Vivek Krishnakumar (10903253) School of Biotechnology SRM University Cell Biology Lab, Pharmacology Division, Indian Institute of Integrative Medicine (CSIR) Jammu Tawi 15/05/2007 Immunomodulatory Activity of Silymarin and its Effect on Stress Advisors Dr. Sarang Bani (Scientist, IIIM, Jammu Tawi) Dr. R. S. David Paul Raj (Asst. Professor, SRM University)

15/05/2007 OVERVIEW 1. Introduction 2. Motivation 3. Objectives of work 4. Immunomodulatory study a. Protocol b. Results 5. Stress related study a. Protocol b. Results 6. in vitro TNFα estimation a. Protocol b. Results 7. Inferences & Conclusion 8. References

Immunomodulation 15/05/2007 Introduction A global term that refers to a wide range of drug or immunologic interventions that alter normal or abnormal immune responses by • Deletion of specific T or B cells • Immune deviation • Modification of various inflammatory pathways (e.g., chemotaxis, adhesins, or intracytoplasmic signaling). Immunomodulators (agents that exhibit immunomodulatory properties) can be categorized into 1. Immunosuppressants 2. Immunostimulants

15/05/2007 Introduction Silybum marianum Silybum marianum (Blessed milk thistle) is a milk thistle which belongs to the Asteraceae family which is a taxon of dicotyledonous plants. A fairly typical thistle with red and purple flowers and shiny green leaves with white veins. Originally, a native of Southern Europe through to Asia, it is now found throughout the world. The medicinal parts of the plant are the seeds and the fruits.

15/05/2007 Introduction Silymarin (C25 H22 O10 ) A plant-derived flavonoid isolated from the seeds and fruits of the Milk thistle, Silybum marianum. Active principle was first isolated and characterized in 1968-1974 (Wagner et al, 1968) Consists of 4 flavonolignan isomers (Wagner at al, 1974): • Silybinin (70-80%) • Isosilybinin (5%) • Silydianine (10%) • Silychristine (20%)

15/05/2007 Silymarin Properties 1. Hepatoprotective (liver protection) 2. Antidepressant (alleviate mental depression) 3. Antioxidant (maintains ROS levels) 4. Anti-inflammatory (inhibits PG, LTB synthesis; inhibits Neutrophil migration) 5. Chemopreventive (anti-cancer properties) 6. Immunomodulator 7. Demulcent (soothes inflamed mucus membranes) 8. Laxative (stimulates bowel evacuation) Introduction

15/05/2007 Motivation The work already done with Silymarin reveals to us its multifarious utilities. We know that the liver consists of Kuppffer cells (specialized macrophages that form part of the RET system). Since Silymarin is known to possess liver restorative capacities, it can directly be linked to be capable of exhibiting immune-stimulating activity. This study focuses on determining the effect of Silymarin on the Immune system and confirm whether it does possess immune-stimulatory activity or not. Introduction Motivation

15/05/2007 Objectives of Work • Study the immunomodulatory activity of Silymarin (in vivo) • Study the effect of Silymarin on stress- induced mice (in vivo) • Study the effect of Silymarin on intracellular TNF-α secretion by isolated murine neutrophils (in vitro) Motivation Objectives of work Introduction

