Soluble CD40L is associated with increased oxidative burst and neutrophil extracellular trap release in Behçet’s disease

This study included three main subject groups: (1) 30 healthy control subjects (HC), (2) 31 patients with iBD, and (3) 30 patients with aBD. All patients with BD met the International Study Group for Behçet’s Disease criteria [17]. Active BD was defined as a score ≥ 2 and iBD as a score of zero on the Simplified Behçet’s Disease Current Activity Form adapted for Portuguese by the Sociedade Brasileira de Reumatologia (sBR-BDCAF) [18]. The HC group comprised healthy adult volunteers from the hospital staff with no evidence or family history of autoimmune disease or immunodeficiency. Additionally, for some experiments, we used samples derived from 25 patients with sepsis (PSs), 27 patients with inactive pediatric systemic lupus erythematosus (iSLE), and 27 patients with active pediatric systemic lupus erythematosus (aSLE). The PS group, a positive control group for neutrophil activation, consisted of adult inpatients meeting the sepsis criteria of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference [19]. The SLE group consisted of pediatric patients meeting the American College of Rheumatology criteria for SLE [20]. Patients scoring ≥ 6 on the Systemic Lupus Erythematosus Disease Activity Index [21] were considered to have active disease, and those scoring zero were considered to have inactive disease. The study was approved by the institutional ethics review boards of Universidade Federal de São Paulo (CEP 0013/11) and Seattle Children’s.

Plasma pools for each group were assembled by mixing plasma samples derived from heparinized blood drawn from each subject enrolled in the study. Additionally, plasma from four different X-linked hyper immunoglobulin M patients collected at the immunology outpatient clinic of the University of Sao Paulo was used to constitute a pool of hyper immunoglobulin M plasma (HIgM). All pools were stored in aliquots at −80 °C.

RNA was isolated by standard RNA extraction using TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA) and transformed into complementary DNA (cDNA) by using the SuperScript One-Step RT-PCR System with Platinum Taq DNA polymerase (Life Technologies) using a thermocycler (Eppendorf, Hamburg, Germany).

After donation, blood samples were immediately spun at 2500 rpm for 15 minutes at room temperature, and 2-ml plasma aliquots with 40 μl of dipeptidyl peptidase-4 protease inhibitor (Merck Millipore, Billerica, MA, USA) were stored at −80 °C. All plasma samples were thawed only once for determination of the concentration of 13 soluble receptors (HSCR-32 K; Merck Millipore) and 64 cytokines (HCYTOMAG-60 K and HCYP2MAG-62 K; Merck Millipore) by addressable laser bead immunoenzyme assay according to the manufacturer’s specifications and read using the Luminex MAGPIX® System 40-072 (Merck Millipore). sCD40L plasma levels were subsequently assessed by enzyme-linked immunosorbent assay (ELISA) using the Human sCD40L Platinum ELISA kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s specifications and read at λ = 450/570 nm in a VICTOR X3 2030 multilabel ELISA reader (PerkinElmer, Singapore).

Neutrophils and PBMCs were separated by density gradient separation using dextran and Ficoll-Hypaque 1077, respectively. Cells were stimulated with (1) 30 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA), (2) 50 μl of pooled plasma (from HCs, patients with iBD, or patients with aBD) with or without 2 μg/ml recombinant CD40 (rhCD40-muIg; (R&D Systems, Minneapolis, MN, USA), (3) 50 μl of pooled plasma from HIgM patients, or (4) 100 ng/ml rh-sCD40L (Life Technologies). Hydrogen peroxide and superoxide production was determined by luminol/lucigenin chemiluminescence intensity and quantified for 2 h at 37 °C during the readout in the VICTOR X3 2030 multilabel plate reader as described previously [22, 23]. Each well was read for 0.5 seconds every 50 seconds, and the results were expressed as a kinetic curve of relative light units on the x-axis and time in minutes on the y-axis. The analysis was based on the AUC.

Gene expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components was determined by qRT-PCR as described previously [22]. (Primers are shown in Additional file 1: Table S1.) Data were acquired and analyzed using a ViiA 7 RT-PCR system (Thermo Fisher Scientific, Waltham, MA, USA).

