Early-Life Microbiota Exposure Restricts Myeloid-Derived Suppressor Cell-Driven Colonic Tumorigenesis.

Gut microbiota and their metabolites are instrumental in regulating homeostasis at intestinal and extraintestinal sites. However, the complex effects of prenatal and early postnatal microbial exposure on adult health and disease outcomes remain incompletely understood. Here, we showed that mice raised under germ-free conditions until weaning and then transferred to specific pathogen-free (SPF) conditions harbored altered microbiota composition, augmented inflammatory cytokine and chemokine expression, and were hyper-susceptible to colitis-associated tumorigenesis later in adulthood. Increased number and size of colon tumors and intestinal epithelial cell proliferation in recolonized germ-free mice were associated with augmented intratumoral CXCL1, CXCL2, and CXCL5 expression and granulocytic myeloid-derived suppressor cell (G-MDSC) accumulation. Consistent with these findings, CXCR2 neutralization in recolonized germ-free mice completely reversed the exacerbated susceptibility to colitis-associated tumorigenesis. Collectively, our findings highlight a crucial role for early-life microbial exposure in establishing intestinal homeostasis that restrains colon cancer in adulthood.

Heterosubtypic influenza protection elicited by double-layered polypeptide nanoparticles in mice.

Influenza is a persistent threat to public health. Here we report that double-layered peptide nanoparticles induced robust specific immunity and protected mice against heterosubtypic influenza A virus challenges. We fabricated the nanoparticles by desolvating a composite peptide of tandem copies of nucleoprotein epitopes into nanoparticles as cores and cross-linking another composite peptide of four tandem copies of influenza matrix protein 2 ectodomain epitopes to the core surfaces as a coating. Delivering the nanoparticles via dissolvable microneedle patch-based skin vaccination further enhanced the induced immunity. These peptide-only, layered nanoparticles demonstrated a strong antigen depot effect and migrated into spleens and draining (inguinal) lymph nodes for an extended period compared with soluble antigens. This increased antigen-presentation time correlated with the stronger immune responses in the nanoparticle-immunized group. The protection conferred by nanoparticle immunization was transferable by passive immune serum transfusion and depended partially on a functional IgG receptor FcγRIV. Using a conditional cell depletion, we found that CD8+ T cells were involved in the protection. The immunological potency and stability of the layered peptide nanoparticles indicate applications for other peptide-based vaccines and peptide drug delivery.

Mild electrical stimulation increases stress resistance and suppresses fat accumulation via activation of LKB1-AMPK signaling pathway in C. elegans.

Electrical current at physiological strength has been applied as a therapeutic approach for various diseases. Several of our works showed that mild electrical stimulation (MES) at 0.1-ms pulse width has positive impact on organisms. But despite the growing evidence of the beneficial effects of MES, its effects on individual animals and the molecular underpinnings are poorly understood and rarely studied. Here, we examined the effects of MES on individual animal and its mechanisms by mainly using Caenorhabditis elegans, a powerful genetic model organism. Interestingly, MES increased stress resistance and suppressed excess fat accumulation in wild-type N2 worms but not in AMPK/AAK-2 and LKB1/PAR-4 mutant worms. MES promoted the nuclear localization of transcription factors DAF-16 and SKN-1 and consequently increased the expression of anti-stress genes, whereas MES inhibited the nuclear localization of SBP-1 and suppressed the expression of lipogenic genes. Moreover, we found that MES induced the activation of LKB1/PAR4-AMPK/AAK2 pathway in C. elegans and in several mammalian cell lines. The mitochondrial membrane potential and cellular ATP level were slightly and transiently decreased by MES leading to the activation of LKB1-AMPK signaling pathway. Together, we firstly and genetically demonstrated that MES exerts beneficial effects such as stress resistance and suppression of excess fat accumulation, via activation of LKB1-AMPK signaling pathway.

Mild electrical stimulation at 0.1-ms pulse width induces p53 protein phosphorylation and G2 arrest in human epithelial cells.

Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53(-/-) cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

Insulin receptor activation through its accumulation in lipid rafts by mild electrical stress.

Insulin resistance is due to the reduced cellular response to insulin in peripheral tissues. The interaction of insulin with its receptor is the first step in insulin action and thus the identified target of insulin resistance. It has been well established that defects or mutations in the insulin receptor (IR) cause insulin resistance. Therefore, an IR activator might be a novel therapeutic approach for insulin resistance. Our previous report showed that mild electrical stress (MES) enhanced the insulin-induced signaling pathway. However, the molecular mechanism of the effect of MES remains unclear. We assessed the effect of MES, which is characterized by low-intensity direct current, on insulin signaling in vitro and in vivo. Here, we showed that MES activated the insulin signaling in an insulin-independent manner and improved insulin resistance in peripheral tissues of high fat-fed mice. Moreover, we found that MES increased the localization of IR in lipid rafts and enhanced the level of phosphorylated Akt in insulin-resistant hepatic cells. Ablation of lipid rafts disrupted the effect of MES on Akt activation. Our findings indicate that MES has potential as an activator of IR in an insulin-independent manner, and might be beneficial for insulin resistance in type 2 diabetes.

Diethyldithiocarbamate induces apoptosis in HHV-8-infected primary effusion lymphoma cells via inhibition of the NF-κB pathway.

Primary effusion lymphoma (PEL) is a subtype of B-cell lymphoma caused by human herpes virus 8/Kaposi sarcoma-associated herpes virus (HHV-8/KSHV), which is mostly found in patients with AIDS and has poor prognosis. Nuclear factor (NF)-κB pathway is constitutively activated in HHV-8-infected PEL cells and plays a crucial role in tumorigenesis. Recently, it has been shown that diethyldithiocarbamate (DDTC), an active metabolite of disulfiram, has apoptotic activity in cancer cells. Here, we investigated the effect of DDTC on PEL using a PEL mouse model generated by intraperitoneal injection of BC-3 cells, a PEL cell line. DDTC ameliorated the symptoms of PEL in these mice, such as development of ascites, splenomegaly and increase of body weight, in comparison with PBS-treated controls. Moreover, we determined in vitro that DDTC suppressed the constitutively activated NF-κB pathway in BC-3 cells. Methylthiotetrazole assay revealed that the cell proliferation of various PEL cell lines was significantly suppressed by the treatment of DDTC. DDTC also induced the expression of cleaved caspase-3, an apoptosis marker, whereas the addition of Q-VD-OPh, a pan-caspase inhibitor, inhibited cell apoptosis induced by DDTC treatment. Together, our results indicated that DDTC induces apoptosis via inhibition of the NF-κB signaling pathway in HHV-8-infected PEL cells. This study suggests the potential use of DDTC as a therapeutic approach for PEL.

Glucose uptake in rat skeletal muscle L6 cells is increased by low-intensity electrical current through the activation of the phosphatidylinositol-3-OH kinase (PI-3K) / Akt pathway.

Activation of Akt by insulin is transmitted via phosphatidylinositol-3-OH kinase (PI-3K) and enhances glucose uptake. The PI-3K/Akt signaling is diminished in insulin resistance. Thus, approaches that activate PI-3K/Akt signaling leading to improved glucose uptake may ameliorate hyperglycemia. Here we showed that low-intensity electrical current or mild electrical stimulation (MES) activated the PI-3K/Akt signaling and increased the glucose uptake in rat skeletal muscle (L6) cells. The glucose uptake enhanced by MES in muscle cells, the major cells involved in glucose disposal, suggests MES may have a possible beneficial effect on hyperglycemia.