Introduction

The 2001 anthrax attacks in Florida, New Jersey, New York and the Hart Senate building in Washington, DC, have intensified the search for new therapeutic strategies and a better understanding of the mechanisms needed for survival following exposure to anthrax. Research with animal models has shown that there are both species-specific and intra-species strain differences in the ability to survive anthrax. These studies have identified both sensitive and resistant individuals, and also point toward the presence (or absence) of cellular factors that influence an individual's response to the effects of anthrax exposure.

The pathogenesis of the disease following exposure to Bacillus anthracis spores is well understood and has been studied for several decades using a variety of animal model systems, including rodents.^{1, 2, 3} While macrophages from some strains are sensitive to the effects of anthrax lethal toxin (LeTx),⁴ the exact mechanism of action for cell death remains to be solved. The host cellular machinery is known to play a key role as demonstrated by the identification of a receptor complex for LeTx^{5, 6, 7} and downstream intracellular signaling pathways.^{8, 9, 10}

Systemic injection of anthrax LeTx in animals and exposure of rodent macrophages to LeTx in vitro are the primary model systems used to study the mechanism of action of anthrax. LeTx consists of two polypeptides, lethal factor (LF) and protective antigen (PA). PA binds to the anthrax toxin receptor complex composed of ATR/tumor endothelial marker 8 (TEM8)⁵ encoded by Anxtr1 and/or capillary morphogenesis protein 2 (CMG2)^{11, 12} encoded by Anxtr2, along with coreceptor low density lipoprotein receptor-related protein 6 (LRP6),⁷ then translocates LF into the cytosol, where it functions as a zinc metalloprotease capable of cleaving mitogen-activated protein kinase kinases.^{13, 14} LF also exhibits several macrophage-specific effects, including a cytolytic effect and induction of cytokines.^{4, 15} Species-specific differences have been reported in rodents in response to LeTx. For example, mortality after systemic administration of LeTx is measured in minutes to hours for rats like the Fischer 344 (F344),¹⁶ but in days for some strains of mice.³ There is also substantial evidence that different strains of mice respond differently to either infection by Bacillus anthracis spores or to systemic injection of anthrax LeTx, indicating that there is a genetic basis for the host response.^{3, 4, 17, 18, 19} Strain differences in sensitivity of macrophages to the lytic effects of anthrax LeTx in mice have recently been linked to sequence differences in Nalp 1b.⁸

Here, we examined whether four progenitor strains of rat and their first filial generation (F₁) offspring exhibit differences in response to anthrax LeTx. The progenitor strains were selected based on their ability to survive anthrax LeTx and by their amount of genetic diversity. By combinatorial breeding of both sensitive and resistant progenitor strains, a test population with six hybrid rats was created. In theory, the rat test population should simultaneously identify differential responses to anthrax LeTx along with determining the mode of inheritance for survival of these strains. We tested this concept by measuring the survival of the rat test population in response to acute administration of various doses of LeTx in vivo, and the ability of LeTx to lyse macrophages derived from these strains in vitro.

Results

Predicting a rat test population for studying anthrax using genomic information

The criteria for selecting the progenitor strains used to breed a rat test population depended on both their sensitivity to anthrax LeTx and their level of genome variance. We reasoned that having both sensitive and resistant progenitors would enable us to determine the mode of inheritance for survival to anthrax LeTx. We initially selected the F344 rat because it is known to be very sensitive to anthrax LeTx.^{1, 2} We then used the simple sequence length polymorphism (SSLP) genetic marker information²⁰ that is accessible through the ACP Haplotyper Tool at the Rat Genome Database (http://rgd.mcw.edu/ACPHAPLOTYPER/) to pick the three other progenitor strains. Using the ACP Haplotyper, we compared nearly 5000 genetic markers from the F344 rat with the markers of 47 other laboratory rat strains and selected the Wistar–Kyoto (WKY), Lewis (LEW) and Brown Norway (BN) rats as the other progenitors. Using these four inbred rat strains to breed the test population, we used a Strain Calculator Tool to calculate that the resultant six strains would capture 57% of the known genome variance found in commercially available strains of rat.

