Tissue-resident memory Tr1 cells, interleukin-10, and the extracellular matrix in airway inflammation

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with pleiotropic effects on antigen presentation, T-cell activation, and other immune phenotypes. IL-10 is a crucial regulator of immune responses, including those involved in type 2 allergic responses, autoimmunity, and inflammation-induced fibrosis. IL-10 is particularly important in allergic asthma, a common, debilitating disease that disproportionately affects children, minorities, and urban populations. Regulatory T cells, including Foxp3+ regulatory T cells (Treg) and type 1 regulatory T cells (Tr1), play a significant role in IL-10 production. These tolerogenic cell types have been proposed to contribute to immune tolerance in different, perhaps distinct contexts. However, while Foxp3+ Treg biology has been investigated extensively, Tr1 biology is relatively poorly understood. Recent efforts have sought to provide IL-10 exogenously or to induce human Tr1 cells for therapeutic treatment of allergic asthma and other disorders. However, these efforts are stymied by limitations in our understanding of Tr1 biology and the factors that govern IL-10 production. Extracellular matrix (ECM) molecules, including hyaluronan (HA) and heparan sulfate (HS), may modulate both Tr1 induction and IL-10 activity and provide a novel potential mechanism for enhancing existing Tr1 cell therapies. HA acts as a tissue integrity signal, capable of promoting homeostatic tolerance in its high molecular weight form or amplifying pro-inflammatory responses in its fragmented low molecular weight form. Heparin and HS can bind a number of soluble growth factors, chemokines, and cytokines, including IL-10, and modulate their activity in vitro and in vivo. In this thesis I have examined the cells and tissue ECM factors that influence Tr1 and IL-10 levels in the lung with an emphasis on allergic inflammation. Moreover, I present investigations into the impact of extracellular matrix molecules on tolerogenic cytokines and regulatory T cells. In Chapter 2, I describe my work characterizing the cells and kinetics involved in IL-10 production in a mouse model of allergic asthma. I report that Tr1 cell frequencies rise with inflammation and comprise the majority of the IL-10-producing cells at the peak of inflammation. These Tr1 cells are capable of forming antigen-specific tissue-resident memory but lose their inhibitory phenotype and propensity to produce IL-10 upon secondary challenge. Continual stimulation is required to maintain expression of the transcription factors Irf1 and Batf and consequent IL-10 production in memory Tr1 cells. This loss of IL-10 production in memory Tr1 cells suggests a critical need for novel approaches to maintain Tr1 phenotype long-term within tissue extracellular matrix. In Chapter 3, I describe the use of high molecular weight hyaluronan (HMW-HA) to promote the induction of IL-10-producing Tr1 cells and tolerance to airway allergens. We created a modified version of hyaluronan cross-linked to antigen, which we call XHA, which allows for the delivery of allergens in the context of anti-inflammatory co-stimulation. Intranasal administration of Ova-loaded XHA in previously sensitized mice reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and Th2 cytokine production. This inhibition was mediated by the suppressive effects of HA on dendritic cell maturation and was dependent on IL-10 production. Importantly, these effects were durable for weeks, providing an advantage over current desensitization protocols. In Chapter 4, I describe the therapeutic delivery of IL-10 using HA and HS hydrogels in a mouse model of bleomycin-induced fibrosis. The inclusion of HS facilitates the slow release of IL-10, prolonging its bioavailability and increasing its efficacy relative to IL-10 administered alone. This work highlights the anti-fibrotic effects of IL-10 and the potential for using ECM for delivering this. Chapter 5 focuses on the role of HS and the enzyme heparanase (HPSE) in regulatory T cell homeostasis. I report that Foxp3+ Treg use HPSE to access IL-2, a tolerogenic cytokine that promotes Treg survival, from the tissue ECM. These investigations highlight the importance of ECM factors in cytokine signaling and immune regulation. Finally, in Chapter 6, I describe the impact of a HS-mimetic, PG545, on lymphocyte polarization. I demonstrate that PG545 promotes Foxp3+ Treg polarization and can ameliorate allergic hypersensitivity in the skin. Together, these studies highlight crucial insights into IL-10 and regulatory T-cell mediated tolerance in airway inflammation. These studies open the door for further study into the roles and potential utilization of extracellular matrix and tissue-specific cues to promote the maintenance of airway tolerance

Keywords: Immunology.
Advisor: Bollyky Paul
Bendall Sean
Davis Mark M.
Nicolls Mark