Growth hormone-releasing hormone receptor antagonist MIA-602 attenuates cardiopulmonary injury induced by BSL-2 rVSV-SARS-CoV-2 in hACE2 mice

Abstract

COVID-19 pneumonia causes acute lung injury and acute respiratory distress syndrome (ALI/ARDS) characterized by early pulmonary endothelial and epithelial injuries with altered pulmonary diffusing capacity and obstructive or restrictive physiology. Growth hormone-releasing hormone receptor (GHRH-R) is expressed in the lung and heart. GHRH-R antagonist, MIA-602, has been reported to modulate immune responses to bleomycin lung injury and inflammation in granulomatous sarcoidosis. We hypothesized that MIA-602 would attenuate rVSV-SARS-CoV-2-induced pulmonary dysfunction and heart injury in a BSL-2 mouse model. Male and female K18-hACE2tg mice were inoculated with SARS-CoV-2/USA-WA1/2020, BSL-2-compliant recombinant VSV-eGFP-SARS-CoV-2-Spike (rVSV-SARS-CoV-2), or PBS, and lung viral load, weight loss, histopathology, and gene expression were compared. K18-hACE2tg mice infected with rVSV-SARS-CoV-2 were treated daily with subcutaneous MIA-602 or vehicle and conscious, unrestrained plethysmography performed on days 0, 3, and 5 (n = 7 to 8). Five days after infection mice were killed, and blood and tissues collected for histopathology and protein/gene expression. Both native SARS-CoV-2 and rVSV-SARS-CoV-2 presented similar patterns of weight loss, infectivity (~60%), and histopathologic changes. Daily treatment with MIA-602 conferred weight recovery, reduced lung perivascular inflammation/pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued altered respiratory rate, increased expiratory parameters (Te, PEF, EEP), and normalized airflow parameters (Penh and Rpef) compared to vehicle, consistent with decreased airway inflammation. RNASeq followed by protein analysis revealed heightened levels of inflammation and end-stage necroptosis markers, including ZBP1 and pMLKL induced by rVSV-SARS-CoV-2, that were normalized by MIA-602 treatment, consistent with an anti-inflammatory and pro-survival mechanism of action in this preclinical model of COVID-19 pneumonia.

Keywords: ARDS; COVID-19; GHRH-R; Necroptosis; SARS-CoV-2.

Fig. 1.

rVSV-SARS-CoV-2 mimics SARS-CoV-2 infectivity. (A) K18-hACE2tg mice were inoculated intranasally with ~10⁵ PFU SARS-CoV-2 (n = 10) or intratracheally with ~2 × 10⁷ PFU IU rVSV-SARS-CoV-2 (n = 8) for 5 d. (B and C) Weight loss was recorded daily and compared between both viruses. (D–G) Similar infection pattern with native SARS-CoV-2 Washington strain and chimeric SARS-CoV-2 in hACE2tg mice after 48 h. N = 3 to 4 mice per group. (Scale bar: 500 μm.) Quantification shown for immunostaining and viral load using the Median Tissue Culture Infectious Dose (TCID50) assay. (H) Electron microscopy shows rVSV-SARS-CoV-2 viral entry into the lung epithelial cell (yellow arrow). Data are means ± SD. The t test or ANOVA with Tukey post hoc correction was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.

rVSV-SARS-CoV-2 induces pulmonary dysfunction. (A) K18-hACE2 mice were inoculated intratracheally with ~2 × 10⁷ PFU rVSV-SARS-CoV-2 (n = 8). On day 0, day 3, and day 5, whole-body plethysmography (WBP) was recorded. (B) Respiratory rate and minute ventilation. (C) Expiratory values (Te: expiratory time; PEF: peak expiratory flow; Tr: relaxation time) and inspiratory values (Ti: inspiratory time; PIF: peak inspiratory flow) (D and E) Schematic expiratory and inspiratory curves to determine airway obstruction measures (Penh: enhanced pause; Rpef: The location into expiration where the peak occurs as a fraction of Te). Data points represent individual animals. Green lines and points represent infected animals. Data are means ± SD. ANOVA with Tukey post hoc correction was used. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.

