Circulating Th17, Th22, and Th1 Cells Are Elevated in the Guillain-Barré Syndrome and Downregulated by IVIg Treatments

The Guillain-Barré syndrome (GBS) is considered a T helper 1 (Th1) cells-mediated acute inflammatory peripheral neuropathy. However, some changes in GBS could not be explained completely by Th1 cells pathogenic role. Recently, Th17 cells have been identified and can mediate tissue inflammation and autoimmune response. Therefore, a study on the role of Th17 and Th22 cells and their cytokines in GBS is necessary for exploring the pathogenesis of GBS. Here, we detected the frequency of Th1, Th17, and Th22 cells by using 4-color flow cytometry and we detected the plasma levels of IL-17 and IL-22 by ELISA in GBS patients, relapsing-remitting multiple sclerosis patients at the acute phase of relapse, viral encephalitis or meningitis patients and healthy controls. Our data showed that the frequency of circulating Th1, Th17, and Th22 cells was significantly increased in GBS patients. The plasma levels of IL-17 and IL-22 in GBS and relapsing-remitting multiple sclerosis at the acute phase of relapse were also markedly elevated. Enhanced circulating Th22 cells were correlated with GBS severity. Intravenous immunoglobulin therapy downregulated Th17, and Th22 cells and the plasma levels of IL-17 and IL-22 in GBS patients. Th17 and Th22 cells may be involved in the pathogenesis of GBS, and intravenous immunoglobulin mediates therapeutic effects by downregulating these cells and their cytokines.


Introduction
The Guillain-Barré syndrome (GBS) is an immune-mediated peripheral neuropathy involving both the myelin sheath and axons. Acute inflammatory demyelinating polyneuropathy (AIDP) and acute motor axonal neuropathy (AMAN) are the most common subtypes of GBS [1][2][3][4]. The pathogenesis of GBS remains still enigmatic, but it is largely accepted that both cellular and humoral immune responses are involved in the pathogenesis of GBS [5,6]. AIDP and its animal model experimental autoimmune neuritis (EAN) have hitherto been classified to Th1 cells-mediated disorders [7][8][9]. Th1 cells, a subset of CD4 + T (T helper) cells, are dominant in the inflamed nerves at the acute phase of GBS [8], which could produce IFN-as a major pathogenic cytokine in GBS, because increased IFN-was seen in the serum of GBS patients at acute phase [10], and higher immunoreactivity for IFN-was showed in sural nerves biopsies of GBS patients [11]. However, some changes in GBS and EAN could not be explained by Th1 cell pathogenic role. Most likely, it is complex and involves multiple factors in GBS.
T helper 17 (Th17) cells have been identified as an obvious distinct Th population and a novel Th lineage mediating 2 Mediators of Inflammation tissue inflammation and autoimmune response in both animal models and humans [12]. Th17 cells can induce local inflammation in the target organs and help B cells to produce antibodies, two of the hallmarks of GBS pathology. Th17 cells mainly produce IL-17A, which could be promoted by IL-23 in vitro [13] and in vivo [14]. In addition to IL-17A, Th17 cells can secrete IL-17F, IL-21, and IL-22 [15], which induces massive tissue reactions by promoting the recruitment of inflammatory cells [16], while IL-22 shows specific biological characters, such as tissue repairing and wound healing [17].
The increased frequency of Th17 and Th22 cells along with higher levels of IL-17A and IL-22 has been found in multiple inflammatory and autoimmune diseases. IL-17A positive cells were enhanced in the lamina propria and epithelium of inflammatory bowel disease [18]. IL-22 presented high quantities in the blood of the patients with Crohn's disease, and IL-22 mRNA expression was elevated predominantly in mouse colitis model [19,20]. Th1, Th17, and Th22 cells and IL-22 level were enhanced in psoriasis [21]. Th17 cell frequency was higher in the cerebrospinal fluid of the patients with relapsing-remitting multiple sclerosis (MS) during the relapse phase [22]. Recently, we reported an aggravated clinical course of EAN in IFN-deficient mice, concomitant with an upregulated level of Th17 cells, indicating a pathogenic role of Th17 in EAN [9]. EAN was attenuated by atorvastatin treatment, a lipid lowering drug with anti-inflammatory properties, and the level of IL-17A was decreased in parallel [23].
However, the role of Th17 and Th22 cells as well as their cytokines in the pathogenesis of GBS is still unclear. Here, we detected the frequency of Th1, Th17, and Th22 cells in the peripheral blood and levels of IL-17 and IL-22 in plasma of GBS patients at the acute and the plateau phases to unravel the mechanisms by which Th17 and Th22 as well as their cytokines may play a pathogenic role in GBS.

Patients and Specimens.
We recruited 29 GBS patients fulfilling international diagnostic criteria for GBS or its variants [24], 32 other neurological inflammatory disease controls (ONIDs), including 15 MS patients and 17 encephalitis or meningitis infected by virus (VEM) patients, and 20 healthy controls (HC). All subjects were from the Department of Neurology, the First Hospital, Jilin University, Changchun, China, during June 2010 to August 2012. All GBS patients were classified electrophysiologically as AMAN ( = 16) and AIDP ( = 13), using motor nerve conduction criteria [1]. Severity of GBS was scored by the use of GBS disability scale scores (GDSs), a widely accepted scoring system to assess the functional status of the patients with GBS [25]. The clinical characteristics of the patients with GBS are shown in Table 1. ONIDs included 15 patients with relapsingremitting MS (RR-MS) meeting the McDonald criteria [26], who showed mono-/multifocal neurological episode lasting for more than 24 hours (h) at the time of sampling after being neurologically stable for more than 30 days and excluding an acute systemic infection (acute relapse; RR-MS/R). The patients with VEM who exhibited the clinical signs and CSF characteristics according to encephalitis or meningitis and positive virus antibodies in CSF detected by enzyme-linked immunosorbent assays (ELISA) as well as excluding other systemic infections were recruited in our study. These patients also did not receive any immune-modulating drugs or other treatments within 3 months. Twenty age-and sex-matched HC (as compared with GBS subjects) were included in the study.
The pretreatment GBS patients were defined as the patients who did not receive any immune-modulating drugs or other treatments within 3 months, and the posttreatment patients were defined as the patients who received treatments with intravenous immunoglobulin (IVIg) at a dose of 0.4 g/kg body weight per day for 5 days consecutively in the acute phase (1-14 days from onset day). Blood was sampled two times at the acute and the plateau phases (15-32 days from onset day) of GBS and one time for ONIDs before any immune-modulating drugs and other treatments. The present study was approved by the Human Ethics Committee of Jilin Province, China, and informed consent was obtained from all patients and HC.

Preparation of Peripheral Blood Mononuclear Cells and
Plasma. Blood samples were taken at 07:00 a.m. each day. Ficoll-Paque (1.077 g/mL, GE Healthcare Bio-Science AB, Uppsala, Sweden) density gradient centrifugation was used to separate peripheral blood mononuclear cells (PBMCs). Mononuclear cells were washed twice in phosphate-buffered saline (PBS), and cell viability measured by Trypan blue exclusion was confirmed to exceed 95%. Plasma samples were also collected and cryopreserved at −80 ∘ C for further ELISA performance.
Subsequently, cells were fixed in 2% paraformaldehyde PBS and stored at 4 ∘ C until flow cytometric analysis by FACSCalibur cytometer using CellQuest software (Becton Dickinson, California, USA). To analyze surface markers in combination with intracellular staining of IFN-, IL-17A, and  and IL-22 were used to plot standard curves using computer software and then were automatically converted to pg/mL by standard curve. In this assay, background absorbencies (wells without coating mAb) were not obvious and were subtracted from the absorbencies of the specimens. All assays were done in duplicate.

