Objectives 1) To evaluate the changes in inflammatory response induced by sorghum polyphenols in activated macrophages and 2) to evaluate possible efficacy of sorghum polyphenols on an opportunistic intracellular pathogen, Legionella pneumophila (LP). Methods Raw 265.7 cells mouse macrophage cells were treated with sorghum phenolic extract (SPE) under control and activating conditions to evaluate the role of SPE in inflammatory and anti-inflammatory processes. Study measured: nitric oxide production in the supernatant, mRNA using qPCR array of 84 genes in activated macrophages. Morphological changes were observed, and LC3 protein expression was measured to test for autophagy using western blot. NF-kB and STAT3 nuclear translocation was measured using a fractionization kit, followed by western blot. The replication of LP was measured within RAW 264.7 cells and in vitro (in media without cell presence). Cytotoxicity assay and a western blot apoptosis marker caspase-3 were used to evaluate the cytotoxicity of SPE on RAW 264.7 cells. Because LP reproduction within cells is greatly attenuated in the presence of Tumor Necrosis Factor (TNF), LP replication was measured under the presence of TNF neutralizing antibodies. Results SPE decreased nitric oxide production in activated LPS/IFNΥ macrophages although not significantly. SPE attenuated Th2 cytokine response in LPS/IFNΥ activated macrophages by decreasing expression of IL-6 and IL-10 while not changing expression of other inflammatory cytokines. Quantitative PCR data confirmed that genes in the IL-10 and IL-6 pathway were downregulated by cotreatment of SPE and LPS/IFNΥ when compared to LPS/IFNΥ alone. Morphological changes observed exhibited formation of large vacuole like structures. Analysis of LC3 confirmed that autophagy was increased in activated cells treated with SPE. Nuclear fractionization confirmed that STAT3 signaling was attenuated by SPE in activated macrophages. SPE significantly reduced the replication of LP in macrophage cells but not in vitro. The attenuation of LP grown in RAW 264.7 cells was independent of TNF presence. Conclusions This data suggests that sorghum phenolic compounds may have potential pharmaceutical/nutritional uses to combat intracellular pathogens. Funding Sources All funding was provided by the United States Department of Agriculture.
Objectives The objective was to evaluate the potential anti-inflammatory effects of Sorghum polyphenolic extracts on raw 264.7 cells. Methods Sorghum polyphenols were extracted using a 70% ETOH and 5% citric acid solvent. Raw 264.7 cells were treated with either vehicle 1.25, or 0.625 mg/ml polyphenol extract from either a novel high polyphenol sorghum or SC84. Supernatant was harvested and Nitric Oxide was measured at a 12 hour time point. ELISA assay was performed to measure the concentrations of 12 anti-inflammatory associated cytokines. Cell morphology changes were observed at 3, 6, 12, and 24 hours using light microscopy. 84 genes associated with inflammation were measured via QPCR. Western blot analysis measured the expression of LC3 as well as STAT1, STAT3 and NF-kB nuclear translocation. Results Nitric Oxide was reduced by the sorghum extract (not significant). Cell morphology changed by developing vacuole like structures and an apparent decrease in cell number. ELISA analysis showed that Il-6 and Il-10 were significantly reduced in all treatments. SC84 extract showed an increase in G-CSF production in activated macrophages. QPCR revealed LPS and IFNY activated cells treated with HP extract showed an increase in the expression of 9 and decreased expression of 14 cytokine related genes compared to cells that had only been activated by LPS and IFN Y. When LPS and IFN Y activated cells were cotreated with SC84 extract, 15 cytokine related genes were upregulated and 16 cytokine related genes were downregulated. LC3 expression was measured via western blot and showed a dose dependent with 1.25 mg/ml showing statistical significance. STAT3 nuclear translocation induce by LPS/IFN Y was attenuated by sorghum polyphenols. Conclusions The sorghum polyphenols modulated immune response via a reduction in Th2 promoting cytokines IL-6 and IL-10. LC3 II expression increased with the concentration of the HP polyphenol extract treatment, suggesting autophagy. Funding Sources All funding was provided by the United States Department of Agriculture.
Polyphenols derived from a variety of plants have demonstrated antimicrobial activity against diverse microbial pathogens. Legionella pneumophila is an intracellular bacterial pathogen that opportunistically causes a severe inflammatory pneumonia in humans, called Legionnaires’ Disease, via replication within macrophages. Previous studies demonstrated that tea polyphenols attenuate L. pneumophila intracellular replication within mouse macrophages via amplification of tumor necrosis factor (TNF) production. Sorghum bicolor is a sustainable and resilient cereal crop that thrives in arid environments and is well suited to continued production in warming climates. Polyphenols derived from sorghum have anticancer and antioxidant properties, but their antimicrobial activity has not been evaluated. Here, we investigated the impact of sorghum polyphenols on L. pneumophila intracellular replication within RAW 264.7 mouse macrophages. We discovered that sorghum high-polyphenol extract (HPE) treatment attenuates L. pneumophila intracellular replication in a dose-dependent manner. Sorghum HPE did not impair bacterial replication in vitro or impact macrophage viability. Moreover, in contrast to tea polyphenols, HPE treatment impaired TNF secretion from infected macrophages. Thus, polyphenols derived from sorghum enhance macrophage restriction of L. pneumophila by a novel mechanism. This work provides a foundation for the use of sorghum as an antimicrobial agent.
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