Intravital imaging of splenic classical monocytes modifying the hepatic CX3CR1+ cells motility to exacerbate liver fibrosis via spleen-liver axis.

CX3CR1+ cells play a crucial role in liver fibrosis progression. However, changes in the migratory behavior and spatial distribution of spleen-derived and hepatic CX3CR1+ cells in the fibrotic liver as well as their influence on the liver fibrosis remain unclear. METHODS: The CX3CR1GFP/+ transgenic mice and CX3CR1-KikGR transgenic mice were used to establish the CCl4-induced liver fibrosis model. Splenectomy, adoptive transfusion of splenocytes, in vivo photoconversion of splenic CX3CR1+ cells and intravital imaging were performed to study the spatial distribution, migration and movement behavior, and regulatory function of CX3CR1+ cells in liver fibrosis. RESULTS: Intravital imaging revealed that the CX3CR1GFP cells accumulated into the fibrotic liver and tended to accumulate towards the central vein (CV) in the hepatic lobules. Two subtypes of hepatic CX3CR1+ cells existed in the fibrotic liver. The first subtype was the interacting CX3CR1GFP cells, most of which were observed to distribute in the liver parenchyma and had a higher process velocity; the second subtype was mobile CX3CR1GFP cells, most of which were present in the hepatic vessels with a faster moving speed. Splenectomy ameliorated liver fibrosis and decreased the number of CX3CR1+ cells in the fibrotic liver. Moreover, splenectomy rearranged CX3CR1GFP cells to the boundary of the hepatic lobule, reduced the process velocity of interacting CX3CR1GFP cells and decreased the number and mobility of mobile CX3CR1GFP cells in the fibrotic liver. Transfusion of spleen-derived classical monocytes increased the process velocity and mobility of hepatic endogenous CX3CR1GFP cells and facilitated liver fibrosis progression via the production of proinflammatory and profibrotic cytokines. The photoconverted splenic CX3CR1+ KikRed+ cells were observed to leave the spleen, accumulate into the fibrotic liver and contact with hepatic CX3CR1+ KikGreen+ cells during hepatic fibrosis. CONCLUSION: The splenic CX3CR1+ monocytes with classical phenotype migrated from the spleen to the fibrotic liver, modifying the migratory behavior of hepatic endogenous CX3CR1GFP cells and exacerbating liver fibrosis via the secretion of cytokines. This study reveals that splenic CX3CR1+ classical monocytes are a key driver of liver fibrosis via the spleen-liver axis and may be potential candidate targets for the treatment of chronic liver fibrosis.

Melittin-Carrying Nanoparticle Suppress T Cell-Driven Immunity in a Murine Allergic Dermatitis Model.

Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are the most common human skin disorders. Although corticosteroids have been widely used to treat ACD and AD, the side effects of corticosteroids encourage researchers to explore new immunoregulatory treatments. Here, an immunomodulatory approach based on lipid nanoparticles carrying α-helical configurational melittin (α-melittin-NP) is developed to overcome T cell-mediated inflammatory reactions in an oxazolone (OXA)-induced contact hypersensitivity mouse model and OXA-induced AD-like mouse model. Intradermal injection of low-dose α-melittin-NPs prevents the skin damage caused by melittin administration alone and efficiently targeted lymph nodes. Importantly, melittin and α-melittin-NPs restrain RelB activity in dendritic cells (DCs) and further suppresses dendritic cell activation and maturation in lymph nodes. Furthermore, low-dose α-melittin-NPs leads to relief of antigen recognition-induced effector T cell arrest in the dermis and inhibited allergen-specific T cell proliferation and activation. Significantly, this approach successfully controls Th1-type cytokine release in the ACD model and restricts Th2-type cytokine and IgE release in the AD-like model. Overall, intradermal delivery of low-dose α-melittin-NPs efficiently elicits immunosuppression against T cell-mediated immune reactions, providing a promising therapeutic strategy for treating skin disorders not restricted to the lesion region.

