The Role Of Th17and Th22Cells In The Pathogenesis Of Guillain-Barré Syndrome

Guillain-Barré syndrome （GBS） is an autoimmune inflammatorydisease affecting both myelin sheath and axons of the peripheral nervoussystem （PNS）. The pathogenesis of GBS remains still enigmatic, mostcases are reported to undergo previous infections, surgery andvaccination. It is clinically characterized by rapidly progressingsymmetrical weakness and hyporeflexia/areflexia followed mostly byrecovery. Meanwhile, the cerebrospinal fluid （CSF） of GBS patientsusually shows characteristic albumin-cytological dissociation. Some ofGBS patients’ recovery can be shortened by plasma exchange （PE） andintravenous immunoglobulin （IVIg） therapy. Acute inflammatorydemyelinating polyradiculoneuropathy （AIDP）, acute motor axonalneuropathy （AMAN）, acute motor sensory axonal neuropathy （AMSAN）and Miller-Fisher syndrome are the most common subtypes of GBS.GBS and its animal model experimental autoimmune neuritis （EAN）have hitherto been attributed to Th1cells-mediated disorders. IncreasedIFN-γ was seen in the serum of GBS patients at acute phase, and higherimmunoreactivity for IFN-γ was showed in sural nerves biopsies of GBSpatients. IL-17-producing T helper （Th） cells or Th17cells have beenidentified subsequently as an obvious distinct Th population and a novelTh lineage mediating tissue inflammation and autoimmune response inboth animal models and human. EAN has been observed with decreasedTh1and Th17cytokines when administered atorvastatin which hasanti-inflammatory properties. Furthermore, a Germany’s group reported acompound, FTY720, attenuated EAN via reducing Th17cells in PNS. These findings suggest that Th17and IL-17paticipate in the inflammationof EAN in addition to Th1and IFN-γ. Recent studies testified thatIL-17/Th17and IL-22/Th22cells play important roles in the pathogenesisof various inflammatory and autoimmune diseases, such as inflammatorybowel disease （IBD）, rheumatoid arthritis （RA）, psoriasis, and multiplesclerosis （MS）.The study on the role of Th17and Th22cells in GBS has not yetestablished so far. In the present study, we detected the frequency of Th1,Th17and Th22cells and their subgroup cells in the peripheral blood andlevels of IL-17and IL-22in plasma of GBS patients at the acute/plateauphase, IL-17and IL-22levels in paired CSF and plasma of GBS patientsat the acute phase, and evaluated the correlations between these twocytokines and GBS functional disability scales （GDSs） as well as variousCSF parameters. These human studies will allow us to understand the roleof Th17and Th22cells as well as IL-17and IL-22in the development ofthe autoimmune response in GBS.Materials and methodsDuring June2010to August2011, we recruited29GBS patients,32other neurological inflammatory disease controls （ONIDs）, including15MS,17encephalitis or meningitis infected by virus （VEM） and20healthy controls （HC）. All subjects are from the Department ofNeurology, the First Hospital, Jilin University, Changchun, China. AllGBS patients were classified electrophysiologically as AIDP （n=13）and AMAN （n=16）, using motor nerve conduction criteria. Blood wassampled two times at acute （1-14days from onset day, GBS-A） andplateau phases （15-32days from onset day, GBS-P） of GBS. The pre-treatment GBS patients were defined as absence of anyimmune-modulating drugs and other treatments within3months, and thepost-treatment patients as treatments with IVIg at a dose of0.4g/kgbody weight per day for5days consecutively in acute phase. In thisstudy, paired samples of CSF and plasma were collected from22GBSpatients and18HC at the Neurological Laboratory of the First Hospital,Jilin University, Changchun, and the Department of Neurology, theAffiliated Hospital of Medical College, Qingdao University, Qingdao,China during June2010to December2011. GBS patients fulfilledinternational diagnostic criteria for GBS or its variants. Severity of GBSwas scored by the use of GDSs.Ficoll-PaqueTMdensity gradient centrifugation was used to separateperipheral blood mononuclear cells （PBMCs）. PBMCs were resuspendedat1×10⁷cells/ml by Trypan blue stain and stimulated with phorbol12-myristate13-acetate （PMA） and ionomycin in the presence ofBrefeldin A in37℃，5%CO₂cell incubation box for4h. We used mouseanti-human monoclonal antibodies （mAbs） of PerCP-conjugated CD4for extra-cellular marker stain and phycoerythrin （PE）-conjugated IL-22,FITC-conjugated IFN-γ, Alexa Fluor○R647-conjugated IL-17A forintra-cellular marker stain. Plasma and CSF samples were also collectedand cryopreserved at-80℃. Then we used ELISA performance to detectthe level of IL-17and IL-22according to the manufacturer’s instructions.Data were expressed as the mean±standard deviation （SD）. Forstatistical analysis, differences of mean values were tested with one wayanalysis of variance （ANOVA） for multiple comparisons and thestudent-t test for two groups. The Pearson or Spearman correlation coefficient was used to analyze correlations depending on datadistribution. Reported P-values are two-tailed and considered statisticallysignificant at P <0.05.ResultsTwenty-nine GBS patients [mean age,41years （20men and9women）] had manifested acutely