Biosorption of high-concentration Cu (II) by periphytic biofilms and the development of a fiber periphyton bioreactor (FPBR).

In this study, a kind of microbial aggregates: periphytic biofilms were used for Cu removal and immobilized onto fiber for developing a novel bioreactor. Results show that periphyton can effectively entrap Cu at initial concentrations of 2-20mgL-1 due to the overproduction of EPS and porous structure of periphyton, and biosorption was the primary mechanism of Cu removal. Cu (mainly Cu3(OH)42+, Cu2(OH)22+ and Cu2+) adsorption onto periphytic biofilms followed the pseudo-second order kinetic model. The biosorption process fitted the Freundlich, Langmuir and Dubinin-Radushkevich Isotherm models well and was thermodynamically spontaneous. The fiber substrate used in the periphyton bioreactor greatly increased the Cation Exchange Capacity (CEC) of the system. This study indicates that immobilization of periphytic biofilms onto fiber for novel bioreactor development is a feasible way of entrapping high-concentration Cu from wastewater.

[Blocking p38MAPK pathway inhibits the proliferation of OT-II cells mediated by splenic dendritic cells].

OBJECTIVE: To investigate the effect of blocking p38 mitogen-activated protein kinase (p38MAPK) pathway on the proliferation of OT-II cells mediated by splenic dendritic cells (DCs). METHODS: Splenic DCs of C57BL/6 mice were purified with anti-CD11c immunomagnetic beads, and OT-II cells were isolated from the splenic of CD4(+)-ovalbumin transgenic mice (OT-IItransgenic mice) by mouse CD4 T cell isolation kits. After being pretreated with SB203580, an inhibitor of p38MAPK, DCs were stimulated with lipopolysaccharides (LPS). Then the expression levels of co-stimulatory molecules (CD80, CD86) and MHCII in DCs, and antigen-presenting ability of DCs treated with the 52-68 fragment of the E alpha-chain of I-E class II molecules (Eα52-68 peptide) were detected by flow cytometry. The protein levels of tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), interleukin 6 (IL-6) and transforming growth factor ß (TGF-ß) in the culture supernatants were measured by ELISA. The proliferation of OT-II cells which were co-cultured with OVA323-339-treated DCs was analyzed by flow cytometry. RESULTS: The purity of both DCs and OT-II cells reached over 90% after isolation. SB203580 downregulated the expressions of CD80, CD86 and MHC II, and suppressed the antigen-presenting ability of DCs. The expressions of TNF-α, IL-1α and IL-6 were downregulated, while the expression of TGF-ß was raised. Finally, SB203580 inhibited DCs-mediated proliferation of OT-II cells. CONCLUSION: Blocking p38MAPK pathway with SB203580 could inhibit DCs-mediated proliferation of OT-II cells, which might be involved in modulating the expressions of CD80, CD86 and MHC II, the antigen-presenting ability, as well as the expressions of pro-inflammatory and anti-inflammatory cytokines.

[Interleukin-37 inhibits the motility and migration of mouse splenic dendritic cells].

OBJECTIVE: To investigate the effect of interleukin-37 (IL-37) on the motility and migration of mouse splenic dendritic cells (DCs). METHODS: Magnetic bead cell sorting was used to isolate mouse splenic DCs, and flow cytometry was used to analyze the purity. DCs were visualized in real time by PerkinElmer laser confocal microscopy and analyzed by Volocity image analysis software. The alteration of chemokine (C motif) receptor 1 (XCR1) mRNA was revealed by microarray analysis and confirmed by real-time PCR and flow cytometry. Migration ability of DCs was detected by Transwell(TM) migration assay. RESULTS: The purity of isolated DCs was over 95%. The treatment of IL-37 reduced the velocity of DCs mobility, decreased XCR1 expression and depressed the cell migration ability. CONCLUSION: IL-37 can inhibit the motility and migration of mouse splenic DCs.

Tsc1 is a Critical Regulator of Macrophage Survival and Function.