Pickering emulsions stabilized by pea protein isolate-chitosan nanoparticles: fabrication, characterization and delivery EPA for digestion in vitro and in vivo.

The work aimed to prepare pea protein isolate-chitosan (PPI-CS) nanoparticles, fabricate PPI-CS nanoparticles stabilized Pickering emulsions (PPI-CS Pickering emulsions) and deliver EPA for digestion in vitro and in vivo. The nanoparticles were characterized by scanning electron microscopy (SEM), and PPI-CS Pickering emulsions were characterized by physicochemical and rheological properties. The results showed that the size of PPI-CS nanoparticles was 194.22 ± 0.45 nm. Rheological measurement showed that the PPI-CS Pickering emulsions possessed a gel-like network. EPA encapsulated Pickering emulsions (EPA-PE, φ = 0.6) exhibited a high retention rate (93%) during storage and performed a lower release rate compared with EPA-PE (φ = 0.4) in vitro digestion. The area under the curve of EPA concentration of EPA-PE group and EPA-emulsions (EPA-Em) group was 1.71 and 1.48, respectively. It demonstrated that PPI-CS Pickering emulsions provided the possibility to deliver EPA for digestive absorption.

DHA loaded nanoliposomes stabilized by ß-sitosterol: Preparation, characterization and release in vitro and vivo.

DHA loaded nanoliposomes, stabilized by ß-sitosterol, were prepared by thin film hydration-sonication method. The characteristics and membranes properties of DHA-NLs with different ß-sitosterol content were measured. The samples with the same formulation were used to measure the resistance of environment stress and controlled release & absorption of DHA in vitro and in vivo. The results showed that the maximal encapsulation efficiency of DHA-NLs was (86.95 ± 0.95)%, when the ratio of soybean lecithin to ß-sitosterol was 5:1. The particle size of all samples was within 200 nm and relative retention rate was more than 60% after 3 weeks storage. The area under the curve of DHA concentration of DHA-NLs and DHA-emulsion groups was 1.32 and 1.08, respectively. In summary, the nanoliposomes were promising to improve the absorption of DHA in form of ethyl ester.

Formation, characterization and application of arginine-modified chitosan/γ-poly glutamic acid nanoparticles as carrier for curcumin.

A novel nanoparticle (NP) delivery carrier for curcumin based on electrostatic 6-deoxy-6-arginine modified chitosan (DAC) assembled by γ-poly-glutamic acid (γ-PGA) was prepared. The NP structure was evaluated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Interactions between DAC and γ-PGA were characterized using Fourier transform infrared spectroscopy (FT-IR). The sustained release kinetics of curcumin-loaded NPs was investigated in simulated gastrointestinal fluids. After exposed to heating, pH, and NaCl aqueous solution, the stabilities of both normal and curcumin-loaded NPs were determined. The results showed that NPs achieved a high encapsulation efficiency (79.5%) and loading capacity (11.31%) for curcumin. The curcumin-loaded NPs displayed a sustained release profile under simulated gastrointestinal conditions. Under certain pH (3-9), salt (0-100 mM), and temperature (30 - 60 °C) conditions, the vehicles of curcumin showed better stability. This demonstrates that NPs can be used as stable carriers for curcumin.

Preparation of Ca-alginate-whey protein isolate microcapsules for protection and delivery of L. bulgaricus and L. paracasei.

To promote the application of probiotics that are beneficial to hosts, calcium-alginate (Ca-Alg) coated whey protein isolate microcapsules were prepared for protection and delivery of L. bulgaricus and L. paracasei. The internal layer was formed with transglutaminase-induced gelation of whey protein isolate (WPI). Sodium alginate (SA) was applied to form outer layer with external Ca2+ gelation method. The microcapsules loaded with probiotics were characterized by scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC). The results showed that the co-encapsulation efficiency was 90.54% and 84.46% of WPI micro-beads and Ca-Alg-coated microcapsules, respectively. The trehalose was added as cryoprotectant to improve the survival rate of probiotics in freeze-dried Ca-Alg-coated microcapsules from 3% to 41.26%. Ca-Alg-coated microcapsules have regular morphology and intensified structure. The protection of Ca-Alg-coated microcapsule for probiotics was improved under simulated gastrointestinal and thermal conditions. Ca-Alg-WPI microcapsules showed a useful way for protection and delivery of L. bulgaricus and L. paracasei.

