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Department of Pathology, University of Utah, Salt Lake City, UT 84132;
Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802; and
Departments of Agronomy and Statistics, Purdue University, West Lafayette, IN 47907
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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60% of untreated individuals developing
arthritis. The murine model of Lyme disease has provided strong
evidence that the genetic composition of the host influences the
severity of arthritis following infection with Borrelia
burgdorferi: infected C3H mice develop severe arthritis while
infected C57BL/6N mice develop mild arthritis. Regions of the mouse
genome controlling arthritis severity and humoral responses during
B. burgdorferi infection were identified in the
F2 intercross generation of C3H/HeNCr and C57BL/6NCr mice.
Rear ankle swelling measurements identified quantitative trait loci
(QTL) on chromosomes 4 and 5, while histopathological scoring
identified QTL on a unique region of chromosome 5 and on chromosome 11.
The identification of QTL unique for ankle swelling or
histopathological severity suggests that processes under distinct
genetic control are responsible for these two manifestations of Lyme
arthritis. Additional QTL that control the levels of circulating Igs
induced by B. burgdorferi infection were identified on
chromosomes 6, 9, 11, 12, and 17. Interestingly, the magnitude of the
humoral response was not correlated with the severity of arthritis in
infected F2 mice. This work defines several genetic loci
that regulate either the severity of arthritis or the magnitude of
humoral responses to B. burgdorferi infection in mice,
with implications toward understanding the host-pathogen interactions
involved in disease development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies with the murine model of Lyme disease have provided evidence that the genetics of the host contributes to the severity of B. burgdorferi-induced arthritis (8). Several substrains of the C3H mice develop severe arthritis 3–4 wk following infection (8, 9). The arthritis, which is representative of human disease, is reproducibly found in the rear ankle joints, where its development can be monitored by measurement of ankle swelling and by histopathological assessment (10). High levels of B. burgdorferi DNA can be detected in arthritic joint tissues (5).
BALB/cAnN and C57BL/6NCr mice are relatively resistant to B. burgdorferi-induced arthritis; however, resistance in these two strains appears to be mediated by distinct mechanisms (11). Arthritis in BALB/cAnN mice is mild when the inoculum dose is small (102–103 B. burgdorferi) but becomes severe at high inoculum doses (104–105). Mild arthritis in BALB/cAnN mice is correlated with 5- to 10-fold fewer spirochetes in joint tissues than found in tissues of severely arthritic mice. Arthritis resistance in C57BL/6NCr mice has distinct characteristics, with mild arthritis seen even in mice infected with very high inoculums of B. burgdorferi (105). Spirochete levels in joint tissues of C57BL/6NCr mice are high at all infectious doses, similar to those found in severely arthritic C3H/HeNCr mice. This suggests that the localized inflammatory responses in infected joint tissues of C57BL/6NCr mice are more tightly regulated than in C3H/HeNCr mice. These observations suggest that genetic traits possessed by C57BL/6NCr mice are responsible for more regulated inflammation seen in their joints and that the responsible genetic loci may be identified by quantitative trait assessment in intercross populations bred from C57BL/6NCr and C3H/HeNCr mice.
In this study, the mode of inheritance of susceptibility to severe arthritis was analyzed by microsatellite mapping of quantitative trait loci (QTL)3 in the F2 intercross of C3H/HeNCr and C57BL/6NCr mice. Several QTL regulating arthritis severity were identified by measurements of rear ankle swelling and by histopathological scoring of joints taken from infected mice. Interestingly, the two measurements of arthritis severity were linked to distinct genomic regions. Additional, distinct QTL were identified that regulate plasma levels of specific and total Igs in infected F2 mice. These results indicate that the severity of arthritis induced by B. burgdorferi infection in mice is regulated by multiple genes, that the magnitude of the humoral response does not regulate arthritis severity, and that distinct genetic loci may be involved in different components of Lyme arthritis development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C3H/HeNCr, C57BL/6NCr, and B6C3F1 (C3H/HeNCr x C57BL/6NCr) mice were obtained from the National Cancer Institute (Bethesda, MD). B6C3F1 males and females were bred to generate F2 mice. All mice were housed in the Animal Resource Center at the University of Utah Medical Center according to guidelines of the National Institutes of Health for the care and use of laboratory animals.
