FC020: The Uraemic Toxin Indoxyl Sulfate is an Activator Of Caspase-4 but not of Caspase-1 in Monocyte-Like THP-1 Cells
Authors: Ulrich Christof , Fiedler Roman , Markau Silke , Girndt Matthias 
Published: May 2022

Abstract

BACKGROUND AND AIMS
Among the plethora of uraemic toxins the protein-bound indoxyl sulphate (IS) plays a prominent role in cardiovascular and renal disease progression. IS also appears to be involved in programmed cell death pathways. Regarding the high inflammatory pyroptosis rates in blood cells of haemodialysis patients (HD), there is the question of whether IS can induce caspase-1 and/or caspases-4/-5, thus triggering pyroptosis-related pathways and the release of proinflammatory interleukin IL-1ß in monocytes.

METHOD
IS-related effects were investigated in the monocytic cell line THP-1. Differentiated THP-1 (100 nM PMA) cells were used to study IS effects in macrophages. IS concentrations in the range of 1 to 4 mM were applied to reveal putative mechanistic effects mediated by IS. Caspase-1, -4 and -5 specific substrates were used to analyse the different caspase activities by flow-cytometry and luminometric assays. IL-1ß activity of THP-1 supernatants was measured in the HEK-Blue™ IL-1ß cell line, which produces secreted embryonic alkaline phosphatase (SEAP) upon induction with active IL-ß.

RESULTS
Flow cytometric analysis demonstrates that pyroptosis (AAD+/caspase-1+) is already increased by the stimulation of THP-1 cells at concentrations of 1 mM IS, a concentration which can be found in HD patients with high IS load. These data were confirmed by a second luminometric assay, which is based on a different caspase-1 specific substrate compared with the flow-cytometric assay (Ac-WEHD-aminoluciferin versus FAM-YVAD-FMK). Further, the caspase-1 activity could be enhanced by supplementing IS-stimulated (2 mM) samples with the caspase-1-dependent NLRP3 inflammasome activator nigericin (21 µM). Taking in mind that especially FAM-YVAD-FMK substrate can be cleaved by caspase-1 and caspase-4/-5, we examined the specificity of the tested substrate using inhibitor probes. Supplementing cells with the caspase-1 specific inhibitor Ac-YVAD-CHO (2 µM), we failed to inhibit IS-mediated effects, whereas the nigericin-driven effect could be blocked. However, the addition of the inhibitory substrate Ac-LEVD-CHO (2µM), which specifically blocks caspase-4 and -5 activities totally decreased IS-derived caspase activity (Control: 100%, IS: 128 ± 12%, IS + Ac-LEVD: 81 ± 22%, IS + AC-YVAD: 107 ± 15%; IS versus IS + Ac-LEVD, P &lt; .001). It is known that IS has a stimulating impact on mRNA expression of pro-IL-1ß in macrophage-like THP-1 cells. When differentiated THP-1 cells were stimulated with 4 mM IS for 24-h we found a two-fold increase in IL-1ß mRNA expression. Further, we find a significant increase in caspase-1 and caspase-4 in these cells. However, IL-1ß protein levels seem to be only marginally elevated in IS stimulated samples. Analysing the biological activity of the THP-1 derived supernatants in the HEK-Blue™ IL-1ß cell line, we did not find a SEAP increase in samples stimulated with the IS-derived THP-1 supernatants.

CONCLUSION
Analysing the effects of indoxyl sulphate in the THP-1 model systems we provide evidence for the first time that the inflammatory caspase-4 and/or caspase-5 is activated in monocyte-like cells without increasing IL-1ß levels. Therefore, one can conclude that IS is not an archetypical NLRP3 inflammasome activator but may contribute to cell death via caspase-4 activation.