Do uric acid crystals activate the NLRP3 inflammasome?

 

Radomir Slominski; Marilyn E. Merriman ; Isidoro Cobo ; Tanecia Mitchell ; Joseph Crivelli; Chander Raman ; Richard Reynolds ; Tony R. Merriman.

 

Affiliation(s):

University Of Alabama At Birmingham

 

 

Background and Objective: Crystallopathies are disorders defined by the formation, deposition, and accumulation of crystals within tissues. A classic example is gout, in which needle-shaped monosodium urate (MSU) crystals that form at neutral pH activate innate immunity via phagocytosis-dependent NLRP3 inflammasome activation leading to Interleukin-1β (IL-1β) secretion. Kidney stone disease represents another crystallopathy, arising from crystal nucleation and aggregation within the renal collecting systems. Uric acid (UA) stones form in an acidic environment (pH <5.5) and exhibit a high rate of symptomatic reoccurrence. One overlooked aspect of uric acid crystal formation in the kidney is the potential of small crystals to induce an inflammatory response. Evidence suggests that the size and morphology of crystals can significantly influence the immune response to these foreign particles. We hypothesize that UA crystals activate the innate immune system through the NLRP3 inflammasome in a size-dependent manner.

Methods: THP-1 cells (a human monocytic cell line) and primary peripheral blood mononuclear (PBMCs) from healthy donors were used for experimental models. THP-1 cells were stimulated with lipopolysaccharide (LPS) for 16 hours and PBMCs for 24 hours, with and without MSU crystals at 300 μg/mL (for comparison) and sonicated and non-sonicated UA crystals, also at 300 μg/mL. UA crystals were sonicated to decrease their size, relative to the non-sonicated ones. Cells were seeded at 5 x 10^5 cells per well in a 48 well plate with four biological replicates and two technical replicates per condition for THP-1 cells and five donor samples and two technical replicates per condition for PBMCs. IL-1β was measured using an enzyme linked immunosorbent assay (ELISA) kit. Statistical significance was assessed using two-way analysis of variance (ANOVA) in SAS, followed by appropriate post-hoc multiple-comparison testing.

Results: In THP-1 cells, two-way ANOVA revealed a significant main effect of sonicated UA crystals on IL-1β response production (p<.0001) and a significant main effect of LPS (p=0.0011), with a trend toward an interaction between LPS response and sonicated UA crystals (p=0.067). Post-hoc pairwise comparisons (Figure 1) showed that IL-1β levels were significantly increased with MSU+LPS (p= 0.0327) and sonicated UA + LPS (p= 0.0031), but not with UA + LPS (p= 0.88). 

In PBMCs, two-way ANOVA revealed a significant main effect of sonicated UA IL-1β crystals on IL-1β response production (p< 0.0019), with a significant main effect of LPS (<.0001), and the LPS response dependent on the presence of sonicated UA crystals (p=0.0042).  Post-hoc pairwise comparisons (Figure 2) shown that IL-1β levels were significantly increased with MSU +LPS (<.0001) and sonicated UA + LPS (p <.0001), but with an insignificant interaction between LPS and UA (p=0.2661).

Thus, in both THP-1 cells and PBMCs, co-stimulation with sonicated UA crystals and LPS induced IL-1β production at levels comparable to those elicited by LPS combined with MSU crystals and significantly higher than LPS alone.

Conclusion and Significance: The results suggest that sonicated UA crystals enhance LPS- induced IL-1β secretion, with a more robust effect observed in PBMCs. These findings raise the possibility that subclinical(asymptomatic) UA crystals may activate innate immune pathways and can contribute to chronic, low-grade kidney inflammation.