recent clinical and experimental studies have indicated that the long - term effects of severe inflammatory events often include the suppression of immune system functions .
the decrease of th1/th2 ratio is one of the major characteristics of immunosuppression in sepsis .
it is reported that membrane adhesive protein annexin - a1 ( anxa1 ) and transcription factor gata-3 , which were both decreased in sepsis patients [ 2 , 3 ] , play important roles in the th1/th2 shift .
many researchers have studied anxa1 and gata-3 , respectively , but the relationship between anxa1 and gata-3 is still unknown . exploring the interactions between anxa1 and gata-3 may provide clues to understand the immunosuppression and improve the treatment effects of sepsis patients . as an anti - inflammatory protein , anxa1 plays a homeostatic role in the innate immune system through mediating immune cells , such as neutrophils and macrophages .
endogenous anxa1 markedly reduced leukocyte adhesion to postcapillary venules through formyl peptide receptor ( fpr ) pathway .
furthermore , anxa1 promotes inflammatory cell apoptosis associated with transient rise of intracellular calcium and caspase-3 activation .
moreover , anxa1 has been recently identified as one of the  eat me  signals on apoptotic cells to be recognized and ingested by phagocytes .
studies on the expression of anxa1 in human and mouse leukocytes have shown that this protein is also expressed at higher levels in cells of the adaptive immune response , such as t and b lymphocytes [ 810 ] .
further research indicates that anxa1 increases t cells activation and favors their differentiation to th1 cells by modulating t cell receptor ( tcr ) signaling .
in addition , analysis of the inflammatory response of anxa1 mice has demonstrated an exquisite role of anxa1 in modulation of tcr signaling by the fpr family .
these findings suggest a potential role of anxa1/fprl-1 pathway in the adaptive immune response . upon antigen stimulation of their tcr by antigen
presenting cells , nave cd4 t cells can differentiate to at least two different types of t helpers , th1 and th2 cells , which were documented to be involved in adaptive immunity .
the transcription factor gata-3 is selectively upregulated during th2 differentiation in vitro [ 13 , 14 ] .
gata-3 is important not only for the transactivation of th2 cytokine genes but also for the suppression of th1 development .
. in vivo experiment from anxa1 mice exhibited that anxa1-deficient t cells expressed th2 skewing .
however , there is not enough evidence to support the interrelation between gata-3 and anxa1 . in our previous studies ,
the expressions of anxa1 and gata-3 were both decreased in the burnt mouse with sepsis .
the purpose of this study is to investigate whether anxa1 and gata-3 interact with each other to influence the immune response in t lymphocyte , as well as exploring the possible molecular mechanisms involved .
our results show that overexpressed anxa1 ( or gata-3 ) represses the expression of gata-3 ( or anxa1 ) , while knockdown of anxa1 ( or gata-3 ) increases the gata-3 ( or anxa1 ) expression .
further studies indicate that anxa1 regulates gata-3 expression through anxa1/fprl-1/erk and pkb / akt signaling pathways , and gata-3 mediates anxa1 transcription activity by binding to anxa1 promoter .
thus this study , together with our previous observations of anxa1 , suggests that the anxa1/fprl-1 axis and gata-3 are potential therapeutic targets of the th1/th2-mediated immunological suppression in sepsis .
anti - mouse cd3e ( clone 145 - 2c11 ) and anti - mouse cd28 ( clone 37.51 ) were purchased from bd bioscience ( san jose , ca ) .
murine il-2 , il-4 , ifn , il-12 , anti - il-4 ( clone 11b11 ) , and anti - ifn ( clone xmg1.2 ) were purchased from ebioscience ( wembley , united kingdom ) .
anti - mouse gata-3 , total and phosphorylated erk1/2 , and akt were purchased from cell signaling ( danvers , ma ) .
anti - t - box transcription factor ( t - bet / tbx21 ) was purchased from abcam ( cambridge , uk ) , anti - fprl-1 purchased from novus ( colorado , usa ) , and anti - anxa1 purchased from proteintech ( manchester , uk ) . unless otherwise specified , all the other reagents were from sigma - aldrich ( st . louis , mo ) .
