Importance of Macrophages, IL-6 and IL-4 in Exercise

Introduction:

Chronic inflammation is linked to sedentary life style such as Obesity, which is an important concern in human health (11,26).The experienced lifestyle of Western societies have shift toward less physical activity, and consumption more of the energy-dense food; in which this lead to increase of obese and overweight people(2,11). Generally, obese is associated with low-grade inflammatory state, which is reflected by the activation of immune system cells in adipose tissue (2). However, in England, statistics of National Health Service in 2013 shows 61.9% of adults and 28% of children which aged from 2 to 15 are overweight (36), and according to Welsh Health Survey in 2012, shows 59% of adults are obese (37). Consequently, this may increase the risk of having type 2 diabetes mellitus and cardiovascular disease (1, 2, 3,26). The contraction of the skeletal muscle lead to producing many protein, but if there is no physical activity may resulting in altering of myokines response(1,19). Therefore, this may give an evidence the association between sedentary behavior and many chronic disease. In addition, exercise play an important role in function and metabolism of many organs (1, 2,3).

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Human health is an essential concern in our life. However, it is very important that we know something that nobody yet has done a research experiment, which help us to focus on something that we need to know, especially the thing which related to human health, so there is currently a gap in our knowledge about something that associated with our body. Therefore, the gap is exercise seems to cause cells such as monocyte-macrophages to produce more IL-4 regulating signaling, but as many studies have been published, showed that exercise is known to cause an increase in IL-6 not in IL-4, so they do not understand why it seem the stimulus response not matchup IL-6, IL-4 stimulus and IL-4 dependent response(2)?

In my project, I cannot replicate the experiment which was done in vivo in living mice, which was already done it, and as I am a student science and working in a biomedical science , I have to think about human(2). However, in vitro study, this review aim is to treat monocyte-macrophages with IL-6 and determine whether Dectin-1 increases, which mean if IL-4 express increase, then that people who did the experiment in mice in 2014 are correct, in which would expect that genes which controlled by IL-4 signaling will also increase(2).

The importance of macrophages and IL-6, IL-4 associated in exercise:

Macrophages are found in the body tissue, in which their function is to ingest and process of dead cells, foreign materials during response to signal of inflammatory. They are normally plasticity in which they can change their job phenotype in response to the signal of the microenvironment (4, 17, 24). Therefore, they are highly heterogeneous cells. Macrophages are released as monocyte, and within a few days, they start their function in the tissues (4, 17). In addition, when monocyte goes from the circulation, they differentiate in to macrophages (4, 17, 23, 24). Monocyte-Macrophages playing an important role in phagocytic cells (4). They are two types of macrophages such as M1 phenotype, which act as pro-inflammatory and a defense from different of viruses, bacteria and protozoa, and another type M2 phenotype, which function as anti-inflammatory and regulating the wound healing (4, 23, 24). Generally, after phagocytosis finished, M1 macrophages begin the polarization to M2 phenotype, in which that M1 phenotype loss their properties of pro-inflammatory (4, 14, 17, 22, 23) Figure (1). Nevertheless, the changes of monocytes in to macrophages in the muscle appear to be the most reaction of the immune cell after the damage of skeletal muscle (4, 23, 24). Generally, Obesity associated inflammation are a central mediator by macrophages (2, 14). So, in my project, my aim to treat and investigate the signaling in these cells by IL-6.

In chronic inflammation, there is an increase in the inflammatory activity, as the inflammatory cells such as the monocyte-macrophages detect inflammation stimuli and either they destroy them, or antigen presenting cells to make the lymphocyte to produce antibodies (1, 2). At certain period, it will cause to increase the activity which is useful to destroy the infectious agent. However, in the acute inflammatory state there is a second phase of process which return the inflammatory back to normal, so there is a load of pro-inflammatory processes and then a set of anti-inflammatory processes (e.g. IL-4), which is a signal molecules produced by cells of macrophages who is functioning is to be anti-inflammatory, in which resulting to an end to anti-inflammatory episodes to come back to normal (1, 2, 3, 4, 5, 13, 14, 20). Therefore, there is a need to increase IL-4 signaling in order to suppress chronic inflammation diseases such as Type 2 diabetic mellitus, cardiovascular disease and Obesity (19). For example, it is important to have exercise, in order to boost IL-4 signaling in monocyte-macrophages. Interestingly, IL-6 release from contracting myocytes is essential for IL-4 induced activation of macrophages (1, 2, 3).