Protein expression of NADPH oxidase subunit protein expression was determined by flow cytometry. Briefly, 100 μl of whole blood was diluted with 100 μl of PBS and incubated with 30 ng/ml PMA or 50 μl of pooled plasma (from HCs, patients with iBD, or patients with aBD) for 6 h in a 5% CO₂ atmosphere at 37 °C. For gp91-phox and p22 subunit evaluation, the samples were stained with 2 μl of anti-gp91-phox-fluorescein isothiocyanate (Santa Cruz Biotechnology, Dallas, TX, USA) and 2 μl of anti-p22-phycoerythrin (PE) (Santa Cruz Biotechnology), respectively, for 30 minutes in the dark. For p40, p47, and p67 subunit evaluation, the samples were initially fixed with 50 μl of BD Cytofix fixation buffer (BD Biosciences, San Jose, CA, USA) and then permeabilized with 50 μl of BD Phosflow Perm Buffer III (BD Biosciences) according to the manufacturer’s instructions. Then, the samples were stained with 2 μl of anti-p40-AF488 (Santa Cruz Biotechnology), 2 μl of anti-p47-AF647 (Santa Cruz Biotechnology), and 2 μl of anti-p67-PE (Santa Cruz Biotechnology). Using a FACSAria III flow cytometer (BD Biosciences), we observed that monocytes and neutrophils were gated by forward and side scatter. Acquired data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

Assessment of NF-κB p65 subunit phosphorylation was performed by flow cytometry as previously described [24] following incubation with 30 ng/ml PMA or 50 μl of pooled plasma (from HCs, patients with iBD, or patients with aBD) for 15 minutes in a 5% CO₂ atmosphere at 37 °C.

NET release was determined as previously described [25] after stimulation with 30 ng/ml PMA, 100 ng/ml rh-sCD40L, or 50 μl of pooled plasma (from HCs, patients with iBD, or patients with aBD) with or without 2 μg/ml rhCD40-muIg. Cells were stained with primary anti-histone H2A/B (kindly provided by Dr. Arturo Zychlinsky, Max Planck Institute, Berlin, Germany) and anti-human neutrophil elastase monoclonal antibodies (Abcam, Cambridge, UK). Bisbenzimide (Life Technologies) was used for DNA labeling. Three random microscopic fields (×400 magnification) were photographed with a × 40 or × 63 lens objective and 17, 20, and 21 high-efficiency filters for each fluorochrome, respectively. Total NET area in each photo was calculated using ZEN lite 2012 (black edition) software (Carl Zeiss, Oberkochen, Germany).

CD40L protein expression was determined by flow cytometry as previously described [14, 26]. Platelets were obtained and assessed by flow cytometry for CD40L protein expression before and after stimulation with 0.2 U/ml thrombin for 5 minutes at 37 °C in a 5% CO₂ atmosphere [14]. CD40L protein expression in monocytes and in CD4⁺ and CD8⁺ T lymphocytes was determined by flow cytometry before and after stimulation with 1.5 μM ionomycin (Sigma-Aldrich) and/or 25 ng/ml PMA [26]. CD40L gene expression in PBMCs was determined before and after stimulation with 10 ng/ml PMA for 3 h using the TaqMan® Gene Expression qRT-PCR Assay for CD40L (Thermo Fisher Scientific). cDNA levels were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (Thermo Fisher Scientific).

Continuous variables were evaluated using the Kolmogorov-Smirnov test and analyzed using Student’s t test (normal distribution) or the Mann-Whitney U test (nonparametric distribution). Qualitative parameters were analyzed using the chi-square test and Fisher’s exact test when appropriate. Multiparametric analyses were conducted with one-way analysis of variance (ANOVA) followed by Bonferroni posttests when appropriate. The statistical inference level was set at 0.05.

