Figure 5. Activation of the inflammasome with TLR ligand-conjugated microparticles.

A) Secretion of IL-1β by human THP-1 macrophages was measured in cell culture media 3 and 24 hr after introduction of LPS-microparticles (pSi-LPS; 1:20; *p<3×10⁻¹¹ compared to no treatment controls). The influence of the caspase inhibitor Z-VAD-FMK (*p<3×10⁻¹⁰, **p<7×10⁻¹¹ compared to pSi-LPS) or cytochalasin B (Cyto; *p<3×10⁻⁶, **p<0.04 compared to pSi-LPS) on IL-1β levels was determined. ATP was used as a positive control for inflammasome activation (*p<2×10⁻⁸, **p<6×10⁻⁸ compared to control levels; n=6 per group). B-C) Secretion of IL-1β by GM-CSF-stimulated bone marrow cells was studied at B) various time points after introduction of free, adsorbed, or conjugated LPS (1:25; *p<0.05; **p<0.005; ***p<0.02 compared to equivalent free LPS), or C) different concentrations of control or ligand-bound microparticles at 24 hr (*p<0.01; **p<0.007; ***p<0.02, compared to same concentration control microparticles). D–E) Secretion of TNF-α and IL-6 by GM-CSF-stimulated bone marrow cells 24 hr after introduction of microparticles titrated at 3 concentrations (*,*p<0.04 1:5 MPL-pSi compared to untreated cells and control microparticles; **p<0.0005; ***p<0.006 LPS-pSi compared to same concentration pSi). F) Serum cytokine levels were measured in mice 24 hr after subcutaneous injection with control or ligand-bound microparticles (10 μg LPS equivalent for 5 × 10⁸ LPS-microparticles per mouse; TNF-α *p<0.05; IL-6 *p<0.03; **p<0.003; ***p<0.001; IL-1β * p<0.0002. **p<0.01 compared to basal levels). G–I) Cytokine production was compared in cells treated with free ligand or microparticle-bound ligand (covalent) at 5 microparticles per cell (see C–E for cytokine levels at higher numbers of microparticles). Free MPL and LPS were presented at 1μg/ml in 2 ml media (2 μg ligand per well).

(5-fold,p<0.04; and 2-fold, p<0.02, respectively