In 2010, researcher published, the gene in which has known to be regulator by IL-4, the expression was went up, so it seem that it was increasing IL-4 signaling, but surprisingly the scientist they couldn’t find IL-4 in the blood stream in the people who have been exercising(3,14). Indeed, they saw an increasing in IL-6. As many studies showed that exercise is linked to increase in IL-6 not in IL-4, so this is the question that it need to focus in my project (1, 2, 3, 8). Another study, in 2011-2012, which state that the genes which controlled by IL-4 increase in expression, which suggest that it can have this beneficial effect, but they couldn’t find IL-4 itself, or there is no increase IL-4 itself (1,2). Basically, the measurement of IL-4 is by ELISA or by looking the amount of IL-4 that cells producing by using RT-PCR (6, 8). Last scientific studies by Mauer et al (2014), where they treated cells with IL-6 to see what result will find. Many studies showed that when we do the exercise our skeletal muscle produce IL-6, but as this study stated an increase of IL-4 also, in which the scientist surprise from this study in 2014(2, 13). They said that IL-6 causes monocyte-macrophages to increase expression of the IL-4 Receptor protein in mice (vivo), in which it can imaging that small amount of IL-4 in the circulation, but the cell have got more of the receptor of IL-4(10). As a result, this lead the cell to be more sensitive to IL-4(1).To be more precise, if a cells which is on its surface is a protein to which IL-4 will bind, and if there is a certain amount of IL-4 in the system, then it will have a certain amount of IL-4 dependent signaling within that cell. Therefore, if IL-4 increase, then it will lead to find more of IL-4 signaling, and these are IL-4 genes which resulting to increase expression (2). For this reason, this will lead to anti-inflammatory effect, in which IL-4 functioning process happen during inflammatory state. In most cases, scientist before they do not see more IL-4, in which these people in 2014 see more of the protein to which IL-4 bind(2). In addition, if the cell has more of the receptor, that will make almost more sensitive to whatever level of IL-4 depending signaling within the cells. However, these researcher did this experiment in macrophages in mice, which is an interesting, but in my side I have to do the experiment in human cells. This will make the macrophages more sensitive to IL-4, rather than producing more IL_4 (2).

IL-6 release from contracting muscle cells:

IL-6 is a type of cytokines and linked to resistance to insulin and Obesity. However, deficiency in IL-6 resulting to adult to begin Obesity, and IL-6 signaling disruption cause resistance to insulin (22). Many studies showed that IL-6 play an essential role in pro-inflammatory, therefore Obesity- associated inflammation is linked by IL-6(2, 5, 7, 10, 12, 18, 21, 22). In addition, pro-inflammatory M1 macrophages which act by IL-6 are a mediators of Obesity- induced inflammation and resistant to insulin (2, 11, 12). A published study by Mauer et al 2014, illustrated that IL-6 as a critical initiator of the macrophages M2 polarization, and it is useful in role of the prevention of obesity which is associated resistance to insulin (2, 5, 19, 22). As a result, the assumption of this find is that IL-6 acts as a pro-inflammatory cytokine (21). Moreover, the observation by Mauer et al 2014, demonstrate that IL-6 act also as anti-inflammatory cytokine in which it limit LPS ( Lipopolysaccharide )-induced endotoxemia, and not only acted to prime myeloid cells by IL-4 signaling during obesity(2). However, more than a decade ago that role was assigned to IL-6 (2).

During muscle contractions the cytokines IL-6 is the first myokines found in the blood stream (1, 5, 13, 20). Generally, healthy individual and patient with Type 2 Diabetes mellitus (T2DM) released IL-6 from human primary muscle cell cultures (1, 5, 18, 22). Nowadays, the dominant source of IL-6 production during exercise is muscle cells (1, 5, 18, 21). However, sometimes during physical activity the IL-6 level increase in the circulation without any sign of muscle damage, and macrophages is responsible for this increase. In addition, the transcription rate of IL-6 and IL-6 mRNA level is increased within 30 minutes of the start of exercise (1, 19, 21, 22). Moreover, the insulin-stimulated glucose, which is uptake in muscle cells in vitro increased by IL-6.As a consequence, treatment with IL-6 will increase the rate of glucose infusion without having any effect on the total glucose production. When human IL-6 infused into a healthy people during low-intensity exercise, in order to mimic the concentration of IL-6 during high intensity exercise, it result that glucose output was as increase as during high-intensity exercise (1, 5, 10, 19, 22).

