damage - associated molecular patterns ( damps ) are endogenous molecules that can perpetuate inflammatory responses during cell stress or injury . the ecm glycoprotein tn - c , is specifically induced upon tissue injury [ 1 , 2 ] and infection [ 3 , 4 ] and upregulated in septic patients . tlr4-mediated tn - c expression induces cytokine production in both human and murine macrophages and in rheumatoid arthritis synovial fibroblasts . importantly , glucocorticoids can inhibit the expression of tn - c in bone marrow stromal cells and fibroblasts . in addition , mice and bone marrow - derived macrophages ( bmdms ) deficient in tn - c display lower production of proinflammatory cytokines such as tnf- during lps - induced sepsis . thus , tn - c has been recognized as a regulator of the early immune response . il-10 is a vital anti - inflammatory cytokine which is required for dampening inflammatory signals and defending the host from excessive inflammation . mice lacking il-10 infected with bacterial pathogens display high mortality , associated with excessive inflammatory responses . low levels of il-10 expression were associated with various inflammatory diseases such as ulcerative colitis , crohn 's disease , and asthma in humans [ 11 , 12 ] . the anti - inflammatory effect of il-10 is mediated through the jak1-stat3 pathway which leads to the inhibition of proinflammatory proteins such as tnf- and il-6 [ 10 , 13 ] . higher expression of il-10 was found in bmdms from tn - c - deficient mice while there was lower expression of proinflammatory cytokines , indicating an anti - inflammatory role of il-10 in the tn - c - mediated inflammatory disease model . carbon monoxide ( co ) is generated as an end product of the oxidative degradation of heme by the enzymatic action of heme oxygenase , which converts heme into biliverdin , free iron , and co . anti - inflammatory effects of co have been evident in murine models of sepsis , postoperative ileus , and organ xenotransplantation [ 15 , 16 ] . in addition , co has been found to be an important regulator in the suppression of inflammatory cytokines and mediators including inducible nitric oxide synthase ( inos ) , tnf- , and il-6 [ 17 , 18 ] as well as induction of the anti - inflammatory cytokine il-10 . to date , there are no reports regarding the effects of co - mediated il-10 production on the regulation of tn - c - mediated inflammation . therefore , in the current study , we examined the effects of co - dependent il-10 generation on tn - c - mediated inflammation in macrophages and in the septic mice model . tenascin - c antibody was purchased from cell signaling technology ( ma , usa ) . -actin , anti - mouse and anti - goat antibodies conjugated to horseradish peroxidase were obtained from santa cruz biotechnology ( santa cruz , ca , usa ) . lipopolysaccharide ( lps ) and protease inhibitor cocktail sets were purchased from sigma - aldrich ( st . louis , mo , usa ) . dulbecco 's modified eagle medium ( dmem ) , fetal bovine serum ( fbs ) , penicillin - streptomycin , and sodium pyruvate were purchased from invitrogen ( grand island , ny , usa ) . raw 264.7 cells and peritoneal macrophages were cultured in dmem ( invitrogen ) containing 10% fetal bovine serum ( fbs ) and 1% penicillin streptomycin at 37c in 5% co2 until 7580% confluence . for preparation of peritoneal macrophages , mice were injected intraperitoneally with 3% thioglycolate for 3 days and cells were collected for culture . cells ( 5  10/ml ) were seeded in 6-well plates and incubated overnight for subsequent experiments . seven - week - old wild type male c57bl/6 mice were pretreated with corm-2 ( 30  mg / kg , i.p . )   or rucl3 ( 30  mg / kg , i.p . ) . and then , mice were injected with lps ( 10  mg / kg , i.p . ) . after 2 hours , blood serum and liver tissues were collected and stored at 80c for protein and rna analysis . all experiments with mice were approved by the animal care committee of the university of ulsan , ulsan , korea . raw 264.7 cells ( 5  10/ml ) were cultured in 6-well plates for 3  h and transfected with il-10 sirna ( 100  nm ) or tn - c sirna ( 100  nm ) from santa cruz biotechnology using lipofectamine 2000 according to the manufacturer 's instructions . after transfection , cells were incubated with or without corm-2 ( 20  m ) and then stimulated with or without lps ( 100  ng / ml ) . cell extracts were lysed using lysis buffer containing ripa buffer , protease inhibitor , and phosphatase inhibitors . after lysis , protein concentration was measured by bca assay ( pierce biotechnology inc . , samples containing equal amounts of protein were subjected to electrophoresis and proteins were transferred to polyvinylidene difluoride ( pvdf ) membranes . membranes were blocked with 5% skim milk for 20  min and