Elevated indoleamine-2,3-dioxygenase enzyme activity in a novel mouse model of HIV-associated atherosclerosis

Abstract

Objective: HIV atherosclerosis and cardiovascular disease (CVD) represent a significant human health burden in the era of combination antiretroviral therapy (cART). The pathogenesis of HIV atherosclerosis is still poorly understood, due, in part, to the lack of a suitable small animal model. Indoleamine-2,3-dioxygenase (IDO) enzyme activity is the first and rate-limiting step in tryptophan catabolism and is measured by the kynurenine to tryptophan ratio (KTR). The serum KTR is a biomarker of inflammation and has recently been implicated as an important risk factor for CVD in patients living with HIV (PLWH) who are virologically suppressed under cART. However, IDO activity in HIV-associated CVD has not been studied in mouse model before.

Design: A novel mouse model of HIV atherosclerosis (Tg26/ApoE) was generated and examined for IDO activity and atherogenesis throughout 8 weeks on a high-fat diet. Tg26/ApoE mice were compared with Tg26 and ApoE single transgenic mice, before and during a high-fat diet.

Method: Serum kynurenine, tryptophan and percentage of aortic plaque formation were measured. Additionally, levels of relevant cytokines were investigated in Tg26/ApoE and ApoE.

Results: Tg26/ApoE developed an accelerated atherosclerosis with increasing levels of KTR that were associated with plaque progression. This accelerated plaque was potentially driven by elevated levels of circulating IL-6.

Conclusion: These results indicate that Tg26/ApoE serve as a new mouse model for HIV-induced atherogenesis, and aid in understanding the role of tryptophan catabolism in the pathogenesis of HIV atherosclerosis/CVD.

Figure 1:. Accelerated Atherosclerosis in Tg26^+/−/ApoE^−/− after 8 weeks on a HFD.

(A) Representative en face Aorta from the ApoE^−/− and Tg26^+/−/ApoE^−/− mice stained with Oil Red O for quantifaction of plaque percentage from arch to ileal bifurcation. (B) Tg26^+/−/ApoE^−/− mice had significantly higher plaque formation as measured as a percentage of en face (13.74 ± 1.349%; N=9) compared to ApoE^−/− controls (9.293 ± 0.7756%; N=8, p<0.05). (C and D) There was no any significant difference in body weight and blood urea nitrogen level between Tg26^+/−/ApoE^−/− and ApoE^−/− mice. (E) Tg26^+/−/ApoE^−/− mice had significantly lower levels of cholesterol than ApoE^−/− mice. However, the result is not reproduced in study from (Kearns et al. submitted: related manuscript attached). (F) There was no significant difference in the levels of serum triglycerides between Tg26^+/−/ApoE^−/− and ApoE^−/− mice. * P<0.05. All data analyzed by unpaired parametric two tailed T-Test and represented as mean ± SEM.

Figure 2:. IDO activity progressively increases in Tg26^+/−/ApoE^−/− fed with HFD over course of 8 weeks.

Littermate Tg26^+/−/ApoE^−/− and ApoE^−/− mice were fed a HFD for 8 weeks starting at 8 weeks of age. Serum was collected at 0,2,4,6 and 8 weeks on the high fat diet for measurement of Kynurenine and Tryptophan concentrations and determination of KTR for analysis of IDO activity (Supplemental figure 1). The levels of serum kynurenine (A), serum tryptophan (B), and KTR (C) between Tg26^+/−/ApoE^−/− and ApoE^−/− mice were compared by Two-way ANOVA followed by bonferroni post hoc test. The horizontal bar at the top of the graph represents any significant difference in the trend of serum levels over time. Any single statistic over the time points represents a significant difference between those two groups at that time point as determined by Bonferroni post hoc. . *P<0.05, **P<0.01, ***P<0.001 vs ApoE^-/−. All data represented as mean ± SEM.

Figure 3:. Elevated serum cytokines in Tg26^+/−/ApoE^−/− after 8 weeks on a HFD.

Serum was collected from mice on HFD for 8 weeks for measurement of IFN-γ, IL-6, M-CSF, and TNF-α. Data analyzed by two tailed T-test with ROUT for outlier removal. (A) Serum IFN-γ (pg/mL) in Tg26^+/−/ApoE^−/− and ApoE^−/− mice with no significant difference. (B) Significant in crease in serum IL-6 (pg/mL) in Tg26^+/−/ApoE^−/− mice (128.9 ± 36.41 pg/mL) vs. ApoE^-/- mice (46.35 ± 9.50 pg/mL; P<0.05). (C) Trending increase in serum M-CSF in Tg26^+/−/ApoE^−/− mice (37.39 ± 11.67 pg/mL) vs. ApoE^-/- mice (22.08 ± 3.163 pg/mL; P=0.06). (D) Trending increase in serum TNF- α in Tg26^+/−/ApoE^−/− mice (19.73 ± 3.60 pg/mL) vs. ApoE^-/- mice (12.17 ± 2.99 pg/mL; P=0.13). *P<0.05. All data represented as mean ± SEM.