the t cell immunoglobulin and mucin domain ( tim ) family is located on chromosome 11b1.1 in mice and consists of several members ( tim-18 ) . in humans
it is located on chromosome 5q33.2 and consists of three members ( tim-1 , 3 , and 4 ) .
individual tim family members may serve as susceptibility markers for asthma , allergies , and autoimmune diseases , as well as potential cell surface markers for t helper ( th ) type 1 and th2 cells [ 1 , 2 ] . therefore , the human tim gene family is critical in the regulation of th1/th2 mediated immunological reactions .
tim-1 was first identified as a hepatitis a virus cellular receptor 1 [ 3 , 4 ] and a kidney injury molecule , kim-1 [ 5 , 6 ] .
tim-1 is expressed on cd4 t cells after activation and its expression is sustained preferentially in th2 but not th1 cells [ 1 , 7 ] .
the high - avidity anti - tim-1 antibody enhances the severity of experimental autoimmune encephalitis by increasing autopathogenic th1 and th17 responses , whereas the low - avidity antibody inhibits autopathogenic th1 and th17 responses .
tim-4 is a natural ligand of tim-1   and is exclusively expressed on antigen - presenting cells , including dendritic cells ( dcs ) and macrophages [ 9 , 10 ] , where it mediates phagocytosis of apoptotic cells and plays an important role maintaining tolerance [ 11 , 12 ] .
tim-1 and tim-4 interact to regulate th cell responses and modulate the th1/th2 cytokine balance .
dc - derived tim-4 maintains tim-1 in th2 cells in a stable status and plays a critical role sustaining th2 polarization .
a higher dose of tim-4-ig consistently leads to an increase in t cell proliferation upon ligation with the t - cell receptor , whereas a lower concentration of tim-4-ig inhibits t cell proliferation .
human tim-1 is also associated with other types of immune dysfunction , such as atopic dermatitis , allergy , rheumatoid arthritis , asthma , and systemic lupus erythematosus ( sle ) [ 1418 ] , suggesting that tim-1 may regulate immune responses .
in addition , tim-4 expression in peripheral blood mononuclear cells ( pbmcs ) also increases in patients with sle .
behet 's disease ( bd ) is a th1-polarized , chronic , multisystemic inflammatory disorder with arthritis , gastrointestinal , mucocutaneous , ocular , vascular , and central nervous system involvement .
the etiology of bd is unclear ; however , viral infection has long been postulated as one of the main factors .
since behet first proposed a viral etiology , his hypothesis has been verified by detecting virus in saliva , intestinal ulcers , and genital ulcers [ 24 , 25 ] of patients with bd .
subsequently , herpes simplex virus ( hsv ) inoculation of the earlobes of icr mice resulted in the development of bd - like symptoms .
manifestations in mice inoculated with hsv include multiple symptoms such as oral ulcers , genital ulcers , skin ulcers , eye symptoms , intestinal ulcers , arthritis , and neural involvement , as well as skin crusting .
the frequencies of these symptoms are similar to those of patients with bd . tim-1 and
tim-4 have not been studied much in bd until now . in this study , we investigated the tim expression in a bd mouse model with bd - like symptoms .
the expression tim-1 and tim-4 was analyzed in bd mice and the changes in bd - like symptoms were observed by regulating of tim-1 or tim-4 expression .
furthermore , the changes in cellular phenotypes and cytokine levels on immune cells were confirmed after upregulation of tim-1 or downregulation of tim-4 in bd mice .
mouse anti - cd4 , anti - tim-1 , anti - tim-4 , anti - cd8a , anti - cd122 , anti - cd11b , anti - cd11c , and anti - cd25 antibodies as well as an anti - foxp3 staining kit were purchased from ebioscience ( san diego , ca , usa ) .
icr male mice ( 4 - 5 weeks old ) were infected with hsv type 1 ( 1  10  pfu / ml , f strain ) grown in vero cells as described previously .
we used anesthetic composed of a mixture of zoletil ( virbac lab , carros , france ) and rompun ( bayer , seoul , korea ) .
the ratio of zoletil and rompun was 1  :  4 , and it was administered at a dose of 40  l / mouse ( tiletamine 10  mg / kg , zolazepam 10  mg / kg , and xylazine hydrochloride 36  mg / kg ) via intramuscular injection .
