The Immunomodulatory Effects of Red Yeast Rice (Ryr) on Activation

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THE IMMUNOMODULATORY EFFECTS OF RED YEAST RICE (RYR) ON ACTIVATION

INDUCED B LYMPHOCYTE PROTEIN EXPRESSION

A RESEARCH PROJECT

SUBMITTED TO THE GRADUATE SCHOOL

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE

MASTER OF ARTS

ROBERT S. BEVARS

DR. HEATHER BRUNS – ADVISOR

BALL STATE UNIVERSITY

MUNCIE, INDIANA

MAY 2018

TABLE OF CONTENTS

TABLE OF CONTENTS ...... 2

LIST OF FIGURES ...... 3

ABSTRACT ...... 4

ACKNOWLEGEMENTS ...... 27

CHAPTER 1

ABSTRACT ...... 4

INTRODUCTION ...... 5

MATERIALS AND METHODS ...... 12

RESULTS ...... 18

DISCUSSION ...... 25

REFERENCES ...... 28

LIST OF FIGURES

Figure 1: RYR cytotoxicity to stimulated B cells increases with treatment Page 21 concentration, but has little toxic effect in comparison to ethanol controls.

Figure 2: Stimulated B cell viability increases with RYR concentrations. Page 22

Figure 3: RYR does not significantly inhibit expression of MHCII on Page 23 stimulated B cells.

Figure 4: Indeterminate effect of RYR on CD40 and CD86 expression on Page 24 stimulated B cells.

CHAPTER 1

1. RYR exhibits little cytotoxicity to stimulated B lymphocytes ...... 18 2. RYR induces the proliferation of stimulated B lymphocytes ...... 19 3. RYR did not significantly alter MHCII expression...... 20 4. Lovastatin did not significantly alter MHCII expression ...... 20

ABSTRACT

RESEARCH PAPER: The immunomodulatory effects of red yeast rice (RYR) on activation

induced B lymphocyte protein expression

STUDENT: Robert S. Bevars

DEGREE: Master of Arts

COLLEGE: Science and Humanities

DATE: May 2018

PAGES: 29

Red yeast rice (RYR) lowers low density lipoprotein (LDL) cholesterol. RYR may also modulate the adaptive immune response. B lymphocytes are responsible for producing immunoglobulin effector functions in adaptive immunity. This work seeks to analyze the activation-induced protein expression of B lymphocytes during exposure to RYR treatments. The research explores if the monacolins in RYR synergize to effectively inhibit various proteins expressed by B cells. RYR effects on activated B lymphocyte protein expression can be assessed by flow cytometry. RYR, in increasing concentration, increased cytotoxicity of LPS-activated B cells at higher concentrations, but contrastingly enhanced total B cell number suggesting it may influence proliferation or survival of B cells. RYR did not significantly affect MHC Class II expression of B cells. Effects on B cell CD40 and CD86 expression were indeterminate. Results from this study further the understanding of RYR effects on B lymphocytes. Future research regarding RYR effects on immunoglobulin production and presentation may provide insight into therapeutic applications for RYR.

INTRODUCTION

The integrated immune response is characterized by innate and adaptive functions. Innate immunity offers protection through a limited amount of invariant receptors to detect foreign antigen. It is often characterized by augmenting physiological barriers in the anatomical construct while subsequently activating adaptive immunity shortly after rapid inflammatory response. Adaptive immunity provides a plethora of recognition functions for host and foreign antigen. Lymphocytes are primarily responsible for adaptive immunity as they clear pathogen through the clonal expansion of individual cells (Turvey and Broide 2010). During lymphocyte development, random somatic recombination of variable, diversity, and joining (VDJ) gene segments allows for highly specific antigen receptors. In this process, VDJ recombination can produce adaptive immunoglobulin and lymphocyte receptors specific to always changing pathogen. After maturation, resting lymphocytes are activated after each initial antigenic exposure. Activated lymphocytes are characterized by their roles in adaptive effector functions

(B cells) or managing the adaptive immune response (T cells) (Parham 2009).

T lymphocytes have influential roles in orchestrating an appropriate immune response through cell-mediated interactions; so they require a specific set of steps to become fully activated. Originating from bone marrow and beginning their differentiation in the thymus, naïve thymocytes mature into cytotoxic or helper T cells based on receptor expression. Cytotoxic T lymphocytes that express the co-receptor CD8 have a T cell receptor (TCR) that recognizes protein peptides bound to a presentation protein called major histocompatibility protein (MHC)

Class I. MHC Class I molecules are located on all nucleated host cells, allowing them to alert and activate CD8+ T lymphocytes when they have become infected or abnormal. These CD8+ T

cells are also called cytotoxic T cells because they initiate apoptotic signaling pathways upon contact with a virally infected cell (Parham 2009).