km male mice with weight of 3550  g were obtained from department of experimental animals , central south university ( changsha , china ) .
animal work has been performed according to the approval of the ethics of xiangya hospital , central south university .
mice were anesthetized with sodium pentobarbital [ 4050  mg / kg intraperitoneal ( i.p . ) ] .
their spleen was teased apart to make a single - cell suspension and stained with apc - conjugated anti - cd4 .
the isolated naive cd4 t cells ( > 95% , 1  10/6 wells ) were cultured in rpmi 1640 ( gibco , grand island , ny ) complete medium .
the culture conditions of t cells are as follows : th0 : anti - cd3/cd28 ( 5  g / ml ) and il-2 ( 50  u / ml ) ; th1 : il-12 ( 2  ng / ml ) , il-2 ( 50  u / ml ) , and anti- il-4 ( 20  g / ml ) and th2 : il-4 ( 1000  u / ml ) , il-2 ( 50  u / ml ) , and anti - ifn ( 10  g / ml ) . the sequences for anxa1 and gata-3 overexpression were artificially synthesized and cloned into the plv ( exp ) puro - ires - egfp vector ( cyagen , guangzhou , china ) .
interference lentiviral ( plent ix1 puro - egfp vector ) against anxa1 and gata-3 was constructed .
the above constructed lentivirus was stably transfected into the t cells with an optimal multiplicity of infection ( moi ) of 100  tu / ml after stimulated with anti - cd3/cd28 ( 5  g / ml ) for 24  h. upregulated ( downregulated ) expressions of anxa1 and gata-3 were confirmed by qrt - pcr and western blot ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/1701059 ) .
the shrna sequences with the maximum of more than 75% knockdown efficiency were selected for further stable anxa1 and gata-3 silencing in t cells .
the most efficient anxa1 shrna sequence of three was constructed as follows : ( shanxa1 - 3 ) 5-gagatctggccaaagacataa-3 ; ( anti - shanxa1 - 3 ) 5-ttatgtctttggccagatct-3. the most efficient gata-3 shrna sequence of three was constructed as follows : ( shgata-3 - 2 ) 5-gctcagtatccgctgacggaa-3 ; ( anti - shgata-3 - 2 ) 5-ttccgtcagcggatactgagc-3. th0 cells ( 1  10/ml ) , obtained after infection with upanxa1 and culturing in th0 conditions with complete rpmi medium for seven days , were stimulated with phorbol 12-myristate 13-acetate(pma ) 10  ng / ml and ionomycin 1  g / ml for 4  h in 6-well plates to produce cytokine .
the ifn and il-4 levels were measured by elisa kits purchased from multi - sciences ( china ) .
qrt - pcr was performed to determine the expression levels of gata-3 , t - bet , and anxa1 .
total rna extraction was performed using simply p total rna extraction kit ( bioflux , europe ) .
the cdna synthesis was performed using all - in - one first - strand cdna synthesis kit ( genecopoeia , maryland , usa ) .
cdna was used to set up a quantitative real - time pcr ( qpcr ) reaction using the all - in - one qpcr mix ( genecopoeia ) .
the expression levels of gata-3 , anxa1 , and t - bet were normalized to actin .
the primer sequences were as follows : mouse gata-3 forward primer : 5-gctggatggcggcaaag-3 , mouse gata-3 reverse primer : 5-gtgggcgggaaggtgaa-3 , mouse anxa1 forward primer : 5-aaggtggtcctgggtcagc-3 , mouse anxa1 reverse primer : 5-tgagcattggtcctcttggt-3 , mouse tbx21/t - bet forward primer : 5-atgttcccattcctgtccttca-3 , mouse tbx21/t - bet reverse primer : 5-aaatgaaacttcctggcgcatc-3 , mouse actin forward primer : 5-catcctgcgtctggacctgg-3 , and mouse actin reverse primer : 5-taatgtcacgcacgatttcc-3. all protocols were carried out according to the manufacturer 's instructions .
