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Background

Autoimmune Disease

Autoimmune diseases encompass a range of disorders that result from the attack of self-tissues by a dysregulated immune system. More than 80 autoimmune diseases have been characterised, each with long-term effects on health, quality of life and cost to healthcare services. These include Crohn’s disease, Coeliac disease, type 1 diabetes, multiple sclerosis, systemic lupus erythematosus and rheumatic disease.

Global Burden of IBD

Inflammatory Bowel Disease (IBD) is a heterogeneous group of autoimmune disorders encompassing Crohn’s disease and Ulcerative Colitis. It is estimated to affect 0.3% of the population in industrialised nations, with over 5 million patients worldwide.

The global prevalence of IBD is increasing rapidly as countries become more developed. In addition, instances of IBD in developing countries are thought to be accelerated by the presence of bacteria, such as segmented filamentous bacteria (SFB), in water sources influencing the gut microbiome and host immune response. Gram-positive SFB have recently been implicated in promoting the development of lymphocytes and the growth of Th17 populations.

An additional effect of the signals required for Th17 differentiation is the inhibition of the production of Treg cells. Once fully differentiated, Th17 cells produce interleukin 17a (IL-17a) a proinflammatory cytokine. Elevated levels of IL-17a are associated with MS and the development of epilepsy and other autoimmune diseases.

Most diagnoses occur between the ages of 20 and 30; the disease presents a significant burden to patients, societies, and healthcare systems. Current treatment is focused on symptom management, frequently resulting in sub-optimal control despite the burden of long-term treatment. Common therapeutics, such as corticosteroids, require regular administration, have many side-effects and are less accessible in the developing world. The aim of our project is to address these problems and meet the notable, yet currently unmet, need for curative treatments for autoimmune diseases. This will be achieved via regulation of Treg and T helper cell populations to ensure immune responses of the correct magnitude. The specific control mechanism is discussed below.

Immune Cell Populations

T helper cells (Th) are a component of the adaptive immune systems and are characterised by the presence of the CD4 protein on the cell surface. CD4+ cells are able to interact selectively with major histocompatibility complex 2 on the surface of antigen presenting cells such as macrophages and dendritic cells. These interactions facilitate the release of small proteins known as cytokines and so Th cells contribute to varying the activity of other cell types. More specifically, Th cells are required for the maturation of B cells to upregulate production of antibodies and the proliferation of cytotoxic T cells, which degrade cancerous or infected body cells. Our project focuses on the detection of a particular subset of T helper cells namely, Th17 cells. Th17 cells produce interleukin- 17 (IL-17) and have a proinflammatory function, as well as regulating the production of neutrophils. Incorrect functioning of these cells is associated with the development of autoimmune diseases. Overactivation of Th17 populations that are responsive to self-antigens causes the accumulation of antigen-antibody complexes, leading to type III hypersensitivity. This causes an inflammatory response and is associated with autoimmune diseases such as lupus and arthritis.

Th17 cells are also closely associated with Treg cells as the chemicals required for Th17 differentiation and maturation are inhibitory with regard to this process for Treg cells. At low concentrations TGFβ associates with IL-6 and IL-21 to promotes Th17 differentiation; however, when TGFβ is at high concentrations, Treg differentiation is favoured as the production of IL-23 is repressed.

In contrast to T helper cells, natural T regulatory cells (Tregs) are characterised by the presence of both the CD4 receptor and CD25. The role of Tregs is the suppression of the action of helper and cytotoxic T cells, for example, by blocking the production of cytokines. They also suppress B cell and dendritic cell function, for example, via the production of inflammatory cytokines. The immunosuppressive role of Tregs is important in regulating the activity of the Th cells and to prevent excessive inflammation and help to reduce self- reactivity, thereby limiting the development of autoimmune diseases.

Pathogenesis

Immunodeficiency is a state where the immune system is unable to fight against infection due to a lack of, or suppression of cells of the innate or adaptive immune responses. Immunodeficiency can be either primary or secondary in nature. Primary immunodeficiency is the result of genetic mutations that lead to reduced immune responses. This is further categorised into B cell and T cell immunodeficiencies, severe combined immunodeficiency, complement defects and phagocyte disorders. Secondary cases are caused by environmental factors or diseases that induce weakening of the immune system.

A reduction in the population size of Th cells, as a method to counteract autoimmune diseases, increases the risk of development of immunodeficiency as a depleted population of Th cells will be unable to resist infection as successfully. Preventing the development of immunodeficiency is of particular importance in regions with poor sanitation where exposure to infection is more common.

To prevent the development of immunodeficiency in individuals that use our treatment, we have created a system that responds to two input values. Upon detection of the size of Th17 and Treg cell populations, the system responds to ensure optimum population sizes. If one cell population exceeds optimum levels, there is a response to restrict a further increase, preventing uncontrollable growth. This mechanism prevents both autoimmune disease and immunodeficiency by maintaining optimum population sizes for both cell types.

Human Microbiome

The microbiota refers to the population of symbiotic microorganisms, typically bacteria, that reside in the gut and are known to play a role in human health. It has been estimated that there are approximately ten times more microbial cells than human cells in adults. There are around 100 species of microorganism in the gut in young infants and this number steadily increases to around 1000 in adults. The populations that compose the community of the microbiota are stable within an individual; however, the composition of the microbiota is highly variable between individuals. The composition of the microbiome is determined as early as birth, for example, by the method of delivery. Babies that are born via the birth canal are exposed to vaginal bacteria and so have a different microbiome than those delivered via C-section, which are typically colonised by bacteria located on the skin. Age, gender, diet, exercise also play a role in determining the composition of the bacterial community within the gut.

Traditionally, the microbiota refers to the microorganisms, for example, bacteria, viruses and fungi that have colonised the human body. In contrast, the microbiome is the total sum of the genes that the species within the microbiota possess.

The microbiota differs between regions of the body. It should be noted that opportunistic pathogens are present in the microbiota alongside non-pathogenic species. High microbial diversity within the gut is known to be associated with good gut health and lower incidence of Inflammatory Bowel Disease.

Our Solution

Our solution is to develop a probiotic strain that restores the Th17/Treg cell balance via secretion of IL-10 in response to nitric oxide in the intestinal lumen. Overshoot is prevented by an adenine riboswitch-sRNA construct which responds to extracellular adenosine, an indicator of Treg cell function. Integration of separate stimuli in a dual feedback loop enables a more dynamic, robust response to the immune state of the body. Various features have been incorporated to maximise biological safety, including an inducible kill switch system. We believe our design offers a non-invasive, self-tuning therapeutic for IBD, with the potential to replace conventional immunosuppressants in the treatment of gastrointestinal autoimmune disorders.

A range of purines are used by Treg cells as immune signals. ATP is released from cells during stress or injury. To prevent it from causing excessive inflammation, ATP is rapidly metabolised into AMP then into Adenosine (Ado) by Treg cell surface enzymes. Ado is detected by GPCRs of Th cells, triggering increased cAMP in the cells. Inflammatory signals are inhibited and IL-10 production is enhanced in mature dendritic cells. Ado acts as an anti-inflammatory metabolite by inducing semi-maturation of immature dendritic cells, in order to increase immunoregulatory activity. Production by Treg cells and its role in regulating T cell populations and IL-10 production make it a suitable marker of Treg function and degree of immunodeficiency.

As well as being a generalised marker of inflammation, NO is thought to be produced by Th cells as a signal to induce differentiation of precursors into Th cells, thereby influencing the relative populations of Th and Treg cells. Continuous production of NO makes it a suitable marker of the size of the population and severity of the autoimmune disease.

References

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