Introduction
14.7-23.5 million people with autoimmune diseases in the US and is among the top ten leading causes of death (Khan, 2015). Immune responses and systems of human and rodent males and females differ in significant ways after puberty. Men and women can get the same types of age-related autoimmune disease, yet males experience them earlier and more dramatically than women (Bupp, 2015). After menopause, the rate of chronic inflammatory disease in women is equal to or greater than men’s (Bupp, 2015). Sex hormones on the immune system, genetic factors, and gender-specific behavior and exposure all contribute to sex-related differences in immune responses (Bupp,2015). Females exhibit higher basal immunoglobulin levels, increased numbers of circulating and resident CD4+ T cells and an increased CD4/CD8 T cell ratio as compared to males (Bupp, 2015). Estrogens (ERs) bind to three distinct receptors of either ER∝ or ERβ (Bupp, 2015). These ER can be found in T cells, B cells, dendritic cells, neutrophils, macrophages, NK cells, thymic stromal cells, and bone marrow (Bupp, 2015). Estrogen bound ERs move to the nucleus to regulate the expression of genes with estrogen responsive elements (EREs) in their promoters or they interact with other transcription factors (Bupp, 2015). In a way estrogen is like a promoter for immune response expression genes. Estrogens influence the development of T and B cells where high estrogen levels induce aging of the immune system which develops and maintains a robust and healthy immune system. Heightened immune reactivity is a double edged sword (Khan, 2015). It may further increase the risk of autoimmunity because over the promotion of extra production of immune cells which increase the escape of negative selection in B and T cells and therefore increases the risk of autoimmune diseases. On a more positive side, increased estrogen allows for enhanced diversity of T cell receptors (Bupp, 2015). Women are exposed to higher levels of estrogen until menopause at which time estrogen concentration in men can actually be greater than in women because of age-associated increased aromatization of testosterone in men (Khan, 2015). The average age of menopause is 51 (Bupp, 2015).
miRNA and the X chromosome are key sex-related factors that contribute to the robustness of the immune system and functions. X chromosomes encodes many immune-regulatory genes (Khan, 2015). The X chromosome has 112 microRNAs while Y chromosomes only has 2 (Bupp, 2015). 15% of X-linked genes escape silencing resulting in increased expression of certain gene products in females; this could be why females produce more immune cells than males in response to infections and pathogens (Bupp, 2015). miRNA are endogenous small noncoding RNA about 22 nucleotides long that function as post-transcriptional gene regulators.They are located in different loci of the genome (Bupp, 2015). They make up about 3% of DNA, but regulate about 30% of human genes (Bupp, 2015). They inhibit RNA by complementing and degrading mRNA or by translation repression. An important miRNA located on the X chromosome is miR-221/222 controls immune cell gene expression and regulation (Khan, 2015).
Men do not experience the same dramatic drop of androgen, a sex hormone, as women do. It steadily decreases at a rate of 1% a year after the age of 30 (Bupp, 2015). Despite this, BMI index has more of an impact of testosterone levels compared to aging (Khan, 2015). Androgens signaling through these receptors in immune cells serves to inhibit both innate and adaptive immunity, whereas estrogen promotes it which may be which females have more robust and active immune systems (Khan, 2015). Like estrogen, androgen also reduces thymic cellularity (state of a tissue or other mass with regard to the degree, quality, or condition of cells present in it) but do so at the expense of CD4/CD8 DP and CD4 SP thymocytes (Bupp, 2015). Aging and decreased autoimmunity is indicated by increased susceptibility to various infections, the reduced efficacy of some vaccinations, and a greater risk of chronic disease driven by chronic inflammation. Older people only produce 25%-50% influenza specific antibodies in response to the influenza vaccine compared to as younger individuals (Bupp, 2015). Males and Females experience the same age-related changes to the immune system, but males experience theme at a more dramatic accelerated rate (Bupp, 2015). After the 6th decade of life, a more dramatic reduction in the total numbers of B cells and T cells occurs in males (Bupp, 2015).
Taken together, sex hormones influence the cellularity and possibly the defense mechanisms of immune responses differently in men and women (Bupp, 2015). However, testosterone inhibits whereas estrogen excites. Impact of gonad-specific hormones on the immune system has been the focus of many studies examining gender differences in immunity, the potential impact of genes encoding on the sex chromosomes and gender-related environmental factors shouldn’t be overlooked. The scientific community has yet to completely explain why this disparity occurs in gene expression. Will microRNA injections excite expression genes in male rats and give them a more robust immune system. Will their immune cell production almost match female rats?