30  g protein was separated by sds - page , transferred onto pvdf membrane , and hybridized with a primary antibody followed by a horseradish - conjugated secondary antibody .
the cells permeabilized with 0.1% triton x-100 were incubated with 2% bovine serum albumin for 30  min and followed by the primary antibodies against gata-3 and anxa1 at 4c overnight .
the slides of cells were subsequently incubated with the corresponding alexa fluor 488-conjugated secondary antibodies for 1  h at room temperature .
the gata-3 penter ( nm-001002295 ) and control penter plasmids were obtained from vigene biosciences ( rockville , md , usa ) .
the pgl3 enhancer vector and prl - tk vector were obtained from promega ( madison , wi , usa ) .
anxa1 ( nm00200.2 ) promoter region 2000  bp base - pairs and firefly luciferase reporter gene were built in pgl-3 enhancer vector and the renilla luciferase reporter gene was built in prl - tk vector .
the renilla luciferase plasmid was used in all the experiment to normalize the efficiency of the transfection .
briefly , on the day of transfection , 5  10 cells were plated in 100  l in each well of a 96-well pate prior to incubation with vigenefection ( vigene , rockville , md ) complexes according to the manufacturer 's instruction using a total of 0.3  g dna and 0.9  l vigenefection . at
48 to 72 hours after transfection , cells were lysed in 20  l reporter lysis buffer ( promega , madison , wi ) for 15 minutes on ice .
add the 100  l luciferase assay reagent ii ( promega ) and record the firefly luciferase activity measurement . and then add the 100  l stop & glo reagent ( promega ) and read the renilla luciferase activity measurement .
at least three independent experiments were performed and the data were presented as mean  sem .
all data were presented as mean  sem and analyzed by student 's t - test .
to explore the expression of anxa1 and/or gata-3 of the th0 , th1 , and th2 cells , immunofluorescence was carried out to confirm that anxa1 and gata-3 expression were located in cell nucleus and cytoplasm ( figure 1 ) . to determine the roles of overexpressed endogenous anxa1 in the balance between gata-3 and t - bet expression , two major transcriptional switches in th1/th2 differentiation
, we examined the differentiation of t cells , which is driven by the strength of overexpressed anxa1 in th0 conditions .
naive t cells from mouse were infected with upanxa1 viruses and cultured in th0 medium for seven days , followed by being exposed to pma ( 10  ng / ml ) and ionomycin ( 1  g / ml ) for 4  h to stimulate the production of cytokines in th1/th2 .
upanxa1 significantly increased the expression of ifn and reduced il-4 expression in t cells , compared with those infected with up - control ( p < 0.01 , figure 2 ) .
anxa1-silenced t cells exhibited decreased production of ifn and increased approximately 50% higher production of il-4 , compared with t cells infected with down - control ( p < 0.01 , figure 2 ) .
these results demonstrated that anxa1 induced th0/th1 differentiation , whereas cells infected with silenced anxa1 promoted th0/th2 differentiation .
the important roles of gata-3/t - bet in influencing differentiation of t cell lineage into th1 or th2 effector cells were well accepted [ 19 , 20 ] .
as shown in figure 3 , the results indicated that anxa1-silenced t cells expressed higher levels of gata-3 and low level of t - bet , compared with the control and upanxa1 groups ( p < 0.01 ) .
considering that erk and pkb / akt are two major downstream transcription factors of fprl-1 , we further investigated the role of fprl-1 signaling pathways in the regulation of anxa1 on gata-3 .
the result showed that anxa1 significantly phosphorylated erk and pkb / akt , while knockdown of anxa1 decreased erk and akt activation .
the expression levels of frrl-1 in all groups , regardless of anxa1 status , were not significantly different ( figure 4 ) . to investigate the effect of gata-3 on anxa1 expression in cd4 t cells ,
cd4 cells with different gata-3 expression levels were used . as shown in figures 5(a ) and 5(b ) , overexpressed gata-3 inhibited anxa1 protein and mrna expression , while silenced gata-3 increased the expression of anxa1 . as a transcription factor , gata-3 mediates anxa1 expression through anxa1 promoter .
anxa1 promoter area about 2000  bp was found in the eukaryotic promoter database and ucsc genome bioinformatics .
