Soy Dietary Intervention in HIV+ ART-Treated Individuals - Preliminary In-Vitro HIV-Uninfected Conditions

Soy Dietary Intervention in HIV+ ART-treated Individuals - Preliminary In-vitro HIV-Uninfected Conditions

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Sylvia Phimsouay, B.S.

Graduate Program in Allied Medicine

The Ohio State University

2017

Thesis Committee:

Nicholas Funderburg, Advisor

Tammy Bannerman

Christopher Taylor

Sylvia Phimsouay

2017

Abstract

The production and dissemination of antiretroviral therapy (ART) has improved the longevity of the ART-treated HIV-infected individual via improved immune system function; it has also raised concerns regarding increased risk of cardiovascular disease (CVD) morbidity and mortality in this specific population. HIV-infection results in chronic immune activation and inflammation, which may play an important role in the increased risk of CVD. Drug intervention with statins has been shown to lower inflammatory markers, decrease carotid intima media thickness (cIMT) - which is significant in determining CVD progression - and improve the lipid profile in an HIV-infected population. A soy-based dietary intervention could serve as a potential non-drug therapy method for improving the morbidity and mortality of CVD in the HIV- infected population and has been shown to act as a natural modulator of lipid profiles and pro- inflammatory cytokines in men with hypercholesterolemia and prostate cancer. We hypothesize that soy treatment will reduce pro-inflammatory lipids, immune activation, and inflammation in HIV-infected ART-treated individuals, and as a correlate, potentially reduce their risk of morbidity due to CVD.

Acknowledgements

I would like to thank all the members of the Funderburg Lab at The Ohio State University for accommodating their space and time for my project as well as every participant who has volunteered for this study. Thank you to my academic supervisor, Nicholas Funderburg, for providing the expertise and necessary materials to perform preliminary analysis in the experiment, Soy Dietary Intervention in HIV+ ART-treated Individuals - Preliminary In-Vitro

Healthy Donor Conditions.

iii

Vita

2016 ...... Medical Technologist, ASCP(CM)

2013 ...... B.S. Biology, The Ohio State University

Field of Study

Major Field: Allied Medicine

Table of Contents

Abstract ...... ii

Acknowledgments ...... iii

Vita ...... iv

Table of Contents ...... v

List of Figures ...... vi

Chapter 1: Introduction...... 1

Chapter 2: Review of the Literature ...... 4

Chapter 3: Methodology ...... 10

Chapter 4: Results ...... 12

Chapter 5: Discussion ...... 24

Chapter 6: Conclusion ...... 30

References ...... 32

List of Figures

Figure 1. Monocyte Flow Cytometry Dot Plot ...... 13

Figure 2. Monocyte %# Monocytes No Stimulation and LPS ...... 14

Figure 3. Monocyte Flow Cytometry Subsets Shifts in HLA-DR MFI and Counts ...... 15

Figure 4. Prostate Cancer Cytokine Mean Fold Change in Soy Bread Study ...... 17

Figure 5. Monocyte Subsets CD69 MFI after TNFα Stimulation ...... 19

Figure 6. Monocyte Subsets HLA-DR MFI after LPS Stimulation ...... 21

Figure 7. LPS Induced TNF-α and IL-6 Cytokine Release ...... 23

Figure 8. Diagram of Cytokine Signaling Cascade ...... 26

Chapter 1: Introduction

Human immunodeficiency virus (HIV) is a derivative of the Simian immunodeficiency virus. Per the World Health Organization, in 2015, there has been a reported 36 million people living with HIV worldwide, with approximately 2 million new cases reported, and 1.1 million people died in 2015 of HIV-related illnesses. The cause of HIV infection is due to direct contact with most bodily fluids from infected individuals and can be spread by sexual transmission, needle sharing or accidental punctures, during child birth and breast-feeding, or by blood transfusions. HIV is incurable; however, it can be managed by antiretroviral therapy (ART). ART affects several stages of the viral life cycle, diminishing the viral load measured in blood and tissues.

Progression of HIV infection is associated with a massive decrease in CD4+ T cells, a cell type that plays an important role in directing the immune system. A healthy individual typically has 500 – 1,500 CD4+ T cells/µL of blood and an ART-naive HIV+ person may have approximately

200 cells/µL or less, leading to Acquired Immunodeficiency Syndrome (AIDS). HIV infects cells that express CD4 and the chemokine receptors, CCR5 and/or CXCR41. Infection and subsequent lysis of target cells by HIV is one cause of the CD4 depletion in infected individuals. To combat viral replication and improve the CD4+ T cell counts, six classes of anti-retroviral therapy (ART) drugs have been developed that affect HIV at different stages of the viral life cycle – fusion

1 inhibitors (FIs), nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), integrase inhibitors (INSTIs), protease inhibitors (PIs), and chemokine receptor antagonists. Improvements in the effectiveness of combination ART regimens and increased dissemination of ART has improved longevity of HIV-infected individuals via improved immune system function; yet, it has also raised concerns regarding cardiovascular disease (CVD) morbidity and mortality in this specific population.