When IL-6 increase during exercise, it followed by the appearance IL-1 receptor antagonist (IL-1ra) and anti-inflammatory cytokine IL-10 (3, 21). To make sure which cells produce the IL-6, Keller et al, used muscle biopsies to isolate nuclei, which they obtained before, during and after exercise. They used a technique of RT-PCR (3). It was showed that the nuclear transcription rate of IL-6 peaked rapidly after onset of exercise (3). In other word, the factor which associated with contraction increase IL-6 transcriptional rate, which then observed that in the monocyte nuclei, the IL-6 protein is expressed within muscle fibers(3). Studies showed that during inflammation condition, IL-6 has a role in the expression of genes and it affect the responsiveness of macrophages to IL-4 (2). However, this illustrates the role of IL-6 which limiting Obesity- associated resistance to insulin and inflammation, and it will decrease the risk of chronic disease and premature mortality (1, 2, 5, 10, 18, 22).

IL-4 triggered signaling mechanisms:

It’s a cytokine that stimulate differentiation of naïve helper T cells in to Th2 cells (8) figure (1). The mechanisms of IL-4 signaling has an important insight on the regulation of immune responses. IL-4 use IL-4R alpha as a receptor component (6, 7, 9). IL-4 activate a lot of common signaling pathways. Therefore, IL-4 is playing an important role in anti-inflammatory cytokines, and it signal through JAK-STAT (Janus kinase/ signal transducer and activator of transcription) pathway (6, 7, 8, 9, 13, 16, 28) Figure (2). However, it has many function such as activation of macrophages, chronic inflammation and healing of the wound. It produced by activated T cells, and its increase associated with allergies. In JAK-STAT signaling, which is used by different cytokines, IL-4 will bind to its receptor IL-4Rα and IL-4Rγ, followed by association to proteins called JAK1 and JAK3, which is moves away next of the receptor. Then, it bind to STAT6 protein. After that, STAT6 translocate to the nucleus, where they bind and recognize DNA sequences which have target genes that are focus for this process. However, at the end you will get mRNA transcription for these genes been produced, but only the target genes which I need to focus in my project (6, 7, 8, 9, 16, 28).

A published study by Mauer et al 2014, showed the expression of IL-4R is induced by IL-6 directly, which then primed macrophages for IL-4 dependent activation of STAT6. Generally, IL-4-STAT6 is the most inducer of the M2 polarization of macrophages, and the activation of macrophages in vitro and in vivo inhibits strongly by the ablation of the genes encoding IL-4Rα or STAT6 (2) Figure (1). Studies showed that during inflammation, the expression of IL-4Rα is unregulated in myeloid effector cells, in which is dependent on IL-6 Figure (2).

Exercise mimetic:

Exercise mimetic define as a mimetic of a natural physical activity. As we know that skeletal muscle is the largest organ in the human body. Generally, doing exercise regularly can promotes metabolic adaptions and favorable structural, particularly in the skeletal muscle (20, 25, 26, 27). Physical exercise enhances the skeletal muscle to burn calories, which is useful in many clinical condition. However, as a general rule, that nothing can fully replace exercise. As a consequence, the exercise mimetic development can enhance the beneficial effects of physical exercise in many experiment, and focused on the substance that mimic the effects of exercise without energy consumption of the actual exercise (27).

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Dectin-1 and its role in the immune response:

Dectin-1 is the most important C-type lectin receptor for beta-glucan, which is naturally occurring polysaccharides that play an essential role in the innate immune system. Dectin-1 is a major receptor of the particles fungal on macrophages (30, 31, 32,33). It is a type II membrane receptor which has C-terminal, NK like (35). The role of Dectin-1 in the immune response, which include the factor production of phagocytosis and pro-inflammatory is that functioning as anti-fungal to eliminate the fungi infection (30, 31, 32, 33). The discovery of Dectin-1 as a receptor of beta-glucan gives the important of Dectin-1 in the sensing processes of beta-glucan, the signaling intracellular and cellular response induction (31). In addition, it provide the advantage of beta-glucan and its function in disease and immunity (31). Moreover, Dectin-1 is play an important role in adaptive immune response as well as immune tolerance and autoimmune disease (32). The stimulation of Dectin-1 to different cellular response through the Syk/CARD9 signaling pathway can involving in phagocytosis, production of cytokine and respiratory burst (34).