the jaspar database was used to predict the binding domain of anxa1 promoter with gata-3 .
the result suggested that gata-3 could combine anxa1 promoter agataaga ( start-281 end-274 ) area on the level of nucleic acid sequences .
the area the same as zinc finger of gata-3 closet to the c terminus binds to the consensus dna sequence area ( a / t)gata(a / g ) . to verify this hypothesis , anxa1 promoter area ( 2000  bp ) and luciferase reporter gene were built in pgl-3 enhancer vector and renilla luciferase reporter gene was built in prl - tk .
three plasmids gata-3 penter , anxa1 promoter pgl3 , and prl - tk were transfected in 293 t cell . as shown in figure 5(c ) , the luciferase activity was significantly increased , compared with up - controls when cotransfected with upgata-3 .
these results demonstrate that gata-3 binds to anxa1 promoter to repress its expression in 293 t cells .
excessive inflammatory responses including the release of a cytokine storm and the activation of a large number of t cells are characteristics of early stage sepsis . in spite of their ability to kill pathogens , they are equally effective in inducing cell and tissue damage . on one hand , the cytokine storm could be protective by activating the host anti - inflammatory proresolving response .
on the other hand , during sepsis , the cytokine storm induces excessive activation induced t cell death ( aicd ) ; the cause of aicd is an aberration in the cell cycle program .
enhanced anti - inflammatory response , characterized by a large number of t cells apoptosis and decreased responsiveness to antigen / mitogen , leads to the immunosuppression .
more patients die in the immunosuppression phase where the risk of a secondary infection increases .
anti - inflammatory therapy can improve the survival rate of patients in hyperinflammatory status , while it may be worse if applied during the sepsis - induced immunosuppression .
the conventional understanding of immune responses of post - sepsis involves a shift away from th1 responses towards th2 responses .
promoting a low ratio of th1/th2 immune responses to th1 skewing might be a treatment of immunosuppression .
studies have shown that anxa1 is an endogenous anti - inflammatory factor in the innate immune system [ 6 , 29 ] .
increasing evidences suggest that anxa1 plays an important role in the regulation of the adaptive immune response .
consistent with above studies , we found that overexpressed anxa1 induced the differentiation of th1/th2 to th1 skewing with increased ifn and reduced il-4 cytokines expression .
in the process of th1/th2 differentiation , another important factor is gata-3 . as a specific transcription factor of th2
, gata-3 activates tcr signaling in pre - t cells and promotes the cd4 t cell lineage differentiation after positive selection .
many researches and our previous studies have found that the anxa1 plasma levels were decreased in the rabbit and patients with severe sepsis [ 2 , 32 ] .
our previous animal model observed that anxa1 and gata-3 both present the same downtrend in mouse with severe sepsis [ 18 , 33 ] ; this phenomenon may be related with a large number of t cells apoptosis .
the major finding of this study is the discovery of a novel role of transcription factor gata-3 regulation on anxa1 in the adaptive immune response and the molecular mechanisms of mutual regulation contributing to immunosuppression in inflammatory processes .
our results indicated that expressions of anxa1 and gata-3 are both located in th0 , th1 , and th2 cells nucleus and cytoplasm .
oliani et al . also found that anxa1 was expressed in the eosinophil cytosol and nucleus .
further study manifested that overexpressed endogenous anxa1 exerted an effect on decline of gata-3 expression and increase of t - bet expression , which may be one reason of anxa1 inducing the differentiation of th1/th2 to th1 skewing .
one viewpoint deems that anxa1 modulates th1/th2 differentiation by promoting t cell activation [ 4 , 30 ] .
but another study finds t cell - expressed anxa1 inhibits t cell activation in the adaptive immune response .
our view is that t cells differentiate into various subtypes according to environmental signals , and anxa1 modulates t cell differentiation through modulating their transcription factors .
it is known that anxa1 is one endogenous ligand of fprl-1 , which belongs to the members of the g protein - coupled receptor family .
exogenous anxa1 protein and its n - terminal peptide ac2 - 26 were demonstrated to activate erk in vitro and in vitro .
