Study On Contribution And Mechanism Of Activated Invariant Natural Killer T Cells In Abdominal Aortic Aneurysm Pathogenesis

PARTIObjectiveAdaptive and innate immunities have been implicated in the pathogenesis of abdominal aortic aneurysm(AAA),and damage and remodeling in the tunica media are a focus of the aneurysm development.Thus,identification of key immune cells or molecules as potential targets for the treatment of AAA is critical.Materials and Materials and MethodsAbdominal aortic aneurysmal wall tissues were harvested in patients with AAA who undergoing open repair,the healthy abdominal aortic tissues were harvested from donations after cardiac death as controls.After transporting to the laboratory,the tissue sample was cut into two pieces,one for the separation of the media and the other for analysis of the whole aortic wall.Then,the media was separated by forceps in the PBS on ice.After The single-cell suspension of vascular tissue was successively prepared,the cells were stained by specific antibody and analyzed the proportion of innate immune cells by flow cytometry.ResultFlow cytometry results of AAA specimens revealed that the numerous types of lymphocytes were enriched in AAA wall tissues and had an obvious increase in proportion compared to levels found in healthy abdominal aortic tissue samples in controls.Apart other lymphocytes,a higher proportion of iNKT cells marked as CD3 and Va24Ja18 invaded into the human AAA wall tissues,which is especially significant in the aortic media.These infiltrating iNKT cells in aneurysmal tissues have a high expression of CD69 which indicating a highly active function.These observations suggested that iNKT cells can successfully enter the media and exert their inflammatory effect,which is different from other lymphocytes.iNKT cells may have a more active role in the injury of the aortic wall.ConclusionsNKT cells play an important and key role in AAA development.iNKT cells,through an unknown pathway,enter the media of the aortic wall as drivers of AAA development and are highly active,as shown by their impressively high expression of CD69,which is different from results seen in peripheral blood.Additionally,the isolated enrichment of iNKT cells showed that iNKT cells may perform irreplaceable functions in the media and may promote the progression of the AAA disease process in the mouse model.PART ⅡObjectiveTo confirm if NKT cells might be the key point on the media damage in AAA,AAA mouse model induced by angiotensin Ⅱ(Ang Ⅱ)infusion were used to reproduce the inflammatory response of AAA in mouse.Materials and MethodsTwenty C57BL/6J ApoE-/-male mice aged 10-12 weeks were infused with angiotensin Ⅱ,which was dissolved in sterile saline and infused using Alzet osmotic pumps.The micropumps were pushed into the subcutaneous space through a small incision in the back of the neck after anesthetizing the mice with isoflurane.The wound sites were routinely disinfected.The standard food and water were used to feed all the mice,and which was monitored twice per day.Meanwhile,the arterial pressure of the mice was measured twice per week.On day 28,mouse serum was subsequently collected and aortas were dissected and harvested.For traditional Ang Ⅱ-induced AAA mouse model and the controls,three biological replicates were selected.The total RNA of tissue was extracted using TRIzol Reagent(Invitrogen),according to the manufacturer’s protocol.The Ribo-Zero rRNA Removal Kit(Illumina,San Diego,CA,USA)was used to reduce the ribosomal RNA content of total RNAs.RNAseq was performed on Illumina Novaseq 6000 platform.To investigate gene expression characteristics,the Cuffdiff package was used for differential mRNA expression analysis in different groups.Next,GO and KEGG pathway enrichment analyses were conducted based on the differentially expressed mRNAs.Pathological aortic structure changes were observed by hematoxylin eosin(HE)staining.Masson staining was used to observe the elastin disruptions in abdominal aortic specimens form different groups.ResultThe ApoE-/-AAA mouse model was made by Ang Ⅱ infusion.Which has been widely used in AAA research because of its pathological similarity to human AAA.Besides,an RNAseq assay was carried out to analyze the Ang Ⅱ AAA mouse model and confirmed the inflammatory response in the abdominal aortic wall.The results showed that the AAA incidence of this Ang Ⅱ-induced AAA mouse model is 30%.The results of HE