Systemic sclerosis (SSc) is a rare autoimmune disease in which the connective tissue in the skin and internal organs of patients thickens and hardens due to the excessive production of extracellular matrix (ECM) proteins. This process is also referred to as the development of fibrosis. Fibrosis affecting the internal organs such as the lungs or the cardiovascular system is a common cause of death for these patients. SSc is a complex disease in which a high degree of heterogeneity is observed in the patient population. What causes the onset of fibrosis is not exactly known, but clinical observations and the results of previous studies indicate the presence of vascular abnormalities and a persistent inflammation before the onset of fibrosis. Although various genetic changes have been identified as risk factors, the heredity of SSc is very limited. In addition to the genetic factors, epigenetic changes probably play an important role in SSc development. These epigenetic changes regulate the expression of genes without changing the genetic code. Examples of such regulatory mechanisms are the expression of non-coding RNAs such as microRNAs and long non-coding RNAs, DNA methylation and histone modifications. The theory behind the development of this deforming disease is therefore that environmental factors cause epigenetic changes in people genetically predisposed to SSc, leading to immune activation and subsequent stimulation of ECM overproduction by fibroblasts.
In recent years, extensive research efforts have been spent to obtaining a better understanding of the pathogenesis of SSc. One of the recurring observations in previous studies is the presence of large amounts of monocytes in the affected skin of patients, which are very likely to contribute to the activation of fibroblasts and thereby stimulate the overproduction of ECM. Additionally, it is known that the concentration of several signaling proteins, including platelet factor 4 (CXCL4) and angiopoietin 2 (ANG2), is increased in the blood of SSc patients. However, the effects of these factors on the different immune cells are not well understood. Finally, the activation of both the innate and adaptive immune systems and the increased expression of type 1 interferon responsive genes, also known as the IFN signature, have been associated with the pathogenesis of SSc.
Aim of this thesis
The aim of this thesis was to study monocyte dysregulation in patients with SSc and to evaluate the effects of this dysregulation on the pathogenesis of SSc. To realize this, we first looked at changes in the frequency of monocytes in the circulation, changes at the level of gene expression in these cells and the histone modifications that may underlie this aberrant gene expression. In the second part of the thesis we studied the response of monocytes to CXCL4 and ANG2, two signaling factors associated with SSc, and looked at how this response can contribute to the inflammation and fibrosis observed in SSc patients.
Summary of the findings
Monocyte dysregulation in SSc patients
In Chapter 2, I describe the results of a study looking at the frequency of 44 different types of circulating immune cells in two cohorts of healthy people, patients with SSc and patients with other autoimmune conditions. The results indicated that an increased frequency of monocytes was present in the circulation of SSc patients compared to healthy people. This increase was not present in patients with other autoimmune diseases. In SSc patients, the frequency of circulating monocytes was directly correlated with the disease severity. For example, the degree of skin fibrosis was found to be related to the monocyte frequency and we found that SSc patients with lung involvement had more circulating monocytes compared with patients without lung involvement. In addition, the increase in monocytes was found to coincide with an increase in two signaling proteins, CXCL10 and CXCL11, which were previously shown to be related to SSc disease progression. The increase in monocytes was also present in so-called "early" SSc patients, indicating that the increased number of monocytes in circulation may be linked to disease progression.
In Chapter 3 we took a closer look at the dysregulation of monocytes in SSc patients. Using a global gene expression analysis, we identified genes with an abnormal expression pattern in patient monocytes compared to those of healthy people. The genes with increased expression were found to mainly play a role in the regulation of cell processes that are part of the immune response system, the production of cytokines and the IFN response. Concomitantly, we looked at epigenetic modifications of the histones that form the packaging material of the DNA and regulate its accessibility. We found that many alterations of these modifications are present in patients and show that these changes are linked to the sustained dysregulation of gene expression. We identified several histone-modifying enzymes whose aberrant gene expression may underlie the observed histone modifications. In a proof-of-concept experiment, we demonstrated that the effects of the altered histone modifications can be reversed with an inhibitor, JQ1, implying that epigenetic changes may be a pharmacological target for restoring homeostasis in the monocytes of SSc patients.
Using a similar method as the one described in Chapter 3, in Chapter 4 we studied the changes of epigenetic histone modifications in monocytes from patients with SSc, systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) compared to healthy people. In this study, we looked at two different modifications, one of which is associated with the activation of gene expression while the other modification inhibits gene expression. In the monocytes of SSc and SLE patients, we found both unique and overlapping differences in these modifications compared with healthy monocytes. The genes associated with an increased amount of the activating modification had, on average, a higher expression level and were found to play a role in cellular processes associated with the pathogenesis of the various autoimmune disorders. Although a decrease in the inhibitory modification does not directly result in increased gene expression, it is known that it allows the expression of nearby genes to be quickly induced upon activation of the cell by exogenous or endogenous factors or during differentiation. In monocytes from SSc patients, genes with the inhibitory modification removed were found to encode pro-fibrotic factors or ECM molecules, such as THBS1, FAM20A and collagens, as well as growth factors such as PDGFB which we investigated further as described in Chapter 6.
Impact of SSc-related factors on monocyte activation
In the second part of this thesis, we looked at the effects of SSc related signaling molecules on monocytes and evaluated how this can contribute to the inflammation and fibrosis which is observed in SSc patients.
In Chapter 5 we investigated the effect of the imbalance between angiopoietin (ANG) 1 and 2 observed in SSc patients on monocytes. Although ANG1 and ANG2 are primarily known as regulators of vascularization by binding to the TIE-2 receptor on endothelial cells, it has recently been shown that monocytes also express these receptors. Our results show that in SSc monocytes, the TIE-2 signaling induced by ANG2 causes the production of the pro-inflammatory cytokines IL-6 and IL-8. We found the same result after the stimulation of healthy monocytes with serum from SSc patients. In addition, our experiments indicated that the neutralization of ANG2 in the serum of patients or the inhibition of the TIE2 receptor could nullify this effect. This shows that ANG2 neutralization could be a promising therapeutic target in inhibiting inflammation to treat SSc.
Finally, Chapter 6 of this thesis describes how CXCL4 leads to activation of monocytes and macrophages and reveals how this activation contributes to inflammation and fibrosis in SSc patients. CXCL4 is a signaling protein of which the concentration is greatly increased in the circulation and affected tissue of SSc patients. The stimulation of monocytes and macrophages with CXCL4 increases the secretion of the growth factor PDGF-BB. This effect appears to be specific for CXCL4 as it was not observed after stimulation of monocytes with interferon, transforming growth factor (TGF) beta or ligands for the Toll-like receptors. In skin fibroblasts, PDGF-BB contributes to the induction of the expression of pro-inflammatory cytokines and proteins that play a role in the chemotaxis of immune cells. In addition, PDGF-BB has a pro-fibrotic function as it stimulates the production of ECM molecules such as collagen and fibronectin in fibroblasts. That the CXCL4 activation of macrophages can directly contribute to fibrosis was shown in experiments where the culture medium of CXCL4- stimulated macrophages was found to induce inflammation and ECM production in fibroblasts. Blocking the PDGF receptor with Crenolanib, a specific inhibitor molecule, prevented this response which implies that intervention with the CXCL4/PDGF-bb signaling axis in monocytes may hold therapeutic value.
In summary, we can state that there is a clear dysregulation of monocytes in patients with SSc. This dysregulation is strongly related to the disease severity and is therefore very likely directly involved in the pathogenesis of SSc. Future research will hopefully show how the prevention of monocyte dysregulation can contribute to curing or preventing SSc.