MicroRNA-155Modulates Th1and Th17Cells Differentiation And Is Associated With Multiple Sclerosis And Experimental Autoimmune Encephalomyelitis (EAE)

Objective：Multiple sclerosis (MS) is a chronic demyelinatingneurodegenerative disease of the central nervous system (CNS). It isbelieved that the disease process starts with increased migration ofautoreactive lymphocytes across the blood–brain barrier (BBB), leadingto axonal demyelination of neurons. Although the exact pathogenesis ofMS remains to be fully elucidated, CD4+T cell mediated autoimmunityhas been accepted as one of the most important aspects of thepathogenesis. The cause of MS is not clear, but according to current data,the disease develops in genetically susceptible individuals with thecontributionsof environmental factors. The knowledge about how theimmune system in MS patients is controlled differently than in unaffectedindividuals is still in its infancy. Thus, it is very useful to understand thefunctional significance of miRNAs with respect to MS pathogenesis.After antigen stimulation, naive CD4+helper T cells differentiateinto effector T cells to participate in the adaptive immune response. IFN-γproducing T helper type1(Th1) cells have been considered the type ofeffector helper T cells that mediate the pathogenesis of MS andexperimental autoimmune encephalomyelitis (EAE); Recent work hasrevealed that T helper17(Th17) cells, are critical mediators of chronic,autoimmune inflammation. Subsequent studies demonstrated thatinhibition of IL-17in mice can ameliorate several autoimmune disordersincluding experimental autoimmune encephalomyelitis (EAE).IL-17-expressing T cells have been found in lesions of brain tissues frompatients with multiple sclerosis.MicroRNAs (miRNAs) are a class of small noncoding RNAs which have recently been discovered to be regulatory modulators of geneexpression post-transcriptionally, either by targeting mRNA degradationor by inhibition of protein translation. MiRNAs directly modulate theexpression of regulatory proteins that are required for normaldevelopment and function of the immune system. Emerging evidenceunderlines an involvement of miRNAs in the pathogenesis of MultipleSclerosis (MS). A number of miRNAs have been found to bedysregulated in blood cells from MS patients, in brain lesions, as well asin biological fluids such as serum and plasma. When poorly regulated,microRNAs are critically involved in a range of human diseases andpotentially serve as good diagnostic markers, prognostic markers ortherapeutic targets.In the present study, we report that miR-155expression wasupregulated in patients with MS, and this correlated with disease severity.Further more，we detected the roles of miR-155in Th1and Th17cellsdifferentiation and cytokines production by over-expression andinhibition of miR-155in EAE.Methods:1. Patients and controls. Sera were collected from MS patients,30patients with immunology disease in neurological systerm and31healthysubjects. Controls were matched with cases in terms of gender and age.All patients had not received any immunomodulatory therapy withinthree weeks prior to blood withdrawal. RNA was isolatedaccording to themanufacturer’s instructions, and stored at-80C until analysis. Expressionof micoRNA in MS patient and controls was detected by real time PCR.2. EAE mode. Mice were injected s.c. in both flanks with100mgmyelin oligodendrocyte glycoprotein (MOG)35–55peptide dissolved inPBS emulsified in an equal volume CFA supplemented with5mg/mlMycobacterium tuberculosis H37Ra and injected twice i.p. with200ngpertussis toxin administered on the day of immunization and48h later.Clinical assessment of EAE was performed daily after disease induction, according to the following criteria:0, no disease;1, tail paralysis;2,hindlimb weakness or partial paralysis;3, complete hindlimb paralysis;4,forelimb and hindlimb paralysis;5, death.3. Expression of miR-155in T cells from mice was detected via realtime PCR, during the acute phase and remitting phase in EAE.4. Mir-155treatment. The oligonucleotides