Background
ESRD patients have a defective T-cell mediated immune system that is clinically characterized by an increased risk of a variety of infections [
1,
2] and impaired response of vaccination [
3‐
7]. Infections are the second leading cause of mortality following cardiovascular disease and a major cause of morbidity in ESRD patients [
8].
Uremia-associated T-cell defects closely resemble premature immunological T-cell ageing [
9]. ESRD patients have a discrepancy of 15–20 years between the immunological T-cell age and their chronological age [
10]. Declined thymic output, more differentiated memory T cells, T cells lacking co-stimulatory molecules like CD28, skewed T cell receptor (TCR) Vβ repertoire diversity and shorter telomere length are observed in ESRD patients compared to age-matched healthy individuals (HI) [
11].
TCR-induced signaling mediates clonal (positive or negative) selection of thymocytes in the thymus and initiates T cell immune responses in the periphery, consisting of T cell proliferation and differentiation [
12]. The mitogen-activated protein kinase (MAPK) pathway is one of the major pathways induced upon TCR stimulation [
13]. Activation of MAPK is mediated by phosphorylation of MAPK and downregulated by MAPK phosphatase resulting in inactive MAPK [
14]. In particular, the extracellular signal-regulated kinase (ERK) pathway is one of the important MAPK pathways. Phosphorylation of ERK can reduce sensitivity of cells to apoptosis and promote cell proliferation [
15]. ERK activity controls the positive feedback loop in the TCR-induced activation cascade and reduced ERK activity affects signal strength and activation of T cells [
16,
17]. Reduced phosphorylation of ERK is associated with decreased interleukin-2 (IL-2) production [
18] and vice versa [
19]. Dual specific phosphatases (DUSPs) represents a family of phosphatases that dephosphorylate phosphor-threonine and phosphor-tyrosine residues on MAPK and that are pivotal regulators of MAPK activities. DUSP6 is a cytoplasmic phosphatase with substrate specificity to dephosphorylate pERK [
20]. Ageing is also associated with defective signaling pathways [
21,
22]. Recently it was shown that decreased phosphorylation of ERK in naive CD4
+ T cells from elderly HI was associated with more time to build up the required signaling strength following stimulation compared to those from young HI. This decreased phosphorylation of ERK can be overcome by inhibiting DUSP6 [
16].
P38 is another pivotal protein in the MAPK pathway [
23] and of interest with respect to age-related changes is T cell activation. Most stimuli, including engagement of TCR, costimulatory receptors, inflammation, stress, growth factors, as well as DNA damage induce phosphorylation of p38 by various pathways [
24,
25]. Although phosphorylation of ERK and p38 from T cells share some upstream molecules after triggering of TCR, such as phosphorylation of CD3 zeta-chain associated protein kinase of 70 kDa (ZAP70) [
26], they each have their unique upstream MAPK kinases (MKKs) [
14]. Highly differentiated CD4
+ T cells lacking expression of CD28 are accumulated in elderly healthy individuals [
27], patients with ESRD [
28], following chronic viral infection [
29], and also in patients suffering from autoimmune disease [
30]. Senescent CD27
−CD28
−CD4
+ T cells employ an MKK-independent mechanism for phosphorylating p38 and depend on 5′ adenosine monophosphate-activated protein kinase (AMPK) and transforming growth factor-β-activated protein kinase 1(TAK1)-binding protein 1(TAB1) ex vivo [
31].
Little is known as to how MAPK signaling pathways in ESRD patients. Understanding MAPK signaling in ESRD patients may increase knowledge about mechanisms of uremia-associated impaired T-cell mediated immunity and offer possibilities for intervention. Here, we demonstrate that TCR-induced phosphorylation of ERK, and not p38, in CD4+ T cells decreases with age and T cell differentiation. This pathway is specifically affected in young ESRD patients and at the level of elderly healthy individuals, compatible with the concept of premature immunological T cell ageing in patients with renal failure. In addition, inhibition of DUSP6 may offer a potential intervention for improving T-cell mediated immunity in ESRD patients.
