Full Paper

Genes and Immunity (2004) 5, 93–100. doi:10.1038/sj.gene.6364042 Published online 11 December 2003

Leishmaniasis host response loci (lmr1–3) modify disease severity through a Th1/Th2-independent pathway

C M Elso1, L J Roberts1, G K Smyth1, R J Thomson1, T M Baldwin1, S J Foote1,2 and E Handman1,2

1The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia

Correspondence: Dr SJ Foote, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Victoria 3050, Australia. E-mail: foote@wehi.edu.au

2These authors have contributed equally to this work.

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Abstract

The severity of disease caused by infection with Leishmania major depends critically on the genetics of the host. Early induction of T helper (Th)1-type immune responses in the resistant C57BL/6 mice and Th2-type responses in the susceptible BALB/c mice are thought to determine cure or disease, respectively. We have previously mapped three host response loci in a genetic cross between C57BL/6 and BALB/c mice, and here we show definitively the involvement of these loci in disease severity using animals congenic for each of the loci. Surprisingly, in the late stage of infection when the difference in disease severity between congenic and parental mice was most pronounced, their cytokine profile correlated with the genetic background of the mice and not with the severity of disease. This indicates that the loci that we have mapped are acting by a mechanism independent of Th phenotype.

Keywords:

Leishmania major, quantitative trait, T-lymphocyte subsets, immune response genes, interleukin-4

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Introduction

The clinical manifestations of human leishmaniasis display a spectrum of disease severity from self-healing cutaneous lesions caused by infection with Leishmania major to visceral disease caused by L. donovani where the parasites spread into the reticuloendothelial system with fatal outcome. Even within the cutaneous syndrome, there is a significant variation in severity.1 As early as 1979, Walton and Valverde2 suggested host genetics as the basis for the spectrum of disease severity in cutaneous leishmaniasis in Central America. This suggestion was also made in relation to the increased susceptibility of Oriental Jews to infection with L. major and L. tropica.3

The disease phenotype observed in human leishmaniasis can be mimicked in the laboratory by infection of different inbred strains of mice with L. major. Thus, the mouse model of the highly susceptible BALB/c at one end of the spectrum, and the resistant C57BL/6 at the other, has been widely used to study both the genetics and biology of the host response phenotype. The murine system offers the advantage of fixing the effect of the parasite genome using cloned parasite lines and studying the host response using inbred strains of mice to minimise environmental effects.

A number of genes mediating host response to infectious disease have been identified, including genes in the interleukin (IL)-12/interferon-gamma (IFN-gamma) pathway mediating resistance to mycobacterial diseases4 and Slc11a1 (previously known as the natural resistance-associated macrophage protein 1 or NRAMP1)5,6 found to control resistance to infection with L. donovani, Salmonella typhimurium and Mycobacterium bovis BCG. These genes act in a monogenic Mendelian manner, which facilitated their identification. However, host response to infection more commonly presents as a complex trait and studies of the murine model of response to L. major have indicated that this phenotype is indeed mediated by multiple genes.7 This has hampered the identification of individual genes affecting the phenotype. Whereas linkage has been found in different studies to loci on chromosomes 5, 6, 9, 10, 11, 15, 17 and X,8,9,10,11,12 so far only a single locus on chromosome 11 has been confirmed as playing a role in response to disease in L. major-infected mice.9

In addition to work carried out on the genetics of host response to L. major, this system has long fascinated immunologists because a marked polarisation in the immune response is observed upon challenge with L. major parasites. BALB/c produces T helper (Th)2-type cytokines, in particular IL-4, which has been shown to be associated with disease progression and susceptibility,13 whereas recovery from infection in resistant C57BL/6 depends critically upon the induction of a Th1-type response resulting in the activation of macrophages to kill the intracellular organisms.14,15 This has led to the intensive study of the regulators of the polarised Th1/Th2 helper T-cell responses by immunologists.

While there is little doubt that these cytokines are necessary for the observed disease manifestations, recent data have challenged the simplicity of this model and have revealed a much greater complexity in the mechanisms of acquired resistance.16

In our studies, we chose to make no assumptions on which genes may be important for susceptibility to disease. Using a genome-wide scan on two large populations of F2 mice created from a cross between the susceptible BALB/c and the resistant C57BL/6 strains, we have shown that susceptibility to disease caused by infection with L. major in these strains is controlled by two autosomal loci: lmr1 on chromosome 17 and lmr2 on chromosome 9,10 as well as a third locus, lmr3 on the X chromosome11. It is interesting to note that loci mapping to similar positions on chromosomes 9 and 17 have also been linked to the severity of tuberculosis in mice.17 M. tuberculosis, like L. major, is an intracellular pathogen whose host cell is the macrophage.

