Neutrophil Lifespan Extension with CLON-G and an <em>In Vitro</em> Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

University of electronic science and technology of China

University of Electronic Science and Technology of China

It has been reported that high mobility group nucleosomal binding domain 2 (HMGN2) is a nucleus-related protein that regulates gene transcription and plays a critical role in bacterial clearance. An elevated level of HMGN2 reduced integrin α5/β1 expression of human pulmonary epithelial A549 cells was demonstrated during Klebsiella pneumoniae infection, thus weakening bacterial adhesion and invasion. However, the mechanism by which HMGN2 regulates integrin expression remains unclear. This study found that a transcription factor-nuclear factor I (NFI), which serves as the potential target of HMGN2 regulated integrin expression. The results showed that HMGN2 was able to promote NFIA and NFIB expression by increasing H3K27 acetylation of NFIA/B promoter regions. The integrin α5/β1 expression was significantly enhanced by knockdown of NFIA/B via a siRNA approach. Meanwhile, NFIA/B silence could also compromise the inhibition effect of HMGN2 on the integrin α5/β1 expression. Mechanistically, it was demonstrated that HMGN2 facilitated the recruitment of NFI on the promoter regions of integrin α5/β1 according to the chromatin immunoprecipitation assay. In addition, it was further demonstrated that the knockdown of NFIA/B induced more adhesion of Klebsiella pneumoniae on pulmonary epithelial A549 cells, which could be reversed by the application of an integrin inhibitor RGD. The results revealed a regulatory role of HMGN2 on the transcription level of integrin α5/β1, indicating a potential treatment strategy against Klebsiella pneumoniae-induced infectious lung diseases.

High mobility group nucleosomal binding 2 reduces integrin α5/β1-mediated adhesion of Klebsiella pneumoniae on human pulmonary epithelial cells via nuclear factor I.

As an important endogenous growth factor, PDGF-BB can effectively promote neurogenesis, thus is considered as a potential agent for Parkinson's disease (PD) therapy. However, the protective function of PDGF-BB on neuronal cells, especially the molecular mechanism, remains less clear, which is needed to explore before its clinical practice. In this study, we investigated the function and mechanism of PDGF-BB against 1-methyl-4-phenylpyridinium (MPP) toxicity in SH-SY5Y cells, a widely used cellular tool for PD-related molecular study. Our results indicated that PDGF-BB exerts a prominent protective effect against neurotoxin MPP-triggered ROS generation and cellular loss. We further dissected the molecular mechanism involved in this process by using specific pharmacological inhibitors and validated that the distinct signaling pathways PI3K/Akt/GSK-3β and MEK/ERK are involved in the process against MPP toxicity upon PDGF-BB treatment. We also detected that activation of upstream PI3K/Akt/GSK-3β and MER/ERK signaling pathways contribute to phosphorylation and nuclear translocation of the downstream effector cyclic response element-binding protein (CREB), a known transcription factor to exhibit neuroprotective and growth-promoting effects. Using genetic approach, we further confirmed that the activation of CREB is involved in PDGF-BB-mediated protection in MPP-exposed SH-SY5Y cells. Together, these data demonstrated the protective effect of PDGF-BB in MPP-mediated toxicity in SH-SY5Y cells and verified the involved molecular mechanism in PDGF-BB-mediated neuroprotection.

Molecular Mechanism of Platelet-Derived Growth Factor (PDGF)-BB-Mediated Protection Against MPP Toxicity in SH-SY5Y Cells.

The effective therapy for lung infectious diseases became more and more difficult since the severe antibiotic resistance of pathogenic microorganisms, it is urgent to develop new antimicrobial agents. Luteolin has been reported to play a crucial part in host immune responses. However, the clinical use of luteolin is impeded due to its hydrophobicity and low oral bioavailability. In this study, we formulated luteolin-loaded Methoxy poly(ethylene glycol)-poly(lactide) micelles (luteolin/MPEG-PLA), to improve the bioavailability of luteolin in lung infectious diseases. The results showed that luteolin/MPEG-PLA treatment could reduce the adhesion of ) to lung epithelial cells and enhance the germicidal ability of macrophages against  compared to untreated group. Meanwhile, luteolin/MPEG-PLA showed stronger adhesion resistance of epithelial cells and germicidal ability of macrophages compared with free luteolin.  study, luteolin/MPEG-PLA administration significantly promoted the clearance of bacteria and reduced inflammatory infiltration of lung tissue in  induced lung infectious mice model. Further studies showed that treatment with luteolin/MPEG-PLA reduced the mRNA expression of LPS-induced inflammatory cytokines and chemokines in macrophages significantly. In general, luteolin/MPEG-PLA can enhance the anti-bacterial ability of lung epithelial cells and macrophages, and has a stronger therapeutic effect than free luteolin in bacterial-induced lung infection. Luteolin/MPEG-PLA may be an excellent potential drug for bacterial-induced lung infectious diseases treatment.

