Inhibition of Alveolar Macrophage Pyroptosis Reduces Lipopolysaccharide-induced Acute Lung Injury in Mice

Background：Pyroptosis is the term for caspase-l-dependent cell death associated with pro-inflammatory cytokines.The role of alveolar macrophage （AM） pyroptosis in the pathogenesis of the acute lung injury and acute respiratory distress syndrome （ALI/ARDS） remains unclear.Methods：C57BL/6 wild-type mice were assigned to sham,lipopolysaccharide （LPS） ＋ vehicle,LPS ＋ acetyl-tyrosyl-valyl-alanyl-aspartyl-chloromethylketone （Ac-YVAD-CMK） and LPS ＋ Z-Asp-Glu-Val-Asp-fluoromethylketone groups.Mice were given intraperitoneal （IP） injections of LPS.Drugs were IP injected 1 h before LPS administration.Mice were sacrificed 16 h after LPS administration,and AMs were isolated.Western blot analysis for active caspase-1 and cleaved caspase-3,evaluation of lung injury and a cytokine release analysis were performed.AMs were treated with LPS and adenosine triphosphate （ATP）;caspase-l-dependent cell death was evaluated using flow cytometry;the apoptosis-associated speck-like protein containing a caspase recruitment domain （ASC） pyroptosomes were examined by immunofluorescence.Results：The expression of activated caspase-1 in AMs was enhanced following LPS challenge compared with the sham group.In the ex vivo study,the caspase-1/propidium iodide-positive cells,caspase-1 specks and ASC pyroptosomes were up-regulated in AMs following LPS/ATP stimulation.The specific caspase-1 inhibitor Ac-YVAD-CMK inhibited the activation of caspase-1 and pyroptotic cell death.Ac-YVAD-CMK also reduced the lung injury,pulmonary edema and total protein in bronchoalveolar lavage fluid （BALF）.In addition,Ac-YVAD-CMK significantly inhibited interleukin-β （IL-lβ） release both in serum and BALF and reduced the levels of IL-18,tumor necrosis factor-α （TNF-α）,High Mobility Group Box 1 （HMGB1） in BALF during LPS-induced ALI/ARDS.Conclusions：This study reported AM pyroptosis during LPS-induced ALI/ARDS in mice and has demonstrated that Ac-YVAD-CMK can prevent AM-induced pyroptosis and lung injury.These preliminary findings may form the basis for further studies to evaluate this pathway as a target for prevention or reduction of ALI/ARDS.

Vitamin B6 regulates IL-33 homeostasis to alleviate type 2 inflammation

Interleukin-33(IL-33)is a crucial nuclear cytokine that induces the type 2 immune response and maintains immune homeostasis.The fine-tuned regulation of IL-33 in tissue cells is critical to control of the type 2 immune response in airway inflammation,but the mechanism is still unclear.Here,we found that healthy individuals had higher phosphate-pyridoxal(PLP,an active form of vitamin B6)concentrations in the serum than asthma patients.Lower serum PLP concentrations in asthma patients were strongly associated with worse lung function and inflammation.In a mouse model of lung inflammation,we revealed that PLP alleviated the type 2 immune response and that this inhibitory effect relied on the activity of IL-33.A mechanistic study showed that in vivo,pyridoxal(PL)needed to be converted into PLP,which inhibited the type 2 response by regulating IL-33 stability.In mice heterozygous for pyridoxal kinase(PDXK),the conversion of PL to PLP was limited,and IL-33 levels were increased in the lungs,aggravating type 2 inflammation.Furthermore,we found that the mouse double minute 2 homolog(MDM2)protein,an E3 ubiquitin-protein ligase,could ubiquitinate the N-terminus of IL-33 and sustain IL-33 stability in epithelial cells.PLP reduced MDM2-mediated IL-33 polyubiquitination and decreased the level of IL-33 through the proteasome pathway.In addition,inhalation of PLP alleviated asthma-related effects in mouse models.In summary,our data indicate that vitamin B6 regulates MDM2-mediated IL-33 stability to constrain the type 2 response,which might help develop a potential preventive and therapeutic agent for allergy-related diseases.