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Evidence supports the theory that bacterial communities colonizing Echinacea purpurea contribute to the innate immune enhancing activity of this botanical. Previously, we reported that only about half of the variation in in vitro monocyte stimulating activity exhibited by E. purpurea extracts could be accounted for by total bacterial load within the plant material. In the current study, we test the hypothesis that the type of bacteria, in addition to bacterial load, is necessary to fully account for extract activity. Bacterial community composition within commercial and freshly harvested (wild and cultivated) E. purpurea aerial samples was determined using high-throughput 16S rRNA gene pyrosequencing. Bacterial isolates representing 38 different taxa identified to be present within E. purpurea were acquired, and the activity exhibited by the extracts of these isolates varied by over 8000-fold. Members of the Proteobacteria exhibited the highest potency for in vitro macrophage activation and were the most predominant taxa. Furthermore, the mean activity exhibited by the Echinacea extracts could be solely accounted for by the activities and prevalence of Proteobacteria members comprising the plant-associated bacterial community. The efficacy of E. purpurea material for use against respiratory infections may be determined by the Proteobacterial community composition of this plant, since ingestion of bacteria (probiotics) is reported to have a protective effect against this health condition.

Our previous studies indicate that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea depends on bacterial components. In the present study, total bacterial load was determined within E. purpurea samples and ranged from 6.4 × 10(6) to 3.3 × 10(8) bacteria/g of dry plant material. To estimate total bacterial load, we developed a PCR-based quantification method that circumvents the problems associated with nonviable/nonculturable cells (which precludes using plate counts) or the coamplification of mitochondrial or chloroplast DNA with the use of universal bacterial primers (which precludes the use of qPCR). Differences in total bacterial load within Echinacea samples were strongly correlated with the activity (NF-κB activation in THP-1 cells) and content of bacterial lipopolysaccharides within extracts of this plant material. These results add to the growing body of evidence that bacteria within Echinacea are the main source of components responsible for enhancing innate immune function.

Previous studies indicate that Immulina®, a commercial extract of Arthrospira (Spirulina) platensis, is a potent activator of innate immune cells and that bacterial Braun-type lipoproteins (a principal toll-like receptor 2 ligand) are the main active components within this product. In the present study, mice fed Immulina® for 30 days prior to infection with influenza A (H1N1) virus exhibited a statistically significant reduction in the severity of infection. Compared to the control group, Immulina®-fed mice exhibited more appetite, reduced weight loss, diminished clinical signs of disease and lower lung histopathology scores. This study adds to the growing body of evidence that oral administration of bacterial components that activate immune cells, whether derived from a bacterial preparation (probiotics or cyanobacterial extracts) or from bacteria associated with plants as endophytes, can exhibit a protective effect against influenza A (H1N1) viral infection. Acknowledgements: This research was funded by Scandinavian Clinical Nutrition AB (Stockholm, Sweden). NDP and DSP were partially supported by the USDA, Agricultural Research Service Specific Cooperative Agreement Nos. 58 – 6408 – 6-067 and 58 – 6408 – 1-603 (NCNPR/University of Mississippi).

This research describes the identification of three new high molecular weight polysaccharide preparations isolated from food-grade microalgae that are potent activators of human monocytes/macrophages: "Immulina" from Spirulina platensis, "Immunon" from Aphanizomenon flos-aquae, and "Immurella" from Chlorella pyrenoidosa. These polysaccharides are structurally complex and have estimated molecular weights above ten million daltons. All three polysaccharides are highly water soluble and comprise between 0.5 % and 2.0 % of microalgal dry weight. Immunostimulatory activity was measured using a transcription factor-based bioassay for nuclear factor kappa B (NF-kappa B) activation in THP-1 human monocytes/macrophages. Using this system the EC(50) values for these microalgal polysaccharides are between 20 and 110 ng/ml (about 10pM). THP-1 activation was confirmed by measuring immune cytokine mRNA induction using reverse transcriptase-polymerase chain reaction (RT-PCR). Each polysaccharide substantially increased mRNA levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). These polysaccharides are between one hundred and one thousand times more active for in vitro monocyte activation than polysaccharide preparations that are currently used clinically for cancer immunotherapy.

