Fig. 7: Recombinant antagonistic GSDMD nanobodies inhibit pyroptosis when administered extracellularly.
A–D PMA-differentiated THP-1 cells were treated with MxiH as described in Fig. 2C (A, B) or with LPS and Nig. as described in Fig. 2D (C, D) in the presence of increasing concentrations (2, 20, 50, 100, and 200 µg/mL) of the indicated recombinant nanobodies. A, C LDH release was measured and normalized to cells lysed in Triton X-100. B, D IL-1β in the supernatant was quantified by HTRF. E, F PMA-differentiated THP-1 cells were treated with MxiH, 100 ng/mL PFO, or 5 µM Stau for 1, 2, 3, 4, and 20 h in the presence of 200 µg/mL of the indicated nanobodies. The reducing capacity as a readout for viability was determined by CellTiter-Blue (CTB) assay and normalized to untreated cells in the presence of VHHNP-1. As a positive control, cells were incubated with 1% Triton X-100. Where indicated, cells were treated for 4 h in the presence of 40 µM caspase-3/7 inhibitor (casp-3/7i) (F). G, H M-CSF-differentiated primary human macrophages from independent donors were treated as in Fig. 7, A and B, and LDH release (G) and IL-1β secretion (H) were quantified as before. I, J THP-1 cells (I) or THP-1 cell lines expressing the indicated HA-tagged VHHs (J) were differentiated with PMA, labeled with CMO, and stimulated with MxiH in the presence of 200 µg/mL VHHNP-1 total (60 µg/mL of the nanobody was AF647 labeled). Where indicated, stimulation was performed in the presence of 40 µM VX (I). Cells were recorded by live cell confocal microscopy including bright field recordings. Cell areas (mostly containing a single cell) were identified using the CMO staining by Imaris. Cell areas were scored as VHH-positive (VHH+) when VHHNP-1-AF647 intensity was at least 80 (corresponding to ca. 75% of the mean intensity outside the cells in the first data set). The fraction of VHH+ cell areas was normalized to the total cell area. Average values from three independent experiments ± SEM are displayed. K Model for the inhibition of pyroptosis by antagonistic GSDMD nanobodies added to the extracellular space. Inflammasome-dependent cleavage of GSDMD allows insertion of monomeric GSDMDNT into the plasma membrane, where monomers oligomerize and assemble pores that penetrate the membrane. This allows influx of extracellular nanobodies into the cytosol (left). These prevent the formation of new GSDMD pores by stabilizing monomers. Existing GSDMD pores are removed by membrane repair mechanisms, including shedding as well as potentially endocytosis, which ultimately prevents cell death by pyroptosis (right). Cells bearing conventional inflammasomes will eventually die by non-inflammatory apoptosis. Data on LDH, IL-1β release, and CTB assays represent average values (with individual data points) from three independent experiments or donors ± SEM. *P  <  0.05, **P  <  0.01, ***P  <  0.001, and ****P  <  0.0001 (unpaired two-tailed Student’s t-test).