virus inoculation was conducted twice at 10 day intervals , after which the animals were observed for 16 weeks .
animals were handled in accordance with a protocol approved by the animal care committee of ajou university school of medicine ( institutional approved number : amc-102 ) .
multiple symptoms were observed in the mice after hsv inoculation , and 12% of the hsv - injected mice developed bd - like symptoms .
disappearance of symptoms or a > 20% decrease in dimension of lesion size was classified as effective .
determination of the bd severity score was followed by determining the value of the bd activity index , as outlined on the bd activity form ( http://www.behcet.ws/pdf/behcetsdiseaseactivityform.pdf ) .
symptoms exhibited by patients , including mouth ulceration , genital ulceration , erythema , skin pustules , skin ulceration , joints - arthritis , diarrhea , blurred or red eye ( right , left ) , reduced vision ( right , left ) , loss of balance , discoloration of skin , and swelling of the face were selected and analyzed in the bd mouse model .
the score of each symptom was one , and the total score was used to determine the bd severity score .
briefly , the tim-1 open reading frame ( excluding the start codon and signal sequence ) was amplified from this clone by polymerase chain reaction and ligated into a pcdef3 expression plasmid .
all plasmids used were purified by two passes through endo - free columns ( qiagen , chatsworth , ca , usa ) as described previously .
cac auc aga uca aca gca guu -3 , and antisense : 5- cug cug uug auc uga ugu gau uua guu -3. the tim-4 sirna with transfection reagent jetpei ( polyplus - transfection , llkirch , france ) was used to inject into mice .
ten g of tim-1 vector was intraperitoneally injected four times at 2 day intervals into bd mice when the bd - like symptoms appeared , followed by 2 weeks of observations .
five g of sirna tim-4 was intraperitoneally injected three times at 2 day intervals into bd mice to downregulate tim-4 , followed by a 2-week observation .
pbmcs and lymph node cells were isolated from mice and erythrocytes were removed from cell suspensions in ack solution , then washed with phosphate buffered saline ( pbs ) .
the cells were surface - stained with anti - mouse antibodies ( cd4 , cd8 , cd11b , cd11c , cd25 , cd122 , tim-1 , and tim-4 ) for 30  min at 4c in the dark .
an anti - mouse foxp3-staining buffer set was used according to the manufacturer 's instructions to detect foxp3 intracellularly .
briefly , cells were fixed using fix / perm buffer after washing with 1 permeabilization buffer and then incubated with anti - mouse foxp3 antibody . for analysis ,
the cells were gated and then the population of stained cells was analyzed by a flow cytometer ( facs canto ii ; becton dickinson , franklin lakes , nj , usa ) with 10,000 gated lymphocytes .
serum was collected 14 days after the first administration of the tim-1 vector and tim-4 sirna into bd mice .
serum was analyzed using commercial elisa kits for the detecting mouse interleukin ( il)-6 ( r&d systems , minneapolis , mn ) , tumor necrosis factor ( tnf)- ( r&d systems ) , il-17 ( r&d systems ) , il-4 ( r&d system ) , and interferon ( ifn)- ( r&d systems ) , according to the manufacturer 's instructions .
the elisa reader was bio - rad model 1706850 microplate reader , and samples were read at a wavelength of 450  nm . all data are expressed as mean  standard deviation .
statistical differences between the experimental groups were determined using student 's t - test with a bonferroni correction .
the frequencies of tim-1 , cd4tim-1 , and cd8tim-1 cells were analyzed in cells from lymph nodes ( ln ) , spleen ( sp ) , and pbmcs of normal healthy ( nor ) and bdn ( hsv-1 was inoculated but no symptoms ) mice and compared with those in bd mice by facs . in ln , the frequencies of tim-1 , cd4tim-1 , and cd8tim-1 cells in bd mice were significantly lower than those in bdn mice ( bdn versus bd ( % ) : tim-1 , 20.1  11.5 ( n = 12 ) versus 11.1  6.7 ( n = 12 ) , p = 0.01 ; cd4tim-1 , 3.4  1.9 ( n = 9 ) versus 2.2  1.6 ( n = 9 ) , p = 0.09 ; cd8tim-1 , 1.9  1.1 ( n = 9 ) versus 1.1  0.6 ( n = 9 ) , p = 0.04 ) ( figure 1(a ) ) . in sp ,
the bd mice showed significantly lower frequencies than nor or bdn mice ( nor versus bdn versus bd ( % ) : tim-1 , 21.3  6.5 ( n = 6 ) versus 18.3  8.5 ( n = 10 ) versus 12.2  5.9 ( n = 10 ) , nor versus bd p = 0.006 , bdn versus bd p = 0.04 ; cd4tim-1 , 3.1  1.6 ( n = 6 ) versus 1.9  1.0 ( n = 8) versus 1.7  0.5 ( n = 8) , nor versus bd p = 0.02 , bdn versus bd p = 0.35 ) .