Figure 1 depicts the combinatorial breeding strategy used to create the rat test population. It also illustrates that the genetic background of each progenitor strain is represented three times in the rat test population. This strategy enabled us to test the influence that multiple allelic combinations have on survival following LeTx administration. As shown in the next section, testing with anthrax LeTx confirmed that the progenitor strains were either sensitive (F344 and BN) or resistant (WKY and LEW) to the effects of the toxin, thereby satisfying the initial criteria we selected for breeding the rat test population.

Figure 1.

Strategy for breeding the rat test population. Shown in the top box are the four inbred rat strains (F344, red; WKY, yellow; BN, green and LEW, blue) selected as progenitors for breeding the test population. The arrows indicate the pairwise combinatorial breeding scheme. The lower box shows the results for generating six F₁ hybrids using this strategy. Each F₁ hybrid is heterozygous and bred each time from the same progenitors. By this process, each genome is represented three times, thereby increasing the number of allelic combinations that can be tested for the effects of anthrax LeTx. Isogenic male rats from each strain were tested with different doses of anthrax LeTx. The F₁ hybrid nomenclature is denoted as female male for breeding purposes.

Full figure and legend (99K)

Survival of inbred rat strains to anthrax LeTx administration

We determined a single effective dose of anthrax lethal toxin needed to differentiate the response of a sensitive versus resistant rat. Since the F344 rats were expected to be sensitive, we tested whether outbred Caesarian-Derived-International Genetic Standard (CD-IGS) rats could survive LeTx and thus serve as a resistant control in our studies. F344 and CD-IGS rats were observed for up to 2 h following systemic injection of LeTx (a 4:1 mixture of PA and LF) through the tail vein. A range of 25–567 g LeTx/kg was tested in the F344 rats (Figure 2a), while a range of 40–340 g LeTx/kg was tested in the CD-IGS rats (Figure 2b). Under these conditions, we found that an average dose of 87 g LeTx/kg was lethal to all of the F344 rats (Figure 2a). The outbred CD-IGS rats were more resistant to the effects of a similar LeTx dose (80 g LeTx/kg), as all of the animals were still alive at 2 h (Figure 2b). In addition, only 75% of the CD-IGS rats survived for the entire 2 h of the test when given an average dose of 225 g LeTx/kg. Overall, about a 2–3-fold higher dose of LeTx was required to kill the CD-IGS rats compared with the F344 rats using this assay.

Figure 2.

Survival of rat strains following challenge with anthrax LeTx. The survival of F344 (a) and CD-IGS (b) rats treated with a range of anthrax LeTx doses is shown. Multiple individuals were tested for each strain at the mean dose of anthrax LeTx as indicated. These data were used to derive a single dose (240 g/kg) for testing the progenitor strains (c) and the rat test population (d), which elucidates the mode of inheritance for survival to LeTx. For all panels, the time post LeTx injection is shown on the x-axis in minutes while the percent survival (%) is plotted on the y axis. The survival curves show the percentage of total animals (n shown for each strain in legend) surviving up to 2 h after treatment with LeTx.

Full figure and legend (41K)

Based on the response of the F344 and CD-IGS rats, the survival of the BN, WKY and LEW rats was tested in the same way. We chose a slightly higher dose of 240 g LeTx/kg of anthrax LeTx because 50% of the CD-IGS rats survived at this dose. The response of the sensitive BN rats was similar to that of the F344 rats as all animals died during the test (Figure 2c). However, the WKY and LEW rats were relatively resistant as all of the animals survived the effects of the anthrax LeTx. In follow-up studies, we found that WKY (n=2) and LEW (n=3) animals survived when given very high doses of LeTx (1.4 mg/kg). Based on these findings, we bred the two sensitive (F344 and BN) and two resistant (WKY and LEW) rats in all combinations to generate the rat test population (Figure 1) and tested the rats for their sensitivity to anthrax LeTx.

