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The Journal of Immunology, 2007, 178: 5069-5075.
Copyright © 2007 by The American Association of Immunologists, Inc.

Description and Mapping of the Resistance of DBA/2 Mice to TNF-Induced Lethal Shock1

Ben Wielockx2, Jan Staelens, Leen Puimège, Ineke Vanlaere, Maarten Van Roy, Philippe van Lint, Frans Van Roy and Claude Libert3

Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Ghent, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In our search for genes that inhibit the inflammatory effects of TNF without diminishing its antitumor capacities we found that, compared with C57BL/6 mice, DBA/2 mice exhibit a dominant resistance to TNF-induced lethality. Tumor-bearing (C57BL/6 x DBA/2)(BXD)F1 mice completely survived an otherwise lethal TNF/IFN-{gamma}-antitumor therapy with complete regression of the tumor. This was not the case for C57BL/6 mice. Genetic linkage analysis revealed that TNF resistance is linked to a major locus on distal chromosome 6 and a minor locus on chromosome 17. Compared with littermate controls, chromosome substitution mice carrying a DBA/2 chromosome 6 in a C57BL/6 background were significantly protected against TNF and TNF/IFN-{gamma}, albeit less so than DBA/2 mice. Definition of a critical region of 13 Mb on chromosome 6 was the highest mapping resolution obtained. Further analysis of candidate genes may provide a powerful tool to control TNF-induced pathologies in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Originally, TNF was identified for its ability to selectively kill tumor cells, especially when used in combination with IFN-{gamma} (1, 2). However, clinical trials with TNF in cancer patients revealed extensive toxic effects including hypotension, hyperglycemia, anemia, bowel necrosis, coagulopathy, and hepatotoxicity (3, 4, 5, 6). Indeed, the systemic administration of TNF to cancer patients or laboratory animals induces a systemic inflammatory response syndrome (SIRS),4 caused by the proinflammatory nature of TNF. Therefore, the application of TNF as an anticancer drug is currently limited to isolated and locoregional perfusions of the limbs, kidney, and liver (7, 8, 9). TNF has also been shown to play a detrimental role in many diverse pathologies, including rheumatoid arthritis (RA) (10), sepsis (11), AIDS (12), Crohn’s disease (13), multiple sclerosis (MS) (14), and hepatitis (15, 16). Although new medical therapies such as the TNF-inhibiting infliximab have forced a breakthrough in the treatment of Crohn’s disease and RA (13), direct inhibition of TNF is not always an advantage, viz, for the treatment of MS (17, 18). Even a single dose is extremely expensive (19) and furthermore some reports demonstrate that this anti-TNF therapy might promote tuberculosis (20, 21, 22). To develop safer and more widely applicable strategies for the treatment of TNF-mediated diseases and to develop TNF as a systemic antitumor drug, it is of utmost importance to find new targets or protective proteins. In this study we describe the dominant resistance of DBA/2 mice (designated D against high doses of systemically injected TNF. Using a backcross (BC) and intercross (IC) strategy, we were able to reveal that this protection is linked to protective loci on distal chromosome (Chr) 6 and on Chr 17. Furthermore, to illustrate the therapeutic relevance of the finding, we show that tumor-bearing (BXD)F1 mice and chromosome substitution CS) mice carrying a DBA/2 (D) Chr 6 in a C57BL/6 (B) background (B.D-Chr 6 CS mice) are protected against the negative side effects of TNF/IFN-{gamma} therapy without compromising the antitumor effects. We describe our efforts to narrow the critical region on Chr 6 and discuss some relevant candidate genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6Jico mice (designated as C57BL/6 or B hereafter) were purchased from Iffa-Credo and DBA/2NCrl mice (designated DBA/2 or D hereafter) were from Charles River Laboratories. (C57BL/6 x DBA/2)F1 hybrid mice were generated by crossing parental strains (C57BL/6 females and DBA/2 males) and are denominated (BXD)F1. All mice were bred in a conventional animal house and received food and water ad libitum. Only female mice 8–12 wk of age were used. To analyze a complex genetic trait we generated CS mice by backcrossing (BXD)F1 mice to the host strain, C57BL/6, and repeatedly backcrossing to the host strain and screening the progeny for a nonrecombined donor chromosome of interest in each generation (23). After the eighth BC generation (N9) when the genome of the progeny theoretically consists of 99.8% host DNA (besides the substituted chromosome), mice containing this chromosome (CS mice) or lacking it (control wild-type counterparts) were selected and used.