cd8tim-1 cells showed similar frequencies among three groups ( figure 1(b ) ) . in pbmcs ,
the frequencies of bd mice were slightly lower than those of bdn , but not significantly ( bdn ( n = 9 ) versus bd ( n = 9 ) ( % ) : tim-1 , 27.6  17.1 versus 23.6  8.4 ,   p = 0.54 ; cd4tim-1 , 8.3  5.9 versus 5.7  4.4 , p = 0.31 ; cd8tim-1 , 7.6  5.0 versus 5.2  4.3 , p = 0.29 ) .
bd mice had significantly higher levels than those of nor mice ( figure 1(c ) ) .
these data indicate that the frequencies of tim-1 expressing cells in bd mice were downregulated compared to those in bdn mice .
the frequencies of tim-4 cells were also analyzed in ln , sp , and pbmcs of nor , bdn , and bd mice . in ln , the frequencies of tim-4 cells in bd mice were significantly higher than those in nor and bdn mice ( nor versus bdn , versus bd ( % ) : 1.3  0.8 ( n = 11 ) versus 3.2  0.9 ( n = 15 ) versus 5.8  2.0 ( n = 15 ) , nor versus bdn p = 0.000005 , nor versus bd , p = 0.0000001 , bdn versus bd p = 0.00003 ) ( figure 2(a ) ) . in sp ,
the frequencies of tim-4 cells in bd mice were also significantly higher than those in nor mice ( nor versus bd ( % ) : 3.9  2.8 ( n = 11 ) versus 6.7  4.3 ( n = 17 ) , p = 0.035 ) .
however , the difference between bdn and bd mice was not significant ( figure 2(b ) ) . in pbmcs ,
the frequencies of tim-4 cells in bd mice were higher than those in nor mice ( figure 2(c ) ) .
moreover , in peritoneal macrophages , the frequencies of tim-4 cells in bd mice were significantly higher than those in nor and bdn mice ( nor versus bdn versus bd ( % ) : 6.1  2.0 ( n = 5 ) versus 8.6  4.0 ( n = 9 ) versus 26.6  17.0 ( n = 5 ) , nor versus bdn ,   p = 0.11 , nor versus bd ,   p = 0.02 , bdn versus bd ,   p = 0.005 ) . no differences between nor and bdn mice were observed ( figure 2(d ) ) .
in addition , we also examined the frequencies of cd11btim-4 and cd11ctim-4 cells in ln and sp . in ln , both markers in bd mice were higher than those in nor and bdn mice ( nor ( n = 5 ) versus bdn ( n = 6 ) versus bd ( n = 5 ) ( % ) : cd11btim-4 , 1.1  0.3 versus 1.7  0.7 versus 3.0  1.4 , nor versus bdn ,   p = 0.05 , nor versus bd ,   p = 0.01 , bdn versus bd ,   p = 0.04 ; cd11ctim-4 , 0.1  0.0 versus 0.5  0.1 versus 0.7  0.5 , nor versus bdn ,   p = 0.00003 , nor versus bd ,   p = 0.01 , bdn versus bd ,   p = 0.14 ) ( figure 2(e ) ) . in splenocytes ,
expression of cd11ctim-4 cells was similar to that in ln in nor ( 0.3  0.1% , n
= 5 ) , bdn ( 0.6  0.4% , n = 6 )  and bd mice ( 1.0  0.6% ,
n = 8) ( nor versus bdn ,   p = 0.07 , nor versus bd ,   p = 0.07 , bdn versus bd ,   p = 0.1 ) .
