Background
Granulomatosis with polyangiitis (GPA) is a severe systemic autoimmune disease of unknown etiology. The hallmark of the disease is the presence of antineutrophil cytoplasmic autoantibodies (ANCAs) mainly directed against protienase-3 (PR3) [
1]. GPA is characterized by necrotizing granulomatosis in the respiratory tract, and a systemic vasculitis preferentially affecting pulmonary and renal small- and medium-sized blood vessels. The abundance of T cells in these vasculitic and granulomatous lesions of GPA patients support their involvement in disease pathogenesis [
2]. There is substantial evidence of activated T cells and antigen-driven T cell responses in GPA [
3‐
5] In addition, remission has been induced with therapeutics directed against T cells in patients with refractory GPA [
6,
7]. These studies strongly indicate a T cell-mediated pathology in this disease.
The involvement of cluster of differentiation (CD)4
+ T helper (T
H) cells in the pathogenesis of GPA has been suggested to depend on disease activity, and whether the disease is localized, i.e. restricted to the respiratory tract, or generalized. Prior to the discovery of T
H17 cells, research in GPA focused on the disturbed balance between T
H1 and T
H2 cells. It was found that GPA patients with active disease demonstrated a dysregulated cytokine prolife of circulating T cells with increased IFN-γ production versus a normal interleukin (IL)-4 production [
8]. Additional studies demonstrated the presence of T
H1-associated markers in the circulation as well as in nasal granulomatous lesions of patients with localized disease, while T
H2-associated markers were dominant in generalized disease [
9‐
11]. More recently, levels of IL-17A, the T
H17-associated cytokine, were found to be elevated in serum of GPA patients irrespective of active or quiescent disease [
12]. In addition, a relative increase in autoantigen-specific T
H17 cells in GPA patients has been reported [
12,
13].
Defects in regulatory T cell (T
REG) function in GPA patients may contribute to abnormal skewing in T
H cell responses and may result in an expansion of the CD4
+ effector memory T (CD4
+ T
EM) cell population [
14]. In addition, altered T
H cell distribution in GPA patients may be in part driven by chronic cytomegalovirus (CMV) infection [
15]. We have demonstrated previously that circulating CD4
+ T
EM cells (CCR7
-CD45RO
+) in GPA patients were proportionally increased during remission [
16], but were decreased during renal active disease upon migration to the inflammatory site [
17]. However, during active renal disease not all circulating CD4
+ T
EM cells tend to migrate to the target tissues [
17]. It is possible that a subset of circulating CD4
+ T
EM cells have a distinct migratory capacity and pathogenic function in GPA patients related to distinct clinical manifestations.
The recruitment of the CD4
+ T
EM cells to inflammatory sites is orchestrated by their chemokine receptors. Analysis of chemokine receptor expression has been instrumental in the characterization of memory T
H subsets with distinct cytokine patterns and antigen responses [
18]. The expression pattern of four chemokine receptors allows the identification of CD4
+ T
EM subsets, which are defined as T
EM1 [C-C chemokine receptor (CCR)6
- CXC chemokine receptor 3 (CXCR3)
+CCR4
+CRTh2
-] T
EM2 (CCR6
-CXCR3
-CCR4
+CRTh2
+), T
EM17 (CCR6
+CXCR3
-CCR4
+CRTh2
-) [
19,
20], and a subset that exhibits both T
H17 and T
H1 features, referred to as T
EM17.1 (CCR6
+CXCR3
+CCR4
- CRTh2
-) [
21,
22].
The aim of the present study was to determine the distribution of circulating CD4+ TEM cell subsets based on chemokine receptor expression in GPA patients. Identification of particular circulating CD4+ TEM subsets may reveal distinct associations of specific CD4+ TEM subsets with clinical manifestations or with autoantibodies in GPA patients.
