Assessing the impact of AGS-004, a dendritic cell-based immunotherapy, and vorinostat on persistent HIV-1 Infection

Approaches to deplete persistent HIV infection are needed. We investigated the combined impact of the latency reversing agent vorinostat (VOR) and AGS-004, an autologous dendritic cell immunotherapeutic, on the HIV reservoir. HIV+, stably treated participants in whom resting CD4+ T cell-associated HIV RNA (rca-RNA) increased after VOR exposure ex vivo and in vivo received 4 doses of AGS-004 every 3 weeks, followed by VOR every 72 hours for 30 days, and then the cycle repeated. Change in VOR-responsive host gene expression, HIV-specific T cell responses, low-level HIV viremia, rca-RNA, and the frequency of resting CD4+ T-cell infection (RCI) was measured at baseline and after each cycle. No serious treatment-related adverse events were observed among five participants. As predicted, VOR-responsive host genes responded uniformly to VOR dosing. Following cycles of AGS-004 and VOR, rca-RNA decreased significantly in only two participants, with a significant decrease in SCA observed in one of these participants. However, unlike other cohorts dosed with AGS-004, no uniform increase in HIV-specific immune responses following vaccination was observed. Finally, no reproducible decline of RCI, defined as a decrease of >50%, was observed. AGS-004 and VOR were safe and well-tolerated, but no substantial impact on RCI was measured. In contrast to previous clinical data, AGS-004 did not induce HIV-specific immune responses greater than those measured at baseline. More efficacious antiviral immune interventions, perhaps paired with more effective latency reversal, must be developed to clear persistent HIV infection

RAGE-induced changes in the proteome of alveolar epithelial cells

The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor and member of the immunoglobulin superfamily. RAGE is constitutively expressed in the distal lung where it co-localizes with the alveolar epithelium; RAGE expression is otherwise minimal or absent, except with disease. This suggests RAGE plays a role in lung physiology and pathology. We used proteomics to identify and characterize the effects of RAGE on rat alveolar epithelial (R3/1) cells. LC-MS/MS identified 177 differentially expressed proteins and the PANTHER Classification System further segregated proteins. Proteins involved in gene transcription (RNA and mRNA splicing, mRNA processing) and transport (protein, intracellular protein) were overrepresented; genes involved in a response to stimulus were underrepresented. Immune system processes and response to stimuli were downregulated with RAGE knockdown. Western blot confirmed RAGE-dependent changes in protein expression for NFκB and NLRP3 that was functionally supported by a reduction in IL-1β and phosphorylated p65. We also assessed RAGE's effect on redox regulation and report that RAGE knockdown attenuated oxidant production, decreased protein oxidation, and increased reduced thiol pools. Collectively the data suggest that RAGE is a critical regulator of epithelial cell response and has implications for our understanding of lung disease, specifically acute lung injury. Significance statement: In the present study, we undertook the first proteomic evaluation of RAGE-dependent processes in alveolar epithelial cells. The alveolar epithelium is a primary target during acute lung injury, and our data support a role for RAGE in gene transcription, protein transport, and response to stimuli. More over our data suggest that RAGE is a critical driver of redox regulation in the alveolar epithelium. The conclusions of the present work assist to unravel the molecular events that underlie the function of RAGE in alveolar epithelial cells and have implications for our understanding of RAGE signaling during lung injury. Our study was the first proteomic comparison showing the effects of RAGE activation from alveolar epithelial cells that constitutively express RAGE and these results can affect a wide field of lung biology, pulmonary therapeutics, and proteomics. © 2018 Elsevier B.V

Angiotensin II regulates δ-ENaC in human umbilical vein endothelial cells

The amiloride-sensitive epithelial sodium channel (ENaC) has been characterized in a variety of non-epithelial tissues. In the current study we sought to understand the effect of angiotensin II on δ ENaC function using human umbilical vein endothelial cells (HUVECs). The δ ENaC subunit is found in humans, but notably absent in rat and most mouse epithelial tissues. In this study we report the presence of δ ENaC in HUVECS with a half-life of ~ 80 min and a change in δ ENaC abundance when HUVECs were treated with angiotensin II. We also observed that angiotensin II increased apical membrane expression of δ ENaC and decreased protein ubiquitination. Equivalent short circuit current measurements showed angiotensin II increased δ ENaC ion transport in HUVEC cells. Treatment with the antioxidant apocynin attenuated angiotensin II mediated effects indicating an important role for angiotensin-derived H2O2 in δ ENaC subunit regulation. Whole cell recordings from oocytes injected with δβγ ENaC shows H2O2-sensitive current. These results suggest that δ ENaC subunits can make up functional channel in HUVEC cells that are regulated by angiotensin II in a redox-sensitive manner. The novel findings have significant implications for our understanding of the role of ENaC in vascular conditions in which oxidative stress occurs. © 2017 Elsevier Inc

Receptor for advanced glycation end-products regulates lung fluid balance via protein kinase C-gp91phox signaling to epithelial sodium channels

The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91phox signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples fromsmokers showed increasedAGEs (9.02 ± 3.03 μg versus 2.48 ± 0.53 μg), lower sRAGE (1,205 ± 292 pg/ml versus 1,9106263 pg/ml), and lower volume(s) of epithelial lining fluid (97 ± 14 ml versus 133 ± 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 ± 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N -oxyl decreased ENaC NPo to 0.10 ± 0.03 ( P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47phox and increased gp91phox dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91phox abrogated the response. Copyright © 2015 by the American Thoracic Societ

H2O2 regulates lung epithelial sodium channel (ENaC) via ubiquitin-like protein Nedd8

Redundancies in both the ubiquitin and epithelial sodium transport pathways allude to their importance of proteolytic degradation and ion transport in maintaining normal cell function. The classical pathway implicated in ubiquitination of the epithelial sodium channel (ENaC) involves Nedd4-2 regulation of sodium channel subunit expression and has been studied extensively studied. However, less attention has been given to the role of the ubiquitin-like protein Nedd8. Here we show that Nedd8 plays an important role in the ubiquitination of ENaC in alveolar epithelial cells. We report that the Nedd8 pathway is redox-sensitive and that under oxidizing conditions Nedd8 conjugation to Cullin-1 is attenuated, resulting in greater surface expression of α-ENaC. This observation was confirmed in our electrophysiology studies in which we inhibited Nedd8-activating enzyme using MLN4924 (a specific Nedd8-activating enzyme inhibitor) and observed a marked increase in ENaC activity (measured as the product of the number of channels (N) and the open probability (Po) of a channel). These results suggest that ubiquitination of lungENaCis redox-sensitive and may have significant implications for our understanding of the role of ENaC in pulmonary conditions where oxidative stress occurs, such as pulmonary edema and acute lung injury. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc