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27 Microalgae and Immune Potential
Rathinam Raja and
Shanmugam Hemaiswarya
Key Points
Immunopotentiating activities have been found in whole cells such as bacteria, mush-•
rooms, algae, lichens, and higher plants.
Microalgae with its long history of food use, easy cultivation, and high nutritional •
content make it a valuable source for immunomodulating studies.
One of the main components that possesses the immunomodulating activity is the •
polysaccharides of the cell.
The basic mechanism of the immunostimulatory activity is through the stimulation •
of the macrophages and modulation of the complement system.
Key Words: Microalgae, immunomodulators, spirulina, immunoglobulin, allergy,
metabolic diseases.
27.1 INTRODUCTION
One of the most promising recent alternatives to classical antibiotic treatment is the
use of immunomodulators for enhancing host defense response (1). Several types of
immunomodulators have been identified including mammalian proteins such as inter-
feron gamma (IFN-g) (2), granulocyte colony-stimulating factor (3), and granulocyte
macrophage colony-stimulating factor (GM-CSF) (4), as well as substances isolated and
purified from microorganisms (1). Immunopotentiating activities have also been found
in whole cells like bacteria, mushrooms, algae, lichens, and higher plants. Microalgae
with its added advantages such as a long history of its food use, easy cultivation, and
high nutritional content make it a valuable source for immunomodulating studies.
Scientists are increasingly turning their attention to algae as ingredient factories, par-
ticularly the nutritional components. The omega-3 fatty acid DHA extracted from
marine algae is already in market, while several companies are offering the carotenoid
and astaxanthin (AX) from other algal sources. Spirulina, Chlorella, and Aphanizomenon
From: Dietary Components and Immune Function
Edited by: R.R. Watson et al., DOI 10.1007/978-1-60761-061-8_27
© Springer Science+Business Media, LLC 2010
Raja and Hemaiswarya
Fig. 27.1. (a–d) Morphological structures of some microalgae. (a) Spirulina sp. ×100. (b) Spirulina
sp. ×45. (c) Chlorella sp. ×100. (d) Haematococcus sp. ×100.
flos-aquae provide cellular protection with exceptional amounts of b-carotene (provita-
min A) and chlorophyll, whereas Dunaliella is the highest known natural source of
b-carotene (5–7). Some microalgae have favorable nutritional profiles for cancer and
immune therapies. Chlorella, the alga to emphasize stimulates immunity in the treat-
ment of all degenerative diseases by means of Chlorella Growth Factor (CGF).
27.2 SPIRULINA
The blue green alga (Cyanobacterium), Spirulina (Arthrospira) platensis Geitler
(Fig. 27.1a, b) has a soft cell wall made up of complex sugars and protein which is eas-
ily digestible. It has 62% amino acid which is a richest natural source of pseudo-vitamin
B
12
with (Vitamins B
1
and B
2
) and a whole spectrum of carotenoids and xanthophyll
phytopigments (8). Hence, a considerable attention has been paid for the cultivation of
Spirulina. Current world production of Spirulina for human consumption is more than
1,000 metric tons annually (9). The USA leads the world for Spirulina products in the
form of pills and spray-dried powder followed by China, India, Israel, Japan, Mexico,
Taiwan, and Thailand in the healthy food market (10). Phycocyanin (PC) extracted from
Spirulina, which is commercially known as lima blue, is used as a blue colorant for food
and cosmetics. The phycobiliproteins (phycoerythrins and phycocyanin) extracted from
Spirulina is used as fluorescence tag in connection with the detection of particular bio-
logical molecules viz., a fluorescent color marker coupled to antibiotics.