The female-to-male ratios were 14:16 in the aBD group and 15:16 in the iBD group, with ages ranging from 20 to 57 years (36.13 ± 11.02) and from 20 to 55 years (40.96 ± 9.72), respectively. The female-to-male ratios were 18:12 in the HC group, 14:11 in the PS group, 18:9 in the iSLE group, and 22:5 in the aSLE group, with age ranges varying from 24 to 62 years (35.63 ± 9.49), 20 to 82 years (46.00 ± 25.76), 6 to 18 years (14.32 ± 2.96), and 7 to 20 years (15.12 ± 3.48), respectively. The HIgM plasma pool was constituted of plasma from four X-linked hyper-IgM male patients aged 4, 5, 7, and 8 years. Patients with BD and control subjects did not differ regarding gender distribution. The HC, iBD, aBD, and PS groups did not differ regarding age distribution. The iSLE, aSLE, and HIgM groups were significantly younger than the other groups.

Miscellaneous comorbidities (osteoarthritis, osteopenia/osteoporosis, glaucoma, arterial hypertension, dyslipidemia, chronic obstructive pulmonary disease, and epilepsy, among others) were equally present in patients with iBD (n = 22; 70.96%) and patients with aBD (n = 19; 63.33%) (p = 0.59). The clinical characteristics and therapeutic regimens of patients with BD are shown in Table 1. There were no significant differences between patients with aBD and patients with iBD regarding previous disease manifestations. All patients were under treatment at the time of the study. As expected, the number of patients with aBD receiving cyclophosphamide or high doses of corticosteroids was higher than among patients with iBD.

Neutrophils from patients with BD showed a higher rate of spontaneous NET release than those from HCs, although no difference was observed after stimulation with PMA (Fig. 1a). When stimulated with plasma, neutrophils from patients with BD released more NET than HCs’ neutrophils (Fig. 1b). Moreover, NET release was more strongly stimulated by plasma from patients with aBD than by plasma from HCs or patients with iBD (Fig. 1c). Figure 2 shows photomicrographs illustrating the results.

Superoxide and hydrogen peroxide production in resting phagocytes was similar in patients with BD and HCs. Plasma from patients with BD, but not from HCs, stimulated oxidative burst in phagocytes from normal individuals and patients with BD (Fig. 3a–d). Interestingly, neutrophils from HCs produced more superoxide (Fig. 3a) and hydrogen peroxide (Fig. 3b) after stimulation with aBD plasma than with iBD and HC plasma. In general, PMA stimulated higher superoxide production in neutrophils than any other stimulus; however, hydrogen peroxide production by aBD neutrophils was higher after aBD plasma than after PMA stimuli, and hydrogen peroxide production by iBD neutrophils after PMA stimulation was equivalent to that observed with iBD or aBD plasma stimuli.

Similar results were also observed in PBMC assays, in which superoxide (Fig. 3c) and hydrogen peroxide production (Fig. 3d) were higher after BD plasma stimulation than after HC plasma stimulation. Remarkably, aBD plasma exerted a stronger effect than iBD plasma on superoxide production by PBMCs from HCs and were even comparable to PMA on aBD cells (Fig. 3c). In all the other comparisons, reactive oxygen species production by PMA-stimulated PBMCs was higher than with any other stimulus.

To determine whether the increased production of reactive oxygen species by BD neutrophils could be a result of increased expression of the NADPH oxidase subunits, we evaluated the level of subunit expression by both messenger RNA (mRNA) and protein analysis. Expression of each NADPH oxidase subunit gene was equivalent in resting neutrophils (Fig. 4a) and PBMCs (Fig. 4b) from HCs, patients with iBD, and patients with aBD. Cells from a group of 25 patients with severe sepsis were used as a positive control and, as expected, exhibited higher expression of gp91-phox, p22, p40, and p67 NADPH oxidase subunits. As for NADPH oxidase protein, the expression of the five subunits in neutrophils and monocytes increased significantly after stimulation with aBD or iBD plasma. Interestingly, plasma from HCs also caused an increase in NADPH oxidase protein expression, but plasma from patients with BD had a significantly stronger effect (Table 2).

