Ex vivo HIV entry into blood CD4+ T cells does not predict heterosexual HIV acquisition in women.

CAPRISA, 2018.Abstract available in pdf

Proteolytic Processing of Nlrp1b Is Required for Inflammasome Activity

Nlrp1b is a NOD-like receptor that detects the catalytic activity of anthrax lethal toxin and subsequently co-oligomerizes into a pro-caspase-1 activation platform known as an inflammasome. Nlrp1b has two domains that promote oligomerization: a NACHT domain, which is a member of the AAA+ ATPase family, and a poorly characterized Function to Find Domain (FIIND). Here we demonstrate that proteolytic processing within the FIIND generates N-terminal and C-terminal cleavage products of Nlrp1b that remain associated in both the auto-inhibited state and in the activated state after cells have been treated with lethal toxin. Functional significance of cleavage was suggested by the finding that mutations that block processing of Nlrp1b also prevent the ability of Nlrp1b to activate pro-caspase-1. By using an uncleaved mutant of Nlrp1b, we established the importance of cleavage by inserting a heterologous TEV protease site into the FIIND and demonstrating that TEV protease processed this site and induced inflammasome activity. Proteolysis of Nlrp1b was shown to be required for the assembly of a functional inflammasome: a mutation within the FIIND that abolished cleavage had no effect on self-association of a FIIND-CARD fragment, but did reduce the recruitment of pro-caspase-1. Our work indicates that a post-translational modification enables Nlrp1b to function

Development and clinical application of a novel ex vivo assay to assess genital HIV susceptibility

A better understanding of the biological factors that promote heterosexual HIV acquisition in the female genital tract (FGT) may inform HIV prevention efforts. The mucosal cellular targets of HIV are not well defined and proposed cellular and molecular correlates include the HIV co-receptor CCR5 and markers of immune activation, including CD69. I established a novel ex vivo HIV entry assay for mucosal studies to enable assessment of ex vivo HIV infection in freshly isolated and unstimulated endocervical CD4+ T cells. HIV susceptibility was enhanced in cervix-derived CD4+ T cells compared to blood. Cervical CCR5+, CD69+, α4β7+ or α4β1+ CD4+ T cells were preferential HIV targets, however, the homing integrins α4β7 or α4β1 did not mediate direct binding to the HIV env or HIV entry into CD4+ T cells, suggesting that α4β7 and α4β1 are unlikely to be HIV entry receptors. Next, I tested whether ex vivo HIV entry predicts subsequent heterosexual HIV acquisition. Ideally, this would be done using fresh genital samples, but these are usually not available. Hence, I performed a nested, retrospective, blinded, case-control study in blood CD4+ T cells obtained from HIV-uninfected South African female participants enrolled in the CAPRISA 004 clinical trial. Virus entry into blood CD4+ T cell subsets did not predict subsequent HIV acquisition, possibly due to the compartmentalization of immune factors in the genital mucosa. One such compartmentalized factor in the FGT is the bacterial microbiota, which is dominated in the majority of women by lactobacilli, while a shift towards diverse gram-negative anaerobic species, defined (based on Gram stain) as bacterial vaginosis (BV), is associated with increased HIV acquisition in women. To investigate underlying biological mechanisms that may increase HIV acquisition risk in women with BV, I assessed the impact of BV treatment using the current standard of care, oral metronidazole, on genital immunology and HIV susceptibility. BV treatment reduced endocervical CD4+ T cell susceptibility to HIV and genital IL-1 levels; however, an unexpected increase in several chemokines previously associated with increased HIV risk suggests that more studies are needed before the treatment and/or prevention of asymptomatic BV can be recommended for HIV prevention.Ph.D

The FIIND Domain of Nlrp1b Promotes Oligomerization and Pro-caspase-1 Activation in Response to Lethal Toxin of Bacillus anthracis

Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like-receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self-associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity.MAS

Nlrp1b1_1100–1233 self-associates and actives pro-caspase-1.

<p>(A) HT1080 cells were co-transfected with Nlrp1b1 truncation constructs, pcDNA3-pro-caspase-1-FLAG and pcDNA3-pro-IL-1β-HA as indicated. After 24 h, cells were lysed and lysates were probed for T7-tagged Nlrp1b truncation mutants and β-actin by immunoblotting; supernatants were immunoprecipitated with anti-HA antibodies and then probed for HA-tagged IL-1β. (B) HT1080 cells were transfected with pcDNA3-His₆-Nlrp1b1-HA and pcDNA3-His₆-Nlrp1b1-T7 tagged Nlrp1b1 truncation constructs as indicated. Cells were lysed 24 h after transfection, and proteins were immunoprecipitated using anti-HA antibody, followed by immunoblotting with anti-T7 antibody and anti-HA antibody. Blots are representative of three independent experiments.</p

The V988D mutation in Nlrp1b1_720–1233 reduces its ability to associate with pro-caspase-1.