B. burgdorferi infection
Mice between 5 and 7 wk of age were infected by intradermal injection in the shaven back with 2 x 103 of the N40 isolate of B. burgdorferi (provided by Dr. Stephen Barthold, University of California at Davis, CA (11) at passage 3 from an infected mouse) and used at passage 5. Spirochetes were grown in BSK-H medium containing 6% rabbit serum (Sigma, St. Louis, MO), enumerated by counting in a Petroff-Hauser chamber, and diluted with sterile medium. Infection was followed in five male and five female mice of each parental strain (C3H/HeNCr, C57BL/6NCr, and B6C3F1) and in 150 F2 mice (mixture of male and female). Mock-infected groups consisted of five male and five female mice of each parental strain that received an intradermal injection of an equivalent volume of BSK-H.
Detection of B. burgdorferi-specific and total circulating Ig levels
Serum samples from infected and control mice were taken from each mouse at the 4-wk sacrifice and analyzed for B. burgdorferi-specific Ab by a quantitative Ab capture ELISA (12). Eleven columns per 96-well plate were coated with 10 µg of B. burgdorferi sonicate per ml. The 12th column was coated with goat anti-mouse IgG, IgM, and IgA (Zymed, South San Francisco, CA) at a concentration of 5 µg/ml. Dilutions of serum were added to wells coated with B. burgdorferi sonicate, and known concentrations of murine IgG and IgM were added to the wells coated with goat anti-mouse Ig to form a standard curve. Unbound samples were removed by washing, and the anti-B. burgdorferi IgG and IgM were detected by the addition of horseradish peroxidase-conjugated Abs to murine IgG or IgM (Boehringer Mannheim, Mannheim, Germany), respectively. Plates were developed by incubation with 0.4 mg of O-phenylenediamine per ml and 0.01% H2O2 (Sigma) and read at an OD of 492 nm with a Vmax 96-well microtest plate spectrophotometer (Molecular Devices, Palo Alto, CA). Comparison of the OD of B. burgdorferi wells with the OD of the standard curve allowed estimation of Ag-specific Ab levels. Sera from mock-infected mice had B. burgdorferi-specific IgG levels <100 ng/ml, whereas sera from infected mice had specific IgG levels >20 µg/ml. This value was also used to confirm that experimental mice had become infected.
The concentration of total circulating IgG and IgM was determined by Ab capture ELISA with plates coated with goat anti-mouse IgG, IgM, and IgA as described above (10). Dilutions of serum were added to wells, and the amount of bound murine Ig was detected by the addition of horseradish peroxidase-conjugated Abs to murine IgG or IgM and read as described above. Ig levels were determined by comparison with a standard of IgG or IgM included on each plate.
Measurement of the ankle joints
Rear ankle joints of mice were measured with a metric caliper
(Mitutoyo, Tokyo, Japan) at sacrifice. Measurements were taken
in the anterior/posterior position, with the ankle extended, through
the thickest portion of the ankle (10). Ankle joints from uninfected
mice have a diameter of 3 mm at this position, with measurements up
to 4.5 mm observed in severely arthritic mice.
Histopathology of ankle joints
The rear ankle joint displaying the greatest swelling at the 4-wk sacrifice was taken from each mouse for histological analysis. Samples were fixed in 10% formalin, decalcified, embedded in paraffin, and sections were stained with hematoxylin and eosin. Slides were viewed in a blinded fashion and given a score for severity (11). A score of 4+ indicated the greatest severity in tissues of the ankle and tibia and was characterized by a large region of edema with many neutrophils present, thickening of the tendon sheath, and evidence of bone and cartilage abnormalities within the tendon sheath. A score of 1+ displayed slight thickening of the tendon sheath, with little edema or neutrophil infiltration. A score of 0 was given to samples indistinguishable from mock-infected controls. Scores of 2 and 3 were assigned to samples with intermediate pathology. Histopathology was independently evaluated by a second pathologist using slightly different criteria for scoring, which placed greater weight on certain aspects of pathology including inflammatory cell infiltrate and bone remodeling. Correlation analysis using the PROC CORR function of SAS found a correlation of 0.7 between the two assignments (13).
A second quantification of histopathological severity was obtained by measurement of the thickness of the tendon sheath of the cranial tibial muscle using Image Pro 1.3 software (Media Cybernetics, Silver Spring, MD) and a Leitz Orthoplan microscope (Leitz, Stuttgart, Germany) fitted with a Sony CCD-iris/RGB color video camera (Sony, Tokyo, Japan). The average of four measurements made at approximately equal spacing was used for QTL analysis.