Cytokines and cell lines

Recombinant mouse TNF (1.0 x 109 IU/mg) and murine IFN-{gamma} (1.1 x 108 IU/mg) were expressed in Escherichia coli and purified in our laboratory. The TNF and IFN-{gamma} preparations contained less then 10 U of endotoxin per milligram of protein as determined in a Limulus amebocyte lysate assay. The murine B16BL6 melanoma subline was a gift from Dr. M. Mareel (University Hospital, Ghent, Belgium) through the courtesy of Dr. I. Fidler (University of Texas M. D. Anderson Cancer Center, Houston, TX).

Injections, blood collection, measurements, and determination of IL-6 and NOx

TNF (or TNF plus IFN-{gamma}) was diluted in endotoxin-free PBS immediately before injection. Injections were given i.p. in a total volume of 250 µl or paralesionally (close to the tumor) in a total volume of 100 µl. Blood was collected by cardiac puncture under Avertin anesthesia. The blood was allowed to clot for 30 min at 37°C and for another hour at 4°C. Serum was prepared and stored at –20°C. Rectal body temperatures were measured with an electronic thermometer (Comark). IL-6 was determined using an IL-6-dependent 7TD1 hybridoma cell line (24). Cells were cultured at 7,000 cells/well in 96-well plates in the presence of serially diluted serum or murine IL-6 as a standard. After 3 days of culture the number of cells was determined using the hexosaminidase colorimetric method. The detection limit of the IL-6 assay is ~l pg/ml. The NOx concentration in the serum, which is the sum of the stable NO metabolites nitrate and nitrite, was determined as previously described (25).

Antitumor model

Approximately 600,000 tumor cells in 100 µl of PBS were injected s.c. in mice on day 0. Treatment was started on day 10 by daily paralesional injection with 10 µg of murine TNF and 5,000 IU of IFN-{gamma}. Tumor size was measured daily and expressed as "a" x "b" (in mm2), "a" being the largest diameter and "b" the largest diameter perpendicular to "a". Deaths were monitored daily.

Mapping of TNF resistance genes

To map the genes responsible for the TNF resistance of DBA/2 mice, we performed a BC ((BXD)F1 x C57BL/6) and an IC ((BXD)F1 x (BXD)F1). We generated 100 BDB (N2) BC and 90 F2 IC mice, and injected them all with 75 µg murine TNF, a dose lethal for C57BL/6 but never lethal for DBA/2 or (BXD)F1. DNA was extracted from the tails of all mice. A genome scan was performed on the DNA samples using 69 robust and unambiguous microsatellite markers spread evenly over the genome. The amount of markers and their positions on the different chromosomes were chosen based on the data available in the online version of Mouse Genetics by L. Silver (http://www.informatics.jax.org/silver/frames/frameb.shtml; see Table IX.4) showing the amount of markers required per individual chromosome for complete coverage at 95% confidence. Finally, after the first genome-wide screen, additional primers were tested in the areas of interest. The sequences were obtained from the Massachussetts Institute of Technology (Cambridge, MA) web page (http://web.mit.edu) and the primers were custom made by Invitrogen Life Technologies. Survival and genotyping data were analyzed using Map Manager QTXb13 (26). Two different approaches were used in the quantitative trait locus (QTL) analysis, namely "marker regression analysis" and "composite interval mapping," the latter allowing for the inclusion of background loci. Threshold values were established by Map Manager QTXb13 using the permutation test method that estimates an empirical genome wide probability for observing a given likelihood ratio statistic (LRS) score by chance through randomization of the phenotypes while simultaneously keeping the genotypes constant, followed by interval mapping. Interval mapping was performed for each chromosome using 1000 permutations to establish critical values for suggestive, significant, and highly significant associations as suggested by Lander and Kruglyak (27).

