Regulatory T cell frequency, but not plasma IL-33 levels, represents potential immunological biomarker to predict clinical response to intravenous immunoglobulin therapy
- Mohan S. Maddur†1, 2, 3, 4, 5,
- Emmanuel Stephen-Victor†1, 2, 3,
- Mrinmoy Das†1, 2, 3,
- Praveen Prakhar6,
- Varun K. Sharma1, 2, 3, 4,
- Vikas Singh6,
- Magalie Rabin3,
- Jamma Trinath6,
- Kithiganahalli N. Balaji6,
- Francis Bolgert7,
- Jean-Michel Vallat8,
- Laurent Magy8,
- Srini V. Kaveri1, 2, 3, 4Email author and
- Jagadeesh Bayry1, 2, 3, 4Email author
© The Author(s). 2017
Received: 27 October 2016
Accepted: 18 February 2017
Published: 20 March 2017
Intravenous immunoglobulin (IVIG) is a polyspecific pooled immunoglobulin G preparation and one of the commonly used therapeutics for autoimmune diseases including those of neurological origin. A recent report in murine model proposed that IVIG expands regulatory T (Treg) cells via induction of interleukin 33 (IL-33). However, translational insight on these observations is lacking.
Ten newly diagnosed Guillain-Barré syndrome (GBS) patients were treated with IVIG at the rate of 0.4 g/kg for three to five consecutive days. Clinical evaluation for muscular weakness was performed by Medical Research Council (MRC) and modified Rankin scoring (MRS) system. Heparinized blood samples were collected before and 1, 2, and 4–5 weeks post-IVIG therapy. Peripheral blood mononuclear cells were stained for surface CD4 and intracellular Foxp3, IFN-γ, and tumor necrosis factor alpha (TNF-α) and were analyzed by flow cytometry. IL-33 and prostaglandin E2 in the plasma were measured by ELISA.
The fold changes in plasma IL-33 at week 1 showed no correlation with the MRC and MRS scores at weeks 1, 2, and ≥4 post-IVIG therapy. Clinical recovery following IVIG therapy appears to be associated with Treg cell response. Contrary to murine study, there was no association between the fold changes in IL-33 at week 1 and Treg cell frequency at weeks 1, 2, and ≥4 post-IVIG therapy. Treg cell-mediated clinical response to IVIG therapy in GBS patients was associated with reciprocal regulation of effector T cells-expressing TNF-α.
Treg cell expansion by IVIG in patients with autoimmune diseases lack correlation with IL-33. Treg cell frequency, but not plasma IL-33 levels, represents potential immunological biomarker to predict clinical response to IVIG therapy.
KeywordsIVIG Guillain-Barré syndrome Treg cells IL-33 TNF-α Clinical response Regulatory T cells
Intravenous immunoglobulin (IVIG) is a polyspecific pooled immunoglobulin G preparation and one of the commonly used immunotherapeutics for the treatment of autoimmune diseases including those of neurological origin [1–4]. Various reports demonstrate that IVIG exerts beneficial effects through several mutually non-exclusive mechanisms including expansion of CD4+Foxp3+ regulatory T cells (Treg cells) [5–10]. Some of these mechanisms are also being explored as biomarkers of IVIG response [11, 12]. Treg cells play an indispensable role in the maintenance of immune tolerance and suppress unnecessary deleterious immune responses, such as autoimmunity . IVIG is shown to expand Treg cells and enhance their functions in experimental animal models and patients with autoimmune diseases [9, 14–17]. Recent studies suggested that IVIG-induced expansion of Treg cells requires the prime role of dendritic cells (DCs), and involves interaction of IVIG with C-type lectin receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and dendritic cell immunoreceptor (DCIR), and is mediated through prostaglandin (PG) E2 secreted by DCs [15, 18]. Interestingly, based on the murine models of autoimmune diseases, i.e., antibody-mediated K/BxN arthritis and T cell-mediated experimental autoimmune encephalitis, a recent report proposed that induction of interleukin 33 (IL-33) in macrophages by IVIG through interaction of α2,6-sialylated crystallizable fraction (Fc) with SIGN-R1 or human DC-SIGN is essential for the expansion of Treg cells [19, 20]. However, translational insight on these observations in patients with autoimmune diseases is lacking.