of Mir-155mimic andinhibitor was synthesized to overexpress or knockdown Mir-155. For invivo mir-155treatment,100ul Entranster TM-in vivo was mixed with50ug mir-155mimic or inhibitor or their controls respectively dissolvedaccording the instruction, and the complexes were administered i.v. toimmunized mice on day5,7,9,11,13,15postimmunization. Clinicalassessment of EAE was performed daily.5. For histopathological studies, spinal cords were dissected fromfemale mice (n=4/group), fixed in10%formalin in PBS, and embeddedin a single paraffin block. The6to10mm thick sections were stainedwith H&E and luxol fast blue, and stained sections were evaluated forimmune cell infiltration and demyelination.6. Mir-155expression was analysied of in spleen，LNs，CNS andliver of Mir-155mimic and control transferred mice via real timeRT-PCR analyses.7. Mice were cued at day13and25repctively. Spleens or draininglymph nodes were harvested and pooled from EAE mice, and single-cellsuspensions were prepared. Cells were cultured at0.5million/well in96-well U-bottom plates with10MOG35-55peptide in RPMI1640medium supplemented with10%FCS. Splenocytes or LN cells were alsocultured in complete RPMI during restimulation with relevant antigens.Splenocytes were labeled in5mM CFSE for10min at37℃, washed andcultured. For ELISA, supernatants were harvested at72h of culture.Intracellular cytokine staining was used to determine the frequency ofTh-1and Th-17cells of in the splenocytes and LNs cells in the fourgroups of miR-155mimic and inhibitor and their respective controls transfected mice. The gene expression of IL-17or interferon-γ (IFN-γ)was analysied in lymph nodes (LNs), spleen. The concentrations ofindicated cytokines were measured by quantitative capture ELISA,according to the guidelines of the manufacturers.8. Splenic mononuclear cells were obtained from mice. Then, usingCD4+T Cell Isolation Kit II naive CD4+T cells were isolated bydepletion of non-CD4+T cells. The purity of CD4+T cells was90.5%.CD4+T cell activation and polarization4h after nucleofection (Mir-155mimic, Mir-155mimic control, Mir-155inhibitor, Mir-155inhibitorcontrol). CD4+T cells were were cultured in complete RPMI, plate-boundCD3antibodies, and soluble CD28antibodies, IL-6and TGF-β for96hr.Intracellular cytokine staining was used to determine the frequency ofTh-1and Th-17cells in the four groups of miR-155mimic and inhibitorand their respective controls transfected mice. The gene expression ofIL-17or interferon-γ (IFN-γ) was analysied and concentrations ofindicated cytokines were measured by quantitative capture ELISA,according to the guidelines of the manufacturers.Results:1. We tested eight immunologically relevant microRNAs in controlsand patients with prelapsing multiple sclerosis. Data showed that fivemiRNAs resulted>1folds up-regulated in MS vs. controls, and mir-155is most up-regulated. The data also showed that miR-155expression wassignificantly higher in serum of patients with MS than in those of controlsor patients with immunology disease in neurological systerm. Detailedanalysis showed that serum of patients with relapsing multiple sclerosishad higher miR-155expression than those of patients with remittingmultiple sclerosis.2. Expression of miR-155from mice with EAE was higher in spleen,lymph node and brain during the acute phase but decreased to normalexpression when disease remitted.3. We transfect miR-155mimic, inhibitor and scrambled control in vivo by injection through the tail vein and assessed the in vivo transfectefficacy by quantitative PCR analysis of miR-155expression in variousorgans. In organs, transfect with miR-155mimic led to nearly two-foldgreater expression of miR-155in spleen and1.75-fold greater expressionin the peripheral lymph nodes, and we detected1.43-fold greaterexpression in the brain.4. Mice transfected with miR-155mimic developed severe EAE,whereas inhibitor transfected mice had somewhat mild EAE (Fig.2C).Histological analysis of spinal cord sections also showed that micetransfected with miR-155mimic developed prominent inflammatoryinfiltration and demyelination, whereas inhibitor transfection miceshowed mild CNS pathology.5. We examined LNs, spleen in the four groups of miR-155mimicand inhibitor and their respective controls transfected mice mice at day25after immunologiation for the presence of IL-17(Th17) or IFN-γ (Th1)producing CD4+T cells during EAE.