Proper functioning of the human immune system is pivotal for keeping various diseases at bay. After many years of research dedicated to understanding what governs immunity, scientists have identified molecules with important roles in the immune response. These molecules, also referred to as immune response biomarkers, are often present at lower or higher-than-normal levels, or produced at altered rates, when the human body is fighting disease.
The human immune system is extremely complex and understanding the intricate details of its functioning in specific conditions, or disease, is still a major challenge for medical researchers. Therefore, it would not be wise to attempt to oversimplify such a topic in a short article. Instead, in the text that follows, we will limit the scope of our discussion to several important immune response biomarkers and their utility in in vitro bioassays designed to assess the effects of plant extracts or natural compounds on immune system function.
Immune response biomarkers. T lymphocytes, or T cells, are types of white blood cells produced in the bone marrow that mature in the thymus. Together with B lymphocytes, T lymphocytes play a vital role in the immune response. When T cells become activated in response to specific pathogens, such as viruses or bacteria, they produce substances called cytokines. Cytokines are regulatory proteins that function as cellular mediators of immune response and inflammatory reactions.
Examples of cytokines include interleukins, such as interleukin-1a (IL-1α), interleukin-1β (IL-1 β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), etc. In certain autoimmune diseases, proinflammatory cytokines such as TNF-a are overproduced, which leads to an imbalance in the body. Therefore, finding effective cytokine inhibitors has become of interest in the search for therapeutic solutions to immune-system-mediated diseases, such as psoriatic arthritis.1
Another category of immune response biomarkers are growth factors. Growth factors are proteins involved in regulation of cellular growth, proliferation and differentiation. Examples of growth factors include Epidermal Growth Factor (EGF) and Vascular Endothelial Growth Factor (VEGF). For illustration, VEGF promotes the growth of endothelial cells and VEGF signaling is required in angiogenesis (blood vessel formation from pre-existing blood vessels) and vasculogenesis (de novo blood vessel formation).2 Scientists have found that VEGF is up-regulated in many tumors where it functions as a promoter of angiogenesis.
Clearly, many immune response biomarkers have implications in the onset and progression of a range of diseases, from autoimmune disorders, across inflammatory conditions and allergies, to various cancers. Therefore, developing assays that can accurately detect the effects of bioactive molecules from plants, on their production/inhibition and/or functioning, is of great interest to researchers. Let’s explore some of the available assays.
The TNF-a inhibition assay. As mentioned earlier, TNF-a is a proinflammatory cytokine, capable of triggering a cascade of cellular signaling reactions governing inflammation. It is overproduced in certain immune-mediated, inflammatory diseases. The TNF-a inhibition assay is a cell-based assay designed to test a compound’s, or extract’s, ability to inhibit the production of TNF-a. In this assay, human cells are first treated with the test compound to allow for its natural absorption into the cells. Following, the cells are then stressed with a TNF-a inducer, which stimulates TNF-a production. Cells used as the control are left untreated. If the test compound has an inhibitory effect on TNF-a production, the level of inhibition will be determined by comparison of TNF-a levels between treated and non-treated cells. Cellular inhibition of interleukins can also be assessed in a similar way in this type of assay.
The Nrf2 activation assay. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor which is often referred to as the ‘’master regulator’’ of the antioxidant response.3 This small molecule has a powerful effect on the human organism at many different levels by modulating the expression of many genes that control immune and inflammatory responses, carcinogenesis/metastasis, tissue regeneration and fibrosis, and even cognitive functioning.3 One previous study has shown that disruption of Nrf2 expression enhances susceptibility to severe airway inflammation in a mouse model of allergic asthma,4 indicating the important protective role that Nrf2 plays in allergic and asthmatic responses of an organism. In the Nrf2 activation assay, the impact of the test compound on cellular Nrf2 expression is studied to assess its potential anti-allergic function.
The T-cell proliferation assay. The effects of plant phytochemicals on T-cell proliferation can be assessed be several related approaches. One of them is to monitor the survival of activated primary human T-lymphocytes in culture. In this assay, purified human T-lymphocytes are pretreated for a defined period of time with the test compound(s) at multiple concentrations. Following preincubation, purified T-lymphocytes are activated with soluble anti-human CD3 and soluble anti-human CD28 antibodies. Cells used as the control are treated with the solvent only. Twenty-four hours later, cell death is assessed by flow cytometry.
Whole blood culture assays. Biomarkers of specific immune pathways can also be assessed in vitro in whole blood cultures. In this type of experiment, stimulated and unstimulated whole blood cultures are incubated with the test compound, and enzyme-linked immunosorbent assays are used to detect various biomarkers of inflammation, such as interleukins and Interferon gamma, in the culture medium.6
Things to consider in immune function testing. Immune system functioning involves a cascade of interactions between a myriad of molecules. Therefore, the best approach to testing the effects of natural compounds on immune system function is to cast a broad net and utilize a range of different assays. This is especially true for those compounds or extracts where limited information is available from previous studies or traditional medicine applications. Each assay can provide useful information about the immune system’s response to the test compound from a different perspective. The results from different assays can help make sense of complex interactions and identify promising biomolecules for further clinical testing.
- 1. Köhm M, Burkhardt H, Behrens F. Anti-TNFa-therapy as an evidence-based treatment option for different clinical manifestations of psoriatic arthritis. Clin Exp Rheumatol. 2015; 33: S109-14.
- 2. Roskoski, Robert Jr. Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev Oncol Hematol. 2007; 62:179-213.
- 3. Hybertson BM, Gao B, Bose SK, McCord JM. Oxidative stress in health and disease: The therapeutic potential of Nrf2 activation. Mol Aspects Med. 2011; 32:234-246.
- 4. Rangasamy T, Guo J, Mitzner WA, et al. Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice. J Exp Med. 2005; 202(1):47.
- 5. Hushmendy S, Jayakumar L, Hahn AB. Et al. Select phytochemicals suppress human T-lymphocytes and mouse splenocytes suggesting their use in autoimmunity and transplantation. Nutr Res. 2009; 29(8):568-578.
- 6. Hendricks R, Pool EJ. The in vitro effects of Rooibos and Black tea on immune pathways. J Immunoassay Immunochem. 2010; 31(2):169-80.
Author Jasenka Piljac Zegarac is a scientist and freelance writer. She holds a PhD in biology and a BS degree in biochemistry, and contributes on a regular basis to several health and science blogs. Her research articles have gathered more than 900 citations. She may be contacted for assistance with a variety of science and medical writing projects. Find Jasenka on LinkedIn.