In the current study, we describe the production of mice congenic for each of these regions singly, and animals congenic for both lmr1 and lmr2, and show that they contribute significantly to the disease phenotype, thus providing proof of their involvement in the response to infection. We also investigate the cytokine profiles produced in the draining lymph nodes during the chronic phase of infection when the difference between the susceptible and resistant mice is most pronounced. The data indicate that the host response loci described above are not responsible for the established, polarised Th1/Th2 immune response.

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Results

Generation of mice congenic for L. major host response loci

Congenic mice were produced by marker-assisted serial backcrossing for at least six generations. Donor loci from both strains were isolated onto their reciprocal background genomes giving rise to animals designated C.B6 where the background genome is BALB/c and the donor interval is from C57BL/6 and B6.C for the reciprocal congenics. After six generations (nine generations for C.B6-lmr3), mice had a fixed background as determined by genotyping polymorphic markers at 15 cMdensity (10cM for C.B6-lmr3). The size and location of the congenic intervals can be seen in Table 1. More dense screening identified small regions of donor genome remaining on chromosomes 8 and 14 in C.B6-lmr2 and C.B6-(lmr1, lmr2) mice. In our earlier studies, we did not observe any contribution of these regions to the disease phenotype caused by infection with L. major, and therefore we consider that they should not affect our analysis.


Effect of the host response loci on the course of disease

Mice individually congenic for C57BL/6 lmr13 or genetically compound for the congenic intervals, lmr1 and lmr2 on the BALB/c background and mice congenic for BALB/c lmr1, lmr2 or both on the C57BL/6 background were infected with 106 L. major promastigotes at the base of the tail and the progression of disease was recorded. A large variation was observed within all groups of congenic mice, illustrated in Figure 1, showing the average weekly increase in lesion size for each group of mice. The average lesion score of each group at each week was calculated in order to compare the progression of disease between the groups (Figure 2). A statistical permutation test was developed to compare pairs of mouse strains over the course of infection. The test statistic (mean t) is the two-sample t-statistic to compare the lesion scores between the two groups at each week, averaged over the course of the infection. This was carried out for weeks 2–4 and 5–12. In the first 4 weeks following infection, there was no difference in the size of the lesion between BALB/c and congenic animals on the BALB/c background. Congenic mice on the C57BL/6 background, especially the mice congenic for both lmr1 and lmr2, showed a slight increase in the average lesion score compared to the parental C57BL/6 mice. As a group, the C57BL/6 mice and congenics on this background had somewhat smaller lesions than the BALB/c and their congenics. However, at the later stages of disease, from 5 to 12 weeks postinfection, the contribution of the different lmr loci to susceptibility became apparent, with significant differences observed on both genetic backgrounds. Thus, the degree of susceptibility to disease as reflected by the size of the developing lesion can be ordered from the most susceptible to the most resistant (Figures 1 and 2): C.B6-lmr3>BALB/c>C.B6-lmr1>C.B6-lmr2>C.B6-(lmr1, lmr2)>B6.C-lmr1> B6.C-(lmr1, lmr2)>B6.C-lmr2>C57BL/6.

Figure 1.
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Average weekly increase in lesion score. The average weekly increase in lesion score is measured for each mouse by a least-squares regression with intercept zero. These data are represented as box plots by strain.

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Figure 2.
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Course of infection in mice congenic for lmr loci. (a) The average lesion score for each strain has been plotted over the course of the infection. Strains are represented as follows: BALB/c, filled square; C.B6-lmr1, filled triangle; C.B6-lmr2, filled circle; C.B6-lmr3, filled down triangle; C.B6-(lmr1, lmr2), filled diamond; C57BL/6, square; B6.C-lmr1, triangle; B6.C-lmr2, circle; B6.C-(lmr1, lmr2), diamond. Error bars are not representative of the analysis carried out and therefore have not been included on this plot. P-values were calculated using a permutation test for the test statistic mean T (see Materials and methods section). (b) Statistical comparison of course of infection between selected strains. Mean T and adjusted P-values for each test.

