deregulation of the inflammatory response contributes to tissue damage in several pathological conditions , including autoimmune and infectious diseases [ 13 ] . to balance inflammation , the innate immune system has developed a regulatory mechanism by which innate immune cells , monocytes , and macrophages in particular display reduced response to subsequent challenges after they have been exposed to low concentrations of endotoxin ( e.g. , lps ) [ 4 , 5 ] , entering in a so - called  tolerant  state . although endotoxin tolerance has been considered as a protective mechanism to regulate overexuberant inflammation , the incidence of endotoxin tolerance has been associated with high risk of secondary infections and an endotoxin - tolerant state is considered to be a clinical phenomenon observed not only in sepsis [ 6 , 7 ] but also in diseases like acute coronary syndrome   and cystic fibrosis [ 9 , 10 ] , and in these pathologies the risk of new infections correlates with a refractory state of innate immune cells . much of our knowledge on the molecular mechanisms responsible for endotoxin tolerance arises from in vitro studies , where monocytes primed with low doses of lps or with il-10 or tgf become tolerant to a subsequent lps challenging and strongly reduce their production of tnf and other proinflammatory cytokines . ifn represents a key negative regulator of lps tolerance , being able to revert tolerance and restore tnf production both in in vitro and in vivo models . the molecular basis of endotoxin tolerance has not been completely elucidated , but it is now clear that it is a dynamic process implying a profound gene reprogramming [ 13 , 14 ] . in particular extensive studies demonstrated the impairment of the toll - like receptor ( tlr ) signaling pathway at multiple levels , with the consequent repression of proinflammatory mediators ( i.e. , tnf , il-6 , and il-12 ) , and the concomitant upregulation of anti - inflammatory molecules , such as il-10 and tgf . functionally , tolerant monocytes also exhibit increased phagocytosis due to increased expression of cd64 and impaired antigen presentation ability due to the downregulation of major histocompatibility class ii ( mhc ii ) , cd86 , and class ii transactivator ( ciita ) [ 9 , 16 , 17 ] . a growing number of mirnas have been reported to be involved in the regulation of the inflammatory response [ 1827 ] , but only recently studies described the differential expression and effects of mirna in the context of endotoxin tolerance [ 28 , 29 ] . mir-146a was the first mirna described as upregulated in tolerant thp-1 monocytic cells after priming with low dose of lps and was shown to partially induce lps desensitization in monocytes [ 28 , 29 ] . evidence suggesting a possible role of mir-155 and mir-125b in tolerance has also been reported . it is still unclear to which degree each mirna contributes to the development of endotoxin tolerance , but of note all these mirnas have been shown to modulate tlr4 signaling pathway by targeting different components of its signaling cascade . in the present study we show the involvement of mir-146b in the induction of endotoxin tolerance by showing its upregulation in lps tolerant monocytes , its induction by the anti - inflammatory stimuli il-10 and tgf , and its negative regulation by ifn. we also demonstrate that tuning mir-146b expression results in the induction or reversion of endotoxin tolerance in the thp-1 monocytic cell line . il-10 , il-4 , il-13 , and tgf were from r&d system ; ifn was from peprotech ; dexamethasone was from sigma aldrich . antibodies anti - pol ii ( n-20 ) , anti - runx3 ( h-50 ) , and anti - stat3 ( c-20 ) for chip experiments were purchased from santa cruz biotechnology and anti - ago2 was purchased from abcam . human monocytes were obtained from healthy donor buffy coats by two - step gradient centrifugation using ficoll ( biochrom ) and percoll ( amersham ) followed by incubation of purified cells in rpmi 1640 ( lonza ) without serum for 10  min at 37c with 5% co2 . adherent monocytes were washed twice with pbs and then cultured with rpmi medium supplemented with 10% fbs and l - glutamine as fully described below . the purity of the monocytes cultures was tested by cd14 staining and flow cytometry analysis , with an average of 90% cd14 cells . monocytes and thp-1 cells ( atcc ) were grown in rpmi supplemented with 10% heat - inactivated fetal bovine serum ( fbs ; lonza ) , 100  u / ml penicillin / streptomycin ( lonza ) , and 25  mm l - glutamine ( lonza ) at 37c with 5% co2 . hek-293 t cells ( atcc ) were grown in d - mem ( cambrex ) supplemented with 10% fbs , 100  u / ml penicillin / streptomycin , and 25  mm l - glutamine at 37c with 5% co2 . 10 human purified monocytes were culture and extracted dna was used to perform qpcr using promoter - specific primers . signals obtained from the chip samples were normalized on those obtained from the corresponding input samples , according to the formula : 100  2 . 