the recruitment and activation of macrophages in response to microbial products such as lipopolysaccharide ( lps ) lead to production of various immune / inflammatory effectors , which are essential for the anti - infectious immune response . interleukin 12 ( il-12 ) , a 70  kd ( p70 ) heterodimeric cytokine comprised of a p40 and a p35 subunit separately , is mainly produced by activated macrophages and plays a pivotal role in connecting innate immunity with adaptive immunity . indeed , various control elements have been revealed in the expression of inducible p40 gene . for example , it was found that a sequence in nf-b ( p50/p65 and p50/c - rel ) complexes was functionally essential for modulating the promoter activity of il-12 p40 in response to several p40-inducing pathogens [ 2 , 3 ] . other regulatory elements , such as c / ebp , ets-2 , and irf1 , have been also implicated in mediating the inducible promoter activity of il-12 [ 1 , 46 ] . however , the upstream signaling events that connect microbial stimulation with downstream transcriptional regulation to control il-12 expression in macrophages remain largely unknown . extracellular calcium influx across the plasma membrane is a major source for the increase of intracellular calcium , which acts as a crucial secondary messenger in regulating macrophage activation , such as phagosomes maturation , activation of nlrp3 inflammasome , and transcriptional control of cytokines [ 79 ] . microbial components ( lps , etc . ) are known to elicit calcium influx in murine macrophages and thereby upregulate the production of mediators like no and il-10 [ 1012 ] . however , it has been found that single lps stimulation is insufficient for the upregulation of il-12 production and there is a negative relationship between the consequences of extracellular derived calcium increase and the insufficient production of il-12 . for example , ligation of fc , complement , or scavenger receptors in macrophage inhibited the induction of il-12 by lps . moreover , further experimental approaches suggested that il-12 inhibition was due to extracellular calcium influxes that occurred after receptor ligation . in our previous study , we demonstrated that clathrin / dynamin - dependent internalization , a process that interplayed with calcium influx , also resulted in the elevation of il-12 secretion in murine macrophages . such results suggest that calcium may activate different intracellular signaling events which may exert an opposite function for il-12 regulation generally , intracellular calcium binds to calmodulin ( cam ) , activates cam kinases , and further regulates transcriptional events in inflammation and immunity . calcium / calmodulin dependent protein kinase kinase  ( camkk ) is a key cam kinase activated by the increased intracellular calcium and plays an essential role in calcium - mediated regulation of inflammation in innate immune cells . amp - activated protein kinase ( ampk ) is a crucial substrate of camkk which mainly functions as a cellular nutrient and energy sensor . recent studies reveal that ampk is also an effective inhibitor for nf-b signaling and associated with inflammatory response in macrophages [ 12 , 16 ] . recently , sirt1 has been identified to be required for ampk - mediated inflammation inhibition . as a nad - dependent deacetylating enzyme , sirt1 could be activated by elevated nad levels and then downregulates inflammation by directly deacetylating subunit of nf-b like p65 and cel [ 18 , 19 ] . in addition , sirt1 could also reciprocally activate ampk or induce phosphorylation of pcg-1 , which thereafter inhibits the rela / p65-mediated nf-b signaling . despite the well known effects of camkk , ampk , and sirt1 on the modulation of inflammation , their concomitant involvement in mediating the regulation of calcium - dependent il-12 production needs to be further studied . in this study , we detected calcium influx across the plasma membrane triggered by lps and observed its inhibitory effect on lps induced il-12 secretion in murine macrophages . we also evaluated the effects of camkk-ampk - sirt1 signaling pathway on regulating nf-b activity and mediating calcium - dependent il-12 inhibition . murine peritoneal macrophages were obtained from peritoneal lavage in balb / c male mice as described previously . briefly , balb / c mice were sacrificed and 1  ml high glucose dmem ( gibco , usa ) was injected into the intraperitoneal cavity . the abdomen was gently massaged for 1  min and the injected medium was aspirated and cell pellets were washed twice with dmem containing 10% fetal bovine serum ( fbs ) . then , peritoneal macrophages were plated in plates or dishes and cultured at 37c in a humidified incubator supplemented with 5% co2 . murine macrophage - like cell line raw 264.7 cells ( atcc , usa ) were directly