Immunomodulatory Effects of Modified Bovine Colostrum, Whey, and Their Combination with Other Natural Products: Effects on Human Peripheral Blood Mononuclear Cells

Highlights • Folk medicine used natural products for the prophylaxis and treatment of infectious diseases for centuries. A few decades ago, we learned that many natural products have immunomodulatory properties. However, the mechanism of immunomodulatory activities was not clear.• In this study, we demonstrate that tested natural product blends modulate the activity of both cells of innate and acquired arms of the immune system. Most of the studied products suppressed the production of inflammatory cytokines by activated PBMCs. The ultrafiltrate colostrum, however, stimulated the production of inflammatory cytokines by LPS-activated PBMCs and suppressed their production by PHA-activated cells.


Introduction
The immune system's primary function is the protection of an organism from infectious agents. 1 The immune system is also involved in maintaining tissue integrity, wound repair, and supporting and regulating the metabolism and functions of different organs and systems. 2 Therefore, support of an adequately balanced immune system function is crucial for maintaining the well-being of people.
4][5] Zinc deficiency, for example, is associated with a reduction of the functions of several immune Bovine colostrum and whey are 2 popular natural products that often undergo manufacturing changes to optimize their immune health benefits.Bovine colostrum may be defatted and ultrafiltered to concentrate the smaller peptides and proteins. 10Whey is usually hydrolyzed and enzymatically digested into bioactive peptides. 11][14] The study presented herein evaluates the effects of colostrumbased natural products, whey extract, and their combinations with egg yolk extract and mushroom extracts on the ability to modulate the immune activity of peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells
Human Peripheral Blood Leukopak was purchased from Stem-Cell Technologies (Vancouver, British Columbia, Canada).The protocol and donor consent form for blood cell collection was approved by our facilities institutional review board. 15The typical Leukopak contains ∼27% CD4 + T cells, ∼16 CD8 + T cells, 8.5% B cells, and ∼26% mono-and granulocytes. 15Collected cells were free of bloodborne infections.The human peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll gradient procedure, 16 washed, and then cryopreserved.
Study products were solubilized in dimethyl sulfoxide (DMSO) and further diluted to the assay concentrations with a complete medium (CM) containing RPMI 1640, 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L-glutamine.The final concentration of DMSO in the culture was 0.1%.

Cell viability assay
AlamarBlue reagent at a 1:10 dilution was added to PBMCs treated with lipopolysaccharide (LPS) or phytohemagglutinin (PHA), or the test compounds plus LPS or PHA (see details below).Because the test products were diluted in DMSO, AlamarBlue reagent was also added to the wells of untreated cells containing 0.1% DMSO to establish baseline cell viability (baseline control).After 4-hour incubation, plates were read on a fluorescence plate reader at 530 nM (530-570 nM filter) for excitation and 595 nM (580-610 nM filter) for emission.Relative fluorescence units were measured.Cell viability was presented as a percentage of control values using the following equation:

Mean relative fluorescence value of test sample × 100
Mean relative fluorescence of baseline control

Cytokine Production by LPS-activated PBMCs
Cryopreserved human PBMCs were drip-thawed, washed in CM, and plated in triplicates at the cell density of 2 × 10 5 cells/well in 150 μL CM into 96-well polystyrene plates (Corning, Glendale, Arizona).Study products were then added to the cells (10 μL) to the final concentrations of 2 μg/mL or 20 μg/mL and incubated in a humidified atmosphere at 37 °C, 5% carbon dioxide for 1 hour.Some cells were cultured in the presence of dexamethasone (Dex), a broad-spectrum anti-inflammatory drug used as a reference control.Dex was added in a volume of 10 μL to a final concentration of 100 nM. 17After the initial 1-hour incubation, PBMCs were stimulated with LPS Escherichia coli O111:B4 (Millipore Sigma, Burlington, Massachusetts) at the final concentration of 50 pg/mL. 18LPSactivated PBMCs untreated with the products were used as a positive control.Plates were incubated in a humidified atmosphere at 37 °C and 5% carbon dioxide for 24 hours.Then, plates were centrifuged at 200 g for 10 minutes; supernatants were collected and stored at -80 °C for later Luminex multiplex cytokine analysis.(EMB Millipore Sigma, Burlington, MA, USA).
Luminex assessment of cytokine levels (human IL-1 β, IL-6, and IL-8, and TNF α) in cell culture supernatants was performed per the manufacturer's protocol using the Human Cytokine/Chemokine Magnetic bead panel from EMD Millipore Sigma (St Louis, Missouri).The samples were diluted 1:8 based on the vendor-specified recommendations.The obtained cytokine values were multiplied by the dilution factor to obtain the final cytokine concentrations per milliliter of the supernatant.

