In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

Background and Objectives: The novel coronavirus disease 2019 (COVID-19) has been a pandemic health issue in 30 January 2020. The mortality rate is as high as 50% in critically ill patients. Stem cell therapy is effective for those who are refractory to standard treatments. However, the immune responses that underlie stem cell therapy have not been well reported, particularly, in patients associated with moderate to severe acute respiratory distress syndrome (ARDS).Methods: On Days 0 and 4, an intravenous infusion of 2 × 10⁷ placenta-derived mesenchymal stem cells (pcMSCs) (MatriPlax) were administered to five severe COVID-19 patients refractory to current standard therapies. Peripheral blood inflammatory markers and immune profiles were determined by multi-parameter flow cytometry and studied at Days 0, 4, and 8. Clinical outcomes were also observed.Results: None of the pc-MSC treated patients experienced 28-day mortality compared with the control group and showed a significant improvement in the PaO₂/FiO₂ ratio, Murray’s lung injury scores, reduction in serum ferritin, lactate dehydrogenase (LDH), and C-reactive protein (CRP) levels. The cytokine profiles also showed a reduction in IL-1β, IFN-γ, IL-2, and IL-6, and an increase in IL-13 and IL-5 type 2 cytokines within 7 days of therapy. Lymphopenia was also significantly improved after 7 days of treatment. Immune cell profiles showed an increase in the proportions of CD4⁺ T cells (namely, CD4⁺ naïve T cells and CD4⁺ memory T cell subtypes), Treg cells, CD19⁺ B cells (namely, CD19⁺ naïve B cells, CD27⁺ switched B cell subtypes) and dendritic cells, and a significant decrease in the proportion of CD14⁺ monocytes (namely, CD16^- classical and CD16⁺ non-classical subtypes), and plasma/plasmablast cells. No adverse effects were seen at the serial follow-up visits for 2 months after initial therapy.Conclusion: pc-MSCs therapy suppressed hyper-inflammatory states of the innate immune response to COVID-19 infection by increasing Treg cells, decreasing monocytes and plasma/plasmablast cells, and promoting CD4⁺ T cells and CD19⁺ B cells toward adaptive immune responses in severely critically ill COVID-19 patients with moderate to severe ARDS, especially those who were refractory to current standard care and immunosuppressive therapies.

Background: Mast cells are implicated in the pathogenesis and severity of asthma in children and adults. The release of proinflammatory mediators and cytokines from activated mast cells (MC) is associated with Type 2 (T2) cell-skewed inflammation. Methods: We obtained the airway tissues of Balb/c mice with or without intra-tracheal diesel exhaust particles (DEP) instillation to measure the extent of tryptase⁺ MCs infiltration and interleukin (IL)-33 expression. Cultured human mast cells (HMC-1) were stimulated with DEP to determine the role of aryl hydrocarbon receptor (AhR) in mediating the synthesis and release of IL-33 and type-2 cytokines. Results: In the control animals, most of the MC accumulated in the submucosal vessels without expression of IL-33. Intra-tracheal DEP installation increased the number of IL-33⁺ MC infiltrating in the epithelial and sub-epithelial areas of mice. Human MC exposed to DEP upregulated mRNA and protein expression of IL-33. These effects were abolished by knockdown of expression of the AhR or AhR nuclear translocator (ARNT) by small interfering (si)RNA transfection. DEP also activated nuclear factor-kappa B (NF-κB) to facilitate nuclear translocation of the AhR. DEP increased MC migration and induced the synthesis and release of IL-4, IL-5, and IL-13 in MCs, and these effects were abolished by anti-ST2 antibodies. Conclusions: Airborne pollutants may activate MCs to produce IL-33 via the AhR/NF-κB pathway, leading to type 2 cytokines production and enhancing MC airway epithelium-shifted migration through the autocrine or paracrine IL-33/ST2 axis.

Background: Circulating fibrocytes, trafficking into bronchial sub-epithelial zone, actively participate in airway remodeling of patients with severe or treatment refractory asthma. IL-4 and IL-13 participate in the pathogenesis of asthma, including reduction in apical junction, and induction of epithelial permeability, goblet cell hyperplasia. Objectives: Production of T2 cytokines by infiltrating fibrocytes underneath airway epithelium may be involved in airway remodeling in T2-high asthma. Methods and Results: We investigated circulating fibrocytes expressing IL-4, IL-5 or IL-13 (T2 fibrocytes) in the peripheral blood of 14 stable (ACT>24) allergic asthmatics (CAA) and 16 uncontrolled (ACT <19) allergic asthmatics (UAA). Patients with UAA had higher number of circulating fibrocytes, and higher proportion of T2 fibrocytes in circulating fibrocytes than CAA. The immune-fluorescence and RT/PCR studies revealed increased expression of IL-4 and IL-13 in fibrocytes from UAA, compared to CAA patients. The bronchial tissue T2 fibrocytes also increased in UAA, compared with CAA. IL-4 and IL-13 produced by T2 fibrocytes of UAA, through IL-4Rα, mediated fibrocyte α-SMA⁺ transformation, CTGF synthesis and CCR7 expression, increased fibroblast proliferation and matrix proteins production, and induced ALI cultured epithelial cells MUC5AC⁺ metaplasia, decreasing junction protein expression, leading to an increased TEER and para-cellular flux of dextran. Conclusion: Circulating T2 fibrocytes of asthmatics migrating to airway sub-epithelial areas contribute to airway remodeling by releasing IL-4 and IL-13 to induce epithelial disruption, mucus metaplasia and increase fibrogenesis.