HIV-infection results in chronic activation of both the innate and adaptive immune responses. Monocytes play a significant role in the innate immune response to microbes and are characterized into three different subsets circulating the bloodstream based on CD14 and

CD16 surface marker expression: traditional, CD14+CD16-; inflammatory, CD14+CD16+; and patrolling, CD14dimCD16+. Several studies have indicated that the classical and inflammatory subsets are functionally different with regard to cytokine production in response to bacterial

Toll-like Receptor (TLR) ligands and antigen presentation26,27, and the patrolling monocytes respond to viral products and home to vascular endothelium28. A study by Funderburg and colleagues demonstrated that proportions of monocyte subsets are altered in HIV-1 disease,

CD16+ monocytes are increased in HIV-infected individuals and in people who have recently experienced acute coronary syndrome (ACS), and that the inflammatory and patrolling subsets have elevated tissue factor (TF) expression in comparison to expression on cells from healthy,

HIV-negative, controls28. Enrichment of these cell subsets in the circulation, and their increased activation status, may make them more likely to home to the vascular endothelium where they can contribute to vascular inflammation and atherosclerotic plaque formation. Identification of mechanisms that may inhibit monocyte activation may lower CVD progression in HIV infection.

Several inflammatory and coagulopathic biomarkers are associated with HIV morbidity and mortality, including: interleukin-6 (IL-6), soluble CD14 (sCD14), C-reactive protein (CRP), and

D-dimer 2,3. Piconi et al also demonstrated that the aforementioned inflammatory markers in

HIV-infected individuals neared normalization with ART, but the inflammatory lipid markers apolipoprotein A (apo-A) and low-density lipoprotein (LDL) remained significantly elevated2.

This recent discovery of inflammatory lipid biomarkers correlating with CVD risk better than inflammatory markers has led to further lipid/metabolic studies, including studies aimed at reducing oxidized low-density lipoprotein (oxLDL) in HIV-infected individuals4. Reducing both hyperlipidemia and the inflammatory markers measured in HIV-infected ART-treated individuals may improve the course of HIV disease and reduce the risk of CVD events in this population. We hypothesize that soy treatment will reduce pro-inflammatory lipids, immune activation, and inflammation in HIV-infected ART-treated individuals, and as a correlate, potentially reduce their risk of morbidity due to CVD.

Chapter 2: Review of Literature

The causes of chronic inflammation and immune activation in HIV-infected individuals may be multifactorial and may include microbial translocation, coinfections, and dyslipidemia.

Microbial translocation in HIV disease is likely driven by the severe CD4+ T cell depletion from the gut-associated lymphoid tissue (GALT). The subsequent weakening of the gut barrier allows for microbial products to penetrate the mucosal barrier, circulate in the bloodstream, and stimulate innate immune cell activation, leading to chronic inflammation7. Additionally, dysbiosis of the gastrointestinal tract has been associated with HIV infection which may further exacerbate microbial translocation induced chronic inflammation33. Alterations in the microbial communities in the gastrointestinal tract may also contribute to changes in metabolic and lipid profiles, thus dysbiosis associated metabolic profiles are of growing interest in understanding

CVD risk in HIV infection34. Increased plasma levels of microbial products, including lipopolysaccharide (LPS), have been measured in HIV-infected individuals; ART causes levels of

LPS to decrease, but these levels do not reach levels measured in HIV-uninfected individuals23.

Lipopolysaccharide can be recognized by Toll-like Receptor 4 (TLR-4), which is expressed on the surfaces of innate immune cells, including monocytes, resulting in cellular activation and cytokine production. Furthermore, proportions of inflammatory monocytes in HIV-infected individuals receiving antiretroviral therapy are directly related to levels of LPS in the plasma28.

Co-infection with other microbes may also cause chronic inflammation; most notably, infection 4 with cytomegalovirus (CMV) may enhance immune activation in HIV-infected individuals7,8 as

CMV is a common co-pathogen in this population. Co-infections may contribute to activation of both the innate and adaptive immune responses, perpetuating cellular activation and inflammation.

Another factor contributing to chronic inflammation in HIV-infected individuals is increased levels of pro-inflammatory lipids. HIV infection can cause metabolic changes to an individual, potentially resulting in increased oxidation of low-density lipoprotein (oxLDL), which is known to stimulate monocyte activation4 and increase the risk of CVD. One study demonstrated that HIV-infected individuals taking protease inhibitors9 resulted in changes in their metabolic lipid profile, suggesting that ART may also contribute to an increased risk for

CVD. A consequence of recognition of LPS or oxLDL by their receptors is increased production of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α); levels of TNF-α, and its soluble receptors, are increased in HIV infection24 and are associated with mortality including deaths related to CVD in HIV-infected individuals25. More studies are being performed to resolve the potential contributors of increased risk for CVD in ART-treated HIV+ individuals, and by understanding the mechanisms that drive inflammation and hyperlipidemia, interventional trials can be designed to reduce inflammation related comorbidities.

