
 francisella 
 tularensis is   a gram - negative facultative intracellular bacterium and the 
 causative agent of tularemia , a systemic infection of many mammals including 
 humans .  
left untreated , the virulent type a 
 subspecies of f. tularensis routinely 
 caused lethal infection in people particularly after aerosol exposure to the 
 pathogen ; as few as 10 virulent type a bacilli can initiate severe disease .
consequently , f. 
 tularensis is considered a category a biological warfare agent . despite its clinical and biosecurity 
 importance
, the molecular basis for the immunopathogenesis of f. tularensis infection , 
 particularly when initiated through the respiratory tract , remains largely 
 unknown .
lymphotoxin- ( lt ) is a member of the tumor 
 necrosis factor ( tnf ) superfamily of cytokines and has two distinct roles : as a membrane - bound heterotrimer in 
 combination with lt , it 
 binds the lt 
 receptor and is critical in the development and maintenance of organized secondary 
 lymphoid organs , and as a soluble homotrimer , it   signals through the tnf receptor pathway and leads 
 to activation of various inflammatory cytokines and chemokines [ 3 , 4 ] .
indeed , lts ( lt and lt ) , together with tnf and light ( lt - related inducible ligand that competes for glycoprotein d binding to herpesvirus 
 entry mediator on t cells ) , form an integrated signaling network which 
 is important for the regulation of both innate and adaptive immune responses . in this
regard , lt has been implicated in the host defense 
 against several different bacterial , viral , and parasitic pathogens ( reviewed in ) .
for instance , several studies 
 have shown that after infection with mycobacterium tuberculosis ,   mice genetically defective in lt ( lt/ mice ) harbour increased bacterial 
 burdens and exhibited a shorter median time to death when compared to lt+/+ mice [ 57 ] .
similarly , mice deficient in 
 lt , lt , and the lt receptor ( ltr ) are more susceptible to listeria 
 monocytogenes infection than wild - type mice [ 6 , 7 ] . given that lt is important in the control of these 
 intracellular bacterial pathogens , in the present study we sought to determine 
 whether it also plays a role in host defense against low - dose aerosol infection 
 with a virulent type a strain of f. 
 tularensis .
eight- to twelve - week old , 
 age - matched b6.129s2-lt/j ( lt/ ) , and wild - type c57bl/6j ( lt+/+ ) mice were used in this study .
the 
 foundation breeding pairs of lt/ mice were purchased from 
 jackson laboratories ( bar harbor , me , usa ) .
mice were bred and housed 
 under specific - pathogen - free conditions in a federally licensed animal 
 biosafety level-3 facility and given free access to sterile water and certified 
 mouse chow .
the animals were maintained and used in accordance with the 
 recommendations of the canadian council on animal care guide to the care and 
 use of experimental animals .
stocks of type a f. tularensis strain fsc33/snmf , 
 originally isolated from a squirrel in georgia , usa . for low - dose aerosol exposure , thawed f. tularensis stocks were diluted in mueller hinton broth 
 containing 20% ( v / v ) glycerol to maintain infectivity at the high - relative 
 humidity employed .
aerosols of f. 
 tularensis strains were generated with a lovelace nebulizer operating at a 
 pressure of 40 p.s.i .
to produce particles in the 46 m range required for inhalation and retention 
 in the alveoli .
mice were exposed to these aerosols for 7 
 minutes ( inhaled dose of ~10 organisms ) using a customized commercial nose only 
 exposure apparatus ( in - tox products , albuquerque , nm ) resulting in the 
 implantation of 1020 organisms into the lungs .
groups of five mice of each strain 
 were sacrificed at 2 and 4 days after inoculation ( dpi ) by co2 asphyxiation . 