Guillain-Barré syndrome (GBS) is an autoimmune disease of neurological origin affecting the peripheral nerves . GBS is believed to be caused by effector T cells and autoantibodies to myelin components [22, 23]. Furthermore, GBS patients display reduced frequency of Treg cells that are required for the prevention of autoimmunity . Amelioration of experimental autoimmune neuritis, an experimental model of GBS, was associated with up-regulation of Treg cells . Currently IVIG is used as a first-line therapy for GBS . Of note, sialylated IVIG has been shown to suppress anti-ganglioside antibody-mediated nerve injury in experimental GBS model and was associated with increased expression of IL-33 mRNA . Sialylated IVIG also inhibited anti-ganglioside antibody-mediated complement deposition in vitro . In view of these reports, we investigated the role of IL-33 towards clinical response to IVIG treatment and Treg cell expansion in GBS patients. We found that kinetics of peripheral Treg cell expansion and improvement of clinical symptoms in GBS patients following IVIG therapy lack correlation with the level of induction of IL-33 in the blood.
Materials and methods
Clinical evaluation and sample collection
Demographics and clinical information of GBS patients
MRC score (pre-IVIG)
Flow cytometry and ELISA
Plasma and peripheral blood mononuclear cells (PBMCs) were separated from the heparinized blood samples. PBMCs were stained for surface CD4 and intracellular Foxp3, and also interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) following stimulation with phorbol myristate acetate (50 ng/mL) and ionomycin (500 ng/mL, Sigma-Aldrich, France), along with GolgiStop (BD Biosciences, France), for 4 h. The intracellular staining was performed using Foxp3 staining kit (eBioscience, France) as per the manufacturer’s instructions. Stained cells were acquired on LSR II (BD Biosciences) flow cytometer and data was analyzed using BD FACS DIVA (BD Biosciences) and FlowJo (FlowJo LLC, USA) softwares. IL-33 was measured in the plasma by ELISA (R&D systems, France). The amount of PGE2 in the plasma samples was estimated by ELISA as described previously .
One-way ANOVA “Kruskal-Wallis test” was used for the analysis of IL-33 in the plasma. Pearson correlation (r) was used to determine the association between MRC or MRS scores and plasma levels of IL-33 and PGE2 and the frequency of Treg cells in the blood of GBS patients at different time points following IVIG treatment. Same assay was also used for determining the association between frequency of Treg cells and CD4+IFN-γ+ and CD4+TNF-α+ T cells.
Results and discussion
In order to explore the role of IL-33 in IVIG-mediated therapeutic effects, we analyzed whether IL-33 induction correlates with clinical response to IVIG therapy and Treg cell expansion . Of note, the fold changes in plasma IL-33 level at week 1 showed no correlation with the MRC scores at weeks 1, 2, and ≥4 post-IVIG therapy (Fig. 1b). Also, the r values are consistently negative at all the time points. On the other hand, Treg cell response following IVIG therapy appears to be linked with clinical recovery from GBS and is consistent with the previous observations on essential role of Treg cells in IVIG-induced protection in mouse models [18, 31]. Yet, we observed no correlation between the fold-changes in Treg cells at week 1 and clinical score at weeks 1, 2, and ≥4 (Fig. 1c). These results suggest that Treg cells, but not IL-33, might predict clinical response to IVIG therapy.