in contrast , the frequencies of cd11btim-4 cells in bd mice were significantly lower than those in nor , but higher than those in bdn mice ( figure 2(f ) ) .
these data suggest that the frequencies of tim-4 expressing cells in bd mice were upregulated compared to those in bdn mice .
we investigated the change in bd - like symptoms according to the regulation of tim-1 expression .
the tim-1 vector was injected intraperitoneally at 10 , 30 , and 50  g / mouse into nor mice twice at 2 day intervals .
the frequencies of tim-1 cells from ln were compared to those of the control vector injected group the day after the last injection .
the expression of tim-1 cells in the 10  g tim-1 vector injected group ( 19.1  5.1% , n
= 4 ) was significantly higher than that in the control vector injected group ( 10  g : 9.4  2.6% , n = 4 , p = 0.01 ; 50  g : 10.4  5.4% , n
however , the 30 ( n = 3 ) and 50  g ( n = 4 ) tim-1 vector injected groups showed lower expression than that in the 10  g injected group .
ten g of tim-1 vector was injected intraperitoneally into bd mice four times at 2 day intervals , followed by observations for 2 weeks .
skin and genital ulcers improved after administering the tim-1 vector when compared to those in the control vector - injected group ( figure 4(a ) ) .
additionally , the severity score was 2.83  0.41 before and 2.83  0.75 at 1 week in the control vector group and 2.67  1.21 at 2 weeks after the first injection of control vector ( n = 6 ) .
the severity score in the tim-1 vector group was 3.17  0.75 before injection , 1.50  1.22 at 1 week after injection ( p = 0.004 ) , and 1.33  1.51 at 2 weeks after injection in bd mice ( p = 0.03 , n = 6 ) ( figure 4(b ) ) .
the severity score decreased after injecting the tim-1 vector , whereas the control vector injection did not result in a difference .
two weeks after the first administration of the tim-1 vector to bd mice , isolated ln and pbmcs were analyzed for tim-1 cells by facs . in ln ,
the frequencies of tim-1 cells in tim-1 vector injected group were slightly higher compared to those in the control vector injected group ( con versus tim-1 ( % ) : 10.3  1.7 ( n = 4 ) versus 11.9  3.2 ( n = 5 ) , p = 0.19 ) .
however , cd4tim-1 and cd8tim-1 cells were similar to those in the control vector injected group ( figure 4(c ) ) .
the frequencies of tim-4 cells were also not different ( figure 4(d ) ) . in pbmcs ,
tim-1 cells were slightly lower in the tim-1 vector injected group , but cd4tim-1 , cd8tim-1 , and tim-4 cells were not different ( figures 4(c)-4(d ) ) .
interestingly , cd4 t cells in the tim-1 vector injected group were slightly higher than those in the control vector injected group ( con versus tim-1 ( % ) : 21.3  10.7 ( n = 8) versus 27.6  13.7 ( n = 8) , p = 0.36 ) ( figure 4(c ) ) . several cellular phenotypes in the ln and pbmcs
the frequencies of cd4cd25 , cd4foxp3 , and cd4cd25foxp3 ( regulatory t , treg ) cells in ln were not significantly changed in the tim-1 vector compared to those in the control vector injected group ( con ( n = 5 ) versus tim-1 ( n = 6 ) ( % ) : cd4cd25 , 6.5  1.6 versus 6.1  2.3 ,   p = 0.35 ; cd4foxp3 , 5.3  1.5 versus 4.7  1.7 , p = 0.29 ; treg , 5.2  1.4 versus 4.7  1.7 , p = 0.3 ) ( figure 5(a ) ) . but , in pbmcs , cd4cd25 , cd4foxp3 , and treg cells in the tim-1 vector injected group were significantly higher than those in the control vector injected group ( con ( n = 8) versus tim-1 ( n = 8) ( % ) : cd4cd25 , 0.31  0.18 versus 0.61  0.21 ,   p = 0.01 ; cd4foxp3 , 0.10  0.10 versus 0.31  0.12 , p = 0.004 ; treg , 0.04  0.08 versus 0.16  0.10 , p = 0.03 ) ( figure 5(a ) ) .
cd8cd122 t cells are newly identified , regarded as treg cells , and are involved in anti - inflammatory responses .