the 
 phenotype of the lt/ mice was confirmed by visual 
 inspection at necropsy to confirm the absence of peripheral lymph nodes .
the 
 lungs and spleens were removed aseptically , cut into small pieces , and then 
 homogenized using an aerosol - proof homogenizer for quantitative bacteriology or 
 fixed immediately by immersion in 10% 
 neutral buffered formalin for histopathology .
for quantitative 
 bacteriology , ten - fold serial dilutions of the tissue homogenates were plated 
 on cysteine heart agar supplemented with 1% ( w / v ) hemoglobin and sulfamethoxazole 
 and trimethoprim .
the tissues were processed by standard 
 paraffin embedding methods ( department of pathology and laboratory medicine , university of ottawa , 
 ottawa , ontario ) . 

sections were cut 4 m thick , stained with haematoxylin - eosin ( he ) 
 and examined by light microscopy . in some experiments ,
the 
 lungs were lavaged with five 1.0-ml aliquots of pbs supplemented with 3 mm edta 
 , and the total lavage cell numbers 
 were counted on a haemocytometer , and differential cell counts were carried out 
 on cytospin preparations stained with hema3 stain set ( fisher scientific , 
 middletown , va , usa ) .
the bronchoalveolar lavage ( bal ) 
 fluid was centrifuged at 3000 xg for 7 minutes , supernatants were removed , sterile - filtered , and 
 stored at 80c .
serum 
 and bal levels of cytokines and 
 chemokines were determined using beadlyte mouse 21-plex cytokine detection 
 system ( upstate , temecuta , calif , usa ) on a luminex 100is system ( luminex , 
 austin , tex , usa ) .
undiluted bal and 1 : 2 diluted serum samples ( 50 l ) were analyzed as specified by the manufacturer ( http://www.millipore.com/userguides/tech1/ proto_mpxmcyto-70k ) . 

the analysis was done in duplicate , and the cytokine / chemokine concentrations 
 were calculated against the standards using beadview software ( ver 1.03 , 
 upstate ) .
all data are presented as mean  standard deviation ( sd ) 
 for each group .
differences between groups were analyzed by two - way anova followed 
 by the bonferroni multiple comparison test ( graphpad prism 4.0 , graphpad software , inc .
initially , a total of 22 lt/ and 14 lt+/+ mice were challenged by 
 low - dose aerosol with virulent type a f. tularensis and their survival monitored . 
 with the exception of two lt/ mice and one lt+/+ mouse , all mice succumbed to 
 infection between day 
 4 and 7 with a median time to death of 5 days ( range 46 days for lt/ mice and 57 days for lt+/+ mice , p > .05 by 
 kaplan - meier survival analysis ) ( figure 1 ) , indicating that lt/ mice are no more susceptible to 
 low - dose aerosol challenge with this strain of the pathogen than control lt+/+ mice .
it has been previously 
 reported that increased susceptibility of certain immunocompromised mice to 
 intradermal infection with the live vaccine strain ( lvs ) of f. tularensis   and oral type a f. tularensis infection   is only apparent when using a 
 very high inoculum . to 
 examine the possible effect of inoculum size on the need for lt expression to control respiratory infection 
 with type
a f. tularensis , groups of lt/ and lt+/+ mice were intranasally 
 challenged with 10 , 100 , and 1000 cfu type a f. tularensis and their survival monitored .
this study revealed 
 that the ld100 of type a f. 
 tularensis for lt/ and lt+/+ mice was comparable in that all mice 
 died of the infection by dpi 5 ( data not shown ) .
these results indicate that lt does not appear to play a significant 
 role in determining the clinical outcome of respiratory infection with various 
 doses of type a f. tularensis in mice . since type a strains of f. 
 tularensis are extremely virulent for mice even at the minimum challenge 
 dose , it remained possible that 
 subtle effects of lt expression were overlooked by the above 
 relatively crude survival experiments .
therefore , we next examined whether lt contributes to the control of f. 
 tularensis replication and systemic dissemination by comparing the 
 bacterial burdens in the lungs and spleens of lt/ and lt+/+ mice at dpi 2 and 4 following aerosol challenge ( figure 2 ) .
there was no difference in the bacterial burdens 
 in the lungs , the primary site of infection , between lt/ and lt+/+ mice at dpi 2 .
however , the 
 bacterial burdens in the spleens   of lt/ mice were about 1 to 1.5 log , but 
 not statistically significant , lower than those in lt+/+ mice at this time point . by dpi 4 ,
lt/ mice had approximately 10-fold more 
 bacteria in their lungs than did lt+/+ mice ( p < .01 ) , and the bacterial 
 burdens in the spleens of lt/ mice were also higher , although not 
 statistically significant , than those in lt+/+ mice .
hence , 
 these data imply that although lt is not sufficient to control virulent f. tularensis infection , it may play a minor 
 role in the initial dissemination of f. 
 tularensis from the lung to spleen and subsequent multiplication of the 
 pathogen in the lungs and spleen .
histopathologically , 
 both lt/ and lt+/+ mice showed moderate 
 inflammatory infiltrations in the livers and spleens and mild , focal 
 bronchopneumonia at dpi 2 , and by dpi 4 moderately severe necrotic hepatitis , 
 lymphoid follicle destruction in the spleen , and bronchopneumonia . 