this figure will be printed in b/w
Chapter 27 / Microalgae and Immune Potential
Spirulina inhibits viral replication and strengthens cellular and humoral immune
system causing regression and inhibition of cancers (11). Several reports revealed that
the extract unique to Spirulina named as Calcium-Spirulan (a polymerized sugar mol-
ecule) inhibits replication of HIV-1, herpes simplex, human cytomegalovirus, influenza
A virus, mumps virus, and measles virus under in vitro conditions and are very safe to
human cells (12). Hamsters treated with this water soluble extract had better recovery
rates when infected with lethal herpes virus. A recent in vitro study showed in vitro that
an aqueous extract of S. platensis inhibited HIV-1 replication in human T-cells, periph-
eral blood mononuclear cells, and Langerhan cells (11). The advantage of using algal
products with proven antiviral properties in fighting certain viruses is that they can
immunomodulate even when an infection is developed. Studies on mice, hamsters,
chickens, turkeys, cats, and fish reveal that Spirulina consistently improves their
immune system functions. The rich brilliant blue polypeptide, Phycocyanin from
Spirulina, stimulates hematopoiesis and emulates the effect of hormone erythropoetin
(EPO). Phycocyanin also regulates the production of white blood cells, even when bone
marrow stem cells are damaged. Although early interest in commercial production of
Spirulina was focused mainly on its nutrient content, recent attention has been given to
its therapeutic properties such as antioxidant effects, immunomodulation, anticancer
potency, antiviral, and cholesterol regulatory properties.
27.3 IMMUNOMODULATION OF MACROPHAGES BY SPIRULINA
Monocytes and macrophages are phagocytes, acting in both nonspecific defense (and
innate immunity) or help to initiate specific defense mechanisms (or adaptive immunity)
in vertebrate animals. Their role is to phagocytose (engulf and then digest) cellular debris
and pathogens either as stationary or as mobile cells, and to stimulate lymphocytes and
other immune cells to respond to the pathogen. In response to antigen, macrophages
secrete mediators such as nitric oxide and cytokines. Cytokines play an important role in
the inflammatory cascade and include proinflammatory cytokines such as TNF, IL-1,
IL-8, and anti-inflammatory cytokines like IL-10 (13). The proinflammatory cytokines
are responsible for initiating an effect against exogenous pathogens and anti-inflamma-
tory cytokines are crucial for downregulating the incremented inflammatory process and
maintaining homeostasis for the correct functioning of vital organs.
Mice fed with microalgal diet showed an increase in the number of splenic antibody
producing cells in response to sheep red blood cells (SRBC). Spirulina enhanced the
macrophage functions and IL-1 production, but there was no change in the IgG-antibody
production (9). IL-1 is a proinflammatory cytokine that has numerous biological effects,
including activation of many inflammatory processes, induction of expression of acute-
phase proteins, an important function in neuroimmune response, and direct effects on
the brain itself (14). Similarly, in chickens fed with the Spirulina diet had macrophages
which exhibited enhanced phagocytic activity and increased nitric oxide synthase activ-
ity (13). The enhanced macrophage phagocytic function has also been shown in cats
(15), dogs (16) as well as in humans (17, 18) fed with Spirulina platensis extract. The
immunomodulatory action has been suggested by some researchers to be mediated
through the innate immune system. All the above studies used Spirulina as powder or
hot water extract and the active components considered were phycocyanin and water
Raja and Hemaiswarya
Table 27.1
Microalgal polysaccharides with immunomodulating activity
Microalgae Polysaccharides Effect References
Aphanizomenon flos-
aquae
Immunon
Increase NF-kB activa-
tion and IL-1b and
TNF-a signaling
(19, 22)
Chlorella pyrenoidosa Immurella (22, 23)
Spirulina platensis Immulina (22)
Chlorella stigmatophora Hydrosoluble
extracts
Anti-inflammatory, anal-
gesic and free radical
scavenging activities
(24)
Phaeodactylum tricor-
nutum
soluble polysaccharides. These components have been known to cause immuno-
modulation via increased proliferation of erythrocytes, granulocyte-monocyte, and
fibroblast lineage cells derived from bone marrow cells of mice (18).
A high molecular weight polysaccharide fraction (Immulina) from Spirulina was a
potent activator of NF-kB and induced both IL-1b and TNF-a mRNAs in THP-1 human
monocytes (19). Immulina dose-dependently increased the expression of chemokines,
namely, interleukin (IL)-8, MCP-1, MIP-1a, MIP-1b, IL-10 as well as the expression of
TNF-a, IL-1b, and COX-2. Thymidine uptake experiments verified that Immulina did
not affect the viability and growth rate of THP-1 cells. The activity in vitro was more
potent than the in vivo effects after oral administration (20). NF-κB activation by
Immulina is suppressed by antibodies to CD14 and TLR2 but not by antibodies to TLR4.
