Introduction
Juvenile idiopathic arthritis (JIA) is a heterogeneous T cell-mediated autoimmune disease. Common to all seven subtypes of JIA, disease onset is prior to the age of 16 years and is characterized by a chronicity of at least six weeks. For adult rheumatoid arthritis (RA), it has been suggested that T cells play an important role in the pathogenesis of the disease. Patients with RA present with immune system abnormalities that resemble the typical characteristics of immune dysfunction described in older patients [
1]. Immunological investigations of patients with RA have revealed disturbed T cell homeostasis and diminished thymus output, which was characterized by decreased amounts of T cell receptor excision circles (TRECs) and compensatory peripheral T cell proliferation with telomere shortening [
2] and loss of the co-stimulatory molecule CD28, a typical sign of replicatively stressed cells [
3]. Patients with JIA demonstrate premature immunosenescence of the CD4
+ naive T cell pool with age-inappropriate low TREC numbers, shortened telomere lengths and increased peripheral replication of peripheral naive T cells [
4]. TRECs are stable deoxyribonucleic acid (DNA) episomes forming during T cell receptor rearrangement. TRECs are not replicated during mitosis and are diluted out during cell divisions. Therefore, TREC counts in naive T cells are not only recognized as a marker for recent thymic emigrants (RTE), but are also influenced by peripheral replication of naive T cells [
5]. Telomeres are TTAGGG-rich repeats located at the ends of chromosomes and play an important role in DNA replication and preservation of chromosome integrity. Telomere erosion has been considered a mitotic clock, with the telomere length approximately reflecting the life history of divisions of individual cells [
6].
The influence of clinical disease activity on these parameters is still unclear in JIA. The present investigation of immunosenescence parameters was performed via comparison of a case of acute exacerbation of JIA against six patients with JIA with disease remission and six age-matched healthy donors (HD) over a follow-up course of 12 months.
Discussion
Our patient’s case gives an impression of disturbed peripheral CD4+CD28+CD45RA+ T cell homeostasis under the condition of acute disease exacerbation and underlines the importance of considering disease activity when evaluating immunological aging markers. Although limited by a small number of patients with JIA, the present study corroborates the fact that markers of immunosenescence such as decreased naive T cells and lower TREC numbers can only be interpreted correctly together with replication markers such as telomere length or Ki67 expression. Intracellular cytokines may be supportive parameters but are shown to be too variable to act as reliable markers to study clinical aspects of disease activity.
Loss of CD28 expression has been suggested as a marker of replicative senescence for T cells [
10]. A clonal expansion of CD8
+CD28
- T cells was found in older people [
10] and individuals with RA [
3]. The data from the follow-up cohort confirm the findings of an accelerated decrease of naive T cells in patients with JIA with advancing chronological age [
4], which was also demonstrated for patients with RA [
1,
2]. However, this comparison is limited as we were not able to provide follow-up data of the HD group. Although acute exacerbation did not change the total counts of CD4
+CD28
+CD45RA
+ or total CD28-expressing T cells, percentages of CD28-expressing CD4
+ T cells were significantly decreased in our patient with acute exacerbation, whereas percentages of CD28-expressing CD4
+ T cells were different between JIA follow-up and HD.
TRECs were evaluated to estimate thymic output and peripheral T cell turnover under the condition of acute disease exacerbation. TREC numbers were lower in patients with JIA compared to HD and unaltered by follow-up in the JIA group. These results corroborate the findings that patients with JIA show age-inappropriate lower TREC numbers but with no correlation with chronological age [
4]. Our patient with acute exacerbation showed relatively low TREC numbers during the acute exacerbation period compared to patients with JIA with disease remission. In our patient with acute exacerbation, enhanced TREC numbers and even higher TREC numbers than the other patients with JIA were found after disease remission, suggesting recovery of thymus function after successful treatment. This is in accordance with findings in children and younger adults with acquired immunodeficiency syndrome (AIDS) under highly active anti-retroviral therapy (HAART) [
11] in whom recovery to higher levels than before HAART was found. The study suggested that ongoing production of naive CD4
+ T cells in the thymus is most active during the earlier stages of HAART and declines later during HAART. However the precise timing of when children show maximum thymic output during HAART remains unclear. An increase in the fraction of TREC
+ T cells per naive T cell, which was equivalent to a faster growth of TREC
+ T cells versus total naive T cells was also found for CD4
+ T cells following initiation of HAART [
12]. When discussing the TREC dynamics in our patient with acute exacerbation, we cannot sufficiently answer the question as to whether our patient already had low TREC numbers before exacerbation, as found in children with AIDS before initiation of HAART.
