Oral delivery of staphylococcal nuclease ameliorates DSS induced ulcerative colitis in mice via degrading intestinal neutrophil extracellular traps.

Recent studies have revealed that neutrophil extracellular traps (NETs) may contribute directly to the initiation of ulcerative colitis (UC), a typical inflammatory bowel disease (IBD) characterized by mucosal damage. Staphylococcal nuclease (SNase), a nonspecific phosphodiesterase, has a strong ability to degrade DNA. Here we investigate whether intestinal NET degradation with an oral preparation of SNase can ameliorate dextran sulfate sodium (DSS)-induced UC in mice. SNase encapsulated with calcium alginate (ALG-SNase) was formulated using crosslinking technology with sodium alginate and calcium chloride. ALG-SNase were orally administered to DSS-induced UC mice, and their therapeutic efficacy was evaluated. The expression of inflammatory cytokines and biomarkers of NETs was also assessed, as well as the intestinal permeability in mice. The results showed that ALG-SNase nanoparticles were successfully prepared and delivered to the colon of UC mice. In addition, oral administration of ALG-SNase nanoparticles decreased NET levels in the colon and effectively alleviated the clinical colitis index and tissue inflammation in UC mice. Moreover, the SNase nanoparticles reduced intestinal permeability and regulated the expression of proinflammatory cytokines. Furthermore, the markers of NETs were strongly correlated with the expression levels of tight junction proteins in colon tissue. In conclusion, our data showed that oral administration of ALG-SNase can effectively ameliorate colitis in UC mice via NET degradation and suggested SNase as a candidate therapy for the treatment of UC.

Evaluation of protein arginine deiminase-4 inhibitor in TNBS- induced colitis in mice.

BACKGROUND AND AIM: Many evidences indicated that neutrophil extracellular traps (NETs) are involved in the pathogenesis of inflammatory bowel disease (IBD). Citrullination of histones by Protein Arginine Deiminase-4 (PAD4) is central for NETs formation. This paper aimed to explore the definite role of NETs in mouse model of Crohn's disease (CD) with 2,4,6-trinitrobenzene sulfonic acid (TNBS). METHODS: The expression of NETs-associated proteins and mRNAs in colon tissue were detected by immunohistochemistry and Real-time Quantitative PCR (QPCR) respectively. Neutrophils were isolated and stimulated in vitro to form NETs. In addition, we also administered Cl-amidine, PAD4 inhibitor, resulting in less NETs formation to investigate protective effect by measuring weight loss, gross bleeding, colon length, myeloperoxidase (MPO) activity, and cytokine expression in mice. RESULTS: The results showed enhanced expression of Ly6G, citrullinated histone H3 (CitH3), and PAD4 in TNBS-induced colitis mice and higher ability of neutrophil to produce NETs in vitro. Blocking NETs formation through Cl-amidine effectively alleviated the clinical colitis index and tissue inflammation in TNBS mice, regulated the expression of pro- or anti-inflammatory cytokines. In addition, Cl-amidine reduced the gene expression of PAD4 and the expression of NETs-associated proteins in the colon of TNBS mice and inhibited the formation of NETs in vitro. CONCLUSIONS: Our data showed that Cl-amidine could alleviate the clinical colitis index in TNBS mice to some extend and suggested blocking NETs formation through inhibition of PAD4 as therapeutic targets for the treatment of CD.

Ameliorating gut microenvironment through staphylococcal nuclease-mediated intestinal NETs degradation for prevention of type 1 diabetes in NOD mice.

AIMS: Recent studies have revealed that neutrophil extracellular traps (NETs) provide negative feedback in the progression to chronic inflammation and contribute to the pathogenesis of multiple autoimmune diseases including type 1 diabetes (T1D). In addition, accumulating evidences suggest that gut immunity play a key role in T1D pathogenesis. Our study aimed to evaluate whether staphylococcal nuclease (SNase) targeting intestinal NETs can ameliorate the intestinal inflammatory environment and protect against T1D development in non-obese diabetic(NOD) mice. MAIN METHODS: Degradation of NETs with SNase in vitro was examined using SYTOX green assay. NOD/LtJ mice were oral administration of Lactococcus lactisl (L. lactis) pCYT: SNase and blood glucose levels were monitored weekly. Several biomarkers of NETs formation, gut leakage and inflammation were determined using a commercial ELISA kit. T Cell phenotypes in peripheral immune system were analyzed in flow cytometry and fecal samples were isolated to investigate intestinal microbiota. KEY FINDINGS: The oral delivery of SNase by L. lactis can decrease the NETs levels and ameliorate inflammation both in the intestine and pancreatic islets and finally effectively regulate the blood glucose levels of NOD mice. Meanwhile, zonulin and lipopolysaccharide levels also reduced in SNase-fed NOD mice, suggesting SNase could improve gut barrier function via intestinal NETs degradation. Furthermore, the abundances of the intestinal microbiota and butyrate-producing gut bacteria were also increased with SNase treatment. SIGNIFICANCE: SNase shows potential for intestinal NETs to prevent T1D based on the gut-pancreas axis.

Two tandem repeats of mHSP70407-426 enhance therapeutic antitumor effects of a recombined vascular endothelial growth factor (VEGF) protein vaccine.

AIMS: Active immunization with human vascular endothelial growth factor (hVEGF) vaccines provides a therapeutic option instead of bevacizumab therapy. However, the immunity to self-molecule is difficult to elicit due to immune tolerance. A bioactive peptide of two tandem repeats of mHSP70407-426 (M2) has exhibited potent adjuvant ability in our previous study, and the aim of this study was to explore whether M2 could assist hVEGF to display enhanced therapeutic anti-tumor effects. MAIN METHODS: The anti-tumor effects of hVEGF-M2 vaccine were evaluated in both H22 hepatocellular carcinoma and Lewis lung tumor models. CD31 analysis of excised tumors was used to evaluate anti-angiogenesis effects. The titers of anti-VEGF antibody was detected by ELISA and verified by western blot analyses, and the effects of immune sera on HUVEC differentiation were investigated by tube formation assay. KEY FINDINGS: M2 could assist hVEGF to exhibit more favorable therapeutic anti-tumor growth and metastasis effects than hVEGF. Meanwhile, high titer of anti-VEGF antibody was detected in hVEGF-M2 immunized mice sera by ELISA and verified by western blot analysis. Sera from hVEGF-M2 immunized mice could more significantly inhibit HUVEC tube formation than hVEGF immune serum. The hVEGF-M2-immune sera could more effectively inhibit H22 tumor growth and extend the survival rates of H22 tumor bearing mice than hVEGF-immune sera. CD31 analysis of the excised tumors verified a significant reduction in vessel density after hVEGF-M2 vaccination. SIGNIFICANCE: M2 could assist hVEGF to display enhanced anti-tumor effects, which are important for the further application of M2 to enhance antigen-specific immune responses.