Beneficial immunostimulatory effect of short-term Chlorella supplementation: enhancement of Natural Killercell activity and early inflammatory response (Randomized, double-blinded, placebo-controlled trial)

Chlorella is an unicellular green algae which contains essential amino acids, protein, minerals, vitamins, dietary fiber, and a wide range of antioxidants, bioactive substances and chlorophylls etc. [1, 2]. It has been a popular foodstuff worldwide, especially in Asian countries i.e. Japan, Taiwan as well as Korea.

Various pharmacological effects of Chlorella have been expressed not only in animal models [3, 4] but also in human experiments [5‐7]. According to Okudo et al [5], the blood cholesterol levels were reduced in hypercholesterolemic patients who consumed Chlorella. Fujiwara et al [6] also showed a beneficial effect of Chlorella consumption on hyperlipemia. Nakamura et al demonstrated that Chlorella intakes reduced blood pressure in mildly hypertensive people [7]. Chlorella supplementation for 6-week, brought a favorable impact on antioxidant status in male smokers [8]. In addition, Chlorella showed a considerable antioxidant effect and a reduction of blood glucose levels in diabetic animal models and stress-induced ulcer mice [9‐15].

In fact, in vitro and animal studies have demonstrated that Chlorella or Chlorella extract are involved in the modulation of immune responses against tumors [16‐18], bacterial and viral infection [19‐23] as well as native Ags i.e. casein [24]. It has been reported that oral administration of Chlrorella extract enhanced the resistance to infection with Listeria monocytogenes, an intracellular bacterium, through the augmentation of T-helper-1 (Th1) cell response both in normal and immunocompromised hosts [21, 22]. However, there were no direct evidences on the effect of Chlorella supplementation on immune response in humans, particularly in uninfected normal people. Therefore, this study aimed to investigate if Chlorella supplementation gives a beneficial immunostimulatory effect to uninfected normal people.

Among the enrolled subjects (n=60), 9 subjects dropped out and 51 subjects completed the study. Among the 9 drop-outs, 7 were in the placebo group (2 for personal problems, 5 for no good-compliance on tablets consumption) and 2 (for personal problem) were in Chlorella supplement group. No serious adverse reactions due to Chlorella supplementation were noted and the compliance was 85%. Participants were supplied with 360 tablets of placebo or Chlorella at 0-week and at 4-week respectively. They were also asked to return unconsumed tablets at the next visit (4- and 8-week respectively). All the participants were reinforced to regularly consume the tablets by a dietitian through every 2 week’s phone-call check during the intervention period.

This present study shows that 8-week supplementation of Chlorella tablets may give a beneficial immunostimulatory effect to normal (uninfected) people by enhancing the NK cell activity and producing INF-γ and IL-12 as well as IL-1β, the Th-1 cell-induced cytokines.

Helper T lymphocytes (Th) are divided into two functional subclasses, Th-1 and Th-2 cells based upon the cytokines that they produce and their effects on cell mediated and humoral immunity [28]. Th-1 cells produce IL-2, INF-γ, TNF-α and IL-12 and enhance cell-mediated immunity, thereby inhibiting cell-mediated immunologic activities. On the other hand, Th-2 cells produce IL-4, IL-5, IL-6 and IL-10, and upregulate humoral immunity. Th-1 and Th-2 derived cytokines also cross-regulate each other in various clinical conditions. Hasegawa et al reported in his mice model infected with Listeria monocytogenes that Chlorella extract (from Chlorella vulgaris, CVE) augmented IL-12 and IFN-γ [29], both of which promote the differentiation of naive CD4+ T cells into Th-1 cells producing IFN-γ, but inhibited the generation of Th-2 cells producing IL-4. In his other report, oral administration of CVE in mouse model inhibited immunoglobulin E (IgE) production against casein together with an impaired Th-2 response [11]. Ewartet al shows that Chlorella extract (from Chlorella pyrenoidosa, CPE) induces a Th-1 patterned cytokine response (IFN- γ and TNF-α) and a strong anti-inflammatory regulatory cytokines response (IL-10) in human PBMC stimulated ex vivo[30]. In our study, subjects who consumed Chlorella tablets for 8 weeks had a significant increase in serum levels of INF-γ and an increasing tendency in the levels of IL-12 compared with those who consumed placebo, which indicates that Chlorella supplementation may induce the production of INF- γ and IL-12 favoring a Th-1 mediated immune reaction in humans.

