IFN-α affects Th17/Treg cell balance through c-Maf and associated with the progression of EBV- SLE.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease, of which the pathogens is remains obscure. Viral infection, particularly Epstein Barr viru (EBV) infection, has been considered a common pathogenic factor. This study suggests that c-Maf may be an important target in T cell differentiation during SLE progression, providing a potentially new perspective on the role of viral infection in the pathogenesis of autoimmune diseases. METHODS: Cytokines of EBV-infected SLE patients were measured by ELISA and assessed in conjunction with their clinical data. IFN-α, c-Maf, and the differentiation of Th17/Treg cells in SLE patients and MRL/LPR mice were analyzed using FCM, WB, RT-PCR, etc. Following the infection of cells and mice with EBV or viral mimic poly (dA:dT), the changes of the aforementioned indicators were investigated. The relationship among IFN-α, STAT3, c-Maf and Th17 cells was determined by si-RNA technique. RESULTS: Many SLE patients are found to be complicated by viral infections; Further, studies have demonstrated that viral infection, especially EBV, is involved in SLE development. This study showed that viral infections might promote IFN-α secretion, inhibit c-Maf expression by activating STAT3, increase Th17 cell differentiation, and lead to the immune imbalance of Th17/Treg cells, thus playing a role in the onset and progression of SLE. CONCLUSION: This study demonstrates that EBV infections may contribute to SLE development by activating STAT3 through IFN-α, inhibiting c-Maf, and causing Th17/Treg immune imbalance. Our work provided a new insight into the pathogenesis and treatment of SLE.

VX-765 inhibits pyroptosis and reduces inflammation to prevent acute liver failure by upregulating PPARα expression.

INTRODUCTION AND OBJECTIVES: As a fatal clinical syndrome, acute liver failure (ALF) is characterized by overwhelming liver inflammation and hepatic cell death. Finding new therapeutic methods has been a challenge in ALF research. VX-765 is a known pyroptosis inhibitor and has been reported to prevent damage in a variety of diseases by reducing inflammation. However, the role of VX-765 in ALF is still unclear. MATERIALS AND METHODS: ALF model mice were treated with D-galactosamine (D-GalN) and lipopolysaccharide (LPS). LO2 cells were stimulated with LPS. Thirty subjects were enrolled in clinical experiments. The levels of inflammatory cytokines, pyroptosis-associated proteins and peroxisome proliferator-activated receptor α (PPARα) were detected using quantitative reverse transcription-polymerase chain reaction (qRT‒PCR), western blotting and immunohistochemistry. An automatic biochemical analyzer was used to determine the serum aminotransferase enzyme levels. Hematoxylin and eosin (HE) staining was used to observe the pathological features of the liver. RESULTS: With the progression of ALF, the expression levels of interleukin (IL) -1ß, IL-18, caspase-1, and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were increased. VX-765 could reduce the mortality rate of ALF mice, relieve liver pathological damage, and reduce inflammatory responses to protect against ALF. Further experiments showed that VX-765 could protect against ALF through PPARα, and this protective effect against ALF was reduced in the context of PPARα inhibition. CONCLUSIONS: As ALF progresses, inflammatory responses and pyroptosis deteriorate gradually. VX-765 can inhibit pyroptosis and reduce inflammatory responses to protect against ALF by upregulating PPARα expression, thus providing a possible therapeutic strategy for ALF.

Eliminating the invading extracellular and intracellular FnBp+ bacteria from respiratory epithelial cells by autophagy mediated through FnBp-Fn-Integrin α5ß1 axis.