Preparation of DNA from infected tissues
Mice were sacrificed at 4 wk following infection, and rear ankle joint tissues and hearts were prepared as previously described (11, 12). Tissues were placed individually in 15-ml polypropylene tubes containing 2.5 ml of a 0.1% collagenase A (Boehringer Mannheim) solution in PBS, pH 7.4. Samples were digested with collagenase for 4 h at 37°C and then mixed with an equal volume of 0.2 mg/ml proteinase K (Boehringer Mannheim) in 200 mM NaCl, 20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1% SDS for 16 h at 55°C. DNA was recovered by extraction with an equal volume of phenol:chloroform and precipitation with ethanol. Following digestion with 1 mg/ml DNase-free RNase (Sigma), the DNA samples were subjected to a second extraction and precipitation, and finally resuspended in 1.5 ml dH2O. The average recovery was 200–400 µg DNA from each heart and ankle, as determined by absorbency at 260 nm.
Quantification of B. burgdorferi DNA in mouse tissues
B. burgdorferi DNA levels were determined in DNA prepared from hearts of infected F2 and parental mice by continuous monitoring PCR using the Light Cycler (Idaho Technologies, Idaho Falls, ID) (14). This technique, which is fully described elsewhere,4 monitors the cycle-by-cycle accumulation of fluorescently labeled product. The cycle at which the product is first detected is used as an indicator of relative starting copy. The chromosomally encoded recA gene was used for quantitation of B. burgdorferi levels in mouse hearts and was normalized relative to 106 copies of a single-copy mouse gene.
Genotyping of F2 mice
To characterize the genetic control of arthritis severity and humoral responses in C3H/HeN mice, we constructed a genetic linkage map of the mouse genome using 150 F2 mice of a C3H/HeNCr and C57BL/6NCr intercross. A total of 195 microsatellite markers that discriminated between C3H/HeNCr and C57BL/6NCr alleles were used to perform a 20-cM scan of the mouse genome. Microsatellite primers were either purchased from Research Genetics (Huntsville, AL) or synthesized according to sequences obtained through the Whitehead Institute/Massachusetts Institute of Technology mouse genome database (www.genome.wi.mit.edu/cgi-bin/mouse/index). Microsatellite typing was done as described (15, 16, 17). PCR was performed using a PTC100 Programmable Thermal Controller (MJ Research, Watertown, MA) with the following program: 1 cycle at 94°C for 3 min; 35 cycles at 94°C for 1 min, 55°C for 2 min, 72°C for 2 min; 1 cycle at 72°C for 10 min. The PCR-amplified microsatellite size variants were resolved by electrophoresis on large-format denaturing 7% polyacrylamide gels and visualized by autoradiography.
Linkage analysis
Linkage maps were estimated using the Kosambi map function within the MAPMAKER/EXP computer package (www.genome.wi.mit.edu/ genome_software/other/mapmaker.html) (18). Quantitative trait linkage analysis was performed by interval mapping using MAPMAKER/QTL under the assumptions of a free genetic model (19). Composite interval mapping was performed using QTL-Cartographer (statgen.ncsu.edu/qtlcart/ cartographer.html) (model 6) when the possibility of multiple loci linked on a single chromosome existed (20, 21, 22). Significant markers from the entire genome were used as a control for the genetic background. A window size of 15 cM was used on either side of the test interval to analyze a particular region independently from the rest of the chromosome.
Significant linkage of QTL to marker loci for all analyses was determined by permutation threshold analysis using QTL-Cartographer (22, 23). This method of analysis takes into account the specifics of the experimental situation and satisfies the multiple testing issues implicit in the genome (23). For this experiment, 1000 permutations of the actual data were generated to provide the sampling distribution of test statistics under a null hypothesis of no linkage. A permutation was generated by randomly reassigning all of the phenotypes from every mouse to each of the genotypes generated by the microsatellite typing. A new test statistic (LOD score) was found at each test site in each permutation, and the highest test statistic from the entire genome was selected. The highest test statistics for each of the 1000 permutations were then ordered, and the threshold for significant experimentwise linkage was taken at the 95% level (24). Loci showing significant linkage were given designations resulting in the nine proposed disease-modifying loci in this study.
