across all examined organs. These Tregs seem to have a tissue Treg phenotype characterized by high Gata3 and interleukin 33 receptor expression. Nevertheless, we did not find any hint for IL-33 being a driver of Treg accumulation in vivo. As the tissue Treg phenotype suggested self-reactivity, we checked whether these mice are protected from autoimmune diseases. Indeed, ablation of RelB in dendritic cells almost completely protects mice from a murine model of multiple sclerosis (MS) in a Treg-dependent manner. These data have been published recently in a peer reviewed journal (Andreas et al., J Immunol. 2019).
As high Gata3 expression in Tregs driven by enhanced interleukin 4 receptor signaling has been proposed to render Tregs pathological cells in food allergy we will now address whether mice lacking RelB in dendritic cells show more severe food allergy including life-threatening anaphylactic reactions. Furthermore, we are currently addressing the underlying mechanisms for the observed changes on T cells in the absence of RelB in dendritic cells.
We currently follow several lines of approaches proposed in the ALLERGUT project. For instance, we have managed to establish TCR sequencing of type 3 Tregs isolated from the intestinal tract of wild type mice at the single cell level. This type of analysis will reveal whether and how overlaps in the TCR repertoire occur and how this depends on the early life colonization with a complex microbiome. Importantly, this is highly relevant for the hypothesis that there is a critical ‘time window of opportunity’ in which immune modulation and susceptibility to allergic disorders even later in life is modulated by microbial entities. We are further pursuing the search for such microbial entities by using defined gnotobiotic mouse models in which we can show how type 3 Treg depend on microbial complexity. As hypothesized within ALLERGUT this frequency is inversely correlated to the frequency of intestinal Th2 T cells. Therefore this system is currently exploited to identify novel bacterial components regulating the balance of type 3 Tregs and Th2 cells within the intestinal tract. As other laboratories worldwide have in the meantime shown the relevance of type 3 Tregs in food allergy, we focus our research now on the identification of T cell-intrinsic or T cell-extrinsic molecular pathways regulating the abundance of type 3 Tregs. This approach will hopefully lead to novel concepts for prevention or treatment of allergic diseases.
Alternative NF-kB pathway within dendritic cells affects different regulatory T cells.