Statistical analysis

Data of final mortality were analyzed using a {chi}2 analysis. Survival curves (Kaplan-Meier plots) were compared by a log-rank test. Other data (means with SD values) were analyzed using a one-tailed Student’s t test. For the calculation of the cumulative p values, the data corresponding to the most interesting markers from the two independent genome-wide screens were pooled into one set of data. The cumulative probabilities for the pooled results at individual marker loci or common map positions were computed as the {chi}2 probability of –2(lnPFormula BDBFormula + lnPFormula F2Formula ) with four degrees of freedom (Fisher’s method) as suggested by Williams et al. (28). These data are combined point-wise probabilities for both groups of progeny (BC and IC) and should not be considered conclusive QTLs. However, the QTLs on Chr 6, Chr 17, and Chr 10 (BC only) have genome-wide probabilities of <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Response of C57BL/6, DBA/2, and (BXD)F1 mice to TNF

In the past, we intensively investigated the response of different mouse inbred strains to lethal doses of exogenously delivered recombinant TNF (29). We found that most strains of mice, e.g., C57BL/6, are sensitive to doses of TNF ranging between 12.5 and 25 µg, and only a few strains were identified as resistant to high doses of TNF. One such strain is SPRET/Ei discussed by Staelens et al. (29) and another is DBA/2. To fully characterize the TNF dose response, groups of C57BL/6, DBA/2 and (BXD)F1 mice were injected with increasing doses of TNF ranging from 15 to 100 µg and mortality was scored over 5 days (Fig. 1A). Almost all C57BL/6 mice died after the administration of 20 µg of TNF, whereas DBA/2 and (BXD)F1 mice survived TNF doses up to 100 µg. To investigate the resistance in more detail, we used a dose of 75 µg of TNF in all other experiments. As shown in Fig. 1B, this dose caused 100% mortality in C57BL/6 mice (n = 7) within 36 h after the challenge but no mortality at all in DBA/2 and (BXD)F1 mice (n = 7) (both p = 0.0002 compared with C57BL/6). The body temperature of C57BL/6 mice decreased dramatically to below 33°C within 7 h of TNF injection. In contrast, no hypothermia was observed in DBA/2 and (BXD)F1 mice (p < 0.0001, compared with C57BL/6 after 7 h) (Fig. 1C). Furthermore, we studied the induction of IL-6 by TNF in the serum of all three strains (n = 5) 2, 4, and 7 h after TNF injection (Fig. 1D). A significant difference in IL-6 concentration between C57BL/6 on the one hand and (BXD)F1 and DBA/2 mice on the other was clear within 2 h after TNF administration (p = 0.032 and p = 0.013, respectively). Four and seven hours after the challenge IL-6 levels in C57BL/6 mice were at least 25 times higher than those in (BXD)F1 or D mice (p < 0.0001). TNF is also known to induce NO, a potent vasodilator (30). Seven hours after TNF challenge serum was collected and NOx was measured. In Fig. 1E we demonstrate that significantly more NOx is induced in C57BL/6 mice compared with DBA/2 mice (p = 0.0025) and (BXD)F1 mice (p = 0.035). The response of the F1 mice, however, was intermediate. A possible explanation for the resistance of DBA/2 mice could be the faster clearance of TNF from the circulation. Therefore, TNF concentrations were determined in all serum samples obtained from the previous experiments (2, 4, and 7 h). However, no difference in TNF concentrations was observed between the three strains at the different time points (data not shown).


Figure 1
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  		FIGURE 1. Response of C57BL/6, (BXD)F1, and DBA/2 mice to TNF. A, Lethal response of C57BL/6, (BXD)F1, and DBA/2 mice to increasing doses of TNF. Lethality deaths occurred for 5 days after injection, after which no further deaths occurred (n = at least 5). B, Lethal mortality response after injection of 75 µg of TNF. All mice (n = 7) were observed for 5 days after injection. C and D, Induction of hypothermia and IL-6 in mice after the injection of 75 µg of TNF. Filled bars, C57BL/6; gray bars, (BXD)F1; and open bars, DBA/2. Body temperature (n = 7) was measured 2, 4, and 7 h after the challenge. For the determination of IL-6 concentrations, serum was collected at the same time points (n = 5) and assayed as described in Materials and Methods. E, Induction of NOx 7 hours after the challenge in C57BL/6 (n = 5), (BXD)F1 (n = 6), and and DBA/2 (n = 5). Statistical significance was calculated vs C57BL/6 mice. *, p < 0.05; **, p < 0.01; ***, p < 0.0001 as described in Materials and Methods.

