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 .
 tenascin - c ( tn - c ) , an extracellular matrix ( ecm ) glycoprotein , is specifically induced upon tissue injury and infection and during septic conditions . 
 carbon monoxide ( co ) gas is known to exert various anti - inflammatory effects in various inflammatory diseases . 
 however , the mechanisms underlying the effect of co on tn - c - mediated inflammation are unknown . in the present study 
 , we found that treatment with lps significantly enhanced tn - c expression in macrophages . 
 co gas , or treatment with the co - donor compound , corm-2 , dramatically reduced lps - induced expression of tn - c and proinflammatory cytokines while significantly increased the expression of il-10 . 
 treatment with tn - c sirna significantly suppressed the effects of lps on proinflammatory cytokines production . 
 tn - c sirna did not affect the corm-2-dependent increase of il-10 expression . in cells transfected with il-10 sirna , corm-2 had no effect on the lps - induced expression of tn - c and its downstream cytokines . 
 these data suggest that il-10 mediates the inhibitory effect of co on tn - c and the downstream production of proinflammatory cytokines . 
 additionally , administration of corm-2 dramatically reduced lps - induced tn - c and proinflammatory cytokines production while expression of il-10 was significantly increased . in conclusion , co regulated il-10 expression and thus inhibited tn - c - mediated inflammation in vitro and in vivo . 
1. Introduction
2. Methods and Materials
3. Results
4. Discussion