T helper cells with the CD4 co-receptor conjugate with the presentation protein MHC

Class II. Antigen presenting cells (APCs) function to sample antigen from sites of infection and traffic to peripheral lymphatic organs. There they present antigenic epitopes on MHC Class II proteins to naïve T lymphocytes. Once activated, T lymphocytes may differentiate into a variety of different Th cells or become T regulatory cells. These different T cell types proliferate following activation, migrate to sites of infection, and release cytokine effectors which influence other cells to specifically respond to the antigenic presence. In this way, Th cells regulate innate immunity along with B lymphocyte activation and functions (Parham 2009).

B lymphocytes are pivotal in humoral immune responses. B cells develop in the bone marrow and migrate to secondary lymphoid tissue, such as the spleen, where they become activated through interaction with antigen via their B cell receptors (BCRs). MHCII, CD80,

CD86, and other interactions/cooperation with T cells fully activates B cells to induce robust proliferation and antibody production. Immunoglobulins are the main effector molecules for B lymphocytes and can either be membrane-bound or secreted in the extracellular space. Secreted immunoglobulins are called antibodies. Antibodies function in binding foreign antigen and pathogens to serve as opsonins, enhancing phagocytosis, or to neutralize pathogen interaction with host cells. Membrane-bound immunoglobulins are functional BCRs used for signal transduction and to bind antigen. Antigen may then be internalized, processed, and used for antigenic epitope presentation on MHC Class II to activate CD4 T lymphocytes (Parham 2009).

Following antigen recognition as presented by MHC Class II proteins by B cells, CD4 T lymphocytes secrete a variety of cytokines, such as TGFβ, IL2, IL10, IL4, IFNγ, as well as

others. In combination with the cytokines produced by CD4 T lymphocytes, the interaction of

CD40L on T cells with CD40 on B cells induces germinal center formation. Germinal centers serve as sites for intense proliferation of B cells. The co-stimulation also provides enhanced antibody production and class switching to different antibody isotypes (Parham 2009). While costimulatory responses are often necessary for development and maintenance of effective immune functions, statin molecules have been shown to interfere with adhesion and co-stimulation of lymphocytes (Sun and Singh 2009).

Statins are a class of pharmaceuticals known for their LDL cholesterol lowering abilities as a result of being 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors. The

HMG-CoA reductase enzyme mediates the rate of isoprenoid production by the cellular metabolic mevalonate pathway (Sun and Singh 2009). The pathway is highly conserved among species and its isoprenoid products are used for essential cell functions including: regulating gene expression and enhancing protein functionality (Tousoulis et al. 2014). Isoprenoids are precursors to many vitamins, hormones, sterols, and steroids (Holstein and Hohl 2004). Statins are highly competitive inhibitors of HMG-CoA reductase because of their β-hydroxy acid conformation being analogous to the structure of an HMG-CoA intermediate (Patakova 2013).

Statins have immunomodulatory properties likely due to their inhibition of an early step in cholesterol biosynthesis which inhibits posttranslational lipid attachments for intracellular signaling molecules; such as Rho GTPases (Tousoulis et al. 2014).

The Rho family of GTPase signaling proteins regulate a variety of cytoskeletal gene expressions through activating transcription mechanisms (Tousoulis et al. 2014). Isoprenylation of Rho is inhibited by several statins; which disrupt its ability to bind membranes. Guanosine triphosphate (GTP) binding transcription protein Rho is involved in expression of pro-

inflammatory cytokines (Rikitake and Liao 2005). By suppressing the inflammatory mechanism, pathogen clearing functionality is reduced (Parham 2009). Statins also effect innate immune responses by suppressing IL-6 cytokine through induction of monocyte chemotaxis and adaptive immunity by blocking IFNγ. Statins also have been shown to inhibit expression of MHC Class II expression and costimulatory proteins (e.g. CD40, CD80, CD86) on APCs (Cȏté-Daigneault et al. 2016). The presence of these proteins is essential for T lymphocyte response in adaptive immunity (Bonetti et al. 2003). As with all biomolecules, the various functional groups attached to a statin molecule can directly influence its metabolic effects.

Building from bicyclic six-member hydrocarbon rings, a variety of functional groups can yield different functional statin molecules (Schachter 2005). Statin drugs include a variety of derivatives including simvastatin, atorvastatin, lovastatin, and others. All of these statins share common effects including: prevention of cardiovascular events, moderation of adhesion molecules, and anti-inflammatory responses (Tousoulis et al. 2014). Aspergillus terreus,

Monascus ruber, and other penicillium species produce secondary metabolites which create lovastatin by fermentation. The biosynthesis of lovastatin is dependent on two polyketide synthases which produce it as a secondary metabolite (Patakova 2013).

