Fig 1.
IL-1β and IL-18 secretion in HIV infection are differentially controlled.
Monocyte derived macrophages (MDMs) (1 x 106) were infected with HIV-1 (MOI = 1) or left untreated for 12hr and 24hr. Supernatant was collected post infection and analysed for IL-1β and IL-18 secretion using ELISA kits (R&D Systems) (A). Confocal microscopy of ASC speck formation and oligomerisation of NLRP3 in MDMs in response to HIV-1 (B). TLR2, TLR3, TLR4 or TLR8 expression in MDMs was knocked down using siRNA (80% inhibition of expression of TLRs) and infected with HIV-1 for 12 hr and subsequently determined pro-IL-1β by ELISA (C). NLRP3, NLRP1, NLRC4, or NLRC5 expression in MDMs was knocked down by siRNA (80% inhibition of expression of NLRs) and the cells were infected with HIV-1 for 12 hr and examined for IL-1β, IL-18 (D), Graphs were normalized to the level of siRNA knockdown. Furthermore NLRP3, NLRP1, NLRC4, or NLRC5 expression was knocked down by siRNA and the cells were infected with HIV-1 for 12 h and the presence of pro-IL1β, pro-IL18, and cleaved IL-1β and IL-18 was investigated via western blotting (E). Quantification of relative levels of pro-IL-1β/IL-1818 and IL-1β/Il-18 was determined by densitometry using Image Studio Lite (Licor) and normalized to internal control (β-actin) from 3 western blots (F). The data represent the mean of three independent experiments ± SD (n = 3 sets of MDMs) yielding consistent results. **, p < 0.005 and ***, p < 0.001 indicate statistically significant differences.
Fig 2.
HIV Vpu and gp41 play a role in inflammasome activation.
Monocyte derived macrophages (MDMs) (1 x 106) were either left untreated or transfected with 2μg of pVpu plasmid expressing Vpu or pgp41 or gp120 plasmid expressing gp120. Supernatants were collected and tested for IL-1β and IL-18 secretion at 12 hr (A) and 24 hr (B). Cells extracts at 12 hr from mock or transfected cells were analysed for the presence of caspase-1 p20 by western blotting, followed by quantification using Image Studio Lite (Licor) and normalized to internal control (β-actin) from 3 western blots(C). MDMs expressing gp41 (D) or Vpu (E) monocytes were silenced for TLR2, TLR3, TLR4 and TLR8 by siRNA. Supernatant was collected at 12 hr and analysed for IL-1β and IL-18 using ELISA. Cell extracts of MDMs stimulated with either Vpu (F) or gp41 (G) were analysed for the presence of pro-IL-1β, pro-IL-18, cleaved IL-1β or cleave IL-18 by western blotting, followed by quantification using Image Studio Lite (Licor) and normalized to internal control (β-actin) from 3 western blots. MDMs expressing Vpu (H-J), gp41 (K-M) or pgmock (N-P) were silenced for NLRP3, NLRP1, NLRC4 or NLRP3 and NLRC4 by siRNA. Supernatant was collected at 12 hr and analysed for IL-1β (Η, Κ, Ν) and IL-18 (I,L,O) using ELISA. All graphs were normalized to the level of siRNA knockdown. Cells extracts were analysed for the presence of caspase-1 p20 at 12 hr by western blotting, followed by quantification using Image Studio Lite (Licor) and normalized to internal control (β-actin) from 3 western blots (J,M,P). The data represent the mean of three independent experiments ± SD (n = 3 sets of macrophages) yielding consistent results. **, p < 0.005 and ***, p < 0.001 indicate statistically significant differences.
Fig 3.
Monovalent Ion channel inhibitors reduce IL-1β dependent inflammasome activation.
Monocyte derived macrophages (MDMs) (1 x 106) were infected with HIV-1 (MOI = 1) for 12 hr were also cultured in the presence or absence of Cathepsin B inhibitor (CA-074) (100 μM), DPI (20μM), NAC (20mM) chloropromazine (50μg/ml). Supernatants were collected and analysed for IL-1β and IL-18 using ELISA (A). MDMs stimulated with either Vpu (50 ng/ml) (B) or gp41 (50 ng/ml) complexed with Lyovec (C)were pre-treated with and cultured in the presence of BAPTA-AM (15 μM), EIPA (25 μM), benzamil (50μM), verapamil (50μM), amantadine (6.25 μM) or rimantadine (6.25 μM) or TEA (10mM) for 12 hrs. Supernatants were collected and tested for IL-1β (B) or IL-18 (C)) secretion using ELISA. IL-1β secretion in response to VpuS24L (D). In addition, MDMs stimulated with Vpu (50 ng/ml) or gp41(50 ng/ml) were treated with and cultured in the presence or absence of MgTX (5nM), Ba2+ (300 mM), 4AP (1mM), Sotalol (100μM), GSK2332816A (350nM) for 12 hrs. Supernatants were collected and tested for IL-1β (E) or IL-18 (F) secretion using ELISA. The data presented is the mean of three independent experiments ± SD ** p < 0.005 and ***, p < 0.001 indicate statistically significant differences.