Cytokine Production by PHA-activated PBMCs
The PBMC culture was set up as described above.After the initial 1-hour incubation with the study products or Dex, PHA was added to the culture at a final 10 μg/mL concentration.PHAactivated PBMCs were used as a control.Cell culture supernatants were collected after 48 hours and stored at -80 °C for later Luminex multiplex cytokine analysis. 19 , 20All samples were diluted based on the vendor-specified dilution recommendations (1:2 dilution for IL-1 β, IL-5, IL-10, and IL-13; and 1:8 dilution for IFN γ , and TNF α).The obtained cytokine values were multiplied by the dilution factor to obtain the final cytokine concentrations per milliliter of the supernatant.

Data analysis
Levels of cytokine in the supernatants were calculated as concentrations based on a 5-point standard curve, a nonlinear regression analysis.Outlier calculations were performed using the Grubbs' Maximum Normed Residual Test available in GraphPad Prism version 8.0 (GraphPad Inc, La Jolla, California).All data are presented as the average of 3 replicates (SD).Due to the moderate cytotoxicity of some test products, the cytokine levels were recalculated per 10 0,0 0 0 viable cells.Statistical differences were calculated by ANOVA test in GraphPad Prism version 8.0.

Cell viability
The analysis of the cytotoxic effects of the study products and other chemicals used in the cell culture is present in Table 2 .When the cytotoxicity of a compound is evaluated, the results of cytotoxic assay need to meet all following criteria: "the signal in the cells treated with the test substance is reduced at least by 20% compared with the negative controls," "a concentration-related reduced signal is observed," and the results are reproducible. 21It has been shown that DMSO at concentrations higher than 1% can influence cell viability. 22The final DMSO concentration in our experiment was 0.1%.The addition of DMSO did not influence cell viability compared with naive cells (data not shown).The activation of PBMCs with LPS resulted in a reduction of cell viability by ∼17%.The addition of study products to LPS-activated PBMCs did not result in a substantial reduction in cell viability compared with LPSactivated cells.The study products demonstrated a degree of cytotoxicity following incubation of PBMCs with PHA and the study products ( Table 2 ).In some instances, the percent of dead cells was ≥30% compared with control.In this assay, the cytotoxicity of the test compounds was not dose-dependent, for example, ultrafiltered colostrum at concentration 2 μg/mL had 84% of viable cells and at concentration 20 μg/mL the viability of cells increased to 108% compared with control with the exception of egg yolk extract and botanical blend products.Due to the decreased cell viability, the cytokine production was calculated per 10 0,0 0 0 viable cells.
All study products modulated the activity of LPS-activated PBMCs ( Figure 1 ).Unfiltered colostrum significantly increased the LPS-stimulated secretion of IL-1 β when used at 2 and 20 μg/mL concentrations and interleukin 6 secretion at the concentration of 20 μg/mL ( Figure 1 ).Ultrafiltered colostrum at the dose of 20 μg/mL stimulated the production of IL-8 and TNF α, but the differences between the production of these cytokines by PBMCs treated with ultrafiltered colostrum plus LPS and PBMCs treated with LPS were not statistically significant.
Egg yolk extract showed overall inhibitory effects on the ability of LPS-activated PBMCs to secret cytokines.( Figure 1 ).Differences were not statistically compared with the cytokine levels produced by LPS-only activated cells except for significant inhibition of TNF α production at the egg yolk concentration of 20 μg/mL.
When used at both concentrations, NC slightly inhibited the secretion of the cytokines (IL-6, IL-8, and TNF α) by LPS-activated PBMCs and did not influence IL-1 β production ( Figure 1 ).The difference between the group pretreated with NC and stimulated with LPS and the group of cells stimulated with LPS-only was not statistically significant.
Whey significantly inhibited the IL-1 β by LPS-activated PBMCs when used at both concentrations and the secretions of IL-8 and TNF α at the concentration of 20 μg/mL ( Figure 1 ).
Ultra-nano colostrum + egg yolk extract inhibited the secretion of all 5 cytokines by LPS-stimulated PBMCs (IL-1 β, IL-6, IL-8, and TNF α) at both concentrations 2 and 20 μg/mL.However, these differences were not statistically significant ( Figure 1 ).Botanical blend demonstrated an overall inhibitory effect on the ability of LPS-stimulated cells to produce cytokines ( Figure 1 ).
The botanical blend significantly inhibited the secretion of IL-1 β at both concentrations, 2 and 20 μg/mL, and TNF α when added at 2 μg/mL to the culture, and the secretion of IL-8 at the concentration 20 μg/mL.
Ultra-nano colostrum + egg yolk extract + botanicals showed a maximal inhibitory effect on the secretion of cytokines by LPS- † Cells were pretreated with the test compounds at 2 or 20 μg/mL, followed by activation with PHA 10 μg/mL for 48 hours or LPS 50 pg/ml for 24 hours.stimulated PBMCs at 2 μg/mL ( Figure 1 ).It significantly inhibited the secretion of IL-1 β and TNF α when used at this concentration.

