Immunotheraphy Of Atorvastatin-modified Dendritic Cells In Experimental Autoimmune Neuritis

Background:Acute inflammatory demyelinating polyneuropathy (AIDP), the most common form of Guillain-Barre syndrome (GBS), is a CD4+T cell-mediated disease of the peripheral nervous system (PNS), with an annual incidence of0.62-2.66cases per100,000population. Experimental autoimmune neuritis (EAN) serves as an animal model for AIDP in humans, which can be induced in susceptible animal species and strains by immunization with an autoantigen emulsified in complete Freund’s adjuvant.Dendritic cells (DCs), being professional antigen-presenting cells, initiate and orchestrate immune responses. In contrast to activated DCs, immature DCs with low levels of major histocompatibility complex (MHC) and co-stimulatory molecules have been implicated in the regulation of immune responses through diverse effector mechanisms. Particularly, immature DCs are able to induce a state of hyporesponsiveness in T cells, and the phenomenon has been used for the control or suppression of the immune response. Therefore, some attempts have been made to modify DCs to retain their immature phenotype for the induction of antigen-specific tolerance and protect from some autoimmune diseases. Statins, including atorvastatin, are competitive inhibitors of HMG-CoA reductase that regulates cholesterol synthesis in the mevalonatepathway. They are widely used in the treatment of atherosclerosis and hypercholesterolemia. Clinical and experimental evidences have demonstrated that statins have pleiotropic effects that include anti-inflammatory and immunomodulatory effects, especially inhibiting DCs differentiation and maturation. Previously, our group found that atorvastatin suppressed the maturation of spleen derived DCs, on which with the lower expression of CD80and CD86, and the statin-modified DCs could ameliorate experimental autoimmune myasthenia gravis by up-regulated Treg cells and shifted Th1/Th17to Th2cytokines. So we presume that statin-modified DCs could also attenuate EAN by immune regulation. In the present study, we transferred statin-modified DCs to EAN rats in the initial phase of the disease to evaluate its possible role and immunomodulatory effects during the process.Objective:The aim of the present study was to analyze the effects of DCs modified with atorvastatin (statin-DCs) on the immune tolerance induction in Lewis rats with EAN.Methods:1. Induction of EAN and assessment of clinical symptoms200μl of the inoculum contained5mg BPM and2mg Mycobacterium tuberculosis emulsified in incomplete Freund’s adjuvant was subcutaneous injected into both hind footpads of the rats to induce EAN. Clinical scores were assessed immediately before immunization (day0) and thereafter every day until day14post immunization (p.i.).The severity of clinical symptoms was scored as follows:0=no illness;1=flaccid tail;2=dragging both hind limbs;3=paralysis of both hind limbs; and4=paralysis of four limbs or death, intermediate scores of0.5increment were given to rats with intermediate signs. The rats were monitored for clinical symptoms of disease by two independent investigators.2. DCs preparation, modification and injection DCs were prepared by the method of Duan et al. with some modifications. The spleens were removed from normal Lewis rats of6-8weeks old under aseptic conditions. Grinding the spleen from individual rats through cell strainers in medium to obtain the mononuclear cells (MNC) suspensions, then erythrocytes were lysed osmotically. DCs were further enriched by differential adherence by incubating cells. After removing the non-adherent cells, new RPMI1640medium containing gentamicin, penicillin and10%fetal bovine serum were added to the flasks. After18h of incubation, atorvastatin dissolved in DMSO was added to the flasks. The same volume of DMSO was added to the other flasks. After incubation, floating cells were collected as a DC-enriched fraction. Then statin-DCs and untreated-DCs were intraperitoneally transferred into EAN rats respectively on day5p.i. And the same volume of medium was transferred into the control rats. For phenotypic analysis, statin-DCs and untreated-DCs were washed with0.5%bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and stained with fluorescein isothiocyanate (FITC)-conjugated anti-rat MHC class Ⅱ, phycoerythrin (PE)-conjugated anti-rat CD80and FITC-conjugated anti-rat CD86antibodies, after resuspending, the cells were analyzed by FACS.3. Preparation of lymph node MNC Inguinal lymph nodes were removed under aseptic conditions when rats were sacrificed on day14p.i. MNC suspensions were obtained by grinding the organs through cell strainers in serum-free medium. Then cells were rediluted to a cell concentration of2*106cells/ml for all experiments.4. Flow cytometric analysis of surface molecules, NK and NKT cells, intracellular cytokines and Treg cells in MNC from lymph nodes MNC suspensions were incubated with PE-labeled anti-rat CD80, FITC-labeled anti-rat CD86, FITC-labeled anti-rat MHC class Ⅱ, FITC-labeled anti-rat CD3, and PE-labeled anti-rat CD161a in the dark, respectively; Lymph node MNC were fixed and permeabilized, then incubated with the PE-labeled IL-10, FITC-labeled IFN-γ, PE-labeled TNF-a and FITC-labeled IL-17A, in the dark respectively; Fixation and permeabilization of lymph node MNC were performed using the eBioscience Foxp3Staining Buffer Set. Then the cells were re-suspended in PBS respectively and analyzed by