In Vitro Immuno-Modulatory Potentials of Purslane (Portulaca oleracea L.) Polysaccharides with a Chemical Selenylation

The soluble polysaccharides from a non-conventional and edible plant purslane (Portulaca oleracea L.), namely PSPO, were prepared by the water extraction and ethanol precipitation methods in this study. The obtained PSPO were selenylated using the Na2SeO3-HNO3 method to successfully prepare two selenylated products, namely SePSPO-1 and SePSPO-2, with different selenylation extents. The assay results confirmed that SePSPO-1 and SePSPO-2 had respective Se contents of 753.8 and 1325.1 mg/kg, while PSPO only contained Se element about 80.6 mg/kg. The results demonstrated that SePSPO-1 and SePSPO-2 had higher immune modulation than PSPO (p < 0.05), when using the two immune cells (murine splenocytes and RAW 264.7 macrophages) as two cell models. Specifically, SePSPO-1 and SePSPO-2 were more active than PSPO in the macrophages, resulting in higher cell proliferation, greater macrophage phagocytosis, and higher secretion of the immune-related three cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β. Meanwhile, SePSPO-1 and SePSPO-2 were more potent than PSPO in the concanavalin A- or lipopolysaccharide-stimulated splenocytes in cell proliferation, or more able than PSPO in the splenocytes to promote interferon-γ secretion but suppress IL-4 secretion, or more capable of enhancing the ratio of T-helper (CD4+) cells to T-cytotoxic (CD8+) cells for the T lymphocytes than PSPO. Overall, the higher selenylation extent of the selenylated PSPO mostly caused higher immune modulation in the model cells, while a higher polysaccharide dose consistently led to the greater regulation effect. Thus, it is concluded that the employed chemical selenylation could be used in the chemical modification of purslane or other plant polysaccharides, when aiming to endow the polysaccharides with higher immuno-modulatory effect on the two immune cells.

Effect of Poly(I:C) and melanoma B16-F10 on the immunophenotype of murine spleen cells

Introduction. It is known that the agonist of TLR-3 Poly(I:C), used as an adjuvant in a number of models of antitumor vaccines, causes inhibition of melanoma B16 growth, but the immunological aspects involved in this process have not been fully studied.The aim of the study was to evaluate changes of the immunophenotype of the spleen cells of C57BL / 6 mice caused by the tumor load and / or Poly(I:C), which is necessary for better understanding of the processes occurring during Poly(I:C) inhibition of melanoma B16-F10.Materials and methods. The immunophenotype of splenocytes of C57Bl / 6 mice was studied by flow cytometry asfollowing: the group 1 was a control (intact animals), the group 2 was mice with subcutaneously transplanted melanoma B16-F10, the group 3 was mice without a tumor treated with Poly(I:C) and the group 4 – mice with subcutaneously transplanted melanoma B16-F10 treated with Poly(I:C).Results. Median values of parameters such as the CD4 / CD8 immunoregulatory index, the percentage of CD69+ CD4+ and CD8+ T cells, the number of B and NK cells for the group of mice with melanoma treated with Poly(I:C) were between the values in the control group and in the group of mice with B16-F10. when comparing the results, the number of B and NK cells, the percentage of CD69+ on CD4+ and CD8+ T cells, their median in the group of mice with melanoma treated with Poly(I:C) was closer to the control than to the values obtained in the B16-F10 group and in the group of healthy mice receiving Poly(I:C). At the same time, we found that the total number of CD3+ cells, the number of naive CD4+ and CD8+ T cells was higher in the group of mice with melanoma treated with Poly(I:C) compared to all other groups.Conclusion. The analysis revealed the changes of the immunophenotype of murine spleen cells (CD4 / CD8, the percentage of CD69+ CD4+ and CD8+ T cells, the number of B and NK cells), which were affected by the tumor load and / or the administration of Poly adjuvant (I:C). Changes in the immunophenotype of murine splenocytes were associated with the tumor load and its size. It was also found that the splenocyte immunophenotype was affected by the repeated administration of Poly(I:C) during the tumor growth.

