Immune-metabolic characterization of a humanized mouse model for translational studies on cardiovascular diseases

Aim: Atherosclerosis is one of the leading cause of cardiovascular diseases and it’s driven by elevated plasma cholesterol levels, paralled by an impaired immune-inflammatory response. Despite effective lipid-lowering therapies, some patients still show a residual inflammatory risk, potentially linked to immune response dysfunctions, thus fueling the interest in developing cardiovascular immunomodulatory approaches. This scenario highlights the need for experimental models that can directly translate both molecular mechanisms and therapeutic strategies of CVD immunomodulation from preclinical to clinical studies. Here we will present the immune-metabolic characterization of an immunodeficient mouse – on an atheroprone genetic background -, whose immune system is reconstituted with human hematopoietic stem cells (CD34). Methods: Humanized TKO-LDLr KO mice (generated by crossing LDLr KO with immunodeficient Rag2-KO/IL2rg-KO/CD47-KO mice, HuTKOL) were generated after low-dose irradiation (250 cGy) of 2-3 days old pups followed by hepatic injection of commercial or iPSCs-derived CD34 (250.000-300.000 cells/mouse). After 12 weeks, human cell engraftment was evaluated by FACS. HuTKOL were then fed 12-week high-cholesterol diet (WTD) to investigate immunometabolic phenotype and atherosclerosis. Results: HuTKOL presented human leukocytes (% hCD45+ cells/total number of live leukocytes: 40,97%, SE±3,46%) in circulation after engraftment with commercial hCD34+ cells, with B cells representing the most abundant population at 8 wks (% hCD19+ cells/hCD45+ cells: 72,81%, SE±2,33%), but they proportion decreased over time (% hCD19+ cells/hCD45+ cells: 13,85%, SE±2,98%). In contrast, T cells show the opposite trend (% hCD3+ cells/hCD45+ cells at 8 wks: 10,93%, SE±3,76%) being the most represented human subset after 12-wks of WTD (% hCD3+ cells/hCD45+ cells: 58,84%, SE±4,86%), similar to human lymphocyte profile. On WTD, HuTKOL developed dyslipidemia (plasma cholesterol levels: 1086,90 mg/dl, SE± 65,15) and atherosclerosis (% aortic sinus plaque occlusion: 24,79%, SE±3,17%; atherosclerotic lesion volume: 0,32 mm3; % fibrosis/plaque area: 33,94%, SE±16,17%), with human immune cell infiltration into the plaques replicating the compostion of human plaques. Moreover, the feeding with high cholesterol diet induced the expansion of hCD4 memory T cells and the production of IgM against atherosclerosis-related antigens, confirming an activation of the human adaptive immune response. Characterization of iPSCs-derived CD34+ cells and their engraftment in TKO-L is ongoing. Conclusion: HuTKOL mice represent a valuable platform to investigate human adaptive immunity dynamics under dyslipidemia and to test immuno-modulation for CVD. Furthermore, we envision that the use of iPCS-derived CD34+ cells would allow to investigate the pathological and pharmacological editing of immune cells for cardiovascular disease prevention.