The first commercially developed statin drug, lovastatin, contains methylbutyric and methyl functional groups (Seenivasan et al. 2008). Simvastatin was a similar derivation of lovastatin; while further remodeling of compactin produced pravastatin. Subsequently, fluvastatin, atorvastatin, pitavastatin, and rosuvastatin were synthetically developed. Differing by hydrophobicity, lipophilic: fluvastatin, atorvastatin, simvastatin, and lovastatin are non-polar compounds. More hydrophilic compounds such as rosuvastatin have a polar methane sulfonamide group (Endo 2010). While all statins differ in reduction of elevated LDL

cholesterol, treatments suggest simvastatin is twice as effective as lovastatin and pravastatin per mg dose. Pravastatin and lovastatin are approximately equipotent in mg/log-linear dose response

(Illingworth and Tobert 1994). While statins are the premier lipid-lowering pharmaceutical on the market, food supplements such as RYR are comparable to higher dose statin drugs (Patakova

2013). This is because a variety of metabolites in RYR share similar structure and functional efficacy as statin compounds (Yang and Mousa 2012).

RYR is a fermented dietary food used widely in Asian cultures. The fermented product of

Monascus purpureus (red yeast) metabolized rice, it has been used as a food preservative, food coloring, nutritional supplement, and to make rice wine for centuries (Patakova 2013, Yang and

Mousa 2012). RYR is known to have alternative therapeutic properties in hypercholesterolemia, dyslipidemia, coronary heart disease, myocardial infarction, liver disease, bone formation, and diabetes (Yang and Mousa 2012). It has been suggested that RYR containing pigments and metabolites can impose immunological modifications provided they are in large enough concentration (Patakova 2013).

Monascidin A (citrinin) is a Monascus metabolite possessing antibiotic and cytotoxic effects (Wong and Koehler 1981). RYR containing Monascus pigments (e.g. monascin, ankaflavin, monascorubin) have also been shown to exhibit antimicrobial properties and reduce the risk of certain cancers (Hong et al. 2008). Most of the major polyketide pigments are aromatic bicyclic azaphilones which may inhibit inflammatory response. Both citrinin and pigment synthesis are inhibited by signal protein G in cyclic adenosine monophosphate (cAMP) mediated protein kinase A (PKA) cell signaling. cAMP concentration inhibits Monascus production of secondary metabolites including monacolin K, pigments, and citrinin (Patakova

2013). RYR is also composed of isoflavones, sterols, and a variety of active monacolins. These

are metabolites resulting from the fermentation process, which include naturally occurring lovastatin. RYR also contains monacolins J, L, M, and carboxylate acid forms which contribute to lipid regulation (Yang and Mousa 2012).

RYR monacolins may exert immunomodulatory properties by suppressing pro- inflammatory cytokines IL-6 and IFNγ (Seenivasan et al. 2008). These monacolins have been implicated in modulating C-reactive protein concentrations; as well as, facilitating disease in renal tissues (Zhao et al. 2004, Patel 2016). RYR monacolins have also been shown to inhibit T cell proliferation. Through reduced isoprenylation of Rho GTPases, RYR monacolins may provide immunomodulatory qualities as well (Bonetti et al. 2003). While these statin derivatives are of lower concentration than therapeutic dosages and function at different rates of reduced

GTPase isoprenylation, they can function together to down-regulate the mevalonate pathway

(Seenivasan et al. 2008, Zhao et al. 2004). Of the monacolins present within RYR, monacolin K is a functional molecule of interest.

Monacolin K, lovastatin, has functionality in cholesterol reduction, but may also mediate intestinal intraepithelial lymphocytes. Lovastatin-based inhibition of the mevalonate pathway may directly influence Th1 cells through suppression of IL-5, IL-4, IL-2, TNFα, and IFNγ cytokines (Zhang et al. 2013). By down-regulating genes from the mevalonate pathway, lovastatin may also inhibit CD40 activation of B lymphocytes. CD80 and CD86 expression may also be reduced in this manner. Reduction in these costimulatory proteins may impair B cell functions in T cell stimulation through APC interactions (Shimabukuro-Vornhagen et al. 2010).

Lovastatin also causes a reduction in B cells in synthesis phase; particularly those undergoing differentiation for antibody secretion. LPS stimulated B lymphocytes experience a lack of proliferation and undergo apoptosis after addition of lovastatin. Suggestions are that larger

concentrations of lovastatin can inhibit the activated B cell response through induced apoptotic mechanisms (Reedquist et al. 1995).