100  ng of total rna was reverse transcribed for quantification of mir expression using taqman mirna reverse transcription kit ( applied biosystems ) , according to manufacturer 's instructions and as previously described [ 20 , 21 ] . mirna expression values were calculated according to the comparative threshold cycle method , using the ubiquitous small nucleolar rna u6 as endogenous reference . to overexpress mir-146b in thp-1 monocytic cells a lentiviral - based system was used , as described elsewhere   briefly , the mir / lentiviral - based expression vector prrl - mir-146b was generated by cloning in the prrlsin.cppt.pgk-gfp.wpre vector ( plasmid # 12252 ; addgene ) a 500  bp region encompassing the pre - mir-146b . the lentiviral construct prrl - ct , encoding for a hairpin yielding a 22-mer rna with no homology to any human gene , was used as mock construct . thp-1 cells were transduced with prrl-146b or prrl - ct vectors and prrl-146b thp-1 gfp cells and prrl - ct thp1 gfp cells were sorted by facs with a 9095% of purity . to knockdown mir-146b-5p expression , thp-1 cells were transduced with the mirzip lentivector - based construct anti - mir-146b and the relative control ( system biosciences ) . transduced gfpmirt-146b and gfpmirt - ct thp-1 cells were sorted by facs with over a 95% of purity . 30  10 stimulated monocytes were used and results were expressed as fold enrichment relative to unstimulated samples . all antibodies and detection reagents were purchased from r&d systems . samples were diluted so that the optical density fell within the optimal portion of a log standard curve . mirt - ct and mirt-146b-5p thp1 cells were cultured in 24-well plates in 500  l rpmi supplemented with 10% fbs and 1% l - glutamine , incubated or not for 18  h with 50  ng / ml tgf or 20  ng / ml il-10 , followed by lps stimulation with 0.1  ng / ml or 20  ng / ml lps . statistical evaluation was determined using either student t - test or one - way anova and p values are reported in figures ( p < 0.05 ; p < 0.01 ) . the mir-146 family is composed by mir-146a and mir-146b , both of which are induced during the inflammatory response and target different component of tlr signaling pathway , thus acting as anti - inflammatory regulators [ 18 , 21 , 33 ] . in previous studies we reported that lps induced the expression of both mir-146a and mir-146b in human monocytes   and demonstrated that mir-146b but not mir-146a induction was dependent on the endogenous production of il-10 subsequent to lps exposure . we here investigated the regulation of mir-146b and mir-146a by anti - inflammatory mediators known to negatively modulate the activation of monocyte / macrophages . we found that , in addition to il-10 , tgf was able to specifically increase the expression of mir-146b but not mir-146a . glucocorticoids ( dex ) , il-4 , and il-13 did not affect mir-146b and mir-146a expression levels ( figure 1(a ) ) . to investigate the functional activity of mir-146b we evaluated its presence into the ago2-risc complex and consistent with the expression data we found an enrichment of mir-146b only when monocytes were treated with lps , il-10 , or tgf ( figure 1(b ) ) . as a control , we measured the relative enrichment of mir-146a and consistent with previous data   we detected mir-146a in ago2-risc complex only when monocytes were stimulated with lps ( figure 1(b ) ) . to elucidate the transcriptional regulation of mir-146b , the cis - regulatory elements in the mir-146b promoter region were characterized . monocytes exposed to tgf or il-10 showed an enrichment of pol ii onto the mir-146b promoter but not on mir-146a promoter ( figures 1(c ) and 1(d ) ) . bioinformatic analysis performed on the 1  kbp promoter region of mir-146b showed the presence of two consensus sites for stat3 and one for runx3 . since we previously demonstrated the binding of stat3 to mir-146b promoter in il-10 stimulated monocytes   ( figure 1(d ) ) we thought to determine whether stat3 is also recruited to the mir-146b promoter region upon monocytes tgf stimulation . we found that recruitment of stat3 is specific to mir-146b but not to mir-146a promoter ( figure 1(c ) ) . interestingly , stat3 has been reported to cooperate with runx3 , which is also directly induced by tgf signaling pathway . given the proximity of the stat3 and runx3 consensus sites on the mir-146b promoter region , we performed chip experiments and found the specific recruitment of runx3 transcription factor to the mir-146b promoter region only in monocytes challenged with tgf ( figures 1(c ) and 1(d ) ) . taken together , these data identify mir-146b as an il-10- and tgf-responsive gene , whose expression in monocytes is specifically driven by stat3 in response to il-10 and by stat3 and runx3 in response to tgf. since mir-146a was shown to be involved in lps tolerance [ 28 , 29 ] and we previously demonstrated that mir-146b is a negative regulator of tlr signaling , we investigated the role of mir-146b in the induction of lps tolerance . we found tnf strongly downregulated in in vitro tolerized monocytes ( figure 2(a ) ) , in agreement with previous reports . interestingly , the decrease of tnf levels was proportional to the dose of lps used to prime cells : its expression was dramatically impaired in monocytes primed with 0.1  ng / ml lps and completely abolished with 10  ng / ml lps ( figure 2(a ) ) . next we analyzed mir-146b expression levels