CD44‐Targeted Monoclonal Antibody is Anti‐Inflammatory in an In‐Vivo Model of Monosodium Urate Crystal Induced Peritonitis

Introduction Gout is a common form of arthritis caused by hyperuricemia and the subsequent deposition of monosodium urate (MSU) crystals in peripheral joints and surrounding tissues, which activate the resident macrophages to produce pro‐inflammatory cytokines such as IL‐1β. In turn, the secretion of these cytokines promotes the infiltration of neutrophils and monocytes into the joint. The interaction of these infiltrated cells with the MSU crystals heightens inflammation leading to escalation of the condition. CD44 is a transmembrane glycoprotein that is expressed in a variety of cell types. CD44 participates in a multitude of critical physiological functions including cell migration and contributing to the ingestion of particles and apoptotic cells. In vivo studies using CD44‐specific monoclonal antibody (IM7) treatment reported anti‐inflammatory effects in animal models of several immune‐mediated human diseases, including rheumatoid arthritis and multiple sclerosis. The objectives of this study are to investigate the role of CD44 receptor in the pathogenesis of crystal‐induced inflammation in‐vivo and provide a proof‐of‐concept for systemic functional blockade of CD44 receptor as a novel treatment strategy for acute gout. Methods 12 to 14‐week‐old wildtype (WT), Cd44 −/− , and Nlrp3 −/− male mice were injected intraperitoneally ( i.p ) with 2 mg MSU crystals in 0.2 ml of sterile PBS or PBS alone. In another set of experiments, WT mice were randomly assigned to the following experimental groups: untreated controls, MSU + vehicle treated, MSU + anti‐CD44 antibody tor MSU + isotypc ontrol (IC) antibody. At 4 hours following i.p. injections, mice were euthanized and peritoneal lavaging was performed. Peritoneal lavage cells were stained with monocyte or neutrophil marker antibodies and analyzed by flow cytometry. Cytokine levels (IL‐1β, IL‐1Ra) in the peritoneal lavage fluids were determined by ELISA. Results Total cell numbers in peritoneal lavage from MSU‐administered WT mice were higher than total cells in Cd44 −/− and Nlrp3 −/− mice (fig. 1A ) . The number of infiltrated neutrophils and monocytes was higher in MSU‐administered WT mice compared to MSU‐administered Cd44 −/− and Nlrp3 −/− mice (fig. 1B–1D ). Lavage IL‐1β levels were higher in MSU‐administered WT mice compared to MSU‐administered Cd44 −/− or Nlrp3 −/− mice (fig. 1E ). IL‐1Ra levels in MSU‐administered WT mice were lower than corresponding levels in Cd44 −/− mice or Nlrp3 −/− mice (fig. 1F ). The IL‐1Ra/IL‐1β ratios in MSU‐administered Nlrp3 −/− and Cd44 −/− mice were higher than corresponding Il‐1Ra/IL‐1β ratio in MSU‐administered WT mice (fig. 1G ). Anti‐CD44 antibody treatment reduced the number of total cells recovered in peritoneal lavages compared to vehicle treatment (fig. 2A ). Anti‐CD44 antibody treatment reduced the number of infiltrated neutrophils and monocytes compared to vehicle and IC treatment groups (fig. 2B –2E). Lavage IL‐1 β levels in MSU + anti‐CD44 antibody group were lower than corresponding levels in MSU + vehicle and MSU + IC treatments (fig. 2F ). Conclusion Cd44 plays a significant role in the pathogenesis of MSU inflammation and consequently constitutes a potential therapeutic target for managing acute gout. Support or Funding Information Supported by NIH R01AR067748 to KE Impact of intraperitoneal administration of MSU crystals on inflammatory cell infiltration and production of IL‐1β and IL‐1Ra in WT, Cd44 − / − and Nlrp3 − / − mice. MSU (2mg in 200μl PBS) or PBS (200μl) were administered via the intraperitoneal route in WT (n=8), Cd44 − / − (n=8) or Nlrp3 − / − (n=6) and peritoneal lavaging was performed at 4 hours. Total peritoneal lavage cell counts were determined. The number of infiltrated neutrophils and monocytes were determined using flow cytometry and probing for neutrophil markers; Ly6B.2 and Ly6G and monocyte markers; Cd11b and Ly6C. Peritoneal lavage interleukin‐1 beta ( IL‐1β) and interleukin‐1 receptor antagonist (IL‐1Ra) levels were determined by ELISA and the IL‐1Ra/IL‐1β ratios were computed. *p<0.001; **p<0.01; ***p<0.05 . A. MSU administration in Cd44 − / − mice did not result in an increase in overall inflammatory cell infiltration. B. Representative flow cytometry plots showing increased percentage of lavage cells positive for Ly6B.2 and Ly6G in MSU‐administered WT mice compared to Cd44 − / − and Nlrp3 − / − mice. C. MSU administration in Cd44 − / − mice did not result in increased neutrophil infiltration. D. MSU administration in Cd44 − / − mice did not result in increased Ly6C hi monocyte infiltration. E. MSU administration in Cd44 − / − mice did not result in increased peritoneal IL‐1β levels. F. MSU administration in Cd44 − / − mice increased peritoneal IL‐1Ra production. G. Lavage IL‐1Ra/IL‐1β ratio was higher in Cd44 − / − mice. Figure 1 Impact of anti‐CD44 antibody treatment on inflammatory cell infiltration and production of IL‐1β in murine peritoneal MSU crystal inflammation model. MSU crystals (2mg in 200μl PBS), vehicle (Veh.; 200μl PBS), anti‐CD44 and isotype control (IC) antibodies (50μg in 200 μl PBS) were administered via the intraperitoneal route. Experimental groups included untreated controls (n=4), MSU+Veh. (n=4), MSU+Anti‐CD44 antibody (n=5) and MSU+IC antibody (n=3). Peritoneal lavaging was performed at 4 hours. Total peritoneal lavage cell counts were determined. The number of infiltrated neutrophils and monocytes were determined using flow cytometry and probing for neutrophil markers; Ly6B.2 and Ly6G and monocyte markers; Cd11b and Ly6C. Peritoneal lavage IL‐1β levels were determined by ELISA. *p<0.001; **p<0.01; ***p<0.05 . A. Anti‐CD44 antibody treatment reduced total cell recovered in peritoneal lavage following MSU administration. B. Representative flow cytometry plots showing reduced percentage of lavage cells positive for Ly6B.2 and Ly6G in anti‐CD44 treated animals compared to vehicle or IC antibody groups. C. Anti‐CD44 antibody treatment reduced neutrophil infiltration in MSU peritoneal inflammation model. D. Representative flow cytometry plots showing reduced percentage of lavage cells positive for cd11b and Ly6C in anti‐CD44 treated animals compared to vehicle or IC antibody groups. E. Anti‐CD44 antibody treatment reduced Ly6C hi monocyte infiltration in MSU peritoneal inflammation model. F. Anti‐CD44 antibody treatment reduced lavage IL‐1β levels compared to vehicle or IC antibody treatments. Figure 2