cultured in dmem supplemented with 10% fbs and under similar environment with primary macrophages . for calcium modulation , macrophages were treated in calcium - free dmem ( gibco ) or dmem with egta ( sigma , usa ) for calcium deprivation . cells were treated with calcium - free dmem plus 2  mm cacl2 ( sigma ) for calcium recovery . then , macrophages were also treated with skf96365 , 2-aminoethyl diphenylborinate ( 2-apb ) ( sigma ) , and lacl3 ( aladdin , china ) for inhibition of store - operated calcium entry ( soce ) or with atp to increase calcium atpase activity . then , lps o55 : b5 ( sigma ) was added and incubated for the indicated time periods before further measurement . for chemical modulation of camkk , ampk , sirt1 , nf-b , and ap-1 , macrophages were pretreated with sto-609 , aicar , compound c , wedelolactone ( sigma ) , sr11302 ( tocris , uk ) , and srt1720 and ex527 ( selleck , usa ) for 1  h and then stimulated with lps before further measurement . time - dependent intracellular calcium detection was performed with a varioskan flash multimode reader ( thermo ) using the fluorescent dye fura-2am ( sigma ) . murine peritoneal macrophages were seeded in 96-well plates and preloaded with fura-2am for 60  min . loaded cells were treated with or without lps ( 1  g / ml ) . online detection of fura-2am in live cells was performed every 10  s for 6  min via fluorescence excited at 340 and 380  nm and filtered at 510  nm . camkk , ampk , and sirt1 were transiently knocked down by sirna transfection following the manufacturer 's instruction . negative control ( nc ) sirna ( santa cruz , usa ) or sirna for camkk , ampk ( ruibo biotech ltd . , china ) , and sirt1 ( santa cruz ) were mixed with lipofectamine 3000 reagent ( invitrogen , usa ) and added to the medium . after 24  h of transfection , the culture medium was replaced and further treatment was performed . raw 264.7 cells were lysed with ripa reagents ( pierce , rockford , il , usa ) containing cocktail protease and phosphatase inhibitors ( roche , switzerland ) . plasma proteins were separated by sds - page and transferred onto pvdf membranes ( millipore , usa ) . blots were blocked with 5% bsa for 1  h and incubated with primary antibodies ( 1  :  1000 dilutions ) for camkk , pcamkk , ampk , pampk , sirt1 , ib , irf3 , pirf3 , p38 , p - p38 , and tubulin ( cell signaling , usa ) at 4c overnight . then , the blots were further incubated with hrp - conjugated secondary igg antibodies ( 1  :  2000 dilutions , cell signaling ) at 37c for 1  h. chemiluminescence images were developed with supersignal sensitivity substrate kit ( pierce ) with a chemidoc xrs imaging system ( bio - rad , usa ) . total rna extracted from raw 264.7 cells by a trizol reagent ( roche ) was reversely transcribed into cdna with a revertra ace--rna easy kit ( toyobo , japan ) . the cdna templates were mixed with sybr green pcr master mix ( toyobo ) and pcr primers for il-12 p35 , il-12 p40 , tnf- , il-6 , camkk , ampk , and -actin ( sequences are listed in table s1 , in supplementary material available online at http://dx.doi.org/10.1155/2016/6152713 ) . quantitative real - time pcr was performed with an icycler thermal cycler ( bio - rad ) . raw 264.7 cells ( 1  10/ml ) were cultured in a 24-well plate and transfected with plasmids pgl - luc2p / nf-bre ( promega , usa ) and pap1-luc and pifn--luc ( genepharma , china ) using x - tremegene hp dna transfection reagent ( roche ) for 24  h. then , the plate was added with lps for 6  h of incubation treatment . the luciferase activity was analyzed with the firefly luciferase assay kit ( beyotime ) and detected in a multimode reader ( thermo ) . supernatants from cultured murine peritoneal macrophages or raw 264.7 cells were directly collected after 24  h of lps treatment or at a given time . the levels of il-12 p40 and il-12 p70 in the supernatant were measured with elisa kits ( ebioscience , usa ) as indicated by the manufacture 's instruction . raw 264.7 cells ( 2  10/ml ) growing in 24-well plates were treated with lps and lps plus calcium - free dmem and calcium - free dmem with cacl2 for 1  h. cells were washed twice with pbs . nad / nadh levels in cell lyses were detected through a nadh quantitative detection kit ( sigma ) . difference with a p value less than 0.05 and 0.01 was considered to be statistically significant . to compare supernatant levels of tnf- , il-6 , il-10 , and il-12 ( p40 and p70 ) , primary murine peritoneal macrophages were treated with 100  ng / ml lps . results showed lps induced dramatic increase of tnf- , il-6 , and il-10 secretion but triggered relatively lower concentration of il-12 p40 and il-12 p70 production ( figure 1(a ) ) . moreover , lps ( 10 , 100 , and 1000  ng / ml ) induced robust increase of tnf- secretion in a significantly dose - dependent manner , but il-12 secretion only increased slightly with elevated lps concentration ( figure 1(b ) ) . in the time course detection , il-12 p40 and il-12 p70 were also elevated time dependently . however , their increases were much weaker ( figure 1(c ) ) . consistent trend was also detected in lps - treated raw 264.7 cells ( figure s1 ) . the influence of extracellular calcium on cytokines production was investigated by treating murine peritoneal macrophages in dmem with or without calcium ( calcium - free ) . the results showed there was no difference in the expression of tnf- or il-6 regardless of whether the medium contained calcium or not ( figures 2(a ) and 2(b ) ) . . similar changes of tnf- and il-12 p40 were detected in raw 264.7 cells ( figure s2 ) . moreover , lps induced production of il-12 p40 was enhanced by cotreatment with ifn-. deprivation of calcium further increased the il-12 p40 production ( figure s3 ) . the specificity of lps induced il-12 alteration due to calcium modulation was also identified by pmb cotreatment in raw 264.7 cells . this specific lps neutralizer effectively suppressed the elevated il-12 p40 and il-6 regardless of whether calcium is present or absent ( figure s4 ) . to fully elucidate the influence of extracellular calcium on il-12 production , murine peritoneal macrophages were treated in calcium - free dmem with or without cacl2 supplementation . calcium - free dmem and egta consistently increased the secretion of il-12 p40 and il-12 p70 proteins . accordingly , mrna expressions of il-12 p35 and il-12 p40 were also significantly upregulated ( figures 2(c ) and 2(d ) ) . the elevated expression of il-12 was terminated when cacl2 was resupplied , suggesting that the inhibitory effect was calcium - dependent . a transient elevation of intracellular calcium levels ( indicated by fura-2am ) was detected after lps stimulation . moreover , lps induced increase of intracellular calcium levels was impaired when extracellular free calcium was removed ( figure 3(a ) ) . these data suggest that transmembrane influx was induced by lps which increased intracellular calcium levels . besides direct extracellular calcium modulation , we further pretreated macrophages with skf96365 and atp , which either inhibited or activated the influx of calcium . in our study , skf96365 treatment attenuated the influx of calcium and then resulted in the elevation of il-12 p40 and il-12 p70 production . in contrast , atp treatment enhanced calcium influx and further decreased il-12 secretion upon lps stimulation . however , tnf- release was not affected by skf96365 or atp ( figures 3(b ) and 3(c ) ) . we further detected the il-12 modulating effects of other soce inhibitors ( 2-apb and lacl3 ) and observed similar effects with skf96365 ( figure s5 ) . these results indicated that lps induced intracellular calcium elevation by triggering extracellular calcium entry , which is also required for the negative regulation of il-12 production in macrophages . to identify the downstream signaling effectors in calcium - mediated il-12 inhibition , we detected camkk activation in raw 264.7 cells . in our study , lps induced phosphorylation of camkk was suppressed in medium without calcium or egta , both of which inhibited extracellular calcium influx . restoration of camkk phosphorylation was observed when extracellular calcium was resupplied ( figure 4(a ) ) . to investigate whether activation of camkk leads to il-12 inhibition , we used chemical inhibitor sto-609 to suppress camkk activation and found that sto-609 increased il-12 production but did not affect the release of tnf- induced by lps ( figures 4(b ) and s6 ) . we further transfected specific sirna to directly downregulate camkk expression ( figure 4(c ) ) . il-12 production significantly increased in camkk sirna - treated groups ( p < 0.01 versus lps , figure 4(d ) ) . such results demonstrate that the activation of camkk is required for calcium associated il-12 suppression in lps - treated macrophages . next , we investigated whether ampk was activated downstream of camkk to mediate il-12 inhibition . in our study , suppressed ampk phosphorylation ( thr 172 ) was detected in raw 264.7 cells stimulated by lps in calcium - free medium . ampk phosphorylation was restored by supplementing cacl2 recovery in the medium ( figure 5(a ) ) . we further treated raw 264.7 cells with aicar ( ampk activator ) or compound c ( ampk inhibitor ) before lps stimulation . whereas compound c caused significant increase of il-12 secretion , aicar further decreased the production of il-12 ( figure 5(b ) ) . then , addition of aicar in calcium - free medium before lps stimulation resulted in suppressed production of il-12 p40 and il-12 p70 ( p < 0.01 versus lps + ca - free ) . we also transfected ampk sirna in raw 264.7 cells to directly decrease