Biologic anti-rheumatic drug treatment effects on telomere length in collagen - induced arthritis
Mahlaule, Rixile Allot
Background. Telomere length is considered a marker of biological ageing. Several disease states have been associated with an increased rate of telomere attrition. Shorter telomere lengths are considered a predictor of cardiovascular disease risk. Chronic inflammation increases cell replication, hence increases telomere attrition. Chronic inflammation also increases cardiovascular disease risk. Recent studies in chronic high grade inflammatory conditions in humans have shown controversial results when comparing telomere lengths of patients to healthy controls. Furthermore, paradoxical relationships between telomere length and sub-clinical cardiovascular disease markers have also been reported in inflammatory conditions. Therefore, this study aimed to determine the effect of high-grade inflammation on leukocyte telomere length and whether inflammation induced changes in telomere lengths are affected by biologic anti inflammatory treatment. Methods. Three-month-old, male and female Sprague-Dawley rats were randomly divided into four groups. Three of the groups were exposed to an arthritis inducing protocol where rats received a subcutaneous injection of bovine type-II collagen emulsified in incomplete Freund’s adjuvant at the base of the tail. The other group served as the control (n = 25) that received a saline injection. After inoculation with collagen and upon signs of inflammation, one group received no treatment (Inflammation group, n = 22), one group received Etanercept, a TNF-α inhibitor (TNF-α blocker group, n = 11) every three days for six weeks and one group received Tocilizumab, an IL-6 receptor blocker (IL-6 blocker group, n = 12) once a week for six weeks. Body weight, blood pressure and arthritis scores in the hind paws were measured every two weeks. After six weeks of treatment, cardiac function was measured by echocardiography and vascular reactivity to vasodilators and vasoconstrictors in renal arteries were measured using a wire myograph. Blood was obtained and serum concentrations of TNF-α, IL-6 and C-reactive protein (CRP) were measured by ELISA. Serum vascular adhesion molecule-1 (VCAM 1), a marker of endothelial activation was measured using ELISA. DNA was extracted from whole blood and from visceral adipose tissue. Quantitative real-time PCR (qRT PCR) was used to determine relative telomere length of leukocyte DNA and visceral adipose tissue DNA. Group differences in body weight, blood pressure and arthritis scores were measured by repeated measures analysis of variance (ANOVA). Group differences in inflammatory cytokine concentrations and relative leukocyte telomere length were determined by two-way ANOVA with a Tukey post-hoc test. Group differences in tissue telomere length was determined by a Kruskal-Wallis test. Associations between relative leukocyte telomere length and inflammatory markers, vascular function markers and cardiac function markers were determined by Pearson’s correlations. Results. Body weight and blood pressure did not change over the course of the study and did not differ between the groups (all p > 0.05). Arthritis scores were higher in all groups inoculated with collagen from six weeks onwards, compared to baseline and to controls. Circulating inflammatory marker concentrations were higher in all rats inoculated with collagen compared to the control group (all p < 0.05). Serum VCAM-1 concentrations were significantly higher in the inflammation group compared to the control (p = 0.0008) and TNF-α blocker groups (p = 0.02). Leukocyte telomere lengths were significantly longer in the inflammation group compared to the control group (p = 0.05). Leukocyte telomere length in the TNF-α blocker and IL-6 blocker groups were not different from controls (p > 0.05). Adipose tissue telomere length did not differ between the groups (p = 0.32) and was not associated with leukocyte telomere length (r = -0.10, p = 0.44). Higher circulating CRP concentrations were paradoxically associated with longer leukocyte telomere lengths (r = 0.47, p = 0.001). Controlling for body mass, sex and anti inflammatory treatment did not materially alter this association. Adipose tissue telomere lengths were not associated with circulating inflammatory markers. Longer leukocyte telomere lengths were paradoxically associated with higher systolic blood pressure (r = 0.29, p = 0.05), higher pulse pressure (r = 0.31, p = 0.04), reduced maximal vascular relaxation (r = -0.55, p = 0.006) and reduced maximal vascular contractile (r = -0.37, p = 0.04) responses. Upon adjusting for body weight and sex, the results were materially unaltered, however, treatment with anti-inflammatory drugs impacted the relationship between telomere length and blood pressure, but not the relationship between telomere length and vascular reactivity. In all groups, longer leukocyte telomere lengths were associated with increased VCAM-1 concentrations (r = 0.29; p = 0.05). Longer telomere lengths were associated with increased left ventricular filling pressure (r = 0.35, p = 0.04) in control and inflammation groups. When adjusting for body weight and sex, leukocyte telomere length was associated with impaired left ventricular relaxation (r = -0.37, p = 0.03) and increased left ventricular filling pressure (r = 0.39, p = 0.02). When including the treatment groups in the model, the associations between leukocyte telomere length and cardiac function markers were no longer significant. Adipose tissue telomere lengths were not associated with any marker of vascular function, but shorter adipose tissue telomere lengths were associated with impaired cardiac systolic function, regardless of drug treatment. Conclusion. Exposure to high grade inflammation paradoxically resulted in longer leukocyte telomere length but did not affect adipose tissue telomere length. Treatment with anti-inflammatory drugs impacted the inflammation induced changes in leukocyte telomere length. In control and inflammation groups only, longer leukocyte telomere length was paradoxically associated with impaired vascular function and cardiac function. When including all groups in the analysis, longer leukocyte telomere length remained associated with impaired vascular function, but not with cardiac function markers. Adipose tissue telomere length was not associated with vascular function, but shorter adipose tissue telomere length was associated with impaired cardiac systolic function. These results suggest that exposure to high grade inflammation may impact leukocyte physiology and that leukocyte telomere length may not be an accurate marker of biological ageing or cardiovascular disease risk in patients exposed to high-grade inflammation or those using anti-inflammatory treatments.
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, School of Physiology, University of the Witwatersrand, Johannesburg, 2020