Similarly, NF-κB directed luciferase expression was enhanced by Immulina treatment
when cells were cotransfected with vectors expressing proteins supporting TLR2- (CD14
and TLR2) but not TLR4-(CD14, TLR4, and MD-2) dependent activation (21). In addi-
tion to Immulina, there are several other microalgae which produce polysaccharides with
immunomodulating activities especially on the macrophages (Table 27.1).
27.4 SPIRULINA AND CELL MEDIATED IMMUNITY
In mice, S. fusiformis (400 or 800 mg/kg body wt.) administration significantly inhib-
ited the humoral immune response, cell-mediated immune response delayed-type
hypersensitivity reaction (DTH) and TNF-alpha in a dose dependent manner (Fig. 27.2).
In vitro, S. fusiformis (50 or 100 mg/mL) decreased the mitogen (phytohemagglutinin)
induced T-lymphocyte proliferation in a concentration dependent manner when
compared with control cells. These observations clearly suggest that S. fusiformis has a
remarkable immunosuppressive effect which provides a scientific validation for the
popular use of this drug (25).
The number of hemagglutinating antibodies in serum was reduced after S. fusiformis
(400 or 800 mg/kg body wt.) dose-related treatment. During the cell-mediated immune
response, the sensitized T-lymphocytes, on being challenged with the antigen, secrete a
number of lymphokines. These lymphokines attract scavenger cells to the site of the
reaction, where they are then immobilized to promote defensive (inflammatory) reaction
(26). Therefore, the inhibition of inflammation observed in our present study indicates
that there might be an inhibition of release of lymphokines on S. fusiformis
Chapter 27 / Microalgae and Immune Potential
Fig 27.2. Hypothesized mechanism of immunomodulation.
administration. A moderate level of inflammatory mediators is essential for host sur-
vival from infection, whereas overproduction has deleterious effects. As a result, syn-
thesis of inflammatory mediators must be tightly governed. The cytokine TNF-a
induces the production of proinflammatory cytokines, active oxygen species, nitric
oxide and matrix metalloproteinases, thereby implicating TNF-a as a therapeutic target
for treating the pain, swelling, and progressive joint destruction caused by rheumatoid
arthritis (27). In the present study, the abundance of TNF-a in arthritic mice joints pro-
vides evidence of its involvement in the disease pathology, which is supported by stud-
ies demonstrating that neutralization of TNF-a leads to decreased production of other
inflammatory cytokines (28). The S. fusiformis significantly decreased the production
of TNF-a in adjuvant-induced arthritic mice in dose dependent manner.
IFN-g is a macrophage activating cytokine that promotes Th1 biased responses asso-
ciated with cell mediated immunity (29). The Th1/Th2 balance is critical in determining
whether an immune response is to be dominated by macrophage activation or by anti-
body production. An increased spleen cell production of IFN-g in mice fed with
Spirulina extract, suggests a shift toward Th1 type cell mediated immunity. It was also
indicated a Th1 bias (increased production of IFN-g) in mice fed with when Spirulina
(30) or when added to cultures of human peripheral blood mononuclear cells (increased
production of IFN-g). In addition, consumption of a hot water extract of Spirulina for
two months by human volunteers resulted in greatly enhanced production of IFN-g by
NK cells in response to IL-12 and IL-18 (17).
Raja and Hemaiswarya
27.5 ENHANCED IMMUNOGLOBULIN PRODUCTION BY Spirulina
Spirulina is known to immunomodulate by activating the IgA secretion. IgA
secreted at mucosal surfaces function to protect against various viral and bacterial
pathogens by its unique nature of agglutination of microorganisms, neutralization of
bacterial enzymes, toxins and inhibition of antigens (31). Several investigators have
observed an increased mucosal IgA response by long-term treatment with Spirulina
or its components or after antigen stimulation. Mice that consumed a chemically
defined chow mixed with an extract containing Immulina exhibited changes in sev-
eral immune parameters. The ex vivo production of IgA and IL-6 from Peyer’s patch
cells was enhanced two fold and (IFN-g) production from spleen cells was increased
four fold in immulina treated mice. Oral consumption of this polysaccharide can
enhance components within both the mucosal and systemic immune systems (21).