BACKGROUND/AIMS: Tuberous sclerosis complex 1 (Tsc1) has been shown to regulate M1/M2 polarization of macrophages, but the precise roles of Tsc1 in the function and stability of macrophages are not fully understood. Here we show that Tsc1 is required for regulating the survival, migration and phagocytosis of macrophages. METHODS: Mice with Tsc1 homozygous deletion in myeloid cells (LysMCreTsc1(flox/flox); Tsc1 KO) were obtained by crossing Tsc1(flox/flox) mice with mice expressing Cre recombinase under the control of Lysozyme promoter (LysMCre). The apoptosis and growth of macrophages were determined by flow cytometry and Real-time PCR (RT-PCR). The phagocytosis was determined using a Vybrant™ phagocytosis assay kit. The migration of macrophages was determined using transwell migration assay. RESULTS: Peritoneal macrophages of Tsc1 KO mice exhibited increased apoptosis and enlarged cell size. Both M1 and M2 phenotypes in Tsc1-deficient macrophages were elevated in steady-state as well as in inflammatory conditions. Tsc1-deficient macrophages demonstrated impaired migration and reduced expression of chemokine receptors including CCR2 and CCR5. Phagocytosis activity and ROS production were enhanced in Tsc1-deficient macrophages. Furthermore, pharmacological inhibition of the mammalian target of rapamycin complex 1 (mTORC1) partially reversed the aberrance of Tsc1-deficient macrophages. CONCLUSION: Tsc1 plays a critical role in regulating macrophage survival, function and polarization via inhibition of mTORC1 activity.

IL-37b gene transfer enhances the therapeutic efficacy of mesenchumal stromal cells in DSS-induced colitis mice.

AIM: To investigate whether the transfer of the IL-37b gene, a newly identified inhibitor of both innate and adaptive immunity, could improve the therapeutic efficacy of mesenchumal stromal cells (MSCs) in inflammatory bowel disease (IBD). METHODS: The expression of IL-37 in biopsied specimens of the patients with active ulcerative colitis (UC) was detected using RT-PCR and immunohistochemistry. Mice were treated with 3% dextran sulfate sodium (DSS) for 8 days to induce colitis. Before DSS treatment, the mice were injected with MSCs, MSC-eGFP or MSC-IL37b. Their body weight was measured each day, and the colons and spleens were harvested on d 10 for pathological and biochemical analyses. RESULTS: In biopsied specimens of the patients with active UC, the expression of IL-37 was dramatically elevated in inflamed mucosa, mainly in epithelial cells and infiltrating immune cells. Compared to MSC-eGFP or MSCs, MSC-IL37b administration significantly attenuated the body weight and colon length reduction, and decreased the histological score in DSS-induced colitis mice. Furthermore, MSC-IL37b administration increased the percentage of myeloid-derived suppressor cells (MDSCs) among total splenic mononuclear cells as well as the percentage of regulatory T cells (Tregs) among splenic CD4+ T cells in the mice. Moreover, MSC-IL37b administration increased the IL-2+ cells and decreased the IFN-γ+ cells among splenic CD4+ T cells. CONCLUSION: IL-37 is involved in the pathophysiology of UC. IL-37b gene transfer enhances the therapeutic efficacy of MSCs in DSS-induced colitis mice by inducing Tregs and MDSCs and regulating cytokine production.

IL-37b suppresses T cell priming by modulating dendritic cell maturation and cytokine production via dampening ERK/NF-κB/S6K signalings.

Interleukin 37b (IL-37b) plays a key role in suppressing immune responses, partially by modulating the function of dendritic cells (DCs). However, the precise mechanisms are still largely unknown. Here, we investigated the effects of IL-37b on DC maturation and T cell responses induced by DCs, and explored the involved signaling pathways. It was found that IL-37b down-regulated the expressions of co-stimulatory molecules CD80 and CD86 on DCs in vitro. At the same time, the expressions of pro-inflammatory cytokines, such as TNF-α and IL-6, were suppressed, while the expression of the T cell inhibitory cytokine TGF-ß was increased in IL-37b-treated DCs. In addition, the activation effect of DCs on T cells was impaired by IL-37b. We further revealed that extracellular single-regulated kinase (ERK), nuclear factor-κB (NF-κB), and mTOR-S6K signaling pathways were involved in the inhibition of DCs induced by IL-37b. This was confirmed by the similarly suppressive effect of chemical inhibitors against NF-κB, ERK, and S6K on the expressions of IL-6 and TNF-α in DCs. In conclusion, these results demonstrated that IL-37b suppressed DC maturation and immunostimulatory capacity in T cell priming by involving in ERK, NF-κB, and S6K-based inhibitory signaling pathways.

Transfer of the IL-37b gene elicits anti-tumor responses in mice bearing 4T1 breast cancer.

