
 multiple sclerosis ( ms ) is a chronic inflammatory disease of the central nervous system ( cns ) .
numerous evidences strongly suggest that it is an autoimmune disease in which activated t cells enter the cns and trigger an inflammatory cascade resulting in demyelination and axonal loss .
different genetic and environmental factors have shown to play a role in ms susceptibility , being class ii alleles of the major histocompatibility complex ( mhc ) , the more closely associated genes , and infectious agents such as epstein - barr virus , the environmental factors that have been more clearly associated with ms susceptibility .
conversely , high serum levels of vitamin d have a protective role , delaying the appearance of a second demyelinating event after a clinically isolated syndrome .
it has been described that this action can be mediated by upregulation of regulatory t cells ( treg )   and b cells ( breg ) .
it was also described that ms patients suffer from a peripheral b cell tolerance defect that may be attributable to impaired treg function   and that proliferation of breg induced by intestinal helminth infections ameliorates ms course .
the role of regulatory cells in demyelinating diseases has been also explored in experimental autoimmune encephalomyelitis ( eae ) , a widely used animal model of ms in which t cell mediates cns demyelination .
eae can be induced in several species by immunization with myelin antigens or via adoptive transfer of myelin - reactive t cells [ 9 , 10 ] .
adoptive transfer of breg reduced significantly the severity of eae by inhibiting th1 and th17 inflammatory responses .
likewise , expansion of treg diminished the infiltration of inflammatory cells into the cns and improved clinical signs of eae .
these data strongly suggest that deficits in regulatory pathways may contribute to the induction of autoimmune reactivity in eae .
these experimental models may be useful tools to identify immunological mechanisms mediating a protective role in demyelinating diseases .
several strains of mice are resistant to develop eae as mice are asymptomatic following immunization with myelin antigens . it was argued that a poor recognition of encephalitogenic peptides by mhc molecules might contribute to resistance to eae .
we explored them in two mouse strains , cd1 and c57bl/6 , that are , respectively , resistant and susceptible to eae induced by the myelin oligodendrocyte glycoprotein 3555 peptide ( mog3555 ) .
since cd1 is an outbred strain , these mice do not share a unique mhc haplotype , which makes this model useful to study mhc - independent mechanisms that mediate resistance to this experimental model of ms .
we explored breg and treg in both strains and studied if variations in regulatory cells associated with downregulation of effector immune mechanisms .
eight - to - ten - week - old female c57bl/6j mice , which are susceptible to eae induction by mog3555 immunization , were purchased from harlan ( barcelona , spain ) .
age- and sex - matched cd1 mice that are resistant to eae induction were obtained from our own conventional barrier protection breeding colony ( cajal institute , csic , madrid , spain ) .
mice were housed under standardized light- and climate - controlled conditions and were fed with standard chow and water ad libitum .
experiments were done in compliance with the guidelines of animal care set by the european union ( 86/609/eec ) , and all animal protocols were approved by the cajal institute animal welfare committee ( protocol n iorg0006540 ) . for immunization c57bl/6 and cd1 mice were injected subcutaneously on day 0 with 200  l of an emulsion containing 300  g of mog3555 ( cnb , csic , madrid , spain ) and 800  g of heat killed mycobacterium tuberculosis h37ra ( difco , bd diagnostics , md ) emulsified in incomplete freund adjuvant oil ( sigma - aldrich , st .
in addition , the mice received 100  ng of bordetella pertussis toxin ( sigma - aldrich ) intraperitoneally on days 0 and 2 after immunization ( p.i . ) .
mice from both strains c57bl/6 or cd1 were immunized in the same way using the 139151 peptide of proteolipid protein ( plp139151 ) ( proteomics section , universitat pompeu fabra , barcelona , spain ) for comparison with mog3555 as c57bl/6 mice do not develop eae following immunization with plp peptide .
mice were monitored daily , and clinical signs of eae were graded as follows : grade 0 , normal ; grade 1 , flaccid tail ; grade 2 , mild hind - limb weakness ; grade 3 , severe hind - limb weakness ; grade 4 , hind - limb paralysis ; grade 5 , hind - limb paralysis and partial fore - limb weakness . on the basis of previous reports   we selected day 21 p.i . as the time point in which experimental procedures were performed .
mice were anaesthetized by intraperitoneal administration of eutalender ( normonlab , madrid , spain ) .
spleens were removed and splenocyte suspensions were generated by grinding spleens through a wire mesh .