Monacolins, including lovastatin, present in RYR constitute a small portion of the RYR components, but they have been shown to lower cholesterol and have immunomodulatory properties (Seenivasan et al. 2008). This may be due to a synergistic effect by the presence of several monacolins and their stereoisomers. Thus, the goal of this study is to investigate the effect of RYR extract on B lymphocyte functions. We are contrasting the individual effects of lovastatin on B cell functionality with that of RYR treatments. We believe RYR inhibits activation-induced protein expression, proliferation, and antibody production by B lymphocytes.

MATERIALS AND METHODS

Red Yeast Rice Extract Synthesis: Red yeast rice (RYR) extract was created by solubilizing 600 mg RYR powder (Solaray, Park City, UT) in 10 mL reagent grade methanol (AAPR,

Shelbyville, KY, Cas#67-56-1). By mixing (28⁰C, 10 m) in a polypropylene tube and then centrifuging (2800 g, 10 m), the supernatant (in 1 mL aliquots) was transferred to 1.5 mL microcentrifuge tubes for drying under nitrogen gas until completely evaporated. The remaining residue was resuspended in Roswell Park Memorial Institute-1640 media (RPMI-1640) (Lonza,

Williamsport, PA, Cat#12167F) containing 1% methanol (AAPR, Cas#67-56-1). The extract underwent analysis by reverse phase high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS) to identify metabolite components and corresponding concentrations (collaboration with Dr. Phillip Albiniak, Department of Chemistry,

Ball State University).

Mice: Adult male and female C57BL/6J mice between the ages of 8-12 weeks, bred from mating pairs purchased from The Jackson Laboratory (Bar Harbor, ME, Cat#000664), were used for each study. Methods involving mice have been approved by the Ball State University Animal

Care and Use Committee.

Splenocyte Isolation: Murine splenocytes were extracted from the spleen of one C57BL/6J M/F mouse (Jackson Laboratories, Cat#000664). They were placed in RPMI-1640 (Lonza,

Cat#12167F) containing 10% fetal bovine serum (FBS) (Gibco, San Francisco, CA,

Cat#16000069), 5000 µg/mL penstrep (Gibco, Cat#15140122), 100mM sodium pyruvate

(Hyclone, Logan, UT, Cat#SH30239.01), 200mM L-glutamine (VWR Life Sciences,

Indianapolis, IN, Cat#10128-832), 1M N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid

(HEPES) (Lonza, Cat#17-737E), 14.3M B-mercaptoethanol (SIGMA, Cat#M3148), and 100X

MEM non-essential amino acids (Gibco, Cat#11140050). After, the spleen was disaggregated with the back of a sterile syringe. Centrifuging the cell suspension (1500 rpm 4⁰C, 5 m) allowed for the supernatant to be discarded and treatment of the cells with 1X red blood cell (RBC) lysis buffer (Invitrogen, Carlsbad, CA, Cat#00-4333-57). Incubation at room temperature (5 m) and washing the cell suspension with complete RPMI-1640 sufficiently isolated the splenocytes in suspension. Cells were counted using a hemacytometer after trypan blue staining to create a

2x106 cell/mL cell suspension in complete RPMI-1640.

Splenocyte Stimulation for Cytotoxicity Analysis: 2x106 cell/mL suspension was put into 18 wells in a 24 well plate (Table 1). Treatments and controls were done in triplicate. Control samples remained untreated and unstimulated. Experimental samples were stimulated with 10 µg/mL lipopolysaccharide (LPS) (SIGMA, Cat#L3024). Experimental wells received treatment with 2.5

µL, 5.0 µL, 10 µL, or 20 µL RYR extract treatment. Incubation (37⁰C/5% CO2) followed for 24 h.

Table 1: Murine splenocyte sample wells used for cytotoxicity analysis of RYR treatment on LPS stimulated B cells. RYR treatments were given in escalating amounts to assess cytotoxicity. All treatments were prepared in triplicate under sterile conditions. 1.0 mL cell 1.0 mL cell 1.0 mL cell suspension+ suspension+ suspension+ 1.0 µL 10 1.0 µL 10 1.0 µL 10 µg/mL LPS µg/mL LPS µg/mL LPS 1.0 mL cell 1.0 mL cell 1.0 mL cell 1.0 mL cell 1.0 mL cell 1.0 mL cell suspension+ suspension+ suspension+ suspension+ suspension+ suspension+ 1.0 µL 10 1.0 µL 10 1.0 µL 10 1.0 µL 10 1.0 µL 10 1.0 µL 10 µg/mL LPS µg/mL LPS µg/mL LPS µg/mL LPS µg/mL LPS µg/mL LPS + + + + + + 2.5 µL RYR 2.5 µL RYR 2.5 µL 10 5.0 µL 10 5.0 µL 10 5.0 µL 10 extract extract µg/mL RYR µg/mL RYR µg/mL RYR µg/mL RYR 1.0 mL cell 1.0 mL cell 1.0 mL cell 1.0 mL cell 1.0 mL cell 1.0 mL cell suspension+ suspension+ suspension+ suspension+ suspension+ suspension+ 1.0 µL 10 1.0 µL 10 1.0 µL 10 1.0 µL 10 1.0 µL 10 1.0 µL 10 µg/mL LPS µg/mL LPS µg/mL LPS µg/mL LPS µg/mL LPS µg/mL LPS + + + + + + 10 µL 10 10 µL 10 10 µL 10 20 µL 10 20 µL 10 20 µL 10 µg/mL RYR µg/mL RYR µg/mL RYR µg/mL RYR µg/mL RYR µg/mL RYR