however , as would be expected from the quantitative 
 bacteriology and survival data , no overt differences in tissue histopathology 
 or blood clinical chemistry ( data not shown ) were observed between lt/ and lt+/+ mice following aerosol exposure 
 to type a f. tularensis . 

early pulmonary recruitment of inflammatory cells and local and 
 systemic production of proinflammatory cytokines are considered important 
 characteristics of innate host responses against respiratory infections including f. tularensis   .
previous studies have shown that 
 respiratory infection of mice with type a and attenuated live vaccine strain of f. tularensis upregulates a number of 
 proinflammatory cytokines , which play important roles in host defense against f. 
 tularensis infection [ 1315 ] .
therefore , we determined total and differential 
 leukocyte counts   in the bal fluid to identify the inflammatory 
 cell influx into the lungs on dpi 2 and 4 . as can be seen in table 1
, low - dose 
 aerosol infection of mice with type a f. 
 tularensis induced no significant difference in either the total 
 cell number or the composition of cell 
 populations ( macrophage , neutrophil , and lymphocyte ) in the lavage fluids of lt+/+ and lt 
 / mice with the exception of a small but not significant 
 increase in lymphocytes in lt/ mice on both dpi 2 and 4 .
this is likely due to a higher baseline number of lymphocytes in the 
 lungs of lt/ mice   rather than a result of f. 
 tularensis infection . to 
 assess whether lt deficiency alters f. 
 tularensis - induced cytokine responses following aerosol challenge with the 
 pathogen , levels of a panel of 21 cytokines and chemokines , including ifn- , il-6 , kc , and mcp-1 , in the bal and the sera of lt/ and lt+/+ mice killed at dpi 2 and 4 were measured
overall , there was little change in the levels of 
 the majority of assayed cytokines in either the bal or the sera at dpi 2 or 4 in either mouse strain ( data not shown ) . however , f. tularensis infection resulted in a substantial 
 increase of mcp-1 and a moderate increase of kc   in bal fluid at dpi 2 ( figure 3(b ) ) and a substantial increase of ifn- , il-6 , kc , and mcp-1 in both bal and sera at dpi 4 ( figures 3(a ) and 3(b ) ) , but again no differences were 
 observed between the two mouse strains with the exception of il-6 , which was 
 significantly higher in bal fluid 
 of lt/ mice than that of lt+/+ mice ( figure 3(b ) , p < .05 ) .
recent studies have established that , in addition to its role in 
 the organogenesis of secondary lymphoid organs , lt plays an important role in host defenses against microbial 
 infections ( reviewed in ) .
however , the role of lt in host defenses against infection appears to be complex and 
 varies from pathogen to pathogen . in this study
, we utilized lt/ mice and performed some preliminary studies to examine the 
 potential role of lt in the host resistance to respiratory infection with virulent 
 type a f. tularensis .
our 
 results showed that lt/ mice had lower bacterial 
 burdens in their spleens on dpi 2 and higher bacterial burdens in their lungs 
 on dpi 4 when compared to lt+/+ mice but showed no overt 
 differences in clinical outcome , tissue damage , or host immune responses to the 
 infection .
although our data suggest that lt exerts some subtle influence over the 
 course of aerosol - initiated tularemia , its mechanism of action remains unknown . 