Survival of a rat test population to anthrax LeTx administration

We tested the survival of six F₁ hybrid strains after anthrax LeTx administration (Figure 2d). Based on the studies with the progenitor and control strains, we selected the same dose of LeTx (240 g/kg) and the 2 h time point to determine the level of sensitivity or resistance of the different rats in the test population. The survival curves show that when the rats were administered 240 g/kg of LeTx, only the LEW WKY F₁ animals survived, while all the rats from the other five hybrid strains died within 2 h (Figure 2d). Our finding that the LEW WKY rats, which are derived from a cross of LEW and WKY strains, survive following administration of LeTx, indicates that these two strains share a common resistance gene with a recessive mode of inheritance. This conclusion is consistent with our findings that the BN WKY, BN LEW, F344 WKY and F344 LEW F₁ strains are sensitive to LeTx and with reports in the literature that refer to a dominant sensitivity to anthrax LeTx in mice.⁴

The toxicity of systemic LeTx administration is reported to cause dramatic pulmonary edema in rat lungs, which is accompanied by an increase in the weight of the lungs.¹ The results of lung to body weight measurements at the end of LeTx treatment corresponded with differences in survival among the strains (data not shown). The effect of LeTx on increased lung weight was significant for all sensitive progenitor and F₁ hybrid rat strains when compared with rats treated with the vehicle alone. However, no significant increases were found in the weight of the lungs in any of the resistant strains (LEW, WKY, LEW WKY and CD-IGS).

Survival of macrophages isolated from progenitor and test population rats to anthrax LeTx

In vitro studies challenging mouse macrophages with anthrax LeTx⁴ have been used as a way to map quantitative trait loci (QTL) for susceptibility (e.g. Ltx1), leading to positional cloning of a candidate gene, Nalp1b,⁸ that influences macrophage survival in the response to anthrax LeTx.⁸ To determine if the rat macrophages also exhibit strain differences that correlate with survival in vivo, we tested the effects of LeTx on bone-derived macrophages from two sensitive (F344 and F344 WKY) and two resistant (WKY and LEW WKY) strains. As control, we first tested sensitive J774A.1 (derived from Balb/c) and resistant IC-21 (derived from C57BL) mouse macrophage cell lines.⁴ Our findings were similar to those previously reported for mouse in that all IC-21 macrophages survived from lysis with a saturating concentration of PA (1 g/ml) and up to 10 g/ml of LF, while all J774A.1 macrophages were lysed at 10 ng/ml LF (data not shown). The rat macrophages isolated from either the WKY or the LEW WKY strains did not lyse when exposed to even the highest concentration of LF (10 g/ml) in the LeTx mixture (Figure 3). As expected, the macrophages obtained from the F344 WKY and F344 rat were sensitive to the effects of LeTx, although a small percentage of the F344 WKY cells did not lyse.

Figure 3.

Survival of macrophages to challenge with anthrax LeTx. Susceptibility of bone-derived macrophages to LeTx was measured in microwells containing approximately 50 000 rat macrophages isolated from the femur of hybrid and progenitor rats. Selected rat strains include those found to either survive (LEW WKY, WKY) or die (F344 and F344 WKY) from anthrax LeTx injection. Each assay well contained a saturating amount of PA (1 g/ml) plus an amount of LF (0–10 000 ng/ml) determined by serial dilution (x axis). Percent viability (y axis) was calculated by dividing the number of live cells (neutral red dye staining) remaining in each well by the number of cells in the wells with only PA (0 ng/ml LF). Means are presented.

Full figure and legend (13K)

Discussion

The present study examined the response of a rat test population, along with isolated macrophages from these rats, to anthrax LeTx. Using a genomic strategy, we identified three inbred rat strains that are genetically distinct from the LeTx-sensitive F344 rat. We confirmed in vivo that one of these strains was sensitive (BN), and two were resistant (WKY and LEW), to intravenous LeTx (240g/kg) administration during a 2-h test. We bred these four strains in all combinations to create a test population of six F₁ hybrid rats. We hypothesized that this new rat model could represent a reproducible way to simultaneously determine whether genetic differences account for strain susceptibilities to anthrax LeTx, and also to determine the mode of inheritance for the response in these strains.