in tolerized monocytes , using mir-146a as positive control . strikingly , both mir-146b and mir-146a showed significant higher expression in tolerized monocytes compared to untolerized control cells ( figures 2(b ) and 2(c ) , resp . ) , thus suggesting mir-146b putative role in lps tolerance . il-10 and tgf act as feedback inhibitors of the lps - mediated inflammatory response and are part of a more complex network of regulation in which ifn operates an opposite function working as a coactivator of the inflammatory pathway triggered by lps . it has also been reported that ifn is able to revert lps tolerance both in human [ 11 , 38 ] and murine [ 39 , 40 ] phagocytes . accordingly , in human monocytes ifn pretreatment strongly enhanced lps - dependent induction of tnf and significantly impaired lps - dependent tolerization effect ( figure 3(e ) ) . interestingly , ifn was able to inhibit the lps - dependent induction of mir-146b when monocytes were challenged with both lps and ifn ( figure 3(a ) ) , whereas it did not affect the expression of mir-146a ( figure 3(b ) ) . moreover , pretreatment with ifn completely abolished the increased expression of mir-146b observed in lps - tolerized monocytes , but it had no effect on the overexpression of mir-146a observed in tolerized monocytes ( figures 3(c ) and 3(d ) , resp . ) . opposite to ifn , the anti - inflammatory cytokines il-10 and tgf support lps desensitization and have been experimentally used to induce hyporesponsiveness to lps . ifn pretreatment , which strongly enhanced lps - dependent induction of inflammatory cytokines , was also able to impair the il-10- and tgf-dependent tolerization effect ( figures 4(a ) and 4(b ) ) . since we have observed that both il-10 and tgf induce the expression of mir-146b ( figure 1(c ) ) , altogether these data candidate mir-146b as an effector of lps tolerance . to determine the role of mir-146b in endotoxin tolerance , we used the thp-1 monocytic cell line , a well - established model for in vitro study of endotoxin tolerance . we showed that both mir-146b and mir-146a were significantly induced by lps ( figure 5(a ) ) . this data were consistent with the expression data of mir-146b and mir-146a in human primary monocytes . in order to discriminate the relative contribution of mir-146b to the establishment of the endotoxin tolerance phenotype , we transduced thp-1 monocytic cells with lentiviral vectors to specifically overexpress ( prrl-146b thp-1 cells ) or inhibit mir-146b isoform ( mirt-146b thp-1 cells ) without affecting mir-146a expression ( figures 5(b)5(d ) ) . as shown in figure 5(e ) , prrl-146b thp-1 cells stimulated with a single dose of lps exhibited lower levels of tnf as compared to prrl - ct thp-1 cells , thus inducing a state of hyporesponsiveness to lps similar to that observed by the subsequent lps stimulation . accordingly , the induction of endotoxin tolerance by priming monocytes with lps , il-10 , or tgf was completely abolished when the endogenous mir-146b expression was inhibited ( figure 5(f ) ) . therefore , lps responsiveness was restored in all the three different in vitro model of endotoxin tolerance , demonstrating that mir-146b is induced during lps tolerance as part of the inhibitory feedback mechanism that take place in monocytes to render cells refractory to subsequent lps challenge . figure 6 depicts the proposal model of mir-146b and mir-146a effect on induction of endotoxin tolerance . deregulation of the inflammatory response contributes to tissue damage in pathological conditions such as autoimmune diseases and cancer [ 3 , 41 , 42 ] . the innate immune system has developed a number of mechanisms , such as endotoxin tolerance , to balance inflammation . endotoxin tolerance is a complex , orchestrated counter - regulatory response to inflammation , during which monocytes undergo a global transcriptional and functional reprogramming and enter into a refractory state , unresponsive to a subsequent lps challenge . a characteristic feature of endotoxin tolerance is the downregulation of several proinflammatory genes and the concomitant upregulation of some anti - inflammatory genes . in particular , the induction of il-10 and tgf in the late phase of the inflammatory response represents a key step for the induction of endotoxin tolerance [ 16 , 21 , 44 ] leading to leukocyte deactivation through the impairment of tlr signaling . defects of tlr4 signaling have been observed at the level of the receptor , adaptors , transcription factors , and signaling molecules . in this regard , recent studies have focus attention on the differential expression and effects of mirna in the modulation of tlr4 signaling in monocytes [ 1820 ] and interestingly , new evidence suggests the involvement of mirna in the regulation of key components of tlr4 pathway during endotoxin tolerance [ 28 , 29 ] . the mir-146 family , composed by mir-146a and mir-146b , has been implicated in the regulation of immune cell signaling [ 45 , 46 ] and more interestingly in the modulation of the inflammatory response in monocytes [ 18 , 21 , 22 ] . in particular , mir-146a was shown to be involved in lps response and in endotoxin tolerance ; recent clinical studies