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Figure 3.
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Ratio of IFN-gamma to IL-4 at a late stage of infection. Expression levels of IL-4 and IFN-gamma were calculated in BALB/c, C57BL/6 and congenic mice at week 14–17 after infection using real-time fluorescence PCR. The ratio was taken and the data were log transformed. Each spot represents the log10 of the ratio between IFN-gamma and IL-4 of an individual mouse.

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Production of Th1/Th2-type cytokines in mice during the late stage of infection

As the polarisation of the disease manifestations in the parental and congenic mice was most apparent during the late stages of infection (Figure 2), we chose to examine the signature Th1/Th2 cytokines IFN-gamma and IL-4 in the draining lymph nodes at 14–17 weeks after infection. RNA from inguinal lymph nodes of 36 mice from each of the following groups: BALB/c, C.B6-(lmr1, lmr2), C57BL/6 and B6.C-(lmr1, lmr2) was analysed for the expression of IL-4 and IFN-gamma by real-time fluorescence PCR. A ratio between the starting template values of IFN-gamma and IL-4 was calculated for each mouse (Figure 3). No difference was observed between the parental and compound congenic strains (BALB/c compared to C.B6-(lmr1, lmr2), P=0.30, C57BL/6 compared to B6.C-(lmr1, lmr2), P=0.27, t-test). In contrast, a significant difference was observed between all mice on the C57BL/6 background and those on the BALB/c background (P<0.001, t-test).

Quantitation of cytokines in the draining lymph nodes of infected congenic mice

Piqued by the similarity in the ratio of IFN-gamma to IL-4 between the congenics and parental mice described above, despite the difference in the lesion score, we set out to quantitate the relative expression of several cytokines and cytokine receptors deemed important for the establishment and maintenance of the Th cell phenotype. RNA from inguinal lymph nodes of six chronically infected mice from each of the following groups: BALB/c, C.B6-(lmr1, lmr2), C57BL/6, B6.C-(lmr1, lmr2) was analysed by real-time fluorescence PCR. IL-4, IL-10 and IL10Ralpha chain thought to be involved in the Th2-type responses, and IFN-gamma, IL-12p35, IL12p40 and IL12Rbeta2 chain important for Th1-type responses were examined (Figure 4). The housekeeping gene, porphobilinogen deaminase (PBGD), was used to normalise differences due to the reverse transcription and the amount of cDNA added to each reaction. The only differences observed were increases in IL-4 and IL-10 mRNA, which were significantly higher in BALB/c and C.B6-(lmr1, lmr2) compared to C57BL/6 mice and B6.C-(lmr1, lmr2) mice. No difference was found between any of the strains for IFN-gamma, IL-12p35, IL12p40, IL12Rbeta2 chain or IL10Ralpha chain.

Figure 4.
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mRNA expression levels of Type 1 and Type 2 cytokines quantitated using real-time fluorescence PCR. Expression of (a) IL-4, (b) IFN-gamma, (c) IL10, (d) IL10Ralpha, (e) IL-12p35, (f) IL12p40, and (g) IL12Rbeta2. These data were normalised to PBGD expression and expressed as log10 fold increase compared to C57BL/6- or BALB/c-uninfected controls. 1=BALB/c, 2=C.B6-(lmr1, lmr2), 3=C57BL/6, 4=B6.C-(lmr1, lmr2)

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In view of the increased level of mRNA for IL-4 in the BALB/c and C.B6-(lmr1, lmr2) compared to C57BL/6 and B6.C-(lmr1, lmr2) mice, the relationship between IL-4 levels and disease severity was investigated. Both background strain and lesion score were found to contribute to IL-4 levels. The logarithm of IL-4 level was found to increase linearly with lesion score, with the rate of increase being the same for each strain (Figure 5). Only background strain, not severity of lesion, was correlated with IL-10 levels (not shown).

Figure 5.
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Correlation between IL-4 and lesion size within strains. The relative increase of IL-4 was plotted against lesion score at weeks 14–17 after infection for each strain. A general linear model was used to ascertain whether strain and/or lesion score correlated with log10 IL-4 levels. The lines plotted on the graph are those predicted by the general linear model. Strains are represented as follows: BALB/c, filled diamond; C.B6-(lmr1, lmr2), triangle; C57BL/6, square; and B6.C-(lmr1, lmr2), filled circle.