staining and Masson staining showed abdominal aortic specimens from the experimental group had a significantly stronger inflammatory reaction than those from control group.ConclusionsThe aneurysmal incidence was 30%in the Ang Ⅱ-induced AAA mouse model,which have enough space for the possible increasement for AAA incidence in the further experiment.The results of RNAseq,HE staining and the Masson staining confirmed that there was obvious inflammatory reaction in the aortic specimens of this AAA mouse model.Which reproduce the inflammatory responses of the AAA formation.PART ⅢObjectiveThe aneurysmal incidence was 30%in the Ang Ⅱ-induced AAA mouse model,ha ve enough space for the possible increasement for AAA incidence in the further exp eriment.The results of RNAseq,HE staining and the Masson staining confirmed tha t there was obvious inflammatory reaction in the aortic specimens of this AAA mous e model.Which reproduce the inflammatory responses of the AAA formation.Materials and Methods48 male mice(C57BL/6J ApoE-/-)were randomly divided into four groups,including:(1)Group 1,the control group,included mice treated with PBS(Phosphate buffer saline)intraperitoneally once a week;(2)Group 2,theα-GalCer only group,included C57BL/6J ApoE-/-mice treated with 2 μg α-GalCer(ab144262,Abeam)intraperitoneally once a week;(3)Group 3,the AngⅡ-pump only group.Osmotic minipumps filled with Ang Ⅱ(1.44 mg/kg·per day)were implanted subcutaneously in the backs of ApoE-/-mice and the mice were also treated with PBS intraperitoneally(IP)once a week;and(4)Group 4,the Ang Ⅱ-pump+α-GalCer group.Osmotic minipumps filled with Ang Ⅱ(1.44 mg/kg·per day)were implanted subcutaneously in ApoE-/-mice and 2 μgα-GalCer(IP,once a week)was administered after surgery.18 On day 28,the whole aorta from the ascending aorta to the bifurcation of the iliac artery was immediately isolated free from surrounding tissue under a dissection microscope after the mice were euthanized.Then aortic section samples were collected serially from the proximal to the distal aorta for morphological and histological analyses or digested into a single cell suspension for inflammatory cell component analyses by flow cytometry.The differences of pathological structure and changes of aortic specimens were showed by HE and Masson staining.ResultThe results showed that the incidence of AAA increased from 31.82%(AngⅡ-pump only)to 61.54%(Ang Ⅱ-pump+α-GalCer)after α-GalCer administration and the pathological changes in the aortic aneurysms became more serious.Meanwhile,the incidence of aortic aneurysm in both the control group andα-GalCer only group was 0%.Elastin staining showed a deranged,fractured and disorganized arrangement of the elastin fibers in the aneurysm tissues.Different from controls,tissues from AngⅡ-pump only group and Ang Ⅱ-pump+α-GalCer group showed significant elastin damage and fracture,especially in the Ang Ⅱ-pump+α-GalCer-treated mice,which showed the most serious elastic fiber damageAdditionally,immunofluorescence staining was used to observe the distribution of inflammatory cell infiltration.In AAA lesions,a large number of CD3+T lymphocytes were found to invade the aortic wall,including the media.The number of CD3+T cells within the suprarenal abdominal aortic wall in mice treated by AngⅡ+α-GalCer was significantly higher than those in other groupsThe results showed the percentages of iNKT cells in the aortic wall tissues in Ang Ⅱ+α-GalCer group increased more than those in the control group(4.14 ±3.37%vs 0.72±0.28%,P<0.001).However,the proportion of iNKT cells in the aortic tissues from control group was still obviously lower than which in α-GalCer only group(0.72±0.28%vs 2.17±1.36%,P=0.0182).There was no statistically significant difference between any other two groups.The flow cytometry was used to determine and analyze iNKT proportion in splenic tissues in different mouse model groups.The results showed the iNKT cell proportion in the spleen from the group treated by the Ang Ⅱ+α-GalCer was also significantly higher than that in control group(0.47±0.21%versus 0.94±0.27%,P=0.0012);The Ang Ⅱ-pump only group and α-GalCer only group did not show significant differences in splenic tissue among any other groups.ConclusionsThese results highly suggest that the active iNKT cells have a great contribution to AAA development and controlling the activation state of NKT cells may present an important therapeutic strategy for AAA.