Statistical
Analysis. Data were expressed as the mean ± SD. For statistical analysis, the differences of mean values were tested with one-way analysis of variance (ANOVA) for multiple comparisons and Student's t-test for two groups, using SPSS software (version 17.0). The Spearman correlation coefficient by rank test was used for correlation analysis between two sets of data. values are two-tailed and are considered statistically significant at < 0.05.

Clinical Profiles.
The clinical characteristics of GBS are presented in Table 1. Twenty-nine GBS patients had manifested acutely progressive weakness of limbs. A history of antecedent illness was present in 62.1% of the patients (upper respiratory tract infectious symptoms in 20.7%, gastrointestinal tract symptoms in 34.5%, and both symptoms in 6.9%). The characteristics regarding the age and gender of subjects within the four groups are presented in Table 2.

Mediators of Inflammation
Both GBS and ONIDs had a higher frequency of Th1/Th17 cells than HC (for GBS, . No significant differences of the frequency of these cells were found among GBS and ONIDs (Figure 2(b)).

Elevated Circulating Th22 Cells Are Correlated with GBS Severity, but Not with GBS Subtypes.
To determine the correlation between the elevated cells and GBS severity, we analyzed our data with GDSs by using Spearman's correlation coefficient. Our data revealed that Th22 cells were correlated with GDSs ( = 0.399, = 0.032) (Figure 3(h)), though there was no quantitative uniqueness for the frequency of CD4 + IL-22 + cells with GBS severity; the elevated CD4 + IL-22 + cells had a tendency for disease severity status in GBS ( = 0.160, = 0.405) (Figure 3(c)); no other cells were correlated with GDSs (Figures 3(a), 3(b), and 3(d)-3(g)). We also compared the frequency of these elevated cells with the GBS subtypes, including AIDP ( = 13) and AMAN ( = 16), but there was no significant difference between the two subtypes (data not shown).

Higher Plasma Levels of IL-17 and IL-22 in GBS.
Plasma levels of IL-17 and IL-22 in all groups were also measured by ELISA. Our data demonstrated that plasma level of IL-17 was significantly elevated in GBS and RR-MS/R compared with HC ( < 0.001 and = 0.01, resp.). Meanwhile, the IL-22 level was higher in GBS and RR-MS/R than in HC ( = 0.009 and = 0.007, resp.). However, there was no significant difference in IL-17 and IL-22 levels between VEM and HC (data not shown) (Figures 4(a) and 4(b)).

IVIg Reduces Inflammatory Cells as well as Plasma Levels of IL-17 and IL-22 in GBS.
To further understand the effect of IVIg treatment on the inflammatory cells and cytokines, we detected the frequency of these cells by flow cytometry and plasma levels of cytokines by ELISA before and after treatment with IVIg in GBS patients ( = 24), who were suffering from more serious clinical signs. Our data showed that the frequency of CD4 + IFN-+ cells, CD4 + IL-17A + cells,  (Figure 5(b)). Clearly, IVIg treatments also declined the levels of IL-17 and IL-22 in plasma after treatment compared with before treatment (for IL-17, < 0.001; for IL-22, = 0.033) ( Figure 5(c)). However, there was no significant difference regarding the levels of these cells and cytokines in GBS patients with (24 cases) and without IVIg treatments (5 cases with slight clinical signs) (data not shown).