the possible reasons are as 
 follows : ( 1 ) lt- is not crucial in host defense against 
 this pathogen ; ( 2 ) the role of lt- can be compensated by other 
 cytokines / chemokines in this infection model ; and ( 3 ) the pathogen is too 
 virulent and even immunocompetent hosts have little resistance to the 
 infection . in this regard
, we have previously shown that a number of 
 immunodeficient mice show similar clinical outcome to the immunocompetent mice 
 .
the lower bacterial burdens in the spleen of lt/ mice at dpi 2 could be 
 explained by a delay / reduction in the dissemination of bacteria from the lung 
 since lt/ mice lack tracheobronchial 
 lymph nodes which normally are the major draining lymph nodes for the lung . 

once f. tularensis reached the spleens , however , bacteria quickly 
 multiplied and by dpi 4 , bacterial burdens were no longer significantly 
 different in the spleens of lt/ and lt+/+ mice .
in fact , lt/ mice actually seem to have 
 slightly higher burdens in their spleens at this time point despite starting on 
 dpi 2 with substantially lower bacterial burdens ( see figure 2 ) . also by this 
 time , lt/ mice harbored significantly 
 more bacteria in their lungs than lt+/+ mice ( p < .01 ) , 
 suggesting that lt may play a role in host 
 defense against f. tularensis infection which is distinct from its role 
 in lymphoid organogenesis . alternatively , the lack of draining lymph nodes in 
 lt/ mice may simply cause a 
 delay in antigen presentation leading to a delayed or otherwise impaired 
 antibacterial host response . in summary , following a low - dose aerosol 
 infection with the highly virulent type a strain of f. tularensis , lt/ mice consistently harbored approximately 10-fold 
 fewer bacteria in their spleens at dpi 2- and 10-fold more bacteria in their 
 lungs at dpi 4 than lt+/+ mice . however , the mortality and median time to death were indistinguishable 
 between the two mouse 
 strains .
in addition , the inflammatory responses to the infection , 
 as reflected by the cytokine 
 levels and leukocyte influx in bal fluid and histopathological analysis , were generally similar between lt/ and lt+/+ mice .
these data suggest that although lt does not contribute significantly to 
 the resistance and host responses of mice to airborne type a f. tularensis infection ; it does play a subtle role in the multiplication / dissemination of f. 
 tularensis .
 this study examined the role of lymphotoxin ( lt)- in host defense against airborne infection with francisella tularensis , a gram - negative facultative intracellular bacterium and the causative agent of tularemia . following a low - dose aerosol infection with the highly virulent type a strain of f. tularensis , mice deficient in lt ( lt/ ) consistently harbored approximately 10-fold fewer bacteria in their spleens at day 2 and 10-fold more bacteria in their lungs at day 4 than lt+/+ mice . 
 however , the mortality and median time to death were indistinguishable between the two mouse strains . 
 in addition , the inflammatory responses to the infection , as reflected by the cytokine levels and leukocyte influx in the bronchoalveolar lavage fluid and histopathological analysis , were generally similar between lt/ and lt+/+ mice . 
 these data suggest that although lt does not contribute significantly to the resistance and host responses of mice to airborne type a f. tularensis infection , it does play a subtle role in the multiplication / dissemination of f. tularensis . 