Methods
Study population
Peripheral blood was collected from 63 GPA patients in remission (r-GPA) and 42 age- and sex-matched healthy controls (HCs) in a cross-sectional study..The r-GPA patients fulfilled the criteria of the American College of Rheumatology and the Chapel Hill Consensus Conference definition for GPA [
23,
24]. Only patients with PR3-ANCA positivity at diagnosis, and complete remission of their disease at the time of sampling, were included in the study. The PR3-ANCA titers were measured by indirect immunofluorescence (IIF) on ethanol-fixed human granulocytes according to the standard procedure as described previously [
25]. ANCA titers lower than 1:20 were considered negative. Complete remission was defined as the complete absence of clinical signs and symptoms of active vasculitis, as indicated by a score of zero on the Birmingham Vasculitis Activity Score (BVAS) [
26]. According to these criteria, blood samples were taken during a visit to our outpatient clinic.
Disease extent was defined as localized when GPA was restricted to the upper and lower respiratory tract and generalized when systemic disease with vasculitis extended to more clinical manifestations including involvement of kidneys, joints, eye, and nervous system. None of the patients and controls experienced an infection at the time of sampling.
Twenty-nine of 63 r-GPA patients were treated with maintenance immunosuppressive therapy at time of blood sampling. Three r-GPA patients received azathioprine, 12 r-GPA patients received azathioprine in combination with prednisolone, six r-GPA patients were treated with low-dose prednisolone, seven r-GPA patients received low-dose prednisolone in combination with mycophenolate mofetil (MMF), and one r-GPA patient was treated with methotrexate.
Detailed clinical and laboratory characteristics of the patients are summarized in Table
1. All patients and healthy volunteers provided informed consent and the local medical ethics committee approved the study.
Table 1
Laboratory and clinical characteristics of r-GPA patients and HC
Subjects, n (% male) | 63 (% 44) | 42 (% 40) |
Age, mean (range) | 62.3 (26.8–85.2) | 57.2 (21.5–86.8) |
PR3-ANCAa, n (% positive) | 39 (% 62) | |
PR3-ANCA titer, median (range) | 1:40 (0–1:640) |
Creatinine umol/L, median (range) | 86 (52–224) |
CRP mg/L, median (range) | 2.7 (0.3–99) |
eGFR ml/min*1.73 m2, median (range) | 64 (21–109) |
CMV seropositive, n (% positive) (N.D.) | 33 (% 54) (2) | 21 (% 58) (6) |
S. aureus, n (% positive) (N.D.) | 27 (% 44) (1) | |
BVAS, mean | 0 |
Disease duration in years, median (range) | 9.6 (1.9–42.7) |
No. of total relapses, median (range) | 1 (0–7) |
Relapserb, n (%) | 43 (% 68) |
Disease type, n (% generalized) | 52 (% 83) |
Treatment at time of sampling, n (%) |
Azathioprine | 3 (% 5) | |
Azathioprine + prednisolone | 12 (% 19) |
Prednisolone | 6 (% 10) |
Mycophenolate mofetil + prednisolone | 7 (% 11) |
Methotrexate | 1 (% 2) |
No immunosupressive treatment | 34 (% 54) |
Co-trimoxazole, high dose/low dose/no dose | 17/15/31 |
No. of organs involved, median (range) | 3 (1–7) |
Clinical manifestations, n (%) |
Renal | 35 (% 56) | |
ENT | 45 (% 71) |
Joints | 36 (% 57) |
Pulmonary | 40 (% 63) |
Nervous system | 20 (% 32) |
Eyes | 24 (% 38) |
Cutaneous | 13 (% 21) |
Other | 7 (% 11) |
Sample preparation and immunophenotyping by flow cytometry
EDTA-anticoagulated peripheral blood was obtained by venipuncture from r-GPA patients and HCs. Whole blood samples were stained within 4 hours after blood withdrawal with appropriate concentrations of fluorochrome-conjugated monoclonal antibodies for cell surface antigens. The samples were immediately processed to obtain the most sensitive detection for the chemokine receptor expression and to minimize cell manipulation. The peripheral blood was stained using the following monoclonal antibodies for cell surface antigens in combination: Alexa Fluor® 700-conjugated anti-CD3, eFluor® 450 (eF450)-conjugated anti-CD4 (both from eBioscience, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-CD45RO, phycoerythrin-cyanin7 (PE-C7)-conjugated anti-CCR7 (both from BD Biosciences, Franklin Lakes, NJ, USA), PE-conjugated anti-CRTh2, allophycocyanin-C7 (APC-Cy7)-conjugated anti-CXCR3, peridin chlorophyll α-protein (PerCP-Cy5.5-conjugated anti-CCR4, and Brilliant Violet 605™ (BV605)-conjugated anti-CCR6 (all from BioLegend, San Diego, CA, USA). The appropriated isotype-matched control antibodies of irrelevant specificity were added to a separate tube as negative controls. Samples were incubated for 15 minutes at room temperature. Afterward, cells were treated with 2 mL diluted FACS lysing solution (BD Biosciences) for 10 minutes. Finally, the samples were washed in PBS containing 1% (w/v) bovine serum albumin (BSA), and immediately analyzed by eight-color flow cytometric analyses on BD™ LSR II flow cytometer. Data were collected for 1.0 *106 events for each sample and plotted using Kaluza v1.2 (Beckman Coulter, Brea, CA, USA). Lymphocytes were gated for analysis based on forward and side scatter properties. Positively and negatively stained populations were calculated by quadrant dot-plot analysis or histograms, determined by the appropriate isotype controls. Within the CD4+ TEM cell subset (CD4+CCR7-CD45RO+) the expression pattern of chemokine receptors CCR6-CXCR3+CCR4-CRTh2-, CCR6-CXCR3-CCR4+CRTh2+, CCR6+CXCR3-CCR4+CRTh2-, and CCR6+CXCR3+CCR4-CRTh2- were used to distinguish TEM1, TEM2, TEM17, and TEM17.1 cells, respectively.
Detection of S. aureus
From 62 r-GPA patients,
S. aureus nasal carriers were determined as described previously [
27]. Briefly,
S. aureus nasal isolates were sampled by rotating a sterile cotton swab in each anterior nary. Swabs were inoculated on 5% sheep-blood and salt mannitol agar for 72 h at 35 °C.
S. aureus was identified by coagulase and DNase positivity. Patients were considered to be chronic nasal carriers when ≥50% of their nasal cultures grew
S. aureus.
CMV ELISA
CMV-specific IgG was determined in serum samples using an in-house enzyme-linked immunosorbent assay (ELISA). In brief, 96-well ELISA plates (Greiner, Kremsmünster, Austria) were coated overnight with lysates of CMV-infected fibroblasts. Lysates of non-infected fibroblasts were used as negative controls. Following coating, serial (1:100–1:3200) dilutions of serum samples were incubated for 45 minutes. Next, goat anti-human IgG-HRP (Southern Biotech, Birmingham, AL, USA) was added and incubated for 45 minutes. Samples were incubated with TBE substrate (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes and the reaction was stopped with sulfuric acid. The plates were scanned on a Versamax reader (Molecular Devices, Sunnyvale, CA, USA). A pool of sera from three CMV-seropositive individuals with known concentrations of CMV-specific IgG was used to quantify levels of CMV-specific IgG in the tested samples.
Statistical analysis
Statistical analysis was performed using SPSS v22 (IBM Corporation, Armonk, NY, USA) and GraphPad prism v5.0 (GraphPad Software, San Diego, CA, USA). Data are presented as median values. Data were analyzed with the D’Agostino-Pearson omnibus normality test for Gaussian distribution. For comparison between r-GPA patients and HCs the unpaired t test was used for data with Gaussian distribution and the Mann-Whitney U test for data without Gaussian distribution. For intra-individual comparison of values at multiple time points during follow-up, repeated measures analysis of variance was used if data were normally distributed and a Friedman test was used if data had a non-Gaussian distribution. The association between clinical parameters and CD4+ TEM cell subsets in inclusion samples of r-GPA patients was investigated using the Spearman’s rank correlation coefficient. In order to account for interactions of CMV and age on the percentage of CD4+T cells subsets and CD4+TEM cell subsets we used a linear (Enter) regression analysis. Non-normally distributed data were log-transformed. Differences were considered statistically significant at two-sided p values equal to or less than 0.05.