Deep-Sea Anemones Are Prospective Source of New Antimicrobial and Cytotoxic Compounds

The peculiarities of the survival and adaptation of deep-sea organisms raise interest in the study of their metabolites as promising drugs. In this work, the hemolytic, cytotoxic, antimicrobial, and enzyme-inhibitory activities of tentacle extracts from five species of sea anemones (Cnidaria, orders Actiniaria and Corallimorpharia) collected near the Kuril and Commander Islands of the Far East of Russia were evaluated for the first time. The extracts of Liponema brevicorne and Actinostola callosa demonstrated maximal hemolytic activity, while high cytotoxic activity against murine splenocytes and Ehrlich carcinoma cells was found in the extract of Actinostola faeculenta. The extracts of Corallimorphus cf. pilatus demonstrated the greatest activity against Ehrlich carcinoma cells but were not toxic to mouse spleen cells. Sea anemones C. cf. pilatus and Stomphia coccinea are promising sources of antimicrobial and antifungal compounds, being active against Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and yeast Candida albicans. Moreover, all sea anemones contain α-galactosidase inhibitors. Peptide mass fingerprinting of L. brevicorne and C. cf. pilatus extracts provided a wide range of peptides, predominantly with molecular masses of 4000–5900 Da, which may belong to a known or new structural class of toxins. The obtained data allow concluding that deep-sea anemones are a promising source of compounds for drug discovery.

In Vitro Immunomodulation of the Polysaccharides from Yam (Dioscorea opposita Thunb.) in Response to a Selenylation of Lower Extent

The immunomodulation of chemically selenylated polysaccharides has been attracting more attention recently, but the corresponding performance of the yam polysaccharides (YPS) with lower selenylation extent remains, thus far, unsolved. In this study, the YPS was selenylated with Na2SeO3 under acidic conditions generated by HNO3 to reach two lower selenylation extents, yielding two selenylated YPSs, namely SeYPS-1 and SeYPS-2 with selenium contents of 715 and 1545 mg/kg, respectively. The results indicated that YPS, SeYPS-1, and SeYPS-2 all had in vitro immuno-modulation when using RAW 264.7 macrophages and murine splenocytes as cell models. In detail, the three polysaccharide samples at dose levels of 5–160 μg/mL showed insignificant cytotoxicity to the macrophages and splenocytes with cell exposure times of 12–24 h, because of the measured values of cell viability larger than 100%. However, Na2SeO3 at dose levels of 1.3–3.25 μg/mL mostly caused obvious cytotoxic effects on the cells, resulting in reduced cell viability values or cell death, efficiently. The results demonstrated that, compared with YPS, both SeYPS-1 and SeYPS-2 at a lower dose level (5 μg/mL) were more active at promoting phagocytosis activity, increasing the CD4+/CD8+ ratio of the T-lymphocyte sub-population in the murine splenocyte, improving cytokine secretion, including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α in the macrophages, or increasing interferon-γ secretion, but suppressing IL-4 production in the splenocytes. Consistently, SeYPS-2 has more potential than SeYPS-1 at exerting these assessed bioactivities in the cells. Thus, we conclude that a chemical modification of YPS using trace element Se at a lower selenylation extent could bring about higher immunomodulatory activity towards macrophages and splenocytes, while selenylation extent of YPS is a critical factor used to govern the assessed activity changes of YPS.

Inhibition of TGF-β Signaling Suppresses Th17 Differentiation and Promotes Treg Numbers but does not Reduces Experimental Arthritis

ObjectivesTGF-β is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-β as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-β inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. MethodsMurine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. FACS analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction.ResultsSB-505124 potently reduced murine Th17 differentiation by decreasing Il7a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants. In vivo, SB-505124 reduced Th17 levels, while increased levels of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction.ConclusionsBlocking TGF-β signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, SB-505124 treatment does not suppress experimental arthritis.

Acetaminophen-Induced Liver Injury Exposes Murine IL-22 as Sex-Related Gene Product

Gaining detailed knowledge about sex-related immunoregulation remains a crucial prerequisite for the development of adequate disease models and therapeutic strategies enabling personalized medicine. Here, the key parameter of the production of cytokines mediating disease resolution was investigated. Among these cytokines, STAT3-activating interleukin (IL)-22 is principally associated with recovery from tissue injury. By investigating paradigmatic acetaminophen-induced liver injury, we demonstrated that IL-22 expression is enhanced in female mice. Increased female IL-22 was confirmed at a cellular level using murine splenocytes stimulated by lipopolysaccharide or αCD3/CD28 to model innate or adaptive immunoactivation. Interestingly, testosterone or dihydrotestosterone reduced IL-22 production by female but not by male splenocytes. Mechanistic studies on PMA/PHA-stimulated T-cell-lymphoma EL-4 cells verified the capability of testosterone/dihydrotestosterone to reduce IL-22 production. Moreover, we demonstrated by chromatin immunoprecipitation that testosterone impairs binding of the aryl hydrocarbon receptor to xenobiotic responsive elements within the murine IL-22 promoter. Overall, female mice undergoing acute liver injury and cultured female splenocytes upon inflammatory activation display increased IL-22. This observation is likely related to the immunosuppressive effects of androgens in males. The data presented concur with more pronounced immunological alertness demonstrable in females, which may relate to the sex-specific course of some immunological disorders.