Methods
Study population
In line with our previous studies, young and elderly patients groups were defined based on their chronological age [
32,
33]. Twenty-four stable ESRD patients, defined as having a glomerular filtration rate of ≤15 ml/min with or without renal replacement therapy (RRT; i.e. dialysis) and 24 HI were included (Study population characteristics are described in Table
1) at the outpatient clinic. Patients with any clinical or laboratory evidence of acute bacterial or viral infection, malignancy, immunosuppressive drug treatment within 28 days prior to transplantation (except for glucocorticoids) were excluded. Lithium-heparinized blood was drawn of ESRD patients and healthy kidney donors. All individuals included gave informed consent and the local medical ethical committee approved the study (METC number: 2012–022). It was conducted according to the principles of Declaration of Helsinki and in compliance with International Conference on Harmonization/Good Clinical Practice regulations.
Table 1
Clinical characteristics of the study population
Number of individuals | 24 | 24 | |
Age groups (years; mean ± SD) |
young | 29,4 ± 5,6 | 34,6 ± 8,0 | ns |
elderly | 70,5 ± 5,8 | 70,8 ± 4,2 | ns |
Sex (% male) | 50 | 79,2 | ns |
CMV IgG serostatus (% pos) | 62,5 | 62,5 | ns |
RRT (number; %) | | 11; (45,8%) | |
Duration of RRT in months (median with range) | | 22 (1—37) | |
Hemodialysis (number) | | 7 | |
Peritoneal dialysis (number) | | 4 | |
Underlying kidney disease (number; %) |
atherosclerosis/hypertensive nephropathy | | 8; (33%) | |
primary glomerulopathy | | 4; (17%) | |
Diabetic nephropathy | | 6; (25%) | |
congenital disorder | | 3; (13%) | |
others | | 2; (8%) | |
unknown | | 1; (4%) | |
PBMCs preparation
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood from HI and ESRD patients as described previously [
34] and then cryopreserved for further analysis.
Phosphorylation-specific flow cytometry
PBMCs were stained with eFluor-450-labeled anti-CD7 (eBioscience, Vienna, Austria), allophycocyanin-Cy7 (APC-Cy7)-labeled anti-CD8 (BD, Erembodegem, Belgium), Brilliant Violet (BV)-510-labeled anti-CD16 (BD) and fluorescein isothiocyanate (FITC)-labeled anti-CCR7 (R&D system, Uithoorn, the Netherlands) for 30 min at room temperature. Then 1 million PBMCs/50 μl were prepared for stimulation by labeling cells with 20 μg/ml mouse anti-human CD3 (BD) and mouse anti-human CD28 (BD) each on ice for 20 min, followed by an incubation with goat-anti mouse IgG (BD) for cross-linking on ice for 20 min. Stimulation was initiated by transferring cells to a 37 °C water bath for 10 min. Cells were fixed using Cytofix (BD) at 37 °C for 10 min and then permeabilized in 70% methanol at -20 °C for 30 min. Subsequently, cells were stained with peridinin chlorophyll (PerCP)-labeled anti-CD4 (BD), phycoerythrin (PE)-Cy7-labeled anti-CD45RO (BioLegend, Uithoorn, Netherlands), PE-labeled anti-phospho-p38MAPK (pT180/pY182) (BD), and Alexa Fluor (AF) 647-labeled anti-phospho-ERK1/2 (pThr202/pTyr204) (BD). Phosphorylation was measured on a BD FACSCanto II flow cytometer (BD) and data were analyzed by Kaluza™ software (Beckman Coulter, Woerden, Netherlands). Median fluorescence intensity (MFI) of phosphorylated ERK or p38, generated by Kaluza™, were multiplied by 256 to make them comparable to the data analyzed by FACS Diva software (linear value instead of log-transformed value) (BD). The MFI obtained for anti-CD3/anti-CD28-stimulation were corrected by subtracting the MFI of the unstimulated condition.
CD69 and IL-2 measurement
PBMCs were either not or stimulated with anti-CD3/anti-CD28 T-cell expander beads (Invitrogen Dynal, Oslo, Norway) at different ratios, i.e. 1 cell / 0.1 bead, 1 cell / 0.5 bead, 1 cell / 1 bead for 6 h in human culture medium (HCM; RPMI-1640 with GlutaMAX, 10% heat-inactivated pooled human serum and 1% penicillin and streptomycin) (Lonza, Breda, Netherlands) with Golgistop (BD). Then cells were stained with AmCyan-labeled anti-CD3 (BD), Pacific Blue-labeled anti-CD4 (BD), APC-Cyanin 7 (APC-Cy7)-labeled anti-CD8 (BD), APC-labeled anti-CD45RO (BD) and PE-Cy7-labeled anti-CCR7 (R&D Systems) antibodies and a live–dead marker ViaProbe (7-aminoactinomycin D; 7AAD; BD). Upon fixation with FACS lysing solution (BD) and permeabilization using FACS permeabilizing solution 2 (BD), cells were stained intracellular using PE-labeled anti-CD69 (BD) and FITC-labeled anti-IL-2 (BD). Percentages CD69-expressing and IL-2 producing CD4+ T cell subsets were evaluated upon measuring the samples on a BD FACSCanto II flow cytometer (BD). Data were analyzed by Kaluza™ software (Beckman Coulter).
