| Goals:This study examined the role of lymphotoxin(LT)-αin host defense against airborne infection and oral infection with Francisella tularensis.At the same time,we examined the susceptibility of mice to intragastric inoculation with virulent type A F. tularensis and characterized the course of infection and the associated host responses. And we examined the effect of virulent strains of F.tularensis infection on the thymus and thymocytes and the potential mechanisms involved.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.Incidental or deliberate contamination of drinking water and the food supply could be an alternative means to initiate infection with this pathogen.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 and intragastric tract,remain largely unknown. Lymphotoxin-α(LTα) 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 LTβ,it binds the LTβreceptor and is critical in the development and maintenance of organized secondary lymphoid organs.In this regard,LTαhas been implicated in the host defense against several different bacterial,viral and parasitic pathogens.Given that LTαis 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 and oral infection with a virulent type A strain ofF.tularensis.Methods:1 Mice and Inoculation C57BL/6,BALB/c,Transgenic mice B6.129S2-Ltαtm1 Dch/J (LTα-/-),B6.129S7-Ifngtml AgtNmg(IFN-γ-/- and B6.129S-Tnfrsflatm1 Imx Tnfrsfibtm1 Imx (TNFR1R2-/-. Type A F.tularensis strain FSC033/snMF(strain FSC033) was originally isolated from a squirrel in Georgia(United States).For oral inoculation, thawed F.tularensis stocks were diluted in phosphate-buffered saline,and 0.5 ml of the inoculum was given to each mouse by using an 18-gauge gavage needle.For aerosol exposure,aerosols of F.tularensis strains were generated with a Lovelace nebulizer operating at a pressure of 40 psi.Mice were exposed to these aerosols for 7 min using a customized commercial nose-only exposure apparatus resulting in the implantation of 10-20 organisms into the lungs.2 The role of LT-αin host defense against aerosol infection with Francisella tularensis Groups of age matched female 22 LTα-/- and 14 LTα+/+ mice were challenged by low-dose aerosol(inhaled dose of～10 organisms) with virulent type A F.tularensis and their survival and median time to death were monitored.To examine the possible effect of inoculum size on the need for LTαexpression to control respiratory infection with type A F.tularensis,groups of LTα-/- and LTα+/+ mice were intranasally challenged with 10,100,and 1,000 cfu type A F.tularensis and their survival monitored.Groups of age matched LTα-/- and LTα+/+ mice were inoculated by low-dose aerosol with type A F.tularensis,and sacrificed at 0,2 and 4 days post inoculation (dpi).The phenotype.of the LTα-/- mice was confirmed by visual inspection at necropsy to confirm the absence of peripheral lymph nodes.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,and the changes in the mean body weights and relative weights of the thymus over the course of infection were assessed.The thymus were removed and weighed in a balance with accuracy to 0.1mg.The organ index was calculated as organ weight(in grams)/body weight(in grams)<100.Blood samples and bronchoalveolar lavage(BAL) fluid were collected for the determination of serum and BAL fluid cytokine levels and for serum clinical chemistry.In some experiments, the lungs,spleens and thymus were removed,homogenized and used for quantitative bacteriology or fixed immediately by immersion in 10%neutral buffered formalin for histopathology.Single thymocyte suspensions were prepared by pressing individual thymus in cold PBS through a 75-μm pore sized Falcon disposable cell strainer.The total thymocyte number in each mouse was determined using a hemocytometer.To determine the potential role of TNF in the development of type A F. tularensis infection-associated thymus atrophy,TNFR1R2-/-mice were challenged by aerosol with low-dose type A F.tularensis.3 Clinical relevance of thymus damage and tularemia in mice We compared the clinical outcome of thymectomized C57BL/6 mice and control C57BL/6 mice following low-dose aerosol infection with type A F.tularensis,monitor the mortality and the median time to death among these two mouse strains.We isolated the thymocytes from the thymus of untreated mice and culture in RPMI-1640 Culture medium with 10%FBS in 24 wells plates.Add fresh culture medium with the corresponding numbers of F.tularensis and negative control.Incubate and observe the cells at different time points.4 The role of LT-αin host defense against oral infection with Francisella tularensis To determine the relative susceptibility of mice to oral infection with type A F.tularensis,groups of LTα-/-mice were gavaged with 104 to 106 CFU and matched LTα+/+ mice were gavaged with 104 to 108 CFU of type A F.tularensis in some separate experiments.In some experiments,mice were fasted overnight and administered 0.2 ml of 3%sodium bicarbonate to neutralize gastric acidity 10 min prior to oral inoculation.Survival of the inoculated mice was monitored for 14 days.Groups of immunocompromised mice(TNFR1R2-/-,IFN-γ-/-,LTα-/-) as well as wild-type(WT) C57BL/6,were inoculated by gavage with 5×105 CFU,and their survival rates and median times were monitored.To examine the effect of high inoculum,groups of immunocompromised mice(TNFR1R2-/-,IFN-γ-/-,LTα-/-) as