during the coagulation cascade , fibrin is generated by the action of thrombin on fibrinogen . although best appreciated for its roles in vascular hemostasis , coagulation leading to fibrin deposition also frequently accompanies type 1 immune responses , including delayed - type hypersensitivity ( 13 ) , autoimmunity ( 410 ) , and graft rejection ( 1113 ) . prior studies indicate that fibrin , and other coagulant products , can exacerbate inflammation and disease in those settings ( 116 ) . coagulation also frequently accompanies infectious disease , and apparently can exacerbate pathology in that context as well ( 16 ) . indeed , administration of activated protein c , a natural anticoagulant protein , reduces septic mortality in humans ( 17 ) . while these and other studies have documented pathological roles for coagulation during immunity and infection , beneficial functions for immune - associated coagulation have yet to be established . toxoplasma gondii is a widely distributed , obligate intracellular , protozoan parasite ( 18 , 19 ) . natural infection is acquired by ingestion of encysted organisms , which are released in the digestive tract , invade intestinal tissues , and then disseminate . in mouse models , t. gondii infection elicits a robust type 1 immune response , which eradicates most parasites within 2 wk of infection . nevertheless , some organisms escape immune surveillance , encyst , and enter a dormant state from which they can emerge if immunity wanes , as frequently occurs in humans suffering from the acquired immunodeficiency syndrome . here , we evaluate roles for fibrin in the context of the strong type 1 immune response prompted by t. gondii infection . in contrast to prior studies of immune - associated fibrin deposition , we demonstrate that coagulation performs a critical protective function in the setting of t. gondii infection by suppressing hemorrhage evoked by ifn- , a critical mediator of type 1 immunity . thus , while coagulant products have the potential to function pathologically , our data indicate that immune - associated coagulative responses can also protect host tissue from collateral damage caused by the immune system as it combats infection . fibrinogen - deficient mice ( reference 20 ; backcrossed seven generations to c57bl/6 mice ) were generously supplied by jay degen ( children 's hospital medical center , cincinnati , oh ) and were bred at the trudeau institute animal breeding facility . animals were housed in a specific pathogen - free facility and cared for according to the trudeau institute animal care and use committee guidelines . t. gondii strain me49 was originally provided by jack remington ( palo alto medical foundation , palo alto , ca ) . me49 cysts were obtained from brains of chronically infected c57bl/6 mice , and infections were initiated by peroral administration of 10 cysts in 0.1 ml of diluted brain suspension using a 19-gauge gavage needle . sham - infected mice received similarly diluted brain suspensions from uninfected mice . where indicated , 2 mg / l warfarin ( 3-(-acetonylbenzyl)-4-hydroxy - coumarin ; sigma - aldrich ) was added to drinking water and replenished every 48 h. the anti ifn- protein levels in sera were determined by sandwich elisa ( bd biosciences ) . nitric oxide levels in plasma were determined as described ( 21 ) , except that plasma was diluted fourfold in water and filtered ( 10000 mwco ; amicon ) before analysis . tissue levels of mrna encoding ifn- , tnf- , il-10 , inducible nitric oxide synthase ( inos ) , and the t. gondii p30 gene were measured by real - time pcr ( perkinelmer ) and normalized to levels of -2-microglobulin ( 2 m ) . for t. gondii , standard curves were prepared by adding known numbers of me49 tachyzoites ( obtained from peritoneal exudate fluid of t. gondii  infected ifn-deficient mice ) to uninfected liver tissue , and then preparing cdna . real - time pcr primer and probe sequences were previously described ( 22 ) , supplied by daniel douek ( nih vaccine research center , bethesda , md ) , or designed using primer express software ( perkinelmer ) . except for p30 p30-forward tttccgaaggcagtgagacg , p30-reverse ggatccgatgccatagcg , p30-probe ttgccgcgcccacactgatg ) ; ifn--forward cattgaaagcctagaaagtctgaataac , ifn--reverse tggctctgcaggattttcatg , ifn--probe tcaccatccttttgccagttcctccag ; tnf--forward catcttctcaaaattcgagtgacaa , tnf--reverse tgggagtagacaaggtacaaccc , tnf--probe cacgtcgtagcaaaccaccaagtgga ; il-10-forward gaagaccctcaggatgcgg , il-10-reverse acctgctccactg - ccttgct , il-10-probe tgaggcgctgtcatcgattt - ctccc ; inos - forward cagctgggctgtacaaaccttc , inos - reverse cattggaagtgaagcgtttcg , inos - probe cgggcagcctgtgagacctttga ; 2m - forward tgaccggcttgtatgctatc , 2m - reverse cagtgtgagccaggatatag , 2m - probe tatactcacgccacccaccggagaa . fibrin levels within tissue samples were quantified essentially as described previously ( 23 , 24 ) . tissue was harvested from anticoagulated mice ( 500 u heparin , intravenously ) , weighed , and stored at 70c . samples were then homogenized in 10 mm sodium phosphate ( ph 7.5 ) containing 5 mm edta , 100 mm -aminocaproic acid , 10 u / ml aprotinin , 10 u / ml heparin , and 2 mm phenylmethanesulfonyl fluoride . after overnight rocking at 4c , insoluble fibrin was pelleted ( 10,000 g , 10 min ) , washed ( 10 mm sodium phosphate , 5 mm edta ) , extracted twice with 3 m urea at 37c , and then solubilized in reducing sds - page buffer at 65c . fibrin levels were then quantified by western blotting , using biotinylated anti- fibrin mab 350 ( 2 ng / ml ; american diagnostica , inc . ) followed by rabbit anti - biotin ( 1:20,000 ; dakocytomation ) , anti rabbit horseradish peroxidase polymer ( 1:40 ; dakocytomation ) , and chemiluminescent detection ( santa cruz biotechnology , inc . ) . to generate fibrin for standard curves , purified mouse fibrinogen ( sigma - aldrich ) statistical analyses were performed using the program instat 3.0 ( graphpad software ) , using either mann - whitney or student 's t tests , as appropriate . fibrinogen - deficient mice ( reference 20 ; backcrossed seven generations to c57bl/6 mice ) were generously supplied by jay degen ( children 's hospital medical center , cincinnati , oh ) and were bred at the trudeau institute animal breeding facility . animals were housed in a specific pathogen - free facility and cared for according to the trudeau institute animal care and use committee guidelines . t. gondii strain me49 was originally provided by jack remington ( palo alto medical foundation , palo alto , ca ) . me49 cysts were obtained from brains of chronically infected c57bl/6 mice , and infections were initiated by peroral administration of 10 cysts in 0.1 ml of diluted brain suspension using a 19-gauge gavage needle . , 2 mg / l warfarin ( 3-(-acetonylbenzyl)-4-hydroxy - coumarin ; sigma - aldrich ) was added to drinking water and replenished every 48 h. the anti ifn- protein levels in sera were determined by sandwich elisa ( bd biosciences ) . nitric oxide levels in plasma were determined as described ( 21 ) , except that plasma was diluted fourfold in water and filtered ( 10000 mwco ; amicon ) before analysis . il-10 , inducible nitric oxide synthase ( inos ) , and the t. gondii p30 gene were measured by real - time pcr ( perkinelmer ) and normalized to levels of -2-microglobulin ( 2 m ) . for t. gondii , standard curves were prepared by adding known numbers of me49 tachyzoites ( obtained from peritoneal exudate fluid of t. gondii  infected ifn-deficient mice ) to uninfected liver tissue , and then preparing cdna . real - time pcr primer and probe sequences were previously described ( 22 ) , supplied by daniel douek ( nih vaccine research center , bethesda , md ) , or designed using primer express software ( perkinelmer ) . except for p30 p30-forward tttccgaaggcagtgagacg , p30-reverse ggatccgatgccatagcg , p30-probe ttgccgcgcccacactgatg ) ; ifn--forward cattgaaagcctagaaagtctgaataac , ifn--reverse tggctctgcaggattttcatg , ifn--probe tcaccatccttttgccagttcctccag ; tnf--forward catcttctcaaaattcgagtgacaa , tnf--reverse tgggagtagacaaggtacaaccc , tnf--probe cacgtcgtagcaaaccaccaagtgga ; il-10-forward gaagaccctcaggatgcgg , il-10-reverse acctgctccactg - ccttgct , il-10-probe tgaggcgctgtcatcgattt - ctccc ; inos - forward cagctgggctgtacaaaccttc , inos - reverse cattggaagtgaagcgtttcg , inos - probe cgggcagcctgtgagacctttga ; 2m - forward tgaccggcttgtatgctatc , 2m - reverse cagtgtgagccaggatatag , 2m - probe tatactcacgccacccaccggagaa . fibrin levels within tissue samples were quantified essentially as described previously ( 23 , 24 ) . tissue was harvested from anticoagulated mice ( 500 u heparin , intravenously ) , weighed , and stored at 70c . samples were then homogenized in 10 mm sodium phosphate ( ph 7.5 ) containing 5 mm edta , 100 mm -aminocaproic acid , 10 u / ml aprotinin , 10 u / ml heparin , and 2 mm phenylmethanesulfonyl fluoride . after overnight rocking at 4c , insoluble fibrin was pelleted ( 10,000 g , 10 min ) , washed ( 10 mm sodium phosphate , 5 mm edta ) , extracted twice with 3 m urea at 37c , and then solubilized in reducing sds - page buffer at 65c . fibrin levels were then quantified by western blotting , using biotinylated anti- fibrin mab 350 ( 2 ng / ml ; american diagnostica , inc . ) followed by rabbit anti - biotin ( 1:20,000 ; dakocytomation ) , anti rabbit horseradish peroxidase polymer ( 1:40 ; dakocytomation ) , and chemiluminescent detection ( santa cruz biotechnology , inc . ) . to generate fibrin for standard curves , purified mouse fibrinogen ( sigma - aldrich ) statistical analyses were performed using the program instat 3.0 ( graphpad software ) , using either mann - whitney or student 's t tests , as appropriate . upon peroral infection with 10 cysts of avirulent strain me49 t. gondii , c57bl/6 mice mount a prototypical type 1 immune response , characterized by robust ifn- production , which soon contains parasite replication ( fig . 1   ; references 19 and 25 ) . however , as reported for many other conditions that elicit type 1 immunity ( 113 ) , we detected markedly elevated levels of fibrin in t. gondii  infected mice ( fig . fibrin deposition within hepatic and intestinal tissues peaked at day 8 after infection , correlating kinetically with peaks in both parasite burdens and hallmarks of type 1 immunity ( fig . 1 , and unpublished data ) . wild - type c57bl/6 mice were infected with t. gondii ( 10 strain me49 cysts , peroral ) . on the indicated days , we measured parasite burdens within liver tissue ( a ) , ifn- protein levels in sera ( b ) , and fibrin deposition within liver tissue ( c ) dashed lines indicate levels of detection , and open circles depict points below that level . t. gondii infection stimulated significant increases in liver fibrin deposition ( p < 0.01 ) , peaking at day 8 d after infection , and correlating with peak parasite burdens and ifn- levels . to evaluate the function of this infection - stimulated coagulative response , we treated mice with warfarin , an orally available pharmaceutical used clinically for the long - term anticoagulation of thrombosis - prone humans . supplementing drinking water with 2 mg / l warfarin did not impair the growth or survival of uninfected mice , but was clinically effective , as it suppressed t. gondii  stimulated fibrin deposition to below the detection limit of our assay ( unpublished data ) . strikingly , warfarin - treated mice acutely succumbed to t. gondii infection , dying around the time of the peak coagulative response of untreated animals ( fig . 2    a ) . that finding suggested that coagulation might function protectively during t. gondii infection . anticoagulated mice succumbed to t. gondii infection significantly earlier than did untreated mice ( p < 0.01 ; n = 5 mice per group ) . ( b ) fibrinogen - deficient ( fib ko ) mice and littermate control fibrinogen - heterozygous ( fib het ) were infected with t. gondii . fibrinogen - deficient mice succumbed to t. gondii infection significantly earlier than did littermate control mice ( p < 0.01 ; n = 5 mice per group ) . similar results to those shown in a and b were independently replicated in two subsequent experiments . to specifically evaluate protective roles for fibrin during t. gondii infection , we examined gene - targeted fibrinogen - deficient mice ( 20 ) , which can not produce fibrin . we found that fibrinogen - deficient mice also succumbed acutely to t. gondii infection ( fig . 1 ) , and acute mortality in both warfarin - treated and fibrinogen - deficient mice ( fig . 2 ) , we conclude that fibrin performs a critical protective function during t. gondii infection . fibrin may protect against infection - triggered mortality via a number of plausible mechanisms . as levels of fibrin and parasitemia correlated ( fig . 