AIM: IL-37b has shown anti-cancer activities in addition to its anti-inflammatory properties. In this study, we investigated the effects of IL-37b on breast carcinoma growth in mice and to determine the involvement of T cell activation in the effects. METHODS: IL-37b gene was transferred into mouse breast carcinoma cell line 4T1 (4T1-IL37b cells), the expression of secretory IL-37b by the cells was detected, and the effects of IL-37b expression on the cell proliferation in vitro was evaluated. After injection of 4T1 cells or 4T1-IL37b cells into immunocompetent BALB/c mice, immunodeficient BALB/c nude mice and NOD-SCID mice, the tumor growth and survival rate were measured. The proliferation of T cells in vitro was also detected. RESULTS: IL-37b was detected in the supernatants of 4T1-IL37b cells with a concentration of 12.02 ± 0.875 ng/mL. IL-37b expression did not affect 4T1 cell proliferation in vitro. BALB/c mice inoculated with 4T1-IL37b cells showed significant retardation of tumor growth. BALB/c mice inoculated with both 4T1 cells and mitomycin C-treated 4T1-IL37b cells also showed significant retardation of tumor growth. But the anti-cancer activity of IL-37b was abrogated in BALB/c nude mice and NOD-SCID mice inoculated with 4T1-IL37b cells. Recombinant IL-37b slightly promoted CD4(+) T cell proliferation without affecting CD8(+) T cell proliferation. CONCLUSION: IL-37b exerts anti-4T1 breast carcinoma effects in vivo by modulating the tumor microenvironment and influencing T cell activation.

[Interleukin-37 inhibits the activation of mouse dendritic cells induced by lipopolysaccharides].

OBJECTIVE: To investigate the role of interleukin-37 (IL-37) in regulating the activation of mouse dendritic cells (DCs) induced by lipopolysaccharides (LPS). METHODS: The mouse bone marrow cells was induced to differentiate into DCs with GM-CSF and IL-4. DCs were purified with anti-CD11c immunomagnetic beads. After pretreated with IL-37, DCs were stimulated by LPS. Then the levels of co-stimulatory molecules CD80 and CD86 on DCs were detected by flow cytometry. The mRNA levels of inflammation cytokines tumor necrosis factor α (TNF-α), IL-6 and IL-1α were determined by real-time quantitative PCR. The protein levels of inflammatory cytokines IL-1α, IL-6, TNF-α were measured by cytometric beads array (CBA) kit. RESULTS: We induced successfully DCs and obtained a high purity of DCs (>90%) with anti-CD11c immunomagnetic beads. After stimulated by LPS, the levels of co-stimulating molecules CD80, CD86 on IL-37-treated DCs were reduced, and the expressions of the cytokines TNF-α, IL-6 and IL-1α were also down-regulated at both mRNA and protein levels. CONCLUSION: IL-37 plays a critical role in inhibiting LPS-induced DCs activation via suppressing production of co-stimulatory molecules CD80, CD86 and pro-inflammatory cytokines.

[Mesenchymal stem cells promote mouse breast tumor progression by inducing the suppressive function of myeloid-derived CD11b⁺ Gr1⁺ cells].

OBJECTIVE: To investigate the role of mesenchymal stem cells (MSCs) in tumor progression by inducing immuno-suppressive function of myeloid-derived CD11b⁺ Gr1⁺ cells (BM-CD11b⁺ Gr1⁺ cells). METHODS: After co-cultured with MSCs, the immunophenotypes of BM-CD11b⁺ Gr1⁺ cells were tested by flow cytometry. The influence of MSCs on CD11b⁺ Gr1⁺ cells was evaluated by T cell proliferation assay after cultured with or without MSCs. The mouse 4T1 breast tumor model was established to explore the effect on tumor growth. RESULTS: MSCs promoted the survival of CD11b⁺ Gr1⁺ cells and induced the generation of CD11b⁺ Gr1⁺ cells from CD11b⁻ Gr1⁻ cells sorted from bone marrow. MSCs also enhanced the ability of BM-CD11b⁺ Gr1⁺ cells to suppress T cell proliferation and activation. CD11b⁺ Gr1⁺ cells after co-cultured with MSCs promoted 4T1 tumor growth and accelerated death of tumor-bearing mice. CONCLUSION: MSCs can promote tumor progression by inducing suppressive function of myeloid-derived CD11b⁺ Gr1⁺ cells from bone marrow.