Interaction between marker loci
Interaction between marker loci was investigated using a general linear model in SAS (PROC GLM) (13). Models were used to analyze the effects of significant loci on several phenotypes associated with infection. Significant markers for ankle swelling (D5 Mit431 and D4 Mit87), histopathology (D11 Mit120 and D5 Mit312), total IgM (D6 Mit194 and D12 Mit139), specific IgM (D6 Mit200, D11 Mit51, and D17 Mit232), and total IgG (D9 Mit48 and D12 Mit139) were analyzed as independent variables using multiple regression analyses with the appropriate phenotype as the dependent variable (25). To investigate possible interactions between significant marker loci for each phenotype associated with infection, two-locus interaction terms were added to the multiple linear regression models as independent variables. The model was made with the dependent and independent terms, and all model assumptions were verified.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C3H/HeNCr, C57BL/6NCr, B6C3F1 (C3H/HeNCr x
C57BL/6NCr), and F2 intercross mice were studied for
arthritis severity and humoral immune response following infection with
B. burgdorferi (characteristics for parental mice are
summarized in Table I
). No differences
were seen between responses of infected parental male and female mice,
allowing data for males and females to be pooled. C3H/HeNCr mice
developed severe arthritis when infected with B.
burgdorferi, with a mean ankle measurement of 4.23 ± 0.23 mm
and an average histopathological score of 3.7 ± 0.48. Infected
C57BL/6NCr mice developed milder arthritis with ankle measurements of
3.01 ± 0.12 mm and histopathological scores of 0.8 ± 0.71.
Arthritis measurements for these parental strains formed nonoverlapping
groups (p < 0.005 for both ankle measurements
and histopathology). Arthritis was intermediate in B6C3F1
mice, with ankle measurements of 3.56 ± 0.22 mm and
histopathology of 1.8 ± 0.86. The complete range of arthritis
severity was observed in the F2 population, as is
graphically displayed for ankle swelling and tendon sheath thickness in
Fig. 1, A and B.
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). Less obvious differences
were also observed in total IgM levels in infected mice of these two
strains (1.1 ± 0.14 mg/ml for C3H/HeNCr and 0.97 ± 0.18
mg/ml for C57BL/6NCr) and in the B. burgdorferi-specific IgG
of the parental strains (76 ± 13 µg/ml for C3H/HeNCr and
44 ± 11 µg/ml for C57BL/6NCr mice). The F2
population of mice displayed a complete range of values for total IgM
(0.26–1.75 mg/ml), B. burgdorferi-specific IgM (0.96–11.08
µg/ml), and B. burgdorferi-specific IgG (21.5–101.0
µg/ml).
B. burgdorferi levels in tissues of infected mice were
determined by quantitative PCR. As found previously, B.
burgdorferi levels in ankle joints of C3H/HeNCr and C57BL/6NCr
mice were similar (11); thus, levels in joint tissues of F2
mice were not assessed. In contrast, hearts from infected C3H/HeNCr
mice had ninefold greater levels of B. burgdorferi DNA than
did hearts from C57BL/6NCr mice, suggesting a genetic regulation of
spirochete persistence in some tissues (Table I). B.
burgdorferi levels in the hearts of F2 mice ranged
from 14 to 6158 DNA molecules per 106 mouse cells.
Arthritis severity maps to chromosomes 4, 5, and 11
To characterize the genetic control of arthritis severity and humoral responses in C3H/HeNCr mice, we constructed a genetic linkage map of the mouse genome using 150 F2 mice of a C3H/HeNCr and C57BL/6NCr intercross. A total of 195 microsatellite markers that discriminated between C3H/HeNCr and C57BL/6NCr alleles were used to perform a full genome scan. Intermarker recombination fractions on different chromosomes ranged from 0.037 to 0.107 with a maximum intermarker distance of between 8 and 18 cM. Three parameters were used to assign arthritis severity: measurement of rear ankle joints, histopathological severity, and thickening of tendon sheath.
The measurement of rear ankle swelling demonstrated significant results
on both chromosomes 4 and 5. The QTL for rear ankle swelling was
significantly detected in genomic intervals defined by (D4
Mit171 and D4 Mit172) and (D4 Mit237 and
D4 Mit26). The LOD scores for these regions were greatest at
4.22 and 4.79, respectively, and were deemed significant by comparison
to 95% experimentwise threshold values estimated for these data (23).