 
Clinical trials in cancer patients and laboratory animals revealed that systemic use of TNF in antitumor therapies is handicapped by the induction of SIRS with TNF doses even lower than the therapeutic dose.

To study the response of TNF hyporesponsive of (BXD)F1 mice in TNF/IFN-{gamma}-based antitumor therapy, syngeneic H-2b-restricted B16BL6 melanoma cells were inoculated in C57BL/6 or (BXD)F1 mice. These tumors were treated daily with TNF/IFN- {gamma}. This treatment led to the complete regression of tumors in both mouse strains. However, nearly all C57BL/6 mice died from the therapy (five of seven), whereas all (BXD)F1 mice survived (zero of seven) (Fig. 2, A and B).


Figure 2
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  		FIGURE 2. Response of B16BL6 tumor-bearing (BXD)F1 mice to TNF/IFN-{gamma} therapy. Tumor size index (A) and survival curve (B) are plotted as a function of time (days after inoculation). Daily treatment of C57BL/6 (n = 7) and (BXD)F1 (n = 7) was started 10 days after inoculation and lasted until day 19. Control mice (both n = 7) were injected daily with PBS. ***, p < 0.0001.

 
Subchromosomal mapping of the TNF resistance

To identify and map the loci conferring TNF resistance on DBA/2 mice, we set up BC and IC experiments. Offspring of both BC (n = 100) and IC (n = 90) mice were injected with 75 µg TNF, a dose that has always proved lethal for C57BL/6 mice but never for (BXD)F1 and DBA/2 mice. Eventually, 43 of 100 BC mice survived (43%), as well as 59 of 90 IC mice (66%). This result illustrates the complex nature of the genetics underlying the resistance. Using DNA samples of all tested mice, we performed two independent genome-wide scans using at least 65 different microsatellite markers distributed over the genome. Linkage analysis was performed by introducing survival data and genotyping data into Map Manager QTX (26), defining a death response as 0 and a survival response as 1. As shown in Table I, the phenotype is strongly linked to a locus on distal Chr 6 (in the BC but especially in the IC experiment) and to a locus on Chr 17 (in both the BC and IC experiments). To take advantage of the two independent crosses, we calculated cumulative probabilities at the best markers for both crosses as described by Williams et al. (28) and confirmed the highly significant linkage of these loci with the resistance (Table I).


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  		Table I. Linkage analysis of the TNF resistance

 
To further locate putative QTLs, interval mapping analysis was performed using the IC data and Map Manager QTX by evaluating the association between the trait and the expected contribution of a hypothetical QTL at multiple analysis points. The results confirm the linkage with the distal part of Chr 6 and the minor linkage with Chr 17 (Fig. 3, A and B). The locus on Chr 6 reached highly significant linkage with a peak LRS score of 18.1 and the Chr 17 locus proved to reach a level of significance between suggestive and significant with a maximum LRS of 7.0.


Figure 3
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  		FIGURE 3. QTL analysis of the intercross data using interval mapping of Chr 6 (top) and Chr 17 (bottom). LRS defines the association of the phenotypic trait (TNF resistance) with any locus on the chromosome (1 cM interval, dominant regression). The three significance levels (*, p = 0.63 (suggestive); **, p = 0.05 (significant); and ***, p = 0.001 (highly significant)) were calculated using 1000 permutation tests (Map Manager QTX13).

