Regulatory T and B cells in pediatric Henoch–Schönlein purpura: friends or foes?

Henoch–Schönlein purpura (HSP) or immunoglobulin A (IgA) vasculitis is the most frequent systemic vasculitis in children mainly aged 4 to 8 years [1, 2] mostly affecting boys (sex ratio 1.2–1.6/1). The annual incidence varies from country to country, 13 to 20/100,000 children [3‐5]. HSP is characterized by a triad of purpura, arthritis or arthralgia, and abdominal pain. Diagnostic criteria were published in 2010 [6, 7]. The prognosis for HSP depends on the presence of renal involvement, occurring in 40% of children [8]. Two to 5% of children with HSP nephritis progress towards renal or end-stage renal failure.

HSP is a non-granulomatous IgA vasculitis of the small blood vessels. Skin biopsy reveals leukocytoclastic vasculitis with fibrinoid necrosis and an inflammatory perivascular infiltrate of neutrophilic polymorphonuclear and mononuclear cells. IgA deposits, the complement fraction C3, and fibrin are observed on the injured vessel walls. Kidney involvement varies from focal or diffuse mesangial proliferation to crescent deposits, with diffuse IgA deposits. Most studies on the pathophysiology of HSP have focused on the dysregulation of IgA production [9]: increased serum IgA levels, IgA-containing immune complexes which alter the vessels, presence of abnormal IgA1 glycosylation in renal disease, abnormalities in the regulation of clearance of IgA from the liver, and coagulation disorders (high d-dimer concentrations) [10‐12].

Many contributing infectious triggering factors have been studied—mainly group A streptococcus infection but also other bacteria and viruses, exposure to drugs or toxic agents, and predisposing genetic factors (human leukocyte antigen [HLA] A2, A11, B35, mutation of the MEFV gene or heterozygous deficit in complement C) [13‐15].

Regulatory T lymphocytes (Tregs) are a subgroup of helper CD4 + T lymphocytes (5–10% blood CD4 + T lymphocytes) able to downregulate immune activation. Tregs inhibit effector T and B cells, natural killer cells (NK), NKT lymphocytes, and antigen-presenting cells. Tregs act by cell-to-cell contact or by secreting anti-inflammatory cytokines such as IL-10 and transforming growth factor-β (TGF-beta). In mice, suppressing Tregs or impairing their function leads to severe autoimmune and inflammatory syndromes with multi-systemic involvement, like IPEX syndrome in humans [16].

In addition to Tregs, it has been shown that other T cells can prevent autoimmunity in rodents. Most of these cells, such as TR1 cells secreting IL-10, helper T3 (Th3) cells secreting TGF-beta, and certain CD4 − CD8 − and CD8 + CD28 − T cells, are adaptive regulatory cells: they acquire regulatory functions according to specific antigenic stimuli and cytokine environments.

TGF-beta induces IgA production by B cells stimulated with lipopolysaccharide (LPS) [17‐19]. Thus, TGF-beta can act as a true isotypic switching factor for IgA production during the HSP acute stage [20].

Finally, a subset of B cells, regulatory B cells (Bregs) expressing IL-10, suppress CD4 + T cell-mediated production of pro-inflammatory cytokines [21‐23]. In adult HSP nephritis patients, the proportion of Bregs was significantly lower than in healthy controls [24]. In HSP children, the Breg percentage and their ability to produce IL-10 were lower in patients with renal involvement and lowest in those with massive proteinuria [25].

The complete pathogenesis of HSP remains unknown. Certain T lymphocyte populations, including Treg and Th3, can modulate the IgA switching factor TGF-beta production. This suggests the possible role of natural and adaptive Tregs in the pathophysiology of HSP. The primary objective of our study was to analyze immune regulation in HSP.

The involvement of Tregs in HSP has often been suspected. By contrast to the literature data, our work highlighted that a higher percentage of Tregs was associated with HSP. Indeed, previous studies described an absence of difference in Treg percentages between HSP and HCs [29], or even a decrease in Treg percentages in HSP compared to HCs [25, 30, 31].