Using the rat test population, we detected intra-species differences in the response to LeTx administration, as some rats survived the lethal effects of this compound better than others. For example, the difference in the lethal dose, when all animals die (LD₁₀₀), between the most sensitive (F344) and resistant (WKY and LEW) strains of rat was greater than 15-fold. We also showed that survival of the rats is directly related to the degree of pulmonary edema measured by lung enlargement, a commonly known part of the anthrax disease physiology.¹ Our in vitro results with bone-derived macrophages from sensitive (F344 and F344 WKY) and resistant (WKY and LEW WKY) rats correlated with the in vivo data. Together, these results are consistent with previous findings demonstrating that there are genetic differences among inbred strains of mice that determine their ability to survive the lethal effects of this compound.^{3, 4}

We found that survival from the effects of LeTx administration in rats exhibits a recessive mode of inheritance. Although we discuss these data in terms of survival, which in these strains is recessive, our results could also be described as showing a dominant sensitivity for mortality. Our results do not agree with a previous report in mice, where mortality to anthrax LeTx was assessed using interval-specific recombinant mouse congenic lines, leading to the identification of three linked QTL with potential candidate genes, Ltxs1 (Kif1c), Ltxs2 (Ity3) and Ltxs3 (Nos2).¹⁷ These studies concluded that resistance of the congenic lines to LeTx is dominant over susceptibility. However, results from a previous mouse macrophage study,⁴ and our in vivo and in vitro studies in rats clearly show that survival is recessive. The finding that both the F344 LEW and F344 WKY, along with the BN LEW and BN WKY strains, were sensitive to the effects of LeTx indicates that this trait (the ability to survive the effects of LeTx in rats) exhibits a recessive mode of inheritance. We also found the resistant strains survived even when exposed to very high doses of anthrax LeTx. For example, we found LEW, WKY and one F₁ hybrid (LEW WKY) strains to be resistant to 1.4 mg/kg LeTx in the 2 h in vivo assay. For the LEW WKY rats, we extended the assay time to 16 h for two individuals exposed to a 1.4 mg/kg dose of LeTx. Both animals survived and exhibited no signs of respiratory distress during the experiment. Since the ability to survive LeTx is recessive, and only one hybrid combination (LEW WKY) survives LeTx, this also suggests that a single gene is responsible for this trait. We base this argument on the low likelihood that LEW and WKY rats would share multiple recessive alleles, located throughout the genome, that would complement each other to protect rats from the effects of anthrax LeTx.

The result of the in vitro studies was consistent with the in vivo data. We demonstrated that bone-derived macrophages isolated from the various strains of rats exhibited large differences to the lytic effects of LeTx. Like the in vivo data, macrophages obtained from the F344 and F344 WKY rats were sensitive to LeTx, while macrophages obtained from the WKY and LEW WKY rats were found to be resistant. This means that genetic mapping studies using rat macrophages in vitro is a feasible way to uncover the mechanism for the intra-species differences found in response to the lytic effects of LeTx. Studies with rat macrophages may also help to confirm findings related to the mechanisms of cell death. For example, the Ltxs1 susceptibility locus was discovered after testing macrophages that were isolated from multiple mouse strains, with LeTx.⁴ These studies eventually led to the recent discovery of the polymorphic gene, Nalp1b, as a candidate for determining LeTx survival in mouse macrophages.⁸ Evidence for a mechanism of macrophage cell death suggests that PA binds to a host receptor complex consisting of TEM8/CMG2 with LRP6 and following furin cleavage, forms a heptameric pore, enabling LF entry into the cell.⁷ A functional Nalp1b allele could confer sensitivity of mouse macrophages in a model where LF activity causes Nalp1b protein to oligomerize bringing Pro-caspase-1 monomers nearby.⁸ Auto-activated caspase-1 could possibly then kill macrophages by processing of pro-interleukin-1 to the inflammatory cytokine IL-1.⁸ However, more host cellular targets might exist since there are known species-specific differences in the response of animal models to anthrax LeTx.³ These results need to be confirmed in different species, like the rat, probably beginning with sequencing of the Nalp1b gene.

