Study On Effects Of IFN In Guillain-barré Syndrome And Multiple Sclerosis

Objective To investigate the possibility of interferon-γ (IFN-γ to induceCD4+CD25-T(Tresp) cells from healthy donors and Guillain–Barré syndrome (GBS)patients to CD4+CD25+regulatory T(Tregs) cells in vitro. To investigate therelationship between changes of signal transduction pathway and the state of diseaseduring IFN therapy of multiple sclerosis(MS); to probe effects on signal pathwayand changes of relative cytokines with different doseage of type ⅠIFN. Methods (1)Tresp and Tregs cells were separated by magnetic cell sorting (MACs) from theperipheral blood mononuclear cells of25GBS patients and19healthy donors. IFNγ,anti-CD3and anti-CD28antibodies were used to stimulate Tresp cells. Thepercentage of induced Tregs cells was detected by flow cytometry. The FoxP3expression of the induced Tregs cells in GBS patients and healthy donors weredetected by flow cytometry and real-time PCR at protein and mRNA levelsrespectively. The induced Tregs cells were co-cultured with autologous Tresp cells toestimate the suppressive ability.(2) Peripheral blood MNC were isolated andcultured from patients with RRMS (n=25), stimulated with different doseage of typeⅠIFN, harvested cells according time course, serum were collected in partialpatients. Western Blot were performed to test proteins which its concentration werecalculated according cells numbers, fluorescence intensity of the targetprotein(P-S-STAT1, U-STAT1, MxA, and β-Actin) were calculated exactly byimaging analytical system. The statistical chart was made according relative amountsof targe proteins with β-Actin as inner reference.(3) Contents of many cytokines andlevels of neurotrophic factors were tested simultaneously by Multiplex Luminex Assay. Results (1) The proportion of Tregs cells is variable with the concentration ofIFNγ. When the concentration of IFNγ is20or40ng/mL, the proportion of Tregscells is the highest and there is no significant difference between the two groups.(2)The stimulated time of IFNγ is effective to the conversion rate, when it is on thethird day, the conversion rate of Tregs cells is the highest.(3) The FoxP3expressionof the Tregs cells induced by40ng/mL IFNγ is the highest no matter in GBS patientsor in healthy donors, while it is still lower than that of nTregs in healthy donors.(4)The induced Tregs cells in GBS patients and healthy donors have the suppressivefunction on the proliferation of autologous Tresp cells. The Tregs cells induced by40ng/mL IFN-γ have the strongest suppressive function and the suppressivefunctions have no significant statistical difference between40ng/mL IFN-γ inducedTregs cells and nTregs of healthy donors.(5) Non-attack RRMS patients, in vivobasal P-S-STAT1level maintain stable and endogenous IFN level is relative lowerbefore IFN injection therapy; singal or double dose IFN injection can induceP-S-STAT1increase moderately, U-STAT1level is increased lightly, MxA(downstream IFN-stimulate gene) protein expression is increased obviously. Thoseindicate that the integral pathway is activated appropriately by IFN injection, andnon-attack RRMS patients have good response to IFN therapy.(6) With-attackRRMS patients, endogenous IFN level is relative higher and in vivo basalP-S-STAT1level has increased before IFN injection therapy; singal or double doseIFN injection can induce P-S-STAT1increase robustly, U-STAT1level is higherthan it in non-attack RRMS patients, but MxA protein expression is inhibitedobviously. Those indicate that type Ⅰ IFN pathway has defect in those with-attackRRMS, the possible spot maybe located in the process of posterior-transcription,translation, and modification.(7) With-attack RRMS patients’ cytokines analysisshow that there are more lower levels of neurotrophic factors and more higher levelsof chemokines in attack phase than in stable phase, these changes are consistent with inflammation and nervous system injuries. The level of TNFα as pro-inflammatoryfactors increased at24h posterior IFN injection significantly, this findings maybeone of the reasons that there is no effect to IFN therapy in with-attack RRMS.Conclusions IFNγ can induce Tresp cells to Tregs cells in GBS patients and healthydonors. When the concentration of IFN-γ is40ng/mL and the culture time is3days,the ratio of conversion is the highest and the induced Tregs cells are similar tonTregs of healthy donors phenotypically and functionally. RRMS patients haveheterogeneity, responders have good treatment response to IFN, can maintain noattacks for a long time; in contrast, non-responders have endogenous IFN signalpathway defect which IFN cannot rectify it, on the contrary IFN induce higherexpression of pro-inflammation factors with more relapse.