sepsis is still a leading cause of death among critical patients in the intensive care units , and the life quality of the survivors would usually be impaired [ 15 ] . there was a serious decrease of immunocytes , including b / t - lymphocytes , dendritic cells ( dcs ) , gastrointestinal epithelial cells , and even thymocytes , at the beginning of sepsis as shown in both animal models and septic patients [ 69 ] . it has been noted that the septic patients would gradually enter immunosuppression after primary hyperinflammatory response , which is defined as immunoparalysis [ 2 , 4 , 6 , 7 ] . in recent years , investigators have become interested in the study of the mechanisms regarding immunosuppression and the development of new methods to regulate immune response during sepsis , including both activation of tregs and apoptotic depletion of immunocytes . as a class of cd4 t cell subsets , tregs are a group of specialized immune cells that play an important role in immune homeostasis . during the development of sepsis , tregs subdue inflammation and tissue damage , and they may also cause immune dysfunction , such as induction of t - lymphocytic apoptosis , inhibition of cd4/cd8 t - lymphocytic function , and mediation of shifting from the helper t cell 1 ( th1 ) to th2 response , especially immunoparalysis via expression of ctla-4 and tgf- , as well as anti - inflammatory cytokines ( il-10 and tgf- ) [ 1217 ] . nrp-1 is characterized as a single - pass transmembrane glycoprotein , which is originally described to be involved in axon guidance , revascularization , and tumorous growth [ 18 , 19 ] . recently , nrp-1 , an essential component of the immunological response in humans and animals , is identified as a potent surface marker for cd4cd25tregs [ 2023 ] . in addition , the expression of nrp-1 on tregs was correlated with the expression of foxp-3 and suppressive capacity . solomon and his colleagues further showed that nrp-1cd4cd25tregs suppressed the proliferation of cd4cd25 t cell more efficiently than cd4cd25tregs and nrp-1cd4cd25tregs in a mouse model of experimental autoimmune encephalomyelitis ( eae ) . in our previous study , we indicated that tuftsin - derived t - peptide , the typical ligand of nrp-1 , had the ability to improve the outcome of septic mice in a dose- and time - dependent manner and was associated with downregulation of the negative immunoregulation of tregs and improvement of the microenvironment of cellular immunosuppression in septic mice . thus , further investigation on the impact of nrp-1 on the negative immunoregulation of tregs will provide a new target for the study of immune regulation in sepsis . in the present study , the objective , using the classical septic model , that is , clp , is to investigate the impact of sepsis on the expression of nrp-1 on cd4cd25tregs and the negative immunoregulation of nrp-1cd4cd25tregs in septic mice , as well as the potential therapeutic value of nrp-1 in sepsis . we demonstrated that the expression of nrp-1 on cd4cd25tregs was significantly upregulated in a grade- and time - dependent manner , nrp-1cd4cd25tregs possessed primary negative immunoregulation in septic response , and recombinant nrp-1 polyclonal antibody markedly downregulated the demethylation of foxp-3-tsdr in the stimulation of lps in a dose - dependent manner . nrp-1 could represent a new potential therapeutic target for the study of immune regulation in sepsis . inbred male balb / c mice ( laboratory animal center of chinese academy of medical sciences , number scxk - jing-2009 - 0007 , beijing , china ) , aged 68 weeks , weighing 20  2  g , were used in the present study . all procedures were undertaken in accordance with the criteria of national institute of health guide for the care and use of laboratory animal and approved by the scientific investigation board , medical college of chinese pla , beijing , china . the medium used throughout the in vitro experiment was rpmi1640 ( containing 100  u / ml penicillin , 100  l / ml streptomycin , and 1.5  mm glutamine ) with 10% heat - inactivated fetal bovine serum ( fbs ) . cd4cd25 regulatory