The formation, stability of DHA/EPA nanoemulsion prepared by emulsion phase inversion method and its application in apple juice.

This study prepared edible docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) nanoemulsion using EPI (emulsion phase inversion) method. The method for preparing DHA and EPA nanoemulsions is safe, convenient, low in energy consumption and can be used for food production. Factors affecting particle size and stability during preparation were investigated. Based on the optimal particle size combination, stability studies including particle size and residual rates of DHA and EPA at different temperature, pH and metal ions. The results showed that the nanoemulsion had good stability at low temperature storage, near neutral pH and in the absence of transition metal ions such as Fe3+, Cu2+, Al3+. The experiment initially studied the effect of nanoemulsion on apple juice beverage on the basic properties of juice itself. It was feasible in practical application of edible nanoemulsion.

Layer (whey protein isolate) -by-layer (xanthan gum) microencapsulation enhances survivability of L. bulgaricus and L. paracasei under simulated gastrointestinal juice and thermal conditions.

In the study, Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and Lactobacillus paracasei subsp. paracasei (L. paracasei) were isolated from food products and identified. The probiotics were co-encapsulated by emulsification technique with transglutaminase-induced gelation of whey protein isolate (WPI). A double layer was formed by xanthan gum (XG). The survival of both free and microencapsulated cells in each step was enumerated. The results showed that high entrapment yield (86.36 ± 1.07%) was achieved, WPI-XG provided significant protection for cells in simulated gastric juice and bile tolerance tests compared with free cells. Besides, the survivability under thermal condition also strengthened. During freeze-drying process, the survival was improved and naked cells cannot be seen on microcapsules surface when trehalose was added into WPI solution as a lyoprotectant. Additionally, the structure changes of WPI/XG in the encapsulation process were investigated by Fourier transform infrared spectrophotometer (FT-IR) and the thermal properties were studied by differential scanning calorimetry (DSC).

Effective degradation of the mycotoxin patulin in pear juice by porcine pancreatic lipase.

Porcine pancreatic lipase (PPL) was used to degrade the mycotoxin patulin (PAT) in pear juice. The dosage of PPL, the initial concentration of PAT, reaction temperature and time were investigated by batch experiments to study the optimal degradation condition. The concentration of PAT in pear juice was determined by high performance liquid chromatography with an ultraviolet detector (HPLC-UV). The results showed that the optimal condition was 0.02 g PPL/mL pear juice at 40 °C for 24 h. The content of organic acids, volatile flavor components, polyphenols, ascorbic acid and the degree of browning reaction in pear juice, relating to the quality of juice, changed insignificantly. Although the initial PAT concentration was very high, the degradation product was confirmed nontoxic by cytotoxicity test of Caco-2 cells. It suggested that PPL could be further considered to be applied in the degradation of PAT in pear juice.

Visualizing DC morphology and T cell motility to characterize DC-T cell encounters in mouse lymph nodes under mTOR inhibition.