In conclusion, a combinatorial breeding strategy was used to create a rat test population, which could be used to identify sensitive or resistant strains to toxins, drugs or other compounds for downstream mechanistic studies and development of therapeutics. We were able to demonstrate that there are genetic differences among strains of rat, which cause different responses to the effects of anthrax LeTx, both in vivo and in an in vitro rat macrophage assay. Taken together, the results provide evidence that there is a gene that induces rat's sensitivity to anthrax LeTx. The gene is likely Nalp1b and the next step would be to sequence this locus in the sensitive (F344 and BN) and resistant (WKY and LEW) progenitor strains used for breeding the rat test population. Polymorphisms may also be identified by sequencing other inbred rat strains that we have tested for their survival to anthrax LeTx (data not shown). In addition, sequencing of Anxtr2¹² in the progenitor strains of rats, along with PA binding assays using isolated rat macrophages from the rat test population, may exclude the anthrax receptor complex as playing a role in the rat survival to anthrax LeTx detected in these studies. Identification of the gene expression differences among the strains in the rat test population following LeTx dosing, along with characterization of sensitive versus resistant haplotypes, may also help to identify other pathways involved in the survival response. Ultimately, this information could be a key for the development of new therapeutic strategies.

Materials and methods

Toxin

LeTx is a mixture of PA and LF, which when administered separately is not toxic. Each compound was received in powdered form from List Biologicals Inc. (Campbell, CA, USA). The stock solution is made by dissolving each in ddH₂O to a final concentration for PA of 2 mg/ml and for LF of 1 mg/ml. Separate frozen aliquots (-80°C) were made and thawed on the day of treatment for both in vivo (rat) or in vitro (macrophage) studies.

Selection of genetically diverse inbred rat strains

We determined the level of genetic diversity in commercially available rat strains using the Strain Calculator, a customized version of the ACP Haplotyper available at the Rat Genome Database (http://rgd.mcw.edu/ACPHAPLOTYPER/). The Strain Calculator creates easily discernible patterns based on differences found within specific regions of the rat genome using SSLP markers. This tool assumes that the SSLPs are highly mutable, hence their utility as genetic markers, and also that the DNA flanking the genetic markers is less mutable than the SSLPs. While individually each genetic marker provides a limited amount of information, a series of consecutive genetic markers found to be identical between two inbred strains is strong evidence that the region of the genome inclusive to them is also identical. The Strain Calculator incorporates hierarchical clustering to group strains based on shared marker data within a region of interest. For the analysis, diversity in commercially available rats was estimated by using nearly 5000 SSLPs against the genomes of 48 common laboratory rats.²⁰ We conditioned the output of the Strain Calculator on the F344 strain, since it is sensitive to LeTx and dies in a relatively uniform and predictable manner.

Generation of the rat test population

The progenitor strains for breeding the rat test population were selected based on the following criteria: (1) past use of the strains in anthrax-related studies,^{1, 2} (2) maximal diversity in the host background determined from the Strain Calculator, (3) estimated separation on the phylogenetic tree^{21, 22} and (4) commercial availability of the strains. Breeding pairs of inbred F344 (F344/DuCRL ordered as CDF), WKY (WKY/NCrl), LEW (LEW/Crl) and BN (BN/Mcwi) rats were purchased from Charles River Laboratories (CRL, Wilmington, MA, USA). Note that the strain of BN rat is the same one used for sequencing the rat genome.²³ To create the test population, the F344, LEW, WKY and BN were all bred in pairwise combinations resulting in the following F₁ hybrid strains: F344 LEW, F344 BN, F344 WKY, LEW WKY, BN WKY and BN LEW. Hybrid strains are denoted by the name of the female progenitor strain first followed by the name of the male progenitor strain (e.g. female F344 male LEW). The F₁ hybrid strains in aggregate capture 57% of the allelic diversity found in commercially available inbred rat strains. Rats were group-housed under a 12 h light–dark cycle in an animal care facility that is accredited by the American Association for Laboratory Animal Care. Food and water were provided ad libitum.

