Unveiling the role of PD-L1 in vascular endothelial dysfunction: Insights into the mtros/NLRP3/caspase-1 mediated pyroptotic pathway.

BACKGROUND: Programmed death ligand-1(PD-L1) has been postulated to play a crucial role in the regulation of barrier functions of the vascular endothelium, yet how this novel molecule mediates dysfunction in endothelial cells (ECs) during acute lung injury (ALI) remains largely unknown. METHODS: PD-L1 siRNA and plasmids were synthesized and applied respectively to down- or up-regulate PD-L1 expression in human lung microvascular endothelial cells (HMVECs). RNA sequencing was used to explore the differentially expressed genes following PD-L1 overexpression. The expression levels of tight junction proteins (ZO-1 and occludin) and the signaling pathways of NLRP-3/caspase-1/pyroptosis were analyzed. A mouse model of indirect ALI was established through hemorrhagic shock (HEM) followed by cecal ligation and puncture (CLP), enabling further investigation into the effects of intravenous delivery of PD-L1 siRNA. RESULTS: A total of 1502 differentially expressed genes were identified, comprising 532 down-regulated and 970 up-regulated genes in ECs exhibiting PD-L1overexpression. Enrichment of PD-L1-correlated genes were observed in the NOD-like receptor signaling pathway and the TNF signaling pathway. Western blot assays confirmed that PD-L1 overexpression elevated the expression of NLRP3, cleaved-caspase-1, ASC and GSDMD, and concurrently diminished the expression of ZO-1 and occludin. This overexpression also enhanced mitochondrial oxidative phosphorylation and mitochondrial reactive oxygen species (mtROS) production. Interestingly, mitigating mitochondrial dysfunction with mitoQ partially countered the adverse effects of PD-L1 on the functionality of ECs. Furthermore, intravenous administration of PD-L1 siRNA effectively inhibited the activation of the NLRP3 inflammasome and pyroptosis in pulmonary ECs, subsequently ameliorating lung injury in HEM/CLP mice. CONCLUSION: PD-L1-mediated activation of the inflammasome contributes significantly to the disruption of tight junction and induction of pyroptosis in ECs, where oxidative stress associated with mitochondrial dysfunction serves as a pivotal mechanism underpinning these effects.

miR-205 Suppresses Pulmonary Fibrosis by Targeting GATA3 Through Inhibition of Endoplasmic Reticulum Stress.

OBJECTIVE: To investigate the role of miR-205 and GATA3 in Pulmonary Fibrosis (PF). METHODS: Bleomycin (BLM) was used to induce PF in SD rats and in vitro PF model was established by using TGFß1-induced RLE-6TN cells. miR-205 mimics were used for the overexpression of miR- 205. The expression of miR-205, GATA3, α-SMA, Collagen I, CHOP and GRP78 were measured using RT-qPCR or western blotting. Dual-luciferase reporter assay was used to confirm binding between GATA3 3'-UTR and miR-205. RESULTS: The expression of miR-205 was significantly down-regulated, while the expression of GATA3 was remarkably up-regulated in the model rats. GATA3 levels were remarkably decreased when miR-205 was overexpressed. When miR-205 was overexpressed, the lung injury by BLM-induced fibrosis was improved. The expression of α-SMA, Collagen I, as well as GRP78 and CHOP, was significantly up-regulated in both in vivo and in vitro PF models, and overexpression of miR-205 remarkably reversed the effects. Dual-luciferase reporter assay showed that miR-205 directly targeted and negatively regulated GATA3. CONCLUSION: miR-205 improved pulmonary fibrosis through inhibiting ER-stress by targeting GATA3.

[Blockade of programmed death-ligand 1 attenuates indirect acute lung injury in mice through targeting endothelial cells but not epithelial cells].

