Search

Regulation of the immune system and of uterine tissue homeostasis, growth, and remodelling are deeply intertwined during pregnancy and are essential for successful reproduction. Recent findings showed that tissue-resident innate lymphoid cells (ILCs) are crucial regulators of both physiology and pathology of the tissues they populate. Uterine natural killer (uNK) cells are a subtype of ILCs known to regulate trophoblast invasion, uterine vascular adaptation to pregnancy, and foetal growth. We recently described additional types of ILCs in the uterus of women and mice. However, the role of these ILCs during reproduction is unknown. Among them, group 2 ILCs (ILC2s) have been previously characterised in other tissues, in which they modulate immune cells and tissue homeostasis by producing type-2 cytokines and growth factors (i.e. IL-4, IL-5, IL-13, and Amphiregulin). Based on these premises, I hypothesized that uterine ILC2s (uILC2s) regulate uterine immune homeostasis and thus contribute to successful reproduction. To test this, I first characterised the uILC subtypes present in humans and mice at various stages of the reproductive cycle. Secondly, I addressed the functional role of uILC2s during pregnancy by taking advantage of a uILC2 knockout mouse model. My results show that uterine ILC2s represent < 1% and < 0.1% of murine and human uterine leukocytes, respectively. However, as they can quickly produce large amounts of cytokines, uILCs are capable of potently affect both other immune cells and the surrounding tissue. Indeed, I found that compared to other tissue-resident ILC2s, uILC2s produce high levels of IL-5 and Areg even in the absence of any stimulation. On the contrary, non-uterine ILC2s mainly produce IL-13, which is lowly expressed by uILC2s. To further characterize the tissuespecific properties of uILC2s, I then performed RNAseq on uILC2s isolated from virgin, midgestation, and term murine uterus, and I compared their transcriptomes with those of ILC2s from lung, intestine, and bone marrow. Interestingly, uILC2s specifically express granzymes and genes typical of regulatory T cells. Therefore, uILC2s have tissue-specific properties and are modulated during pregnancy. Furthermore, the ability of uILC2s to produce IL-5 and Areg suggests that they may be crucial in the regulation of uterine type-2 immunity. I then studied the phenotype of $Rora^{flox/flox}Il7ra^{cre/wt}$(ILC2KO) mouse models, as well as that of mice lacking the ILC2 activating cytokine IL-33 ($IL33^{cit/cit}$; IL33KO). I examined the immune microenvironment in both the myometrium and decidua in ILC2KO mice and found alterations in type-2 cytokines and myeloid cell homeostasis. In particular, in absence of ILC2s, IL-4 and IL-5 are dramatically reduced, IL-13 is absent, and decidual inflammatory cytokines IL1β and IL-6 are increased. Furthermore, uterine dendritic cells (uDC), uterine macrophages (uMac), and uterine neutrophils (uN) increase, while uterine eosinophils (uEo) are virtually absent. These results show that uILC2s regulate uterine type-2 immunity, suggesting that uILC2s could be important during pregnancy. Accordingly, I found that lack of uILC2s leads to insufficient spiral artery remodelling and restricted foetal growth. Type-2 cytokines and in particular IL-4 regulates alternative activation of Macrophages (Mac) and Dendritic Cells (DCs), which promote the development of an anti-inflammatory environment and facilitate tissue remodelling. I hypothesised that similar mechanisms occur in the uterus and that uILC2s have a central role in the polarisation of the immune response. To explore this, I studied in more detail the characteristics of uEo, uMac, and uDCs dissected from wild type and ILC2KO mice. I found a reduction in genes associated with alternative activation in uMac and uDCs in the uterus of pregnant ILC2KO mice. Additionally, I showed that uEo are the main producers of the IL-4. This demonstrates that uILC2s promote alternative activation of myeloid cell population by modulating the uterine immune microenvironment. I then assessed the role of uILC2s-dependent type-2 immunity in inflammatory pathology following a type-1 response to bacterial infection. When challenged with LPS, pregnant ILC2KO mice showed more pronounced foetal demise. Therefore, uILC2s regulate uterine type-2 immune homeostasis and this prevents inflammatory pathology. Collectively, my work advances our knowledge of the innate immune mechanisms that control physiological and pathological events during pregnancy. These findings have implications to the field of immunology of pregnancy and may lead to clinical progress in diagnosis and prevention of infection-induced abortion in human pregnancies.

The recognition of foreign molecules by the canine immune system and mounting a protective response to viral, bacterial, and parasitic pathogens requires intricate crosstalk between different immune cells and molecules, and inappropriate activation of these pathways can lead to allergic inflammation. The health of our canine companions is of great intrinsic value and dogs serve as a unique model for the study of naturally occurring complex diseases, such as allergies, and the study of host-pathogen interactions. In these studies, we examined the immune mechanisms associated with two canine diseases of major relevance in clinical practice - canine atopic dermatitis and canine parvovirus infection. Canine atopic dermatitis (CAD). To study the role of important immune cell types associated with excessive type 2 inflammation in allergic disease, we used new methods to describe a previously uncharacterized canine group 2 innate lymphoid cell-like (ILC2-like) population and canine T helper 2 (Th2) cells that express the transcription factor Gata3 and produce type 2 cytokines. We found that increased Th2 cells in the peripheral blood, but not ILC2-like cells, were associated with canine atopic dermatitis. Using single-cell RNA sequencing, we could show a unique gene expression signature in T cells from allergic dogs. Canine parvovirus (CPV) infection. To study the canine antibody repertoire to CPV, we developed new methods for the isolation of antigen-specific B cells and the cloning of CPV-specific monoclonal antibodies directly from dogs. We identified a vaccine-induced canine anti-CPV antibody that bound to an epitope on the capsid that includes the critical antigenic, receptor-binding and host-range residue VP2 300. We also cloned and expressed anti-CPV scFvs previously developed by others and established methods for isolating polyclonal antibody Fab fragments from dog blood. Cryo-EM analysis by members of Dr. Susan Hafenstein’s laboratory at Penn State University of the scFv:capsid and Fab:capsid complexes showed that canine-derived antibodies target two common antigenic sites on the parvovirus capsid, and that many canine antibody binding sites overlap the capsid region that binds to the host cell receptor, the transferrin-receptor type-1. The results in this dissertation highlight the importance of Th2 cells in chronic CAD, and also shed light on the canine antibody repertoire to CPV and the complex interactions between viral capsid structures, ligand binding, and natural variation.

In humans, allergic disease bears striking resemblance to the host response to helminth infection. Thus, we studied N. brasiliensis infection in mice to identify factors that initiate host immunity and identified three that bind the environmental polysaccharide chitin. We further demonstrated that purified chitin acutely induces lung inflammation in mice, similar to that observed in human allergic disease, dependent on resident group 2 innate lymphoid cells (ILC2s). Given their number, distribution and function, lung ILC2s likely coordinate responses with resident epithelial cells that monitor barrier interactions with the environment. In this thesis, we demonstrate that alveolar type II (ATII) cells are the main source of interleukin (IL)-33 and thymic stromal lymphopoietin (TSLP) generated in response to chitin or N. brasiliensis challenge. IL-33 and TSLP synergistically activate ILC2s to produce IL-5, IL-13 and IL-9, and autocrine IL-9 promotes an ILC2 transcriptional program associated with rapid and robust cytokine production necessary to optimize epithelial gene responses in the conducting airways. Thus, ILC2s link a specialized, distal, epithelial population involved in alveolar function with more proximal epithelia that regulate airway flow, revealing a key role for ILC2s in tissue homeostasis that might underlie similar organizational hierarchies for innate lymphoid cells in other organs.