ampk expression ( figure 5(c ) ) . accordingly , the release of il-12 p40 and il-12 70 induced by lps increased with ampk downregulation ( p < 0.01 versus lps , figure 5(d ) ) . these data collectively demonstrate that ampk is activated downstream of camkk and required for calcium - induced inhibition of il-12 production in lps - treated murine macrophages . sirt1 is a substrate of ampk in the regulation of inflammation , so we investigated whether sirt1 was also required in mediating il-12 inhibition . as sirt1 activation is nad - dependent , we first detected the nad / nadh ratio in the presence or absence of extracellular calcium . in our study , lps treatment alone induced elevated nad levels , which was dampened by using calcium - free medium and restored by addition of cacl2 ( figure 6(a ) ) . then , expressions of sirt1 were upregulated by both lps and ampk activator aicar in raw 264.7 cells ( figure 6(b ) ) . in accordance with nad detection , furthermore , the sirt1 inhibitor , ex527 , markedly increased the production of il-12 p40 and il-12 p70 while its agonist , srt1720 , suppressed the il-12 secretion induced by lps ( figure 6(c ) ) . when srt1720 was added in calcium - free medium before lps stimulation , the enhanced il-12 production was inhibited ( figure 6(d ) ) . in addition , knockdown of sirt1 by sirna also markedly enhanced il-12 production upon lps stimulation in macrophages ( figure 6(e ) ) . these data verify that sirt1 may be essentially responsible for calcium - mediated il-12 suppression . as calcium deprivation affects activation of camkk , ampk , and sirt1 , we next used combined modulator of these kinases to identify their connection in mediating il-12 regulation . our results demonstrated that ampk and sirt1 activation impaired the increased il-12 production due to camkk inhibition . moreover , sirt1 activation suppressed il-12 released due to ampk inhibition while sirt1 inhibition partly restored il-12 production suppressed by ampk activation ( figure 7(a ) ) . sirt1 suppresses inflammation driven by interfering with nf-b - activation , so we next detected whether nf-b pathway was specifically targeted by calcium and sirt1 to downregulate il-12 expression . in our study , the transcriptional activity of nf-b induced by lps was enhanced in calcium - free medium . cotreatment with cacl2 , aicar , or srt1720 in calcium - free medium , which restored calcium influx or activated ampk and sirt1 , suppressed the elevated nf-b activity ( figure 7(a ) ) . however , the absence of extracellular calcium did not affect the transcriptional activity of irf3 and c - jun ( a subunit of ap-1 ) induced by lps ( figure 7(b ) ) . moreover , a significant decrease of il-12 production was detected by the cotreatment of nf-b inhibitor wedelolactone with lps . however , ap-1 inhibitor , sr11302 , did not alter il-12 levels . in a parallel study , both wedelolactone and sr11302 significantly attenuated il-6 production in raw 264.7 cells ( figure 7(c ) ) . furthermore , we observed similar modulation on il-12 p40 and il-6 by sr11302 and wedelolactone in the absence of calcium ( figure 7(d ) ) . these results suggest that lps induced calcium influx mediates il-12 inhibition by ultimate suppression of the nf-b signaling pathway . il-12 is essentially involved in host immune response and increased evidences have demonstrated the critical effects of il-12 deficiency that result in immunosuppression and increased susceptibility to infection [ 1 , 21 , 22 ] . however , the precise regulatory mechanisms of il-12 expression remain unclear and thereby are worthy of investigation . in the present study , we report the finding of a calcium - dependent mechanism that may possibly explain the insufficient il-12 expression in lps - stimulated murine macrophages . this newly identified mechanism is verified to be based on observations that lps stimulation induces extracellular calcium influx which activates camkk , ampk , and sirt1 , dampens nf-b activation , and eventually downregulated il-12 production . macrophages are primary sources for the production of inflammatory cytokines when activated by pathogenic molecules such as lps . however , levels of these cytokines can vary greatly due to the complicated and diversified regulatory mechanisms . in our study , lps - triggered production of il-12 p40 and il-12 p70 was significantly lower than those of tnf- , il-6 , and il-10 secreted in the same cell population of either primary or cell line of murine macrophages . previously , it was found that lps could activate the calcium - dependent signaling and regulate cytokine production in innate and adaptive immune cells [ 2426 ] . additional studies provided more specific data that calcium might influence the production of il-12 [ 12 , 13 ] . in our study , the removal of extracellular calcium