Winnie Vos

The immune system is a large network of organs, tissues, immune cells, and proteins that function to protect its host against a broad spectrum of harmful pathogens, including bacteria, viruses, fungi and parasites. In addition, it plays a critical role in identifying and eliminating malignant cells, as well as facilitating tissue repair. Chapter 1 provides an elaborate introduction on the immune system and its role in inflammatory diseases, particularly atherosclerosis. Atherosclerosis is a chronic inflammatory disease in which the arteries narrow due to the buildup of plaque in the vessel walls. This plaque consists of a mixture of fats, (immune) cells, and connective tissue. The accumulation of plaque narrows the arteries and restricts blood flow, increasing the risk of a heart attack or stroke.

In this thesis, the therapeutic potential of immune checkpoints, which are crucial regulators of the immune response, is explored in various preclinical inflammatory diseases.

Chapter 2 explores the role of three E3 ubiquitin ligases – CBL-B, Itch, and GRAIL – as potential targets for immunotherapy in treating atherosclerotic cardiovascular disease. E3 ubiquitin ligases are enzymes that regulate protein degradation, influencing various cellular processes, including immune responses. The review suggests that targeting specific E3 ubiquitin ligases can modulate inflammatory pathways involved in atherosclerosis, potentially offering new therapeutic strategies. By fine-tuning immune responses, such therapies could reduce plaque formation and stabilize existing plaques, lowering the risk of cardiovascular events.

Chapter 3 examines the effects of deleting the E3 ubiquitin ligase Casitas B-lineage lymphoma-b (CBL-B), specifically in T cells, on atherosclerosis development. The study shows that deleting CBL-B in T cells reduces the overall burden of atherosclerosis. However, this deletion also leads to an increase in the number of T cells infiltrating the atherosclerotic plaque and heightens systemic T cell activation. This indicates that while a CBL-B deficiency may limit atherosclerosis progression, it simultaneously promotes a more active immune response, raising questions about the complex role of CBL-B in regulating immune function in atherosclerosis.

The CD40/CD40L dyad is a critical signalling pathway in the development and progression of atherosclerosis. CD40 is a co-stimulatory immune checkpoint protein expressed on various cell types involved in atherosclerosis, including endothelial cells, smooth muscle cells, and immune cells such as macrophages. CD40L, primarily found on activated CD4+ T cells and platelets, binds to CD40, initiating a cascade of pro-inflammatory signals that contribute to plaque formation and plaque instability. CD40L is often considered as merely a facilitator of CD40 signalling, but Chapter 4 shows that CD40L can also reverse signal and modulate T cell activation. A novel binding partner for CD40L, protein receptor for activated C kinase 1 (RACK1), is identified. Furthermore, CD40L signalling has a significant role in the polarisation of CD4+ T cells to a pro-inflammatory T helper 1 phenotype.

Immune checkpoint inhibitors improve the ability of the immune system to fight tumours by blocking co-inhibitory immune checkpoint proteins that normally reduce immune activity. This helps the immune system to recognize and eliminate malignant cells more effectively. While immune checkpoint inhibitors have revolutionized cancer treatment, they have unintended effects on cardiovascular health, particularly in the context of atherosclerotic cardiovascular disease. By stimulating the immune system, immune checkpoint inhibitors can exacerbate inflammation, which is a key driver of atherosclerosis. This heightened inflammatory response accelerates plaque formation and destabilization, increasing the risk of myocardial and cerebral infarction in patients with underlying cardiovascular conditions. In Chapter 5 a systematic review and meta-analysis are conducted to evaluate the effects of immune checkpoint modulation on atherosclerosis progression in preclinical studies. The inhibition of co-inhibitory immune checkpoint proteins accelerates atherosclerosis, potentially by increasing T cell infiltration in the plaques. This observation provides insights in the elevated cardiovascular risk observed in patients with cancer treated with immune checkpoint inhibitors.

While immune checkpoint inhibitor therapies show remarkable success in various cancers, their efficacy is not universal, and many patients do not achieve a complete or lasting response. Consequently, researchers are exploring strategies to enhance the effectiveness of immune checkpoint inhibitors, aiming to overcome these barriers and improve outcomes for a broader range of patients. Chapter 6 explores the potential of combining immune checkpoint inhibitors with statins, a commonly prescribed drug for cholesterol reduction, to enhance cancer immunotherapy outcomes. Preclinical studies indicate that statins may boost the effectiveness of immune checkpoint inhibitors in cancer treatment, though robust clinical trials are still lacking. Given the well-established safety profiles and availability of statins, they present a promising and accessible strategy to augment immune checkpoint inhibitor efficacy in cancer therapy.

Targeting macrophage inflammation in atherosclerosis is an emerging therapeutic strategy aimed at reducing the chronic inflammatory response that drives plaque formation and progression. Macrophages contribute significantly to inflammation by releasing cytokines and other pro-inflammatory molecules. Controlling this macrophage-driven inflammation could stabilise plaques and prevent cardiovascular events. One promising approach is to disrupt the CD40-TRAF signalling pathway. CD40, a key co-stimulatory immune checkpoint protein on macrophages, interacts with TRAFs, an adaptor protein, to amplify inflammatory signals. By developing a small molecule inhibitor that specifically blocks the CD40-TRAF interaction, known as TRAFstop, a reduction in macrophage activation and inflammation has been achieved without broadly suppressing the immune system. In Apoe-/- mice, TRAFstop successfully inhibited the progression of atherosclerosis, while minimizing side effects. In Chapter 7, TRAFstop is further evaluated for its potential in reducing atherosclerosis progression in a more human-relevant animal model, the hypercholesterolaemic pig. Initial data indicates successful induction of atherosclerosis in these animals, but further analysis will determine whether TRAFstop effectively reduces atherosclerosis progression.

Secondary haemophagocytic lymphohistiocytosis (sHLH) is a severe acute inflammatory disease characterised by an overwhelming immune response that can rapidly become life-threatening. The disease triggers excessive activation of immune cells, particularly macrophages and T cells. As a result, there is an uncontrolled release of pro-inflammatory cytokines that cause significant tissue damage and, if untreated, can progress to multiple organ failure. sHLH can be triggered by infections, malignancies, or autoimmune diseases and often presents with symptoms such as fever, liver or spleen enlargement, and cytopaenias. Chapter 8 describes a brief literature review and a pilot study which investigates the role of the co-stimulatory immune checkpoint CD40 in sHLH. Initial data show that CD40-/- mice with sHLH have reduced levels of several serum cytokines compared to wild-type mice with sHLH. These data underscore the need for further investigation into the role of CD40 in sHLH and the potential of CD40-targeted therapies. TRAFstop emerges as a promising therapeutic option, specifically targeting macrophage activation to mitigate the cytokine storm associated with sHLH. This targeted approach could offer a more effective and specific treatment, potentially improving outcomes for patients suffering from this life-threatening condition.

Chapter 9 presents a comprehensive discussion of the findings presented in this thesis. The results are summarized and contextualized within a broader framework. Furthermore, the discussion emphasizes future directions and the additional research required to advance immune checkpoint modulation as a therapeutic strategy for treating inflammatory diseases.