Mammalian target of rapamycin (mTOR), a serine/threonine kinase orchestrating cellular metabolism, is a crucial immune system regulator. However, it remains unclear how mTOR regulates dendritic cell (DC) function in vivo, especially DC-T cell encounters, a critical step for initiating adaptive immune responses. We dynamically visualized DC-T contacts in mouse lymph node using confocal microscopy and established an encounter model to characterize the effect of mTOR inhibition on DC-T cell encounters using DC morphology. Quantitative data showed mTOR inhibition via rapamycin altered DC shape, with an increased form factor (30.17%) and decreased cellular surface area (20.36%) and perimeter (22.43%), which were associated with Cdc42 protein downregulation (52.71%). Additionally, DCs adopted a similar morphological change with Cdc42 inhibition via ZCL278 as that observed with mTOR inhibition. These morphologically altered DCs displayed low encounter rates with T cells. Time-lapse imaging data of T cell motility supported the simulated result of the encounter model, where antigen-specific T cells appeared to reduce arrest in the lymph nodes of rapamycin-pretreated mice relative to the untreated group. Therefore, mTOR inhibition altered DC morphology in vivo and decreased the DC-T cell encounter rate, as well as Cdc42 inhibition. By establishing an encounter model, our study provides an intuitive approach for the early prediction of DC function through morphological quantification of form factor and area.

JNK1/ß-catenin axis regulates H2O2-induced epithelial-to-mesenchymal transition in human lens epithelial cells.

Epithelial-mesenchymal transition (EMT) is the main cause of fibrotic cataracts. Oxidative stress was recently shown to trigger epithelial-mesenchymal transition in human lens epithelial cells (hLECs). However, the underlying mechanism is not fully understood. Here we reported that exposure to low doses (100 µM) of H2O2 led to EMT in hLECs, as indicated by simultaneous down-regulated of E-cadherin and ZO-1, and up-regulated of alpha smooth muscle actin (α-SMA). H2O2-induced EMT was accompanied by accumulation of phosphorylated JNK1. In contrast, knockdown of JNK1 via siRNA reversed H2O2-induced EMT. Of interest, in human lens capsules of anterior subcapsule cataracts, the expressions of JNK1, as well as ß-catenin and its downstream effectors cyclin D and c-Myc, were augmented compared to that in normal lens capsules. Mechanistically, activated JNK1 dislodged ß-catenin from the cell membrane, which subsequently translocated to the nuclei and triggered transcription of its effectors. Nuclei ß-catenin, cyclin D and c-Myc were accumulated in H2O2-induced EMT and JNK1 depletion abrogated these trend in hLECs. In conclusion, our data suggest that JNK1 is essential for H2O2-induced EMT in hLECs via mediating the translocation of ß-catenin.

Wnt5a Contributes to the Differentiation of Human Embryonic Stem Cells into Lentoid Bodies Through the Noncanonical Wnt/JNK Signaling Pathway.

Purpose: Recent work has indicated that Wnt5a has a critical role in embryonic development. We investigate whether the Wnt5a-activated noncanonical Wnt pathway is capable of promoting embryonic lens differentiation. Methods: A "three-stage" protocol was used to induce lens differentiation of human embryonic stem cells (hESCs) in vitro, and Wnt5a levels were modified by addition of exogenous protein and RNA interference. SP600125 was adopted to inhibit JNK cascades. The number and size of lentoid bodies obtained were measured, and quantitative RT-PCR, Western blotting, and immunofluorescence were used to detect gene and protein expression. Results: The quantity and size of lentoid bodies generated were significantly increased by addition of exogenous Wnt5a. Moreover, expression of lens-specific genes, including CRYAA, CRYAB, BFSP1, and MIP, and the lens fiber differentiation regulator PROX1 were prominently increased. We also observed activation of noncanonical Wnt signaling via upregulation of Dvl2, Rac1, and JNK. When Wnt5a-knockdown hESCs were induced to differentiate, fewer and smaller lentoid bodies resulted. In addition, expression of genes specific to lens was decreased and noncanonical Wnt/JNK pathway activity was downregulated. Accordingly, inhibition of JNK cascade suppressed the formation of lentoid bodies as well, consistent with that of Wnt5a-knockdown group. Conclusions: Wnt5a can promote the differentiation of hESCs into lentoid bodies through the noncanonical Wnt/JNK signaling pathway, thereby contributing to the study of human lens development and moreover the underlying etiology congenital cataracts.

The effect of immunosuppressive drug cyclosporine A on myeloid-derived suppressor cells in transplanted mice.