1 ) , we considered the possibility that coagulation might function to physically limit parasite dissemination ( 14 ) . however , parasite numbers within liver , intestine , peyer 's patch , spleen , and brain were not significantly increased in fibrinogen - deficient mice ( fig . fibrinogen - deficient ( white bars ) and littermate control fibrinogen - heterozygous mice ( black bars ) were infected with t. gondii . parasite numbers ( p30 mrna in liver ) , ifn- ( protein in sera , mrna in liver ) , nitric oxide ( nitrite / nitrate in sera ) , inducible nitric oxide synthase ( inos , mrna in liver ) , il-10 ( mrna in liver ) , and tnf- ( mrna in liver ) were assayed on days 4 and 8 after infection . bars depict average  sd of 8 mice per group ( day 0 ) , 4 mice per group ( day 4 ) , or 14 to 16 mice per group ( day 8) . aberrant levels of il-12 , ifn- , tnf- , il-10 , nitric oxide , or neutrophils can impair survival of t. gondii - infected mice ( 19 ) . however , using elisa , real - time pcr , and/or histologic assays , we never observed significant differences in levels of those mediators when comparing t. gondii - infected fibrinogen - deficient and littermate control mice ( fig . 3 , and unpublished data ) . thus , fibrin probably does not function protectively during t. gondii infection by influencing levels of critical immune mediators . in experimental models , acute mortality during t. gondii infection can result from either a failure to control parasite numbers , or excessive immunopathology ( 19 ) . during the course of our studies we noted exacerbated gross pathology in liver tissue of t. gondii  infected fibrinogen - deficient mice ( fig . microscopic examination revealed intense infiltration of mixed inflammatory cells and areas of hepatocellular necrosis in livers of t. gondii  infected control mice ( fig . in fibrinogen - deficient mice , infiltrates tended to be more diffuse , and necrosis was more extensive and frequently associated with hemorrhage ( fig . 4 d ) . exacerbated hepatic pathology in t. gondii - infected , fibrin - deficient mice . representative liver lobes harvested at day 8 after t. gondii infection of littermate control fibrinogen - heterozygous ( a ) and fibrinogen - deficient ( b ) mice . hemorrhagic foci are evident in livers of t. gondii  infected fibrinogen - deficient mice . ( c and d ) micrographs ( 200 ) depicting hematoxylin and eosin stained tissue samples isolated from t. gondii  infected mice . note that areas of hepatocellular necrosis , which are characteristic of t. gondii infection in fibrinogen - sufficient mice ( arrow in c ) , are largely replaced by hemorrhagic pathology in fibrinogen - deficient mice ( arrow in d ) . to quantitatively assess whether infection - stimulated fibrin formation protects against hemorrhagic events , we evaluated hematologic parameters in t. gondii  infected mice ( fig . 5 )   . in wild - type mice , we observed significant reductions in hematocrits , red cell numbers , and hemoglobin levels during the course of t. gondii infection , first evident around day 8 after infection and peaking around day 10 ( fig . 5 a , and unpublished data ) . compared with control heterozygous mice , t. gondii  infected fibrinogen - deficient mice displayed significantly greater reductions in hematocrits , red cell counts , and hemoglobin levels at day 8 after infection ( all p < 0.05 ; fig . treatment of wild - type mice with warfarin similarly exacerbated infection - associated red cell loss ( unpublished data ) . evaluation of peripheral blood smears revealed elevated numbers of reticulocytes , suggesting that red cell loss likely reflected infection - stimulated hemorrhage , rather than decreased erythropoiesis . together , our histologic and hematologic observations indicate that coagulation protects against hemorrhage during t. gondii infection . ( a ) wild - type c57bl/6 mice were infected with t. gondii . on the indicated days , blood was collected and hematocrits , red cell numbers ( unpublished data ) , and hemoglobin levels ( unpublished data ) were determined using a coulter counter ( beckman coulter ) . compared with uninfected mice , each of those parameters were significantly reduced by day 10 after infection ( p < 0.001 ; n = 5 mice per group ) . ( b ) fibrinogen - deficient and littermate control heterozygous mice were infected with t. gondii or were sham infected . on day 8 after infection , hematocrits were significantly reduced in t. gondii  infected control mice ( * p < 0.02 ; n = 5 mice per group ) . compared with infected control mice , t. gondii  infected fibrinogen - deficient mice displayed even greater hematocrit reductions ( * * p < 0.005 ; n = 5 mice per group ) . ( c ) wild - type c57bl/6 and c57bl/6-backcrossed ifn-deficient mice were infected with t. gondii . 