Cytotoxicity Analysis: Following incubation, samples were harvested and centrifuged (1500 rpm

4⁰C, 5 m). Cells were washed twice with fluorescence-activated cell sorter (FACS) buffer containing 2% bovine serum albumin (BSA) (VWR Life Sciences, Cat#0332) and 1X phosphate- buffered saline (PBS) (Gibco, Cat#10010031). Cells were incubated with 30 µg/mL CD19-FITC fluorochrome conjugated anti-mouse antibody (MACS Miltenyl Biotec, Cat#130-102-494) to identify B cells in the cell suspension. Antibody incubation (4⁰C, 10 m) followed with a FACS buffer wash. Centrifugation (1500 rpm 4⁰C, 5 m) preceded suspending in FACS buffer. 30 sec before each flow cytometer analysis, 3 µL 7-AAD (Tonbo Biosciences, Cat#136993-T500) was added to each sample and then immediately analyzed using a BD Accuri C6 flow cytometer (BD

Biosciences). The logarithmic mean fluorescence intensity (MFI) of 7-AAD for CD19+ B cells was recorded for each sample. Treatment groups were done in triplicate. MFI for the 3 samples

in each group were averaged +/- the standard error of the mean (SEM). Differences in 7-AAD

MFI between treatment groups were determinant of RYR cytotoxicity. CD19+ B cells without 7-

AAD fluorescence was counted and averaged +/- SEM. Differences in non-7AAD fluorescent

CD19 cells helped determine viable B lymphocytes within each cytotoxicity treatment group.

Splenocyte Stimulation for Protein Expression Analysis: Cell suspension was made by use of the previous splenocyte isolation protocol. 1x106 cell/mL suspension was added to 18 wells in a 24 well plate (Table 2). Treatments and controls were done in triplicate. Control samples remained untreated and unstimulated. Making two experimental sets, one was unstimulated while the other was stimulated with 10 µg/mL LPS (SIGMA, Cat#L3024). Experimental wells received RYR extract treatment and another set 20 µM lovastatin (SIGMA, Cat#PHR1258). Incubation

(37⁰C/5%CO2) followed for 24 h.

Table 2: Murine splenocyte sample wells used for comparison of RYR to lovastatin in LPS stimulated and non-stimulated B cells. Treatment concentration of RYR extract is still being determined. All treatments were prepared in triplicate under sterile conditions. 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell suspension suspension suspension suspension + suspension suspension 1 µL 10 + + µg/mL LPS 1 µL 10 1 µL 10 µg/mL LPS µg/mL LPS 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell suspension + suspension + suspension + suspension + suspension + suspension + 5 µL RYR 5 µL RYR 5 µL RYR 5 µL RYR 5 µL RYR 5 µL RYR extract extract extract extract+ extract+ extract+ 1 µL 10 1 µL 10 1 µL 10 µg/mL LPS µg/mL LPS µg/mL LPS 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell suspension + suspension + suspension + suspension + suspension + suspension + 20 µL RYR 20 µL RYR 20 µL RYR 20 µL RYR 20 µL RYR 20 µL RYR extract extract extract extract+ extract+ extract+ 1 µL 10 1 µL 10 1 µL 10 µg/mL LPS µg/mL LPS µg/mL LPS 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell 0.5 mL cell suspension + suspension + suspension + suspension + suspension + suspension + 1 µL 20 µM 1 µL 20 µM 1 µL 20 µM 1 µL 20 µM 1 µL 20 µM 1 µL 20 µM lovastatin lovastatin lovastatin lovastatin+ lovastatin+ lovastatin+ 1 µL 10 1 µL 10 1 µL 10 µg/mL LPS µg/mL LPS µg/mL LPS