Monocyte activation and perturbations in lipid profiles are also hallmarks of CVD risk in

HIV-uninfected populations, and recent research has identified similarities in these indices among individuals with CVD and those with chronic HIV infection. Piconi et al. highlighted that metabolic lipid profiles, LDL, ApoA, and ApoB, were better correlated with CVD risk than are inflammatory markers, in ART-treated HIV-infected individuals2. These lipid markers were consistently elevated during ART-treatment, whereas ART initiation often reduces the 5 inflammatory markers, IL-6, CRP, and soluble CD14 (sCD14). Elevated expression of vascular adhesion molecules among CD14+ monocytes has also been reported in ART-treated HIV- infected individuals; this may be important as these cells are involved in plaque build-up within the blood vessel wall. Measurement of the carotid intima media thickness (cIMT) can indicate the severity of CVD progression; ART-treated patients had a higher cIMT, or plaque build-up, when compared to ART-naïve patients. This supports that prolonged ART treatment may contribute to CVD.

Zidar et al. have proposed that oxLDL plays a critical role in HIV-associated monocyte activation by examining relationships among levels of LDL, oxLDL, and markers of monocyte activation – tissue factor (TF), CD36, Toll-like Receptor 4 (TLR-4), and sCD14. Levels of oxLDL were increased in the HIV infected population compared to levels in uninfected donors. In-vitro exposure of monocytes to LPS and oxLDL, and not LDL, resulted in increased TF expression on these cells, indicating that LPS and oxLDL may contribute to monocyte activation and chronic inflammation. OxLDL is a pro-inflammatory form of LDL that may contribute to the generation of foam cells, or high fat concentrated macrophages that contribute to atherosclerosis.

Reducing oxLDL levels can be a potential target to combat CVD in both HIV-infected and uninfected individuals. Receptors for oxLDL, including CD36 and TLR-4 are differentially expressed on monocyte subsets from HIV infected donors compared to expression on these cells from uninfected controls4. Additionally, levels of oxLDL, but not native LDL, were directly correlated with a monocyte activation marker, sCD14. Therefore, reducing levels of oxLDL in HIV infection may reduce immune activation and CVD risk4.

Statin treatment may reduce levels of oxidized LDL10. A study with HIV-infected patients demonstrated that atorvastatin lowered circulating oxLDL levels and lowered levels of coronary atherosclerosis, indicating that oxLDL may be a significant marker for coronary plaque development. Nou and colleagues theorize that a reduction in oxLDL may be a beneficial effect of statins on reducing atherosclerosis in HIV-infected individuals10, in which improves the CVD risk in this population. Additionally, the Stopping Atherosclerosis and Treating Unhealthy bone with Rosuvastatin in HIV (SATURN-HIV) study presented findings that Rrosuvastatin treatment reduces multiple inflammatory markers in HIV-infected patients and that many of these changes, including improvement in cIMT, were related to reduction in oxLDL levels11,21, supporting the theory that oxLDL levels may contribute to inflammation and CVD risk in HIV infection11. These results indicate that an intervention in ART-treated HIV-infected individuals that reduces oxLDL levels could change the severity of their disease course, lowering the risk of mortality.

The oxLDL-signaling cascade consists of recognition of oxLDL by the heterotrimer of

CD36/TLR-4 and TLR-6, which triggers both a pro-inflammatory and pro-atherosclerotic response4,12. CD36 is expressed on multiple cellular surfaces, notably cardiac myocytes, adipocytes, macrophages, and platelets. The overall mechanism of action consists of cellular exposure to oxLDL, resulting in activation of a transcription factor, PPAR-. This increased activity of PPAR- further drives the expression of CD36. When oxLDL levels are in abundance, macrophage CD36 expression is elevated and “loads” the macrophages with cholesterol, producing “foam cells.” These “foam cells” are early contributors to coronary atherosclerosis12.

Understanding the oxLDL and CD36 relationship, has led to studies targeting the regulation of

CD36 and PPAR-. Regulation of oxLDL levels and subsequent cell signaling has been 7 investigated via administration of soy isoflavones. Soy is hydrolyzed in the gut to produce three metabolites – daidzein, genistein, and glycitein. Genistein has been shown to inhibit atherogenesis similarly to known inhibitors that impact signaling cascades downstream of LPS and oxLDL receptors (p38 MAP kinases, TLR intermediates, and the inflammasome20,32).

Genistein down-regulates PPAR- transcriptional activity, leading to fewer “foam cells” and overall reduced chances of atherosclerosis13. Furthermore, genistein inhibits the Lyn tyrosine kinase, which plays a pivotal role in cellular activation downstream of the oxLDL receptor, CD36.