We have found that the bacterial components lipopolysaccharides (LPS) and Braun-type lipoproteins are responsible for the majority of the in vitro macrophage activation exhibited by extracts of Echinacea and other immune enhancing botanicals. Furthermore, we show that endophytic bacteria are the principal source of these components. We also show that total bacterial load within Echinacea samples are strongly correlated with the innate immune stimulatory activity (NF-κB activation in THP-1 cells) and content of bacterial LPS within extracts of this plant material. Differences in the levels of these two bacterial components are responsible for the substantial variation observed (˜200-fold) in innate immune cell activation from E. purpurea material obtained from six major growers/commercial suppliers in North America. This substantial variability in bacterial component content/activity could be a factor contributing to the inconsistent outcomes exhibited in Echinacea clinical trials. We also demonstrate that the oral consumption of extracts rich in these bacterial components is therapeutically relevant with respect to enhancing immune parameters and for impacting disease outcomes in both animal models and in humans. Acknowledgements: This research was partially funded by grants from the National Institutes of Health R21AT01207, R01AT002360 and R01AT007042 (NCAAM) to DSP and by the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58 – 6408 – 02 – 1-612.

Immulina®, a commercial extract of Arthrospira (Spirulina) platensis is a potent activator of THP-1 monocytes and CD4+ T cells IN VITRO and enhances several immunological functions in mice. We further characterized Immulina® by determining that Braun-type lipoproteins are responsible for a major portion of the IN VITRO monocyte activation exhibited by this material. In order to understand the effect of Immulina® on NK cell activity, a pilot study was conducted on ten healthy North American individuals who supplemented their diet with Immulina® (400 mg/day) for seven days. We observed a 40% average increase in the killing of K562 tumor cells by NK cells (p 0.01) after Immulina® supplementation. In a separate placebo-controlled, crossover study involving 11 healthy Danish subjects, we observed increased mRNA expression of the NK cell marker NKG2D by 37% (p = 0.02) and by 55% (p = 0.0003) after administration of Immulina® (200 mg and 400 mg per day, respectively) for seven days. The mRNA expression of the NK- and T-cell marker perforin increased by 75% (p = 0.008) after administration of 400 mg Immulina® per day. Both markers displayed significant dose-dependent effects (p = 0.0003 and p = 0.02, respectively). The ratio between CD56 (bright) and CD56 (dim) NK cells was not affected by Immulina® administration. In summary, two independent studies showed enhancement of NK cell activity following administration of Immulina® for seven days.

Immulina is a commercial extract of Spirulina (Arthrospira) platensis that is standardized by biological activity. We previously reported that this extract is a potent activator of THP-1 monocytes in vitro and that oral consumption enhanced several immunological functions in mice [1]. In this study we further characterized Immulina by determining that Braun type lipoproteins are responsible for a major portion of the in vitro monocyte activation exhibited by this material. In order to understand the effect of Immulina on the human immune system, a pilot study was conducted on ten healthy individuals who supplemented their diet with Immulina (400 mg/day) for seven days. Blood was drawn from the participating individuals at two time points: before and after seven days of Immulina supplementation. Changes in mononuclear and polymorphonuclear phagocytosis were determined in heparinized whole blood as well as the cytotoxicity exhibited by natural killer (NK) and lymphokine activated killer cells. We observed statistically significant increases both in tumor cell killing by NK cells (p = 0.0019) and in phagocytosis by blood mononuclear cells (p = 0.0124) after Immulina supplementation. Acknowledgements: This research was partly funded by a USDA, Agricultural Research Service Specific Cooperative Agreement No. 58-6408-012. Immulina capsules were supplied by Scandinavian Clinical Nutrition Denmark A/S, Greve, Denmark. References: [1] Balachandran P, et al. (2006), Inteternational Immunopharmacology, 6: 1808–1814.