OBJECTIVE: To examine the expression profile of programmed death-ligand 1 (PD-L1) on lung endothelial or epithelial cells, and to determine the specific role of PD-L1 in mouse model of indirect acute lung injury (i-ALI). METHODS: Eighty male C57BL/6 mice were randomly divided into two parts (both n = 40). The effects of different administration routes on the expression of PD-L1 were observed. The mice in each part were randomly divided into sham, i-ALI, i-ALI+small interfering RNA (siRNA) random sequence control, and i-ALI+PD-L1 siRNA which could specifically inhibit PD-L1 expression groups, with 10 mice in each group. i-ALI was reproduced in a mouse model of hemorrhagic shock in combination with a subsequent cecal ligation and puncture (CLP). In sham group, only bilateral femoral arteries were ligated without catheterization or bleeding, and only cecum was separated but perforation was not ligated. Intravenous or intratracheal delivery of PD-L1 siRNA was performed 2 hours following the resuscitation to suppress the expression of PD-L1 on lung endothelial or epithelial cells. The mice in i-ALI+siRNA random sequence control group were given siRNA random sequence without inhibition effect on PD-L1 expression, and those in sham group and i-ALI group were given 100 µL phosphate buffered saline (PBS). The mice were sacrificed at 24 hours after CLP, and samples of blood, lung tissue and bronchoalveolar lavage fluid (BALF) were harvested. Expressions of PD-L1 were determined with flow cytometry. Cytokines and chemokines in plasma, lung tissue and BALF were determined by enzyme linked immunosorbent assay (ELISA). The protein concentration in plasma and BALF and the activity of myeloperoxidase (MPO) in lung tissue were quantitatively measured. The pathological changes in lung tissue were observed under light microscope. RESULTS: (1) Compared with sham group, PD-L1 expression on lung endothelial or epithelial cells were significantly elevated in i-ALI group [endothelial cells: (27.88±1.53)% vs. (19.64±1.03)%, epithelial cells: (58.70±8.21)% vs. (29.23±3.94)%, both P < 0.05]. (2) Mice received intravenous delivery of liposomal-encapsulated siRNA had significantly lower expression of PD-L1 on lung endothelial cells as compared with that of i-ALI group [(21.37±0.76)% vs. (27.88±1.53)%, P < 0.05]. Intratracheal delivery of naked PD-L1 siRNA mainly inhibited the PD-L1 expression on epithelial cell as compared with that of i-ALI group [(31.23±4.71) % vs. (58.70±8.21) %, P < 0.05]. The expression of PD-L1 in pulmonary microvascular endothelial cells or pulmonary epithelial cells of i-ALI mice was not affected by siRNA random sequence. (3) PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to a lower protein ratio of BALF/plasma [(4.48±0.35)×10-3 vs. (6.11±0.56)×10-3, P < 0.05] and a decreased MPO activity in lung tissue (U×µg-1×min-1: 2.48±0.47 vs. 4.56±0.52, P < 0.05) as compared with that of i-ALI group. Moreover, inflammatory mediator levels such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) in lung tissue or plasma were significantly reduced following PD-L1 suppression on endothelial cells as compared with those of i-ALI group [IL-6 (ng/g): 177.4±23.2 vs. 287.9±57.3, MCP-1 (ng/g): 839.6±91.7 vs. 1 395.7±211.9, MIP-2 (ng/g): 923.7±107.3 vs. 1 700.9±240.2 in lung tissue; IL-6 (ng/L): 950.2±192.7 vs. 1 828.2±243.6, TNF-α (ng/L): 258.7±29.1 vs. 443.0±58.1, MCP-1 (ng/L): 2 583.8±302.3 vs. 4 328.1±416.4, MIP-2 (ng/L): 1 512.9±165.6 vs. 2 005.9±85.7 in plasma, all P < 0.05], however, there was no significant change in the levels of inflammatory factors in BALF. It was shown in lung tissue histology that PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to lessened pulmonary edema and reduced immune cells emigration. Intratracheal delivery of PD-L1 siRNA for PD-L1 suppression on epithelial cells had minimal effects on protein ratio of BALF/plasma, MPO activity, inflammatory mediator expressions in lung tissue, plasma, and BALF as well as lung tissue histology. CONCLUSIONS: PD-L1 silencing on endothelial cells but not epithelial cells protected mice against hemorrhagic shock-sepsis induced i-ALI.

Lycium barbarum polysaccharide reduces hyperoxic acute lung injury in mice through Nrf2 pathway.

INTRODUCTION: The disruption of the balance between antioxidants and oxidants plays a vital role in the pathogenesis of acute lung injury (ALI). Evidence has shown that Lycium barbarum polysaccharide (LBP) has antioxidant feature. We examined the efficacy and mechanisms of LBP on hyperoxia-induced acute lung injury (ALI) in the present study. MATERIALS AND METHODS: C57BL/6 wild-type (WT) mice and nuclear factor erythroid 2-related factor 2 (Nrf2)-deficient (Nrf2-/-) mice were used in the present study. LBP was fed by gavages once daily for 1 week. Then, the mice were exposed to hyperoxia or room air for 72 h. Additional dosage of LBP was given per 24 h. RESULTS: Reactive oxygen species production was increased in WT mice exposed to hyperoxia. Inflammatory cytokines including interleukin (IL)-1ß as well as IL-6, and inflammatory cells were increased infiltration in the lung after 3 days hyperoxia exposure. Hyperoxia exposure also induced pulmonary edema and histopathological changes. These hyperoxia-induced changes were improved in LBP treated group. Moreover, elevated activities of heme oxygenase-1 and glutathione peroxidase and enhanced activation of Nrf2 were observed in mice treated with LBP. However, the benefit of LBP on hyperoxic ALI was abolished in Nrf2-/- mice. Moreover, our cell study showed that the LBP-induced activation of Nrf2 was dampened in pulmonary microvascular endothelial cells when the AMPK signal was inhibited by siRNA. CONCLUSIONS: LBP improves hyperoxic ALI via Nrf2-dependent manner. The LBP-induced activation of Nrf2 is mediated, at least in part, by AMPK pathway.

CD86 polymorphism affects pneumonia-induced sepsis by decreasing gene expression in monocytes.

Sepsis, a clinical syndrome occurring in patients following infection or injury, is a leading cause of morbidity and mortality worldwide. CD86 (B7-2) is a costimulatory molecule on antigen-presenting cells and plays critical roles in immune responses. In the current study, we investigated the association of two CD86 polymorphisms, rs1129055G/A and rs17281995G/C, with susceptibility to pneumonia-induced sepsis and examined the effects of these two polymorphisms on gene expression in monocytes. CD86 rs1129055G/A and rs17281995G/C were identified in 192 pneumonia-induced septic patients and 201 healthy controls. Data showed that frequencies of the rs1129055GA and AA genotypes were significantly lower in patients than in controls (odds ratio [OR]=0.57, 95 % confidence interval [CI], 0.35-0.93, p=0.023, and OR=0.40, 95 % CI, 0.23-0.71, p=0.002). Interestingly, the other polymorphism, rs17281995G/C, revealed significantly increased numbers in pneumonia-induced sepsis compared to controls (OR=1.85, 95 % CI, 1.07-3.20, p=0.025). Further analyses about CD86 gene expression revealed that both messenger RNA (mRNA) and protein levels of CD86 were downregulated in monocytes from controls carrying rs17281995GC genotype than those carrying wild-type rs17281995GG genotype (p=0.022 and p=0013). These results suggest that polymorphisms in CD86 gene have diverse effects on the pathogenesis of pneumonia-induced sepsis, in which rs17281995G/C may increase the risk of the disease by interfering gene expression of CD86 in monocytes.