Cytokine production by PHA-activated PBMCs
PHA activation of PBMCs induced robust production of all tested cytokines.Dex treatment significantly inhibited PHA-induced cytokine production ( Figure 2 ).
The treatment with ultrafiltered colostrum had an overall immunosuppressive effect on cytokine production by PHA-activated PBMCs ( Figure 2 ).Ultrafiltered colostrum significantly inhibited PHA-induced IFN γ and TNF α production at both concentrations and IL-5 and IL-13 production at the concentration of 2 μg/mL.
The treatment with nano-colostrum reduced the production of all tested cytokines by PHA-activated PBMCs ( Figure 2 ).Nanocolostrum significantly inhibited IL-5 and IL-13 production by PHAactivated PBMCs when used at concentrations 2 and 20 μg/mL, IL-10 at 2 μg/mL, and TNF α production when used at a concentration of 20 μg/mL only.
The treatment with fermented whey had a mixed effect on cytokine production by PHA-activated PBMCs ( Figure 2 ).The treatment had no effect on TNF α production, reduced IFN γ , IL-5, and IL13 productions at both concentrations and stimulated IL-10 production at 20 μg/mL.Similar to fermented whey treatment, colostrum + egg yolk treatment had a mixed effect on PHA-induced cytokine production by PBMCs ( Figure 2 ).The treatment with colostrum + egg yolk significantly downregulated the production of IL-10 when used at 2 and 20 μg/mL concentrations and IL-5 production at 2 μg/mL concentrations only.IFN γ and IL-13 production were upregulated when PHA-activated cells were treated with 20 μg/mL colostrum + egg yolk.
The botanical blend had mainly immunosuppressive effects on cytokine production by PHA-activated PBMCs ( Figure 2 ).The botanical blend significantly inhibited IL-5 and IL-13 production when used in both concentrations and IFN γ , IL-10, and TNF α when used at the concentration of 2 μg/mL only.