THP-1 cells:

It is one of the most widely cell lines used to investigate the regulation and function of monocyte and macrophages in cardiovascular system (29). Tsuchiya et al identified human THP-1 in 1980. However, THP-1 play an important role in the interconnection between monocyte-macrophages and other vascular cells during inflammation, especially in obesity and atherogenesis (29). Studies by Zhenyn Qin in 2011, demonstrated that TPH-1 not only resemble monocyte –macrophages which is isolated from donors with disease such as diabetes mellitus or healthy donors, but it also mimic the in situ of macrophages in obese or atherosclerotic lesions in the adipose tissue (29). Studies shows that THP-1 resemble primary monocyte-macrophages in differentiation and morphology. THP-1 is usually round cells, single with clear monocytic markers. In the culture plates, THP-1 begin to adhere by differentiation into macrophages phenotype, with change in the morphological. Generally, there are two classic agent which is used to differentiate the THP-1 in to macrophages phenotype, PMA (phorbol-12-myristate-13-acetate) and 1α, 25-dihydroxy vitamin D3. However, these classic agent used to regulate different signaling pathways, and treatment with PMA resulting to more phenotype with higher rate of adherence. In addition, treated PMA with TPH-1 cells are basically used to study the function of macrophages (29).

There are some advantages of THP-1 cells over human primary monocyte-macrophages (29). Firstly, the homogeneous genetic background of THP-1 minimizes the variability degree in the cell phenotype (29). Secondly, human primary monocyte and macrophages have problem in transfection efficiency, whereas the genetic of THP-1 by small interfering RNA to regulate the specific protein expression is easy. Thirdly, THP-1 cells could be stored in liquid nitrogen without any effect on monocyte-macrophages cell viability and features. Fourthly, the monocyte-macrophages polarized activation is divided in to M1, M2a, M2b and M2c cells. Therefore, many studies reported that THP-1 used in the fields which related to inflammation atherosclerosis and diabetes. In my project, I will use THP-1 cells and grow in a cell culture.

Conclusion:

As general rule, muscle is the largest organ in the body, and it is need to understand that it has an important impact in a field of scientific community. Therefore, physical inactivity or lack of exercise may lead to an impaired or an altered myokine response, which then resulting to have chronic inflammation, which is linked to sedentary lifestyle such as Obesity, and it will increase the risk rate of diseases, including T2DM ( Type 2 Diabetes mellitus) and cardiovascular disease (1,20). As many scientific papers showed that cytokine IL-6 produce by skeletal muscle and act as pro-inflammatory (1, 20). In contrast, IL-4 which is produce by macrophages act as anti-inflammatory. However, in my project, I have to focus on the gene that cause an end to the inflammatory episode. Consequently, if these genes increase in tissue culture experiment, it would assuming that in a real life person the same effect will happen, and that will be linked to clinical beneficially anti-inflammatory effects, in which not looking in a patient, instead I have to do my experiment in a culture cell that are act as mimic the effect of exercise.

However, the scientist until now they do not know why exercise produce wrong cytokines or wrong signaling molecules. It should produce IL-4 because of its effect of dependent depicts. Researcher by (Mauer et al 2014), give answer in mice, but I have to do my experiment in human by a cell culture. Therefore, when I will start in the laboratory, I will grow the monocyte- macrophages in cell culture, and treat those cells with IL-6 as a stimulus, and obtain RNA from these cells, and then see whether the RNA for Dectin-1 increases, in which it play an important role in anti-inflammatory.

The last paper which was reported on 2014 by Mauer et al, have been shows a weakness and strength. In that review talk about muscle, exercise and obesity, in which the strengthen is relevant to the strength to my project, but the weakness, is that they focused in skeletal muscle rather than macrophages, and this is essential in anti-inflammatory. Also the weakness, they did the experiment in vivo in mice not in human. So, as I am in a biomedical science, I have to do my experiment on human in vitro by using culture human monocyte-macrophages.

To sum up, because of the clinical importance of the subject, I propose to carry after project, in which I will test the hypothesis in vitro, if I treat monocyte-macrophages by IL-6, then it will cause expression of Dectin-1 to increases.

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