15/05/2007 Animals Before Immunophenotyping or Intracellular Cytokine estimation can be performed, the animals need to be prepped. Grouped into cages with 5/6 animals per cage and marked with picric acid to differentiate between the mice. Grouping Head, Back, Tail Head-Back, Back-Tail, No Color Proper handling of mice Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Protocol for drug administration Ø Challenge with antigen: Challenging activates the immune system. Ø Day 0: Animals were challenged by i.p. injection of SRBC (5 × 109 cells) Ø Drug preparation: Using DW and Gum Acacia Ø Drug Admin: 2hrs after challenge. For a period of 7 days. Ø Dosage is measured as mg/kg body wt. of the mice. Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Protocol (contd.) Ø Challenge with SRBC. Performed to assess primary response. Ø Cyclosporine (a known immunosuppressant) and Levamisole (a known immunostimulant) used as negative and positive controls respectively. Ø 48hrs after challenge (Day 8), blood drawn from the ROP. Ø Blood processed as per the standardized protocol and the sample analyzed by Flow Cytometry. Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Study of Immunomodulatory activity Immunomodulatory activity of Silymarin was studied by using the following technique: IMMUNOPHENOTYPING AND INTRACELLULAR CYTOKINE ESTIMATION BY FLOW CYTOMETRY Both IMMUNOPHENOTYPING and INTRACELLULAR CYTOKINE ESTIMATION focus on the assessment of lymphocyte populations in Acquired immunity. Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Flow Cytometry PRINCIPLE • Uses principles of light scattering, light excitation and emission of flourochrome molecules to generate specific multi-parametric data from particles and cells in the size range of 0.5µm to 40µm diameter. • Lasers are most often used as a light source WORKING • Each suspended particle passing through the beam scatters the light in some way. A combination of scattered and fluorescent light is picked up by the detectors and by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is then possible to extrapolate various types of information about the particles. Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Immunophenotyping A method that involves determining the cells of the immune system by phenotyping them based on the cell surface markers. The surface markers analyzed in this study were: 1. CD8+ (T-cytotoxic cells) 2. CD4+ (T-helper cells) To determine the presence of a particular type of cell, the surface markers are tagged with flourochromes. When run in a flow cytometer, the cells are separated and counted based on the attached flourochrome. Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Intracellular Cytokine Estimation Cytokines : Signaling compounds secreted by Immune cells, which help in regulating the activity of the immune system by autocrine, paracrine and endocrine routes. Estimation of the secreted cytokines (extracellular) is not practically possible because they are not localized. On the other hand, intracellular cytokines are localized within the Golgi and hence can be targeted with Flourochrome + MAbs directed toward the cytokines. The cytokine analyzed in this study was IFNγ. Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 The flourochromes used for the experiments were: 1. Flourescein Iso Thio Cyanate (FITC) 2. Phycoerythrin (PE) The flourochromes were attached to monoclonal antibodies directed against the cell surface markers, namely, CD4+, CD8+ and CD244 and the cytokines, namely, IL12, IFNγ and TNFα The combinations of flourochromes and MAbs used were: • FITC – anti-CD4+ • PE – anti-CD8+ • FITC – anti-IFNγ Flourochromes Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Results Immunophenotyping Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Results Intracellular Cytokine Estimation (IFNγ) Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Stress Stress The constellation of events that begins with a stimulus (stressor), which precipitates a reaction in the brain (stress perception) that subsequently results in the activation of certain physiologic systems in the body (stress response). It refers to the condition where coping with the various actual or perceived stimuli alters the homeostatic state of the organism including behavioral, endocrine and immunological changes. 2 types of stress are: a. Acute Stress b. Chronic Stress Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Stress & the Immune System All lymphocytes have adrenergic receptors. Differential density and sensitivity of these receptors on lymphocytes may affect responsiveness to stress Sympathetic fibers release a wide variety of substances that influence immune responses by binding to receptors on white blood cells. Acute stress generally has positive effect while chronic stress typically provokes immunosuppression. Psychological stress raises catecholamine levels, which suppresses the immune system. Stress Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Protocol Ø Mice subjected to restraint stress in well-ventilated 50 ml polypropylene tubes for 6 hours during the light cycle during the experimental period of 12 days. Ø After 6 hours of restraint, mice removed from tubes. 2 hours after de-stressing, drug is administered. Ø After 12 days, blood drawn from ROP and processed as per the standardized procedure and analyzed by flow cytometry. Stress Immunomodulatory Study Motivation Objectives of work Introduction Ø Groups: Naïve control (not subjected to any stress); Restraint stress (RST) control; RST + Drug

15/05/2007 Results Immunophenotyping (CD8+/CD4+) Stress Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Immunophenotyping (CD244) Results Stress Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Intracellular Cytokine Estimation (IFNγ, IL12) Results Stress Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Intracellular Cytokine Estimation (TNFα) Results Stress Immunomodulatory Study Motivation Objectives of work Introduction

15/05/2007 Motivation Objectives of work Introduction in vitro TNF-α secreted by Neutrophils Neutrophils are the most abundant type of WBCs and are the first immune system cells to rush to the site of an infection. Neutrophils are known as phagocytes because they engulf foreign organisms and use prepackaged chemicals to destroy them. TNFα (Tumor Necrosis Factor) is a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction. On the in vitro activation of neutrophils by an agent, TNFα is secreted. The measure of the amount of TNFα secreted is a good indicator of the level of activation of the immune system. Stress Immunomodulatory Study in vitro TNFα

15/05/2007 Motivation Objectives of work Introduction Protocol Blood is drawn from naïve mice for this purpose. It is processed and the neutrophils are isolated. The isolated supernatant is incubated along with LPS (Lipopolysaccharide - derived from E. coli) and the drug at varying doses in a CO2 incubator. A separate group called LPS control was taken, to which no drug was added. Following the 3 hr incubation, the flourochrome tagged anti – TNFα MAb was added and the sample was run in the flow cytometer to determine TNFα levels. Stress Immunomodulatory Study in vitro TNFα

15/05/2007 Motivation Objectives of work Introduction Results Stress Immunomodulatory Study in vitro TNFα

15/05/2007 Motivation Objectives of work Introduction Results Stress Immunomodulatory Study in vitro TNFα

15/05/2007 Immunomodulatory Study Motivation Objectives of work Introduction Inferences & Conclusion From the Immunophenotyping and Intracellular Cytokine estimation data, Silymarin displayed commendable dose dependent immunopotentiating activity. Most effective dose was 100 mg/kg body wt. Its immune-potentiating activity was confirmed by studying TNF-α secretion by activated Neutrophils in vitro. These studies actuated the test of its activity on mice subjected to stress. The deleterious effect of chronic stress was countered by Silymarin, which was able to hype up the immune parameters to respectable levels, thus demonstrating good stress-busting activity. Stress in vitro TNFα Conclusion

15/05/2007 Stress Immunomodulatory Study Motivation Objectives of work Introduction in vitro TNFα References References Conclusion Wagner H., Horhammer L., Munster R., 1968, The chemistry of silymarin (silybin), the active principle of the fruits of Silybum marianum (L.) Gaertn. (Carduus marianus) (L.). Arzneimittelforschung, 18, 688-696 Wagner V.H., Diesel P., Seitz M., 1974, Chemistry and analysis of silymarin from Silybum marianum (L.) Gaertn, Arzneimittelforschung, 24, 466-471

15/05/2007 Acknowledgements Dr. K. Ramasamy Dr. R. S. David Paul Raj Dr. Sarang Bani Ms. Beenish Khan Ms. Prabhavathy Devan Mr. Deepak Bhagwat Ms. Anjali Pandey Ms. Kiranjeet Ms. Romaisa Latef Thank you !!