Another receptor affected by genistein is TLR-414. TLR-4 ligands may interfere with macrophage cholesterol metabolism17; suggesting TLR-4 interference can positively affect the disease course of ART-treated HIV-infected patients with dyslipidemia.

Another biomarker that has been associated with CVD risk and vascular inflammation is lipoprotein-associated phospholipase A2 (Lp-PLA2); this enzyme is of interest because it is produced by macrophages/foam cells and it hydrolyzes oxLDL15,18. The SATURN-HIV trial11 demonstrated that in 48 weeks, ART-treated HIV-infected patients undergoing Rosuvastatin therapy had reduced inflammatory markers, including decreased levels of sCD14 and Lp-PLA2.

Numerous studies have demonstrated that a soy-diet improves the metabolic lipid profile - LDL, HDL, cholesterol, and triglycerides - in dyslipidemia individuals. A cohort study, designed by collaborators here at OSU, has demonstrated that a soy-based functional food intervention acted as a natural modulator of pro-inflammatory cytokines and improved lipid parameters in men with hypercholesterolemia and prostate cancer5,6. Further investigation is needed to evaluate if a soy based dietary intervention, or exposure to individual soy isoflavones can reduce cardiovascular disease risk and vascular inflammation in HIV-infected individuals.

We hypothesize that soy treatment will reduce pro-inflammatory lipids, immune activation, and inflammation in HIV-infected ART-treated individuals, while also reducing their risk of morbidity due to CVD.

Chapter 3: Methodology

Patients

All participants in the study provided written informed consent in accordance with the

Institutional Review Board at The Ohio State University/The Ohio State University Wexner

Medical Center (IRB Protocol Number: 2014H0001). All participants were uninfected with HIV

(healthy donors) and were recruited from The Ohio State University population.

Sample Collection and Incubation

Fresh whole blood from 11 healthy donors (10 male and 1 female; 21 years to 50 years of age)was drawn into EDTA coated tubes and pre-incubated at 37 °C in 25-mL polypropylene

Falcon tubes (BD Biosciences) for 1 hour with individual inhibitors: 10 µM SB203580 (InvivoGen,

25 mM), 20 µM Z-VAD-FMK (InvivoGen, 20 mM), 50 µM MYD88 inhibitor peptide and control set

(NovusBio, 5 mM), or 50 µM TRIF Blocking Peptide (NovusBio, 0.2 mg/mL); or with individual soy isoflavones at a 100 mM concentration: genistein, daidzein, and glycitein (all LC Laboratories, 50 mM). After 1 hour, stimulants were added and incubated for 3 hours: 100 ng/mL LPS from

Escherichia coli (Sigma-Aldrich, 10 ng/μL), 10 µg/mL Pam3CSK4 (InvivoGen, 1 mg/mL), or 10 ng/mL TNF-α (InvivoGen, 10 ng/uL). Whole blood specimens containing no stimulant or DMSO were used for controls. Plasma was isolated by centrifugation at 800 x g for 15 minutes and stored at -80 °C, then thawed once in batch, for analysis. 10

Flow Cytometry

Monocyte subsets were identified by size, granularity, and positive expression of surface markers CD14 and CD16. Expression of monocyte activation markers was measured in-vitro by flow cytometry (MACs Quant 10; Miltenyi Biotec) after staining cells with the following fluorochrome-labeled antibodies: anti-CD14 pacific blue, anti-CD16 phycoerythrin (PE), anti-

HLA-DR APC-cy7, anti-CD69 PE-cy7, anti-CD86 FITC, and the appropriate isotype control monoclonal mouse antibodies (all BD Biosciences).

Whole blood samples were incubated on ice for 15 minutes with FACS Lyse buffer (BD

Biosciences) and then washed in flow wash buffer (phosphate-buffered saline with 1% bovine serum albumin and 0.1% sodium azide). The cells were then stained with monocyte surface marker antibodies for 30 minutes in the dark on ice, washed in flow wash buffer, fixed in 1% paraformaldehyde, and then analyzed. Analysis of data was performed using a MACs Quant software (Version 2.10; Miltenyi Biotec) and Prism 6.0 GraphPad software.

Measurement of Cytokines

Levels of IL-6, IL-1β, and TNF-α in plasma samples, collected as listed above, were measured using Quantikine ELISA Kits from R&D Systems (Human IL-1β/IL-1F2 Immunoassay

Catalog Number SLB50, Human IL-6 Immunoassay Catalog Number S6050, & Human TNF-α

Immunoassay Catalog Number STA00C). Plasma was diluted 1:5 for IL-1β and TNF-α measurements and 1:10 for IL-6 cytokine measurements.