GHRH-R antagonist attenuates rVSV-SARS-CoV-2-induced pulmonary dysfunction. (A) K18-hACE2tg mice were inoculated intratracheally with ~2 × 10⁷ PFU rVSV-SARS-CoV-2 (n = 15). Animals received daily subcutaneous GHRH-R antagonist (MIA-602; 10 μg/mouse) or vehicle. On day 0, day 3, and day 5, whole-body plethysmography (WBP) was recorded. (B) Weight loss curve. (C) Respiratory rate and minute ventilation. (D) Expiratory values (Te: expiratory time; PEF: peak expiratory flow; Tr: relaxation time; EEP: end expiratory pause) and inspiratory values (Ti: inspiratory time; PIF: peak inspiratory flow; TP: Duration of pause before inspiration) (E) Airway measures (PAU: Pause; Penh: enhanced pause; Rpef: The location into expiration where the peak occurs as a fraction of Te) and EF50: Expiratory flow at 50% expired volume; and TB: Duration of breaking. Data are means ± SD. ANOVA with Tukey post hoc correction was used. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

GHRH-R antagonist reduces rVSV-SARS-CoV-2-induced lung damage. (A) K18-hACE2tg mice were inoculated intranasally with ~10⁵ PFU SARS-CoV-2 (n = 10). Shown are representative H&E images of the lungs with perivascular lymphocytic inflammation and reactive endothelium (yellow arrow). (B) K18-hACE2tg mice were inoculated intratracheally with ~2 × 10⁷ PFU rVSV-SARS-CoV-2 (n = 15). Animals received daily subcutaneous GHRH-R antagonist (MIA-602; 10 μg/mouse) or vehicle. Shown are representative H&E images of the lung bronchial structure compared with the normal PBS/no-virus group. rVSV-SARS-CoV-2+ vehicle: Bronchus with marked intraluminal and peribronchial inflammation. The inflammation spills into the pulmonary interstitium. rVSV-SARS-CoV-2 + MIA-602: Normal bronchial structure and parenchyma. (C) H&E images of the lungs from the rVSV-SARS-CoV-2+ vehicle group show perivascular inflammation with endothelial reactive change (yellow arrows). There is type 2 pneumocytes hyperplasia (green arrows in the left panel) and reactive type 2 pneumocytes (green arrows in the right panel). (D) Histopathological findings using standard pathology grading were ranked from 0 to 4 with zero being absent and 4 being marked/severe. The ranks for each of the 15 mice are displayed as representative colors in a heatmap. (E) Acute Lung injury score using guidelines of the American Thoracic Society (n = 5 to 8 mice per group). (F and G) Immunostaining for ICAM-1 expression in the lung and heart (n = 3 mice per group). (Scale bar: 100 μm.) Data are means ± SD. ANOVA with Tukey post hoc correction was used. *P < 0.05; **P < 0.01.

Fig. 5.

GHRH-R antagonist reduces rVSV-SARS-CoV-2-induced lung necroptosis. (A) K18-hACE2tg mice were inoculated intratracheally with ~2 × 10⁷ PFU rVSV-SARS-CoV-2. Animals received daily subcutaneous GHRH-R antagonist (MIA-602; 10 μg/mouse) or vehicle. After 5 d, lung samples were collected for transcriptomics (n = 3 mice per group). Enrichment analysis from RNASeq reveals Toll-like receptor, type II Interferon, and ADAR1Editing Deficiency pathways as the three main pathways regulated by MIA-602. Shown are normalized representations of the transcripts in each pathway by heatmap and violin plots. (B and C) Protein expression and quantification confirm INF-γ, ZBP1, and SOCS1 regulation by MIA-602 (n = 3 mice per group). (D) Protein expression of necroptosis markers pMLKL and MLKL (n = 3 mice per group). (E and F) DAB immunostaining and quantification for ZBP1 expression in lungs (n = 3 mice per group). (Scale bar: 100 μm.) (G) Immunofluorescence for ZBP1 expression in lungs. The inset shows cytoplasmic expression of ZBP1 in perivascular infiltration. (H) Electron microscopy reveals programmed cell death by necroptosis in an epithelial lung cell (red arrow). Data are means ± SD or ±SEM. ANOVA with Tukey post hoc correction was used. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

Functional and histopathological correlation and prediction analysis. (A) The respiratory parameters and histopathology scores were subjected to Spearman correlation analysis, averaged, and converted to a heat map. (B) Shown are the top four prediction accuracies of histopathologic parameter output using a random forest model with all the WBP parameters as input. (C) Shown are the importance ranking of WBP parameters that contributed to the accurate prediction of the histological scores, in the machine learning model. MVb: minute ventilation (mL/min). PEF: peak expiratory flow; EEP: end expiratory pause; PIF: estimated peak inspiratory flow; TP: duration of pause before inspiration (%); TB: duration of breaking, percentage of the breath occupied by transitioning from inspiration to expiration (%); PAU: pause; Penh: enhanced pause; Rpef: The location into expiration where the peak occurs as a fraction of Te; EF50: expiratory flow at 50% expired volume; and RH: relative humidity (%).