Protein Expression Analysis: Following stimulation, samples were harvested and centrifuged

(1500 rpm 4⁰C, 5 m). Cells were then washed twice in FACS buffer containing 2% bovine serum

albumin (BSA) (VWR Life Sciences, Cat#0332) and 1X phosphate-buffered saline (PBS)

(Gibco, Cat#10010031). Following, cells were incubated with diluted normal rat serum (Jackson

ImmunoResearch, Cat# ) for 5 min at 4⁰C. A wash was done with FACS buffer. Cells were

incubated with: 19 µL 0.5 µg/mL MHC Class II-FITC (affymetrix eBioscience, Santa Clara, CA

Cat#12-5321-81), 9.5 µL 0.2 µg/mL CD40-PE (BioLegend, San Diego, CA, Cat#124610), 9.5

µL 30 µg/mL VIO700 CD86, (MACS Miltenyl Biotec, Cat#130-105-135), 9.5 µL 30 µg/mL

APC CD19 (MACS Miltenyl Biotec, Cat#130-102-546), and 200 µL FACS buffer used to target proteins expressed by B cells. Antibody incubation (4⁰C, 10m) followed with a FACS buffer wash. Centrifugation (1500 rpm 4⁰C, 5m) preceded suspension in FACS buffer for flow cytometer analysis using a BD Accuri C6 flow cytometer (BD Boiosciences). The logarithmic

MFI for MHC Class II, CD40, and CD86 was recorded for each sample. Treatment groups were done in triplicate. MFI for the 3 samples were averaged +/- SEM. Mean fluorescence intensity was normalized to the background fluorescence intensity of the negative control. Differences in

MFI between treatment groups were used to determine the immunomodulation of RYR.

RESULTS

RYR monacolins have been shown to lower cholesterol through inhibition of HMG-CoA reductase. Through mediation of the HMG-CoA pathway RYR may also have immunomodulatory properties. Immunomodulation can be assessed through many physiological processes including: protein expression, proliferation, and overall immune cell viability. To study effects RYR may have on B lymphocytes, splenocytes were extracted from C57BL6/J mice and stimulated with LPS in the presence or absence of RYR extract. This research is focused on how RYR affects B lymphocyte function, but for comparison, the research also examines how the presence of lovastatin may alter B lymphocyte function. This will aid in determining if other metabolites in RYR have an effect differing from that of lovastatin. All treatment incubations occurred for 24 hrs. Antibody staining with MHCII, CD40, and CD86 assessed protein expression. 7-AAD staining was used to determine RYR cytotoxicity of activated B cells. CD19 staining was used to delineate B cells from T cells during both cytotoxicity and protein expression analyses.

In order to study if RYR is overly toxic to activated B cells, cytotoxicity was analyzed.

RYR cytotoxicity to LPS stimulated B lymphocytes was assessed using 7-AAD staining during flow cytometry. 2.5 µL, 5.0 µL, 10 µL, and 20 µL RYR treatments were not significantly different from another in stimulated B cell groups (Fig 1). 10 µL and 20 µL treatments of RYR exhibited significantly more 7-AAD fluorescence than LPS controls; demonstrating an increase in B cell cytotoxicity at that volume. Background cytotoxic effects, observable in 0.0 µL

RYR+LPS, were likely resultant of cell handling and competitive influences during incubation

(Johnston 2009). Difference was apparent between the RYR treated groups and ethanol control

in regards to cell viability (Fig 1). Overall, RYR extract became cytotoxic to activated B lymphocytes when used at 10 µL and 20 µL volumes.

In spite of increased cytotoxicity of B cells treated with 10 µL and 20 µL volumes of

RYR extract, compared with the LPS controls, total numbers of B lymphocytes increased following LPS stimulation and increasing volumes of RYR extract. The LPS-stimulated B cells in 20 µL RYR were significantly different to those in 0.0 µL RYR, 2.5 µL RYR, 5.0 µL RYR, and 10 µL RYR concentrations (Fig 2). In LPS-stimulated samples, 10 µL RYR and 20 µL RYR samples exhibited significant difference from negative controls. These results demonstrate total viable B cell numbers increasing from both 10 µg/mL LPS stimulation and increasing RYR treatment. The total viable B cell numbers, in conjunction with the cytotoxicity assays, help determine that RYR treatments under 10 µL will avoid unintended cell death from RYR treatment.

