Evaluation of protein kinase D auto-phosphorylation as biomarker for NLRP3 inflammasome activation

Abstract

Background: The NLRP3 inflammasome is a critical component of sterile inflammation, which is involved in many diseases. However, there is currently no known proximal biomarker for measuring NLRP3 activation in pathological conditions. Protein kinase D (PKD) has emerged as an important NLRP3 kinase that catalyzes the release of a phosphorylated NLRP3 species that is competent for inflammasome complex assembly.

Methods: To explore the potential for PKD activation to serve as a selective biomarker of the NLRP3 pathway, we tested various stimulatory conditions in THP-1 and U937 cell lines, probing the inflammasome space beyond NLRP3. We analyzed the correlation between PKD activation (monitored by its auto-phosphorylation) and functional inflammasome readouts.

Results: PKD activation/auto-phosphorylation always preceded cleavage of caspase-1 and gasdermin D, and treatment with the PKD inhibitor CRT0066101 could block NLRP3 inflammasome assembly and interleukin-1β production. Conversely, blocking NLRP3 either genetically or using the MCC950 inhibitor prevented PKD auto-phosphorylation, indicating a bidirectional functional crosstalk between NLRP3 and PKD. Further assessments of the pyrin and NLRC4 pathways, however, revealed that PKD auto-phosphorylation can be triggered by a broad range of stimuli unrelated to NLRP3 inflammasome assembly.

Conclusion: Although PKD and NLRP3 become functionally interconnected during NLRP3 activation, the promiscuous reactivity of PKD challenges its potential use for tracing the NLRP3 inflammasome pathway.

Fig 1. PKD auto-phosphorylation coincides with NLRP3 activation.

(A) IL-1β secretion measured using THP-1 cells primed with 100 nM PMA and stimulated with nigericin (15 μM), ouabain (1 uM), gramicidin (10 uM) or niclosamide (1uM) after the indicated time course. This is one of two experiments with similar results. Statistical significance was calculated with a Student T-test. (B) Stimulation time course using THP-1 cells treated with either nigericin, gramicidin, or niclosamide followed by whole cell lysates analysis by immunoblotting to detect PKD activation (auto-phosphorylation) and NLRP3 inflammasome activation (caspase-1 and gasdermin D cleavage). Tubulin levels were used as loading controls. This is one of two (gramicidin) or three (nigericin, niclosamide) experiments with similar results. An additional experiment with a prolonged time course in the presence of nigericin was implemented to allow for detecting NLRP3 activation (lower panel). (C) Stimulation time course using THP-1 cells treated with MSU crystals at 250 μg/ml followed by whole cell lysates analysis as done in B; this is one of two experiments with similar results. Total PKD3 levels (reported to be the only PKD family member expressed in THP1 cells [33]) were also monitored.

Fig 2. PKD kinase inhibition down-regulates NLRP3 activation.

(A) THP-1 cells were treated with CRT0066101 (10 μM) or 0.1% DMSO (vehicle control) for one hour before challenge with nigericin (15 μM) for 20 min. Immunoblot of whole cell lysates probed for auto-phosphorylated PKD. Tubulin is used as loading control. One of three experiments with similar results is shown (B) Left, FACS analysis of ASC speck formation in THP-1 cells treated with 15 uM nigericin for up to 3h in the presence of 20 μM CGP084892 (used to prevent pyroptosis and the resulting speck leakage), in the presence or absence of CRT0066101 (10 μM), MCC950 (1 μM) or vehicle control. Average values of two determinations ±SD; FACS plot data are provided as supplementary material (S1 Fig). Right, IL-1β secretion measured using PMA-primed THP-1 cells, treated with CRT0066101 (10 μM), MCC950 (1 μM) or vehicle control for one hour, before a time course of stimulation with nigericin (15 μM). This is one of three experiments with similar results. Statistical significance between inhibitor-treated samples and DMSO-treated samples was calculated with a Student T-test (C) Left, IL-1β secretion measured in THP-1 cells after 3h of incubation with a concentration range of nigericin; corresponding immunoblots displaying auto-phosphorylated PKD and cleaved gasdermin D levels obtained upon a kinetics of stimulation with 5 μM and 15 μM nigericin. One of two similar experiments is shown. Right, Effect of concentration ranges of MCC950 and CRT0066101 on IL-1β secretion, tested upon stimulation with nigericin (5 μM for 5h, or 15 μM for 3h). Means of triplicate determinations ±SEM. IC₅₀ (μM) for both compounds under both experimental conditions are indicated in the table. Statistical significance was calculated with a Student T-test.

Fig 3. NLRP3 blockade prevents PKD auto-phosphorylation.

(A) Immunoblot analysis of whole cell lysates from THP-1 cells treated with nigericin for the indicated times ± MCC950 (1 μM), AFN700 (3 μM) or CGP084892 (20 μM), monitoring total PKD3 as well as auto-phosphorylated PKD and gasdermin D cleavage. (B) Immunoblot analysis of whole cell lysates from THP-1-WT or NLRP3-KO cells treated with nigericin for the indicated times, auto-phosphorylated PKD, as well as full and N-terminally cleaved gasdermin D.

Fig 4. PKD auto-phosphorylation is not a selective inflammasome marker.

(A) Time course of stimulation of THP1-WT and THP1-NLRC4 cells upon stimulation with PrgI (3 μg/ml). Immunoblot analysis of whole cell lysates showing total and auto-phosphorylated PKD, as well as gasdermin D cleavage. (B) Time course of stimulation of THP1-WT with PrgI (3 μg/ml) ± MCC950 (10 μM). Nigericin stimulation ± MCC950 (10 μM) serves as positive control. Immunoblot analysis of whole cell lysates showing total and auto-phosphorylated PKD, as well as gasdermin D cleavage. Tubulin levels are used for comparison of PrgI and nigericin stimulatory conditions. (C) Time course of stimulation of THP1-WT with BAA473 (100 μM) ± MCC950 (10 μM). Nigericin stimulation ± MCC950 (10 μM) serves as positive control. Immunoblot analysis of whole cell lysates showing total PKD2/3 levels and auto-phosphorylated PKD, as well as gasdermin D cleavage. Tubulin levels are used for comparison of BAA473 and nigericin stimulatory conditions. (D) Time course of stimulation of U937-WT and U937-Pyrin cells with BAA473 (100 μM) ± MCC950 (10 μM). Immunoblot analysis of whole cell lysates showing total PKD2/3 levels and auto-phosphorylated PKD, as well as gasdermin D cleavage. Lower tubulin levels in pyrin-overexpressing cells at 45 min account for cell death triggered by BAA473.