another type of treg cells , cd8cd122 t cells , were also analyzed in ln of the tim-1 vector injected group ( con ( n = 5 ) versus tim-1 ( n = 6 ) ( % ) : 1.3  0.3 versus 1.6  0.7 ,   p = 0.25 ) ( figure 5(b ) ) . in pbmcs ,
cd122 and cd8cd122 t cells in the tim-1 vector injected group were also higher than those in the control vector injected group ( con ( n = 8) versus tim-1 ( n = 8) ( % ) : cd122 , 1.2  1.0 versus 9.8  7.9 ,   p = 0.04 ; cd8cd122 , 1.9  1.5 versus 2.4  2.1 ,   p = 0.58 ) .
our study indicated that the tim-1 vector upregulated treg cells and cd122 cells in bd mice .
up - regulation of these cellular phenotypes may be involved in improved bd - like symptoms after injection of the tim-1 vector .
sera were analyzed by elisa at 2 weeks after tim-1 vector administration into bd mice to determine the levels of cytokines .
the il-17 level in tim-1 vector injected group was significantly lower than that in the control group ( con ( n = 7 ) versus tim-1 ( n = 7 ) ( pg / ml ) : 11.53  4.45 versus 6.84  2.17 , p = 0.03 ) .
tnf- also decreased significantly in the tim-1 vector injected group compared to that in the control group ( con ( n = 11 ) versus tim-1 ( n = 11 ) ( pg / ml ) : 16.5  13.8 versus 6.9  5.1 , p = 0.04 ) .
the il-6 level in the tim-1 vector injected group decreased ( con ( n = 8) versus tim-1 ( n = 8) ( pg / ml ) : 145.7  176.9 versus 72.2  38.8 , p = 0.27 ) .
in contrast , il-4 increased slightly in the tim-1 vector injected group ( con ( n = 8) versus tim-1 ( n = 9 ) ( pg / ml ) : 9.3  5.4 versus 10.8  5.0 , p = 0.53 ) .
but , ifn- levels did not differ between the tim-1 and control vector injected group ( figure 6 ) .
consequently , these results indicate that the tim-1 vector might downregulate proinflammatory cytokines in bd mice . in bd mice ,
the frequencies of tim-4 cells were higher than those in nor and bdn mice in ln cells and peritoneal macrophages ( figure 2 ) .
sitim-4 was injected into nor mice intraperitoneally to downregulate tim-4 cells , and the frequencies of tim-4 cells were analyzed in peritoneal macrophages by facs .
tim-4 sirna ( sitim-4 ) at 2 , 5 , or 10  g / mouse or negative control ( nc ) scrambled sirna ( 2 and 5  g / mouse ) was injected ( nc : 5  g versus sitim-42 , 5 , and 10  g ( n = 4 ) : 8.9  1.4% versus 6.6  3.4% ( p = 0.42 ) , 3.7  0.9% ( p = 0.013 ) , 2.8  2.2% ( p = 0.04 ) ) ( figure 7(a ) ) .
sitim-4 downregulated tim-4 macrophages dose dependently and showed a significant difference in the 5  g and 10  g administered groups .
therefore , 5  g injections were used for the following experiment . to determine the time - dependent efficacy of tim-4 sirna ,
5  g of sitim-4 was injected , and the frequencies of tim-4 macrophages were analyzed by facs after 24 , 48 , and 72 hours .
the frequencies of tim-4 at 48 hours were lowest ( nc versus sitim-4 ( n = 2 ) : 24  h , 7.9  0.6% versus 4.9  0.2% , p = 0.02 ; 48  h , 8.9  1.4% versus 2.9  0.4% , p = 0.03 ; 72  h , 15.4  0.1% versus 4.2  3.0% , p = 0.03 ) ( figure 7(b ) )
. five g of sitim-4 was injected intraperitoneally three times at 2 day intervals into bd mice and the mice were observed for 2 weeks ( figure 8(a ) ) .