t cell isolation kits , goat anti - rabbit igg microbeads , rabbit anti - mouse nrp-1 , and ld / sm columns were purchased from miltenyi biotec gmbh , bergisch gladbach , germany . fluorescein isothiocyanate- ( fitc- ) conjugated annexin - v apoptotic kit was purchased from nanjing keygen biotech , nanjing , china . purified hamster anti - mouse cd3e and cd28 were purchased from bd pharmingen , san diego , ca . antibodies used for flow cytometric analysis , including fitc - conjugated anti - mouse / rat - foxp-3 , fitc - conjugated anti - mouse cd152 ( ctla-4 ) , and allophycocyanin- ( apc- ) conjugated anti - mouse / rat - tgf- , were purchased from ebioscience , san diego , ca . apc - conjugated anti - mouse / rat - nrp-1 and recombinant nrp-1 polyclonal antibody were purchased from r&d systems , minneapolis , mn . enzyme - linked immunosorbent assay ( elisa ) kits for interferon- ( ifn- ) , il-2 , il-4 , il-10 , and tgf- were purchased from excell biol , shanghai , china . ketamine and su - mianxin - ii ( containing 2,4-xylazole , ethylenediaminetetraacetic acid , dihydroetopine , and haloperidol ) were purchased from china academy of military medical sciences , beijing , china , and they were used as the anesthesia for animals . spleens were harvested and prepared into single cell suspension by passing through a 30  m stainless steel mesh twice and then treated with ficoll - paque density gradient centrifugation . cd4cd25tregs and cd4cd25 t cells were isolated from mononuclear cells using mouse cd4cd25treg isolation kit and minimacs separator ( miltenyi biotec gmbh , bergisch gladbach , germany ) according to manufacturer 's instructions . cd4cd25tregs were incubated with a rabbit anti - mouse nrp-1 antibody ( abcam , cambridge , ma ) for 20 minutes at 4c , washed and incubated with goat anti - rabbit igg microbeads for 30 minutes at 4c , and selected for nrp-1cd4cd25tregs and nrp-1cd4cd25tregs by minimacs separator according to manufacturer 's instructions . isolated cells were cultured in rpmi 1640 supplemented with 10% fcs . after being anesthetized , a 0.5  cm incision was made on the abdomen of mouse , and the cecum was exposed . the cecum at the designated position between its distal pole and ileocecal junction was ligated for the desirable degree of sepsis : 1/3 for low - grade sepsis , 2/3 for midgrade sepsis , and ligated ileocecal junction for high - grade sepsis . the diameter of puncture needle was 0.6  mm , and it was used to induce clp in the experiment . the mice were given a subcutaneous injection of 0.9% sterile saline solution with an amount of 40  ml / kg body weight after clp . 150 mice were used to investigate the severity - dependent response between the expressions of nrp-1 and foxp-3 of tregs , and they were divided into five groups : control group , sham group , and three different clp groups ( low - grade , midgrade , and high - grade ) , with 30 mice in each group . with the optimal degree of sepsis , another 150 mice were employed to observe the time - dependent response between the expressions of nrp-1 and foxp-3 of tregs , and they were divided into five groups : control group and clp with four interval groups ( 12 , 24 , 48 , and 72 hours ) , with 30 mice in each group . the survival time and rate of various groups the optimal grade and time point were used to investigate the impact of nrp-1 on the negative immunoregulation of tregs in sepsis . they were divided into four groups with the same cell number : control group and clp with three different subtype tregs ( cd4cd25tregs , nrp-1cd4cd25tregs , and nrp-1cd4cd25tregs ) . they were cocultured with conventional cd4cd25 t cells for 24 hours in a ratio of 1  :  1 and treated with anti - cd3 ( 5  g / ml ) and anti - cd28 ( 2  g / ml ) antibody for polyclonal activation of t cells , respectively [ 1416 ] . the proliferative activity , apoptotic rate , and secretive ability ( including interferon- ( ifn- )  and il-4 ) of cd4cd25 t cells and the expression of foxp-3/ctla-4/tgf-/nrp-1 , apoptotic rate , and secretive ability ( including il-10 and tgf- ) of every subtype of tregs were determined . 