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Discussion

The polarisation of Th cell responses into the inflammatory Th1 and the humoral Th2 response has been implicated in the pathology of many diseases including infectious diseases, allergies and autoimmune diseases.18 Therefore, the molecules and mechanisms controlling the establishment of a particular Th phenotype have been the focus of many studies. Murine cutaneous leishmaniasis has provided much insight into this model with the extreme case of polarisation of C57BL/6 mice displaying a Th1 response associated with cure, and the BALB/c mice with a Th2 response and progressive systemic disease.13,16 Support for this idea was provided by studies showing that the manipulation of key cytokines such as IL-4, IFN-gamma and IL-12 and their signalling pathways early in infection can alter the disease phenotype.14,15,19 However, it now appears that the IL-4 component of the immune response is not sufficient, and in some cases not necessary for susceptibility, suggesting that other pathways exist to promote parasite survival.16,20

The host response to many infectious diseases is controlled by complex genetics and several studies in mice have shown that resistance to Leishmania is multigenic and it is possible that some of the genes may interact with each other. Based on the biology of the resistant C57BL/6 and the susceptible BALB/c mice, we set out to map the genes involved in this phenomenon without making any assumptions on their potential function.10,11 In that study, three major loci involved in host response to L. major were identified in a cross involving C57BL/6 and BALB/c mice. This current work confirms the important role that these loci play in regulating disease outcome. Through the breeding of animals congenic for these loci, definitive genetic proof of their role in both the response to disease and the biological correlates of this response has been established. The availability of the congenic mice will also allow us to fine map the loci in order to identify the causative genes.

We have used an accelerated breeding program to produce the mice congenic for lmr13. Mice containing the desired regions and fixed for background genome by the sixth backcross (ninth for lmr3) were intercrossed. Using conventional backcrossing methods, approximately 3% of donor genome (excluding the region selected for) would be predicted to be remaining by the sixth backcross. With the active selection for fixation of background strain genotypes over three generations, it could be confidently assumed that much less actually remains in mice bred using marker-assisted breeding.21,22,23 After dense genotyping of the C.B6-lmr2 strain, we identified two small regions of contaminating donor DNA on chromosomes 8 and 14. These were also identified in the C.B6-(lmr1, lmr2) strain derived from the same congenic strain. These regions were assumed to have no effect in our system, as they have not been implicated in any genetic study of response to L. major infection.8,9,10,11,12

Many comparisons were made between the different groups of mice in these experiments. We have employed a rigorous and honest statistical approach, carefully choosing levels of significance taking into account project-wide multiple testing. Therefore, while differences between two groups may differ 'significantly' if only this single or a small number of comparisons was used to determine a significance threshold, significance may not be reached when all tests performed are considered.

Infection of parental BALB/c and C57BL/6 mice led to the well-documented early dichotomy of response, with larger lesions developing at week 3 in the BALB/c mice but not in the C57BL/6. In contrast, little difference was seen between congenic mice and their parental controls in the first 4 weeks of infection. This indicates that the effects of lmr1–3 are not manifest early in infection. This is not surprising as the phenotype used to map these loci was a measure of disease severity over the entire course of the infection.

Analysis of the disease manifestations from week 5 to 12 confirmed the linkage results from our earlier studies, with a significant effect conferred by the C57BL/6 lmr2 allele on a BALB/c background. The predicted disease response was suggested when C57BL/6 lmr1 or lmr3 alleles were bred on a BALB/c background; however, statistical significance was not reached when adjusted for multiple testing. On the C57BL/6 background, only the susceptible BALB/c allele of lmr1 was able to confer susceptibility. This may be due to the observed strong epistatic interaction between the susceptible BALB/c lmr1 and C57BL/6 lmr3 alleles.11 The BALB/c lmr2 allele on its own was unable to overcome resistance on a C57BL/6 background due to either its effect being overshadowed by other C57BL/6 genes or because the BALB/c lmr2 allele may be a neutral allele, not itself contributing to susceptibility. This proposition is supported by the observation that the phenotype measured in the original F2 population was markedly skewed towards resistance.10

