severe sepsis remains a challenging clinical problem that accounts for 1.3% of all hospitalizations and is the leading cause of mortality and morbidity in intensive care units [ 1 , 2 ] . the most life - threatening complication of sepsis is the development of the multiorgan dysfunction syndrome ( mods ) , which most commonly affects the lungs and kidneys . acute lung injury ( ali ) occurs in approximately 30% of septic patients , and unfortunately there are no specific treatments available to reduce mortality other than low tidal volume ventilation . sepsis occurs when infections trigger the systemic inflammatory response syndrome in the host . as a central regulator of inflammation , nuclear factor-b ( nf-b ) has long been considered to be a critical pathway involved in the pathogenesis of sepsis [ 4 , 5 ] . available information suggests that inhibition of nf-b could be a promising therapeutic target in sepsis . a number of studies have investigated the effects of manipulating nf-b activity using genetic or pharmacologic approaches during sepsis and/or endotoxemia [ 612 ] . in general , nf-b downregulation has been successful in inhibition of inflammation and reduction of nf-b - mediated cytokines in these studies [ 69 ] . however , we and others have shown that nf-b plays an important role in host defense against bacterial infections in a variety of experimental models [ 13 , 14 ] , raising concern that therapeutic nf-b inhibition might have the unwanted effect of impairing bacterial clearance . a recent study showed that early neutrophil activation and infiltration ( within 6 hours ) are important for bacterial clearance and survival in septic animals . therefore , we reasoned that early nf-b activation and production of nf-b - dependent cytokines in sepsis might be sufficient for host defense , whereas prolonged nf-b activation could contribute to organ injury and dysfunction . to address this hypothesis , we investigated the impact of inhibition of nf-b activation for a defined period of time after the onset of sepsis . we used bms-345541 , a highly selective ikk inhibitor , for these studies . due to its high oral bioavailability and relatively short half - life , bms-345541 can be delivered orally and is effective for short - term inhibition of nf-b in vivo . age- and gender - matched fvb mice between 8 and 16 weeks old were used for these studies . mice were kept in a temperature - controlled room with light and dark cycles every 12 hours . all procedures were approved by the institutional animal care and use committee at vanderbilt university and complied with all state , federal , and nih regulations . after mice were anesthetized with isoflurane ( 2 - 3% ) , the surgical area was disinfected with betadine and 70% alcohol and a midline laparotomy was performed . the cecum was then identified and the distal 50% of the cecum was ligated with 4 - 0 silk suture followed by puncture with one pass of a 21-gauge needle . after surgery , 0.5  ml of sterile normal saline was administered by intraperitoneal injection . two hours after clp , bms-345541 ( 50  g / g body weight , obtained from bristol - myers squibb ) formulated in 3% tween 80 and sterile water or an equivalent volume of vehicle ( without bms-345541 ) was administered by oral gavage . mice were subjected to laparotomy without clp ( sham ) , laparotomy with clp followed by vehicle delivered by gavage ( clp ) , and laparotomy with clp plus bms-345541 administration ( clp+bms ) . mice were evaluated daily and were euthanized at 7 days after clp or earlier if they appeared moribund or in distress . differential leukocyte counting was performed by manual counting of wright - giemsa - stained ( diff quick ; baxter scientific products ) peripheral blood smears . after euthanasia , lungs were perfused with normal saline and inflated with 1  ml of 10% neutral buffered formalin . lungs were then removed en bloc after tracheal ligation , preserved in 10% neutral buffered formalin for 24 hours at room temperature , and subsequently embedded in paraffin . the degree of parenchymal distortion in the alveolar tissue was assessed on 20 sequential high power fields per lung section and graded as follows : 0 : normal ; 1 : < 50% of interalveolar septa ( ias ) have increased thickness due to edema and/or inflammatory cell infiltration ; 2 : > 50% of ias have increased thickness ; 3 : > 50% of ias have increased thickness and inflammatory cells are present within alveolar space ; 4 : consolidated infiltrate with distortion of normal alveolar architecture . the mean score bronchoalveolar lavage ( bal ) fluid was centrifuged at 400  g for 10 minutes to separate cells from supernatant . cells were resuspended in cold pbs and total cell counts were measured on a grid hemocytometer . differential cell counts were enumerated by counting at least 200 cells in sequential fields across the slide . lungs were then placed in an incubator at 65c for 48 hours and the dry weight was recorded . cytokines in bal fluid and serum were measured using a mouse cytokine 20-plex panel ( invitrogen ) that detects basic fgf , il-1 , il-10 , il-13 , il-6 , il-12 , il-17 , mip-1 , gm - csf , mcp-1 , il-5 , vegf , il-1 , ifn , tnf , il-2 , ip-10 , mig , kc , and il-4 . measurements of nf-b dna - binding activity were performed on lung nuclear protein extracts obtained using ne - per nuclear and cytoplasmic extraction reagents ( thermos ) . ten g of nuclear protein extract from tissues was used for measuring dna - binding activity of rela ( p65 ) ( transam elisa kit , active motif ) according to the manufacturer 's instructions . ten microgram of nuclear protein extract from lung tissue homogenates was separated on a 10% acrylamide gel ( nupage , invitrogen ) . western blot analyses were performed with antibodies against rela ( p65 ) , tbp ( santa cruz biotech ) , and assessed using the odyssey infrared system ( li - cor ) . the right middle lobe , peritoneal fluid , and blood ( obtained by cardiac puncture ) were used for bacterial colony counts . lung homogenates , peritoneal fluid , and serum were diluted into serial tenfold dilution using sterile pbs and plated on lb agar at 37c for 2448 hours , after which bacterial colonies were counted . raw264.7 macrophages or cells obtained from peritoneal lavage at 3 hours after clp were seeded into a 24-well plate at a concentration of 5  10 cells / well . after attachment , cells were treated with e. coli lipopolysaccharide ( lps , 200  ng / ml ) , bms-345541 ( 1  m ) , or lps+bms-345541 . one hour after lps treatment , e. coli dh5a , carrying the green fluorescent protein ( gfp)-mut2 encoding plasmid pcd353 ( e. coli - gfp ) , was added at a bacteria / leukocyte ratio of 20  :  1 and incubated at 37c for 1 hour . extracellular fluorescence was quenched by trypan blue , and the numbers of positive cells were counted using fluorescence microscopy , as previously described . to evaluate differences between groups , analyses were performed with graphpad instat software using an unpaired t - test ( two groups ) or anova ( multiple groups ) followed by the tukey - kramer multiple comparisons posttest . nonparametric anova with kruskal - wallis posttest was used to evaluate histological scoring of lung injury . survival data were plotted using kaplan - meier curves and analyzed by the log - rank test . based on previous work using bms-345541 to block nf-b activation in vivo , we developed a protocol to target nf-b activation by beginning bms-345541 treatment after the initiation of systemic inflammation following clp . mice were treated with bms-345541 or vehicle by peroral gavage beginning at 2 hours after clp . multiple doses were used because of the short half - life of the compound in vivo . to evaluate the efficacy of this intervention , we measured nuclear rela ( p65 ) levels by western blot analysis at 24 hours following clp . as shown in figures 1(a ) and 1(b ) , bms-345541 treatment blocked rela ( p65 ) nuclear translocation in spleens and lungs at this time point . in addition , nf-b dna - binding activity was reduced in the lungs of bms-345541-treated mice compared to mice that received clp and vehicle ( figure 1(c ) ) . together , these findings confirmed that bms-345541 blocks clp - induced nf-b activity in our model .   after showing that bms-345541 treatment attenuated nf-b activation , we investigated the impact of this treatment on clp - induced lung and systemic inflammation . consistent with prior studies [ 19 , 20 ] , clp led to a decrease in peripheral wbc counts and an increase in the percentage of neutrophils at 24 hours compared to sham surgery controls ( figure 2 ) . treatment with bms-345541 did not affect total or differential wbc counts at 24 hours after clp . similar findings were obtained from mice in the different treatment groups at 48 hours after clp ( not shown ) .   to evaluate the effects of nf-b inhibition on the course of lung inflammation in this model , we measured total inflammatory cells , neutrophils , cytokines , and chemokines in bal fluid at 24  h after clp . we found a significant reduction in the total number of bal cells , as well as bal neutrophils , in the clp+bms group compared to the clp group ( figures 3(a ) and 3(b ) ) . total bal cells and neutrophils were similar between the clp+bms group and sham laparotomy controls . at 48 hours after clp , we found a similar pattern of cells in bal with a reduction in total cells and neutrophils in the clp+bms group compared to the clp group ( data not shown ) . consistent with reduced neutrophilic lung inflammation , levels of the nf-b - dependent neutrophil chemotactic chemokine kc were lower in serum and bal fluid in the clp+bms group compared to the clp group ( figures 3(c ) and 3(d ) ) . of other cytokines tested , mcp-1 was reduced , while il-10 was increased in serum from mice treated with bms-345541 following clp compared to mice that underwent clp followed by vehicle ( figures 3(e ) and 3(f ) ) . we also found a trend towards decreased il-6 , il-17 , and mig in serum from mice treated with clp+bms compared to mice treated with clp ( + vehicle ) ( data not shown ) . in bal fluid , levels of these cytokines were similar in both groups ( clp+bms and clp ) at 24 hours after clp ( data not shown ) . since bms-345541 inhibited nf-b activation and downstream proinflammatory cytokine