Functional Proteomic Analysis for Regulatory T Cell Surveillance of the HIV-1-Infected Macrophage

Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States.
Journal of Proteome Research (Impact Factor: 4.25). 10/2010; 9(12):6759-73. DOI: 10.1021/pr1009178
Source: PubMed


Regulatory T cells (Treg) induce robust neuroprotection in murine models of neuroAIDS, in part, through eliciting anti-inflammatory responses for HIV-1-infected brain mononuclear phagocytes (MP; macrophage and microglia). Herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that Treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected MP, and inducing phenotypic cell switches. Changes in individual Treg-induced macrophage proteins were quantified by iTRAQ labeling followed by mass spectrometry identifications. Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity. Treg killed virus-infected macrophages through caspase-3 and granzyme and perforin pathways. Independently, Treg transformed virus-infected macrophages from an M1 to an M2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. Taken together, Treg affects a range of virus-infected MP functions. The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection.

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Article: Functional Proteomic Analysis for Regulatory T Cell Surveillance of the HIV-1-Infected Macrophage

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    • "Differences in protein expression among samples are determined by comparing the intensities of the different iTRAQ reporter ions in the MS/MS peptide fragmentation spectra (Zieske 2006). iTRAQ has been recently used to study topics ranging from cellular responses to HIV infection to the composition of HIV-1 virions (Huang et al. 2010; Linde et al. 2013; Navare et al. 2012; Pendyala et al. 2010; Shetty et al. 2011). For HAND research, iTRAQ was recently used to study changes in oxidative stress proteins in the brains of HIV-infected individuals (Uzasci et al. 2013). "
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    ABSTRACT: We identified and measured proteins in the cerebral spinal fluid (CSF) involved in HIV-associated neurological disorders. Protein levels were determined by mass spectrometry (MS) in pooled CSF taken from three patient groups (human immunodeficiency virus (HIV)-1-infected patients that developed HIV-associated neurocognitive disorders (HANDs), HIV-1-infected patients without HAND, and healthy controls). Pools were generated from 10 patients each per group. CSF from individual patient groups were digested with trypsin and separately labeled using with isobaric tags for relative and absolute quantitation (iTRAQ). After combining all samples in one, peptides were extensively fractionated by offline two-dimensional separation and identified by tandem MS. One hundred and ninety three proteins were deemed to be interpretable for quantitation based on permutation tests with a 95 % confidence interval with a p value ≤ 0.05. Using a cutoff of 1.5-fold for upregulation and 0.6 for downregulation, 16 proteins were differentially expressed in HIV + HAND (reporter p value ≤0.05) with seven of them previously described as HIV-interacting proteins: endoplasmin, mitochondrial damage mediator-BH3-interacting domanin death agonist, orosomucoid, apolipoprotein E, metalloproteinase inhibitor 2, peroxiredoxin-2, and the nuclear protein, ruvB-like 2. Several previously unidentified proteins with possible neurological implication in HIV patients include forming-binding protein 1, C-reactive protein, leukocyte-associated immunoglobulin receptor 1, renin receptor, mediator of RNA polymerase II transcription subunit 14, multimerin-2, alpha-N-acetylglucosaminidase, caldesmon, and cadherin EGF LAG G-type receptor. Our results suggest that not only a few but possibly a combination of biomarkers that are highly correlated can predict neurocognitive status in HIV-infected patients and might be involved in monocyte or macrophage activation.
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    • "This study showed that adoptively transferred T reg attenuated microgliosis and astrogliosis, increased expression of BDNF and GDNF expression , and down-regulated proinflammatory cytokines, oxidative stress, and viral replication, whereas effector CD4 þ T cells were not therapeutic (Liu et al. 2009). T reg inhibit release of viral particles from HIV-1-infected human mononuclear phagocytes (MP), kill infected cells, and induce phenotypic changes in MP (Huang et al. 2010). Up-regulation of the antiviral ubiquitin-like protein, interferon-stimulated gene 15 (ISG15) was concordant with the decrease in viral release from MP , and implicating caspase-3 and granzyme/perforin pathways in MP killing. "
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    • "Indeed, other IFN response inhibitors have also been reported in studies from SIVinfected macaques (Akhtar et al. 2010). However, upregulation of an interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity was recently found in Treg-MDM co-cultures with novel proteomic approaches (ITRAQ) indicating a modulation of IFN responses in MDM by T-regs (Huang et al. 2010). Taken together, these proteomics studies have deepened our current understanding of pathways in HIV-infected MDM, and thus could provide better targets for elimination of HIV reservoirs in the future. "
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    ABSTRACT: Mononuclear phagocytes (monocytes, macrophages, and microglia) play an important role in innate immunity against pathogens including HIV. These cells are also important viral reservoirs in the central nervous system and secrete inflammatory mediators and toxins that affect the tissue environment and function of surrounding cells. In the era of antiretroviral therapy, there are fewer of these inflammatory mediators. Proteomic approaches including surface enhancement laser desorption ionization, one- and two-dimensional difference in gel electrophoresis, and liquid chromatography tandem mass spectrometry have been used to uncover the proteins produced by in vitro HIV-infected monocytes, macrophages, and microglia. These approaches have advanced the understanding of novel mechanisms for HIV replication and neuronal damage. They have also been used in tissue macrophages that restrict HIV replication to understand the mechanisms of restriction for future therapies. In this review, we summarize the proteomic studies on HIV-infected mononuclear phagocytes and discuss other recent proteomic approaches that are starting to be applied to this field. As proteomic instruments and methods evolve to become more sensitive and quantitative, future studies are likely to identify more proteins that can be targeted for diagnosis or therapy and to uncover novel disease mechanisms.
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