it is reported that erk suppresses many t cell differentiation related transcription factors , including gata-3 and foxp3 in the t cells [ 33 , 38 ] .
interestingly , some studies show that erk - mapk cascade controls gata-3 stability through the ubiquitin / proteasome - dependent pathway .
these studies further verify the important roles of activated erk - mapk cascade in the gata-3 phosphorylation and its ubiquitin - mediated degradation through the 26 s proteasome .
so erk - mapk is essential for gata-3 activation and stability to avoid gata-3 excessive expression in stimulated t cells .
meanwhile , erk pathway is also essential for t cell proliferation and th1 skewing by inducing the tbx21 expression .
akt is a gata-3 phosphorylase kinase and the activation of akt1 induces derepression of tbx21 and ifn expression in th2 cells .
akt phosphorylates gata-3 and induces the dissociation of hdac2 from the gata-3 complex , resulting in a failure in the repression of ifn expression in th2 cell .
these findings highlight a pivotal role of posttranscriptional modification of gata-3 , which is a kind of negative feedback regulation mechanism of th2 differentiation to avoid excessive differentiation to th2 cell .
therefore , it is proposed that the enhanced anxa1 regulates gata-3 expression to induce th1/th2 shift by erk and pkb / akt to influence the inflammatory response and balance the early proinflammatory function .
another interesting discovery in this study is that the gata-3 also downregulated anxa1 expression in cd4 t cells .
further research indicated that gata-3 exerted a transcriptional - level control by combining with the promoter of anxa1 in 293 t cells .
this experimental conclusion needs to be verified in cd4 t cells and determined further by electrophoretic mobility shift assay or chromatin immunoprecipitation . in the early sepsis stage , tcr trigger gata-3 upregulation via pi3k - mtor dependent pathways .
moreover , gata-3 is suggested to mediate notch signaling that is important for promoting t cell development and proliferation . in our studies , if we downregulate gata-3 expression , the level of anxa1 expression increased . on one hand
, it can remit th1/th2 toward to th2 skewing to prevent immunosuppression . on the other hand , as an anti - inflammatory protein , anxa1 can balance the early proinflammatory function . in the immunosuppression stage
gata-3 can strengthen the tcr activation to improve adaptive immune   and decreased anxa1 can reduce anti - inflammatory response at a certain extent for better care of patients .
in conclusion , our results suggest that gata-3 binds to anxa1 promoter area to inhibit anxa1 expression and functions .
anxa1-mediated gata-3 and t - bet might favor th1 differentiation through erk and akt pathway , reversing tendency characterized by th2 skewing of immunosuppression .
based on the th1/th2 mediation , our discoveries might provide a potential treatment of immunosuppression after sepsis .
 decreased th1/th2 ratio is one of the major characteristics of immunosuppression in sepsis . 
 both membrane adhesive protein annexin - a1 ( anxa1 ) and transcription factor gata-3 have been reported to play important roles in t cell differentiation . 
 however , the relationship between anxa1 and gata-3 in th1/th2 shift is unknown . 
 our study investigated the interaction effects of anxa1 and gata-3 to influence t cell differentiation in cd4 + t cells . 
 we found that gata-3 and anxa1 were coexpressed on th0/th1/th2 cytoplasm and nuclear . 
 overexpressed anxa1 significantly increased the expression of ifn and reduced il-4 expression in t cells , while anxa1-silenced t cells exhibited decreased production of ifn and increased production of il-4 . 
 knockdown of anxa1 promoted higher expression level of gata-3 and low level of t - box transcription factor ( t - bet / tbx21 ) . 
 further study demonstrated that anxa1 regulated gata-3 expression through the formyl peptide receptor like-1 ( fprl-1 ) downstream signaling pathways erk and pkb / akt . 
 these results suggested that anxa1 modulates gata-3/t - bet expression induced th0/th1 differentiation . 
 moreover , we found that gata-3 inhibited anxa1 expression by binding to its promoter for the first time . 
 it is proposed that the interactions between anxa1 and gata-3 may provide clues to understand the immunosuppression and have potential as new therapeutic targets in immunotherapy after sepsis . 