then incubated at 4c overnight with primary antibodies , followed by secondary antibodies against tn - c and -actin conjugated with horseradish peroxidase . the enhanced chemiluminescence ( ecl ) western blotting detection system ( ge healthcare life sciences , buckinghamshire , uk ) was used to visualize the immunoreactive bands . total rna isolation was performed from raw 264.7 macrophages using trizol reagent ( invitrogen ) according to manufacturer 's instructions . briefly , total rna ( 2  g ) was used to prepare cdna by using m - mlv reverse transcriptase ( promega corporation , madison , wi , usa ) and oligo ( dt ) 15 primer ( promega ) . the resulted cdna was subjected to pcr for mouse gapdh ( 5-aggccggtgctgagtatgtc-3 , 5-tgcctgcttcaccttct-3 , 530  bp ) , ho-1 ( 5-tcccagacaccgctcctccag-3 , 5-ggatttggggctggtttc-3 , 313  bp ) , tn - c ( 5-caggtacttcttcacggagc-3 , 5-gcagtcttccccagtgaaac-3 , 834  bp ) , tnf- ( 5-agcccacgtcgtagcaaaccaccaa-3 , 5-acacccattcccttcacagagcaat-3 , 421  bp ) , il-6 ( 5-gtggaaatgagaaaagagttgt-3 , 5-cctcttggttgaagatatgaat-3 , 283  bp ) , and il-10 ( 5-gacaataactgcacccactt-3 , 5-tcaaatgctccttgatttct-3 , 250  bp ) , and gapdh was used as internal loading control . total rna was extracted from raw 264.7 peritoneal macrophages / liver tissues using trizol reagent ( invitrogen ) according to the manufacturer 's instructions . in addition , cdna was prepared by using m - mlv reverse transcriptase ( promega ) and oligo ( dt ) 15 primer ( promega ) . the formulated cdna was subjected to real time rt - pcr using sybr green qpcr master mix ( 2x ) ( usb products , affymetrix ) on an abi 7500 fast real - time pcr system ( applied biosystems ) for mouse gapdh ( 5-gggaagcccatcaccatct-3 , 5-cggcctcaccccatttg-3 ) , tn - c ( 5-accatgctgagatagatgttccaaa-3 , 5-cttgacagcagaaacaccaatcc-3 ) , tnf - a ( 5-agaccctcacactcagatcactttc-3 , 5-ttgctacgacgtgggctaca-3 ) , il-6 ( 5-cgatgatgcacttgcagaaa-3 , 5-tggaaattggggtaggaagg-3 ) , il-10 ( 5-actgctatgctgcctgctcttact-3 , 5-gaattcaaatgctccttgatttct-3 ) , and ho-1 ( 5-tcagtcccaaacctcgcggt-3 , 5-gctgtgcaggtgttgagcc-3 ) . macrophages on 6-well plates were incubated overnight and then pretreated with corm-2 for 1  h followed by stimulation with lps for 24  h. in addition , mice were administrated with corm-2 for 2  h and then sepsis was induced by lps injection . after 2  h , supernatants collected from various samples or blood serum collected from different mice were assayed for tnf- and il-6 by using a mouse elisa kit ( biolegend ) . statistical differences between groups were evaluated by one - way anova ( nonparametric ) or student 's t - test when multiple groups were compared . differences were considered to be significant when p < 0.05 , p < 0.01 , and p < 0.001 . stimulation of macrophages with gram - negative bacterial lps can enhance the expression of tn - c . tlr4 was involved in the induction of tn - c and subsequent cytokine synthesis in both human and murine macrophages   and human chondrocytes . in the present study , we examined inflammatory responses in murine raw 264.7 macrophages treated with lps ( 100  ng / ml ) . tn - c mrna and protein expression increased at 4 and 8  h after lps treatment ( figures 1(a ) and 1(b ) ) , respectively . therefore , in subsequent experiments , we measured tn - c mrna and protein expression at 8  h. furthermore , lps dose - dependently increased tn - c mrna and protein expression at 8  h ( figures 1(c ) and 1(d ) ) . these results suggest that lps induces tn - c expression in a time- and dose - dependent manner in raw 264.7 macrophages . the anti - inflammatory , antiapoptotic , and cytoprotective properties of co are well known . furthermore , it has been reported that the generation of endogenous co was necessary for il-10-dependent inhibition of tnf- expression . co can be generated pharmacologically from co - releasing molecules ( corms ) , which consist of a heavy metal such as ruthenium surrounded by carbonyl groups [ 26 , 27 ] . in brain endothelial cells , the lps - induced activation of inflammatory signals such as nf-b ( p65 ) , cox-2 expression , and pge2 production was inhibited by corm-2 pretreatment . in the present study , we investigated the effects of co on tn - c - mediated inflammation . we found that corm-2 significantly and dose - dependently suppressed the expression of lps - stimulated tn - c expression ( figure 2(a ) ) . in addition , pretreatment with corm-2 significantly reduced the mrna and protein levels of proinflammatory cytokines such as tnf- and il-6 ( figures 2(b ) , 2(c ) , and 2(d ) ) , respectively . to confirm the effects of co on tn - c - meditated inflammation , cells were pretreated with or without corm-2 or rucl3 ( negative control for corm-2 ) and then stimulated with or without lps . interestingly , corm-2 