Chapter 4: Results

Activation of Monocytes – Monocyte Subset by Flow Cytometry

Monocyte subset representation was measured (traditional, CD14+CD16-; inflammatory,

CD14+CD16+; and patrolling, CD14dimCD16+, see figure 1) in whole blood samples and comparisons were made between no stimulation samples and samples that were exposed to stimulants. A significant decrease in traditional monocytes (p=0.005) and a significant increase in inflammatory monocytes (p=0.05) was observed between no stimulation and 100 ng LPS stimulation (see figure 2). LPS stimulated whole blood may reflect the in-vivo consequences of microbial translocation in HIV-infected people. Exposure to LPS induces pro-inflammatory cytokine release and monocyte activation markers within 3 hours. LPS is a TLR-4 agonist, which is involved in pro-inflammatory cytokine release. Additional stimulants, Pam3CSK4, a TLR-1/2 agonist, and TNF-α were used for comparison purposes, as these molecules also induce monocyte activation and may be important in monocyte activation in HIV infection. Oxidized

LDL was also used as a stimulant of monocyte activation, but a reliable and consistent stock of oxLDL was not available from manufacturers, and as a result, oxLDL activation was removed from the analysis.

Figure 1. Monocyte subset representation in a flow dot plot (Traditional CD14+CD16-; Inflammatory CD14+CD16+; patrolling CD14dimCD16+) demonstrating a shift in subset counts from no stimulation and LPS stimulation (100 ng/mL), and minimal inhibition with genistein pre- incubation and LPS (100 mM + 100 ng/mL).

Figure 2. Monocyte subset representation (Traditional CD14+CD16-; Inflammatory CD14+CD16+; patrolling CD14dimCD16+) of whole blood between no stimulation and 100 ng/mL LPS from Escherichia coli (Sigma-Aldrich, 10 ng/μL) after 3-hour incubation. A significant decrease in the tradition monocytes (p=0.005) and a significant increase in inflammatory monocytes (p=0.05) induced by LPS, is shown. No significant inhibition in monocyte subsets was observed with genistein and other isoflavones.

Specific monocyte activation surface markers, CD86, HLA-DR, and CD69 were measured after stimulation. These markers are increased among monocyte subsets in HIV-infected individuals, compared to levels measured on these cells in HIV-uninfected populations29. After individual stimulation of LPS, Pam3CSK4, or TNF-α, the average traditional cells within the

“traditional” monocyte subset had a significant increase in expression levels of HLA-DR

(measured by mean fluorescence intensity, MFI, p=0.0004, p=0.0004, & p=0.007 respectively) compared to the expression measured on unstimulated cells. Exposure to these molecules also resulted in significant increases in HLA-DR expression on inflammatory monocytes (p=0.006, p=0.0008, & p=0.05 respectively). Surface expression of the monocyte activation marker CD69 was also increased significantly by all three stimulants (LPS, Pam3CSK4, and TNF-α) on traditional

(p=0.01, p=0.005, & p=0.007 respectively) and inflammatory monocytes (p=0.001, p=0.007, & p=0.008, respectively). Figure 3 is a visual representation of the shift in counts and HLA-DR expression with LPS stimulation and genistein.

Figure 3. Monocyte subset representation for HLA-DR MFI in a histogram overlay. Demonstrating a shift in subset counts and MFI from no stimulation and LPS stimulation (100 ng/mL), and minimal deviation with genistein pre-incubation and LPS (100 mM + 100 ng/mL).

Activation of Monocytes – Cytokine Release by ELISA

We also measured in-vitro activation of monocytes by release of pro-inflammatory cytokines following 3-hour in-vitro stimulation with LPS, Pam3CSK4, or TNF-α. The diluted plasma was analyzed for levels of the cytokines IL-6, TNF-α, and IL-1β; which are indicative of increased monocyte activity, and these cytokines have been shown to be increased in plasma samples from HIV-infected individuals25,30 and are associated with disease pathogenesis and CVD risk. Levels of the pro-inflammatory cytokines, TNF-α and IL-6, increased significantly after 100 ng LPS stimulation (p=0.02 and p=0.003, respectively). These in-vitro experiments have allowed us to establish culture conditions that recapitulate some of the monocyte activation phenotypes we have measured directly ex-vivo in samples from HIV-infected subjects (increased inflammatory monocyte proportions, increased surface activation markers, and increased production of inflammatory cytokines). We next wanted to explore the in-vitro effects of soy- based inhibitors on these markers of cellular activation.

Figure 4. Lesinski et al. analyzed plasma from 23 patients after a soy bread diet from day 0 and day 566. The mean fold change of 54 soluble cytokines are displayed, and most notable are reduced levels of the pro-inflammatory cytokines (IL-1β, TFG-β, TNF-α, INF-γ; shown in blue).