Ingestion of S. platensis by men for more than a year enhanced salivary S-IgA levels
while treatment for less than a year had no effect. Mice that ingested this extract
exhibited enhanced spleen cell IFN-g production ex vivo. Interestingly, the enhanced
production of IFN-g by spleen cells followed a time course similar to that exhibited
for production of IL-6 and IgA by Peyer’s patch cells. The similar time course may
indicate that a common cell type is initially targeted by the extract and is mediating
both the effects locally on Peyer’s patches as well as systemically on spleen cells.
The influence of Spirulina on IgA levels in human saliva and demonstrated that it
enhances IgA production, indicating an important role of microalga in mucosal
immunity (32).
27.6 Spirulina IN ALLERGY AND RHINITIS
It has been well documented that Spirulina exhibits anti-inflammatory properties by
inhibiting the release of histamine from mast cells (33, 34). In a recent randomized
double blind placebo-controlled trial (35), individuals with allergic rhinitis was fed
daily, either with placebo or Spirulina for 3 months. Peripheral blood mononuclear
cells were isolated before and after the Spirulina feeding and levels of cytokines [IL-4,
IFN- and IL-2] which are important in regulating immunoglobulin (Ig)E-mediated
allergy were measured. The study showed that high dose of Spirulina significantly
reduced IL-4 levels by 32%, demonstrating the protective effects of this microalgae
toward allergic rhinitis.
A Japanese team identified the molecular mechanism of the human immune capac-
ity of Spirulina by analyzing blood cells of volunteers with pre- and post-oral admin-
istration of hot water extract of Spirulina platensis. IFN-g production and Natural
Killer (NK) cell damage were increased after administration of the microalgal extracts
to male volunteers (17). In a recent double blind, placebo controlled study from
Turkey evaluating the effectiveness and tolerability of Spirulina for treating patients
with allergic rhinitis, Spirulina consumption significantly improved the symptoms
and physical findings compared with placebo, including nasal discharge, sneezing,
nasal congestion, and itching (36). It is well understood that deficiency of nutrients is
responsible for changes in immunity which manifests as changes in production of
T-cells, secretory IgA antibody response, cytokines, and NK-cell activity. The above
Chapter 27 / Microalgae and Immune Potential
studies suggest that Spirulina may modulate the immune system by its role in cover-
ing nutritional deficiencies.
27.7 PHYCOCYNANIN FROM Spirulina
Phycocyanin (PC), a water soluble protein pigment, is one of the major constitutes
of Spirulina platensis. It is in association with the outer face of the photosynthetic II
light harvesting apparatus. Some characteristics of PC make it well suitable for fluores-
cence analyses in flow cytometry, histochemistry, immunoassay, and detection of reac-
tive oxygen species (37). PC can be used as natural dyes and its medicinal and
pharmaceutical properties especially antioxidant (38), antitumor, and immunity boost-
ing effects are the focus of research (39) for a long time. Recent studies suggested that
PC inhibited cancer cell growth via the induction of apoptosis (40). It has been known
already that S. platensis is a good candidate for selenium (Se) enrichment and also
serves as a promi sing source of dietary Se supplementation (41, 42). However, very
limited information on the antiproliferative activity of selenium containing phycocyanin
(Se-PC) and the underlying mechanism is available. An influence of selenium enriched
phycocyanin on anaphylactic reaction severity and circulating antibody response against
model allergen – hen’s egg white ovalbumin was studied in rats. Rats receiving Se-PC
demonstrated significantly increased specific IgG response (43). Phycocyanin can pro-
mote the expression of CD59 protein, reduce the reproduction of Hela cells. With an
ascendance of phycocyanin concentration, the expression quantities of CD59 protein
and apoptosis inducing Fas protein increased and the multiplication activity of Hela
cells declined (44).