the splenocytes were seeded in 96-well plates at a cell density of 2  10 cells / well in iscove 's modified dulbecco 's medium ( imdm ; paa laboratories gmbh , pasching , austria ) supplemented with 10% hyclone fetalclone i ( thermo fisher scientific , waltham , ma , usa ) , 50  mol / l of 2-mercaptoethanol ( sigma chemical ) , 2  mmol / l of glutamine , 50 u / ml of penicillin , and 50  mg / ml of streptomycin ; the last three chemicals were obtained from gibco brl ( paisley , uk ) . for splenocyte activation , we used 5  g / ml of mog3555 , plp139151 , or phytohaemagglutinin ( pha ; sigma chemical ) .
cells were incubated in a humidified atmosphere at 5% co2 and 37c for 3 days , the last 1820  h in the presence of 1  ci / well of [ h]-thymidine ( perkinelmer inc . , usa ) .
the levels of incorporated radioactivity were determined using a beta - scintillation counter ( wallac , turku , finland ) . the stimulation index ( si )
was expressed as the mean of the counts per minute ( cpm ) of five replicates from each mouse and culture condition divided by the mean cpm of the baseline control replicates .
the results are expressed as the mean value    standard error of the mean ( sem ) of the si per group of mice .
we performed this study in series of seven mice per condition . to study lymphocyte subsets ,
cells were resuspended at 10/ml and incubated with rat anti - mouse cd16/32 fc receptor ( fcr ) ( cd16/cd32 ) during 10 minutes to avoid unspecific staining .
then , they were incubated with the appropriate combinations of monoclonal antibodies ( bd pharmingen , san diego , ca ) for 20 minutes at 4c .
cells were washed with pbs and flow - cytometry analysis was performed on facscanto ii ( becton dickinson ) and analyzed using the diva software ( becton dickinson ) .
as strategy for flow - cytometry analysis , an initial region was set around cells expressing intermediate to high cd45 with low - to - intermediate side scatter ( p1 ) and then a second region was set on the forward / side scatter dot plot to exclude debris or apoptotic cells ( p2 ) .
we used the following monoclonal antibodies : anti - cd4-fitc , anti - cd8a - pe , anti - cd45-cy5.5percp , anti - cd25-apc , anti - b220-fitc , anti - cd1d - pe , and anti - cd5-apc , and isotype controls conjugated with fitc , pe , cy5.5percp , and apc .
splenocytes were resuspended ( 10 cells / ml ) in complete medium [ rpmi supplemented with 2  mm glutamine , 2  mg / ml gentamycin ( all from gibco brl ( paisley , uk ) ) , and 10% fetal calf serum ( biowhittaker technologies , md , usa ) ] and the cells were cultured for four hours with pma ( 50  ng / ml ; sigma - aldrich , st .
louis , mo ) , ionomycin ( 500  ng / ml ; sigma - aldrich ) , and monensin ( 2  m ; ebioscience , san diego , ca ) . for il-17a and ifn - gamma detection ,
fcr were blocked with anti - cd16/cd32 antibody ( bd pharmingen ) for 10 minutes at room temperature before cell surface staining .
cells were stained with antibodies against surface antigens for 30  min at 4c using predetermined optimal concentrations of each antibody .
stained cells were fixed and permeabilized using a cytofix / cytoperm kit ( bd pharmingen ) , according to the manufacturer 's instructions , and stained with anti - il-17a - pe and anti - ifn - gamma - apc for 30  min at 4c . after washing cells ,
flow - cytometry analysis was performed on facscanto ii ( becton dickinson ) and analyzed using the diva software ( becton dickinson ) .
serum cytokines were determined by a milliplex map mouse cytokine panel ( millipore , billerica , ma ) following manufacturer instructions .
maxisorp microtiter plates ( nunc , roskilde , denmark ) were coated overnight at 4c with 100  l / well of mouse mog3555 peptides ( cnb , csic ) at a concentration of 10  g / ml in 0.1  m carbonate buffer , ph 9.7 .
plates were washed with pbs containing 0.02% tween 20 and blocked with 0.1  m carbonate buffer , ph 9.7 , containing 5% bsa for 1  h at 37c .
then , plates were incubated with triplicate serum samples diluted at 1/200 for 1  h at 37c and washed and incubated with biotinylated anti - mouse igm ( sigma - aldrich ) or anti - mouse igg ( jackson immunoresearch , suffolk , uk ) for 1  h at 37c .
after this , they were washed and incubated with streptavidin - horseradish peroxidase ( roche , basel , switzerland ) for 30  min at 37c .
after a final wash , the reaction products were visualized using opd ( sigma - aldrich ) as a substrate and read at 492  nm with a microplate reader ( thermo fisher , cambridge , uk ) .