responses , we asked whether bms-345541 treatment would reduce clp - induced lung injury . mice treated with clp developed characteristic histological changes of lung injury at 48 hours , including inflammatory cell infiltration , vascular congestion , alveolar edema , interalveolar septal thickening , and hemorrhage ( figures 4(a ) and 4(b ) ) . in contrast , mice treated with bms-345541 following cpl showed preserved alveolar architecture with minimal edema and septal thickening . to quantify lung edema , we measured wet - to - dry ratios in lungs following clp at 24 and 48 hours . the increased wet - to - dry ratio observed in clp group was significantly attenuated in the clp+bms group , consistent with the histological changes observed in the lungs of mice from each treatment group ( figures 4(c ) and 4(d ) ) . together , these studies indicate that pharmacologic nf-b inhibition initiated 2 hours after clp reduces lung inflammation and prevents injury . although nf-b pathway activation plays an important role in host defense , we postulated that short - term nf-b inhibition initiated after the onset of sepsis - induced inflammation would limit downstream organ injury without impairing host defense against bacterial infection . to test this hypothesis , we performed bacterial colony counts from blood ( n = 20 per group ) , lung tissue ( n = 20 per group ) , and peritoneal fluid ( n = 11 per group ) at 24 hours in mice treated with clp+bms or clp ( + vehicle ) . we found no significant differences in colony counts from blood , lung tissue , or peritoneal fluid between bms-345541- or vehicle - treated mice following clp ( figures 5(a)5(c ) ) . these findings suggested that host defense was not impaired by bms-345541 treatment . because macrophages play an important role in host defense against bacterial peritonitis we wondered whether nf-b inhibition affects bacterial uptake and killing by macrophages . in preliminary studies , we found that bms-345541 ( 1  m ) inhibited lps - induced nf-b activation without affecting cell survival . pretreatment with lps substantially increased phagocytosis of e. coli - gfp by raw264.7 macrophages , but bms-345541 treatment did not alter phagocytosis with or without lps pretreatment ( figure 6(a ) ) . since neutrophils are also important for bacterial killing , we conducted an ex vivo experiment to evaluate the ability of innate immune cells in the peritoneum to phagocytose bacteria . peritoneal cells ( approximately 50% macrophages , 40% neutrophils at this time point after clp ) were harvested at 3 hours after clp and incubated with e. coli - gfp+bsm-345541 or e. coli - gfp for 1 hour . as shown in figure 6(b ) together , these studies show that short - term inhibition of nf-b does not impair bacterial clearance following clp and suggest that bacterial phagocytosis by innate immune cells is not impaired by nf-b inhibition . although death in mouse sepsis models is thought to be predominantly due to shock rather than acute lung injury , we wanted to make sure that short - term treatment with bms-345541 did not have a negative impact on survival after clp . in his study , 25 mice per group were subjected to clp ( + vehicle ) , clp+bms , or sham laparotomy and followed for one week . at 7 days after clp , all mice in the sham ( control ) group survived , compared to 12/25 in the clp group and 18/25 in the clp+bms group ( figure 7 ) . this study shows that systemic nf-b inhibition for a defined period of time after the onset of polymicrobial sepsis results in a trend towards improved survival , as opposed to a detrimental effect that would be expected if nf-b was required for bacterial clearance in this model . in these studies , we investigated the effects of inhibition of nf-b over a short , defined time period after the onset of sepsis . we found that a specific ikk inhibitor ( bms-345541 ) effectively suppressed nf-b activation and attenuated lung inflammation and injury . importantly , bacterial clearance was not impaired by inhibiting nf-b during this time frame , and there was a trend towards increased survival after bms-345541 treatment . together , these findings suggest that inhibition of nf-b activation after the onset of sepsis might be an option for suppressing the systemic inflammatory response and downstream organ injury caused by microbial infection .   common models of sepsis include administration of exogenous toxins such as lps , administration of viable bacteria , and polymicrobial infection resulting from clp . in contrast to the rapid inflammatory response induced by systemic lps treatment , clp results in prolonged cytokine production resulting from peritoneal infection by enteric bacteria , which mimics abdominal sepsis in humans . in previous studies , targeting nf-b during sepsis has resulted in variable outcomes that may be due in part to the different animal models used in these studies . for example , downregulation of nf-b activity can lead to improved survival in noninfectious endotoxemia models . in combination with antibiotics , , nf-b inhibition in infection - related models can impair host defense , particularly in genetic models with constitutive defects in nf-b signaling , and reduce survival [ 14 , 23 ] .   