Logic and Reasoning
The questions that may be answered in this investigation are “Will microRNA injections excite expression genes in male rats and give them a more robust immune system. Will their immune cell production almost match female rats?”
In Khan et. al’s study featured in the Cellular Immunology scientific journal, they studied the different ways microRNA are expressed in autoimmune and control mice as well as male and female lupus-prone mice (Khan, 2015). They performed orchiectomies on a group of male mice, removing the testicles, injected them with estrogen, and found that they exhibited earlier expression and severity of lupus compared to their male counterparts that did not have their testicles removed and weren’t injected with estrogen.
Despite the study not mentioning the exact details of the procedure, I derived my experiment based off of their general experiment design. I want to feature healthy rats instead of lupus-prone rats, because I plan to study the general function of expression of microRNA in reaction to Influenza, which is also a more prevalent disease. Instead of looking at which genes and microRNA are expressed and what the sexual bias is, I will be focusing on the expression of miR-221/222 and manipulating the male rat immune system to see if it can fight a disease better by producing more immune cells that almost match female immune cell counts. While Khan’s study focuses on patterns, I plan on focusing in on the experimental aspects of increasing microRNA in male rats. Their study also looks at estrogens, which bind to certain receptors and are found in immune system; I want to focus specifically on microRNA expression and function, because male rats can’t exactly produce estrogen as much as female rats generally, because of the differential structure and function of gonads in males and females.
I hope to understand if microRNA could be a feasible solution to fighting diseases such as bacterial and viral infections instead of just antibiotics. If we find that injecting certain microRNA that boosts the immune system response in male rats could help them fight influenza better than without the microRNA, it could lead to further studies that explore options that cut antibiotics out completely. Taking antibiotics out of the equation may lead to a decrease prevalence of superbugs. Although my experiment is small, if my hypothesis is confirmed the field of studying microRNAs could be further explored. The only drawbacks are microRNAs could end up being as effective, similar to gene therapy. Additionally, more robust or reactive immune systems are also susceptible to autoimmune diseases as Khan’s study showed with estrogen.
In order to see if miRNA from the X chromosome injected in male rats works to age the immune system that would elevate their immune cell count to a closer ratio compared to females. The miRNA I’m focusing on is miR-221/222, which controls immune cell gene expression and regulation. The disease used in this experiment is a strain of influenza.
I would be looking at the results of the cell counts and compare the ratios based on sex of before influenza cell counts and after influenza cell counts in young, mature, and aged rates. A multifaceted statistical analysis for age and sex is required to see the results and establish a conclusion. A one sample, two sample, and matched pairs t-test would be best used to analyze the data.
Methods & Proposed Results
In order to see if miRNA from the X chromosome injected in male rats works to age the immune system that would elevate their immune cell count to a closer ratio compared to females. The miRNA I’m focusing on is miR-221/222, which controls immune cell gene expression and regulation. The disease used in this experiment is a strain of influenza.
The measurement of immune cell counts of T cells, B cells, dendritic cells, neutrophils, macrophages, NK cells, thymic stromal cells, estrogens, androgens, and testosterone must be taken before the injection of influenza, right after influenza is injected, and every 6 hours following the first measure after the injection for 7 days. Once the rats are injected with influenza, all of the male and female rats will be injected with the same amount of a placebo liquid. The same amount of influenza will be injected healthy young rats, healthy mature rats, and healthy old rats of both sexes. The rats should be treated as humanely as possible and feed them all the same amount of food and water. This would be a control group, since they will receive no treatment (miR-221/222).
The measurement of immune cell counts of T cells, B cells, dendritic cells, neutrophils, macrophages, NK cells, thymic stromal cells, estrogens, androgens, and testosterone must be taken before the injection of influenza, right after influenza is injected, and every 6 hours following the first measure after the injection for 7 days. Once the rats are injected with influenza, all of the male rats will be injected with the same amount of miR-221/222 delivered by viral vectors while all the female rats will be injected with the same amount of a placebo liquid. The same amount of influenza will be injected healthy young rats, healthy mature rats, and healthy old rats of both sexes. The rats should be treated as humanely as possible and feed them all the same amount of food and water. This would be the experimental group, since they will receive the treatment.