DUSP6/1 inhibition
PBMCs were pre-incubated in HCM including 50 μM (E)-2-benzylidene-3-(cyclohexylamino)-2, 3-dihydro-1 H-inden-1-one (BCI) (Merck – Millipore, Amsterdam, Netherlands) at 37 °C for 1 h. BCI has been shown to be an inhibitor of DUSP6 and DUSP1 activity. PBMCs were subsequently washed 3 times and then stimulated by CD3/CD28 antibodies (as described previously) for 10 mins and then MFI of pERK of BCI-pretreated T cells was measured by Phosphorylation-specific flow cytometry as described previously.
DUSP6/1 measurement
PBMCs were stained with AmCyan-labeled anti-CD3 (BD), Pacific Blue-labeled anti-CD4 (BD), APC-Cy7-labeled anti-CD8 (Biolegend); APC-labeled anti-CD45RO (BD) and PE-Cy7-labeled anti-CCR7 (R&D Systems) antibodies and 7-AAD for 30 min at 4 °C. Upon fixation and permeabilization using Fix/Perm buffer (eBioscience), 1% bovine serum albumin (Zwijndrecht, Netherlands) was used to block Fc receptors. Then cells were further stained with AF647-labeled anti-DUSP6 (Santa Cruz Biotechnology, Heidelberg, Germany) and PE-labeled anti-DUSP1 (Santa Cruz Biotechnology) for 30 min at 4 °C. MFI of DUSP6 and DUSP1 was measured on a BD FACSCanto II flow cytometer (BD) and data were analyzed using FACS Diva software version 6.1.2 (BD).
Statistical analyses
Data were analyzed by Graphpad Prism 6 (GraphPad Software, CA, USA). Comparison between two groups (non-parametric data) were using Mann Whitney test. Comparison in multiple groups were using Friedman test followed by Dunn’s Multiple Comparison T test or repeated ANOVA test followed by Bonferroni’s multiple comparison test. Comparison between DUSP6-treated and non-treated conditions was done by Paired T- test. All reported P-values are two-sided and were considered statistically significant when P < 0.05.
Discussion
The main observation of this study was that TCR-mediated phosphorylation of ERK in CD4+ T cells of young patients was in between young and old HI. Phosphorylation of ERK decreased in highly differentiated T-cell subsets compared to naive T cells. This defective TCR-mediated phosphorylation was specific as it could be restored by addition of a DUSP6 inhibitor. TCR-induced p38 phosphorylation was comparable between ESRD patients and HI.
Beyond midlife, the immune system shows age-related features and its defensive capabilities becomes impaired [
35]. The uremia-associated inflammatory environment present in ESRD patients accelerates this age-related immune senescence process. In addition to declined thymic output, accumulation of highly differentiated T cells, short telomere length [
10,
33,
36] and narrowed TCR-Vβ repertoire diversity [
32], this study indicates that young ESRD patients also have a defective CD4
+ TCR activation judging from the reduced capacity to phosphorylate ERK upon TCR-triggering. ERK activity is critical for TCR threshold calibration, as it controls positive feedback loops in TCR-induced activation [
17]. Reduced ERK activity impairs TCR signal strength and activation, and favors T cells with higher affinity to antigen to be activated, leading to a contracted immune response to a given antigen [
16]. The ERK phosphorylation upregulation of early activation marker CD69 on T cells ensures a proper inducing activation of T cells in the lymph node [
37], and also play an important role in T cell proliferation [
38] and IL-2 production [
39,
40]. In addition, ERK activation impacts cellular apoptosis as it inhibits Fas-mediated apoptosis in T cells [
41]. Evaluating ERK phosphorylation is a valuable tool to study more upstream molecules in the defective T-cell mediated immune system from ESRD patients. T cells from rheumatoid arthritis (RA) patients exhibit several defects which can also be viewed as premature immunological ageing [
42]. However, ERK phosphorylation in CD4
+ T cells of RA patients selectively increased [
43]. This increased ERK activation lowers the TCR threshold in T cells of RA patients to respond to self-antigens, which may partly explain the adaptive immune system of RA patients to exhibit abnormalities that go beyond the local inflammatory response in the synovium [
44].