directly caused a selective suppression of il-12 production upon lps stimulation . therefore , these data suggest calcium may act as an essential negative regulator for the production of il-12 . extracellular calcium flux in response to microbial stimuli has been demonstrated in both mouse macrophages and mouse dcs [ 27 , 28 ] . more specifically , calcium influx mainly contributes to the increase of intracellular calcium in lps - treated macrophages . in our study , we consistently observed extracellular calcium influx induced by lps treatment in murine peritoneal macrophages and also in raw 264.7 cells ( data not shown ) . such a transmembrane transportation of calcium was then demonstrated to be associated with il-12 regulation , as enhanced il-12 production was detected in macrophages cultured in medium without calcium or with lack of free calcium ( egta ) . it has been found that calcium influx is regulated by clathrin / dynamin - dependent endocytosis , calcium channel regulators , and calcium atpases . for example , the dynamin inhibitor dynasore could block trpv5/v6 activity and inhibit calcium influx in jurkat cells . we found previously that inhibition of endocytic pathway significantly suppressed lps induced production of il-12 p40 . a number of direct channel blockers , including la , skf96365 , and 2-apb , were discovered and designed to inhibit soce [ 30 , 31 ] . the cytoplasmic calcium changes and calcium influx evoked by adenosine triphosphate ( atp ) were widely observed in many different cell types . therefore , the soce based modulators are with opposite function in modulation calcium influx and thereby may facilitate the study of calcium - dependent regulation for il-12 . camkk is a key signaling molecule activated by increased intracellular calcium . in immune cells , camkk expression is only found in monocyte / macrophage lineage and responsible for regulation of inflammation and immunity . more recently , studies performed on myocytes and macrophages have reported that camkk phosphorylation could be induced by calcium influx or lps stimulation . in our study , we found that phosphorylation of camkk was induced by lps stimulation and dependent on the increased calcium influx . to investigate the direct involvement of camkk in regulation of il-12 production , we used its specific chemical inhibitor ( sto-609 ) and sirna to interfere with camkk activation and observed the marked increase of il-12 release in lps - stimulated murine macrophages . however , other authors reported that camkk was essentially involved in macrophage - mediated release of inflammatory cytokines . our finding demonstrated for the first time that camkk activation selectively suppressed lps induced il-12 production in murine macrophages . therefore , the nature of camkk based regulation on inflammation and immunity still requires further study . whereas ampk is a crucial sensor for energy balance , it has been also demonstrated that ampk activation exerts significant anti - inflammatory and immunosuppressive effects on a variety of cell types . recently , ampk has been identified as a direct substrate of camkk for inflammation modulation in macrophages and this is currently accepted molecular pattern that explains the anti - inflammatory efficacy of camkk . the enhanced ampk phosphorylation could be achieved by camkk activation with or without lps [ 35 , 36 ] . however , we demonstrated that lps - stimulated ampk activation depends on calcium influx and camkk activation . moreover , ampk activation was necessary for calcium - dependent inhibition of lps induced il-12 production . firstly , ampk activation by aicar further inhibited the suppression of il-12 production , while inhibition of ampk by compound c resulted in a significant elevation of il-12 release . in ampk knockdown macrophages , secondly , the increase of il-12 production due to calcium depletion was abrogated when aicar was given simultaneously . so these results support our presumption that ampk is activated by the influx of calcium and activation of camkk to mediate il-12 suppression . emerging evidences demonstrated that ampk can inhibit nf-b activation and suppress the expression of inflammatory genes . ampk could also modulate its downstream targets including sirt1 , pgc - l , p53 , and foxo3a , either directly or indirectly . recently , ampk has been verified in coordination with another metabolic sensor , nad - dependent type iii deacetylase sirt1 , to regulate energy metabolism and inflammation [ 18 , 38 ] . ampk regulates sirt1 by increasing the nad / nadh ratio , which acts as a key upstream regulatory event that modulates the activity of sirt1 and could be achieved by lps induced tnf- release in proinflammatory macrophages [ 38 , 39 ] . sirt1 expression could be modulated by lps stimulation in macrophages / monocytes