FACS.5. Analysis of cell viability MNC of rats in the same group were mixed at equally number and incubated with CFSE in the dark, then stained by adding ice-cold1640and incubated on ice, after concentration and suspension, the mentioned cells suspended in1000μl aliquots containing1x106cells were cultured in triplicates in flat-bottomed24-well microtiter plates in the presence of BPM (15μg/ml) or Concanavalin A (ConA,5μg/ml). After incubation in a humidified atmosphere of95%air and5%CO2at37℃, the cells were collected respectively in the tube and incubated with PE-labeled anti-rat CD4antibody in the dark. Lastly, the cells were washed and re-suspended in PBS. Then the cells were analyzed by FACS.6. Histopathological assessment After the rats killed on day14p.i. Sciatic nerve segments were excised close to the lumbar spinal cord, then fixed in10%paraformaldehyde and embedded in paraffin. Multiple longitudinal sections of sciatic nerves were stained with hematoxylin and eosin for evaluation of the inflammatory cells by light microscopy.7. Immunohistochemistry Thymuses obtained from the rats on day14p.i. were excised and fixed in10%paraformaldehyde, then embedded in paraffin. Paraffin tissue sections were deparaffinized and hydrated. And in order to block the endogenous peroxidase activity, the sections were treated with0.3%hydrogen peroxide. After antigen retrieval and incubated with rat anti-mouse/rat foxp3antibody, the sections were stained with HRP-conjugated goat anti-rat secondary antibody, followed by development with diaminobenzidine (DAB) substrate to detect the number of Foxp3+cells. As negative controls for immunostaining, the primary antibodies were omitted.8. Statistical analysis The statistics were calculated and evaluated by the SPSS17.0computer program. Differences among different groups were tested by one-factor analysis of variance (ANQVA) followed by Least Significant Difference (LSD) test as a post-hoc test. Results were presented as mean±SD and a level of p<0.05was considered significant.Results:1. Effects of atorvastatin on the phenotype of DCs The results showed that the expression of CD80and CD86was inhibited on statin-DCs when compared with those on untreated-DCs.2. Statin-DCs suppress severity of clinical EAN The rats in the statin-DC group exhibited lower clinical scores when compared with the rats in control group and untreated-DC group. While it has no significant difference of the clinical scores between untreated-DC group and control group. On the day of the experiment termination, the clinical score of the rats in the statin-DC group averaged1.55±0.45, meanwhile, the clinical scores in the control and the untreated-DCs group averaged2.7±0.57and2.4±0.42.3. Statin-DCs decrease the number of inflammatory cells in the PNS The rats in statin-DC treated group had fewer inflammatory cells in sciatic nerves than the rats in the untreated-DC and control group.4. Statin-DCs inhibit the expression of co-stimulatory molecules and MHC class II in MNC from lymph nodes The expression of CD80, CD86and MHC class II in lymphocytes was inhibited in the statin-DC group when compared with those in the control group and untreated-DC group but without significant difference. Moreover, the expression of CD80and CD86in lymphocytes between the control and the untreated-DC groups did not differ significantly, but the expression of MHC class II was lower in the untreated-DC group when compared with that in the control group.5. Statin-DCs decrease the Th1and Th17cytokines in MNC from lymph nodes IFN-y, TNF-a and IL-17A were decreased in the statin-DC group when compared with those in the control group. IL-17A and TNF-a were decreased in the statin-DC group compared with those in the untreated-DC group, however there were no significant differences. At the same time, IFN-y and TNF-a were lower in the untreated-DC group when compared with those in control group, and there was no significant difference of IL-17A between these two groups. There was no statistical difference for IL-10among the three groups.6. Statin-DCs increase the numbers of NK and NKT cells in MNC from lymph nodes The percentage of CD3+CD161a+NK cells was increased in the statin-DC group when compared with that in the control and the untreated-DC group, and the percentage of CD3+CD161a+NKT cells was increased in the statin-DC group when compared with that in the control as well as the untreated-DC group but without any statistical difference.7. Statin-DCs increase the number of CD4+Foxp3+T cells in MNC of the lymph nodes Statin-DCs increased the percentage of CD4+Foxp3+T cells among lymph node MNC when compared with untreated-DCs and control.8. Statin-DCs increase the number of Foxp3+cells in the thymus The number of Foxp3+cells in thymus of statin-DCs treated rats was increased when compared with those in the other two groups. There was no significant difference between the control group and untreated-DCs group.9. Statin-DCs suppress lymphocyte proliferation in EAN Lymphocyte proliferation was significantly decreased in the statin-DC group compared with those in the control and untreated-DC group with BPM or without BPM. There was no significant difference between the control and untreated-DC group.Conclusions:1. DCs treated with atorvastatin (statin-DCs) can be induced into tolerogenic DCs.2. Administration of statin-DCs ameliorate the clinical symptoms in EAN.3. Statin-DCs induce both central (thymus) and peripheral immune tolerance (lymph node) in EAN.