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).