Assessment of the immunomodulatory capacity of RYR was determined through treatment of stimulated B lymphocytes in vitro. The effect of RYR extract was examined on unstimulated and LPS-stimulated B cells. Resting state samples served as an internal control against stimulated treatments. Although cells were stimulated, as shown by a significant increase in MHC Class II expression following LPS treatment, RYR and lovastatin treatments did not affect the expression of MHC Class II (Fig 3). Significant difference was not determined between stimulated B cells compared to unstimulated controls (Fig 4). CD86 expression in 20 µL

RYR was not significantly different to untreated/unstimulated controls. CD86 expression in unstimulated B cells was not significantly different among 5 µL RYR, 20 µL RYR, and 20 µM lovastatin treatments when compared to 10 µg/mL LPS controls. Both results for CD86 expression and CD40 expression did not show significant differences between treatment groups.

The large variability in CD40 and CD86 standard error amounts may have complicated direct comparisons between LPS-activated groups.

MHC Class II expression for LPS-stimulated B cells were compared between RYR and

20 µM lovastatin treatment. MHCII Class II results were defined based on significant difference among LPS-stimulated samples and controls. Neither 5 µL RYR+LPS nor 20 µL RYR+LPS were significantly different to 10 µg/mL LPS controls (Fig 3). There was no significant difference between 5 µL RYR+LPS or 20 µL RYR+LPS. The data suggests no relevant distinction to MHC Class II modulation by RYR extract. MHC Class II expression was comparable among RYR treatments and 20 µM lovastatin. Activated B lymphocytes treated with

20 µM lovastatin were not significantly different from 10 µg/mL LPS controls. This is indicative of a lack of immunomodulatory influence from lovastatin on MHCII expression during a B cell adaptive immune response. These results are not reflective of a previous lovastatin study; wherein, 48hr 10 nM lovastatin treatment inhibited LPS-stimulated B cell proliferation and induced apoptosis. The study suggested larger concentrations of lovastatin to have even greater effect on activated B lymphocytes (Reedquist et al. 1995). If lovastatin does induce cell death in activated B cells a decrease in expression values would be anticipated for lovastatin-treated LPS samples in comparison to stimulated controls. Our study may be similar to other research; wherein, 18-24 hr simvastatin treatment was done prior to 12-24 hr bacterial infection of macrophages. Results from this study also revealed no alteration of MHC Class II, CD40, CD80, or CD86 expression (Burns et al. 2015). Overall, alterations in protein expression that might suggest possible variation in B lymphocyte function were not observed. MHC Class II, CD40, and CD86 expression, in 10 µg/mL LPS, were not significantly different from stimulated controls.

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Figure 2: Total B cell numbers increase with RYR concentrations. Extracted and purified splenocytes were stimulated with 10 µg/mL LPS and treated with increasing concentrations of RYR extract. Cells were stained with antibodies to CD19 for detection of B cells from morphologically similar T cells. 7-AAD staining was done to determine overall RYR cytotoxicity to CD19 stained B cells. Non-7-AAD fluorescent CD19+ B cells were detected and counted using an Accuri C6 flow cytometer. Experiments were performed in triplicate. Statistical differences were determined by one-way ANOVA with a Tukey’s multiple comparisons test using Prism software. Statistically significant results marked with * p ≤ 0.05 0 µL RYR+LPS. Statistically significant results marked with + p ≤ 0.05 20 µL RYR+LPS.

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S S S S x R R P P P P T Y Y A L L L L + o R R V + + O R R A N L L V u u L Y Y R R O 5 0 M 2 L L L u 0 u u M 2 5 0 u 2 0 2 * = s ig n ific a n t d iffe re n c e to N o T x g ro u p Figure 3: RYR does not significantly inhibit expression of MHC Class II on stimulated B cells. Extracted and purified+ splenocytes = s ig n ific a nweret d if feithere re n c stimulatede to L P S g withro u p 10 µg/mL LPS or left unstimulated. All samples were treated with 5 µL RYR, 20 µL RYR, or 20 µM lovastatin (LOVA). Non-treatment was used for positive and negative controls. Cells were stained with antibodies to CD19 for detection of B cells from morphologically similar T cells. Staining with antibodies to MHC Class II was done to determine relative expression in both stimulated and unstimulated B cells. An Accuri C6 flow cytometer was used for analyzing protein expression. Relative expression was determined by normalization of mean fluorescence intensity (MFI) to negative controls. Experiments were done in triplicate. Statistical differences were determined by one-way ANOVA with a Tukey’s multiple comparisons test using Prism software. Statistically significant results marked with * p ≤ 0.05 no treatment controls. Statistically significant results marked with + p ≤ 0.05 10 µg/mL LPS.