 
Generation and testing of D.B CS mice

To be able to quantify the influence of the single chromosomes that modulate the TNF resistance of DBA/2 mice, we decided to generate CS mice until the N9 generation as described in Materials and Methods. These mice carry either one or two copies of Chr 6 or Chr 17 from DBA/2 in a C57BL/6 background. We injected groups of CS mice heterozygous or homozygous for the selected chromosome, as well as littermate controls, with 30 µg of TNF and scored lethal over 5 days. In this study, we show that B.D-Chr 6 CS mice (homozygous and heterozygous) are significantly better protected compared with their B6 controls (p < 0.0001) (Table II, Chr 6). Furthermore, serum IL-6 concentrations 8 h after the injection of 30 µg of TNF were significantly higher in the wild-type controls compared with B.D-Chr 6 CS mice (p = 0.02) (data not shown). However, the magnitude of protection was much less than that of the DBA/2 vs C57BL/6 mice. Moreover, there was no dose of TNF found that conferred absolute protection of the B.D-Chr.6 (D) mice and in the meantime caused 100% mortality in B.D.-Chr.6 (B) or control mice. It was surprising that B.D-Chr 17 CS mice were not at all protected when only one copy of the chromosome was present but were significantly protected when this chromosome was homozygous DBA/2 (p = 0.0003). Finally, mice carrying one copy of DBA2 Chr 6 and one of Chr 17 (B.D-Chr 6 + 17 (H) CS) did not survive better than single CS mice (data not shown).


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  		Table II. TNF-induced mortality in B.D chromosome substitution mice (CS)a

 
Moreover, B.D-Chr 6 (D) CS mice and their wild-type littermates were inoculated with B16BL6 melanoma cells and treated 10 days later with TNF/IFN-{gamma} for 10 consecutive days. This led to a complete regression of tumors in all treated mice (Fig. 4A). However, B.D-Chr 6 (D) CS mice did significantly better than wild-type mice (p = 0.04) (Fig. 4B). Here too, however, the magnitude of protection was far less prominent compared with the protection observed in the (BXD)F1 mice.


Figure 4
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  		FIGURE 4. Response of B16BL6 tumor-bearing B.D-Chr 6 mice to TNF/IFN-{gamma} therapy. Tumor size index (A) and survival curve (B) are plotted as a function of time (days after inoculation). Daily treatment of homozygous B.D-chromosome 6 (C57BL/6) mice and homozygous B.D-Chr 6 (DBA/2) mice was started 10 days after inoculation and lasted until day 19. Control mice were injected daily with PBS. *, p = 0.04.

 
Attempts to identify the locus on distal Chr 6 conferring protection to B.D-Chr 6 mice

To narrow down the Chr 6 locus that confers protection against TNF, B.D-Chr 6 heterozygous (H) CS mice were backcrossed to C57BL/6, all offspring were screened with at least five microsatellite markers in and around the region of interest, and mice were intercrossed to obtain congenic mice. Sensitivity for TNF had to be tested in a congenic but not in BC mice because of the narrow window of resistance left over in the B.D-chr6 CS mice. As shown in Fig. 5, three distinct groups of congenic B.D-Chr 6 CS mice (at least three mice per group), all containing a particular part of the DBA/2 genome on distal Chr 6, were identified and subsequently injected with 30 µg of TNF. All groups responded either as B.D-Chr 6 (B) or (H) (see Fig. 5) and as a result the locus of interest was narrowed down to <13.2 Mb, namely in between the marker D6Mit54 (position 113.2 Mb from the centromere) and D6Mit254 (at 126.2 Mb from the centromere).


Figure 5
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  		FIGURE 5. Response of B.D-Chr6 congenic mice to TNF-induced lethal shock. B.D-Chr.6 (H) N9 mice were backcrossed to B mice and all individual offspring were genotyped using at least five markers in the distal part of Chr 6. All mice having a comparable genetic reorganization were grouped (A-B-C; n = at least 3) and compared on sensitivity for TNF with control mice (B.D-Chr.6 (B) and B.D Chr.6 (H)). The groups that responded as B.D-Chr.6 (B) or B.D-Chr.6 (H) were designated as sensitive (S (S) or resistant (R), respectively. Color codes: red represents part of the chromosome coming from B; green represents part of the chromosome being heterozygous, and yellow represents part of the chromosome that can be either B or H because it is unknown where the recombination in that particular area occurred. The consensus of the three independent groups is drawn at the right hand side of the figure.

 
Investigating the role of candidate genes in the 13.2 Mb region on Chr 6

Based on a single nucleotide polymorphism (SNP) analysis we performed using the CELERA database, we investigated the role of some candidate genes within the region of 13.2 Mb. A very interesting candidate was the 5-lipoxygenase (5-LO) encoding gene (Alox5 or 5-LO) from which the SNP (Val to Ile) was reported to lead to a 90% reduction of the biological activity (31). Therefore, we injected the 5-LO-deficient mice vs wild-type counterparts (C57BL/6 background) with a dose of TNF that was sublethal to the controls (25 µg i.p.). As shown in Table III it is clear that we could not confirm a function for this gene in TNF-induced lethality mortality, because all 5-LO mice died in the experiment.