<p>(A) HT1080 cells were transfected with pcDNA3-pro-caspase-1-T7 and pcDNA3-pro-IL-1β-HA and co-transfected as indicated with pNTAP-Nlrp1b1_720–1233-HA, pNTAP-Nlrp1b1_720–1233-T7, pNTAP-Nlrp1b1_720–1233-V988D-HA, and pNTAP-Nlrp1b1_720–1233-V988D. Approximately 24 h after transfection, cell lysates were collected and probed for HA-tagged pro-IL-1β and β-actin; cell supernatants were collected and immunoprecipitated with anti-HA antibodies and probed for HA-tagged IL-1β by immunoblotting. (B) Cells were transfected with indicated combinations of pNTAP-Nlrp1b1_720–1233-HA and pNTAP-Nlrp1b1_720–1233-T7 WT or V988D as well as with pcDNA3-pro-caspase-1-C284A-FLAG and pcDNA3-pro-IL-1β-HA. Approximately 24 h following transfection, cells were lysed and immunoprecipitated using an anti-T7 antibody. Proteins were immunoblotted with antibodies against HA, caspase-1, T7 and β-actin. Blots are representative of three independent experiments.</p

Mutation of amino acids 1100–1106 impairs FIIND self-association, FIIND cleavage, and Nlrp1b1 activity.

<p>(A) Nlrp1b1_1100–1233 or Nlrp1b1_1100–1233-7A (containing alanine substitution mutations of amino acids 1100–1106) was expressed with pro-caspase-1 and pro-IL-1β-HA in HT1080 cells. Cells were lysed 24 h after transfection. Cell lysates were probed for T7-tagged Nlrp1b1 constructs and for β-actin by immunoblotting; supernatants were immunoprecipitated with anti-HA antibodies and then probed for HA-tagged IL-1β. (B) T7-tagged and HA-tagged Nlrp1b1_1100–1233 or Nlrp1b1_1100–1233-7A were expressed in HT1080 cells. HA-tagged proteins were immunoprecipitated from cell lysates and the immunprecipitates were probed for T7- and HA-tagged proteins. (C) HA-tagged Nlrp1b1_1100–1233, Nlrp1b1_1100–1233-7A and Nlrp1b1_1142–1233 were co-expressed with pro-caspase-1-C284A-T7. Immunoprecipitations were performed with anti-T7 or control anti-GFP antibodies and immunoblotted for HA and T7 epitopes. Cell lysates (right panel) were immunoblotted as indicated. (D) Nrlp1b1 and Nrlp1b1-7A were expressed with pro-caspase-1 and pro-IL-1β-HA in HT1080 cells. Cells were treated with LeTx for 3 h and supernatants were probed for IL-1β-HA as above. (E) Cell lysates from (D) were probed for Nlrp1b1. Blots are representative of three independent experiments.</p

Nlrp1b1 is cleaved within the FIIND.

<p>(A) Untransduced J774A.1 cells, or J774A.1 cells stably expressing scrambled shRNA or Nlrp1b shRNA were treated with LeTx (10⁻⁹ M LF and 10⁻⁸ M PA) for 4 h and cell supernatants were assayed for LDH activity as an indication of cell death. Error bars indicate SEM of three independent experiments. (B) HT1080 cells were either transfected with pNTAP empty vector or pNTAP-Nlrp1b1. Approximately 24 h after transfection, cells were lysed and TAP-tagged proteins were precipitated with streptavidin resin. Precipitated TAP-tagged proteins and lysates from J774A.1 cells stably expressing either scrambled shRNA or Nlrp1b shRNA were subjected to immunoblotting using an anti-Nlrp1b1 antibody. Arrows indicate two bands present in control, but not in Nlrp1b1 knockdown lysates. (C) HT1080 cells were transfected with either pNTAP empty vector, pNTAP-Nlrp1b1, or pNTAP-Nlrp1b1-TAP. Approximately 24 h following transfection, cells were lysed and TAP-tagged proteins were precipitated with streptavidin resin and immunoblotted using an antibody directed against the TAP tag. Arrows indicate full-length Nlrp1b1 and two cleaved fragments of Nlrp1b1.</p

The cleaved products of Nlrp1b1 remain associated.

<p>(A) HT1080 cells were transfected with the indicated Nlrp1b1 plasmids. Approximately 24 h following transfection, cells were treated with LeTx (10⁻⁸ M LF and 10⁻⁸ M PA) for the indicated times. Cells were lysed and TAP-tagged proteins were precipitated with streptavidin resin. Proteins from the precipitates and the lysates were immunoblotted with antibodies against HA and β-actin. Blots represent three independent experiments.</p

The V988D mutation in Nlrp1b1 eliminates both cleavage and inflammasome activity.

<p>(A) Alignment of allele 1 with allele 3 from amino acid 969 to 1112 of Nlrp1b1. Highlighted amino acid differences use numbering from allele 1. (B, D, and F) HT1080 cells were transfected with pNTAP plasmids encoding the indicated Nlrp1b proteins. Approximately 24 h following transfection, cells were lysed and TAP-tagged proteins were precipitated with streptavidin resin and immunoblotted using an Nlrp1b antibody. (C, E, and G) Cells were transfected with pNTAP plasmids encoding the indicated Nlrp1b constructs, as well as with pcDNA3-pro-caspase-1-T7 and pcDNA3-pro-IL-1β-HA. Approximately 24 h after transfection, cells were treated with LeTx (10⁻⁸ M LF and 10⁻⁸ M PA) for 3 h. Cell lysates were collected and probed for HA-tagged pro-IL-1β and β-actin; cell supernatants were collected and immunoprecipitated with anti-HA antibodies and probed for HA-tagged IL-1β by immunoblotting. Blots are representative of three independent experiments.</p