动物造模,脂肪组织,传染性SIV z-bo 2023-01-18 572

　　Background: Memory CD4 T cells and macrophages are the main host and cell bank of humans and HIV in non-human primates. The anatomical structures of the infected immune cells include lymph nodes (lymph nodes, spleen, thymus, bone marrow, and GALT) and non-lymphatic tissues (lung, skin, liver, kidney, reproductive and nervous system) tissues. Antiretroviral therapy (ART) reduces the systemic viral load in most patients below the detection limit of clinical testing, but if ART is interrupted, viral replication usually resumes. Due to certain unique cell disorders, ART is not completely effective in certain locations such as CNS tissues and lymph nodes. Therefore, understanding the cellular and anatomical reservoirs of HIV and how they prevent adequate tissue penetration of ART drugs is critical to achieving treatment. So far, a major organ that has been neglected in the pathogenesis of HIV is adipose tissue. As we all know, a series of immune cells reside or migrate into adipose tissue and affect their metabolic signals, but fat cells and other adipose tissue-resident cells interact with CD4T cells and macrophages, which is of great significance to the pathogenesis of HIV. Anatomically, adipose tissue is mainly located under the skin (subcutaneous fat) and around the abdominal organs (visceral fat). However, fat cells are also closely related to most lymphoid tissues. Lymph nodes are tightly wrapped by fat tissue. Fat cells are abundant in bone marrow, and the thymus gland is gradually filled with fat cells in adulthood. This complex arrangement is important in infection or immune response, because fat cells are the main source of energy and the survival signal of immune cells. At the cellular level, adipose tissue is heterogeneous, consisting of mature adipocytes (the main part containing lipid droplets and triglycerides) and the stromal blood vessel part (AT-SVF), mainly including pre-adipocytes (adipocyte precursors) ) And fibroblasts, mesenchymal stem cells CE. LLS (MSC), endothelial cells and immune cells. Extensive studies in humans and mice have shown that under normal circumstances, almost every kind of inherent and adaptive white blood cells exist in adipose tissue, and their composition and function change significantly with changes in disease and inflammation. Importantly, for HIV infection, the CD4 T cells residing in adipose tissue are mainly activated memory CD4 T cells (CD45 RO+CD69 + HLA+DR+CD25+). The phenotype is similar to that of HIV persisting in other tissues. Those ones. Recently, we first confirmed the existence of memory CD4 T cells and HIV proviral DNA in the interstitial blood vessels of patients treated with virus-suppressed ART. In addition, we show through in vitro co-culture experiments that primary human adipocytes enhance HIV replication in CD4 T cells. Since adipocytes are widespread endocrine cells that widely regulate immunity and diseases, these findings require further research on the role of adipose tissue in HIV replication and persistence.

　　SIV infection rhesus monkey is still the best animal model of HIV infection and viral pathogenesis. In this study, adipose tissue specimens were collected from the cadavers of rhesus monkeys and infected with SIV-SVF162P3 for 4 weeks (n=8) or SIVMAC251 for 38 weeks (n=8) without antiretroviral treatment. We hypothesized that the memory CD4 T cells in adipose tissue contained infectious viruses, and these untreated infected rhesus monkeys would have metabolic complications similar to those of HIV-infected humans. Although the original purpose of these infected monkeys did not include the study of adipose tissue, examination of adipose tissue showed that the infiltration of adipose tissue by CD4 T cells infected with infectious virus is a regular event in the process of SIV infection. Long-term infection of monkeys also produces some metabolic abnormalities similar to those of HIV patients. The results of this study emphasize the prevalence and stability of the virus in adipose tissue, and provide new evidence for the metabolic dysfunction caused by the virus.

　　Result: The establishment of memory CD4 T cells and SIV pools in adipose tissue during the first infection. Virus eradication is challenged by the rapid spread of SIV in lymphatic tissues (within 7 days) and the establishment of a stable SIV pool within 3 days after macaque infection. Adipose tissue inflammation and dysfunction usually involve the accumulation and regulatory activity of many innate and adaptive immune cells. Especially pro-inflammatory memory T cells, macrophages and NKT cells. Primary infection also includes the spread and establishment of viruses in adipose tissue, because fat cells are intricate with most lymphoid tissues. In order to study the distribution of white blood cells and proviruses in adipose tissue during the initial infection, adipose tissue samples of acute infection (SHIV-SVF162P3) macaques were studied for the first time. Figure 1 shows the plasma viral load of nine rhesus monkeys after intrarectal infection, one of which was unsuccessful. Rhesus monkeys died 4 weeks after infection, and 5 to 15 grams of adipose tissue samples were collected from the subcutaneous and internal organs of the abdomen. AT-SVF cells are isolated from adipose tissue. Use flow cytometry to detect the effect of visceral AT-SVF cells on activated memory T cells (CD3, CD4, CD8, CD95, CD25 and CD69), NKT cells (CD3, CD16, CD27, CD56, GRZA and GRZB), B cells (CD19 And CD80) and macrophages (CD14 and HLA.DR). In uninfected healthy monkeys, CD3+ T cells accounted for 9.8%, SIV-infected monkeys accounted for 6.1%, and uninfected monkeys with chronic enteritis accounted for 15.3%. In addition, the levels of CD8 and CD4 T cells in AT-SVF of uninfected healthy monkeys were similar, and the ratio of AT-SNF CD8 and CD4 T cells increased during infection and disease. For uninfected healthy and SIV-infected rhesus monkeys, almost all AT-SVF CD4 and CD8 T cells are memory T cells (>94% CD95 +), which express tissue resident and activation markers (62-84%) CD69 + and 3-13% CD25+). However, CD4 T cells express higher CD25 (12-13%) than CD8 T cells (3-4%), which may indicate a Treg subpopulation.

