(549) FOX signaling mediates PLSCR1 and cytokine-induced inflammation of the lung in long-COVID

We reported an increase in NMDA receptors 1/2 and pathological changes in the lungs post-acute and long-term infection of mice in our established murine hepatitis virus-1 (MHV-1) model of COVID-19 (AJOG, 2023). Since glutamatergic signaling is known to influence forkhead box (FOX) transcription factors and the FOX family of proteins and phospholipid scramblase 1 (PLSCR1) are known to stimulate inflammation in other lung pathologies, we examined whether: 1) MHV-1 impacts FOX signaling, and PLSCR1, which may lead to chronic inflammation in long-term post-infection; 2) SPIKENET (SPK), our COVID therapeutic invention, reverses inflammation (Front Pharmacol, 2022).

Study Design:

Female A/J mice (Jackson Laboratory, ME, USA) equivalent to human adult age (8-week-old) were divided into 3 groups (n=3/group): 1) control (CON; no infusion); 2) MHV-1 5000 PFU inoculation; and 3) MHV-1 + SPK 5mg/kg/d s.c. on days 2,4,6. Mice were sacrificed 12 months post-inoculation. Protein levels of FOXP1,2,3,4, PLSCR1, and cytokines IL-6, IL-1β, IL-10, IL-18 and TNF-α were measured by immunofluorescence. Data were analyzed by Tukey’s multiple comparisons test using GraphPad 9.5.1 software and presented as % over control.

Results:

Alveolar FOXP1 and FOXP2 protein levels are increased (short arrows in CON, long arrows in MHV-1) 12 months post-MHV-1 infection (109.61% and 291.78%, resp., over CON; p< 0.0001) (Fig. 1) (Scale bar=35 µm). FOXP4 levels are decreased (78.4% reduction, as compared to CON; p< 0.0001 vs. CON) (Fig. 1). In MHV-1-infected mice, PLSCR1 levels are increased (384.4% over CON; p< 0.0001). IL-18 and TNF-α showed the greatest increase (110.30 and 274.12%, resp., over CON; p< 0.0001) (Fig. 1). SPK normalized all aberrant changes (arrowheads) (Fig.1).

Conclusion:

Increased FOXP1, FOXP2, IL-18, and TNF-α, are associated with the development of chronic lung injury, and reduced FOXP4 levels might be the major driving factor. Moreover, the protective effect of SPK strongly suggests the potential therapeutic benefit of SPK in counteracting lung dysfunction in long COVID.