Inhibition of TGF-β Signaling Suppresses Th17 Differentiation and Promotes Treg Numbers but Does Not Reduce Experimental Arthritis.

ObjectivesTGF-β is an important growth factor to promote the differentiation of T helper 17 (Th17) as well as regulatory T cells (Treg). Due to its dual role, the potential of TGF-β as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. In this study, we investigated the effect of TGF-β inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. MethodsMurine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 on Th17 differentiation was studied. RA synovial biopsies were cultured for 24h in the presence or absence of SB-505124. Experimental arthritis models were induced in C57Bl6 mice, and were treated daily with SB-505124. FACS analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction.ResultsSB-505124 potently reduced murine Th17 differentiation by decreasing Il7a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by RA synovial explants. In the Th17-driven arthritis model, SB-505124 reduced Th17 levels, while increased levels of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction in this model.ConclusionsBlocking TGF-β signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, SB-505124 treatment does not suppress experimental arthritis, and is therefore not an adequate way to target Th17-driven inflammation.

Abstract MP52: Hypertensive Stimuli Indirectly Stimulate Mouse Mesometrial Lymphangiogenesis Through Immune Cells

We previously reported increased renal lymphatic density in multiple mouse models of hypertension, and further augmenting renal lymphatics lowers blood pressure. However, whether interstitial levels of hypertensive stimuli have a direct effect on lymphatics or an indirect effect through secreted immune cell factors has not been examined. We hypothesized that hypertensive stimuli directly increases lymphatic endothelial cell (LEC) proliferation and increases sprouting of mouse mesometrial lymphatic vessels. Murine LECs were cultured and treated with angiotensin II (angII), salt, and asymmetric dimethylarginine (ADMA) for 24 hours. To mimic the in vivo environment, a lymphatic-specific reporter mouse (Prox1-tdTomato) mesometrium tissue explant was treated with either the same hypertensive stimuli or with hypertensive conditioned media for 8 days. Mesometrial vascular beds were cultured in DMEM supplemented with 20% fetal bovine serum to induce lymphatic sprouting and this was replenished every day. The conditioned media was made by treating murine splenocytes for 24 hours with the same hypertensive stimuli. These stimuli had no effect on murine LEC proliferation. Hypertensive stimuli significantly decreased mesometrial lymphatic vessel sprout length (SL) and sprout number (SN) compared to controls (control SL in pixels by ImageJ analysis: 34.0 ± 2.6, angII: 3.7 ± 2.6, salt: 2.67 ± 2.18, ADMA: 9.06 ± 5.12, all p<0.05; control SN: 7 ± 3, angII: 0 ± 0, salt: 0 ± 0, ADMA: 1 ± 1, all p<0.05). Conditioned media treatment normalized SL and SN by day 8 for all hypertensive stimuli except salt. In conclusion, hypertensive stimuli directly inhibit mesometrial lymphangiogenesis, but this was mitigated by hypertensive stimuli induced immune cell secreted factors.

Lipofection with Synthetic mRNA as a Simple Method for T-Cell Immunomonitoring

The quantification of T-cell immune responses is crucial for the monitoring of natural and treatment-induced immunity, as well as for the validation of new immunotherapeutic approaches. The present study presents a simple method based on lipofection of synthetic mRNA in mononuclear cells as a method to determine in vitro T-cell responses. We compared several commercially available transfection reagents for their potential to transfect mRNA into human peripheral blood mononuclear cells and murine splenocytes. We also investigated the impact of RNA modifications in improving this method. Our results demonstrate that antigen-specific T-cell immunomonitoring can be easily and quickly performed by simple lipofection of antigen-coding mRNA in complex immune cell populations. Thus, our work discloses a convenient solution for the in vitro monitoring of natural or therapy-induced T-cell immune responses.