OBJECTIVE: Myeloid-derived suppressor cells (MDSCs) play important roles in preventing graft rejection. Immunosuppressive drug cyclosporine A (CsA) is widely used in clinics to treat patients with allografts and autoimmune diseases. However, the effect of CsA on CD11b(+)Gr1(+) MDSCs has not been studied. SUBJECTS: The subjects of the study include BALB/c skin-grafted C57BL/6 mice and the in vitro MDSCs induction system. TREATMENT: Skin-grafted mice were treated with CsA (30 mg/kg, i.p.) or control buffer daily. 0.01 µg/ml CsA was added during MDSC induction. METHODS: Flow cytometry was used to check cell phenotypes and proliferation. Real-time PCR was used for gene expressions. Inducible nitric oxide synthase iNOS-knockout mice were used for the role of iNOS in the immunosuppression of MDSCs. RESULTS: CsA in MDSC-induction system significantly increased the number of CD11b(+)Gr1(+)MDSCs without detectable effects on the expressions of CD31, CD115 and CD274. However, GM-CSF + CsA-induced MDSCs express higher iNOS than control MDSCs. Blocking iNOS activity by inhibitor or gene deletion significantly reversed the inhibitory effects of GM-CSF + CsA-induced MDSCs on T cell proliferation. Importantly, CsA treatment significantly increased the number and the immunosuppressive ability of CD11b(+)Gr1(+)MDSCs in allogeneic skin-grafted mice. CONCLUSIONS: CsA promotes MDSC induction and immunosuppressive function, which might be of clinical importance in treating graft rejection and autoimmune diseases.

TNFα-induced M-MDSCs promote transplant immune tolerance via nitric oxide.

UNLABELLED: Efficient induction of functional competent myeloid-derived suppressor cells (MDSCs) will be critical for the clinical application of MDSCs to treat autoimmune diseases and to induce transplantation immune tolerance. In the present study, we tried to establish the MDSC induction system with M-CSF and tumor necrosis factor α (TNFα) and investigated the immunosuppressive function of M-CSF + TNFα-induced MDSCs in transplant mouse models. Monocytic MDSCs (M-MDSCs) were induced by culture of the non-adherent mouse bone marrow cells with M-CSF or M-CSF + TNFα, respectively, for 7 days. Phenotype analysis revealed that the majority of M-CSF- and M-CSF + TNFα-induced MDSCs express F4/80. The addition of TNFα in the induction period increased Gr-1, Ly6C, CD80, and CD274 expressions on these cells. M-CSF + TNFα-induced M-MDSCs showed poor TNFα, IL-12, and IL-6 expressions after lipopolysaccharide (LPS) stimulation and decreased arginase 1 (Arg-1) and Fizz expressions after IL-4 stimulation compared with M-CSF-induced M-MDSCs. M-CSF + TNFα-induced M-MDSCs showed enhanced ability to suppress T cell proliferation and cytokine production than M-CSF-induced M-MDSCs. M-CSF + TNFα-induced M-MDSCs express high levels of inducing nitric oxide synthase (iNOS) and blocking iNOS activity by a chemical inhibitor or gene deficiency significantly reversed the inhibitory effects of M-CSF + TNFα-induced M-MDSCs on T cells. Adoptive transfer of M-CSF + TNFα-induced M-MDSCs promoted immune tolerance in a male-to-female skin-grafted mice, but M-CSF + TNFα-induced iNOS-deficient M-MDSCs failed to do so. Thus, M-CSF + TNFα-induced M-MDSCs have powerful immunosuppressive activity, which is mediated by an iNOS-dependent pathway. M-CSF + TNFα-induced M-MDSCs can promote immune tolerance to donor antigens in a transplant mouse model. KEY MESSAGE: The combination of M-CSF and TNFα efficiently induces functional M-MDSCs in vitro. M-CSF + TNFα-induced M-MDSCs promote immune tolerance in a transplant mouse model. The immunosuppressive ability of M-CSF + TNFα-induced M-MDSCs is dependent on iNOS.