after administration of sitim-4 , bd - like symptoms , such as skin and genital ulcers , were compared to the control group . in the negative control group , the severity score was 2.25  0.46 before , 2.00  1.07 at one week ( p = 0.35 ) , and 1.88  1.13 at 2 weeks after the first injection of bd mice ( p = 0.28 ,
n = 8) . in the sitim-4 treated group , the score was 2.63  0.52 before , 1.25  0.81 at 1 week ( p = 0.001 ) , and 1.25  0.89 at 2 weeks after injection into bd mice ( p = 0.001 , n = 8) ( figure 8(b ) ) . at 1 and 2 weeks after the first administration of sitim-4 to bd mice
the frequencies of tim-4 cells at 1 and 2 weeks after sitim-4 treatment tended to be lower than those in the negative control group , but the difference was not significant ( 1 week : 18.6  4.8% versus 15.7  3.7% , p = 0.31 , 2 weeks : 20.7  6.5% versus 18.8  3.3% , p = 0.42 ) ( figure 8(b ) ) .
however , in ln , tim-4 , cd11btim-4 , cd11ctim-4 , tim-1 , cd4tim-1 , and cd8tim-1 cells in the sitim-4 treated group were similar to those in the control treated group at 1 week after the first sitim-4 injection ( figures 8(c)-8(d ) ) . even at 2 weeks after the first injection , those markers were not different ( data not shown )
. in these results , bd - like symptoms improved with sitim-4 treatment and decreased the severity score .
the frequencies of cd4cd25 , cd4foxp3 , and treg ( cd4cd25 foxp3 ) cells were analyzed by facs at 1 and 2 weeks after the first treatment with sitim-4 in bd mice ( figure 9 ) .
after 1 week , treg cells increased slightly in sitim-4 treated bd mice compared with those in the nc treated group ( nc versus sitim-4 : cd4cd25 , 5.82  2.01% versus 6.84  2.03% , p = 0.45 , n = 5 ; cd4foxp3 , 4.64  1.9% versus 5.82  2.4% , p = 0.41 ,
n = 5 ; treg , 3.28  1.57% versus 4.08  1.73% , p = 0.47 , n = 5 ) .
after 2 weeks , treg cells were significantly higher in sitim-4 treated bd mice than those in the nc treated group ( nc versus sitim-4 : cd4cd25 , 4.8  0.6% versus 5.9  1.2% , p = 0.03 , n = 6 ; cd4foxp3 , 4.1  0.7% versus 5.2  1.2% , p = 0.03 , n = 7 ; treg , 2.7  0.5% versus 3.5  0.9% , p = 0.04 , n = 7 ) .
these results suggest that the increased number of treg cells is associated with knock down of tim-4 in bd mice . after administering sitim-4 to bd mice ,
the serum level of il-17 was analyzed by elisa and compared with nc sirna treated bd mice .
the level of il-17 tended to decrease in the sitim-4 treated group compared to that in the nc treated group , but the difference was not significant ( nc versus sitim-4 ( n = 8) : 19.4  11.5  pg / ml versus 15.6  8.1  pg / ml , p = 0.25 ) ( figure 10 ) .
bd mice downregulated tim-1 levels and upregulated tim-4 levels in ln , sp , pbmcs , and peritoneal macrophages compared to those in bdn mice .
administration of the tim-1 vector upregulated the frequencies of tim-1 cells , and tim-4 sirna downregulated the frequencies of tim-4 cells .
administering the tim-1 vector improved bd - like symptoms , such as genital and skin ulcers , and decreased the severity score .
tim-1 expression is lower in patients with active sle compared to that in inactive patients .
human tim-1 is associated with immune dysfunction , such as atopic dermatitis , allergy , rheumatoid arthritis , asthma , and sle [ 1418 ] .
our data indicate that the tim-1 related t cells phenotype did not change much after administering the tim-1 vector .
interestingly , the frequencies of cd4 t cells in pbmcs increased in the tim-1 vector injected group compared to those in the control vector injected group .
actually , tim-1 is expressed on cd4 t cells after activation and its expression is preferentially sustained on th2 but not th1 cells [ 1 , 7 ] .
reported that tim-1-fc triggers a significant increase in the frequencies of cd4 t cells . in our study
other treg cells , cd8cd122 t cells , were also higher in pbmcs and ln of the tim-1 vector injected group compared to those in the control group . cd8cd122 t cells are newly identified and regarded as treg cells   and have an effect on anti - inflammatory responses .
our results suggest that the function of tim-1 influenced the bd - like symptoms and is associated with treg and cd8cd122 t cells .
