Results
KVX-053 modulates Spike Protein Subunit 1-induced alveolar Inflammation and cytokine storm. Mice instilled with the S1SP displayed severe alveolar inflammation, as indicated by the increased leukocyte and protein concentrations in the BALF, that were significantly reduced in mice treated with the PTP4A3 inhibitor KVX-053 (10 mg/kg i.p./day) (Fig. 2, A and C). Mononuclear cells, i.e., monocytes and macrophages, and neutrophils, were significantly elevated in the S1SP group compared to the control group. Intratracheal instillation of S1SP did not affect the content of lymphocytes (Fig. 2 B). KVX-053 reduced neutrophil infiltration, BALF protein and white blood cell (WBC) content induced by S1SP exposure.
A wide range of pro-inflammatory cytokines were found elevated in BALF 72 h after S1SP instillation (Fig. 3). KVX-053 (10 mg/kg i.p.), administered every day, successfully reduced or completely blocked the release of cytokines into the alveolar space, with notable effects on interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ) and interleukin 12 (IL-12).
Histopathological analysis of lung sections taken from spike protein-instilled mice showed multiple focal accumulation of inflammatory cells, predominantly neutrophils either in alveolar or interstitial space, along with alveolar septal thickening and alveolar edema (Fig. 4). Mice, treated with KVX-053 (10 mg/kg i.p./day) demonstrated improvement of all signs of lung inflammation, reflected in 2-fold reduction of lung injury score.
KVX-053 inhibits Spike Protein Subunit 1-induced activation of pro-inflammatory pathways. Western blot analysis of lung homogenates showed significant increases in the phosphorylation of both STAT3 and IkBα, the cytosolic inhibitor of NF-κB, in K18-hACE2 mice instilled with S1SP compared to control mice. The inflammasome NLRP3 was also activated 72 h after S1SP exposure. Mice instilled with S1SP and treated with the PTP4A3 inhibitor KVX-053 (10 mg/kg i.p./day) showed attenuation of these proinflammatory biomarkers compared to animals instilled with S1SP and treated with vehicle (Fig. 5).
KVX-053 inhibits S1SP-Induced pulmonary overexpression of PTP4A3. Lung sections of control mice or mice instilled with S1SP and treated with KVX-053 (10 mg/kg i.p./day) or vehicle were stained for PTP4A3. Control mice, instilled with saline, displayed a barely noticeable expression of PTP4A3 in the vascular endothelium. Conversely, S1SP-instilled mice exhibited increased expression of the phosphatase in the lung vascular endothelium, the alveoli, the bronchial epithelium and the lung interstitium, saturated with inflammatory cells (Fig. 6). KVX-053 blocked the overexpression of PTP4A3 throughout the lung sections.
KVX-053 protects against S1SP-induced lung dysfunction. Changes in lung mechanics were analyzed 72 h after S1SP-instillation. Transgenic mice, instilled with S1SP showed a downward shift in PV loops (Fig.7A), as well as significantly reduced Crs and Cst (Fig. 7B). Total Rrs, Ers, G, and Rn increased in S1SP- instilled mice both at baseline and in response to methacholine (Fig. 7C). Treatment with KXV-053 restored all parameters to physiological levels.
Along with pathological changes in the respiratory system, induced by intratracheal instillation of SARS-CoV-2 S1SP, we observed signs of systemic inflammation. The decline in body weight, observed in our previous study [1], was significantly improved within 48 h after KVX-053 administration (Fig. 8A). The inflammatory cytokines IL-1β, IFNγ, TNFα and interferon gamma-induced protein 10 (IP-10) were increased in blood serum of S1SP-instilled K18-hACE2 mice (Fig. 8B). Animals, instilled with spike protein and treated with PTP4A3 inhibitor KVX-053 (10 mg/kg i.p./day) exhibited lower levels of serum cytokines compared S1SP-instilled mice treated with vehicle.
KVX-053 pretreatment ameliorates Spike Protein-induced Pulmonary Microvascular Endothelial Barrier Disfunction. HLMVECs were seeded on gold electrode arrays connected to the ECIS instrument. Cells were then exposed to 10 nM S1SP in either pre-treatment or post-treatment assays with KVX-053. KVX-053 (12.5 µM) administered 2 hours before S1SP exposure or 5 h after, prevented and repaired S1SP-induced endothelial dysfunction and barrier hyperpermeability (Fig. 9A-B). This was associated with KVX-053-mediated attenuation of S1SP-induced activation of inflammatory pathways STAT3 and AKT in HLMVEC (Fig 9C).
Endothelial cells were seeded onto glass slides and allowed to grow for 96 h until >90% confluence was reached. They were then pretreated with KVX-053 (12.5 µM) or vehicle (10% DMSO) for 2 h and then exposed for 4 h to 0.01% DMSO (control) or 10 μM of S1SP. The cells were then fixed with paraformaldehyde, permeabilized and stained with VE-cadherin antibody, followed by F-Actin and DAPI counterstaining. S1SP profoundly reduced cortical VE-cadherin staining and disrupted the integrity of the cell monolayer. KVX-053 protected the vascular endothelium and preserved the expression of VE-cadherin (Fig. 10). Importantly, KVX-053 preserved multiple focal structures between cells, that are responsible for the maintenance of normal transendothelial electrical resistance, as shown above (Fig.9).