in our studies , we used short - term targeted nf-b inhibition with the goal of reducing acute lung injury following clp . acute lung injury is a major clinical problem in the setting of human sepsis that results in respiratory failure and lacks specific treatments other than lung protective ventilator strategies . in rodent models of clp , lung injury is relatively mild and is not thought to substantially contribute to mortality , which is primarily due to shock . therefore , it is not surprising that protection from acute lung injury following this dosing regimen of bms-345541 was not associated with a reduction in mortality . viewed in this context , our studies provide proof - of - concept that short - term nf-b inhibition ( after onset of sepsis but prior to development of lung injury ) could be beneficial in preventing acute lung injury without negative effects on bacterial clearance or mortality . consistent with our findings , it has been suggested that partially inhibiting nf-b activation using nonspecific methods , such as elevated hsp70 expression and glutamine treatment , could result in reversal and/or prevention of lung injury in animals subjected to clp [ 2426 ] . in our studies , first , treatment with bms-345541 was begun at 2 hours after clp and likely required several hours after the onset on infection to achieve substantial nf-b inhibition . therefore , one would not expect the production of important proximal cytokines like tnf and il-1 to be reduced . the early wave of cytokines production , which occurs within the first 1 - 2 hours after infection , likely leads to early neutrophil recruitment and activation of mononuclear phagocytes that play critical roles in host defense . second , only a partial and transient nf-b activation was achieved in these studies . when administered by the oral route , bms-345541 has near complete absorption and bioavailability with a 2.2-hour half - life . the relatively short half - life of bms-345541 in vivo necessities repetitive dosing , as it was done in this study , and limits the ability to provide prolonged , high - level nf-b inhibition in tissues . although this is a potential limitation of currently available compounds targeting the nf-b pathway , it may be beneficial in maintaining host defense functions . differences in the severity of the clp procedure can result in different degrees of peritonitis and different survival rates . although we did not test this possibility , nf-b inhibition might be detrimental in the setting of overwhelming peritoneal sepsis . fourth , we found that nf-b inhibition with bms-345541 did not affect basal or inducible phagocytosis of bacteria by macrophages and neutrophils . together , a number of factors may account for the preservation of bacterial host defense in our studies despite the functional inhibition of nf-b . in these studies , we only tested one regimen of nf-b inhibition using a single agent . in the future , a more comprehensive assessment is warranted using a variety of sepsis models to better define the optimal timing and degree of nf-b inhibition to minimize lung injury while preserving host defense . in conclusion , our study indicates that inhibition of nf-b for a defined time period after the onset of sepsis using a specific ikk inhibitor ( bms-345541 ) is beneficial in limiting lung injury without compromising host defense even in the absence of concurrent antibiotic treatment . we suggest that this approach could hold promise in humans with sepsis , where targeted therapies to limit the down - stream organ injury are needed .
introduction . 
 since the nf-b pathway regulates both inflammation and host defense , it is uncertain whether interventions targeting nf-b would be beneficial in sepsis . 
 based on the kinetics of the innate immune response , we postulated that selective nf-b inhibition during a defined time period after the onset of sepsis would reduce acute lung injury without compromising bacterial host defense . 
 methods . 
 mice underwent cecal ligation and puncture ( clp ) . 
 an nf-b inhibitor , bms-345541 ( 50  g / g mice ) , was administered by peroral gavage beginning 2 hours after clp and repeated at 6 hour intervals for 2 additional doses . 
 results . 
 mice treated with bms-345541 after clp showed reduced neutrophilic alveolitis and lower levels of kc in bronchoalveolar lavage fluid compared to mice treated with clp+vehicle . in addition , mice treated with clp+bms had minimal histological evidence of lung injury and normal wet - dry ratios , indicating protection from acute lung injury . 
 treatment with the nf-b inhibitor did not affect the ability of cultured macrophages to phagocytose bacteria and did not alter bacterial colony counts in blood , lung tissue , or peritoneal fluid at 24 hours after clp . 
 while bms-345541 treatment did not alter mortality after clp , our results showed a trend towards improved survival . 
 conclusion . 
 transiently blocking nf-b activity after the onset of clp - induced sepsis can effectively reduce acute lung injury in mice without compromising bacterial host defense or survival after clp .