 in vitro , nrp-1cd4cd25tregs were isolated from normal mice spleen and cultured with different doses ( including 1 , 10 , 100 , 1000 , and 10000  g / ml ) of recombinant nrp-1 polyclonal antibody in the stimulated lps ( 1000  ng / ml ) for 24 hours . the demethylation level of foxp-3-tsdr was determined by demethylation - sensitive rt - pcr , which had been recounted by tatura et al . . the proliferative activity of cd4cd25 t cells was determined by cck-8 according to protocols provided by the manufacturer . the absorbance was read in microplate reader ( spectra mr , dynex , richfield , mn ) at od 450  nm . cd4cd25tregs were stained with fitc - conjugated anti - mouse - ctla-4 , apc - conjugated anti - mouse / rat - tgf- , or apc - conjugated anti - mouse / rat - nrp-1 for 30 minutes at 4c in the dark based on the same cell number . for determination of intranuclear foxp-3 , cd4cd25tregs were suspended in 1  ml fixation / permeabilization solution for 2 hours at 4c in the dark . after washing cells with permeabilization buffer twice , cd4cd25tregs were stained with fitc - conjugated anti - mouse / rat - foxp-3 for 30 minutes at 4c in the dark . after washing cd4cd25tregs with phosphate - buffered saline ( pbs ) twice , cells were analyzed with flow cytometer ( bd biosciences , mountain view , ca ) . 5  101  10 cd4cd25tregs or cd4cd25 t cells were washed with pbs twice ; cells were suspended in 200  l binding buffer , followed by 10  l fitc - conjugated annexin - v to stain for 30 minutes at 4c or 15 minutes at 25c in the dark . 300  l binding buffer and 5  l pi were added to stain for 5 minutes at 25c in the dark again , and they were subjected to flow cytometric analysis by flow cytometer . the supernatants were collected for measuring the levels of ifn- , il-4 , tgf- , and il-10 by elisa kits , strictly according to the manufacturer 's protocols . the standard concentration curves for ifn- , il-4 , tgf- , and il-10 were plotted from 0 to 1000  pg / ml . data was represented as mean  standard deviation ( sd ) and analyzed by spss 17.0 software with a one - way anova . survival rate in septic mice was evaluated by kaplan - meier via the log - rank test . as shown in figures 1(a ) and 1(e ) , cd4cd25tregs were isolated from spleens at 24 hours after clp , and it was found that , compared with the control group , expressions of nrp-1 and foxp-3 of cd4cd25tregs were significantly promoted by sepsis ( p < 0.01 ) . compared with low - grade septic group , the expressions of nrp-1 and foxp-3 were further promoted in mid- and high - grade septic groups ( p < 0.01 ) , and the difference between mid- and high - grade septic groups was not statistically significant ( p > 0.05 ) . as shown in figure 1(b ) , the survival rate of low- , mid- , and high - grade septic groups was 86.67% , 66.67% , and 33.33% , respectively . the differences between them were statistically significant according to kaplan - meier analysis ( p < 0.01 ) . in figures 1(a ) , 1(b ) , and 1(e ) , we noticed that sepsis promoted the expression of nrp-1 along with the severity of sepsis and the upregulation of cytoactivity of cd4cd25tregs . we used midgrade septic model to further observe the time - dependent impact of sepsis on the expression of nrp-1 and foxp-3 . as shown in figures 1(c ) and 1(f ) , compared with the control group , the expressions of nrp-1 and foxp-3 were significantly promoted at every point after clp ( p < 0.01 ) . compared with 12-hour group , expressions of nrp-1 and foxp-3 were markedly enhanced at 24 and 48 hours ( p < 0.01 ) , but they were downregulated at 72 hours , and the difference between them was not statistically significant ( p > 0.05 ) . the survival rate of 12- , 24- , and 48-hour groups was 93.33% , 66.67% , and 26.67% ( figure 1(d ) ) , respectively , and the differences between them were statistically significant according to kaplan - meier analysis ( p < 0.01 ) , but the difference between 48- and 72-hour ( 20% ) groups was not statistically significant ( p > 0.05 ) . as shown in figures 2(a ) , 2(b ) , and 2(c ) , midgrade septic model was used to evaluate the impact of nrp-1 on the negative immunoregulation of tregs at 24 hours after clp . compared with the control group , sepsis significantly upregulated the expression of foxp-3/ctla-4/tgf- on cd4cd25tregs and nrp-1cd4cd25tregs ( p < 0.01 ) , but the expression of foxp-3/ctla-4/tgf- on nrp-1cd4cd25tregs was significantly downregulated ( p < 0.01 ) . compared with cd4cd25tregs , as shown in figure 3 , with the midgrade septic model at 24 hours , compared with the control group , the apoptotic rates of cd4cd25tregs and nrp-1cd4cd25tregs were markedly reduced ( p < 0.01 ) , but the apoptotic rate of nrp-1cd4cd25tregs was increased ( p < 0.05 ) . in contrast to cd4cd25tregs , as shown in figure 4 , with the midgrade