significantly downregulated lps - induced tn - c as well as proinflammatory cytokines expression whereas rucl3 did not have any effect ( figures 2(e ) , 2(f ) , 2(g ) , and 2(h ) ) . to confirm the effects of co consistently , co gas dramatically reduced lps - stimulated expression of tn - c ( figure 2(i ) ) , as well as tnf- ( figure 2(j ) ) and il-6 ( figure 2(k ) ) . to further confirm the effects of co on lps - induced tn - c expression and proinflammatory cytokines , mouse peritoneal macrophages were pretreated with corm-2 at various concentrations and incubated with lps . we found that corm-2 dramatically decreased lps - induced tn - c ( figure 3(a ) ) and its downstream cytokines ( figure 3(b ) ) . in contrast , rucl3 did not reduce the expression of tn - c and proinflammatory cytokines ( figures 3(c ) and 3(d ) ) . low doses of co suppressed inflammatory responses in a murine model of sepsis through inhibition of inflammatory cytokines production [ 18 , 29 ] as well as increased lps - induced expression of the anti - inflammatory cytokine il-10 in various cell types [ 19 , 29 ] . in addition , mice deficient with tn - c displayed lower levels of tnf- and downstream cytokine production in lps - treated septic mice and bone marrow - derived macrophages ( bmdms ) . in our study , we investigated the effects of co on tn - c - induced proinflammatory cytokines expression and the expression of anti - inflammatory il-10 in raw 264.7 and peritoneal macrophages . corm-2 significantly and dose - dependently induced the expression of il-10 in lps - stimulated macrophages ( figures 4(a ) and 4(b ) ) . furthermore , treatment with co gas significantly increased levels of il-10 ( figure 4(c ) ) in lps - stimulated raw 264.7 macrophages . however , treatment with rucl3 did not have any effect on il-10 expression in these cells ( figures 4(d ) and 4(e ) ) . these observations indicate that the anti - inflammatory effects of co are mediated by il-10 in lps - stimulated macrophages . the incubation of raw 264.7 cells with tn - c sirna significantly suppressed the effects of lps on tn - c and proinflammatory cytokines production ( figures 4(f ) , 4(g ) , and 4(h ) ) , whereas it had no effect on il-10 expression ( figure 4(i ) ) , suggesting that il-10 is regulated independently of tn - c and its downstream cytokines . to confirm the function of co - induced il-10 on tn - c - mediated inflammation , macrophages were transfected with il-10 sirna and treated with corm-2 prior to lps - stimulation . we found that il-10 sirna reversed the inhibitory effect of corm-2 on tn - c expression and inflammatory cytokines production in lps - stimulated macrophages relative to control sirna ( figures 4(k ) , 4(l ) , and 4(m ) ) . pretreatment of recombinant il-10 with or without lps stimulation showed the same efficiency of corm-2 to significantly decrease tn - c expression ( figure 4(n ) ) . also , ho-1 increases il-10 production . according to inoue and colleagues , overexpressions of ho-1 thus , we examined the expression of anti - inflammatory gene ho-1 under these conditions . interestingly , we found that corm-2 significantly increased the level of ho-1 expression ( figure 4(o ) ) , whereas rucl3 did not have any effect on ho-1 expression ( figure 4(o ) ) and conversely decreased the expression levels of tn - c . the inhibition of ho activity using znppix , however , did not reverse the effects of corm-2 on tn - c expression ( figure 4(p ) ) indicating that corm-2 mediated suppression of tn - c is independent of ho activity in lps - stimulated raw 264.7 macrophages . based on these results , we conclude that co - induced il-10 inhibits tn - c - mediated inflammation . sepsis , a systemic inflammatory response , results from excessive production of proinflammatory cytokines by lps stimulation . in addition , proinflammatory cytokines such as tnf- , il-1 , and il-6 have been found at higher levels in septic patients [ 32 , 33 ] . administration of lps in mice revealed that tn - c expression is necessary for proinflammatory signaling . furthermore , application of exogenous co inhibits lps - induced production of tnf- while it increases il-10 production in vitro and in vivo . however , there are no reports regarding the effects of co - mediated il-10 production in relation to the regulation of tn - c and inflammation in a septic mouse model . in our study , to examine the in vivo effects of co using corm-2 on lps - induced endotoxemia and tn - c - mediated inflammatory cytokines expression , we pretreated mice with corm-2 ( 30  mg / kg , i.p . ) or rucl3 ( 30  mg / kg , i.p . ) for 2  h and lps ( 10 mg / kg , i.p . ) for 2  h. interestingly , corm-2 significantly decreased tn - c ( figure 5(a ) ) , tnf- and il-6 mrna expression ( figure 5(b ) ) , and protein secretion ( figure 5(c ) ) and simultaneously increased il-10 expression ( figure 5(d ) ) in liver tissue from lps - induced endotoxemic mice . also , the levels of il-10 ( figure 5(e ) ) were increased and reversely tn - c levels ( figure 5(f ) ) were decreased in the serum of mice treated with corm-2 . therefore , the results from in vivo experiments suggest that co inhibited tn - c and its downstream inflammatory cytokines whereby il-10 expression was upregulated in a septic mice model . tn - c is unique in its distinct pattern of expression . upon tissue injury tn - c is transiently expressed , whereas its expression is reduced after the tissue is repaired . moreover , persistent tn - c expression occurs during chronic inflammation . in addition , tn - c is absent in most healthy adult tissues   whereas high levels are found during infection and in patients with sepsis . however , tn - c is expressed at sites of inflammation regardless of the location or type of causative insult , indicating its capability to participate in the global inflammatory response . tn - c can increase the synthesis of cytokines in human chondrocytes   and myeloid cells   in a tlr4-dependent manner and also activate murine myeloid cells [ 35 , 36 ] . additionally , tn - c expression is transiently induced by lps in innate immune cells in a nf-b - dependent manner [ 7 , 37 ] and its dysregulation is observed in both autoimmune and inflammatory diseases , such as sepsis . in the present study , we found that lps significantly increased tn - c and proinflammatory cytokines production in macrophages as well as in a mouse model . therefore , understanding which compounds can inhibit tlr - mediated tn - c expression and cytokines production may refine strategies to manipulate excessive inflammation . recently , researchers reported that co gas can exert beneficial effects in various cell and animal models . co plays an important role in preventing apoptosis in several cell types such as endothelial cells , fibroblasts , and pancreatic -cells   and inhibits the proliferation of smooth muscle cells , thus preventing atherosclerotic lesions . in animal models , corm compounds provide a reliable source of co that can mimic co gas in many biological functions [ 26 , 45 ] . therefore , corms represent important tools to understand the biological significance of co in physiology and disease . corm-2 was the first compound used to deliver co in biological systems in a controlled manner . in the current study , to examine the effects of co , we pretreated macrophages and mice with corm-2 in an lps - stimulated inflammation model . interestingly , corm-2 significantly decreased lps - induced tn - c and proinflammatory cytokines production in vitro and in vivo . similarly , pretreatment with co gas significantly decreased lps - stimulated tn - c and cytokines production in macrophages , supporting the direct effects of co on tn - c - mediated inflammation . in addition , rucl3 , a negative control for corm-2 , did not affect lps - mediated tn - c , tnf- , and il-6 expression in macrophages or in septic mice . these results confirm that co inhibits lps - mediated tn - c and proinflammatory cytokines production . the anti - inflammatory cytokine il-10 plays a crucial role in dampening toll - like receptor ( tlr ) signaling - induced proinflammatory genes . interestingly , co was found to increase the levels of anti - inflammatory , il-10 , while the levels of proinflammatory cytokines were decreased in several in vitro systems [ 18 , 29 ] . additionally , corm-2 was also found to regulate inflammatory responses through decreasing il-1 expression and increasing il-10 expression . in a sepsis model , co - mediated activation of the mkk3/p38 mapk signaling pathway was involved in the induction of il-10 . in our investigation , we determined that the effects of co significantly increased il-10 expression under lps - stimulated conditions while rucl3 had no effect in vitro or in vivo . furthermore , il-10 sirna significantly reversed the effects of corm-2 on tn - c and proinflammatory cytokines production whereas tn - c sirna significantly decreased proinflammatory gene expression levels without having an effect on co - mediated il-10 expression . this evidence suggests that co - mediated il-10 expression was involved in inhibition of tn - c - mediated inflammation . in summary , we identified that co - induced il-10 was involved in the inhibition of tlr4 signaling - dependent tn - c expression and thus inhibited the inflammatory response in vitro and in vivo . this study describes a novel co - dependent il-10 signaling pathway responsible for the inhibition of tn - c - driven inflammation and potentially provides the rationale for novel therapeutic strategies for the treatment of inflammatory diseases .