Discussion
In this study, we aimed to determine the distribution of circulating CD4+ TEM cell subsets based on chemokine receptor expression in GPA patients. We demonstrated a significant increase in the proportion of TEM17 cells with a concomitant decrease in the proportion of TEM1 cells in peripheral blood of patients with r-GPA. Increased proportions of TEM17 cells were more pronounced in r-GPA patients with systemic manifestations, whereas r-GPA patients with local manifestations showed a remarkable increase in TEM1 cells. Interestingly, CMV seropositivity appeared to modulate the disturbed balance of TEM1 and TEM17 cells in r-GPA patients.
The decreased proportions of T
EM1 cells in r-GPA patients compared to HCs reflect an aberrant T
EM1 response in patients. It has been demonstrated that GPA patients with active or localized disease show a polarization toward a T
H1-type response [
8,
11,
31]. These studies showed an abundant T
H1 cytokine (IFN-γ) and chemokine (CCR5) pattern on circulating T cells, as well as in granulomatous lesions compared to patients in remission or with generalized disease [
11,
31]. It has been suggested that the disturbed T
H1 response might play a role in the initiation of GPA. The disease can progress into a generalized GPA with a less prominent T
H1-type response. The majority of r-GPA patients included in this study present generalized disease with a median disease duration of 9.6 years. This might explain the decreased proportion of circulating T
EM1 cells in our r-GPA patients. However, one may also argue that the relative decrease in circulating T
EM1 cells is due to an increased tissue migration of these cells. In GPA patients with generalized disease it has been reported that renal lesions show polarization toward T
H1 type-responses [
32]. However, we did not observe an association of T
EM1 cells with renal involvement in r-GPA patients.
Our results regarding the increase in T
EM17 response in GPA patients are in line with previous reports on increased T
H17-associated activity in these patients. It has been reported that antigen-specific T
H17 cells are expanded in GPA patients, irrespective of disease activity and maintenance therapy [
13,
33]. In addition, serum IL-17A levels are also found to be elevated in active GPA patients and remained elevated in GPA patients recovering from active disease [
12]. In line with this result, we observed a sustained T
EM17 expansion over a period of 6 months in our r-GPA patients. Altogether, the involvement of T
H17 cells in the immunopathology in GPA appears to be well established, although presently it remains unclear which mechanisms initiate T
H17 responses in GPA. Possible explanations for the expanded T
EM17 population might be related to the presence of granulomas, or chronic nasal carriage of
S. aureus in GPA.
Granulomas are sophisticated and highly organized structures that typically consist of a sphere of highly activated macrophages surround by T lymphocytes. They provide a specialized niche for macrophage-T cell interaction, contributing to the differentiation and maturation of T cells [
34]. The pro-inflammatory cytokine environment in granuloma may contribute to the aberrant T
H1 and T
H17 cell distribution found in the circulation. Since, granulomas are common clinical manifestations in GPA patients they may provide an ideal environment for T
EM17 cell expansion. Engagement of CD4
+T
EM cells with IL-6/TGFβ-producing macrophages may promote CD4
+T
EM cell differentiation into T
EM17 cells. In addition, macrophages also secrete IL-23, which sustains the T
H17 population. Indeed, elevated serum levels of TGFβ, IL-6, and IL-23 have been reported in GPA, and, importantly, elevated levels of IL-23 correlated with disease severity in patients with GPA [
12].