DUSP6 is a cytoplasmic phosphatase with substrate specificity for phosphorylated ERK. In elderly individuals, silencing of DUSP6 increased the expression of T cell activation markers, such as CD69 and CD25, IL-2 production as well as proliferative response [
16]. Inhibition of DUSP6 could be a potential intervention to increase CD4
+ TCR-sensitivity by enhancing ERK phosphorylation in ESRD patients. BCI (an inhibitor of DUSP6 and 1) enhanced TCR-induced pERK in CD4
+ T cells from elderly HI, young and elderly ESRD patients, but not young HI, implying a role for DUSP6 and/or DUSP1 in regulation of ERK phosphorylation. Based on the age- as well as differentiation-related expression of DUSP6, but not DUSP1, in our HI, a potential role for DUSP6 may be present in defective TCR-induced ERK phosphorylation, especially in elderly HI and young patients. This needs to be confirmed in a larger cohort. Furthermore, use of siRNA specific for DUSP6 is required to draw a more definite conclusion with respect to the role of DUSP6 in defective TCR-induced phosphorylation of ERK in ESRD patients. The lack of age-related effects on pERK in CD8
+ T cells of ESRD patients and HI, the latter confirming observations done by another study [
16], as well as absence of effects of BCI on pERK levels in CD8
+ T cells, indicates a different role for DUSP6 in CD8
+ T cells compared to CD4
+ T cells. We did not observe an association between DUSP6 expression and ERK phosphorylation in CD4
+ T cells in ESRD patients. This could be due to the small cohort size or imply other DUSPs (e.g. 2, 4 or 5) [
45‐
49] or upstream signaling molecules to contribute to this defective TCR-induced ERK phosphorylation in ESRD patients.
ERK over-phosphorylation might be as bad as defective ERK phosphorylation. ERK over-activation from kidney cells occurs in the physiologic setting in some chronic kidney diseases, such as compensatory kidney hypertrophy and in pathologic conditions for example glomerular disease [
50]. Increased ERK phosphorylation in T cells predisposes for autoimmunity for example rheumatoid arthritis [
43]. Over-expression of DUSP6 is also reported to impair T-cell function in chronic viral infections such as hepatitis C virus infection [
51]. Therefore, more research is warranted evaluating inhibition of DUSP in the setting of defective T-cell mediated immunity in ESRD patients.
We analyzed the effect of latency for CMV as it represents chronic antigenic stimulation of T cells, but ERK- or p38-activation of CD4
+ or CD8
+ T cells was not different between the CMV-IgG seropositive population and CMV-IgG seronegative population following CD3/CD28 stimulation. Highly differentiated memory CD4
+ and CD8
+ T cells may accumulate in CMV seropositive individuals and are functional CMV-specific T cells [
29,
52,
53]. The results of our study show that non-specific TCR stimulation does not identify a defect p38 and ERK signaling associated with CMV seropositivity. In accordance with the results of a previous study, uremia is the major determinant affecting MAPK pathway parameters in ESRD patients and not RRT [
9,
11,
33].
In the present study, we induced phosphorylation of p38 in T cells via triggering CD3 [
54] and CD28 [
26]. Lack of CD28 may only partly explain the decreased p38 activation in more differentiated CD4
+ T cells. In addition to that, senescent human CD27
−CD28
− CD4
+ T cells lack several essential upstream components including ZAP70 and the loss of TCR signaling machinery in those cells was associated with a defective calcium influx [
31], which may indicate the decreased response of TCR-mediated activation in the more differentiated T cells. Interestingly, in contrast to the p38 activation following CD3/CD28 stimulation, baseline levels (spontaneous phosphorylation) of p38 increased during T cells differentiation [
55]. This might be caused by DNA damage in these more differentiated T cells and mediated by TAB1 (MKK-independent molecule), a key molecule involved in this auto-phosphorylation [
31].
Acknowledgements
Not applicable.