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  		Table III. TNF-induced mortality in 5-LO deficient mice versus WT counterpartsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The impressive antitumor activity of TNF has been known for more than two decades. However, exploiting this cytokine in systemic antitumor therapies is impossible due to the massive induction of SIRS accompanied mainly by a blood pressure drop and hepatotoxicity (4, 32). In contrast, TNF also plays a detrimental role in the progression and final outcome of many important diseases such as RA, MS, hepatitis, Crohn’s disease and many others (33). To turn TNF into a safer antitumor drug on the one hand and to inhibit TNF-mediated pathologies on the other hand, it is of great importance to identify and understand the genes that modulate the response to TNF. In 2002 we described how most mouse strains are rather sensitive to TNF-induced mortality death caused by TNF (LD100 ~20 µg) and reported that a few strains display significant resistance against high doses of TNF (LD100 > 100 µg) (29). We found that the DBA/2 strain is among the protected strains. DBA/2 mice resist up to 100 µg of TNF, which is about five times the dose lethal for C57BL/6. With a dose of 125 µg TNF per mouse, deaths were sporadic (not shown). In this study we demonstrate that the TNF-protection of DBA/2 mice is a dominant trait because (BXD)F1 hybrid mice are as resistant to TNF as DBA/2 mice. Furthermore, we demonstrate that several TNF-induced metabolic changes are strongly attenuated in DBA/2 and (BXD)F1 mice. Though the NOx concentration in the serum of (BXD)F1 mice displays an intermediate phenotype, all other parameters were similar in DBA/2 and (BXD)F1 mice and significantly lower than those measured in C57BL/6 mice. To map the DBA/2 TNF resistance genes, we made use of two independent crosses, a BC and an IC; apart from increasing the number of tested individuals this is one way to improve the signal to noise ratio. The advantage is that the identified loci (QTLs) are much more robust and significant (28). Using this strategy we identified two resistance loci, one located on distal Chr 6 and another on distal Chr 17. Cumulative p values were in the order of 10–5 and 10–3 for the loci on Chr 6 and Chr 17, respectively. An interval mapping approach confirmed that the locus on Chr 6 is almost highly significantly linked to the phenotype. Furthermore, we found no linkage whatsoever with any locus on Chr 12. This was a surprise, because in a previous report we described the identification of a strong QTL (named "TNF-protection" or "TNFp") in DBA/2J mice (purchased from The Jackson Laboratory) on distal Chr 12, using the complete set of (BXD) recombinant inbred strains (34). However, the fold protection of the DBA/2J mice was only double compared with C57BL/6J mice such that a BC or IC experiment with these mice as parents was not possible because in this case the survival/lethality window was too small. The reason for the higher resistance of DBA/2Crl compared with DBA2/J and the lack of linkage with Chr 12 might be explained by the fact that both strains (DBA/2J and DBA/2NCrl) already diverged in 1948, when G. B. Mider distributed DBA/2 mice to The Jackson Laboratory (DBA/2J) and three years later to National Institutes of Health, who passed these through to the Charles River Laboratories (DBA/2NCrl) in 1974 (see www.jax.org).