Mice congenic for combined lmr1 and lmr2 were bred to test whether the presence of resistance or susceptibility alleles at all three loci identified in our linkage study could recapitulate the disease phenotype of the parental strains. An epistatic interaction between these loci was predicted based on observations in the original F2 mice. Mice with a C57BL/6 background containing the BALB/c lmr1 and lmr2 alleles (B6.C-(lmr1, lmr2)) have susceptibility alleles at all three of the candidate loci, as C57BL/6 lmr3 is the susceptibility allele in the presence of BALB/c lmr1. As a group, these mice were more susceptible than the C57BL/6 mice. However, only 12% of the mice displayed 'BALB/c-like' disease severity. B6.C-(lmr1, lmr2) mice may be slightly less susceptible than B6.C-lmr1; however, this does not reach statistical significance. On the BALB/c background, the presence of all three 'resistance' alleles (C.B6-(lmr1, lmr2)) conferred resistance on the congenic group. Of these mice, 30% have a 'C57BL/6-like' course of disease, completely curing their lesion. This value is an overestimate of the effects of the congenic regions because the parental BALB/c mice often exhibit up to 10% resistant phenocopy.10,24 C.B6-(lmr1, lmr2) was more resistant than either of the congenics containing a single congenic interval; however, only the comparison with C.B6-lmr1 was statistically significant after adjustment for multiple testing. These results suggest that the predicted epistatic interaction between lmr1 and lmr2 may occur, but may not have a large effect on disease severity.

These data validate our original mapping observations, implicating these three loci in the host response to infection with L. major. However, they do not determine the entire phenotype. It is therefore likely that there are additional host response genes segregating in this system that were not identified in our analysis. A calculation of the total percent of the genetic variance contributed by lmr1–3 would suggest that other loci individually contribute less to the total genetic variance than the three loci discussed here.

Much emphasis has been placed on the involvement of the cellular immune response in the causation of disease phenotype in this model. However, some evidence has been reported that the pathology of murine leishmaniasis and the cellular immune response may be dissociated.12 We were therefore interested in comparing the cellular immune responses in the congenic mice to the parental mice and to correlate these with disease severity. In other words, did the host response loci contribute to severity through an altered Th cell response? Following established procedures, cytokines typical of a Type 1 or Type 2 response were measured from RNA extracted from the draining lymph nodes of mice at a time point when the polarised Th response was clearly established. We compared the levels of IL-4 and IFN-gamma using a real-time fluorescence PCR assay.25,26 Mice exhibiting a Th2 response would be expected to produce large amounts of IL-4 and therefore the IFN-gamma to IL-4 ratio would be low. Conversely, mice with a Th1-type response would have a high ratio of IFN-gamma to IL-4.27 This is exactly what was found in the C57BL/6 and BALB/c parental animals. However, contrary to predictions, the C.B6-(lmr1, lmr2) mice had a low ratio of IFN-gamma to IL-4 similar to the parental BALB/c mice and B6.C-(lmr1, lmr2) mice followed the parental C57BL/6 phenotype, exhibiting a high ratio of IFN-gamma to IL-4 mRNA. This indicated that the congenic mice, while differing in disease severity from their parental strain, were displaying the same parental Th-type of cytokine response.

In order to investigate this phenomenon further, several genes encoding products considered important in the establishment and maintenance of the Th responses were more fully analysed. The signature Th2 cytokine IL-4 was one of these molecules, since its presence in the first few hours after infection has been shown to play a determining role in the susceptibility to disease.15 IL-12 has been shown to play a key role in initiating Th1 responses, with IL-12Rbeta chain shown to be downregulated in the susceptible BALB/c mice after the initial burst of IL-4.28,29 IFN-gamma is important in mediating the Th1 response,30 whereas IL-10 and its receptor have been implicated in the Th2 response, although its specific involvement in the response to L. major is controversial.31,32 IL-10 has recently been shown to be required for the persistence of parasites in mice after cure of visible disease.33 Although the IL-10Ralpha chain gene localises to proximal chromosome 9, it is not included in the lmr2 congenic interval.

As anticipated, BALB/c mice exhibited very large lesions, showed signs of progressive disease and had elevated IL-4 levels, whereas all C57BL/6 mice cured their lesions with concomitant low IL-4 levels. The congenic mice as a group exhibited no difference in IL-4 production compared to the parental strain of origin of the backcross genome. This is in the context of a very significant difference in disease course between these groups. However, within each group, there was a correlation between IL-4 level and lesion size of individual mice. IL-4 has previously been shown to be a marker for disease at this late stage of infection.16,34 No difference in the level of Th1-type cytokines was observed between the strains, suggesting that these have already been downregulated in the cured mice and suppressed in the mice exhibiting the Th2-type response.