However, TREC numbers are also influenced by peripheral proliferation and, therefore, dilution of TRECs may be caused by increased replication of peripheral CD4
+ naive T cells, which may be confirmed by enhanced Ki67-expressing T cells during acute exacerbation in our patient. Additionally, decreased thymic output will induce compensatory autoproliferation, a process that contributes to TREC dilution. Chronic T cell stimulation induces increased T cell turnover in patients with JIA, as demonstrated by age-inappropriate short T cell telomere lengths in patients with JIA. Age-inappropriate thymic involution and increased peripheral T cell turnover may each contribute to the lower TREC numbers observed in patients with JIA. These data highlight the pitfalls of interpretation of TREC data [
13], therefore, replication markers such as Ki67 expression and RTL were considered in our study. TREC numbers were calculated to CD4
+ naive T cell counts, the latter unaltered by acute exacerbation. In view of these results it has to be considered that the peripheral naive T cell pool consists of two subsets of cells, namely RTE and peripheral replicating cells. Increased replication of naive T cells may be the crucial factor in acute exacerbation and disease activity. However, despite high replication activity increased amounts of naive T cells were not found in the peripheral blood samples from our patient with acute exacerbation, possibly due to accumulation in other compartments, (for example, the inflamed joints). Maturation of naive T cells to CD45RO
+ memory T cells may be a possible explanation for disappearance of peripheral blood naive T cells despite increased replicative activity. This was also assumed for a model describing naive T cell dynamics in children and younger adults receiving HAART [
12]. Increased priming into memory T cells may be also true for our patient with acute exacerbation, who showed markedly increased CD8
+CD45RO
+ counts. During remission, enhanced thymus output of RTE, decreased replication activity of naive T cells, priming into memory T cells and/or depletion of peripheral blood naive T cells by accumulation in other sites may have contributed to the increased TREC numbers in naive T cells seen in the 12-month follow-up of our patient.
There was a tendency to lower RTL in the 12-month follow-up of patients with JIA compared to HD. Although limited by missing follow-up data from HD, these results fit with the tendency of increased Ki67-expressing CD4
+ naive T cells in patients with JIA compared to HD [
4]. Telomeric erosion was also clearly accelerated in patients with RA [
2], which was explained as a consequence of compensatory cell renewal.
Cytokines have been implicated in driving homeostatic T cell replication. Their most likely disease-related production could enhance T cell proliferation and exhaust the replicative potential [
14]. JIA has been associated with high serum or intra-articular levels of proinflammatory cytokines such as IL-6, IL-1α, IL-1β or TNFα [
15]. In fact, our study demonstrated an increase in the levels of TNFα, IFNγ and IL-2 in both CD45RA
+ and CD45RO
+, CD4/CD8 T cell subpopulations in patients with JIA. Moreover, high levels of these pro-inflammatory cytokines were observed in our patient with acute exacerbation during the exacerbation state, suggesting a Th1 cytokine-driven mechanism supported by an accumulation of CD45RO
+ memory T cells and the reduction in the naive T cell population. Interestingly, classical cytokines for memory and effector T cell responses, such as TNFα and IFNγ, were increased in the CD45RA
+ T cells. One may ask the question whether CD45RA
+ alone is a reliable marker for naive T cells, as effector T cells also express CD45RA but lose CD28 surface expression. For this reason it may be more likely that the proportions of increased TNFα and IFNγ within the CD45RA
+ T cell pool are predominately caused by activation of effector T cells.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GA performed the flow cytometry analysis and interpretation of data. MZ recruited patients and handled clinical data. CK performed the telomere length analysis and interpretation of telomere data. AB performed the Ki67 analysis and interpretation of proliferation data. VJ performed the lymphocyte isolation, autoMACS™ separation and DNA preparation. CDu performed the analysis of intracellular cytokines by flow cytometry. CDe analysed and interpreted the intracellular cytokine data. JB recruited patients and helped with interpretation of clinical data. MP designed the study and wrote the paper. All authors read and approved the final manuscript.