The Role of NLRP3 Inflammasome in Pericarditis: Potential for Therapeutic Approaches

Abstract

Human samples of patients with chronic pericarditis and appropriate control subjects were stained for the inflammasome components. A mouse model of pericarditis was developed through the intrapericardial injection of zymosan A. Different inflammasome blockers were tested in the mouse model. Patients with pericarditis presented an intensification of the inflammasome activation compared with control subjects. The experimental model showed the pathological features of pericarditis. Among inflammasome blockers, NLRP3 inflammasome inhibitor, anakinra, and interleukin-1 trap were found to significantly improve pericardial alterations. Colchicine partially improved the pericardial inflammation. An intense activation of the inflammasome in pericarditis was demonstrated both in humans and in mice.

Keywords: IL, interleukin; IL-1 trap; IL-1α; IL-1β; NLRP3 inflammasome; NLRP3inh, NLRP3 inflammasome inhibitor; anakinra; colchicine; pericarditis.

Conflict of interest statement

This investigator-initiated study was supported by funding from Kiniksa Pharmaceuticals Ltd. Dr. Abbate has served as a consultant for AstraZeneca, Janssen, Kiniksa Pharmaceuticals, Merck, Novartis, Olatec, and Serpin Pharma. Dr. Toldo has received research support from Kiniksa, Serpin Pharma, and Olatec. Drs. Bonaventura and Vecchié have received travel grant support from Kiniksa Pharmaceuticals to attend the 2019 American Heart Association Scientific Sessions (Philadelphia, Pennsylvania). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

© 2021 Published by Elsevier on behalf of the American College of Cardiology Foundation.

Figures

Graphical abstract

Figure 1

Schematic Representation of the Experimental Mouse Model of Acute Pericarditis Under general anesthesia, 1 mg of zymosan A (in 50 μl of sterile NaCl 0.9%) was injected in the pericardial sac of the mouse on day 0. Sham control procedures were performed injecting an equal volume of sterile NaCl 0.9% in the pericardial sac. Selected drugs were administered after the surgery was completed until day 7. On day 7, mice underwent echocardiography and were euthanized. IL = interleukin; NLRP3 = NACHT, leucine-rich repeat, and pyrin domain-containing protein 3; NSAID = nonsteroidal anti-inflammatory drug.

Figure 2

Upregulation of NLRP3 Inflammasome Components in Human Pericardial Specimens of Patients Affected by Pericarditis (A) Immunohistochemistry reveals upregulation of the NLRP3 protein following pericarditis in human samples (original magnification 40×). (B) Caspase-1 upregulation detected by immunohistochemical staining (original magnification 40×). C) Immunofluorescence staining of ASC (apoptosis-associated speck-like protein containing a COOH-terminus caspase activation and recruitment domain) (red) showing aggregates in the pericardium. Counterstaining of the nuclei achieved with DAPI (4′,6-diamidino-2-phenylindole) (blue) (original magnification 40×). Abbreviations as in Figure 1.

Figure 3

Assessment of Pericarditis in Mice (A) Pericardial effusion was measured at 7 days by transthoracic echocardiography as echo-free space between the 2 layers of the pericardium. Instillation of zymosan A produces a significant increase in pericardial fluid compared with sham control subjects injected with saline. (B) Pericardial thickening was measured at day 7 by histological assessment of hematoxylin and eosin–stained heart slides. Zymosan A increased significantly the thickness of the visceral pericardial layer compared with sham (original magnification 40×). (C) Immunofluorescence staining for ASC (red) and cardiac α-actin (green) in the pericardium of mice with experimental pericarditis. DAPI was used to counterstain the nuclei (blue) (original magnification 40×). Abbreviations as in Figure 2.

Figure 4

Assessment of IL-1α and IL-1β in Mice (A) IL-1α and IL-1β messenger RNA (mRNA) relative expression in the parietal pericardium was measured at 3 days by real-time polymerase chain reaction. Mice treated with intrapericardial instillation of zymosan A showed a significant increase in mRNA expression of both IL-1α and IL-1β compared with sham control subjects injected with saline. (B) Pericardial expression of IL-1α was measured at day 3 by immunofluorescence staining. Zymosan A–treated mice had a higher expression of IL-1α compared with sham. IL-1α positive cells are stained in red, while DAPI was used to counterstain the nuclei (blue). The myocardium appears green by natural autofluorescence. Original magnification 40×. (C) Immunofluorescence staining for IL-1β (red) in the pericardium of mice with experimental pericarditis demonstrated a higher expression of this cytokine in zymosan A–treated mice compared with sham control subjects. IL-1β is stained in red, while DAPI was used to counterstain the nuclei (blue). The myocardium appears green by natural autofluorescence. Original magnification 40×. Abbreviations as in Figures 1 and 2.

Figure 5

Effects of Ibuprofen Treatment in the Experimental Model of Pericarditis in Mice (A) Pericardial effusion was measured at 7 days following daily treatment with ibuprofen (100 mg/kg). Daily injection with ibuprofen significantly reduced fluid accumulation. (B) Treatment with ibuprofen did not reduce pericardial thickness. (C) ASC (apoptosis-associated speck-like protein containing a COOH-terminus caspase activation and recruitment domain) aggregates were not reduced by treatment with ibuprofen. ∗p < 0.05, ∗∗∗p < 0.001 vs. sham-treated mice; ###p < 0.001 vs. vehicle-treated mice.

Figure 6

Inhibition of the NLRP3 Inflammasome in Mice With Acute Pericarditis (A) Pericardial effusion measured at 7 days following daily treatment with colchicine (100 μg/kg) and NLRP3 inhibitor 16673-34-0 (100 mg/kg). Colchicine and 16673-34-0 significantly reduced pericardial effusion in mice with pericarditis. (B) Treatment with the NLRP3 inhibitor 16673-34-0, but not colchicine, showed a significant reduction of the pericardial thickness following acute pericarditis in mice compared with the vehicle-treated group. (C) ASC aggregates in the pericardium of mice with acute pericarditis were significantly reduced by colchicine and 16673-34-0. ∗p < 0.05, ∗∗∗p < 0.001 vs. sham-treated mice; ##p < 0.01, ###p < 0.001 vs. vehicle-treated mice. Abbreviations as in Figures 1 and 2.

Figure 7

IL-1 Blockade in Experimental Acute Pericarditis Pericardial effusion, thickness, and formation of ASC aggregates were measured 7 days after twice daily treatment with anakinra (100 mg/kg) or IL-1 trap. Anakinra significantly reduced (A) pericardial fluid accumulation, (B) thickening of the pericardium, and (C) ASC aggregation following acute pericarditis in mice. A dose response of IL-1 trap was tested in mice subjected to experimental pericarditis. Treatment with IL-1 trap every 48 h significantly reduced (D) pericardial effusion, (E) pericardial thickness, and (F) inflammasome formation with each dose tested. ∗p < 0.05, ∗∗∗p < 0.001 vs. sham-treated mice; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. vehicle-treated mice. Abbreviations as in Figures 1 and 2.