The above results indicate a possible soluble factor carried by the plasma from patients with BD that would be responsible for activating NET release and oxidative burst. In an effort to identify this factor, we determined the concentration of 64 cytokines and 13 soluble receptors in the plasma from patients with BD and from HCs by multiplex technology (Additional file 2: Table S2). Among the several factors that showed the greatest differences between HC and BD plasma, sCD40L was markedly higher in samples from patients with BD (Fig. 5a). This result was confirmed by ELISA, which demonstrated that sCD40L plasma levels from patients with aBD are significantly higher than those from HCs and patients with iBD (Fig. 5b). To gauge the comparative level of CD40L expression in patients with BD, we quantified sCD40L in patients with iSLE and patients with aSLE, whose CD40L expression is known to be highly upregulated in CD4⁺ T cells and even aberrantly expressed on CD8⁺ T cells, B cells, and monocytes [27, 28]. Despite sCD40L levels being markedly elevated in aSLE, the levels observed in aBD were even higher. In patients with iBD, sCD40L plasma levels were comparable to those in patients with aSLE and higher than those in HCs and patients with iSLE (Fig. 5b). It is remarkable that patients with aBD presented a wide range of sCD40L plasma levels, suggesting some heterogeneity in the pathophysiology of the disease. Of note, nonautoimmune diseases previously associated with high levels of sCD40L, such as myocardial infarction [29], atherosclerosis [30], and diabetes mellitus [31], were not observed in our cohort. Furthermore, the distribution of other risk factors for cardiovascular diseases, namely arterial hypertension (iBD = 9.67% vs aBD = 20%; p = 0.3) and dyslipidemia (iBD = 22.58% vs aBD = 6.66%; p = 0.14), was not different between the two groups of patients.

We then investigated the hypothesis that sCD40L was the stimulating factor responsible for the increased NETosis and oxidative burst after stimulation with BD plasma. In in vitro experiments, we stimulated cells with recombinant sCD40L or with plasma from patients with BD in the presence or absence of rhCD40-muIg used as a specific sCD40L blocking agent. NET release was significantly reduced after sequestering sCD40L with rhCD40-muIg and increased when stimulated with recombinant sCD40L (Fig. 5c). Similarly, superoxide (Fig. 5d and f) and hydrogen peroxide (Fig. 5e and g) production by neutrophils (Fig. 5d and e) and PBMCs (Fig. 5f and g) from patients with iBD, patients with aBD, and HCs was also significantly stimulated by sCD40L and decreased by sCD40L blockade. In addition, sCD40L-poor plasma obtained from patients with CD40L deficiency (hyper-IgM plasma) was not able to increase oxidative burst (Fig. 5d–g).

To determine the source of the elevated sCD40L in the plasma of patients with BD, we evaluated expression by the normal cell sources of CD40L in peripheral blood. Although platelets are a major source of sCD40L, no difference in CD40L protein expression was observed in platelets from patients with BD compared with those of HCs (Fig. 6a). In contrast, on one hand, CD40L gene expression was strikingly increased in resting PBMCs from patients with BD compared with HCs, but there was not an additional increase after PMA stimulation (Fig. 6b). On the other hand, the number of CD4⁺CD40L⁺ T cells from patients with BD was significantly higher after combined PMA-ionomycin stimulation, suggesting that there is a larger preexisting pool of CD40L protein in T cells (Fig. 6c). Interestingly, contrary to previous observations in patients with lupus [27, 28], CD40L expression on CD8⁺ T cells and monocytes from patients with BD was not different from that observed in HC cells (Additional file 3: Figure S1). Additionally, macrophage integrin Mac-1, but not CD40 protein, was overexpressed in resting neutrophils (Fig. 6d) and monocytes (Fig. 6e) from patients with BD compared with those from HCs. Finally, phosphorylation of the NF-κB p65 subunit was constitutively increased in neutrophils and monocytes from patients with BD, suggesting chronic baseline activation (Fig. 6f) of these cells. Importantly, phosphorylation of the NF-κB p65 subunit was significantly increased in normal neutrophils and monocytes after stimulation with iBD or aBD plasma (Fig. 6g).