alpha1-Antitrypsin regulates CD14 expression and soluble CD14 levels in human monocytes in vitro

The recognition of bacterial lipopolysaccharide (LPS) is principally mediated by either membrane-bound or soluble form of the glycoprotein CD14 and CD14-associated signal transducer, toll-like receptor 4 (TLR4). Recent findings indicate that the serine protease inhibitor, alpha1-antitrypsin (AAT), may not only afford protection against proteolytic injury, but may also neutralize microbial activities and affect regulation of innate immunity. We postulated that AAT affects monocyte responses to LPS by regulating CD14 expression and soluble CD14 release. Here we show that a short-term (up to 2h) monocyte exposure to AAT alone or in combination with LPS leads to a remarkable induction of CD14 levels. In parallel, a short-term (2h) cell exposure to AAT/LPS significantly enhances LPS-induced NF kappaB (p50 and p65) activation in conjunction with increased TNFalpha, IL-1 beta and IL-8 release. In contrast, longer term incubation (18 h) of monocytes with combined AAT/LPS results in a significant reduction in expression of both CD14 and TLR4, inhibition of LPS-induced TNFalpha, IL-1 beta and IL-8 mRNA and protein expression. These findings provide evidence that AAT is an important regulator of CD14 expression and release in monocytes and suggest that AAT may be involved in LPS neutralization and prevention of over-activation of monocytes in vivo.     

Effects of itaconic acid on neuronal viability and brain mitochondrial functions

Recent studies have identified that under stimulation by bacterial lipopolysaccharide mammalian macrophages produce itaconic acid. Yet, it is unknown whether itaconate has any effect on viability of brain cells. Here we used extracellularly added itaconate to investigate its effects on viability of cerebellar granule cells (CGC) in cultures and respiratory functions of these cells and isolated brain mitochondria. We found that 3–5 mM itaconate had no effect on the viability of neurons, but 10 mM itaconate was toxic and induced neuronal apoptosis. Removal of itaconate after 24 h incubation resulted in further decrease in viability and number of neurons. Respiration of intact neurons was not affected by itaconate, but permeabilized cells as well as isolated brain mitochondria demonstrated decreased rates of respiration in the presence of itaconate. Using isolated adult rat brain mitochondria we found that itaconate decreased mitochondrial phosphorylating respiration, mitochondrial calcium retention capacity, production of reactive oxygen species with Complex I and Complex II substrates as well as inhibition of Complex I, Complex IV and ATP synthase. In conclusion, the results suggest that itaconic acid at millimolar concentrations affects mitochondrial functions and viability of neurons.