Soy Metabolite and Inhibitors Results – Flow Cytometry

In studies by members of our team involving men with prostate cancer who were administered a soy-based dietary intervention6, treatment with soy resulted in decreased plasma levels of cytokines, including IL-6, TNF-α, and IL-1β (see figure 4). Furthermore, the specific soy metabolites that may be responsible for the decreased inflammatory cytokines in the Lesinski study were measured by Ahn-Jarvis et al.; which demonstrated that the soy isoflavones, genistein and daidzein, were in high concentration in 24-hour urines of individuals consuming bread enriched with soy5,6. Glycitein is another known primary isoflavone of soy. We therefore wanted to explore the effects of pre-exposure of whole blood samples to these soy isoflavones to determine if they could inhibit induction of monocyte activation and cytokine expression by LPS, Pam3CSK4, or TNF-α in-vitro. The concentration of soy isoflavones to be used was determined using whole blood samples, pre-incubated with soy isoflavones at 50 mM and

100 mM. A concentration of 100 mM demonstrated a robust inhibitory effect on monocyte surface markers without being toxic to the cells. After pre-incubation with 100 mM of the individual soy isoflavones – genistein, daidzein, and glycitein – there were no significant inhibition in the activation of the three monocyte subsets induced with LPS and Pam3CSK4 stimulation as measured by flow cytometry. However, daidzein inhibited the activation of inflammatory monocytes induced by TNF-α (measured by decreased MFI for CD69, p=0.03).

Similarly, genistein may potentially inhibit the activation of inflammatory monocytes induced by

TNF-α exposure (p=0.06, figure 5), but this effect was not as significant as the inhibitory effect of daidzein. A larger sample population will be needed to determine the significance of genistein inhibiting the activation of monocyte subsets upon TNF-α exposure.

Figure 5. Monocyte subsets and MFI (mean fluorescent intensity) for monocyte surface marker, CD69, after 1-hour ex-vivo pre-incubation with soy metabolites and stimulation with 10 ng/mL TNF-α for 3 hours. CD69 MFI in the inflammatory monocytes significantly decreased with 100 mM daidzein (p=0.03), while 100 mM genistein decreased, but not as significant (p=0.06).

Known inhibitors that affect molecules downstream of TLR-4 – MYD88 inhibitor,

SB203580 p38 MAP kinase inhibitor, and Z-VAD-FMK were used as controls and for comparison to the effects of the soy metabolites. The MYD88 inhibitor peptide and SB203580, which blocks p38/MAP kinase signaling, affect the LPS signaling transduction pathway and Z-VAD-FMK is a pan-caspase inhibitor known to reduce monocyte cell death, which can cause an increase in inflammatory cytokine release. A TRIF blocking peptide was used in this preliminary study in replacement of a TRIF inhibitor, which also affects the LPS signaling transduction pathway. The results showed inhibition of monocyte activation, induced by LPS, with a significant decrease in

MFI for surface marker HLA-DR with several of the known inhibitors. The TRIF blocking peptide and MYD88 inhibitor significantly decreased the MFI for HLA-DR (p=0.01 & p=0.05, respectively) and the SB203580 p38 MAP kinase inhibitor potentially inhibited monocyte activation with a decrease in MFI for HLA-DR, (p=0.06), see figure 6. As reported above, exposure of whole blood samples to TNF-α resulted in increased expression of CD69 and exposure to LPS increased the expression of HLA-DR. Pre-exposure to these cells with the soy isoflavones resulted in no significant decrease in monocyte surface markers, which further indicates no inhibition of the three monocyte subsets.

CD14+CD16- Traditional

CD14+CD16+ Inflammatory

CD14DimCD16+ Patrolling

Figure 6. Monocyte subsets and the MFI for HLA-DR, after 1-hour ex-vivo pre-incubation with soy isoflavones or known inhibitors and stimulation with 100 ng/mL LPS for 3 hours. Inflammatory monocytes MFI for HLADR significantly decreased with the TRIF blocking peptide and MYD88 inhibitor (p=0.01 & p=0.05, respectively) and decreased average with SB203580 p38 map kinase inhibitor (p=0.06). A larger sample size would validate the decrease in MFI for HLADR with SB203580 p38 MAP kinase inhibitor.

Exposure of whole blood samples to LPS (100ng/mL) resulted in significantly increased levels of pro-inflammatory cytokines, including TNF-α and IL-6. Pre-exposure of whole blood samples to the individual soy isoflavones genistein and daidzein resulted in significant reduction in levels of TNF-α (p=0.005 & p=0.01, respectively) while glycitein also reduced concentrations but the changes were not as statistically significant (p=0.07). Additionally, pre-exposure of whole blood samples to SB203580, a p38 MAP kinase inhibitor, or Z-VAD-FMK also resulted in significant reduction in TNF-α (p=0.01 & p=0.006, respectively), see figure 7A. In contrast, pre- exposure of whole blood samples to individual soy isoflavones did not result in the reduction of

IL-6, however, IL-6 induction was blocked by SB203580, the TRIF blocking peptide, Z-VAD-FMK, and the MYD88 inhibitor (p=0.004, p=0.005, p=0.03, & p=0.01, respectively), see figure 7B.