septic model at 24 hours , cd4cd25tregs , nrp-1cd4cd25tregs , and nrp-1cd4cd25tregs were cocultured with cd4cd25 t cells for 24 hours in a ratio of 1  :  1 , respectively . the production of il-10 and tgf- was significantly increased from cd4cd25tregs and nrp-1cd4cd25tregs compared with that of the control group ( p < 0.01 ) , especially for nrp-1cd4cd25tregs as compared with that of cd4cd25tregs ( p < 0.01 ) . however , il-10 and tgf- levels were significantly descended in nrp-1cd4cd25tregs ( p < 0.01 ) . cd4cd25tregs , nrp-1cd4cd25tregs , and nrp-1cd4cd25tregs were isolated from the midgrade septic model at 24 hours and cocultured with conventional cd4cd25 t cells for 24 hours with a ratio of 1  :  1 , respectively . compared with the control group , the proliferation ( figure 5(a ) ) of cd4cd25 t cells was significantly suppressed , and the apoptosis ( figures 5(b ) and 5(c ) ) of cd4cd25 t cells was significantly increased when cocultured with cd4cd25tregs and nrp-1cd4cd25tregs ( p < 0.01 ) , but the proliferation of cd4cd25 t cells was obviously upregulated , and the apoptosis of cd4cd25 t cells was obviously downregulated , when cocultured with nrp-1cd4cd25tregs ( p < 0.01 ) . compared with cd4cd25tregs , the proliferation of cd4cd25 t cells was further significantly inhibited , and the apoptosis of cd4cd25 t cells was further significantly increased when cocultured with nrp-1cd4cd25tregs ( p < 0.05 or 0.01 ) . as shown in figure 6 , cd4cd25tregs , nrp-1cd4cd25tregs , and nrp-1cd4cd25tregs were , respectively , isolated from the midgrade septic model at 24 hours and cocultured with cd4cd25 t cells for 24 hours in a ratio of 1  :  1 . in comparison to the control group , secretion of ifn- of cd4cd25 t cells was decreased , but release of il-4 was increased when cocultured with cd4cd25tregs ( p < 0.05 ) . nrp-1cd4cd25tregs significantly suppressed the secretion of ifn- and il-4 from cd4cd25 t cells as compared with the control group and cd4cd25tregs ( p < 0.05 or 0.01 ) . the formation of ifn- and il-4 from cd4cd25 t cells was significantly upregulated when cocultured with nrp-1cd4cd25tregs ( p < 0.01 ) . as shown in figure 7 , in vitro , the demethylation level of foxp-3-tsdr in nrp-1cd4cd25tregs was significantly increased in the stimulated lps compared with control group for 24 hours ( p < 0.01 ) ; recombinant nrp-1 polyclonal antibody had an obvious ability to downregulate the demethylation level of foxp-3-tsdr , and in a dose - dependent manner , especially 10000  g / ml ( p < 0.05 or 0.01 ) . foxp-3 , which is still the main intracellular marker for identification of tregs , is a distinctive transcriptional factor of tregs , and it is also critical for their function , differentiation , and maintenance [ 11 , 13 , 26 ] . our previous study demonstrated that a significantly increased expression of foxp-3 in tregs was positively correlated with the mortality of burn - induced septic mice [ 1417 ] . recently , nrp-1 , which was highly expressed on natural tregs but lowly expressed on induced tregs and not expressed on cd4cd25 cells , is a good marker to distinguish natural and induced tregs [ 2024 ] . more interestingly , another study showed a population of nrp-1tregs in human lymph nodes together with the positive expression of foxp-3 that inhibited the proliferative activity of t cells . in the current study , we first reported that sepsis per se markedly promoted the expression of nrp-1 on cd4cd25tregs in a grade- and time - dependent manner , the expression of nrp-1 on tregs was obviously correlated with the expression of foxp-3 and the mortality of clp - induced septic mice , and nrp-1 had prevented ability to preserve the negative immunoregulation of tregs in sepsis . it has been known that immune dysfunction of cd4 t - lymphocytes was one of the primary cellular mechanisms in sepsis - induced immunosuppressive state . immediate observation of specimens from spleen , thymus , and lung in septic patients who died in intensive care units , or those from murine clp model , showed a profound , progressive , apoptosis - induced loss of adaptive immunocytes , thereby resulting in a decrease in ability of producing antibodies and clearing life - threatening pathogens [ 6 , 7 , 2931 ] . it is well known that the activated cd4 t cells can mainly differentiate into th1 and th2 , and they mainly produced ifn- and il-4 , respectively . a shift to th2 response was noted to be corroborated sepsis - induced immunosuppression , and the secretion of th1 associated cytokines was thereby impaired , and , on the other hand , the secretion of th2 associated cytokines was increased during sepsis , and these phenomena were obviously correlated with the outcome of septic complications [ 7 , 31 ] . t cells are anergic when they respond to their specific antigens , which are