and upregulated sirt1 expression was observed in resveratrol treated endothelial cells via the prka - ampk pathway [ 40 , 41 ] . therefore , ampk may act as an essential modulator for sirt1 expression and we mainly focus on ampk induced nad elevation and sirt1 activation in this study . in a previous study , an enhancement of sirt1 activity in response to zymosan stimuli , we identified that lps induced the increase of nad / nadh ratio in murine macrophages and upregulated the expression of sirt1 , both indicating the elevated activity of sirt1 . we also verified the requirement of calcium in facilitating lps induced sirt1 activation which was abolished in medium without calcium . this presumption was further supported by our findings that selective activation of sirt1 ( srt1720 ) activity suppressed il-12 production but selective inhibition of sirt1 ( ex527 ) increased il-12 levels upon lps treatment [ 43 , 44 ] . moreover , the increased il-12 production due to calcium absence was abolished by sirt1 activation . to further clarify the regulatory patterns of camkkb - ampk - sirt1 signaling cascade , we used combined manipulation of agonist and antagonist for camkkbeta - ampk and sirt1 . results demonstrated that camkk couples with ampk and sirt1 to mediate transcriptional inhibition on il-12 production induced by lps . to further establish the camkkb - ampk - sirt1 signaling cascade activation which further mediates the inhibition of nf-b activation by lps , sirt1 could negatively regulate inflammation by interfering with transcription events mediated by rela / p65 , pgc-1 , and ppar . it has also been found that the activity of sirt1 is closely related to nf-b complex in controlling the resolution of inflammation . in our study , depletion of extracellular calcium facilitated activation of nf-b , which was abolished by the presence of cacl2 , ampk activation ( aicar ) , or sirt1 activation ( srt1720 ) . in contrast , the activation of irf3 or ap-1 was not affected in either group as compared with lps alone . in our study , the ib- based nf-b inhibitor wedelolactone   effectively attenuated il-6 and il-12 production in raw 264.7 cells . the ap-1 inhibitor sr11302   only attenuated il-6 production while it did not affect il-12 secretion , although there exist different interpretations for il-12 production mediated by ap-1 as well as its upstream kinases such as erk1/2 , p38 , and jnk . our results indicated that nf-b may be more importantly required for il-12 p40 production , especially in the case that calcium influx was inhibited . however , further study is still necessary to determine transcriptional details involved in the ultimate regulation of il-12 transcription by calcium - camkk-ampk - sirt1 pathway . we demonstrate in this study that lps induced production of il-12 is affected by the increased intracellular calcium derived from extracellular influx . such effects were mediated by activation of camkk , ampk , and sirt1 which ultimately lead to the suppression of nf-b and downregulation of il-12 production . depletion of calcium influx inhibits those signaling events and thus increases lps induced il-12 production . our findings discover a new connection between calcium , cellular energy metabolism , and inflammation , which may provide therapeutic targets for the precise regulation of inflammatory disorders .
activated macrophages are the primary sources of il-12 , a key cytokine bridging innate and adaptive immunity . 
 however , macrophages produce low amounts of il-12 upon stimulation and the underlying regulatory mechanism remains unclear . in this study 
 , we found a new calcium - dependent mechanism that controlled il-12 production in lps - treated murine macrophages . 
 first , lps was demonstrated to induce extracellular calcium entry in murine peritoneal macrophages and inhibition of calcium influx resulted in marked enhancement in il-12 production . 
 then , withdrawal of extracellular calcium was found to suppress camkk and ampk activation triggered by lps while chemical inhibition or genetic knockdown of these two kinases augmented lps induced il-12 production . 
 ampk activation increased the nad+/nadh ratio and activated sirtuin 1 ( sirt1 ) , a nad+-dependent deacetylating enzyme and negative regulator of inflammation . 
 chemical inhibitor or sirna of sirt1 enhanced il-12 release while its agonist suppressed il-12 production . finally , it was found that sirt1 selectively affected the transcriptional activity of nf-b which thereby inhibited il-12 production . 
 overall , our study demonstrates a new role of transmembrane calcium mobilization in immunity modulation such that inhibition of calcium influx leads to impaired activation of camkk-ampk - sirt1 signaling pathway which lifts restriction on nf-b activation and results in enhanced il-12 production .