Background: We previously found that the respiratory epithelial cells could eliminate the invaded group A streptococcus (GAS) through autophagy induced by binding a fibronectin (Fn) binding protein (FnBp) expressed on the surface of GAS to plasma protein Fn and its receptor integrin α5ß1 of epithelial cells. Is autophagy initiated by FnBp+ bacteria via FnBp-Fn-Integrin α5ß1 axis a common event in respiratory epithelial cells? Methods: We chose Staphylococcus aureus (S. aureus/S. a) and Listeria monocytogenes (L. monocytogenes/L. m) as representatives of extracellular and intracellular FnBp+ bacteria, respectively. The FnBp of them was purified and the protein function was confirmed by western blot, viable bacteria count, confocal and pull-down. The key molecule downstream of the action axis was detected by IP, mass spectrometry and bio-informatics analysis. Results: We found that different FnBp from both S. aureus and L. monocytogenes could initiate autophagy through FnBp-Fn-integrin α5ß1 axis and this could be considered a universal event, by which host tries to remove invading bacteria from epithelial cells. Importantly, we firstly reported that S100A8, as a key molecule downstream of integrin ß1 chain, is highly expressed upon activation of integrin α5ß1, which in turn up-regulates autophagy. Conclusions: Various FnBp from FnBp+ bacteria have the ability to initiate autophagy via FnBp-Fn-Integrin α5ß1 axis to promote the removal of invading bacteria from epithelial cells in the presence of fewer invaders. S100A8 is a key molecule downstream of Integrin α5ß1 in this autophagy pathway.

Bombyx batryticatus Cocoonase Inhibitor Separation, Purification, and Inhibitory Effect on the Proliferation of SMCC-7721 HeLa-Derived Cells.

The present study was to isolate and purify Bombyx batryticatus cocoonase inhibitor (BBCI) and to evaluate its inhibitory effect on the proliferation of SMCC-7721 cells. BBCI was purified from the crude proteins of Bombyx batryticatus using affinity chromatography with cocoonase as the ligand, its N-terminal amino acid sequence was determined using the Edman degradation method, and its inhibiting activity on SMCC-7721 cell proliferation was detected in vitro using the MTT method and in vivo in tumor-bearing nude mice. The purified BBCI presented as a single band in SDS-PAGE, the molecular weight determined by time-of-flight mass spectrometry was 13,973.63 Da, and its N-terminal amino acid sequence was VRNKRQSNDD. BBCI was a noncompetitive cocoonase inhibitor with an average Michaelis constant of 76.50, and it inhibited cocoonase activity with an inhibition ratio of 1 : 1 (molar). BBCI could inhibit the proliferation of SMCC-7721 cells in vitro with the IC50 being about 260.52 µg/ml within 36 h of treatment and inhibit the SMCC-7721 tumor growth in nude mice by subcutaneous injection of BBCI around the tumor, where the tumor inhibitory effect was dose dependent. BBCI did not significantly influence the spleen coefficient of the mice. In conclusion, to the best of our knowledge, the present study is the first to report that BBCI, which was purified from Bombyx batryticatus, was a serine proteinase inhibitor with antitumor activity.

Macrophages Inhibit Ciliary Protein Levels by Secreting BMP-2 Leading to Airway Epithelial Remodeling Under Cigarette Smoke Exposure.

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease with high morbidity and mortality worldwide. So far, smoking is still its leading cause. The characteristics of COPD are emphysema and airway remodeling, as well as chronic inflammation, which were predominated by macrophages. Some studies have reported that macrophages were involved in emphysema and chronic inflammation, but whether there is a link between airway remodeling and macrophages remains unclear. In this study, we found that both acute and chronic cigarette smoke exposure led to an increase of macrophages in the lung and a decrease of ciliated cells in the airway epithelium of a mouse model. The results of in vitro experiments showed that the ciliary protein (ß-tubulin-IV) levels of BEAS-2B cells could be inhibited when co-cultured with human macrophage line THP-1, and the inhibitory effect was augmented with the stimulation of cigarette smoke extract (CSE). Based on the results of transcriptome sequencing, we focused on the protein, bone morphogenetic protein-2 (BMP-2), secreted by the macrophage, which might mediate this inhibitory effect. Further studies confirmed that BMP-2 protein inhibited ß-tubulin-IV protein levels of BEAS-2B cells under the stimulation of CSE. Coincidentally, this inhibitory effect could be nearly blocked by the BMP receptor inhibitor, LDN, or could be interfered with BMP-2 siRNA. This study suggests that activation and infiltration of macrophages in the lung induced by smoke exposure lead to a high expression of BMP-2, which in turn inhibits the ciliary protein levels of the bronchial epithelial cells, contributing to the remodeling of airway epithelium, and aggravates the development of COPD.