　　adipose tissue CD4 T cells infected with infectious SIV: In order to evaluate the infectivity of SIV-infected CD4 T cells in adipose tissue, we performed a virus growth test on AT-SVF purified CD4 T cells from chronic SIVMAC251 infected monkeys (38 weeks after infection). CD4T cells purified from peripheral blood or AT-SVF cells were serially diluted six times, activated with PHA+IL-2 for 2 days, and then SIV induced by M8166 cells (measured by extracellular P27) was added. For five infected monkeys, the virus was induced from peripheral blood CD4 T cells parallel to AT-SvF CD4 T cells. For two infected monkeys, peripheral blood was not available, and virus induction was detected only from AT-SvF CD4 T cells. In the seven rhesus monkeys studied, infectious SIV was induced by AT-SvF CD4 T cells. On the basis of five monkeys, the infection rate of peripheral blood and AT-SVF F CD4 T cells at 3 to 4 weeks is basically the same. We also assessed whether the infectious virus can be separated from the fat cells in the fat tissue The mature fat cells are isolated or captured, because some people think that HIV may not produce infected fat cells. From three infected monkeys, 6×106 M8166 cells were rotated at 37°C and suspended in 5-8 ml of visceral floating fraction suspension for 8 hours, then centrifuged, washed, and applied density gradient The dead M8166 cells were removed by centrifugation. Then 3×106 M8166 cells were cultured for 3 weeks, and extracellular P27 was measured. No p27 was detected, indicating a lack of isolation of infectious virus particles or capture of fat cells. In order to further evaluate the function of adipose tissue CD8 T cells, we tried to use the total AT-SVF cells of three SIV-infected rhesus monkeys to detect the virus growth test. The replication ability and infectivity of CD4 T cells in the adipose tissue of rhesus monkeys infected by SIV are affected, but this virus inducibility does not occur in the presence of CD8 T cells in adipose tissue.

　　In the absence of antiretroviral drugs, SIV infection induces metabolic disorders: metabolic disorders (such as dyslipidemia, hyperlipidemia, and decreased production of leptin and adiponectin) and adipocyte abnormalities (such as due to inactivated expression of key adipogenic transcription factors) The resulting blockade of differentiation) is common during HIV infection. Although some of these deficiencies have been attributed to the adverse effects of ART drugs, similar complications also occur in untreated HIV patients. In addition, viral proteins such as Vpr, NEF and TAT directly affect fat cell function. To determine whether SIV infection induces fat metabolism defects in rhesus monkeys, we studied the mRNA expression of visceral adipocytes of C/EBPα, C/EBPβ, PPARγ2, leptin, adiponectin, and GLUT4, as well as serum total cholesterol and lipid ( Triglycerides and free fatty acids), leptin and adiponectin. When adipocytes interact extensively with T cells, we also studied the factors that regulate T cell stimulation, survival and migration (IL-2, IL-7, IL-15/IL-15Rα, IL-6, TNFα, CCl 2 , CCl 5, CC19, CL21) in adipocytes. For adipocyte mRNA analysis, visceral adipose tissue was obtained from three uninfected healthy rhesus monkeys and compared with three acutely infected and five chronically infected monkeys. Compared with uninfected monkeys, PPARγ2, C/EBPa, C/EBPβ, leptin and GLUT4 were differentially expressed in the fat cells of infected monkeys. Compared with uninfected rhesus monkeys, acutely infected PPARγ2 expression increased 30.2 times, chronically infected monkeys PARRγ expression increased 3 times, and C/EBPa reduced acute infection by 2.9 times. It is reduced by 2.5 times, and C/EBPβ is reduced by 4.3 times in chronic infections. Infected monkeys, leptin decreased 4.5 times, acutely infected and 3.1 times chronically infected monkeys, GLUT4 decreased 4.1 times for acutely infected patients, and chronically infected monkeys decreased by 2.6 times. The expression of adiponectin in adipocytes is similar between uninfected and infected rhesus monkeys. In combination with unexpressed adipocytokines, the adipocytes of uninfected and infected rhesus monkeys also express important immune regulatory factors. Compared with uninfected monkeys, the expression of IL-2, IL-7 and CCR19 in the fat cells of infected monkeys increased by 1.6 to 3 times, while the expression of other cytokines and chemokines was between the uninfected and infected rhesus monkeys It is similar, which suggests that the factors expressed by adipocytes may contribute to the storage and survival of infected CD4 T cells in adipose tissue.

　　Conclusion: In this study, adipose tissue infected with SIV monkeys proved to be rich in memory CD4 T cells, highly infectious viruses and pro-inflammatory immune cells. SIV infection causes metabolic complications in the absence of antiretroviral drugs, some of which are similar to the metabolic status of HIV patients.