well as WT control mice,were gavaged with 5×107 CFU(～50 LD50) of type A F. tularensis,and their survival rate were compared.In order to determined the bacterial burdens and examined the associated pathology in different organs of C57BL/6 mice and LTα-/- mice,C57BL/6 mice and LTα-/- mice were gavaged with 5×105 CFU of type A F.tularensis and C57BL/6 mice were gavaged with 5×107 CFU over the course of infection.The mice killed at dpi 0,2 and 3.Mesenteric lymph nodes(except LTα-/- mice),spleens,and lungs were aseptically removed and homogenized in aerosol-proof homogenizers,and the number of viable bacteria in the respective organs was determined by quantitative bacteriology.The sera were collected.The serum levels of cytokines and chemokines were determined by using the mouse multiplex cytokine detection system 2 and the mouse chemokine 5-Plex on a Luminex 100 IS system.Additional groups of mice were sacrificed and the lung and spleen was removed from each animal;fixed immediately in 10%neutral buffered formalin;and processed by standard paraffin-embedding methods to do the histopathology assay.5 Statistical analyses Data are presented as means±the standard deviation for each group.Differences in quantitative measurements were assessed by one-way or two-way analysis of variance,followed by post hoc multiple comparison tests. Differences in nonparametric data were analyzed by the Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney U test.The Fisher exact test and the chi-square test were used for comparison of categorical variables.Differences were considered significant when the P value was<0.05. 1 Survival rate after aerosol infection A total of 22 LTα-/- and 14 LTα+/+ mice were challenged by low-dose aerosol with virulent type A F.tularensis and their survival monitored.With the exception of two LTα-/- mice and one LTα+/+ mouse,all mice succumbed to infection between day 4 and 7 with a median time to death of 5 days(range 4-6 days for LTα-/- mice and 5-7 days for LTα+/+ mice,p>0.05 by Kaplan-Meier survival analysis),indicating that LTα-/- mice are no more susceptible to low-dose aerosol challenge with this strain of the pathogen than control LTα+/+ mice.To examine the possible effect of inoculum size on the need for LTαexpression to control respiratory infection with type A F.tularensis,groups of LTα-/- and LTα+/+ mice were intranasally challenged with 10,100,and 1,000 cfu type A F.tularensis and their survival monitored.This study revealed that the LD100 and LD1000 of type A F. tularensis for LTα-/- and LTα+/+ mice were comparable in that all mice died with a median time of 5 days.These results indicate that LTαdoes 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.2 Low-dose aerosol infection of mice induces severe thymus atrophy and thymocytes depletion After the Low dose aerosol infection,the thymus size of LTα+/+ mice decreased slightly at dpi 2 and drastically by dpi 4(24-48 h prior to expected death),concurrent with the decrease in the thymus size,the total number of thymocytes in the infected mice dropped by 85%at dpi 4.The Thymus weight index of LTα-/- mice decreased slightly at dpi 4 and the total number of thymocytes in the infected mice dropped by 30%.There are significant difference between two mice strain at dpi4 in Thymus weight index and the total number of thymocytes.Compared to low-dose type A F.tularensis infected conventional B6 mice at dpi 4, TNFR1R2-/-mice did not show any thymus changes.3 Bacterial burdens in different tissues after aerosol infection We examined whether LTαcontributes to the control of F.tularensis replication and systemic dissemination by comparing the bacterial burdens in the lungs and spleens of LTα-/-and LTα+/+ mice at dpi 2 and 4 following aerosol challenge.There was no.difference in the bacterial burdens in the lungs,the primary site of infection,between LTα-/- and LTα+/+ mice at dpi 2.However,the bacterial burdens in the spleens of LTα-/- mice were about 1 to 1.5 log lower than those in LTα+/+ mice at this time point.By dpi 4, LTα-/- mice had approximately 10-fold more bacteria in their lungs than did LTα+/+ mice,and the bacterial burdens in the spleens of LTα-/- mice were also higher, although not statistically significant,than those in LTα+/+ mice.We have cultured large numbers of F.tularensis consistently from the thymuses of our infected LTα+/+ mice and LTα-/- mice at dpi 4 and LTα-/- mice had approximately 10-fold more bacteria in their lungs than did LTα+/+ mice.The subtle differences in bacterial burdens were consistently observed in three independent experiments.4 Pathology,clinical chemistry,Cytokine and chemokine responses after aerosol infection Histopathologically,both LTα-/- and LTα+/+ 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 were observed between LTα-/- and LTα+/+ mice following aerosol exposure to type A F.tularensis.