Improving Anti-Inflammatory Effect of Luteolin with Nano-Micelles in the Bacteria-Induced Lung Infection.

N6-Methyladenosine (m6A) is the most prevalent internal modification in messenger RNAs (mRNAs) of eukaryotes and plays a vital role in post-transcriptional regulation. Recent studies demonstrated that m6A is essential for the normal function of the central nervous system (CNS), and the deregulation of m6A leads to a series of CNS diseases. However, the functional consequences of m6A deficiency within the dopaminergic neurons of adult brain are elusive. To evaluate the necessity of m6A in dopaminergic neuron functions, we conditionally deleted Mettl14, one of the most important part of m6A methyltransferase complexes, in the substantia nigra (SN) region enriched with dopaminergic neurons. By using rotarod test, pole test, open-field test and elevated plus maze, we found that the deletion of Mettl14 in the SN region induces impaired motor function and locomotor activity. Further molecular analysis revealed that Mettl14 deletion significantly reduced the total level of m6A in the mRNA isolated from SN region. Tyrosine hydroxylase (TH), an essential enzyme for dopamine synthesis, was also down-regulated upon Mettl14 deletion, while the activation of microglia and astrocyte was enhanced. Moreover, the expression of three essential transcription factors in the regulation of TH including Nurr1, Pitx3 and En1, with abundant m6A-binding sites on their RNA 3'-untranslated regions (UTR), was significantly decreased upon Mettl14 deletion in SN. Our finding first confirmed the significance of m6A in maintaining normal dopaminergic function in the SN of adult mouse.

Conditional deficiency of m6A methyltransferase Mettl14 in substantia nigra alters dopaminergic neuron function.

The neurotropic growth factor PDGF-BB was shown to have vital neurorestorative functions in various animal models of Parkinson's disease (PD). Previous studies indicated that the regenerative property of PDGF-BB contributes to the increased intensity of tyrosine hydroxylase (TH) fibers in vivo. However, whether PDGF-BB directly modulates the expression of TH, and the underlying mechanism is still unknown. We will carefully examine this in our current study.

Platelet-derived growth factor (PDGF)-BB protects dopaminergic neurons via activation of Akt/ERK/CREB pathways to upregulate tyrosine hydroxylase.

N6-methyladenosine (m6A), the most prevalent form of internal mRNA modification, is extensively involved in Treg cells differentiation and function. However, the involvement of m6A in functional Treg cells for transplantation tolerance remains to be elucidated. By using an experimental transplantation mouse model, we found that m6A levels in Treg cells were altered during the induction of transplant tolerance by performing a dot blotting assay. Subsequently, we used the heterogenic Treg-specific Mettl14 knockout mice (Foxp3-Mettl14 cKO) to reduce METTL14 expression and performed islets allograft transplantation. Our result revealed that reduced expression of METTL14 prevented Treg cells expansion and promoted the infiltration of CD4 and CD8 T cells around the allograft, which led to rapid allograft rejection in Foxp3-Mettl14  cKO mice. The expression of regulatory cytokines including IL-10 and TGF-β was significantly decreased in Foxp3-Mettl14  cKO mice, and the suppressive function of Treg cells was also abrogated. In addition, an analysis of RNA-seq data revealed that the SOCS family (SOCS1, SOCS2 and SOCS3) is the subsequent signaling pathway affected by the METTL14 mediated m6A modification in Treg cells to modulate the suppressive function after transplantation. Taken together, our study showed for the first time that the METTL14-mediated m6A modification is essential for the suppressive function of Treg cells in transplantation and may serve as a regulatory element of Treg cell-based therapy in transplant medicine.

Mettl14-mediated m6A modification enhances the function of Foxp3 regulatory T cells and promotes allograft acceptance.

[This corrects the article DOI: 10.3389/fimmu.2022.1022015.].

Yan Teng

Sichuan Provincial People's Hospital

University of electronic science and technology of China

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

Yan Teng

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University of electronic science and technology of China

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

Sichuan Provincial People's Hospital