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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 18, 2004 as doi:10.1096/fj.04-1572fje. |
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,
,||,1
* Transplantation Biology and the Departments of
Immunology,
Medicine,
Surgery, and
|| Pediatrics, Mayo Clinic College of Medicine, Rochester, Minnesota, USA
1Correspondence: Transplantation Biology, Mayo Clinic College of Medicine, 200 First St. SW, 2-66 Medical Sciences Building, Rochester, Minnesota, MN 55905, USA. E-mail: platt.jeffrey{at}mayo.edu
SPECIFIC AIMS
The objective of this study was to determine whether mammalian Toll-like receptor 4, the homologue of Toll, could play a role in establishing and/or maintaining the body plan.
PRINCIPAL FINDINGS
1. As C3H/HeJ mice with mutant TLR4 age they develop bones with higher mineral content, size, and density than wild-type mice
To test whether TLR4 affects bone development, we compared the bone mineral content of mice, which have a loss-of-function (Pro712His) mutation in TLR4 (C3H/HeJ), with strain-specific control mice. To assess the mineral content of bones we analyzed whole mice with dual-energy X-ray absorptiometry (DEXA) (Fig. 1
). TLR4 mutant mice had greater bone mineral content (P<0.001) and larger bones (P<0.001, as measured by bone area) than wild-type (WT) mice of the same genetic background; these differences increased as the mice aged (P<0.001, and P<0.01, respectively). Mutant mice had significantly more bone mineral density after 20 wk of age (P<0.001); this difference increased with age (P<0.001) (Fig. 2)
. Increased bone area, mineral content, and density were confirmed by DEXA analysis of tibias (P<0.05, 0.001, and 0.01, respectively) and femurs (P<0.001, 0.001, and 0.05, respectively).
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2. As C57Bl/10ScNCr mice with a deletion of TLR4 age they develop bones with higher mineral content, size, and density than WT mice
To determine whether differences in bone in C3H/HeJ mice result from loss of TLR4 function or a novel signaling pathway specific to TLR4 with a Pro712His mutation, we asked whether C57Bl/10ScNCr mice, which have a deletion of the entire TLR4 gene, have the same phenotype. TLR4-negative mice had larger bones (P<0.05) and at 20–24 wk greater bone mineral content (P<0.05) than strain-specific controls (Fig. 2)
. Like C3H/HeJ mice, divergence of C57Bl/10ScNCr mice from controls increased with age (P<0.05 for bone mineral density, P<0.001 for bone mineral content, P<0.01 for bone area) (Fig. 2)
.
3. By 6 wk of age Balb/c mice with mutant TLR4 develop larger bones than WT mice
To our knowledge, C3H/HeJ and C3H/HeSnJ (or C57Bl/10ScNCr and C57Bl/10SnJ) mice have no genetic differences other than in TLR4. To exclude involvement of loci other than TLR4, we studied C.C3H-TLR4lpsd mice, which have the Pro712His mutation but a Balb/c genetic background. C.C3H-TLR4lpsd mice had larger bones (P<0.05) than controls; bone mineral content, though greater than controls, was not significant by 6 wk of age (P=0.16) (Fig. 2)
. These data in three strains of mice with two different mutations confirm that mice with loss of TLR4 function have larger bones, with more mineral content.
4. All mice continue to gain weight and body fat as they age; TLR4 mutant mice gain less body fat than WT mice
Application of stress to bones, such as carrying increased body mass, leads to larger, denser, and stronger bones; thus, obesity is associated with denser bones and reduced risk of fracture in postmenopausal woman. To determine whether mice with mutations in TLR4 have an increased body mass as one explanation for larger bones with more mineral content, we compared the body weight of various mice. All mutant and control strains of mice tested continued to gain weight as they aged, but at varied rates. Mice with a TLR4 mutation in the C3H background were smaller than controls at all ages tested, as measured by DEXA (Fig. 2)
. To determine whether mice with increased bone mineral content in association with TLR4 mutations have increased (or decreased) body fat, we analyzed fat mass and fat-free mass by DEXA and calculated % body fat. Surprisingly, as TLR4 mutant mice in all three strains grew, they gained up to 70% less body fat than controls (P<0.001 for both); only the C3H/HeJ strain developed less lean (fat-free) body mass. These differences in body fat and % body fat increased with age (P<0.001 for both; C.C3H-TLR4lpsd mice were not studied over time). These data show that differences in body mass do not explain the changes observed in bone.
5. The phenotype of CD14 knockout mice mimicks the bone and fat changes seen in TLR4 mutant mice
CD14 is required for signaling of free agonists via TLR4 unless the dose of agonist is very high, and CD14 is not thought to interact directly with TLR4 except perhaps when TLR4 is actively signaling. Although CD14 is required for responses to free agonists of TLR4, CD14 is not required for the activation of TLR4 by whole bacteria, which contain LPS on their surface. We studied B6.129S-Cd14tm1Frm mice, which have a targeted deletion of CD14 and were backcrossed 20 times onto the C57Bl/6J strain. Although CD14 is a coreceptor for TLR2 and is not required for TLR4 to respond to bacterial infections, the phenotype of CD14 knockout mice is remarkably similar to the phenotype of TLR4 mutant mice (Fig. 1)
. Thus, CD14 knockout mice had increased bone area (P<0.001), bone mineral content (P<0.001), and bone density (P<0.001), less fat mass (P<0.01 at 6–9 wk of age), less % fat (P<0.01), and more lean body mass (P<0.001) than controls. To verify the differences in bone parameters seen by DEXA, we analyzed female CD14 knockout and control mice with peripheral quantitative computed tomography (pQCT). pQCT data reaffirmed the increase in bone area (P<0.01) and mineral content (P<0.01) measured by DEXA but not the increase in cortical bone density.
6. TLR4/CD14 may act together to regulate the body plan
To address whether the phenotypic differences in bone and fat were independent of background genetics, we studied 163 mice, taking into account strain-specific differences. Loss of TLR4 signaling through mutations in TLR4 or CD14 resulted in increased bone density (P<0.05), coronal bone area (P<0.001), and bone mineral content (P<0.001), decreased fat (P<0.001) and % fat (P<0.001); these differences increase as mice age (P<0.001 for all 5) independent of strain.
7. Mutant mice have stronger bones
C3H/HeJ mice have a higher resistance to fracture than WT mice. If the resistance of bone to fracture in C3H/HeJ mice reflects TLR4 deficiency, then this phenotype should be found in CD14 knockout mice. CD14 knockout and control mice were compared in biomechanical 3-point bending analysis. Bones from CD14 knockout mice withstood greater maximum forces before fracture (P<0.001) and had higher overall bending rigidity (P<0.01) than controls. The increased bone strength was consistent with that predicted by pQCT, showing no significant differences in the elastic modulus of the cortical bone tissue. These results support the idea that the CD14 knockout mice have normal bone composition. The increased strength of the tibias of CD14 knockout mice likely reflects in part the larger circumference (P<0.001) and increased content of trabecular bone (P<0.01). However, the tibias had similar length as controls (0.3% larger, P=0.53).
8. TLR4 mutant mice are not more physically active
Although the studies demonstrate that TLR4 affects body plan, the effect could be indirect. The most obvious indirect mechanism would be if absence of TLR4 caused an increase in activity, which in turn caused progressive decrease in body fat and an increase in bone strength. To test this, we monitored female C3H/HeJ and control mice in a photobeam activity monitoring system. C3H/HeJ mice had the same or less physical activity than control mice, as indicated by the number of horizontal, vertical, and stereotypic movements and total distance traveled. This finding is remarkable because only a few phenotypes of high bone mass and low body fat in the absence of increased physical activity have been reported—individuals receiving estrogen replacement and ill and short-lived mice, like the A-ZIP/F-1 transgenic mice, being two exceptions.
CONCLUSIONS AND SIGNIFICANCE
Here we report that TLR4/CD14 complexes, once thought to function only in inflammation and immunity, profoundly affect the body plan, and these effects increase with age. With high bone mineralization and low adiposity, TLR4 and CD14 mutant mice have characteristics of "Adonis," a youth beloved by Venus for his beauty. Yet in contrast to those in whom thin body habitus is acquired, these mutant mice do not exhibit increased physical activity. The connection of TLR4/CD14 function with the properties of bone and fat brings the function of TLRs in mammals much closer to that of Toll in the fly, where Toll is known to be critical for establishment of the body plan (Fig. 2
).
How mutations in TLR4 and CD14 effect changes in bone and fat is not known. Bone growth might be regulated by TLR4 on osteoblast, osteoclasts, or stromal cells, common precursors of osteoblasts and adipocytes. One might imagine that mediators such as TNF
or leptin produced in response to TLR4/CD14 activation could affect bone and fat, but we did not find significant differences in basal serum TNF or leptin between mutant and control mice by ELISA. These or other mediators could increase physical activity, reducing body fat and increasing bone density, but we found no evidence of increased physical activity. The absence of TLR4/CD14 might allow chronic infection, which in turn might decrease body mass; however, the combination of changes we observed has never been reported in chronic infection. The mutant mice exhibited a strikingly uniform increase in bone mineral content (Fig. 1)
, with no change in elastic modulus.
Also uncertain is how TLR4/CD14 might be stimulated in WT mice that would lead to changes in bone density and body fat. TLR4 in WT mice could be stimulated by bacteria or other organisms that colonize the gut or by endogenous agonists; this stimulation could affect the body plan (increase fat, decrease bone density, etc). This manner of stimulation would be lacking in TLR4-deficient mice. That the changes we observed progress with age is consistent with either possibility. Analysis of germ-free animals or those with altered gut flora might help identify the source of the agonist(s) of TLR4 stimulation that changes body habitus. The phenotype is unlikely to be the result of significant systemic activation of TLR4/CD14 (e.g., by infection), since systemic activation of TLR4 causes generalized loss of bone, muscle, fat, and total body weight, and all mice strains tested continued to gain bone, muscle, fat, and body weight as they aged (data not shown). It is unlikely that the phenotype owes to infection (as opposed to colonization), because TLR4-deficient mice should be more susceptible than WT mice to infection and the variation from animal to animal at each time point is relatively narrow. Moreover, CD14 is not required for TLR4 activation by whole bacteria; unlike TLR4 mutant mice, CD14 knockout mice actually resist infection with LPS-containing bacteria better than WT mice. We think it most likely that TLR4/CD14 complexes are activated by endogenous agonists, such as fragments of heparan sulfate proteoglycan and hyaluronic acid, in local tissue environments during development and in normal physiology.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1572fje; doi: 10.1096/fj.04-1572fje
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