I would be looking at the results of the cell counts and compare the ratios based on sex of before influenza cell counts and after influenza cell counts in young, mature, and aged rates. A multifaceted statistical analysis for age and sex is required to see the results and establish a conclusion. A one sample, two sample, and matched pairs t-test would be best used to analyze the data.
I predict that the results will show that the amount of immune system cells in young, mature, and old male rats will have immune cell counts closer to that of females after they receive the microRNA injection. We might see that mature rats, specifically female rats, may produce more immune cell counts across all female rats. We will see that old male rats may produce more immune cells than old female rats. We will see that across the board, old rats produce less immune cell counts compared to young and mature in their respective groups. Other patterns could arise in the statistical analysis, which could indicate that microRNA could have a statistically significant impact on gene expression and production of immune system cells. Alternatively, any difference between the control group male rats and the experimental group male rats would expand our knowledge of the function of this specific and important microRNA.
If this procedure fails, it should be adapted and another trial should be conducted. At least 3 trials should be run.
Abstract
14.7-23.5 million people with autoimmune diseases in the US and is among the top ten leading causes of death (Khan, 2015). Immune responses and systems of human and rodent males and females differ in significant ways after puberty. Men and women can get the same types of age-related autoimmune disease, yet males experience them earlier and more dramatically than women (Bupp, 2015). miRNA expression and the X chromosome are key sex-related factors that contribute to the robustness of the immune system and functions. X chromosomes encodes many immune-regulatory genes (Khan, 2015). The X chromosome has 112 microRNAs while Y chromosomes only has 2 (Bupp, 2015). 15% of X-linked genes escape silencing resulting in increased expression of certain gene products in females; this could be why females produce more immune cells than males in response to infections and pathogens (Bupp, 2015). An important miRNA located on the X chromosome is miR-221/222 controls immune cell gene expression and regulation (Khan, 2015). In order to see if miRNA from the X chromosome injected in male rats works to age the immune system that would elevate their immune cell count to a closer ratio compared to females. The miRNA I’m focusing on is miR-221/222, which controls immune cell gene expression and regulation. The disease used in this experiment is a strain of influenza. I hope to find out if microRNA injections excite expression genes in male rats and give them a more robust immune system and if their immune cell production almost match female rats with miR-221/222 injections. This will be quantified by the measurement of immune cell counts of T cells, B cells, dendritic cells, neutrophils, macrophages, NK cells, thymic stromal cells, estrogens, androgens, and testosterone. I would be looking at the results of the cell counts and compare the ratios based on sex of before influenza cell counts and after influenza cell counts in young, mature, and aged rates. A multifaceted statistical analysis for age and sex is required to see the results and establish a conclusion. A one sample, two sample, and matched pairs t-test would be best used to analyze the data.
The results may show that the amount of immune system cells in young, mature, and old male rats will have immune cell counts closer to that of females after they receive the microRNA injection. Overall, I hope to understand if microRNA could be a feasible solution to fighting diseases such as bacterial and viral infections instead of just antibiotics. If we find that injecting certain microRNA that boosts the immune system response in male rats could help them fight influenza better than without the microRNA, it could lead to further studies that explore options that cut antibiotics out completely. Taking antibiotics out of the equation may lead to a decrease prevalence of superbugs. Although my experiment is small, if my hypothesis is confirmed the field of studying microRNAs could be further explored. The only drawbacks are microRNAs could end up being as effective, similar to gene therapy. Additionally, more robust or reactive immune systems are also susceptible to autoimmune diseases as Khan’s study showed with estrogen.
Literature Cited
Bupp, M. R. (2015). Sex, the aging immune system, and chronic disease. Cellular Immunology,294(2), 102-110. doi:10.1016/j.cellimm.2015.02.002
Khan, D., Dai, R., & Ahmed, S. A. (2015). Sex differences and estrogen regulation of miRNAs in lupus, a prototypical autoimmune disease. Cellular Immunology,294(2), 70-79. doi:10.1016/j.cellimm.2015.01.004
Kovats, S. (2015). Estrogen receptors regulate innate immune cells and signaling pathways. Cellular Immunology,294(2), 63-69. doi:10.1016/j.cellimm.2015.01.018