Ikbkb Gain-of-Function in human disease

Abstract

The NFkB signaling pathway is important in regulating numerous immune and inflammatory responses. NFkB family members can modulate the transcription of cytokines, as well as regulate genes involved in cellular differentiation, survival, proliferation, and immune cell function. Multiple groups have identified hypomorphic mutations in IKBKB (encodes IKKB) in patients suffering from immunodeficiency, where impaired NFkB activity was observed. My PhD has focused on the effects of an IKKB gain-of-function mutation and its role in human disease. In this thesis I describe experiments conducted to investigate the effects of a novel heterozygous de novo missense mutation that we identified in a proband with immunodeficiency. The mutation was found to result in a valine to isoleucine amino acid substitution within the kinase domain of the IKKB domain sequence, and resulted in a gain-of-function effect on IKKB. This enabled overactivation of the NFkB signaling pathway. To prove causation of this mutation, we generated a CRISPR-cas9 mouse model carrying the orthologous mutation. Biochemical and cellular analysis revealed similarities between the human and the mouse, therefore conferring a causative role of the mutation in the proband's immunodeficiency (Chapter 3). Through aging and observation of the mouse model, we identified the development of an inflammatory condition that involved the skin and bone/joints, and closely resembled the IL-17-mediated human disease, psoriatic arthritis (PsA) (Chapter 4). A gene dosage effect was evident where a skin-only disease was present in mice that were heterozygous for the IkbkbV203I variant, whilst a skin and systemic inflammatory illness developed when mice carry a double dose of the IkbkbV203I variant. The Ikbkb gain-of-function mutation generated a remarkable Treg population that abnormally produces increased IL-17 both in lymphoid tissues and at the sites of inflammation (Chapter 5). Single-cell RNA sequencing enabled the identification of an abnormally abundant Treg cluster within the spleen and bone marrow of mice homozygous for the IkbkbV203I. This cluster resembled a gene signature similar to an established non-lymphoid tissue Treg population, as well as a strong NFkB signature mediated by the gain-of-function mutation (Chapter 6). In this thesis I have investigated and identified the effects of an overactive IKKB protein within the immune system to result in primary immunodeficiency disease (PID) and the IL-17-mediated inflammatory condition, psoriatic arthritis in a mouse model. Our findings provide evidence that a fine balance of the NFkB is required to maintain immune system homeostasis. Furthermore, we have identified a biomarker for progression from skin-only inflammation to psoriatic arthritis through the presence of IL-17+ Tregs, mediated by GoF IKKB. We expect that these findings will be important in better defining diagnosis methods, as well as novel therapeutic targets for PIDs and PsA.