ADAM17/MMP inhibition prevents neutrophilia and lung injury in a mouse model of COVID-19

Abstract

Severe coronavirus disease 2019 (COVID-19) is characterized by lung injury, cytokine storm, and increased neutrophil-to-lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe COVID-19 in infected individuals. Angiotensin-converting enzyme-2 (ACE2) is the receptor for SARS-CoV-2, the virus causing COVID-19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates COVID-19-related lung inflammation. We employed a preclinical mouse model using intratracheal instillation of a combination of polyinosinic:polycytidylic acid (poly(I:C)) and the receptor-binding domain of the SARS-CoV-2 spike protein (RBD-S) to mimic lung damage associated with COVID-19. Histologic analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion, and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill COVID-19 patients. Administration of the ADAM17/MMP inhibitors apratastat and TMI-1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of proinflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM-1 and VCAM-1. Additionally, the NLR was significantly reduced by ADAM17/MMP inhibition. Thus, we propose inhibition of ADAM17/MMP as a novel promising treatment strategy in SARS-CoV-2-infected individuals to prevent the progression toward severe COVID-19.

Keywords: IL-10; MMP; TACE; TNF-α; acute lung injury; endothelium; lymphocyte; neutrophil.

FIGURE 1

Preclinical mouse model of COVID‐19‐related lung inflammation. (A) Cartoon depicting the model consisting of intratracheal (i.t.) instillation of poly(I:C) and RBD‐S, intraperitoneal (i.p.) or intranasal (i.n.) treatment with apratastat, TMI‐1 or vehicle (DMSO), and harvesting of samples for the indicated analyses. (B–J) Comparison of lung inflammation induced by poly(I:C) alone or the combination of poly(I:C)/RBD‐S in terms of frequency of Ly6G⁺CD45⁺ neutrophils (B), F4/80⁺CD45⁺ macrophages (C), and CD3⁺CD45⁺ T cells (D) in the lungs. Cell frequencies were determined by flow cytometry 24 h after surgery. Total numbers of cells in the bronchoalveolar lavage fluids (BALF) were determined 24 h after surgery: Ly6G⁺ neutrophils (E), F4/80⁺ macrophages (F), and CD3⁺ T cells (G). Frequency of Ly6G⁺CD45⁺ neutrophils (H) and frequency of CD3⁺CD45⁺ T cells (I) in the peripheral blood 24 h after surgery. (J) Neutrophil–lymphocyte ratio (NLR) in the peripheral blood 24 h after surgery. Data are represented as mean ± SEM of at least 4 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 2

Lung injury is reduced upon ADAM17/MMP inhibition in poly(I:C)/RBD‐S‐induced lung inflammation. (A) Representative images of H&E‐stained lung tissue cross‐sections showing normal tissue morphology, and absence of edema, fibrosis, and vascular congestion in the sham control (10×, upper left panel, black arrow = blood vessel, asterisk = bronchiole; 40× magnifications of the boxed areas, lower panel, blue arrow = alveoli, green arrow = alveolar septa). The poly(I:C)/RBD‐S + vehicle group shows major inflammatory changes such as alveolar wall/septa thickening (bold black arrow) and alveolar space closing and fibrosis (arrowhead), leukocyte infiltration (+), and vascular congestion (star). The lung tissues of poly(I:C)/RBD‐S + apratastat and + TMI groups clearly show less inflammatory damage and overall a better‐preserved tissue morphology. (B) Histologic scores of the lung tissues based on the extent of inflammatory changes with 0 = absence, 1 = low, 2 = moderate, 3 = high. Data are shown as mean ± SEM of images from at least 4 independent tissue preparations. *p < 0.05, ****p < 0.0001

FIGURE 3

Neutrophil influx into lungs is reduced upon ADAM17/MMP inhibition. (A) Frequency of Ly6G⁺CD45⁺ cells in the lungs of the indicated groups. (B) Frequency of F4/80⁺CD45⁺ cells in the lungs. (C) Frequency of CD3⁺CD45⁺ cells in the lungs. Cell frequencies were determined by flow cytometry 24 h after surgery. Total numbers of Ly6G⁺ neutrophils (D), F4/80⁺ macrophages (E), CD3⁺ T cells (F) from bronchoalveolar lavage fluids (BALF) 24 h after surgery. Data are shown as mean ± SEM of at least 6 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001

FIGURE 4

Poly‐I:C/RBD‐S‐induced neutrophilia is prevented upon inhibition of ADAM17. Frequency of Ly6G⁺ CD45⁺ neutrophils (A) and CD3⁺CD45⁺ T cells (B) in the peripheral blood 24 h after surgery. (C) Neutrophil–lymphocyte ratio (NLR) in the peripheral blood 24 h after surgery. Data are represented as mean ± SEM of at least 6 mice per group. * p < 0.05; **p < 0.01

FIGURE 5

ADAM17 inhibition affects mRNA levels of cytokines and adhesion molecules. Apratastat and TMI‐1 ameliorate the poly‐I:C/RBD‐S‐induced increases in TNF‐α mRNA levels (A), and TNF‐α serum protein levels (B). (C) ADAM 17 inhibitors induce an increase in IL‐1β mRNA. (D) IL‐6 mRNA levels are significantly reduced only after TMI‐1 treatment. (E) Apratastat and TMI‐1 further increase mRNA levels of IL‐10 compared with vehicle‐treated inflamed mice. The poly‐I:C/RBD‐S‐induced increase in mRNA levels of ICAM‐1 (F) and VCAM‐1 (G) are significantly reduced by treatment with both apratastat and TMI‐1. Gene expression was analyzed using quantitative real‐time RT‐PCR with β‐actin as housekeeping gene. Data are represented as mean ± SEM of at least 4 independent cDNA preparations per group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

FIGURE 6

Intranasal administration of apratastat reduces neutrophil and macrophage presence in poly(I:C)/RBD‐S‐inflamed lungs. (A) Percentage of Ly6G⁺CD45⁺ neutrophils, (B) F4/80⁺CD45⁺ macrophages, and (C) CD3⁺CD45⁺ T cells in lung tissue 24 h after surgery as determined by flow cytometry. (D) Total Ly6G⁺ neutrophils, (E) total F4/80⁺ macrophages, and (F) total CD3⁺ T cells in the bronchoalveolar lavage fluids (BALF) as determined by flow cytometry 24 h after surgery. Results are represented as mean ± SEM of at least 5 mice per group. *p < 0.05, ***p < 0.001, and ****p < 0.0001

FIGURE 7

ICAM‐1 expression in lung endothelial cells is reduced after intranasal administration of apratastat. (A) Representative ICAM‐1/CD31 plots from lungs show a clear shift toward higher ICAM‐1 levels in CD31⁺ endothelial cells in poly‐I:C/RBD‐S‐inflamed and vehicle‐treated lungs. This shift is reverted in apratastat‐treated inflamed mice. (B) Quantification of the MFI of the ICAM‐1 signal from CD31⁺ICAM1⁺CD45^– cells. Results are represented as mean ± SEM of at least 5 mice per group. i.n.: intranasal. ***p < 0.001 and ****p < 0.0001