Role Of Monocytes In AIDS And As Targets For Antiviral Therapy

对艾滋病毒的天然防御 粘膜相关淋巴组织是HIV在早期感染和疾病发展过程中的主要靶标，也可以在高效抗逆转录病毒治疗期间提供病毒安全避难所。在这些组织中，扁桃体作为逆转录病毒感染的主要和/或继发部位的地位仍然是个谜，特别是因为经口传播艾滋病毒似乎很少见。为了分析在这个隔室中可获得性和对HIV易感性的机制，我们用激光捕获显微解剖(LCM)解剖了上皮细胞，并进行了微阵列基因表达分析。我们的研究表明，扁桃体上皮是一个独特的部位，与其他口腔上皮相比，它表现出更高的HIV传播潜力，可能部分是由于HIV辅助受体CXCR4的增加和抗病毒SLPI的减少。为了确定有利于扁桃体淋巴样细胞作为病毒库的选择性因素，我们比较了分离的扁桃体细胞和血淋巴细胞的敏感性。在这些研究中，我们确定了扁桃体微环境的几个独特方面，可能支持其允许的性质，包括与PBMC相比，Th2细胞因子水平升高和Th1细胞因子有限。尽管抗病毒干扰素水平升高，但扁桃体在体外不能在暴露于HIV后产生有效的抗病毒反应，在体内也不明显，这可能反映了干扰素信号负调控因子的结构性表达，如SOCS。SOC似乎有助于阻断扁桃体中的干扰素信号级联反应，明显表现为STAT1的磷酸化减少，这可能是局部脱离干扰素信号和降低抗病毒活性的原因。在循环模式中，SOCS3也可能促进Th2极化。这样的免疫调节与控制免疫激活的必要性是一致的，但在不断受到抗原轰炸的环境中保持准备状态。在正在进行的研究中，产生惊人数量艾滋病毒的扁桃体多核细胞和单核细胞正在被分离和研究，以及Treg作为病毒调节器和靶点的潜在参与。进一步了解这些人群以及影响他们对艾滋病毒致病易感性的免疫调节途径，对于保护脆弱的粘膜隔膜不被用作病毒储存库是重要的。 在早期的研究中，我们证明了一种人工合成的三萜类和过氧化物酶体增殖物激活受体伽马(PPARGamma)配体，2-氰基-3，12-二氧乙烯-1，9-二烯-28-OIC酸(CDDO)，已知可以影响p21激酶抑制剂的表达(与病毒生命周期有关)，抑制巨噬细胞和PBMC中的病毒复制。最近，一种新的CDDO甲酯衍生物(CDDO-Me)已经被合成，它是唯一的口服生物利用度，在癌症患者的初步I期试验中，它被证明没有显著的毒性，这与在HIV患者中测试这种试剂的可能性是一致的。令人感兴趣的是有证据表明这种甲酯衍生物针对的是组织，而不是血液，因此我们专注于确定它在扁桃体组织来源的CD4 HIV靶细胞中抑制HIV的能力。由于抗逆转录病毒治疗的特点往往是高毒性，并经常导致耐药病毒株的出现，因此识别可单独使用或与现有抗病毒药物联合使用的针对宿主细胞分子的新的抗病毒药物将为治疗艾滋病毒感染提供额外的资源。 在其他临床研究中，我们一直在与NIAID的研究人员合作，用干扰素治疗艾滋病患者，试图确定干扰素抑制HIV负担的抗病毒机制。与我们的体外数据一致的是，干扰素有效地上调固有细胞内蛋白胞苷脱氨酶家族的成员，我们发现这也发生在接受治疗的患者的PBMC中。载脂蛋白B基因编码酶催化类多肽3G(APOBEC3G)是一种对HIV具有致死活性的胞苷脱氨酶，它被包装在子代病毒粒子中，导致HIV基因的降解。作为反击，HIV病毒感染性因子(Vif)以APOBEC3G为靶标进行蛋白酶体蛋白分解，从而排除了其与萌芽病毒粒子的结合。我们发现，干扰素治疗增加了APOBEC3G和其他家庭成员，同时降低了患者的病毒载量。病毒下降的最大幅度出现在治疗后的第一周，特别是在病毒载量较低的患者，与早期的蛋白水解酶抑制剂相当。因此，进一步考虑干扰素在低病毒载量情况下作为诱导治疗的作用可能是有必要的，其他临床研究正在进行中。此外，通过使用微阵列，我们已经确定了更多的干扰素分子靶点，这些靶点可能有助于其抗病毒作用，并可能被认为是额外的干预方法。

Innate defense against HIV Mucosal associated lymphoid tissues are major targets of HIV during early infection and disease progression, and can also provide a viral safe haven during highly active antiretroviral therapy. Among these tissues, the tonsils remain enigmatic regarding their status as primary and/or secondary sites of retroviral infection, particularly since oral HIV transmission appears rare. To dissect the mechanisms underlying accessibility and susceptibility to HIV in this compartment, we dissected the epithelium with laser capture microdissection (LCM) and performed microarray gene expression analyses. Our studies demonstrated that the tonsil epithelium is a unique site and exhibits heightened potential for HIV transmission compared to other oral epithelia, likely due in part, to increased HIV co-receptor CXCR4 and reduced antiviral SLPI. To identify selective factors that favor the tonsil lymphoid cells as a viral reservoir, we compared isolated tonsil cell susceptibility to that of blood lymphocytes. In these studies, we identified several unique aspects of the tonsil micromilieu that may support its permissive nature, including elevated levels of Th2 cytokines and limited Th1 cytokines compared with PBMC. Despite elevated levels of antiviral IFN, the tonsil was not able to mount an effective antiviral response after exposure to HIV in vitro, nor apparently in vivo, which may reflect constitutive expression of negative regulators of IFN signaling, such as SOCS. SOCS appear to contribute to the blockade of IFN signaling cascades in the tonsil, evident by the reduced phosphorylation of Stat1, which may account for the local disengagement of