Figure 7. A) Levels of TNF-α production induced by LPS were reduced by soy isoflavones and known inhibitors of TLR-4 signaling. Significance of p=05 was used for this study, however, due to low sample population, p=0.07 could potentially be significant if a larger population was studied. B) Levels of IL-6 release induced by LPS were reduced by known inhibitors of TLR-4 signaling.

Chapter 5: Discussion

Monocyte activation is an important indication of inflammation and atherosclerosis in all individuals, regardless of HIV-status. Infection with HIV and subsequent treatment with ART is associated with an increased risk for CVD, potentially due to chronic inflammation and immune activation25. This in-vitro soy isoflavone study was performed to evaluate modulation of inflammatory biomarker expression by soy isoflavones – genistein, daidzein, and glycitein – in comparison to known inhibitors downstream of Toll-like receptor 4 (TLR-4).

Lipopolysaccharide is a component of microbial cell walls and stimulates an immune response through activation of TLR-4. An increase in LPS levels in the human body can upregulate inflammatory cytokines and further invoke chronic inflammation and chronic immune activation in individuals infected with HIV24. We know that 100 ng/mL of LPS produces a strong monocyte stimulation from previous studies and decided to continue with it as the primary stimulant for preliminary soy metabolite studies to mimic the inflammation seen in HIV- infected people. Additional stimulants, Pam3CSK4, a TLR-1/2 agonist, and TNF-α were used for comparison purposes, as these molecules also induce monocyte activation and may be important in monocyte activation in HIV infection. Further study will be needed to evaluate how ox-LDL may stimulate monocyte activation, and possibly how this activation may be reduced with soy metabolites, in-vitro. This preliminary soy metabolite data indicates donor

24 variability in response to the addition of soy phytochemicals after LPS stimulation. Some factors that may explain the variability may be due to ethnicity, sex, and age of donors.

Cytokine analysis can provide great insight into what monocyte signaling pathways may be affected from individual soy isoflavones. The significant reduction in LPS induced levels of

TNF-α as a result of soy metabolite exposure indicate that these isoflavones could be affecting signaling cascades downstream of TLR-4. Several key mediators involved in expression of TNF-α have been identified (see figure 8). Nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), a major regulator of TNF-α, could have been affected by these soy isoflavones.

Furthermore, p38 MAP kinase is also downstream of the TLR-4 and the TNF-α receptor. This kinase can also regulate NF-κB activation within cells. As demonstrated in this study, the p38

MAP kinase inhibitor, SB203580, significantly reduced TNF-α production to a much greater extent than the individual soy isoflavones. This demonstrates that the efficacy of certain known inhibitors may be higher than the inhibition provided by individual soy isoflavones. Further studies should be done with a combination of the isoflavones, to determine whether the efficacy of soy compounds have a synergistic effect in the human body.

Figure 8. A cytokine signaling cascade from Tedgui and Mallat22. This diagram displays the possible areas of inhibition the soy metabolites could interfere (blue box added) and may explain the decrease in TNF-α secretion in the cytokine ELISA assays.

Furthermore, there may be differences in the ability of these soy isoflavones to inhibit induction of specific cytokines. While the soy isoflavones were able to inhibit production of

TNF-α as a consequence of LPS exposure, these isoflavones did not inhibit induction of IL-6.

There was little to no decrease with daidzein and glycitein; genistein does decrease IL-6 secretion, but not to the same extent as the known inhibitors. Again, there may be a factor of drug efficacy of the known inhibitors in comparison to soy isoflavones as a means of pro- inflammatory cytokine release. Exploring signaling molecules that are important for TNF-α production, but not for production of IL-6, downstream of TLR-4 may also provide insights into the specific molecules that may be inhibited by the soy isoflavones. If a signaling molecule is identified that may be important for TNF-α production, but not IL-6 production, we will assess the ability of soy isoflavone exposure to block activity of this molecule by more specific assays, i.e. western blots to detect phosphorylation of this signaling molecule.

Additionally, the flow cytometry analysis may demonstrate that the soy isoflavones do not inhibit signaling cascades involved in regulating surface expression of monocyte activation markers. As shown in figure 5, monocyte surface marker HLA-DR was not significantly affected by soy isoflavones, indicating that surface markers of monocyte activation were not as affected, in contrast to TNF-α release shown in figure 7A. As stated earlier, the isoflavones did not show inhibiting effects with IL-6 secretion. This may further indicate that the soy isoflavones effect a specific molecule upstream of TNF-α production that is not involved in production of IL-6 or

HLA-DR expression. This finding is of interest, as both TNF-α and IL-6 were reduced in the intervention trial where soy products were given to men with prostate cancer5,6. The results from the Lesinski et al. and Ahn-Jarvis prostate cancer trials in conjunction with this soy isoflavone study may indicate that combinations of soy isoflavones could potentially reduce 27 inflammatory cytokines. Furthermore, the in-vivo metabolism of the soy isoflavones by the microbial flora of the gastrointestinal tract of the men with prostate cancer may be involved in the reduction of multiple inflammatory cytokines in comparison to the in-vitro study and deserves further investigation.