Discussion
The immunomodulatory properties of natural products are well known.In addition to modulating immune responses, many of these products also influence other organ and system functions.Several natural compounds (eg, flavonoid, nonflavonoid polyphenols, mycrosporine-like amino acids, and terpene) have ultraviolet light absorption properties and antioxidant and anti-inflammatory properties. 23Moringa oleifera extracts demonstrate antioxidant, hepatoprotective, radioprotective, and immunomodulatory activities. 24Glucans, the major bioactive compound in mushrooms, exhibit immunomodulatory and anticancer properties. 25n this study, we investigated the effects of multicomponent blends on the ability of LPS or PHA-activated PBMCs to produce an array of cytokines.Cytokines are small proteins produced by immune cells, usually upon cell activation.Cytokines facilitate communication between innate and adaptive arms of the immune system and between immune and nonimmune cells. 26It appears that study products had diverse effects on the ability of PBMCs to produce cytokines upon activation.Most of the studied products demonstrated anti-inflammatory properties.In 1 set of experiments, PBMCs were activated with LPS.LPS is a component of the outer membrane of gram-negative bacteria that stimulates monocytes, macrophages, and B cells to produce inflammatory cytokines. 27Activated phagocytic cells release a specific set of cytokines that help to destroy pathogens or repair damage by inflamed tissues. 27Activation of B cells with LPS results in cell differentiation to antibody-producing cells. 28In vivo, LPS stimulation also results in T cell activation, but it is likely through its effect on antigen-presenting cells like dendritic cells or macrophages. 29herefore, in our experiments, when PBMCs were activated by LPS, the cytokine levels detected in the supernatants resulted from the complex activation of monocytes, B cells, and potentially T cells.
Ultrafiltered colostrum showed a significant stimulatory effect on the ability of PBMCs to produce IL-1 β and IL-6 when PBMCs were stimulated with LPS.IL-1 β is a proinflammatory cytokine produced mainly by monocytes and macrophages at the early stages of infections. 26This cytokine stimulates the expansion and differentiation of CD4 + T cells.IL-6 may demonstrate anti-inflammatory and proinflammatory properties. 26IL-6 stimulates B cells' differentiation into plasma cells and cytotoxic T lymphocyte activity while suppressing regulatory T cells.Bovine colostrum has been shown to increase the number of CD14 + IL-12 + monocytes following PBMC treatment with IFN γ and LPS ac-tivation. 30The same study reported that bovine colostrum enhanced IFN γ secretion in response to weak antigenic stimulation by, for example, cytomegalovirus and inhibited IFN γ production in response to strong antigenic stimulation.This may suggest that bovine colostrum could exert its immunomodulatory effects by boosting the cytokine when an immune response is suboptimal and suppressing the same cytokine production when the immune system is hyperactivated.In our study, bovine colostrum enhanced the secretion of IL-10 and IL-2 while suppressing the secretion of IFN γ and TNF α when human PBMCs were stimulated with PHA (unpublished data).The composition of ultrafiltered colostrum is different from that of bovine colostrum due to processes of ultraand nanofiltration.Following the ultrafiltration process, the final product contains bioactive products with molecular sizes smaller than 10,0 0 0 Daltons.Bovine colostrum contains immunoglobulins (secretory immunoglobulin A, M, and G) with molecular weight ranging from approximately 150,0 0 0 to 970,0 0 0 Daltons. 31 , 32ther tested products showed a suppressive effect on cytokines produced by LPS-activated PBMCs.The botanical blend and colostrum + egg yolk + botanical blend significantly suppressed productions of IL-1 β and TNF α following treatment at low concentration (2 μg/mL).The botanical blend contains medicinal mushrooms Grifolda frondosa , Lentinus edodes , Cordyceps sinensis , and