8 d later , hematocrits were significantly reduced in t. gondii  infected wild - type control mice ( * p < 0.005 ; n = 5 mice per group ) , but not in the ifn-deficient mice . ( d ) wild - type c57bl/6 received a single injection ( 1 mg , intraperitoneal ) of control rat igg1 mab or anti  ifn- mab ( ifn ) . the next day , mice were sham infected or infected with t. gondii . 8 d later , hematocrits were significantly reduced in t. gondii  infected control mab - treated mice ( * p < 0.008 ; n = 5 mice per group ) , but not in the anti - ifn-treated mice . ( e ) fibrinogen - deficient and littermate control mice received control rat igg1 mab or anti - ifn- mab ( 1 mg , intraperitoneal ) , and were infected with t. gondii the following day . anti - ifn- mab treatment significantly protected against hematocrit reductions ( * p < 0.01 ; n = 4 mice per group ) . notably , the hematocrits of anti - ifn- mab - treated fibrinogen - deficient and littermate control mice were not significantly different . similar results to those shown were replicated in two ( a ) or more ( b  e ) independent experiments . t. gondii infects all nucleated cells ( 18 ) , and thus has the potential to directly damage vascular cells , thereby causing hemorrhage and activating a coagulative response . however , parasite - stimulated immunopathology could also prompt hemorrhage leading to coagulation , as levels of ifn- and its potentially destructive downstream product , nitric oxide , both peak around the time of the acute death of coagulation - impaired t. gondii  infected animals ( fig . 1 , and unpublished data ) . notably , ifn- also up - regulates cellular expression of procoagulant molecules ( 2628 ) , thereby potentially amplifying fibrin production . to decisively evaluate roles for ifn- in t. gondii  stimulated hemorrhagic pathology , we infected gene - targeted ifn-deficient mice . prior studies established that ifn- is a major mediator of resistance to t. gondii , as its neutralization leads to increased parasite burdens and acute mortality ( 25 ) . indeed , compared with wild - type mice infected in parallel , we found that t. gondii parasite numbers were elevated more than 1,000-fold in ifn-deficient mice at day 8 after infection ( p < 0.008 ; n = 5 ; wild - type 77  36 parasites / mg liver tissue , ifn-deficient = 79,000  52,000 parasites / mg liver tissue ) . nevertheless , hematocrits , red cell counts , and hemoglobin levels were not significantly reduced in t. gondii  infected ifn-deficient mice ( fig we observed similar results upon depletion of ifn- in wild - type c57bl/6 mice using a specific neutralizing mab ( fig . 5 d ) . as parasite burdens were elevated , but red cell loss was not , we conclude that parasites do not directly cause significant hemorrhage . rather , our data suggest that fibrin functions to suppress hemorrhagic pathology caused by ifn- , a product of type 1 immunity . to formally establish that fibrin functions to suppress ifn-stimulated hemorrhagic pathology , we evaluated ifn-depleted , t. gondii  infected , fibrinogen - deficient mice . we found that neutralization of ifn- in fibrinogen - deficient mice suppressed the t. gondii  stimulated gross liver pathology ( unpublished data ) and hematocrit reductions ( fig . importantly , hematocrits in ifn-depleted , t. gondii  infected , fibrinogen - deficient , and littermate control mice were not significantly different , establishing that the exacerbated hemorrhagic pathology observed in fibrinogen - deficient mice , like the lesser pathology observed in wild - type mice , is caused by ifn-. numerous prior studies concluded that immune - associated fibrin deposition exacerbates inflammation and pathology . studies using factors isolated from snake venoms that transiently generate a fibrinogen - deficient state suggested pathological roles for fibrin in animals models of glomerulonephritis ( 4 ) , arthritis ( 7 ) , encephalomyelitis ( 9 , 10 ) , and transplant rejection ( 12 ) . studies using mice with impaired fibrin degrading capacities ( i.e. , gene - targeted plasminogen- or plasminogen activator - deficient mice ) confirmed such pathological roles for fibrin during glomerulonephritis and arthritis ( 68 ) . studies using fibrinogen - deficient mice revealed pathological roles for fibrin(ogen ) during atherosclerosis ( 29 ) , and demonstrated that fibrin can up - regulate expression of inflammatory cytokines and chemokines in vivo ( 30 ) . finally , studies using anticoagulated or congenitally fibrinogen - deficient subjects established that fibrin also has inflammatory properties in humans ( 2 , 3 ) . in contrast to those prior reports , our studies demonstrate that coagulation can also function protectively during immunity . specifically , we found that fibrin can prevent infection - stimulated blood loss , thereby performing a protective function that is essential for survival . remarkably , fibrin does not simply protect against vascular damage caused directly by the infectious agent , but rather , protects against hemorrhage evoked by ifn- , a critical mediator of type 1 immunity . notably , ifn- also stimulates expression of genes that favor and/or stabilize fibrin deposition ( 2628 ) , suggesting that type 1 immunity may have evolved means to locally up - regulate coagulant activity , thereby preemptively protecting against its own destructive power . our observations indicate that the pathological potential of immune - associated coagulation is counter - balanced by a critical protective function . such findings help to explain why type 1 immunity frequently elicits fibrin deposition ( 113 ) , and suggest that clinicians should be cautious in ascribing pathological assessments to immune - associated fibrin deposits . while the inflammatory activities of fibrin and other coagulant products may function pathologically in certain contexts ( 116 ) , immune - associated coagulative responses notably , our data do not exclude the possibility that fibrin may also function protectively through direct effects on immunity and/or inflammation . in comparison to littermate control mice , there were no significant differences in pathogen dissemination or levels of immune mediators in t. gondii  infected fibrinogen - deficient mice ( fig . however , the robust type 1 immune response that accompanies t. gondii infection may have masked immune - related impacts of fibrin - deficiency . indeed , while own prior work demonstrated that thrombin can stimulate fibrin - dependent secretion of inflammatory cytokines / chemokines in vivo ( 30 ) , subsequent studies found that endotoxin- or t cell  stimulated secretion of those same cytokines / chemokines is fibrin - independent ( unpublished data ) . thus , we suspect that infection - stimulated immunity may well mask fibrin 's inflammatory activities during t. gondii infection . our data suggest that fibrin - unrelated coagulation deficiencies , whether genetic or pharmacologic , may also exacerbate immunopathology . as such we are also working to define precisely how ifn- evokes pathology necessitating a protective coagulative response . we anticipate that those studies may suggest novel therapies for pathologies characterized by extensive fibrin deposition , including transplantation and many forms of autoimmunity , and may also impact treatments for the numerous virulent human pathogens that elicit deleterious coagulative and/or hemorrhagic responses .
fibrin , a product of the blood coagulation cascade , accompanies many type 1 immune responses , including delayed - type hypersensitivity , autoimmunity , and graft rejection . in those settings 
 , fibrin is thought to exacerbate inflammation and disease . here 
 , we evaluate roles for coagulation during infection with toxoplasma gondii , a pathogen whose control requires robust type 1 immunity . 
 we establish that fibrin prevents infection - stimulated blood loss , thereby performing a protective function that is essential for survival . 
 remarkably , fibrin does not simply protect against vascular damage caused directly by the infectious agent , but rather , protects against hemorrhage evoked by interferon- , a critical mediator of type 1 immunity . 
 this finding , to our knowledge , is the first to document a beneficial role for coagulation during type 1 immunity , and suggests that fibrin deposition protects host tissue from collateral damage caused by the immune system as it combats infection .