The lineage stability and suppressive program of regulatory T cells require protein O-GlcNAcylation.

Regulatory T (Treg) cells control self-tolerance, inflammatory responses and tissue homeostasis. In mature Treg cells, continued expression of FOXP3 maintains lineage identity, while T cell receptor (TCR) signaling and interleukin-2 (IL-2)/STAT5 activation support the suppressive effector function of Treg cells, but how these regulators synergize to control Treg cell homeostasis and function remains unclear. Here we show that TCR-activated posttranslational modification by O-linked N-Acetylglucosamine (O-GlcNAc) stabilizes FOXP3 and activates STAT5, thus integrating these critical signaling pathways. O-GlcNAc-deficient Treg cells develop normally but display modestly reduced FOXP3 expression, strongly impaired lineage stability and effector function, and ultimately fatal autoimmunity in mice. Moreover, deficiency in protein O-GlcNAcylation attenuates IL-2/STAT5 signaling, while overexpression of a constitutively active form of STAT5 partially ameliorates Treg cell dysfunction and systemic inflammation in O-GlcNAc deficient mice. Collectively, our data demonstrate that protein O-GlcNAcylation is essential for lineage stability and effector function in Treg cells.

HIV-1 induces cytoskeletal alterations and Rac1 activation during monocyte-blood-brain barrier interactions: modulatory role of CCR5.

BACKGROUND: Most HIV strains that enter the brain are macrophage-tropic and use the CCR5 receptor to bind and infect target cells. Because the cytoskeleton is a network of protein filaments involved in cellular movement and migration, we investigated whether CCR5 and the cytoskeleton are involved in endothelial-mononuclear phagocytes interactions, adhesion, and HIV-1 infection. RESULTS: Using a cytoskeleton phospho-antibody microarray, we showed that after co-culture with human brain microvascular endothelial cells (HBMEC), HIV-1 infected monocytes increased expression and activation of cytoskeleton-associated proteins, including Rac1/cdc42 and cortactin, compared to non-infected monocytes co-cultured with HBMEC. Analysis of brain tissues from HIV-1-infected patients validated these findings, and showed transcriptional upregulation of Rac1 and cortactin, as well as increased activation of Rac1 in brain tissues of HIV-1-infected humans, compared to seronegative individuals and subjects with HIV-1-encephalitis. Confocal imaging showed that brain cells expressing phosphorylated Rac1 were mostly macrophages and blood vessels. CCR5 antagonists TAK-799 and maraviroc prevented HIV-induced upregulation and phosphorylation of cytoskeleton-associated proteins, prevented HIV-1 infection of macrophages, and diminished viral-induced adhesion of monocytes to HBMEC. Ingenuity pathway analysis suggests that during monocyte-endothelial interactions, HIV-1 alters protein expression and phosphorylation associated with integrin signaling, cellular morphology and cell movement, cellular assembly and organization, and post-translational modifications in monocytes. CCR5 antagonists prevented these HIV-1-induced alterations. CONCLUSIONS: HIV-1 activates cytoskeletal proteins during monocyte-endothelial interactions and increase transcription and activation of Rac1 in brain tissues. In addition to preventing macrophage infection, CCR5 antagonists could diminish viral-induced alteration and phosphorylation of cytoskeletal proteins, monocyte adhesion to the brain endothelium and viral entry into the central nervous system.

Dysregulation of claudin-5 in HIV-induced interstitial pneumonitis and lung vascular injury. Protective role of peroxisome proliferator-activated receptor-γ.

RATIONALE: HIV-1-induced interstitial pneumonitis (IP) is a serious complication of HIV-1 infection, characterized by inflammation and cellular infiltration in lungs, often leading to respiratory failure and death. The barrier function of the pulmonary endothelium is caused in part by tight junction (TJ) proteins, such as claudin-5. Peroxisome proliferator-activated receptor (PPAR)-γ is expressed in lung tissues and regulates inflammation. We hypothesize that HIV-1 induces vascular lung injury, and HIV-1-mediated damage of the pulmonary endothelium and IP is associated with dysregulation of PPAR-γ. OBJECTIVES: Investigate the effects of HIV-1 infection on the pulmonary microvasculature and the modulatory effects of the PPAR-γ ligands. METHODS: Using human lung tissues, we demonstrated down-regulation of claudin-5 (marker of pulmonary barrier integrity), down-regulation of PPAR-γ transcription, and expression in lung tissues of HIV-1-infected humans with IP. MEASUREMENTS AND MAIN RESULTS: Human lung microvascular endothelial cells expressed the TJ proteins claudin-5, ZO-1, and ZO-2; HIV-1 decreased TJ proteins expression and induced nuclear factor-κB promoter activity, which was reversed by PPAR-γ agonist. Using two murine HIV/AIDS models, we demonstrated decreased claudin-5 expression and increased macrophage infiltration in the lungs of HIV-1-infected animals. Activation of PPAR-γ prevented HIV-1-induced claudin-5 down-regulation and significantly reduced viremia and pulmonary macrophage infiltration. CONCLUSIONS: HIV-induced IP is associated with injury to the lung vascular endothelium, with decreased TJ and PPAR-γ expression, and increased pulmonary macrophage infiltration. PPAR-γ ligands abrogated these effects. Thus, regulation of PPAR-γ can be a therapeutic approach against HIV-1-induced vascular damage and IP in infected humans. Removal of Expression of Concern: Issues leading to the previous expression of concern for this article have been resolved after further revisions and editorial review. No further concerns exist.

Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells.

Despite the successes of antiretroviral therapy (ART), HIV-associated neurocognitive disorders remain prevalent in infected people. This is due, in part, to incomplete ART penetration across the blood-brain barrier (BBB) and lymph nodes and to the establishment of viral sanctuaries within the central nervous system. In efforts to improve ART delivery, our laboratories developed a macrophage-carriage system for nanoformulated crystalline ART (nanoART) (atazanavir, ritonavir, indinavir, and efavirenz). We demonstrate that nanoART transfer from mononuclear phagocytes (MP) to human brain microvascular endothelial cells (HBMEC) can be realized through cell-to-cell contacts, which can facilitate drug passage across the BBB. Coculturing of donor MP containing nanoART with recipient HBMEC facilitates intercellular particle transfer. NanoART uptake was observed in up to 52% of HBMEC with limited cytotoxicity. Folate coating of nanoART increased MP to HBMEC particle transfer by up to 77%. To translate the cell assays into relevant animal models of disease, ritonavir and atazanavir nanoformulations were injected into HIV-1-infected NOD/scid-γ(c)(null) mice reconstituted with human peripheral blood lymphocytes. Atazanavir and ritonavir levels in brains of mice treated with folate-coated nanoART were three- to four-fold higher than in mice treated with noncoated particles. This was associated with decreased viral load in the spleen and brain, and diminished brain CD11b-associated glial activation. We postulate that monocyte-macrophage transfer of nanoART to brain endothelial cells could facilitate drug entry into the brain.

Pharmacotoxicology of monocyte-macrophage nanoformulated antiretroviral drug uptake and carriage.

Limitations inherent to antiretroviral therapy (ART) in its pharmacokinetic properties remain despite over 15 years of broad use. Our laboratory has pioneered a means to improve ART delivery through monocyte-macrophage carriage of nanoformulated drug-encapsulated particles (nanoART). To this end, our prior works sought to optimize nanoART size, charge, and physical properties for cell uptake and antiretroviral activities. To test the functional consequences of indinavir, ritonavir, and efavirenz formulations we investigated relationships between human monocyte and macrophage cytotoxicities and nanoART dose, size, surfactant, and preparation. Wet-milled particles were more cytotoxic to monocytes-macrophages than those prepared by homogenization; with concurrent induction of tumor necrosis factor-alpha. Interestingly, pure suspensions of indinavir and ritonavir at 0.5 mM, and efavirenz at 0.1 mM and 0.5 mM also proved cytotoxic. Individual surfactants and formulated fluconazole neither affected cell function or viability. Although nanoART did not alter brain tight junction proteins ZO-2 and occludin, 0. 5mM ritonavir formulations did alter brain transendothelial electric resistance. These results underscore the potential importance of evaluating the physicochemical and functional properties of nanoART before human evaluations.

Cross-talk between STAT1 and PI3K/AKT signaling in HIV-1-induced blood-brain barrier dysfunction: role of CCR5 and implications for viral neuropathogenesis.

How neuroinflammation affects signaling pathways leading to human blood-brain barrier (BBB) dysfunction during HIV-1 infection is incompletely understood. We previously demonstrated that signal transducers and activators of transcription-1 (STAT1) signaling is involved in HIV-1 induced BBB damage and is relevant to viral neuropathogenesis. The objective of this study was to delineate the signaling pathways upstream and downstream of STAT1 involved in HIV-1-induced endothelial dysfunction. We show that HIV-1 activation of STAT1 and STAT3 in human brain microvascular endothelial cells (HBMEC) is associated with induction of promoter activity of the interferon-stimulated response element (ISRE)/interferon-γ-activated sequence (GAS). The STAT1 inhibitor fludarabine diminished HIV-1-induced ISRE/GAS promoter activity. CCR5 neutralizing antibodies and the phosphoinositide-3-kinase (PI3K) inhibitor LY-294002 diminished HIV-1-induced phosphorylation of STAT1 and STAT3, significantly diminished HIV-1-induced ISRE/GAS promoter activity, and diminished virus-induced monocyte adhesion and transendothelial migration. HIV-1 infection did not phosphorylate janus kinases but induced activation of the phosphoinositide-dependent kinase-1 (PDK1) and the serine-threonine protein kinase AKT, both downstream effectors of PI3K. CCR5 antibodies also diminished virus-induced phosphorylation ofPDK1 and AKT. These results suggest that the chemokine receptor CCR5 is partially involved in HIV-1 binding to HBMEC and show cross-talk between STAT1 and PI3K pathways in HIV-1-induced BBB dysfunction.