Anthrax LeTx challenge of rat strains

Dose ranging studies were initially conducted to determine the level of sensitivity or resistance of rats to anthrax LeTx using the F344 (25–567 g LeTx/kg) and CD-IGS (40–340 g LeTx/kg) strains. Each dose of anthrax LeTx mixture (a 4:1 ratio of PALF) was administered via the tail vein and survival was measured for up to 2 h. Similar survival experiments were performed on 8–15 male rats weighing between 200 and 300 g from the rat test population. With these rats, a 240 g/kg dose of anthrax LeTx was selected because it killed all of the inbred F344 rats studied within a 2-h period. However, the same dose only killed 50% of the outbred CD-IGS (CRL:CD®IGS) rats that we used as a resistant control. The end point of the experiment was the time post injection when respiration and heart rate ceased or when the time limit of 2 h was exceeded. All animals that survived this LeTx challenge were killed with an intraperitoneal injection of pentobarbitol (100 mg/kg). After killing the animals, the lungs were removed, blotted with gauze to remove excess blood and weighed to assess the degree of pulmonary edema.

Isolation of macrophages

The femurs of the rats were isolated, rapidly immersed in 70% ethanol (5 s) and then placed in 15 ml conical tubes containing 12 ml rat macrophage media (RMM) comprised of Rosewell Park Mermorial Institute-1640 (RPMI-1640) (Life Technologies, Gaithersburg, MD, USA) containing antibiotics/antimicotics (100 U/ml penicillin, 100 g/ml streptomycin and 0.25 g/ml amphotericin B), 200 M L-glutamine and 10% heat-inactivated fetal bovine serum. The femurs were flushed with RMM and marrow cells isolated by centrifuging at 60 g for 2 min. After centrifugation, each femur cell pellet was resuspended in 12 ml RMM containing 2 ng/ml granulocyte–macrophage colony stimulating factor (GM-CSF). A six-well plate was seeded with 2 ml of cell supernatant per well and placed in an incubator at 37°C. After 3 days, an additional 2 ml of RMM containing 2 ng/ml GM-CSF was added and cell cultures were allowed to incubate for another 7–14 days, allowing for macrophage differentiation from the stem cells before testing viability with LeTx.

Anthrax LeTx challenge of primary macrophage cultures

A 100 l volume of a solution containing 500 cells/l was placed into each well of a 96-well plate and incubated at 37°C for 24 h before cell viability assays with anthrax LeTx as previously described.⁴ Positive control experiments were initially conducted using the mouse macrophage cell lines (IC-21 and J774A.1).⁴ Each assay on rat cultures was performed in triplicate with 25 l of LeTx (1 g/ml PA with serial dilutions of LF ranging from 0 to 10 000ng/ml) added to the macrophage cells. Treated cells were incubated for 4 h at 37°C after which 20 l of neutral red diluted in RMM (1:30) was added to each well and cells were further incubated for an additional 2.0 h at 37°C. Cell viability was calculated as the percentage of live cells (red staining) remaining in toxin containing wells compared with control wells with only PA. Since macrophages were obtained from 3 to 5 individuals of each strain and varying numbers of fields were counted for each well of the assay, we randomly selected a single field for each animal at each LF dilution by using Microsoft Excel's random number function.

Notes

Duality of interest

RJ Roman and HJ Jacob are PhysioGenix founders and major stock holders.

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Acknowledgements

The authors are grateful to Jordan Baye, Sally Korb and Yvette Harrington for excellent technical assistance. We thank Eric Boyden at Harvard University for assistance with the rat macrophage assay and Dr Simon Twigger at the Medical College of Wisconsin for development of the Strain Calculator. We thank Dr Ty Shockley for reviewing the manuscript. This work was funded through a grant provided by the National Institutes of Allergy and Infectious Diseases.