Discussion
In the present study, our results showed that circulating Th1, Th17, and Th22 cells as well as the levels of IL-17 and IL-22 in plasma were obviously elevated in GBS at the acute phase and IVIg treatments could downregulate these cells and their cytokines at the plateau phase of GBS. Thus, it is speculated that Th17 and Th22 cells as well as IL-17/IL-22 are involved in the initiation and development of GBS and IVIg treatments effectively reduce their levels and attenuateclinical signs of GBS.
Th1 and its cytokines are the pathogenic molecules in GBS as we reported previously [7,8]. In the present study, we found that Th17 and Th22 cells and their cytokines may also contribute to the pathogenesis of GBS. Our data showed that, among all Th17 subgroups, the CD4 + IFN-− IL-17A + IL-22 + cell population is a relatively small portion compared to CD4 + IL-17A + and CD4 + IFN-− IL-17A + IL-22 − subgroups. Th17 cell lineage produces not only IL-17A but also IL-22, both of which contribute to the controlling of extracellular bacterial infection by the induction of a powerful immune 8 Mediators of Inflammation response [27]. Although both Th17 and Th22 cells can produce IL-22, they are different T helper cell lineages. Th17 cells play an important role in host defense against infections and in tissue inflammation during autoimmunity [28], while Th22 cells are important in epithelial cell homeostasis, as well as in tissue repair and wound healing [17]. IFN-has an inhibitory effect on the production of IL-17 [29,30]; conversely, in similar conditions, Th22 cells mainly coproduce IFN-rather than IL-17 [31].
We delineated several potential mechanisms by which Th17 and Th22 cells and IL-17/IL-22 could participate in the pathogenesis of GBS. Firstly, higher levels of them showed an apparent enhancement of T cell responses in the acute phase of GBS. Secondly, the quantity of Th22 cells had a positive correlation with disease severity of GBS, which suggested that Th22 may play an important role in the development of GBS. In previous studies, Th22 cells and IL-22 often showed to correlate with disease severity, such as in the patients with rheumatoid arthritis [32] and psoriasis [21]. Th17 and Th22 cells of GBS patients at acute phase could express an appropriate cytokine profile, like IL-17, IL-22, and others (IL-6 and TNF-), which can enhance the inflammatory and autoimmune response and conduce to the development of GBS [33,34]. Finally, increased IL-17 levels in plasma of GBS patients are enhanced homing of inflammatory cells to the peripheral nervous system, which might contribute to the pathogenesis of GBS.
However, in our study, increased Th1, Th1/Th17, Th17, and Th22 cells were also found in ONIDs, and there were no significant differences among GBS and the groups of ONIDs, which indicated that, although the increased cells are associated with GBS, they are also related to other inflammatory and autoimmune disorders; therefore, the increased cells in GBS are not specific for GBS. Similarly, increased inflammatory cytokines and other molecules can be seen in both GBS and ONIDs. It is speculated that Th17 and Th22 cells are not unique pathogenic cells and there could be an intricate network of inflammatory molecules in the pathogenesis of GBS.
High-dose IVIg therapy is an effective treatment in GBS; however, its anti-inflammatory mechanisms remain elusive. It has been reported that IVIg can block the mononuclear phagocytic system, neutralize autoantibodies by anti-idiotype antibodies, interrupt the complement activation cascade, and influence the effect of Fc receptor-mediated activity of immunologically relevant cells [35,36]. IVIg can inhibit T cell proliferation and its cytokine production [37,38]. It also can inactivate, silence, or bring about programmed T cell death [39]. Recent studies showed that IVIg inhibits the differentiation and amplification of Th17 cells and the production of their cytokines IL-17A, IL-17F, IL-21, and CCL20 [40]. Moreover, the effect of IVIg is more prominent on the inhibition of IL-17 production from Th17 cells [41]. Unfortunately, so far, the effect of IVIg treatment on Th22 cells differentiation and function has not been explored. However, Sugita et al. reported that, in the presence of anti-TNF-and anti-IL-6 antibodies, Behçet's disease Th22type T cells failed to produce IL-22 [42]. Thus, it is speculated that IVIg treatment with anti-TNF-property could reduce or inhibit Th22 function probably, which needs to be explored further. In the present study, we firstly reported that IVIg downregulated Th17 and Th22 cells and their effector cytokines IL-17 and IL-22 in GBS patients.

Conclusions
In summary, elevated circulating Th17 and Th22 cells may contribute to the pathogenesis of GBS. IVIg mediates its therapeutic effects by downregulating these cells and their cytokines in GBS. Our data suggest that antagonists of Th17 and Th22 cells and their cytokines may have therapeutic potentials for alleviating GBS in humans.