(1) Intracellular cytokine stainingshowed that both the frequency of Th-1and Th-17cells of in thesplenocytes and LNs cells were higher in miR-155mimic transfectedmice but only the frequency of Th-17cells was lower in miR-155inhibitor transfected mice than the respectively controls.(2)The study ofCCK8shows that CD4+T cells in miR-155mimic transfected miceunderwent more cell divisions in vitro with MOG35-55, whereas CD4+Tcells in miR-155inhibitor transfected mice had a reduced proliferative.(3)IL-17A and IFN-γ production were assayed by ELISA in this issueculture supernatants response to MOG35-55stimulation72hr.Splenocytes of miR-155inhibitor transfected mice showed decreaseproduction of both of IL-17A and IFN-γ compared to their controls,whereas the production of these cytokines increase in splenocytes ofmiR-155inhibitor transfected mice, which further demonstrating adefective of CD4+T cell in drivening recall response to antigen.(4)Consistent with that, the genes relative expression of IL-17and IFN-γ were significantly upregulated in miR-155mimic transfected mice, butdownregulated in miR-155inhibitor transfected mice.6. Mice were harvested on day13postimmunization withMOG35-55.(1). Intracellular cytokine staining showed that both theproportion of Th-1and Th-17cells in the splenocytes were higher inmiR-155mimic transfected mice and lower in miR-155inhibitortransfected mice than their respectively controls. In LNs cells, Th17andTh1cells were present at lower proportion among CD4+T from miR-155inhibitor transfected mice, whereas the frequency of Th1cells amongCD4+T was equivalent between miR-155mimic transfected mice andcontrols.(2) The recall response to MOG35-55was also tested withsplenocytes from day13EAE mice via CCK8essays. Splenocytesexhibited diminished proliferation in MiR-155inhibitor transfected mice,whereas MiR-155mimic transfected mice exhibited increasedproliferation in splenocytes.(3) Splenocytes of miR-155inhibitortransfected mice showed decrease production of both of IL-17A andIFN-γ compared to their controls, whereas the production of thesecytokines increase in splenocytes of miR-155inhibitor transfected mice.Similar deficiencies in Th1and Th17cells were also observed in the LNs.(4) Th17and Th1expression was detected by quantitative PCR (qPCR) inthe four groups. Consistent with that, the genes relative expression ofIL-17and IFN-γ were significantly upregulated in miR-155mimictransfected mice, but downregulated in miR-155inhibitor transfectedmice.7. CD4+Splenic T cells were isolated from mice and cultured in thepresence of CD3and CD28antibodies with and without the addition ofthe Th17cell skewing factors IL-6and TGF-β. CD4+T cell transferedwith miR-155inhibitor defective in their ability to produce Th17cellscompared to miR-155mimic transfected T cell and controls as assayed byintracellular staining of IL-17A. The same cell populations and cultureconditions produced similar amounts of IFN-γ+Th1cells in the4groups. We detected expression of miR-155in CD4+T cells grown in conditionsthat promote Th17cell development. Reduced expression of IL-17AmRNA was measured in miR-155inhibitor transferred T cell under Th17cell skewing conditions compared to miR-155mimic group and controls.These results reveal a T cell-intrinsic role for miR-155in promoting thein vitro development of Th17cells.Conclusion: we report that miR-155expression was upregulated inpatients with MS, and this correlated with disease severity. Further more，we detected the roles of miR-155in Th1and Th17cells differentiationand cytokines production by over-expression and inhibition of miR-155in EAE. These results reveal a T cell-intrinsic role for miR-155inpromoting the in vitro development of Th17cells.