BM214 Immunology Lab 2

Immunology Lab 2: The Role of Macrophages in Protection from TB

Learning Objectives

1. Determine the importance of nitric oxide production from macrophages in response to Mycobacterium tuberculosis (M. tuberculosis).

2. Establish the roles of cytokines Interferon-gamma (IFNγ) and Tumor Necrosis Factor-alpha (TNFα) in stimulating nitric oxide production.

3. Learn to produce a standard curve in Excel using linear regression analysis.

4. Use the derived equation to determine nitrite concentrations from experimental data.

Macrophages

• Function and Role:

  • Macrophages are immune cells that recognize pathogens through Pattern Recognition Receptors (PRRs).

  • They can also recognize pathogens that are coated with antibodies or complement components.

  • Protect against infections primarily through phagocytosis.

• Host Cell for TB:

  • Macrophages serve as host cells for Mycobacterium tuberculosis.

  • Utilize two essential microbicidal mechanisms to destroy phagocytosed pathogens:

  • Superoxide production.

  • Nitric oxide (NO) production.

  • Activation of macrophages is required for nitrogen oxide production, necessitating interactions with T cells and stimulation from cytokines (specifically TNFα and IFNγ).

  • This activation leads to the formation of a granuloma at the infection site.

Interaction of TB and Macrophages

• M. tuberculosis can evade macrophage defenses and replicate within them using various virulence strategies, specifically by preventing the formation of phagolysosomes.

• Nitric Oxide Production:

  • Production occurs through Toll-like receptor (TLR) activation.

  • Requires expression of inducible nitric oxide synthase (iNOS), which catalyzes the reaction converting the amino acid L-arginine to NO.

Investigative Protocols

• Suspicion of TB in Students Returning from South Africa:

  • Initial actions include:

  • Take blood samples to collect and count peripheral blood leukocytes (PBLs).

  • Culture PBLs in the laboratory and stimulate with TB antigen.

  • Collect and freeze media from stimulated cells:

    • Stimulated PBLs will release immune mediators into the culture medium.

    • Test levels of cytokines or other immune mediators through immunological assays to evaluate impacts on other cells.

Experimental Setup for the Griess Assay

• Objective: Test for nitric oxide production from macrophages through the Griess assay, which is a sensitive colorimetric assay for measuring nitrite.

• Two Primary Experiments:

  1. Assess if supernatants from PBL cultures can activate macrophages to release nitric oxide.

  2. Conduct experiments using blocking antibodies to ascertain the cytokines involved in NO production.

Griess Assay Details

• Main Steps in the Griess Reaction:

  • Nitric oxide is a highly reactive species with a brief half-life, rapidly converted to nitrite (NO2-) and nitrate (NO3-).

  • **Griess reagent composition:

  • Sulphanilamide

  • Phosphoric acid

  • Naphthylethylenediamine dihydrochloride**.

  • In acidic conditions, sulfanilic acid reacts with nitrite to form a diazonium salt, coupled with N-(1-naphthyl)ethylenediamine, producing a purple-red compound, quantifiable by absorbance at 570 nm.

Experiment One: Macrophage Activation

• Key Hypothesis:

  • Can PBLs activate macrophages to produce nitric oxide?

  • Requires PBLs to secrete specific cytokines that enhance macrophage antimicrobial mechanisms, resulting in nitric oxide production.

  • Measurements will utilize the Griess assay to quantify the production of NO.

• Cell Culture Setup:

  • Cultures derived from healthy individuals and suspected TB patients, grown for 72 hours under varying conditions:

  • Presence or absence of TB antigen.

  • Presence of Concanavalin A (ConA), which activates T cells and NK cells.

  • Labels:

  • H1 for healthy controls

  • P1 for suspected TB patients.

  • Each group has 5 samples with 4 replicates each.

• Standard Curve Creation for Nitrite:

  • A standard nitrite solution of 100 µM will be utilized, with serial dilutions prepared in a 96-well plate.

  • Procedures for adding media and creating doubling dilutions should be meticulously followed, ensuring accurate pipetting.

Experiment Two: Cytokine Activation of Macrophages

• Objective:

  • Identify which cytokines from T cells activate macrophages to produce NO.

  • Utilization of blocking antibodies to neutralize PBL-derived cytokines, determining their specific contributions to NO release, measured by Griess assay results.

• Layout for Second Plate:

  • Distinct structures for healthy controls (H), TB antigen exposure (H+), and patient samples (P and P+).

  • Inclusion of media only, isotype control antibodies, and various cytokine-specific blocking antibodies.

Data Analysis Methods

• Display one of the standard curves generated from either task as a part of analysis:

  • Present the correlation coefficient and trend line equation on the graph utilizing the format y=mx+c.

  • Extract quantitative data from all patient results on plates 1-5 from MyPlace.

  • Compile findings in a summary table that averages technical replicates and calculates standard deviations (SD) and standard error of means (SEM).

  • Perform a Mann-Whitney U test to compare significance between the healthy control and patient data sets.

Plotting a Standard Curve Using Excel

• Instructions for Standard Curve Creation:

  • Insert charts and select the scatter plot for known concentrations (x-values) against optical density (y-values) measured at 570 nm.

  • Ensure proper labeling of axes and include a trend line on the chart.

Workshop Information

• Griess Assay Workshop:

  • Scheduled for Monday, November 10th with two session options (9-11 am & 1-3 pm).

  • Students should bring devices for calculations, along with relevant data analysis work from the lab. Recommendations include addressing data analysis promptly after the lab session.

  • Support will be available during the workshop, with resources provided through MyPlace.