Integrin α5ß1, as a Receptor of Fibronectin, Binds the FbaA Protein of Group A Streptococcus To Initiate Autophagy during Infection.

Group A Streptococcus (GAS), one of the most common extracellular pathogens, has been reported to invade epithelial and endothelial cells. Our results reveal that M1 GAS strain SF370 can be effectively eliminated by respiratory epithelial cells. Emerging evidence indicates that autophagy is an important strategy for nonphagocytes to eliminate intracellular bacteria. Upon pathogen recognition, cell surface receptors can directly trigger autophagy, which is a critical step in controlling infection. However, the mechanisms of how cells sense invading bacteria and use this information specifically to trigger autophagy remain unclear. In this study, we stimulated cells and infected mice with M and FbaA mutants of M1 GAS strain SF370 or with purified M and FbaA proteins (two critical surface structural proteins of GAS), and found that only FbaA protein was involved in autophagy induction. Furthermore, the FbaA protein induced autophagy independent of common pattern recognition receptors (such as Toll-like receptors); rather, it relies on binding to integrin α5ß1 expressed on the cell surface, which is mediated by extracellular matrix protein fibronectin (Fn). The FbaA-Fn-integrin α5ß1 complex activates Beclin-1 through the mTOR-ULK1-Beclin-1 pathway, which enables the Beclin-1/Vps34 complex to recruit Rab7 and, ultimately, to promote the formation of autophagosomes. By knocking down integrin α5ß1, Fn, Atg5, Beclin-1, and ULK1 in Hep2 cells and deleting Atg5 or integrin α5ß1 in mice, we reveal a novel role for integrin α5ß1 in inducing autophagy. Our study demonstrates that integrin α5ß1, through interacting with pathogen components, initiates effective host innate immunity against invading intracellular pathogens.IMPORTANCE Autophagy is generally considered a strategy used by the innate immune system to eliminate invasive pathogens through capturing and transferring them to lysosomes. Currently, researchers pay more attention to how virulence factors secreted by GAS regulate the autophagic process. Here, we provide the first evidence that the structural protein FbaA of M1 GAS strain SF370 is a potent inducer of autophagy in epithelial cells. Furthermore, we demonstrate that integrin α5ß1 in epithelial cells in vitro and in vivo acts as a receptor to initiate the signaling for inducing autophagy by binding to FbaA of M1 GAS strain SF370 via Fn. Our study reveals the underlying mechanisms by which pathogens induce Fn-integrin α5ß1 to trigger autophagy in a conserved pattern in epithelial cells.

M Protein of Group a Streptococcus Plays an Essential Role in Inducing High Expression of A20 in Macrophages Resulting in the Downregulation of Inflammatory Response in Lung Tissue.

Group A streptococcus (GAS), a common pathogen, is able to escape host immune attack and thus survive for longer periods of time. One of the mechanisms used by GAS is the upregulated expression of immunosuppressive molecules, which leads to a reduction in the production of inflammatory cytokines in immune cells. In the present study, we found that macrophages produced lower levels of proinflammatory cytokines (IL-1ß, TNF-α, IL-6) when challenged with GAS than they did when challenged with Escherichia coli (E. coli). Simultaneously, in a mouse model of lung infection, GAS appeared to induce a weaker inflammatory response compared to E. coli. Our data also indicated that the expression of the A20 transcriptional regulator was higher in GAS-infected macrophages than that in macrophages infected with E. coli, and that high expression of A20 correlated with a reduction in the production of TRAF6. SiRNA targeting of A20 led to the increased production of TRAF6, IL-1ß, TNF-α, and IL-6, suggesting that A20 inhibits synthesis of these key proinflammatory cytokines. We also investigated the pathway underlying A20 production and found that the synthesis of A20 depends on My88, and to a lower extent on TNFR1. Finally, we showed a significant reduction in the expression of A20 in macrophages stimulated by M protein-mutant GAS, however, a speB-GAS mutant, which is unable to degrade M protein, induced a greater level of A20 production than wild type GAS. Collectively, our data suggested that M protein of GAS was responsible for inducing A20 expression in macrophages, which in turn down-regulates the inflammatory cytokine response in order to facilitate GAS in evading immune surveillance and thus prolong survival in the host.