Chronic carriage of
S. aureus constitutes a risk factor for the development of exacerbations in GPA. We have previously shown that the frequency of chronic nasal carriage of
S. aureus is higher in GPA patients compared to HC [
30]. Moreover, it was shown that nasal
S. aureus carriage is associated with increased risk of relapse [
30,
35]. Staphylococcal superantigens act as potent immune stimulators for T cells, resulting in polyclonal T cell proliferation and pro-inflammatory cytokine production [
36]. In vitro studies demonstrated that stimulating T cells with staphyloccal exotoxins (alpha-toxin and SEB), strongly induced IL-17A-secreting T cells [
13,
37]. Therefore, the involvement of T
H17 cells in GPA may possibly be driven by chronic nasal carriage of
S. aureus. However, we did not observe increased frequencies of T
EM17 cells in GPA patients carrying
S. aureus. This observation is in line with earlier studies in GPA patients in which no correlation between the presence of staphylococcal superantigens and the expansion of T cell subsets in peripheral blood was found [
27].
Remarkably, we observed that the proportion of T
EM17 cells in r-GPA patients was highly associated with CMV serostatus with frequencies of T
EM17 cells being decreased in CMV-seropositive r-GPA patients as compared to seronegative r-GPA patients. These observations indicate that latent infection with human CMV modulates the distribution of T
EM cell subsets, although the underlying mechanisms are unclear. For instance, CMV seropositivity is strongly associated with the presence of memory T cells. It has been demonstrated that only CMV-seropositive individuals possess significant numbers of CD4
+CD28
- T cells and many of these T cells respond to CMV [
38]. In fact, the expansion of CD4
+CD28
- T cells in GPA is suggested to be driven by CMV infections, and is associated with increased risk of infection and mortality [
15]. However, the precise role of CMV infection in T
H1 and T
H17 responses is poorly understood. Previous studies indicate that T cells expressing CXCR3 (T
H1 type) arise during primary CMV infection and are maintained during latency [
39]. In line with this study, we observed increased proportions of T
EM1 cells in the circulation of CMV-seropositive r-GPA patients. The skewing toward a T
EM1 response in CMV-seropositive r-GPA patients could also explain the decrease in the proportion of T
EM17 cells since these two T
EM cells subsets inversely correlate with each other. Importantly, the difference in T
EM1 cells between r-GPA patients and HCs was not influenced by CMV and age. Additionally, CMV serostatus did not influence the proportions of T
EM1 cells in HCs whereas in r-GPA patients CMV serostatus had a major impact on both the proportions of T
EM1, and T
EM17 cells.
T
H17 cells may also induce autoimmune responses. Very recently, it was shown that the frequency of T
H17 cells (CCR6
+) in rheumatoid arthritis (RA) patients is associated with anti-citrullinated protein antibodies (ACPA) status [
28]. In particular, CCR6
+ T
H cell proportions were higher in ACPA-positive RA patients in comparison to ACPA-negative RA patients, and inversely correlated with disease duration in ACPA-negative patients. If this were the case in GPA patients, one may argue that the increase in T
EM17 cells might be associated with ANCA status and could be a tool to discriminate ANCA-positive patients from those that are ANCA-negative. In contrast to the data in RA patients, we did not observe any association regarding ANCA status with the frequency of T
EM17 cells in r-GPA patients. This is possibly due to the fact that ANCA titers in GPA patients fluctuate during the disease course, whereas ACPA-positive RA patients consistently remain ACPA-positive over time. On the other hand, we found that T
EM17 cells in GPA patients showed a positive association with organ involvement, whereas T
EM1 cells were negatively associated with organ involvement. This suggests a more severe disease course in individuals with a high frequency of T
EM17 cells. Furthermore, we observed that persistent T
EM17 expansion is associated with a higher tendency to relapse.
The current study was designed as a cross-sectional study using peripheral blood of quiescent GPA patients and HCs. The main limitations are the lack of absolute lymphocyte counts and study samples from GPA patients with active disease. Therefore, the current data only provides observational information of proportions of circulating CD4+ TEM cell subsets in r-GPA patients. Further studies are warranted to assess blood samples from patients during active disease and to study the distribution of infiltrated TEM cell subsets in nasal and renal biopsies to elucidate distinct migratory capacities of TEM1 and TEM17 cells and to confirm their role in inflamed target tissues in GPA. Since TEM cells also appear in the urine during active renal GPA disease, analysis of urine samples might aid in demonstrating which distinct TEM subsets are possibly involved in renal injury.