IFN signaling and reduced antiviral activity. In a cyclic pattern, SOCS3 may also promote Th2 polarization. Such a profile of immune regulation is consistent with the necessity to control immune activation, yet maintain a state of readiness in an environment constantly bombarded with antigens. In ongoing studies, the tonsil multinucleated and mononuclear cells that produce striking amounts of HIV are being isolated and studied, as is the potential involvement of Treg as regulators and targets for the virus. Further understanding of these populations and the immunoregulatory pathways that influence their susceptibility to HIV pathogenesis are important for protecting vulnerable mucosal compartments from serving as reservoirs for the virus. In earlier studies, we demonstrated that a synthetic triterpenoid and peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), known to influence p21 kinase inhibitor expression (linked to viral life cycle), suppressed viral replication in macrophages and PBMC. Recently, a new methyl ester derivative of CDDO (CDDO-Me), which is uniquely orally bioavailable, has been synthesized and in preliminary phase I trials in cancer patients, it has been shown to not have significant toxicities, consistent with the possibility of testing this agent in HIV patients. Of considerable interest is the evidence that this methyl ester derivative targets the tissues, rather than the blood, and therefore we have focused on defining its ability to inhibit HIV in tonsil tissue derived CD4+ HIV target cells. As anti-retroviral therapy is often characterized by high toxicity and frequently results in the emergence of drug resistant virus strains, the identification of new anti-viral agents targeting host cell molecules that can be used independently or in conjunction with current anti-viral drugs will provide additional resources in the treatment of HIV infection. In additional clinical studies, we have been collaborating with investigators at NIAID in the treatment of AIDS patients with IFN in an attempt to define the antiviral mechanisms by which IFN suppresses HIV burden. In concordance with our in vitro data that IFN potently upregulates members of the cytidine deaminase family of innate intracellular proteins, we show that this also occurs in the PBMC of treated patients. Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G), a cytidine deaminase with lethal activity against HIV is packaged into progeny virions and leads to HIV cDNA degradation. As a counterattack, HIV virion infectivity factor (Vif) targets APOBEC3G for proteasomal proteolysis, thus excluding its incorporation into budding virions. We found that IFN treatment increased APOBEC3G and other family members, while reducing patient viral load. The maximal magnitude of viral decline occurred during the first week after treatment, especially in patients with lower viral load, comparable to that of early protease inhibitors. Thus, further consideration of a role for IFN as an inductive therapy in the context of low viral loads may be warranted and additional clinical studies are in progress. Moreover, through the use of microarrays, we have identified additional molecular targets of IFN that may contribute to its antiviral effects and may be considered as additional intervention approaches.