Because of the limited capability of interval mapping to detect
multiple traits on a chromosome, composite interval mapping was used on
chromosome 4 (20, 21). A single significant region between D4
Mit87 and D4 Mit145 was found (maximal LOD = 4.70,
95% threshold = 2.53), implying a single locus or region of
related genes as being significant. The locus in this region was
designated Bb1 for B. burgdorferi-associated
arthritis phenotype 1 (Table II).
Bb1 represents a C3H/HeNCr allele with a dominant influence
on susceptibility to severe arthritis. An additional QTL associated
with ankle swelling was detected (LOD 4.07) on chromosome 5 in the
interval (D5 Mit24 and D5 Mit98) (Table II). This
locus was designated Bb2, and was attributed to C3H/HeNCr
alleles with a recessive influence on susceptibility to severe
arthritis.
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). The
thickness of tendon sheaths was also associated (LOD 4.07) with this
interval (D5 Mit308 to D5 Mit91) on chromosome 5
(Table II). This locus was designated Bb3, with alleles from
C3H/HeNCr mice having a dominant effect on arthritis severity.
Histopathological scoring also demonstrated significant association
with the interval (D11 Mit350 and D11 Mit120) of
chromosome 11. The region demonstrating greatest linkage (LOD 3.91) was
termed Bb4. Alleles of Bb4 from the C3H/HeNCr
mouse contribute to severe arthritis and are recessive. The slides were
scored independently by two individuals, and in both cases linkages to
these regions of chromosomes 5 and 11 were obtained. A summary of the
linkages to arthritis severity are included in Tables II and
III. Quantitative analysis of humoral response to B. burgdorferi in F2 intercross mice
As described above, quantitative differences were found between the humoral immune response of C3H/HeNCr and C57BL/6NCr mice to B. burgdorferi. A locus regulating the elevation in total circulating IgM and in B. burgdorferi-specific IgM was identified on chromosome 6 between markers D6 Mit105 and D6 Mit15. This locus was termed Bb5, and alleles from C57BL/6NCr mice contribute in a recessive manner to lower levels of IgM production (Tables III and IV). A second locus that influences the levels of total circulating IgM and IgG was identified on chromosome 12 between D12 Mit121 and D12 Mit133. This locus was termed Bb6, and alleles from C3H/HeNCr mice contributed to elevated levels of IgG and IgM in a recessive manner. Two additional loci were mapped that influenced B. burgdorferi-specific IgM. Bb7 was identified on chromosome 11 between D11 Mit82 and D11 Mit235, and its effect was identified as a dominant influence leading to lower Ig levels by alleles from C57BL/6NCr mice. Bb8 was identified on chromosome 17 between markers D17 Mit175 and D17 Mit215, and mice heterozygous for Bb8 had the lowest levels of specific IgM. A second locus was also identified that regulated the level of total IgG. This locus, termed Bb9, mapped between D9 Mit48 and D9 Mit73 on chromosome 9. The presence of C57BL/6NCr alleles of Bb9 had a dominant effect resulting in reduced levels of total IgG, as summarized in Tables III and IV.
Correlation between disease severity and humoral response to B. burgdorferi
The outer surface lipoproteins of B. burgdorferi are potent B cell mitogens, suggesting that the degree of humoral response in infected mice could reflect an important aspect of infection influencing disease severity (26). Analyses were performed to determine whether levels of total Ig or B. burgdorferi-specific Ig were correlated with arthritis severity in the F2 population. Statistically significant differences in Ig levels were found in the parental strains, and the entire range of responses were identified in the F2 population. However, there was no significant correlation between arthritis severity (either ankle swelling or histopathological score) and specific or total Ig levels (r2 < 0.18 for all comparisons).
Genetic regulation of levels of B. burgdorferi in hearts from F2 mice
Assessment of levels of B. burgdorferi DNA in the hearts of F2 mice revealed suggestive linkage to chromosome 4. This linkage was below the threshold of statistical significance and will require further studies for confirmation. It is interesting to note that the linkage to chromosome 4 is in the region of Bb1, which regulates ankle swelling. However, strong correlation was not observed between spirochete levels in the hearts of F2 mice and ankle swelling (r2 = 0.1).
Assessment of interaction of loci associated with infection and disease in F2 mice
Because the regulation of arthritis severity and humoral responses were found to be multigenic traits, possible interactions between significant loci for each trait were determined using the most significant marker associated with each QTL (27). Two interactions were suggested: one for ankle swelling measurements between Bb1 and Bb2 on chromosomes 4 and 5; and the second for levels of total IgG between Bb9 and Bb6 on chromosomes 9 and 12. No statistically significant interactions were noted for significant markers for histopathology score, total IgM, specific IgM, or specific IgG.
The significant interaction found for the ankle swelling phenotype was
between the markers D4 Mit87 (Bb1) and D5
Mit431 (Bb2), (F = 6.48,
p < 0.001, R2 = 28.35). The
interaction between Bb1(D4 Mit87) and
Bb2 (D5 Mit431) for ankle swelling was further
studied by plotting the genotypes at these two loci (Fig. 2A). F2 mice that
were homozygous for C3H/HeNCr alleles at Bb1 (D4
Mit87) or Bb2 (D5 Mit431) had severe ankle
swelling, regardless of the genotype at the second locus
(Bb2 or Bb1, respectively). Additionally,
F2 mice homozygous for C57BL/6NCr alleles at either
Bb1 or Bb2 were not protected from severe ankle
swelling conferred by the presence of a single C3H/HeNCr allele at the
second locus, (Bb2 or Bb1, respectively).
F2 mice heterozygous at both loci have increased ankle
swelling, supporting an interaction between C3H/HeNCr alleles at the
two loci.
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B). Those F2 mice
with one or two C57BL/6NCr alleles at Bb9 exhibited low
levels of total IgG. However, an increase in total IgG was seen if two
C3H/HeNcr alleles were present at Bb6 along with one or two
C3H/HeNCr alleles at Bb9. Interestingly, animals which were
heterozygous for the two alleles at Bb6 had a lower level of
total IgG than did mice with any other allelic combination at
Bb6 or Bb9. This suggests a more complex
interaction exists between alleles at these two loci or that
there may be an additional (three-way) interaction with a separate
locus in the genome. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table I). The fact that some humans infected with B.
burgdorferi develop little to no arthritis while others display
severe symptoms suggests that common pathways may regulate the severity
and progression of Lyme disease in mice and humans.
In this study, arthritis severity was determined 4 wk following
infection with B. burgdorferi in 150 mice from an
F2 intercross of C3H/HeNCr and C57BL/6NCr mice. Three
distinct chromosomal regions that regulate arthritis severity in the
F2 population were identified by linkage analysis
(summarized in Table III). QTL that regulate the degree of ankle
swelling were mapped to chromosomes 4 (Bb1) and 5
(Bb2). Two other QTL that were identified from the
histopathological scores or measurements for the ankle joints mapped to
chromosomes 5 (Bb3) and 11 (Bb4). Bb2
and Bb3, which were identified by different measurements of
arthritis severity, map to adjacent regions of chromosome 5.
Interestingly, the identification of QTL unique for ankle swelling on
chromosome 4 (Bb1) and unique for histopathological severity
on chromosome 11 (Bb4) suggests that different processes are
involved in these two arthritis-associated developments. The loci
controlling ankle swelling could be expected to regulate edema and
inflammatory cell infiltrate, whereas the loci regulating
histopathologically determined severity may be required for synovial
hyperproliferation and other abnormalities associated with the tendon
sheath. This suggests that ankle edema and synovial hyperproliferation
are not sequential steps, but rather they are independently controlled
events contributing to the development of Lyme arthritis.
Interestingly, Anguita and colleagues found that Ab neutralization of
IL-12 reduces B. burgdorferi-induced histopathology with
little effect on ankle swelling (28, 29), consistent with our
findings that these two components of Lyme arthritis development are
under distinct genetic control (Table II).
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It is noteworthy that the tick component of natural infection has been eliminated from these studies, and that any contribution the tick might make to the regulated expression of B. burgdorferi Ags or to the magnitude or type of immune response cannot be assessed (40). Thus, it is possible that additional QTL might be identified in intercross populations infected by ticks. Further, the presence of Abs to specific Ags was not assessed in this study, but could provide additional information about regulation of immune responses: others have identified MHC regulation of murine serological responses to B. burgdorferi Ags (41).
The level of B. burgdorferi DNA present in hearts of F2 mice was determined as an indication of the generalized level of spirochete persistence in tissues of these animals (5, 11, 12). Although a suggestive linkage (90% experimentwise permutation threshold) to the region on chromosome 4 that regulates ankle swelling was detected, there was not a strong correlation between B. burgdorferi levels in hearts and ankle swelling. Also surprising was the lack of correlation between tissue spirochete levels and the magnitude of humoral responses in F2 mice. This suggests that the regulation of the magnitude of humoral responses to B. burgdorferi infection is not dependent on the quantity of spirochetes in tissues.
Previous studies on the genetic contribution to arthritis severity have indicated that components of the inflammatory response may be very important in the outcome of disease. Arthritis develops in mice with the scid mutation, indicating that B and T lymphocytes are not required for disease development (42, 43). C3H mice with the scid mutation also develop more severe arthritis than do CD-17 mice (BALB/c congenic) with the scid mutation, further indicating that differences in arthritis severity are not dependent on B and T lymphocytes (42). This is further supported by experiments using both MHC congenic mice and mice genetically deficient in class II MHC molecules. These studies have indicated that class II-dependent responses, and, therefore, CD4+ T cells, are not involved in arthritis development or resolution (10, 44, 45). It should be noted that in humans an association has been made between HLA-DR4 alleles and the development of treatment-resistant arthritis in a small subgroup of Lyme patients (46). This appears to be mediated by a distinct mechanism from the treatable, subacute arthritis observed in most patients and studied in mice.
Our mapping studies support the prediction that host elements other
than the acquired defenses regulate the severity of Lyme arthritis as
QTL for arthritis severity were not found within loci encoding Igs, the
TCRs, or H-2. The assignment of significant linkage to chromosomal
regions also allows speculation about previously identified genes lying
within the linkage interval that could be involved in the severity of
arthritis development in the mouse (Table V). Several of these genes are directly
involved in inflammation or inflammatory control, including:
Gro1, the GRO1 oncogene that is involved in chemotaxis of
neutrophils, macrophages, and lymphocytes; Pdgfra, the
platelet-derived growth factor receptor; and Pafaha,
platelet-activating factor acetylhydrolase. Other genes of interest are
involved in the responses of PMNs or macrophages to microbial and
inflammatory stimuli: Lps, the LPS response gene;
Nos2, the inducible nitric oxide synthase gene;
Tnfip1, TNF-induced protein 1; and Spp1, secreted
phosphoprotein 1, which determines innate resistance to
Rickettsia tsutsugamushi. C3H mice expressing different
alleles of the Lps gene (C3H/HeJ,
lpsr and C3H/HeN, lpss)
do not display any differences in response to B. burgdorferi
infection, including arthritis severity and kinetics, spirochete
dissemination and persistence, or humoral response (5, 9). Studies, in
which the production of nitric oxide, the product of the enzyme encoded
by Nos2, has been blocked in infected mice, have
indicated this molecule is not involved in arthritis development or
host defense in C3H/He and BALB/c mice (12). However, it is still
possible that subtle variations in these genes could contribute to
differences in disease severity on some backgrounds.
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This study is the first attempt to address the complexity of the
genetic regulation of arthritis development induced by B.
burgdorferi. Multigenic control of arthritis development was
demonstrated, with two loci responsible for joint swelling and two loci
responsible for histopathological severity. Additional loci were
identified that regulate humoral responses to B.
burgdorferi. A more complex interaction between loci may also
determine the severity of arthritis and the magnitude of the humoral
response. These studies provide an objective estimation that four
different genes regulate arthritis severity in mice. This finding will
allow further analysis of the actual genes involved in arthritis
development and a detailed assessment of the pathways involved, with
implication for understanding disease development in humans. The fact
that only a portion of humans not treated for early symptoms of Lyme
disease go on to develop severe arthritis strongly supports a role for
genetic regulation of disease severity in humans. The analysis of
syntenic regions in the human genome (Table III) will provide further
opportunity for characterization of candidate genes involved in the
regulation of Lyme arthritis in humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Janis J. Weis, Department of Pathology, University of Utah School of Medicine, 50 N. Medical Drive, Salt Lake City, UT 84132. E-mail address:
3 Abbreviations used in this paper: QTL, quantitative trait loci; LOD, logarithm of the odds favoring linkage; CM, centimorgan.
4 T. B. Morrison, Y. Ma, J. H. Weis, and J. J. Weis. Rapid and sensitive quantification of Borrelia burgdorferi-infected mouse tissues using continuous fluorescent monitoring of PCR. Submitted for publication.
Received for publication June 18, 1998. Accepted for publication September 30, 1998.
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B. Infect. Immun. 64:3845.[Abstract]
in modulation of immunity to Borrelia burgdorferi in mice. J. Immunol. 155:2020.[Abstract]
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