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.There is no significant difference in either the total cell number or the composition of cell populations(macrophage,neutrophil,and lymphocyte) in the lavage fluids of LTα+/+ and LTα-/- mice with the exception of a small but not significant increase in lymphocytes in LTα-/- mice on both dpi 2 and 4.To assess whether LTαdeficiency alters F.tularensis-induced cytokine responses following aerosol challenge with the pathogen,levels of a panel of 21 cytokines and chemokines,including IFN-γ,IL-6, KC and MCP-1,in the BAL and the sera of LTα-/- and LTα+/+ 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. However,F.tularensis infection resulted in a substantial increase of MCP-1 and a moderate increase of KC in BAL fluid at dpi 2 and a substantial increase of IFN-γ, IL-6,KC and MCP-1 in both BAL and sera at dpi 4,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 LTα-/- mice than that of LTα+/+ mice.5 Clinical relevance of thymus damage and tularemia in mice We compared the clinical outcome of thymectomized C57BL/6 mice and control C57BL/6 mice following low-dose aerosol infection with type A F.tularensis.There was no significant difference in the mortality or the median time to death among these two mouse strains.In vitro exposure of cultures of thymocytes,isolated from untreated mice,to F.tularensis failed to induce thymocyte death.6 LD50 to oral inoculation and resistant to acid stress To determine the relative susceptibility of mice to oral infection with type A F.tularensis and the effect on the LT-a,groups of 8- to-10-week-old female C57BL/6 mice and matched LTα-/- mice were gavaged with various numbers of type A F.tularensis(104 to 108 CFU and104 to 106).Survival of the inoculated mice was monitored for 14 days.Oral gavage of mice with 106 CFU of virulent type A F.tularensis resulted in slightly more than 50% mortality by day 5 after inoculation.All C57BL/6 and LTα-/- mice except one of each group that received the lowest inoculum(104 CFU) survived the infection,whereas all mice that received the highest inoculum(108 CFU) died by dpi 5.A similar susceptibility was also observed in BALB/c mice.These results suggest that the 50% lethal dose(LD50) for oral type A F.tularensis infection in mice is about 106 CFU.We find that neutralization of gastric acidity with 3%sodium bicarbonate does not increase the susceptibility of mice to the infection.Type A F.tularensis is relatively resistant to acid stress in vitro in that there was no substantial reduction in the bacterial viability of the pathogen at different pH.7 Susceptibility of immunocompromised mice to oral inoculation Groups of immunocompromised mice(TNFR1R2-/-,IFN-γ-/-,LTα-/-) as well as wild-type(WT) C57BL/6,were inoculated by gavage with 5×105 CFU,and their survival rates and median times were monitored.Immunocompromised mice displayed similar susceptibilities to oral inoculation with this dose of type AF tularensis.To examine the effect of high inoculum,those mice were gavaged with 5×107 CFU(～50 LD50) of type A F.tularensis,and their survival rate were compared.In contrast to WT mice, which showed an MTM of 5 days,mice with various immunodeficiencies had a shortened MTM(3 to 4 days).8 Bacterial burdens in different tissues after oral infection When inoculated by gavage with 5×105 CFU,small numbers of F.tularensis were cultured from the MLN,spleen,and lung of C57BL/6 mice at dpi 2.F.tularensis was not cultured from the lung at dpi 2.The bacterial numbers continued to increase significantly in the spleen at dpi 4.The bacterial numbers in lung and spleen of LTαmice are higher than C57BL/6 mice at dpi2.The bacterial numbers continued to increase significantly in the spleen at dpi 4.But there is no significant difference between those two strain of mice and between the dpi.When inoculated by gavage with 5×107 CFU,the number of bacteria in the MLN peaked at dpi 2,and then fell to undetectable levels in most of the mice by dpi 3.F.tularensis,was present in one mouse at lung at dpi 2,and was only detected occasionally in small numbers at dpi 3.The bacterial numbers continued to increase significantly in the spleen between dpi 2 and 3.9 Cytokine and chemokine responses after oral infection In the C57BL/6 mice and LTα-/- mice,the serum levels of IFN-γ,IL-6,TNF-α,IL-12,IP-10,MIG,and MCP-1 were significantly increased;the KC,MIP-1α,IL-1β,and IL-10 levels were moderately increased at dpi 2;and most of these cytokines or chemokines maintained similar levels at dpi 3 except IL-6 and MCP-1,which increased further.On the other hand,there was little or no change in the serum IL-2,IL-4,IL-5.But To most of the cytokines or chemokines,there is no significant difference between those two mice strains.Conclusions1.) Type A,F.tularensis cause severe reduction in thymus weight and destruction of thymocytes and depletion of thymocytes.2.) The 50%lethal dose(LD50) for oral type A F.tularensis infection in mice is about 106 CFU.Neutralization of gastric acidity does not increase the susceptibility of mice to the infection.3.) Immunodeficiencies display similar susceptibilities to oral inoculation with 5× 105 CFU and increase susceptibility to high dose of type A F.tularensis.4.) The gastrointestinal tract and lungs,acts primarily as a portal of entry for F. tularensis rather than as the critical site of infection.It is the disseminated rather than the pulmonary and gastrointestinal infection that kills the host.5.) LTαdoes play a subtle role in the multiplication/dissemination of F.tularensis.6.) The lack of draining lymph nodes may simply cause a delay in antigen presentation leading to a delayed or otherwise impaired antibacterial host response.7.) IFN-γmay play a divergent role in the pathogenesis of oral infection with virulent type A F.tularensis. |
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