progressive weakness of limbs. Ahistory of antecedent illness was present in62.1%of the patients （upperrespiratory tract infectious symptoms in20.7%, gastrointestinal tractsymptoms in34.5%, and both symptoms in6.9%）.By flow cytometry, our data showed that Th1（CD4+IFN-γ+）, Th17（CD4+IL-17A+）, Th22（CD4+IL-22+） cells and Th1/Th17subgroup（IFN-γ+IL-17A+）, Th17subgroup （IL-17A+IFN-γ-, IL-17A+IL-22-andIL-17A+IL-22+） as well as Th22subgroup （IL-22+IL-17A-and IL-22+IFN-γ-） cells were significantly increased in GBS-A and ONIDscompared with HC［Th1cells （GBS-A: P <0.001; RR-MS/R: P=0.007;VEM: P=0.002）；Th17, Th22cells, and Th1/Th17subgroup, Th17subgroup, Th22subgroup cells P <0.001)］. The frequency of Th1subgroup （IFN-γ+IL-17A-） was elevated in GBS-A （P=0.014） comparedwith HC. Interestingly, the intravenous immunoglobulin （IVIg） therapydown-regulated these cells in GBS-P compared with GBS-A （Th1subgroup：P=0.044，others: P <0.001）. There was no significantdifference of elevated Th1, Th17and Th22cells between AIDP andAMAN. Though there was no quantitative uniqueness for the frequencyof Th1, Th17and Th22cells between GBS subtypes or no correlationwith GBS-A severity, the elevated Th22cells had a tendency withdisease severity status in GBS-A. There was no significant difference with regard to circulating Th1, Th17and Th22cells as well as Th17andTh22subgroup cells between GBS patients （both GBS-A and GBS-P）with and without prodromal infections （P>0.05）.Plasma levels of IL-17and IL-22in all groups were also measuredby ELISA. Our data demonstrated that plasma levels of IL-17and IL-22were significantly elevated in GBS-A and RR-MS/R compared with HC［IL-17（GBS-A：P <0.001，RR-MS/R：P=0.01）；IL-22（GBS-A：P=0.009，RR-MS/R：P=0.007）］. Clearly, IVIg treatments declined thelevels of these two cytokines in GBS-P compared with GBS-A （IL-17：P<0.001；IL-22：P=0.025）. The levels of IL-17and IL-22in paired CSFand plasma were elevated in all GBS patients. The elevation was mostobvious for IL-22in CSF （P <0.001）, followed by IL-17in CSF （P=0.023）, IL-17in plasma （P=0.011）, and IL-22in plasma （P=0.019）,when compared with HC. IL-17and IL-22levels in CSF had positivecorrelation with GDSs （P=0.040; P=0.012, respectively）. Althoughthere was no statistical significance, the elevated level of IL-22in plasmahad a tendency toward positive correlation with GBS severity. Thesignificant positive relationships were found between the levels of IL-17in CSF and IL-22in CSF （P=0.026）, as well as the levels of IL-22inCSF and IL-22in plasma （P=0.039）.DiscussionIn the present study, our results show that circulating Th1, Th17andTh22cells as well as their subgroups and IL-17, IL-22in CSF andplasma levels were obviously elevated in GBS at the acute phase. Thus itis speculated that Th17and Th22cells as well as IL-17/IL-22areinvolved in the initiation and development of GBS. IL-6in the presence of TGF-β1, and IL-6, IL-23in combinationwith IL-1β had been proved to be the effective factors responsible for thedifferentiation of Th17cells and the production of IL-17. Thesecytokines milieux may also facilitate the expression of IL-22. IL-6, IL-1β,IL-23and TNF-α are all pro-inflammatory cytokines and play animportant role in the pathogenesis of GBS. Therefore, it is safe toconclude in our study that the extensive network of IL-17and IL-22incoordination with these inflammatory cytokines is associated with thepathogenesis of GBS. IL-17and IL-22might be important effectors inaddition to Th1cells and Th1cytokines in autoimmune-mediatedresponses of GBS.Our data suggest that the elevation CSF levels of IL-17and IL-22inGBS might be related to PNS local inflammation that causesdemyelination and axon degeneration. The levels of them, respectively,were correlated with GDSs at the acute phase of GBS, and there was apositive correlation between them. Nevertheless, the levels of IL-22inCSF and IL-22in plasma also had a positive correlation. This indicatesthat the elevation of IL-17and IL-22in CSF is related to the severity ofinflammation in the spinal roots. Since their elevations in CSF aresynchronous, IL-17and IL-22may coordinate in the pathogenesis of thedisease and may be a biomarker for indicating disease severity orprognosis.High-dose IVIg therapy is a fundamental effective treatment in GBS.Recent studies showed that IVIg inhibited the differentiation andamplification of Th17cells, as well as the production of their effectorcytokines IL-17and IL-22. In this study, our data showed that IVIg treatments could down-regulate these cells and their cytokines at theplateau phase and attenuates clinical signs of GBS. Our data suggest thatantagonists of Th17, Th22cells and their cytokines may have therapeuticpotentials for alleviating GBS.ConclusionsIn summary, increasing circulating Th17, Th22cells and theireffector cytokines may be involved in the pathogenesis of GBS. IL-17and IL-22levels in CSF are correlated with GDSs of GBS patients,respectively. The significant positive relationships could be foundbetween the levels of IL-17and IL-22in CSF, as well as IL-22in CSFand plasma of GBS patients, IVIg mediates its therapeutic effects bydown-regulating these cells and their cytokines in GBS patients.