CD44 Receptor Mediates Uptake of Uric Acid Crystals by Macrophages: Impact of Antibody‐Mediated Receptor Shedding on NLRP3 Inflammasome Activation and Interleukin‐1 Beta Production
Authors: Bousoik Emira , Elsaid Khaled 
Published: June 2021

Introduction

Gout is a chronic inflammatory disease due to monosodium urate (MSU) crystal deposits in joints. MSU phagocytosis by resident macrophages triggers NLRP3 inflammasome activation and downstream expression of interleukin‐1 beta (IL‐1β). The mechanism of MSU phagocytosis by macrophages is poorly understood. Transmembrane CD44 receptor is highly expressed in macrophages and plays an important role in inflammation. The objectives of this study is to evaluate the role of CD44 in mediating MSU crystal uptake by macrophages and assess the utility of antibody‐mediated receptor shedding as a novel therapeutic approach in gout.

Methods

Bone marrows were harvested from Cd44+/+ and Cd44−/− male mice (n=8 in each genotype; 8–10 weeks old) and bone marrow derived macrophages (BMDMs) were generated using established protocols. All animal tissue harvests were approved by the IACUC committee at Chapman University. Cd44+/+ and Cd44−/− BMDMs were incubated with pyrogen‐free MSU suspension (100μg/ml; Invivogen) for 6 hours followed by determining the percentage of cells with intracellular MSU crystals. In another set of experiments, BMDMs from both genotypes were incubated with MSU followed by measuring IL‐1β expression and protein levels. Human THP‐1 monocytes (ATCC) were differentiated into THP‐1 macrophages using established protocols. MSU phagocytosis by THP‐1 macrophages ± anti‐CD44 or isotype control antibody treatments (2μg/ml for both treatment for 6 hours) was determined using flow cytometry. Soluble CD44 and IL‐1β levels were determined using ELISA (R&D Systems). NLRP3 inflammasome activation was determined using western blot analysis of the inflammasome components: NLRP3, procaspase‐1, activated caspase‐1 and ASC‐1 proteins. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey's post‐hoc test.

Results

Cd44+/+ BMDMs had enhanced MSU phagocytosis compared to Cd44−/− BMDMs (fig. 1A and 1B; p<0.01). MSU exposure induced higher IL‐1β expression and production in Cd44+/+ BMDMs compared to Cd44−/− BMDMs (fig. 2A and 2B; P<0.05; p<0.001). Anti‐CD44 antibody treatment reduced MSU phagocytosis by THP‐1 macrophages as determined by a reduction in cell side scattering compared to MSU alone or MSU+IC treatments (fig. 1C and 1D; p<0.01). Anti‐CD44 treatment resulted in shedding of CD44 extracellular domain (fig. 2C; p<0.01). MSU exposure reduced procaspase‐1 and increased activated caspase‐1 protein levels in THP‐1 macrophages (fig. 2D and 2E; p<0.001). Capsase‐1 activation resulted in enhanced IL‐1β production in MSU‐stimulated THP‐1 macrophages (fig. 2F; p<0.01). Anti‐CD44 antibody treatment reduced caspase‐1 activation (fig. 2E, p<0.05) and downstream IL‐1β production (fig. 2F; p<0.01) in MSU‐stimulated THP‐1 macrophages.

Conclusion

CD44 receptor mediates MSU crystal phagocytosis in murine and human macrophages. Antibody‐mediated CD44 receptor shedding inhibited NLRP3 inflammasome activation and reduced downstream IL‐1 β production. Targeting CD44 receptor is a potential novel therapeutic strategy to treat inflammation in gout.

Support or Funding Information

Supported by NIH R01AR067748.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.