Fig 4.
Vpu targets Kv1.3 channels to induce inflammasome activation.
Monocyte derived macrophages (MDMs) were transfected with pVpu at 24 h post transfection cells were fixed and permeabilised in PBS/0.02% BSA, prior to fixation with 4% formaldehyde for 15 min. Cells were then stained with GM130 mAb to label the Golgi, a rabbit anti-Vpu antibody to label Vpu or a Kv1.3 mAb followed by the appropriate secondary conjugated to Alexa 546 (red) or Alexa 488 (green). The nucleus was stained with TOPRO-3. Cells were imaged using a Zeiss 710 confocal microscope. Bars shown are 10 μm. The data presented are at least in 20 cells over four independent experiments. The merged images show extensive colocalization between Kv1.3 and the Golgi R(obs) of 0.715 as well as between Vpu and Kv1.3 R(obs) of 0.689 respectively (A,B). The degree of colocalization was determined using ImageJ software via the Costes’ method. Data presented are representative images of n = 3 biological replicates, with at least 20 technical repeats. Findings were consistent across all replicates. SIM super resolution imaging of Kv1.3 distribution at Golgi organelles is also shown. As well as Vpu and Kv1.3 distribution in macrophages transfected with pVpu at 24 h post transfection (C,D). Quantification (right) of the relative fluorescence of the target structures where regions of interest (ROI) were selected for the target areas are shown. The distribution/position of receptors was obtained with Zeiss Zen black or ImageJ software. Cells transfected with pVpu at 24 h post transfection cells were treated with 10μg/ml Brefeldin. Golgi was stained with Anti GM130 goat antibody conjugated to Alexa 546. An anti Vpu antibody conjugated to Alexa488 was used to label the Vpu protein. Anti Kv1.3 rabbit polyclonal IgG was followed by donkey anti rabbit IgG-Alexa 633. Images of before and after treatment are depicted (E,F). Supernatants were also collected and tested for IL-1β secretion using ELISA (G). The data presented is the mean of three independent experiments ± SD ** p < 0.005 and ***, p < 0.001 indicate statistically significant differences.
Fig 5.
Monocyte-derived macrophages (MDMs) (1 x 106) were infected with HIV-1 (MOI = 1), left untreated or pre-treated with nystatin for 12 hr. Molecular interactions between gp41 and lipid raft (GM-1 ganglioside), NAIP, NLRC4 and NLRP3 were assessed using Fluorescence resonance energy transfer (FRET) (A). MDMs stimulated with gp41 for 12 hr were treated with different concentrations of nystatin. Supernatants were also collected and tested for IL-18 secretion using ELISA (B). MDMs silenced or not for NAIP and then stimulated with gp41. Supernatant was collected and analysed for IL-18 using ELISA (C). Lysates from MDMs transfected with pgp41-FLAG (Myc-DDK tag) were subjected to IP using anti-gp41 antibody or using control IgG or using anti FLAG (D, top panel) and then analysed by western blotting using anti-NAIP antibody. Lysates from MDMs infected with HIV-1 were subjected to IP using anti-gp41 antibody or using control IgG (D, bottom panel) and then analysed by western blotting using anti-NAIP antibody. (D) The data presented is the mean of three independent experiments ± SD ** p < 0.005 and ***, p < 0.001 indicate statistically significant differences. Super-resolution images of gp41 and NAIP as well as NAIP and NLRC4 in cells infected with HIV-1 are also shown (E) Cells were labelled with anti gp41-Atto488 (green), anti NAIP-Alexa546 (red) (top panel) or anti NLRC4-Alexa546 (red) and anti NAIP–Atto488 (green) (bottom panel), fixed, imaged, and processed for dSTORM. Colocalization of receptors is presented in arbitrary units (AU) as mean fluorescence intensity of the red and green signal. Representative regions (right) of the relative fluorescence of the target structures where regions of interest (ROI) were selected for the target areas is also shown and co cluster analysis using bivariant Ripley’s L function. The distribution/position of receptors was obtained with Zeiss Zen black and ImageJ software.
Fig 6.
NAIP has an affinity for the CT domain of gp41.
A schematic view of gp41 showing important functional regions, including the fusion peptide (FP), the transmembrane region (TM)) and cytoplasmic tail (CT). The domains are drawn approximately to scale. (A), Estimation of NAIP binding affinity to gp41 domains by the BIAcore system. NAIP protein was immobilized on the matrix of the chip and gp41 proteins were injected at flow rate 10 ml/min for 240 s. The binding ability of NAIP to each host protein was monitored and presented as a sensogram (plotted as RU versus time). For kinetic studies, gp41 proteins with increasing concentrations (500ng, 1μg, 5μg, 10 μg) were injected over the sensor chip. (B). Theoretical model of mechanisms of inflammasome activation by HIV Vpu and gp41 (C).
Table 1.
Binding constants of gp41 domains to NAIP.
The binding affinity is expressed as KD.