Human CD59 is a plasma membrane anchored glycoprotein containing a 4 kDa
N-linked carbohydrate chain that functions as an inhibitor of the CD5b-9 membrane
attack complex (MAC) of human complement (45, 46). Thereby, it restricts the cytolytic
activity of the CD5b-9 complex to protect human blood and vascular cells from autolo-
gous complement attack (47, 48). CD59 plays a pivotal role in regulating immunity and
suppressing the hyperacute rejection (HAR) in xenograft transplantation (49). It was
reported that CD59 has close relationship with the occurrence of hurt, inflammation,
and tumor (50, 51). PC has an antitumor activity, and probably it acts as a kind of mito-
sis depressor able to combine with the receptor of mitosis depressor on the surface of
tumor cells. Through the linkage of receptor and the activation of cell death promoting
protein kinases by complicated pathway, the transcription and expression of CD59 gene
was promoted. Meanwhile excessively expressed CD59 protein can induce Fas protein
expression on the surface of Hela cells and then combine with Fas antigen which can
activate death domain and activate the conduction of proapoptosis signal in tumor cells.
In the end, the proliferation of tumor cells is held back and cells go to die. The studies
provide us a new idea about the molecular mechanism of the antitumor activity of PC
(44). In another study, after two weeks the white blood cells (lymphocyte activity) of a
PC group were higher than the control group and higher than or equal to a normal group
without cancer. This suggests that PC raises lymphocyte activity (21). These results
imply that phycocyanin has anticancer activity and also strengthens the body’s resist-
ance through increasing general immunity. Experts deem that taking a small dosage of
PC daily can prevent generation of malignant cancer or can inhibit its recurrence (29).
Raja and Hemaiswarya
27.8 Chlorella
One of the most powerful algae against cancer is Chlorella (Fig. 27.1c) – an unicel-
lular, green algae containing the highest chlorophyll level per ounce of any plant, as
well as protein (nearly 58%), carbohydrates, all of the vitamins B, C, and E, amino
acids, enzymes, and rare trace minerals (9). In a study, lab mice were supplemented with
Chlorella for ten days, and injected the mice with three types of cancer. According to
Moss, over 70% of the Chlorella strengthened mice did not develop cancer while all of
the untreated mice died within twenty days. Research regarding Chlorella’s immune
boosting effect is not limited to animal studies. According to another study, 15 glioblas-
toma patients were treated with powdered and liquid Chlorella along with standard
chemotherapy or radiation therapy. Although glioblastoma patients normally display a
2 year survival rate of 10%, the 15 Chlorella treated patients exhibited a survival rate of
40%. This is only one of the many successful studies linking Chlorella to strengthened
immune response thus making Chlorella a necessary component of effective and well-
rounded cancer treatment. It acts as both a powerful nutrient and a detoxifying food.
An experimental study was designed to assess the effects of oral administration of
Chlorella protein hydrolysate (Cv-PH) on the recovery of both innate and specific
immune responses of undernourished mice (52). The facts in support of the hypothesis
are as follows: the treatment of starved mice with Cv-PH provided benefits in terms of
(a) hemopoiesis (recovery of bone marrow cellularity and the lymphocyte pool), (b)
macrophage activation and phagocytosing capacity, and (c) stimulation of both humoral
and cell immune functions such as antibody response and the reconstitution of delayed-
type hypersensitivity response. Different immune cell populations might be induced
after activation in the gut-associated lymphoid tissue. Chlorella and its hydrophilic
extracts have been shown to possess many physiological functions, including immune
system improvement, hypoglycemic effects, lowering hyperlipidemic state in high fat
fed animals, etc. However, lipophilic extract of Chlorella (LEC) is less appreciated in
terms of its physiological actions. In the concentration ranges that were devoid of cyto-
toxicty, LEC produced a dose dependent (between 0.25 and 0.0315 mg/mL) inhibition
on LPS-induced nitric oxide production. The study shows LEC effectively block LPS-
induced nitric oxide production, is through blockage of expression of iNOS mRNA.
Chlorella powder was tested in 118 in vitro enzyme assay systems. The powder
showed potent inhibitions of peptidase cathepsin S, thromboxane A (2) synthase, and
cyclooxygenase-2 in a dose concentration manner. Other activities observed were inhi-
bitions of tumor necrosis factor alpha converting enzyme, protein tyrosine phosphatase
(SHP-2), calpain, protein kinases, and protein tyrosine phosphatases. Chlorella powder
had no significant effect on cyclooxygenase-1 (53). These actions to inhibit cyclooxy-
genase-2 and thromboxane synthase could contribute to the purported anti-inflamma-
tory and antithrombotic effects of Chlorella.
27.9 haematoCoCCuS
The green alga, Haematococcus pluvialis (Fig. 27.1d) accumulates high amounts
of AX under adverse environmental conditions, and it is the world’s richest source
of astaxanthin (54). AX plays an important role in protecting the alga against UV
Chapter 27 / Microalgae and Immune Potential
light damage and photo-oxidation of the polyunsaturated AX. It exhibits strong free
radical scavenging activity, protects against lipid peroxidation and oxidative damage
of LDL-cholesterol, cell membranes, cells, and tissues. Several studies have clearly
shown the effectiveness of astaxanthin as a cancer preventive agent in rats and mice.
The effect of AX on colon cancer in male rats is well executed (55). A recent study
with rats indicated that astaxanthin is effective at ameliorating retinal injury, and it
is effective in protecting photoreceptors from degeneration. AX was also found to
easily cross the blood–brain barrier (unlike b-carotene) and did not form any crystals
in the eye (56).
Immune response cells are particularly sensitive to oxidative stress and membrane
damage by free radicals because they rely heavily on cell to cell communications via
cell membrane receptors. Furthermore, the phagocytic action of some of these cells
releases free radicals that can rapidly damage these cells if they are not neutralized by
antioxidants (57). AX significantly influences immune function in several in vitro and
in vivo assays using animal models. Other evidences also pointout to the immunomodu-
lating activity of astaxanthin on the proliferation and functions of murine immuno
competent cells (58). Finally, studies on human blood cells in vitro have demonstrated
enhancement by AX of immunoglobulin production in response to T-dependent stimuli.
AX increases the production of T-helper cell antibody and increases the number of
antibody secretory cells from primed spleen cells (59). The above study discovered the
effect of AX in the production of immunoglobulins in vitro by human blood cells and
found that it increases the production of IgA, IgG, and IgM in response to T-dependent
stimuli (60). Other studies performed in vivo using mice have shown the immunomodu-
lating action of AX and other carotenoids for humoral responses to T-dependent anti-
gens and suggested that the supplementation with carotenoids may be useful to restore
immune responses (61). In agreement with the above results, various foods and drinks
with added AX have been prepared to increase the immune response mediated by
T-lymphocytes and NK cells to alleviate or prevent the decrease of immunological func-
tions caused by stress (62). Due to its immunomodulating action, AX has also been
utilized as a medication for the treatment of autoimmune diseases such as multiple
sclerosis, rheumatoid arthritis, and Crohn’s disease (63).
Malnutrition induced by dietary restriction produces a series of metabolic changes
that lead to depression of immunocompetence and several studies have assessed the
effects of nutritional support on immunity (64). Algal protein hydrolysates possess
various biological activities and have been administered to patients with different
protein metabolic diseases (65), but there are no reports on its immunomodulating
properties. Immunomodulation by using natural products can provide an alternative
to conventional therapy for a variety of diseases, especially when the host defense
mechanism has to be activated under the conditions of impaired immune response or
when a selective immunosuppression is desired in situations like autoimmune disor-
ders. Though enhancement of immune function has been claimed by various natural
products, very few have been subjected to randomized clinical trails. Recently, novel
vaccine delivery system has been developed using single-celled alga, Chlamydomonas
reinhardtii. Microalgae in particular, Chlamydomonas have many features that are
desirable for vaccine delivery systems, including its ease for genetic manipulation,
Raja and Hemaiswarya
inexpensive to produce and nontoxic. The first antigen to be tested was the p57
antigen, the causing agent of bacterial kidney disease. It is caused by Renibacterium
salmoninarum and infects all wild and farmed salmonids. The vaccine was well
expressed in the algae and effects in the immune functions were noted in the immu-
nized animals such as fish and rabbits. Issues that remain to be addressed include
expression of the antigen, posttranslational modifications, antigen immunogenicity,
and effective means of production.
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Author Query
Chapter No.: 27
Query Details Required Author’s Response
AU1 Please provide text for “Conclusions and Perspectives.