showed increased expression of mir-146a in monocytes isolated from cll patients , challenged with lps ex vivo . a correlation between mir-146a despite these first studies highlighting the important role of mir-146a in endotoxin tolerance , the possible contribution of mir-146b in the regulation of the lps response has not yet been addressed potentially based on the report by taganov et al . , indicating that mir-146a was the major mirna of the mir-146 family induced by lps in thp-1 monocytes . we previously described mir-146b as the other mir-146 family member able to negatively modulate the inflammatory response in phagocytes by targeting multiple components of the tlr signaling pathway . since defects in tlr signaling and in the consequent release of proinflammatory cytokines are characteristics of endotoxin tolerance , we thought to shed light on the potential role of mir-146b in the induction of the tolerant state . we first demonstrate that both mir-146b and mir-146a are expressed in lps - stimulated human primary monocytes and in thp-1 monocytic cells . secondly , their expression is induced in lps - tolerized monocytes . since both mirnas are abundantly expressed in this cellular context and have overlapping target genes , to discriminate the relative contribution of mir-146b to the establishment of the endotoxin tolerance phenotype , we transduced thp-1 cells with a mir-146b lentiviral vector , which specifically inhibits mir-146b isoform without affecting mir-146a expression . we found that inhibition of endogenous mir-146b levels in lps tolerized cells completely restored the levels of tnf protein , here used as readout of endotoxin tolerance . accordingly , the enforced expression of mir-146b in thp-1 monocytes mimicked the effects of lps priming , inducing a state of tolerance comparable to that observed by the sequential stimulation with lps in control cells . remarkably , mir-146b but not mir-146a expression levels were further induced by lps when monocytes were primed with tgf- and il-10-tolerized monocytes and this is consistent with the specific upregulation of mir-146b in anti - inflammatory conditions . indeed , characterization of mir-146b transcriptional regulation by bioinformatics analysis and chip experiments showed that runx3 , an important mediator of tgf signaling , is specifically recruited to mir-146b promoter in monocytes challenged with tgf , while stat3 drives the expression of mir-146b in both tgf and il-10 stimulated monocytes . it is known that ifn can prevent or revert endotoxin tolerance through the inhibition of tgf and il-10 signaling , although the precise molecular mechanism responsible for ifn-dependent negative feedback loop is still incompletely elucidated . importantly , the direct induction of mir-146b by il-10 or tgf as well as its indirect induction after lps challenge was completely abolished by ifn. altogether our data propose mir-146b as a key anti - inflammatory mirna , which acts together with mir-146a in mediating endotoxin tolerance . notably , we demonstrated a differential regulation of mir-146b and mir-146a expression , suggesting that mir-146b could be one of the molecular tools used by il-10 and tgf to induce an lps refractoriness state . we also suggest that the two mir-146 family members may represent the components of a relay team in which the two isoforms succeed each other to control expression of proinflammatory genes . although the observed mir-146b effect in the induction of endotoxin tolerance is consistent with the previously shown regulation of the tlr signaling pathway by mir-146b , it is known that endotoxin tolerance is a more complex process , involving a wider cellular regulation . other biological processes , such as phagocytosis or antigen presentation , should be investigated in in vivo models of endotoxin tolerance to shed light on the effective impact of mir-146b and mir-146a deregulation in the context of endotoxin tolerance . moreover , due to the large clinical implications of endotoxin tolerance it would be interesting to test the impact of mir-146b deregulation in a clinical context . further investigations covering all these aspects are needed to candidate mir-146b as a possible therapeutic tool for treating several clinical conditions , such as sepsis and other inflammatory - related disorders .
a proper regulation of the innate immune response is fundamental to keep the immune system in check and avoid a chronic status of inflammation . 
 as they act as negative modulators of tlr signaling pathways , mirnas have been recently involved in the control of the inflammatory response . however , their role in the context of endotoxin tolerance is just beginning to be explored . 
 we here show that mir-146b is upregulated in human monocytes tolerized by lps , il-10 , or tgf priming and demonstrate that its transcription is driven by stat3 and runx3 , key factors downstream of il-10 and tgf signaling . 
 our study also found that ifn , known to revert lps tolerant state , inhibits mir-146b expression . 
 finally , we provide evidence that mir-146b levels have a profound effect on the tolerant state , thus candidating mir-146b as a molecular mediator of endotoxin tolerance .