A multigenic trait is difficult to study because of genetic and phenotypic heterogeneity and epistasis. To investigate the potential role of the individual loci and to be able to identify and isolate the genes, we generated heterozygous CS mice for the two candidate chromosomes (B.D-Chr 6 CS and B.D-Chr 17 CS) as described by Nadeau et al. (23). One of the advantages of this approach is that CS strains represent a nearly inbred resource and allow improved statistical power to detect linkage. Indeed, both heterozygous and homozygous B.D-Chr 6 CS mice were significantly protected against moderate doses of TNF in comparison to littermate control animals. The dominant nature of the resistance of DBA/2 mice is thus reflected in the C.S-chr6 CS mice only, although it was also clear from the BC and IC experiment that mice surviving the TNF injection were preferentially homozygous D rather then heterozygous around the identified QTL on distal chromosome 6 (data not shown). The B.D-chr6 CS mice also displayed some protection in a TNF/IFN-based tumor treatment. However, it is clear that the strong resistance to TNF or to TNF/IFN by the DBA/2 parental mice is largely gone. Furthermore, B.D-Chr 17 CS mice displayed protection only when both copies of the DBA/2 chromosome were present. This result is hard to explain because the data obtained with the BC and IC do not support the notion that the locus on chromosome 17 is recessive. On the one hand, the dramatic reduction of the protection in B.D-chr6 CS mice compared with DBA/2 mice illustrates that the major locus linked to resistance depends on the full activity of the epistatic interaction. On the other hand, it makes fine mapping of the responsible gene problematic and therefore less attractive. Indeed, fine mapping was only possible using newly generated congenic mice, which led to a maximal resolution of 13.2 Mb.

Within this region of 13.2 Mb, several candidate genes can be suggested based on both the literature and the SNP analysis we performed using the CELERA database. In this analysis, SNPs found in the coding regions, the 5' untranslated regions (UTRs) and the 3' UTRs were listed between D6Mit54 and D6Mit254. A small number of genes were studied further, but firm conclusions could not be reached. For example, the SNP in the 5-lipoxygenase encoding gene (Alox5) was reported to lead to a dramatic reduction in biological activity (31), but experiments with Alox5-deficient mice could not confirm a function of this gene in TNF-induced lethality mortality (data not shown). Even so, the SNP in the {alpha}-2-macroglobulin-encoding gene (A2m) was of interest because we showed previously that A2m-deficient mice are somewhat resistant to TNF (35), but equal amounts of A2M were found in DBA/2 and C57BL/6 mice (data not shown) and no information is available on the effect of the Thr to Lys point mutation. Finally, very close to the marker D6Mit254 is located tnfrsf1, the gene encoding the major receptor for TNF (TNFR1). However, we believe that this gene is not a candidate because it falls outside the critical region and congenic mice were protected with a clear B copy of the gene. Furthermore, no sequence variance or difference in expression was found between C57BL/6 and DBA/2 mice (data not shown).

Interestingly, within our critical region is located a marker (D66Mit150) that was recently shown to regulate the expression of no fewer than1650 genes in the mouse brain (36). Chesler et al. demonstrate in their Nature Genetics article (36) the identification of this locus using BXD recombinant inbred strains. Although we could not find consistently altered expression of some of these genes (e.g., gelsolin, the IL-15 receptor, hepatocyte growth factor, and tissue factor protease inhibitor) in relevant organs (e.g., liver, small intestine, and kidney) between DBA/2 and C57BL/6 mice, it is quite possible that this locus, through its potential effect on so many other genes, controls the response of mice against TNF.

In conclusion, we demonstrate that DBA/2 mice are significantly protected against TNF- induced lethal shock and that the main locus linked to this trait is located on distal chromosome 6. Identification of this locus has not been possible so far, and we speculate that the relevant genetic element may be identical to the one described by Chesler et al. (36) that controls the expression of hundreds of genes involved in brain function but perhaps also in the inflammatory response to TNF.


   Acknowledgments
 
We thank A. Bredan for editing the manuscript, J. Vanden Berghe, L. Van Geert, and M. Goessens for excellent technical assistance and animal care, and Drs. R. Spanbroek and B. Koller for providing us with the 5-LO deficient mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. B.W. is a postdoctoral fellow at the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. Back

2 Current address: Technical University of Dresden, Dresden, Germany. Back

3 Address correspondence and reprint requests to Dr. Claude Libert, Laboratory of Molecular Biology, Ledeganckstraat 35, Gent, Belgium. E-mail address: Claude.Libert{at}Ugent.be Back

4 Abbreviations used in this paper: SIRS, systemic inflammatory response syndrome; B, C57BL/6; BC, backcross; CS, chromosome substitution; Chr, chromosome; D, DBA/2; IC, intercross; 5-LO, 5-lipoxygenase; LRS, likelihood ratio statistic; MS, multiple sclerosis; QTL, quantitative trait locus; RA, rheumatoid arthritis; SNP, single nucleotide polymorphism. Back

Received for publication September 27, 2006. Accepted for publication January 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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