experiments were always performed in two independent series of mice using four animals per condition .
we explored regulatory and effector immune mechanisms in cd1 and c57bl/6 mice immunized with mog3555 , using as controls nonimmunized mice and mice immunized with plp139151 , a myelin peptide that is not capable of inducing eae in any of those mouse strains .
we tested the polyclonal and antigen - specific proliferative capacity of splenocytes 21 days after the immunization .
a diminished proliferation showing a clear statistical trend ( p = 0.056 ) was detected in the resistant strain when a polyclonal stimulus such as pha was used ( figure 1 ) .
moreover , upon antigen - specific stimulation , the splenocytes from cd1 resistant mice did not proliferate , while those of c57bl/6 susceptible mice did ( p = 0.016 , figure 1 ) .
control study using irrelevant plp139151 peptide as stimulus did not render proliferation in any of the strains .
all these data indicate that despite their heterogeneous mhc antigens , all cd1 resistant mice are unable to develop a t cell response against the mog3555 encephalitogenic peptide . we next aimed to explore immunological mechanisms that contribute to this phenomenon . we first examined b1 and b2 subsets ( figures 2(a ) and 2(b ) ) .
we did not found differences in the percentages of t cell dependant b2 cells ( b220+cd5 ) between resistant and susceptible mice ( nonimmunized or immunized with mog or plp peptides ) .
conversely , the percentage of innate b220+cd5 + b cells was downmodulated in c57bl/6 mice after eae induction by mog3555 ( p = 0.005 ) when compared with cd1 resistant mice immunized with the same peptide .
when exploring these b cells , we detected a decrease of breg in c57bl/6 mice suffering eae ( p = 0.02 , figures 2(c ) and 2(d ) ) .
conversely , resistant cd1 strain upregulated this subpopulation after immunization with mog3555 ( p = 0.03 ) .
these differences between the two strains ( p = 0.001 ) were only observed upon mog3555 immunization . by contrast
, breg remained unchanged in either resistant or susceptible mice immunized with the plp139151 irrelevant peptide .
this strongly suggests that breg are specifically triggered to prevent the autoimmune process and have an important role in inducing resistance to eae .
we also explored this t cell subset ( cd4 + , cd25 ) in our series of mice ( figure 3 ) . upon immunization with
no differences were found in this t cell subset between resistant and susceptible strains when nonimmunized mice or mice immunized with the plp139151 peptide were studied .
these observations indicate that treg may also play a role in resistance to eae shown by cd1 mice immunized with mog3555 .
we did not find differences in the levels of igm or igg anti - mog3555 antibodies or in total cd4 + and cd8 + t cell percentages between the resistant and the susceptible mice ( table 1 ) . to further discriminate different t cell responses , we analyzed th1 and th17 subsets .
we explored the percentage of cd4 + t cells showing intracellular production of ifn - gamma ( figure 4 ) and il-17 ( figure 5 ) upon stimulation with phorbol-12-myristate-13-acetate ( pma ) and ionomycin .
only susceptible mice that developed eae in response to mog3555 showed significant increases in the secretion of ifn - gamma ( p = 0.02 ) and il-17 ( p = 0.009 ) compared to resistant mice . by contrast , resistant mice were unable to mount any of these responses after the same immunization protocol .
in addition , when studying serum inflammatory cytokines , we detected a significant increase of il-17 in susceptible mice immunized with mog3555 ( figure 4(c ) ) .
this shows that downmodulation of regulatory b and t cells is related to the induction of th1/th17 responses in mog - induced eae .
the understanding of the immune mechanisms involved in resistance / susceptibility to eae is important , as it may contribute to identification of new therapeutic targets in ms .
the role of mhc by inducing defective or appropriate presentation of the encephalitogenic peptide has been previously demonstrated . however , different evidences suggest that induction of breg may also contribute to the resistance to eae .
moreover , depletion of breg exacerbates eae symptoms and increases encephalitogenic t cell influx into the cns .
in addition , induction of breg in mog - immunized - c57bl/6 mice treated with antibiotics ameliorates eae course , and this effect can be transferred to other c57bl/6 mice by passive transfer of these b cells . here , we aimed to explore if breg may be a mhc - independent mechanism for inducting resistance to mog - induced eae .
we explored this in c57bl/6 , a congenic mouse strain susceptible to mog3555-induced eae , and in cd1 an outbred strain resistant to mog3555-induced eae in an mhc - independent manner .