We also found the increased levels of serum IL-1β which is a member of the IL-1 cytokine family and produced by activated macrophages [31]. This cytokine is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis [31]. IL-1β stimulates multiple cells to act as immune or inflammatory response effector cells. Hunter et al[32] reported in his severe combined immuno-deficient mice models that IL-1β is required for IL-12 to stimulate production of IFN-γ by NK cells [32]. According to Tominaga et al[33], IL-12 and IL-1β synergistically induce T cells to proliferate and produce the IFN-γ and IL-12-stimulated T cells which responded to IL-18 or IL-1β by their proliferation and IFN- γ production, although the levels of IL-1β-induced responses were lower. Our result may be partially in accordance with the report of Ishikawa et al showing that a polysaccharide-rich fraction from Chlorella pyrenoidosa induced IL-1β and TNF-α in macrophage cells [34]. Taken together, Chlorella cells appear to contain immunostimulatory principles that stimulate a Th-1 based response. However, we did not measure the Th-2 derived cytokines such as IL-4 or IL-6, thus we may need to confirm these cytokines in further studies.

In our study, people who consumed Chlorella had a significant increase in Natural Killer (NK) cell activities. This result might be partly in accordance with the report of Dantas et al showing that oral administration of Chlorella extract (CVE) significantly increased the NK cell activity in normal (non-infected) mice as well as in the Listeria monqtogenes infected mice [35]. In addition, the CV-treated animals presented a dose-related increased survival rate. NK cells were first identified by their cytotoxtc activity against tumor cells, suggesting a role in immunological surveillance against neoplasia. Emerging evidences demonstrated that NK cells are important mediators of innate resistance against a variety of pathogenic intracellular microorganisms [36]. Their main function is the production of early IFN-γ, which is crucial to activate antimicrobial macrophage functions [37‐42]. The kinetics of IFN-γ production by NK cells following infection is extremely fast, providing a source of functional cytokine at the critical time of the T-cell expansion [39, 43, 44]. In our study, changed level of NK cell activity after the intervention shows a significant positive relationship with that of serum IFN-γ. Signficanty strong positive correlations were also observed among the changed levels of IFN-γ, IL-12 and IL-1β, Th-1 patterned cytokines.

We may need to consider further study with increased number of study subjects and for longer period in the future, to confirm and clarify the result pattern. As shown in the Figures, serum levels of INF- γ and IL-12 were significantly decreased in the placebo group after 8 weeks, even though the net change of these cyotkines were significantly bigger in the Chlorella group than in the placebo group. We tried to search the factors which might affect the decrease of these parameters in the placebo group, but it is not easy to address the reason in a word. First, we thought the possibility of lactose, the contents in the placebo affecting cytokine levels, but it seemed not influence the cytokine levels. Second, there were no significant difference in the baseline value between the two groups, and the net change values were generally much greater in the Chlorella group than those in the placebo group, even though the direction for increment or decrement were not the same between the two groups. However, the correlation pattern among NK cell activity and cytokines were consistently in the positive direction, which may indicate that the measurements and the results are reliable.

This study specifically focused on a normal healthy (non-infected) Koreans, so the results cannot be generalized to patients, other ethnic or geographical groups whose biochemical characteristics may differ from those in our subjects. Despite these limitations, 8-weeks of Chlorella intake in healthy Koreans increased the NK cell activity and produced INF-γ and IL-12 as well as IL-1β, the Th-1 cell-induced cytokines. In addition, changes in the NK cell activity positively correlated with those in the cytokines after the intervention. These results add to the growing literature on the beneficial immunostimulatory effect of Chlorella supplementation through a clinical human study.