These results demonstrate that at the time when susceptibility phenotypes are at their most extreme in the BALB/c and C57BL/6 animals, and at a time when differences in clinical outcome are seen between the congenic animals and their backcross parental strains, there is no difference in IL-4 levels. This argues strongly for the case that the Th phenotype has little effect on clinical outcome at this time in infection in these congenic animals. This proposition is also strongly supported by the correlation between lesion score and IL-4 levels in individual, genetically identical mice, arguing again for the IL-4 level being a consequence of the severity of clinical phenotype rather than its cause, at least at this late stage of infection.

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Materials and methods

Breeding of congenic mice

Congenic mice were produced by serially backcrossing BALB/c times C57BL/6 F1 mice to either BALB/c or C57BL/6 mice, with breeders being chosen at generations N3, N4 and N5 based on retention of the interval of interest from the donor parent and with the fewest donor parent alleles elsewhere in the genome. Male mice were genotyped at 30 cM intervals at backcross generations 3 and 4, offset by 15 cM. N5 and N6 mice were then genotyped for retention of the region of interest and in order to check any region not fixed for the background strain allele in previous generations. These mice were intercrossed to produce mice homozygous for the appropriate congenic interval. This was carried out for the lmr1 and lmr2 regions on both the BALB/c and C57BL/6 backgrounds. The single congenics were then intercrossed and bred to homozygosity to produce mice congenic for both lmr1 and lmr2. C.B6-lmr3 mice were intercrossed at N9 after genotyping at a 30 cM density at N3, N5 and N7, offset by 10 cM each time.

After several generations, congenics were genotyped at an average density of 5.5 cM to analyse the background of these mice more closely. Genotyping was performed using PCR with primers from the Whitehead Institute SSLP library fluorescently labelled with one of three dyes. Products were pooled on the basis of size, and dye and products were separated on a denaturing polyacrylamide gel. Fluorescence data were collected on an ABI 377 automated sequencer (PE Applied Biosystems, Foster City, CA, USA) and analysed using the software packages Genescan 2.1 and Genotyper 2 (PE Applied Biosystems). Genotypes were read manually.

Parasites

Parasites were a virulent cloned line, V121 derived from the L. major isolate LRC-L137 (MHOM/IL/67/JerichoII). Promastigotes were maintained in vitro at 26°C in the biphasic blood agar culture NNN and used in the stationary phase of growth.35

Infection with L. major

In all, 25–36 female mice at 7–10 weeks of age from each of the following groups were injected intradermally at the base of the tail,36 with 106 L. major V121 promastigotes in the stationary phase of growth–Experiment 1: BALB/c, C.B6-lmr1, C.B6-lmr2, C.B6-lmr3, C.B6-(lmr1, lmr2); Experiment 2: C57BL/6, B6.C-lmr1, B6.C-lmr2; and Experiment 3: C57BL/6, BALB/c, B6.C-(lmr1, lmr2), C.B6-(lmr1, lmr2). Lesion progression was followed weekly for 12 weeks. A score from 0 to 4 was given to each mouse at each weekly time point as follows: 0=no lesion; 1=a small lump; 2=an open lesion less than 5 mm diameter; 3=an open lesion 5–10 mm diameter; 4=an open lesion >10 mm diameter. After mice reached a lesion score of 4, they were killed. These mice were given a score of 5. This project was approved by the Melbourne University Intercampus Animal Ethics Committee, and all experiments were carried out in compliance with the Australian National Health and Medical Research Council 'Code of Practice for the Care and Use of Animals in Research in Australia', in line with the guidelines of the US National Institutes of Health.

The average weekly increase was measured for each mouse by a least-squares regression with intercept zero. Box plots by strain of the average weekly increase in lesion score experienced by each mouse were plotted. A statistical permutation test was developed to compare pairs of mouse strains over the course of infection. The test statistic (mean t) is the two-sample t-statistic to compare the lesion scores between the two groups at each week, averaged over the course of the infection. Weeks 2–4 and 5–12 were analysed separately in these experiments. A P-value was obtained for the test statistic by simulation. Mice were randomly allocated to each of the two groups and the mean t was recalculated for 10 000 data sets permuted in this way. The P-value is the proportion of permutations, where the mean t is greater in absolute value than the mean t for the original data set. Pairwise comparisons were performed between the nine mouse strains, resulting in 36 pairwise tests and P-values. The 36 P-values were adjusted for multiple testing using a step-down Bonferonni procedure.37 No difference was observed when controls from independent experiments were compared with like controls.

Cytokine assays

In order to determine the ratio of mRNA between IFN-gamma and IL4, RNA was extracted from the inguinal lymph nodes of 36 mice from each of the following groups: BALB/c and C.B6-(lmr1, lmr2) at week 14 postinfection and C57BL/6 and B6.C-(lmr1, lmr2) at week 17 postinfection, challenged in two separate experiments. For the relative quantitation assays, RNA was extracted from the inguinal lymph nodes of six mice from each group (BALB/c, C.B6-(lmr1, lmr2), C57BL/6, B6.C-(lmr1, lmr2)) after 12 weeks infection as well as uninfected parental BALB/c and C57BL/6 mice using Trizol (Life Technologies). Total RNA (1 or 2.5 mug) was reverse transcribed with Superscript II Reverse transcriptase (Stratagene) and oligodT primer in a 20 or 50 mul reaction volume. cDNA was then aliquoted in order to reduce effects due to freeze–thawing. A single aliquot was used for each assay. Real-time fluorescence PCR assays were carried out on the LightCycler (Roche Molecular Biochemicals) using the Faststart DNA Master SYBR Green kit (Roche Molecular Biochemicals). Master mix contains Taq polymerase, buffer, dNTPs and 1 muM MgCl2. Primers were designed to span an intron, to have a melting temperature (Tm) of approximately 60°C and to amplify a product between 97 and 107 bp in length. HPLC-purified primers were obtained from Sigma Genosys. Cytokines and cytokine receptors analysed include IL-4, IFN-gamma, IL-12p35, IL12p40, IL12Rbeta2 chain, IL-10, IL-10Ralpha chain and the housekeeping gene PBGD.38 Optimal magnesium concentrations for each primer set were determined and used in subsequent reactions. Products were analysed for specificity by melting curve analysis and gel electrophoresis (not shown). Primer sequences, magnesium concentrations used, product lengths and Tm's can be seen in Table 2.


A PCR product amplified with each set of primers was purified using Qiagen MinElute PCR purification kit and then quantified by densitometry. A dilution series was made for use as a template for a standard curve, also aliquoted to minimise freeze–thawing effects. For each assay, PCR was carried out under the following conditions: 10 min 95°C, then 40 cycles of 95°C, 10 s; 60°C, 5 s; 72°C, 4 s. A fluorescence reading was taken at the end of each elongation step. Melting curve analysis was performed to ascertain the specificity of the reaction. A standard curve was calculated using the Roche LightCycler software (V3.39) after manual setting of a noise band. The standard curve compared the log copy number of the starting template versus the cycle number at which the sample reached a threshold fluorescence level. Starting copy numbers were calculated for the unknown samples, for each of the products analysed. Either the ratio between levels of IFN-gamma and IL-4 were taken or samples were normalised to PBGD expression and a fold increase over uninfected C57BL/6 or BALB/c mice (calibrator) was calculated as follows: (sample PBGD/calibrator PBGD)/(sample product/calibrator product). The increase in normalised samples from infected mice compared to normalised samples from uninfected mice was used rather than absolute quantitation as the inter-run variation was large as has been previously reported,39 and did not allow comparison of absolute values between runs. Log-transformed results were plotted and analysed using analysis of variance and the t-test. The results were corrected for multiple testing using the Bonferroni correction. The relationship between strain, lesion size and IL-4 or IL-10 mRNA levels was investigated by fitting a general linear model using the statistics software MiniTab 10.5. This allowed the relationship between mRNA levels and lesion size to be compared between the strains.

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References

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Acknowledgements

This work was supported by the National Institute of Health, USA, the Howard Hughes Medical Institute and the National Health and Medical Research Council, Australia. Colleen Elso was supported by an Australian Postgraduate Award. We thank Joan Curtis and Lynn Buckingham for technical assistance.

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