N o rm B C e ll C D 4 0 E x p re s s io n 9 -1 3 -1 7 a nNd o9r-m2 7 -B1 7C e ll C D 8 6 E x p re s s io n 9 -1 3 -1 7 a n d 9 -2 7 -1 7

1 .0 0 .8 *

M E

E 0 .8 S

0 .6

0 .6 6

0 8

4 0 .4

C C

0 .4

F M

M 0 .2

l 0 .2

R R 0 .0 0 .0

S S S S S S S S x R R P P P P x R R P P P P T Y Y A L L L L T Y Y A L L L L R R + + + + o V o R R V + + N L L O R R A O R R A L Y Y V N L L V u u u u L Y Y 5 0 R R O R R O M L 5 0 M 2 u L L 2 L u u u L L 0 M u u M 2 5 0 u 0 2 2 5 0 u 0 2 0 2 2 N o s ig n ific a n t d iffe re n c e s * = s ig n ific a n t d iffe re n c e to N o T x g ro u p Figure 4: Indeterminate effect of RYR on CD40 and CD86 expression on stimulated B cells. Extracted and purified splenocytes were either stimulated with 10 µg/mL LPS or left unstimulated for comparison. All samples were treated with 5 µL RYR, 20 µL RYR, or 20 µM lovastatin (LOVA). Non-treatment was used for positive and negative controls. Cells were stained with antibodies to CD19 for detection of B cells from morphologically similar T cells. CD40 and CD86 antibody staining was done in conjunction with flow cytometry to determine CD40 and CD86 expression of B cells. Relative expression was determined by normalization of mean fluorescence intensity (MFI) to negative controls. Experiments were done in triplicate. Statistical differences were determined by one-way ANOVA with a Tukey’s multiple comparisons test using Prism software. Statistically significant results marked with * p ≤ 0.05 no treatment controls. Statistically significant results marked with + p ≤ 0.05 10 µg/mL LPS.

DISCUSSION

RYR treatments were assessed for amounts cytotoxic to the activated B lymphocytes (Fig

1). 10 µL and 20 µL treatments of RYR exhibited significantly more 7-AAD fluorescence than

LPS controls. This demonstrated toxicity of B cells at that volume. Cytotoxicity analyses demonstrated that RYR induced toxic effects to activated B cells, but at significantly reduced amounts than ethanol controls. Total B lymphocytes also increased following LPS stimulation and increasing volumes of RYR extract (Fig 2). 10 µL RYR and 20 µL RYR samples exhibited significant difference from negative controls; signifying an increase to viable B cell amount. 7-

AAD uptake at 10 µL and 20 µL RYR volumes, without a decrease in total cell numbers, is most likely a consequence of activation-induced cell death (AICD). AICD may result in semi- permeability of membranes; allowing for 7-AAD leakage despite the inability to adequately pump the dye out from the intracellular space. B cells expressing CD95 and Fas may interact with Fas ligand on activated T cells for regulation. APCs undergo caspase-3 induced apoptosis through this interaction of Fas and Fas ligand. The AICD mechanism is used for preservation of homeostasis and preventing autoimmunity (Maher et al. 2002).

Expression of MHC Class II, CD40, and CD86 proteins are critical to activated B lymphocyte functionality; as well as, costimulatory interactions with T lymphocytes (Trickett and Kwan 2003). MHC Class II, CD86, and other proteins provide costimulatory interactions.

CD40/CD40 ligand induce B cell activation and proliferation (Parham 2009). Lack of change in

CD40 expression suggests that the increasing total B cell counts, demonstrated in the cytotoxicity assays, were not a consequence of proliferation (Fig 2)(Fig 4). A lack of RYR modulation in MHCII and CD86 expression suggests T cell/B cell interactions were not affected

(Fig 3)(Fig 4). A lack of change among B lymphocyte protein expression suggests RYR is not

immunomodulatory. Given these results, RYR may be used for its propensity to lower cholesterol without affecting immune responses.

RYR extract treatments were analyzed for various effects on activated B lymphocytes.

This work demonstrated that 10 µL RYR extract exhibits B cell cytotoxicity. Total B cell counts taken during cytotoxicity assays were most likely a consequence of AICD prior to 7-AAD staining. RYR extract also demonstrated no discernable effect on B lymphocyte activation/proliferation nor costimulatory influences between B and T cells. Future experiments into RYR modulation on B cell immunoglobulin production and T cell regulation could verify its effect on adaptive immunity. RYR effects on macrophages and dendritic cells could discern innate immunity during treatment. Taken together, complete immunomodulatory capabilities by

RYR could be verified or refuted in this way. Overall, this study suggests that RYR may serve as a natural HMG-CoA inhibitor while avoiding some immunomodulatory side effects common with statin pharmaceuticals.

ACKNOWLEDGEMENTS

This study was supported by the Ball State University Department of Biology. We would like to formally thank Dr. Susan McDowell and Dr. Jennifer Metzler for their critical review of this manuscript.

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