identified as failure to proliferate or secrete cytokines . in the present study , the results suggested that nrp-1cd4cd25tregs had the strongest ability to inhibit the proliferation and increase the apoptosis , as well as inhibiting the cytokine secretion of cd4cd25 t cells , but nrp-1cd4cd25tregs had opposed ability on cd4cd25 t cells ; thus , we conjectured that nrp-1cd4cd25tregs had a potential ability to prevent cellular immunosuppression in sepsis . in the development of sepsis , tregs can mainly inhibit the activation of t - lymphocytes , especially cd4 t - lymphocytes through various suppressive mechanisms , including inhibitory cytokines ( such as il-10 and tgf- ) and cell - to - cell contact ( such as ctla-4 and tgf- ) , inhibition of cytolysis ( especially granzymes ) , downregulation of metabolic disruption , and suppression of maturation and function of antigen presenting cells , as well as upregulation of antiapoptotic ability [ 12 , 1417 ] . accumulated evidence has shown that a combination of foxp-3 , ctla-4 , tgf- , and inhibitory cytokines ( il-10 and tgf- ) might serve as active markers for tregs in the process of sepsis [ 6 , 7 , 13 , 29 , 30 ] . we used a midgrade septic model reproduced 24 hours after clp to investigate the impact of nrp-1 on the negative immunoregulation of tregs in the setting of sepsis . it was found that sepsis could obviously upregulate the negative immunoregulation of cd4cd25tregs and nrp-1cd4cd25tregs , especially nrp-1cd4cd25tregs , but downregulated the negative immunoregulation of nrp-1cd4cd25tregs , which correlated with the expressions of foxp-3/ctla-4/tgf- and the apoptotic ability , as well as the secretion of il-10 and tgf-. we have reported that the percentage and stability of tregs were higher in septic patients and mice septic models than in those without sepsis . a reduction in the percentage and stability of tregs was accompanied by an improvement in survival rate and immune dysfunction of t - lymphocytes in septic mice [ 1417 ] . it has been documented that the stability of tregs includes the stability of foxp-3 expression and negative immunoregulatory function in sepsis , which is crucially dependent on the demethylation status of the foxp-3-tsdr . in the current study , we found that recombinant nrp-1 polyclonal antibody could downregulate the demethylation of foxp-3-tsdr in the presence of lps ; thus , nrp-1 could represent a new potential therapeutic target , at least via regulating the stability of tregs , for the study of immune regulation in sepsis . nrp-1cd4cd25tregs showed strong resilience to apoptosis and cytokine secretive ability in sepsis , and they possessed a strong ability to inhibit the proliferation and increase the apoptosis , as well as secreting cytokines of cd4cd25 t cells . recombinant nrp-1 polyclonal antibody had the ability to downregulate the demethylation of foxp-3-tsdr in the presence of lipopolysaccharide ( lps ) . nrp-1cd4cd25tregs might exhibit primary negative immunoregulation in sepsis , and nrp-1 could represent a new potential therapeutic target for the study of immune regulation in sepsis .
regulatory t cells ( tregs ) appear to be involved in sepsis - induced immune dysfunction ; neuropilin-1 ( nrp-1 ) was identified as a surface marker for cd4+cd25+tregs . in the current study , we investigated the negative immunoregulation of nrp-1highcd4+cd25+tregs and the potential therapeutic value of nrp-1 in sepsis . 
 splenic cd4+cd25+tregs from cecal ligation and puncture ( clp ) mouse models were further segregated into nrp-1hightregs and nrp-1lowtregs ; they were cocultured with cd4+cd25  t cells . 
 the expression of forkhead / winged helix transcription factor-3 ( foxp-3 ) , cytotoxic t - lymphocyte associated antigen-4 ( ctla-4 ) , membrane associated transforming growth factor- ( tgf-m+ ) , apoptotic rate , and secretive ability [ including tgf- and interleukin-10 ( il-10 ) ] for various types of tregs , as well as the immunosuppressive ability of tregs on cd4+cd25  t cells , were determined . meanwhile , 
 the impact of recombinant nrp-1 polyclonal antibody on the demethylation of foxp-3-tsdr ( treg - specific demethylated region ) was measured in in vitro study . 
 sepsis per se markedly promoted the expression of nrp-1 of cd4+cd25+tregs . 
 foxp-3/ctla-4/tgf-m+ of nrp-1hightregs were upregulated by septic challenge . 
 nrp-1hightregs showed strong resilience to apoptosis and secretive ability and the strongest immunosuppressive ability on cd4+cd25  t cells . in the presence of lipopolysaccharide ( lps ) , 
 the recombinant nrp-1 polyclonal antibody reduced the demethylation of foxp-3-tsdr . 
 nrp-1hightregs might reveal primary negative immunoregulation in sepsis ; nrp-1 could represent a new potential therapeutic target for the study of immune regulation in sepsis .