Iloprost modulates the immune response in systemic sclerosis

Systemic sclerosis (SSc) is a disease whose extremely complex, multifaceted pathogenesis cannot be referred to a single hypothesis, even if fundamental abnormalities in three or more types of cells are involved: fibroblasts, endothelial cells and cells of the immune system, namely T and B lymphocytes [1]. Mononuclear cells (mainly T cells) in the SSc skin infiltrates produce cytokines and growth factors responsible for the onset and progression of fibrosis and microvascular damage. The role of this immune dysfunction is not fully clear. Altered cellular immunity is revealed by aberrant T cell biology in both the skin, where the lesions display various features consistent with T cell activation [2‐4], and the circulation, where CD4+ T cells are increased [2, 5], and natural killer T cells are decreased [6]. Lastly, circulating T cells from SSc patients produce more inflammatory mediators compared to those from healthy controls [7, 8].

An important role as controllers of self-reactivity [9] has recently been defined for T regulatory cells (Treg) in autoimmune disease. These cells are defined as CD4+/CD25^bright/FoxP3+. In SSc, both an increase in their absolute number and a reduction of their function have been demonstrated [10].

A high proportion of SSc patients suffer from Raynaud's phenomenon. This is a vasospastic disorder that causes discoloration of the fingers, toes and occasionally other extremities, and its persistence may result in acral ulcer and significantly reduce the quality of life [11]. Prostaglandin (PG) analogues, particularly the PGI2 analogue iloprost, are widely used as vasodilators to treat this disorder [12].

Patients with SSc receiving Iloprost report a reduction in skin tightness, suggesting that this drug inhibits skin fibrosis. It has been suggested that iloprost reduces skin fibrosis by reducing collagen synthesis [13, 14], the pro-fibrotic cytokine connective tissue growth factor [15] and the fibrotic response [16].

Iloprost has since been shown to possess anti-inflammatory and immunomodulating actions, both in vitro [17‐20] and in vivo [21‐24]. Of the two studies in humans [21, 22], only Filaci et al. [22] evaluated the effect of iloprost in SSc. Its potential in this disease is thus an open question.

Here we demonstrate that iloprost decreases the production of TNF α by acting on T cells, ameliorates Treg function, and increases IL-2.

Prostaglandin I2 is a metabolite of arachidonic acid that has been shown to have anti-inflammatory functions [21, 28, 29], however, the cellular and molecular mechanisms of PGI2-mediated anti-inflammatory effects remain to be determined. Here we evaluated the effect of iloprost, a PGI2 analogue, widely used in the treatment of SSc, on T cells sub-populations and on pro-inflammatory cytokines.

This study shows that infusion of iloprost, according to the standard protocol to treat Raynaud's phenomenon in SSc patients, reduces T cells in the peripheral blood, especially TNF α producing T cells, according to previous in vitro [17‐19] and in vivo [21] studies. The mechanism underling this effect was shown by Jorres et al. [19] that found that iloprost reduces TNF α transcriptional activity and decreases TNF α mRNA stability in human peripheral blood mononuclear leukocytes stimulated with LPS; Our study is the first evaluation of the effect of iloprost on TNF α production and T cell subsets in SSc. Our finding that iloprost acts on T cells only and not on monocytes is in contrast with Di Renzo et al.'s observation of a reduction of both monocytes and T cells [21]. This discrepancy may be attributable to the fact that they infused iloprost for 16 h/day for 7 days as required for critical limb ischemia. Our data show that iloprost reduces both the total amount of TNF α positive T cells and their ability to secrete TNF α after an antigenic stimulus. The role of TNF α in autoimmune diseases is well known, and it is currently a target for monoclonal antibody in diseases such as rheumatoid arthritis [30]. Its reduction by iloprost may thus be an important plus in the evaluation of therapeutic options.

Our study demonstrates a decrease in Treg after 5 days of iloprost infusion in vivo, associated with enhancement of their ability to produce TGF β1 in response to immune stimulation. This effect could be due to a generic immunomodulation by iloprost through a systemic increase in IL-2 production, as it is well known that this cytokine is deeply involved in the control of T cell development and function [25], and it controls Treg suppressor function [38]. Our study demonstrates that iloprost consistently increases serum IL-2 and that this increase persists 28 days after the last infusion. Here we show that IL 2 effect on TGF β1 production is not direct, but may be mediated trough other cell types in vivo. Recently a role for dendritic cells in the mediation of iloprost anti-inflammatory activity has been postulated [31, 32]

The role of TFG β in determining the suppressor ability of Tregs is currently under debate [33], TGF β is crucial in determining the ability of Tregs to convert Foxp3- T cells into Foxp3+ T cells by a mechanism of infectious tolerance [34]. Recent data suggest an important role for Treg cells in the pathogenesis of autoimmune diseases [35], and it has been suggested that their number is increased, whereas their function is impaired, in SSc [10, 30]. It may thus be supposed that the reduction in Treg number coupled with the increase in their TGF β production may be critical for SSc therapy.

We measured the levels of RANKL in serum and in supernatants because this molecule is important in inflammation and in the control of immune function [24] and expected they would be reduced after iloprost similarly to the reduction in TNF. After iloprost instead we found an increase in RANKL levels both as protein and as gene copies number. This could be interpreted as a class effect of prostaglandins, since recent studies [34‐36] have demonstrated an increase in RANKL in cells after incubation with PGE2; moreover Conaway and colleagues demonstrated that both PGE2 and PGI2 were able to enhance osteoclast activity that is mainly under RANKL control [36]. However the RANKL increase is not due to IL-2 increase as demonstrated by the lack of IL-2 effect on in vitro PBMCs.

Our data demonstrated that iloprost does not influence IFN γ production. This cytokine is particularly involved in the inflammatory reaction of monocytes/macrophages and induces their TNF α secretion [26]; hence the lack of effect of iloprost on IFN is in line with the absence of its effect on monocyte TNF α production.

This study suggests that iloprost has specific anti-inflammatory and immunomodulating effects: it reduces TNF α production by T cells without influence on TNF α monocyte production; it reduces the Tregs number, but ameliorates their function probably trough an increase in IL-2 and directly increases RANKL. Hence the therapeutic use of iloprost in SSc patients may be useful not only as vasodilator and antifibrotic, but also as immunomodulator agent.