We also have planned to explore the in-vivo effects of a soy bread product on inflammatory molecules in ART-treated HIV-infected individuals, or HIV-uninfected controls.

This study is currently enrolling. Based on our in-vitro studies here and the work by Lesinski et al., we hypothesize that soy intervention will lower TNF-α levels in HIV-infected participants, and potentially decrease proportions of CD16+ monocyte subsets28. This study may give us insights into the immunomodulatory effects of soy isoflavones in the setting of HIV infection, but we must also consider the effects of microbial translocation and dysbiosis33 in this population. We propose that alterations in the gut microbiome will likely result in changes in the soy isoflavones produced following soy-intervention. Therefore, we will compare the soy metabolite profiles in

HIV+ subjects to profiles in HIV- subjects following soy intervention. If dramatically different profiles are observed in our donor groups, this may have an effect on modulation of inflammatory markers. Based on the soy isoflavones we measure in samples from HIV+ participants, we may want to follow-up with more in-vitro studies using isoflavone combinations measured in HIV+ subjects.

A larger sample population and more monocyte surface marker antibodies were originally used within this study. Due to a massive recall of several antibodies associated with this experiment by the manufacturer Becton Dickenson, the experiments affected by this recall were not included in the results, lowering the sample size and our statistical power.

Furthermore, due to variability and unreliability in the oxLDL stocks used in the early phases of the in-vitro analysis, oxLDL data was excluded. Further studies using oxLDL from a different vendor, or by generating oxLDL in house, we hope to explore the effects of soy isoflavones on oxLDL induced monocyte activation. Despite these issues with our reagents, this study did provide results that demonstrated significant effects of soy-isoflavones on induction of inflammatory cytokine expression.

Chapter 6: Conclusion

Here, we provide evidence that soy isoflavones, including genistein, daidzein, and glycitein, can inhibit signaling cascades that result in production of TNF-a following LPS stimulation. This may be an important finding, as HIV-infected individuals have increased levels of LPS in their plasma as a result of microbial translocation. An increase in LPS levels in the human body can upregulate inflammatory cytokines and perpetuate chronic inflammation and chronic immune activation in individuals infected with HIV24. Chronic inflammation is associated with increased risk of cardiovascular disease in HIV infected subjects before and during suppressive antiretroviral therapy25. Several studies also discovered that oxLDL levels may play a critical role in monocyte activation and chronic inflammation4, and are also known to contribute to atherosclerosis. Further study evaluating monocyte activation with oxLDL and the response to soy isoflavones should be performed to explore the potential inhibitory effects of soy proteins on cytokine release and monocyte activation induced by oxLDL.

This preliminary soy study can be taken in many directions to discover the benefits of soy proteins. Further study will be needed to access the stages of intervention soy isoflavones inhibit along the cytokine signaling transduction pathway. As discussed earlier, the individual isoflavones do not inhibit cytokine release to the extent of other known inhibitors. A limiting factor can be that this in-vitro experiment with 3-hour stimulation may not fully recapitulate the

30 potential beneficial effects seen in-vivo with subjects consuming soy products and a 24-hour experiment should be done to evaluate the differences. Also, in-vitro experiments of a combination of the soy isoflavones may provide more information on their inhibition of monocyte activation. In the Lesinski soy study, a reduction in inflammatory cytokines was observed after the participants consumed soy bread6, which was formulated by Ahn-Jarvis and contained a mixture of soy isoflavones31. We could also measure more biomarkers that are known be associated with atherosclerosis and vascular inflammation, including Lp-PLA2, which hydrolyzes oxLDL.

Furthermore, this in-vitro study will need to be tested in an HIV-infected population to evaluate if soy isoflavones have similar effects in this population. An in-vivo clinical trial pilot is underway, and we hope to have results within the next year regarding levels of soy isoflavone metabolites in the blood and urine of HIV-infected participants, as well as if the soy intervention alters levels of immune activation in this population. If proven beneficial in the population of

HIV-infected individuals taking ART drugs, this soy study will allow them to have an opportunity to reduce CVD risk without adding to their pill burden with statins.

In closing, results from our in-vitro stimulation assays with LPS and soy isoflavones reported here, and the clinical trial results by Lesinski et al. demonstrate that soy isoflavones may improve the inflammatory profile of individuals by inhibiting TLR-4 signaling and reducing

TNF-α production. There may be a synergistic effect of soy isoflavones and this possibility should be tested in the next phase of study. This preliminary analysis can then expand to the

HIV-infected individuals taking ART to study the in-vivo inhibitory effects on their chronic immune activation, inflammatory lipid profile, and chronic inflammation.

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