recent studies suggest that il-17 may play a dominant role in provoking chronic autoimmune inflammation and is considered essential for t cell - mediated colitis and promotion of inflammation [ 3436 ] .
the association of il-17 and il-22 was also reported in patients with bd [ 37 , 38 ] .
tnf- overexpression has been implicated in acute and chronic inflammatory diseases , such as septic shock , bowel disease , crohn 's disease , rheumatoid arthritis , atopic dermatitis , psoriasis , and bd .
overproduction of il-6 plays a role in rheumatoid arthritis , juvenile idiopathic arthritis , inflammatory bowel disease , and sle .
the anti - tim-1 antibody ( high avidity / agonistic ) is a down - regulator of pro - inflammatory th-17 cells .
tim-1-tim-4 interaction is involved in the regulation of th cell responses and modulation of the th1/th2 cytokine balance .
our data also indicate that administering the tim-1 vector decreased proinflammatory cytokines , such as il-17 , tnf- , and il-6 , but the th2 type cytokine il-4 was upregulated in the tim-1 vector injected group . recently ,
upregulated cytokine il-22 in ocular bd patients was also downregulated by anti - il-6 and anti - tnf- .
bd mice displayed markedly increased tim-4 levels in ln and peritoneal macrophages compared to those in nor and bdn mice .
the frequencies of tim-4 cells were downregulated when tim-4 sirna was administered to nor mice compared to those in the nc group .
sitim-4 administered bd mice displayed improved symptoms such as skin and genital ulcers and arthritis and showed a decreased severity score .
tim-4 is a ligand for tim-1   and is not expressed in t cells but is exclusively expressed in antigen - presenting cells , including dcs and macrophages [ 9 , 10 ] .
two groups have reported that tim-4 deficient mice with a 129 or b6 background develop little or no autoimmunity [ 9 , 43 ] . in conjunction with improved symptoms , treg cells in the sitim-4 treated group
were significantly upregulated compared to those in the control group , and il-17 level decreased . in our previous study ,
in addition , the frequencies of treg cells are lower in patients with autoimmune and inflammatory diseases , such as crohn 's disease , multiple sclerosis , and sle , than those in healthy controls and inactive patients . the tim-1-tim-4 interaction is important for regulating t cell proliferation and modulating the th1/th2 cytokine balance .
our data suggest that upregulated treg cells induced by sitim-4 might contribute to improve bd - like symptoms . in conclusion ,
the frequencies of tim-1 cells in bd mice were downregulated compared to those in bdn mice . administering the tim-1 vector to bd mice improved the bd - like symptoms and decreased the severity score by up - regulating treg cells and down - regulating pro - inflammatory cytokines .
in addition , treatment with sitim-4 upregulated treg cells and improved bd - like symptoms , which were related to the tim-1 and tim-4 expression and the tim-1-tim-4 interaction . furthermore , tim-4 sirna downregulated the level of il-17 , which may have been involved in improving the bd - like symptoms .
consequently , regulation of tim-1 and tim-4 was effective for improving bd - like symptoms in mice .
 the t cell immunoglobulin mucin ( tim ) proteins regulate t cell activation and tolerance . 
 tim-1 plays an important role in the regulation of immune responses and the development of autoimmune diseases . 
 tim-4 is a natural ligand of tim-1 , and the interaction of tim-1 and tim-4 is involved in the regulation of t helper ( th ) cell responses and modulation of the th1/th2 cytokine balance . 
 behet 's disease ( bd ) is a chronic , multisystemic inflammatory disorder with arthritic , intestinal , mucocutaneous , ocular , vascular , and central nervous system involvement . 
 tim-1 expression was lower in a herpes simplex virus - induced bd mouse model compared to that in asymptomatic bd normal ( bdn ) mice . 
 tim-4 expression was higher in bd mice than that in bdn mice . in this study , we investigated the tim expression in a bd mouse model with bd - like symptoms . tim-1 and 
 tim-4 expression was regulated by an expression vector or sirna injected into the bd mouse model . the tim-1 vector injected into bd mice resulted in changes in bd - like symptoms and decreased the severity score . 
 treatment with tim-4 sirna also improved bd - like symptoms and decreased the severity score accompanied by upregulation of regulatory t cells . 
 we showed that regulating tim-1 or tim-4 affected bd - like symptoms in mice . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion