Date of Award

12-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Weikuan Gu, PhD

Committee

Elizabeth A. Fitzpatrick, PhD Kui Li, PhD Linda K. Myers, MD Ryan Yates, PhD

Keywords

arthritis, BCP, bone, mouse model, vitamin C

Abstract

Bone diseases and phenotypes are affected in multiple ways. We focused on studying the effects of genetic and environmental factors, especially their impact on bone properties. Firstly, we investigated the effects of β-caryophyllene (BCP), a naturally occurring dietary cannabinoid, on protecting bone from vitamin D deficiency in mice fed on a diet lacking or supplemented with vitamin D (VD). We found that the VD-deficient diet enhanced the length of femur and tibia bones (P<0.05), and increased bone volume (BV; P<0.01) and the trabecular bone volume fraction (BV/TV; P <0.01) compared to the D+ diet. When given BCP-containing diet, mice exhibited higher BV and bone mineral density (BMD; P<0.05) than the control group. The trabecular and cortical bone were also affected by VD and BCP. In addition, the inclusion of dietary BCP improved the serum concentrations of klotho (P < 0.05). In summary, these data indicate that BCP enhances the level of klotho in the serum, leading to improved bone properties and mineralization in an experimental mouse model. Under conditions lacking UV light, the D-deficient diet could affect multiple properties of bone, including trabecular and cortical bone, in mice. The D-deficient diet can also result in weight loss in mice.

My second project is to evaluate the bone properties in a mouse model with Il-1rn mutation. When knockout for IL-1rn, mice of Balb/c genomic background exhibited susceptibility to spontaneous arthritis disease (SAD), while those of a DBA/1 background were resistant to developing SAD. Our progress on the study of SAD suggested that some of the bone phenotypes, BMD, BV, tibia length, and cortical thickness, were different between wildtype and IL-1rn knockout mice both in Balb/c and DBA/1 strains. The two congenic mouse strains were also evaluated for bone properties. The results revealed that IL-1rn affected BMD differently between Balb/c and DBA/1 mouse strains. The absence of IL-1rn decreased BMD in Balb/c mice and increased BMD in DBA/1 -/- mice compared with wildtype animals. QTL in DBA.B -/- which affect arthritis in congenic strains also regulated BMD, with interferon activated gene 202b (Ifi202b) being the most favored candidate gene for BMD. Our data suggest some of the bone phenotypes are affected by the regulation of gene expression in the context of IL-1ra loss. To our knowledge, this is the first study to investigate the relationship of gene interaction in bone phenotypes with the loss of IL-1ra in an animal model.

In my third study, we studied the different effects between endogenously produced and diet-supplied vitamin C on spontaneous arthritis disease susceptibility. In this study, we sought to investigate whether the source of vitamin C (endogenously produced or exogenously supplied) influences the development of inflammatory arthritis using a mouse model of SAD. SAD-susceptible Balb/c IL-1rn-/- mice were bred with vitamin C-deficient Sfx mice to produce a double mutant (SAD-susceptible, vitamin C-deficient) mouse strain. The three strains were raised, with the double mutant and Sfx mice supplied with vitamin C in drinking water, and mice’s arthritis severity scores were measured biweekly. Incidence and average severity for each strain were calculated. At four months of age, the mice were sacrificed, and body measurements and leg samples were collected. X-ray microcomputed tomography was used to scan the legs to characterize the bone profile. Femur length, tibia length, and bone volume were found to be significantly lower in double mutant mice than in Balb/c IL-1rn-/- mice. There was no significant difference in bone mineral density and femur thickness between the two arthritis-susceptible strains. The double mutant mice had an earlier onset of arthritis as well as a more severe disease than that of the Balb/c knockout (KO) strain. Our findings suggest that the source of vitamin C could affect both the susceptibility of mice to SAD and the severity of disease.

In addition, we examined the sex differences in several mouse models of inflammatory arthritis. To understand the basis for these differences we conducted analysis of several mouse models of inflammatory arthritis. The study of whether there are gender and symmetry differences in experimental arthritis expression in the mouse models may be of significance to the study of human rheumatoid arthritis. Our observations and statistical analyses on the incidence of arthritis in four different animal models incorporated relatively large numbers of mice allowing for robust conclusions. Our research showed that there is a sexual dimorphism for arthritis incidence and severity of arthritis in mice harboring specific genetic modifications. For F2 population the incidence of arthritis was 57.1% in female mice and 75.6% in male mice. There was a difference in severity related to sex in two populations: B6.DR1/ B6.DR4 (P < 0.001) and F2 (P = 0.023). Among these populations, scores for the right hindlimbs were significantly higher than those for the left hindlimbs in males (P<0.05). When examining disease manifestation using the collagen induced arthritis model with DBA/1 mice, sex-dimorphism did not reach statistical significance. However, left hindlimbs showed a tendency toward greater disease expression over the right. Our results suggest that sex difference of arthritis exists in animal models not only in terms of gender, but also of left and right limbs. Using animal models, this work has laid the foundation for future research on gender differences in rheumatoid arthritis.

In conclusion, our studies on genetic and environmental factors regulating bone phenotypes and diseases have significant implications. Not only have they raised the concept of sexual dimorphism in disease, but they have highlighted the influence of genetic background on bone disease. The link between different sources of vitamins and immune-mediated disease is intriguing and warrants further research, as is the bone protective effect of BCP.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

http://orcid.org/0000-0002-3323-7835

DOI

10.21007/etd.cghs.2021.0553

Additional Files
2021-024-Dong-DOA.pdf (180 kB)
Declaration of Authorship
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