Correlation between MRS scores at different time points (weeks 1, 2, and ≥4) after initiation of IVIG therapy and the immunological parameters such as IL-33, Treg cells, and PGE2 at week 1. The values in the parenthesis denote statistical significance
MRS (week post-IVIG)
In the previous report , we analyzed IL-33 in the plasma of patients with rheumatic disorders as early as 48–72 h following initiation of IVIG therapy. With the exception of two, the remaining 14 patients had IL-33 levels below 500 pg/ml of plasma following IVIG therapy. Although the underlying pathologies in that report and current study are different, we found that at 1 week following initiation of IVIG therapy, IL-33 level in the plasma was more than 1000 pg/ml in nine GBS patients. Furthermore, in majority of the patients (seven), IVIG was given daily for five consecutive days. Therefore, lack of consistent association between the fold changes in IL-33 level and Treg cell frequency following IVIG therapy might not be due to loss of IL-33 subsequent to proteolytic cleavage and binding to ST2 receptor. Also, IL-33 levels observed at first week are maintained in majority of the patients even after 4 weeks post-IVIG. Whether polymorphisms in either ST2 receptor, IL-1RL1, and/or IL-33 itself, skew the plasma kinetics and/or downstream effects of IL-33 remains to be determined [35, 36].
Current literature suggests that IVIG can induce Treg cell expansion by several mutually non-exclusive mechanisms [9, 14, 16, 17, 31] that may vary among the pathologies and even among the patients with same underlying pathology. This might explain the lack of significant correlation between PGE2 and Treg cells. As we restricted our study only to treatment-naïve GBS patients, difficulty to get large number of patients provides an alternative explanation for the lack of significant positive correlation between changes in the IL-33 and PGE2, and Treg cells. Role of IL-33 in the microenvironment towards DC-mediated human Treg expansion requires further exploration.
Because of its role as promoter of type 2 immune responses and regulator of innate and adaptive immune responses, IL-33 has been explored as therapeutic option in pre-clinical models of Alzheimer’s disease , stroke , cerebral malaria , and transplantation . In addition, IL-33 was also reported to be essential to attenuate viral-induced encephalitis . In line with these reports, enhanced expression of IL-33 in mice was suggested to mediate Treg cell expansion and protection by IVIG [19, 20, 27]. Translation of these results to human is complicated and requires thorough investigation. Indeed, we found that up-regulation of plasma IL-33 does not correlate with the clinical response to IVIG therapy and expansion of Treg cells in GBS patients. Treg cells on the other hand are negatively correlated to effector T cells expressing TNF-α. Thus, the mechanism of IL-33 induction by IVIG and its role in Treg cell expansion observed in mouse models might not be applicable to human. Small size of the patients’ cohort represents limitation of this study. Nevertheless, based on these findings, Treg cell frequency, but not plasma IL-33 levels, represents potential immunological biomarker to predict clinical response to IVIG therapy.
Dendritic cell immunoreceptor
Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin
- MRC score:
Medical research council score
Modified Rankin score
- PGE2 :
Tumor necrosis factor alpha
- Treg cells:
Foxp3+ regulatory T cells
We thank Ms. A Karnam for the support.
Supported by Institut National de la Santé et de la Recherche Médicale, Université Pierre et Marie Curie, Université Paris Descartes (SVK and JB), Centre National de la Recherche Scientifique (SVK), European Community’s Seventh Framework Programme [FP7/2007-2013] under grant agreement 260338 ALLFUN (JB), and fellowship from Journées de Neurologie de Langue Française (MR). KNB is a J. C. Bose National fellow of Department of Science and Technology, New Delhi, India.
The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this published article as figures.
MSM, ES-V, MD, PP, VKS, VS, MR, and JT performed research. FB, J-MV, and LM contributed analytical tools and evaluated the clinical scores. MSM, KNB, SVK, and JB analyzed the data. MSM and JB designed the research and wrote the paper. All authors read and approved the final manuscript.
This work is partially supported by financial support from CSL Behring, France.
Consent for publication
Ethics approval and consent to participate
The study was approved by relevant ethical committee (84-2012-08, CHU Limoges), and the consent was obtained from the enrolled patients.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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