In adults, age, sex, and ethnicity have emerged as major factors contributing to variations in lymphocyte phenotype composition [32, 33]. For example, the reference range of Tregs proposed for adult Chinese and Italian populations is different (2.17–7.94% vs. 0.59–0.79%). In childhood, Treg percentages are similar in the male and female groups (personal data not shown). The absolute number of lymphocytes drastically decreases with age with a significant slope in both male and female groups. However, there is no correlation between Treg percentages and age (personal data not shown).

One explanation for these divergent data may be how Tregs are identified [34, 35]. Another possibility is the clinical forms of the disease analyzed. The timing of sampling relative to the onset of disease may also influence the results of immune parameters. In two of three children, there are no recurrent episodes [1]. Furthermore, one retrospective study found no biological differences between patients with only one HSP flare and those with HSP recurrence [36].

In our HSP population, there was an increase in the IL-17A serum level and a similar trend for IL-1beta and IL-8 serum levels, as reported earlier [29, 31, 37]. This suggests that the action of Treg and Th3 is insufficient to control inflammation.

In our population, Breg percentages tended to be lower in HSP nephritis compared to HSP without kidney involvement. Previously, in HSP children compared to HCs, Yang et al. found a decrease in Breg frequencies in HSP with kidney impairment compared to HSP without kidney impairment and HCs, and no difference in Breg frequencies between HSP on remission and HCs [25]. In an adult population with non-treated HSP nephritis compared to HCs, Breg frequencies and IL-10 levels appeared lower, yet, on treatment, both parameters were restored [24]. This leads to the interesting hypothesis that Bregs might play a role in preventing nephritis in HSP.

As already described, we found an increase in serum IgA levels [9]. In our study, like others [9, 20, 37], serum TGF-beta levels were higher in the HSP population. Li et al. described a tendency towards an increase in TGF-beta levels [29]. This is in line with the fact that TGF-beta induces an IgA switch [17‐19]. Indeed, mice deficient in TGF-beta or receptor II TGF-beta have low levels of IgA [38, 39].

A striking observation in our study is the paradoxical effect of patients’ Tregs on IgA-secreting cells in vitro. Physiologically, Tregs promote an IgA switch. Thus, in mice, Treg depletion reduces circulating IgA levels, and the transfer of Tregs promotes IgA production via TGF-beta [26, 27]. The effect of Tregs on IgA is the same in IgA nephropathy, since IgA serum levels of rats that received Tregs from patients with an IgA nephropathy were significantly higher than in rats that received Tregs from a control group [40]. In the HCs of our study, Treg depletion did reduce IgA production. However, we observed that HSP patients’ Tregs depletion favored IgA production. Our data are in line with the inverse correlation between the number of circulating Tregs and serum IgA concentrations noted in patients with ankylosing spondylitis [41]. This suggests that, in HSP, Tregs might be trying to dampen IgA synthesis rather than induce it. One hypothesis is that activated Th3 cells are responsible for an IgA overproduction/switch that Tregs try to dampen. Another hypothesis is that it is the response of immunoglobulin A-secreting cells to Tregs which is modified in HSP. Further studies are needed to identify whether this is a regulatory deficit due to Tregs or to the response to Tregs.

Based on these results, we propose the following regulatory pattern for HSP (Additional file 3: Fig. S1): Following immune stimulation by a potential viral or bacterial infection [15], the antigen-presenting cells activate the Th3 cells which, by secreting TGF-beta, lead to IgA overproduction by B cell lineage. The IgA produced are deposited on the vessels leading to vasculitis and tissue damage. The Tregs which are not deficient try to dampen the inflammation and, surprisingly, IgA production during the acute phase. In patients with Breg deficiency, the uncontrolled production and deposition of probably abnormally glycosylated IgA [10] will lead to kidney damage.

To summarize, we observed an increase in Tregs and Th3 cells apparently failing to inhibit immune activation. Remarkably, Breg cells are fewer in the HSP population with nephropathy. Finally, we unveiled the unusual negative effect of Tregs on IgA production which is also insufficient.