Agaricus blazeii . It has been reported that β-glucans isolated from
Grifolda frondose or Lentinus edodes have anti-inflammatory properties. 33Cordyceps sinensis extracts may demonstrate either proor anti-inflammatory properties. 33The immunomodulatory properties of Cordyceps sinensis extracts depend on whether it is an aqueous or alkaline extract.The colostrum + egg yolk + botanical blend showed a similar to the botanical blend immunomodulatory profile, possibly due to the presence of mushroom extracts.Our data indicate that the test products (eg, ultrafiltered colostrum, colostrum + egg yolk, and colostrum + egg yolk + botanical blend) stimulate human NK cell activity and IFN γ and granzyme B productions (unpublished data).
PBMCs were activated by PHA in another set of experiments.Typically, T cells present in the peripheral blood respond to PHA activation. 34PHA is a potent mitogen that can induce the proliferation of T cells and cytokine secretion. 35It has been reported that PHA activates both Th cells and cytotoxic T cells. 36 , 37T cells are part of the adaptive arm of the immune system and play an essential role in shaping immune responses to microbial infections. 1 Following microbial infection, dendritic cells process microbial antigens and present them as peptides in the context of Major Histocompatibility Complex (MHC) class II to Th cells.Th cells, upon activation, release cytokines and aid other immune cells recruited to the site of infection to eliminate the pathogens.The processed antigens presented to cytotoxic T cells activate these cells and stimulate their cytotoxic activity against pathogen-infected cells.
The study products significantly influenced the adaptive arm of the immune system, evidenced by changes in the levels of cytokines produced by PHA-activated PBMCs.The treatment with ultrafiltered colostrum inhibited PHA-stimulated PBMC production of IFN γ , IL-5, IL-10, IL-13, and TNF α.IL-5 is produced by Th cells and is a potent activator of eosinophils. 38IL-5 is critical in the pathogenesis of asthma, eosinophilic granulomatosis, and eosinophilic chronic rhinosinusitis. 38Anti-IL-5 monoclonal antibodies are proven effective in treating these diseases; therefore, suppression of IL-5 production by ultrafiltered colostrum may ameliorate eosinophil-mediated conditions.IFN γ regulates immune responses against intracellular microorganisms and controls tumor immunity. 39NK and T cells are the primary producers of IFN γ . 39It appears that murine NK cells are the targets of ultrafiltered colostrum and fermented whey. 13TNF α is among the inflammatory cytokines regulating cell proliferation and survival, leukocyte adhesion, blood coagulation, and some metabolic processes. 40Dysregulated signaling through TNF receptor 1 (TNFR1) and the production of TNF α may lead to the development of rheumatoid arthritis, inflammatory bowel disease, and psoriasis. 41ano-colostrum significantly inhibited IL-5 and IL-13 production by PHA-stimulated PBMCs.IL-13 regulates the differentiation of alternative (M2) macrophages. 42Similar to IL-5, IL-13 is involved in modulations of allergic inflammatory responses.These findings suggest that Nano-colostrum may help support sinus health in people prone to allergic reactions.
The botanical blend, whey protein, and other natural products' combinations had mostly anti-inflammatory effects by suppressing inflammatory cytokines produced by PHA-activated PBMCs.Mushrooms are part of the botanical blend.Amino acids present in mushrooms are mainly responsible for their anti-inflammatory properties via modulating prostaglandin metabolism. 43Our findings are in good agreement with previously published data that whey proteins decrease inflammatory cytokine production via suppression of nuclear factor kappa beta expression. 44Additionally, whey proteins may stimulate immunoglobulin G production via modulation of Th cell functions. 45The review of preclinical and clinical trial results conducted by Hetland et al 46 showed that medicinal mushrooms possess anticancer and antiallergenic properties in addition to anti-inflammatory properties.It appears that mushrooms' anticancer and antiallergenic properties are exerted mainly via modulation of T cell functions.

Study limitations
The results of this in vitro study cannot be generalized to humans.More studies are needed to understand how the products modulate innate and adaptive immune responses.Clinical trials are necessary to confirm the observed immunomodulatory effects in humans.

Conclusions
Most of the study products demonstrated anti-inflammatory properties suppressing the production of cytokine by either LPS or PHA-activated PBMCs.The products' immunomodulatory properties depended on the PBMC pretreatment dose.An outstanding product is ultrafiltered colostrum.It stimulated cytokine production by LPS-activated PBMCs (likely monocytes and B cells) and suppressed the production of cytokines by PHA-activated PBMCs (likely T cells).The study presented evidence that these products might be used to support overall immune health.

Declaration of Competing Interest
This study was funded by 4Life Research, LLC (Sandy, Utah) .The authors have indicated that they have no other conflicts of interest regarding the content of this article.

Table 1
Study products used for PBMC treatment.
DMSO = dimethylsulfoxide; LPS = lipopolysaccharide; PHA = phytohemagglutinin.*Values are presented as mean (SD) mean is percent of viable cells.Cell viability is presented as the percentage of untreated control + 0.1% DMSO from triplicate measurements