Semaphorin 3A is a marker for disease activity and a potential immunoregulator in systemic lupus erythematosus

Semaphorins are a large family (classified into eight subclasses) of secreted and membrane bound proteins originally discovered in the nervous system, which are involved in repulsive axon guidance during nervous system development [1‐3]. They relate to two families of receptors: the neuropilins (NP-1 and NP-2) as the primary ligand binding sites and plexins, as the signal-transducing components [4‐6]. Recent data have pointed to the involvement of NP-1 and semaphorins in the regulation of the immune system, and are thus denoted as 'immune semaphorins' [7‐9].

Semaphorin 3A (sema3A), a secreted member of this family, is well reported as a potent immuno-regulator during all immune response stages, namely, the early initiation and the late phase of inflammatory processes [10]. The expression of sema3A, NP-1, NP-2, and plexins were found to be increased on differentiating macrophages and on activated T cells, suggesting that they have a role in modulating inflammatory conditions [11]. The addition of sema3A to dendritic cell (DC)/T cell co-cultures significantly inhibited allogeneic T cell proliferation. Moreover, neutralization by blocking antibodies against endogenous sema3A resulted in a profound increase in T cell proliferation [12]. Both NP-1 and sema3A expression was recognized on T regulatory cells as a suppressive marker, contributing to the regulatory properties of these cells [13‐16]. In another study, the known immunosuppressive function of bone marrow mesenchymal stem cells (MSCs) was attributed in part to their ability to secrete sema3A which, by binding to NP-1, was found to inhibit T cell proliferation [17]. Becoming a frontier player in the regulation of immune responses and the maintenance of self-tolerance, sema3A should be expected to be involved in the pathogenesis of many autoimmune diseases [18, 19]. The study by Catalano [20] was the first to report on the defective expression of sema3A in CD4 T cells derived from patients with rheumatoid arthritis (RA). In this study, sema3A was shown to enhance the suppressive ability of CD4⁺NP-1⁺ T cells by increasing their IL-10 expression and their regulatory function on effector CD4⁺ T cells. Their altered expression on T cells was shown to correlate with the progression of RA. In consideration of the above, we initiated this current study to assess the possible role of sema3A in systemic lupus erythematosus (SLE), namely to measure its serum level in SLE patients and assess whether this level correlates with disease status. In addition, we sought to assess the possible in vitro immuno-modulatory effect of sema3A on B cells of SLE patients.

The involvement of sema3A in regulating both in vitro and in vivo inflammatory responses has been previously suggested and shown to be of high relevance. The pleiotropic effects of sema3A have been shown to involve a variety of cells, such as T cells, monocytes, macrophages and endothelial cells (ECs) [24‐27]. In this respect, sema3A was shown to inhibit Th1 mediated inflammatory responses in RA patients, that is, reducing the production of pro-inflammatory cytokines such as IFN-γ and IL-17 [15, 20].

This study is the first in which sema3A is shown to play a role in the pathogenesis of SLE, that is, a role in humoral responses, as well as a role in modulating the autoimmune properties of B cells in SLE. Serum sema3A levels were demonstrated to be low in SLE patients, in negative correlation with disease activity, renal damage and the presence of anti-cardiolipin antibodies. It is thus suggested that sema3A is a candidate to become a useful marker for SLE disease activity and renal damage. Though of high significance, our results on sema3A serum levels in SLE patients should be strengthened with a larger number of patients in future studies. In addition, sema3A serum levels should be assessed when patients are in remission. As B cells are the source of autoantibodies and pro-inflammatory cytokine production in SLE, a correlation with sema3A expression on B cells in any relation to these activities was assessed. Indeed, it was found that sema3A expression as well as the expression of its receptor NP-1 was significantly decreased on CD19/CD25^high (Breg) cells from SLE patients when compared to that on this subset of cells of normal individuals.

The observation that sema3A expression is mainly present on CD19⁺CD25^high cells suggests sema3A as a unique marker for this subset of cells. It may be speculated that when sema3A expression is diminished, B cells may lose their regulatory signal and escape self-tolerance becoming auto-reactive and autoantibody producers. NP-1 down-regulation on B cells may also contribute to the development of B cell auto-reactivity and auto-immunity in SLE. Indeed, earlier studies have contributed to the understanding that NP-1 expression may be important for regulatory responses. In this respect, NP-1 positive CD4 T cells have been designated as CD4⁺CD25⁺Foxp3⁺NP-1⁺ [20]. Our finding of decreased NP-1 expression, along with decreased sema3A expression on B cells in SLE further suggests that this defect may play an important role in the pathogenesis of SLE. Assuming that sema3A and NP-1 are essential for the regulatory function of B cells, one would expect to see that when their expression on B cells is decreased, B cells may shift, to become more pro-inflammatory rather that regulatory, and contribute to the development of SLE. There remains still a lack of understanding as to what causes sema3A and NP-1 to be altered in SLE. The milieu of increased pro-inflammatory cytokines such as IFN-γ and IL-6 may be a factor.

Further, sema3A has been reported to be important in the induction of the apoptosis of many immune cells, such as monocytes and macrophages, when these were found to be resistant to Fas-induced apoptosis [28]. Therefore, when sema3A expression is altered auto-reactive B cells escape apoptosis and survive to overproduce autoantibodies, thus contributing to autoimmunity in SLE. The association between TLR-9 expression in B cells, especially memory B cells and the production of IL-10 and IL-6 cytokines in SLE was previously shown by many [29‐31]. The over-expression of TLR-9 in memory B cells was also shown to be in correlation with anti-dsDNA antibody production. Thus, modulation of TLR-9 expression has been suggested as a therapeutic target, aiming to reduce its expression in SLE to be followed by a reduction of IL-10 production which has been demonstrated to be associated with SLE disease activity. Considering sema3A to be an important regulator in SLE, we hypothesized that co-culturing sema3A with B cells of SLE patients could possibly reduce TLR-9 expression. Indeed, it was demonstrated in the present study that the addition of sema3A, in vitro, to B cells of SLE patients, significantly reduced ODN-CpG induced TLR-9 expression in memory B cells, supporting sema3A as a regulator of autoimmunity in SLE. These findings along with the observation of sema3A being reduced in SLE patients, in correlation with disease severity and autoimmunity, and memory B cells being beneficially responsive to sema3A suggest this regulatory molecule as a therapeutic agent for SLE to be assessed in the future.

Sema3A is a good marker for SLE disease activity. It has a regulatory mode of action, with proven abilities of decreasing TLR9 expression in memory B cells of SLE patients. In addition, sema3A is shown to enhance regulatory properties of B cells. Further studies will focus on the therapeutic potential of sema3A in SLE.