we immunized both strains of mice with the encephalitogenic peptide and observed that cd1 splenocytes were incapable of proliferating when stimulated with mog3555 .
we next explored immunological mechanism associated with this phenomenon and observed that susceptible c57bl/6 mice developing the disease after mog3555 immunization downregulate breg , while cd1 resistant mice show a significant increase of these cells upon immunization with the same peptide .
this strongly suggests that breg play a role in the induction of resistance to eae in cd1 mice .
in addition , an increase of treg may prevent the onset of eae . in the present study we found that , in parallel with breg findings , resistant mice upregulated treg .
this seems to indicate that treg also have a role in resistance to mog - induced eae .
however , the immunization of c57bl/6 mice with the plp139151 peptide , which is incapable of inducing eae in this strain in an mhc - dependent manner , did not induce significant changes in breg or treg subsets .
this clearly shows that when mhc molecules impede the correct peptide presentation , regulatory responses are not triggered as it happens in the outbred mice . to further study differences between resistant and susceptible mice , we explored effector responses in the same series of mice .
it has been reported that antibodies may have beneficial or detrimental effects in demyelinating diseases [ 20 , 21 ] . in mog - induced eae model
they are not related to the initiation of the disease and it remains controversial if they can influence disease severity [ 22 , 23 ] .
we tested the presence of anti - mog3555 igm and igg antibodies in serum of resistant and susceptible mice .
there were no significant increases in anti - mog3555 igg or igm responses in mog3555-immunized mice of either susceptible or resistant strains .
this shows that igm or igg anti - mog3555 antibodies do not play a role in inducing susceptibility to mog3555-induced eae .
previous reports showed that cd4 + t cells are required for the initial induction of progressive eae and that the disease is downregulated by cd8 + t cells . here , we did not find differences in total cd4 + and cd8 + t cell percentages between any of the groups of resistant and susceptible mice .
to further discriminate different t cell responses , we analyzed effector th1 and th17 subsets .
it has been reported that il-17 impairs the integrity of the blood brain barrier in eae   and that a rise of spleen th17 cells prolongs the disease . moreover ,
a decrease in il-17 levels in supernatants of mog3555-activated splenocytes runs in parallel with reduced demyelination and axonal damage in mice with eae .
we did not find significant differences in the percentages of cd4 + cells secreting ifn - gamma or il-17 in nonimmunized mice .
conversely , we observed that susceptible mice that developed eae in response to mog3555 showed significant increases in the secretion of ifn - gamma and il-17 .
by contrast , resistant mice were unable to mount any of these responses after the same immunization protocol .
in addition , serum levels of il-17 were increased in susceptible mice immunized with mog3555 .
these data confirm the importance of the induction of th1/th17 responses in mog - induced eae . in conclusion ,
our data show that upregulation of b and , to a lesser extent , of treg , is closely associated with mhc - independent resistance to mog - induced eae in cd1 mice and with the abrogation of both th1 and th17 responses , which have a critical role in the development of the disease .
these data help to ascertain the regulatory mechanisms than can downmodulate inflammatory responses in demyelinating diseases .
 
 background and objectives . 
 resistant and susceptible mouse strains to experimental autoimmune encephalomyelitis ( eae ) , an inducible demyelinating experimental disease serving as animal model for multiple sclerosis , have been described . 
 we aimed to explore mhc - independent mechanisms inducing resistance to eae . 
 methods . for eae induction , female c57bl/6 ( susceptible strain ) and cd1 
 ( resistant outbred strain showing heterogeneous mhc antigens ) mice were immunized with the 3555 peptide of myelin oligodendrocyte glycoprotein ( mog3555 ) . 
 we studied t cell proliferation , regulatory and effector cell subpopulations , intracellular and serum cytokine patterns , and titers of anti - mog serum antibodies . 
 results . upon immunization with mog3555 , t lymphocytes from susceptible mice but not that of resistant strain were capable of proliferating when stimulated with mog3555 . accordingly , resistant mice experienced a rise in regulatory b cells ( p = 0.001 ) and , to a lower extent , in regulatory t cells ( p = 0.02 ) compared with c57bl/6 susceptible mice . as a consequence , mog3555-immunized c57bl/6 mice showed higher percentages of cd4 + t cells producing both ifn - gamma ( p = 0.02 ) and il-17 ( p = 0.009 ) and higher serum levels of il-17 ( p = 0.04 ) than resistant mice . conclusions . 
 expansion of regulatory b and t cells contributes to the induction of resistance to eae by an mhc - independent mechanism . 
1. Introduction
2. Materials and Methods
3. Results 
4. Discussion