MicroRNAs and Immunity

MicroRNAs (miRNA) are small noncoding double-stranded RNA molecules, which play an important role in regulation of gene and protein expression, at the transcriptional and translational levels, affecting important biological processes involved in health and diseases.

MicroRNA processing
The precursors of miRNAs are transcribed from DNA. The processing of miRNAs from primary miRNA transcripts (pri-miRNA) into precursor miRNAs (pre-miRNA) and then into mature miRNAs are mediated by the enzymes Drosha in the nucleus and Dicer in the cytoplasm espectively. The mature miRNA will then associate with a complex called RNA Induced Silencing Complex (RISC). Picture from ambion.com demonstrates the main miRNA processing pathway. Quite recently, a dicer-independent pathway for maturation of miRNA has also been reported (Nature, 29 Apr 2010).

Mechanism of action
Posttranscription
The translational repression has been accepted as the main mechanism by which mature miRNAs contribute to the regulation of endogenous genes' activities. This is mainly via targeting specific region in the 3’untranslated regions (UTR) of messenger RNAs (mRNA), which are usually partially complementary to miRNAs. There are evidences supporting the idea that miRNAs can also positively regulate protein expression.

Transcription
Apart from their roles as posttranscriptional regulators, miRNAs have been shown to exert direct effects on the gene expression via histone modification and DNA methylation of target genes' promoters. MicroRNAs may also indirectly regulate the transcriptional activation of a gene via targeting the related transcription factors and even coactivators.

MicroRNAs and Immune System
MicroRNAs are involved in the regulation of immune system including the development of lymphocytes, the generation of cytokines and antibodies, and proliferation of monocytes and neutrophils etc. It has been reported that disruption of Dicer gene in mice leads to lethality early in development, indicating the importance of miRNAs in embryonic development. However, specific inactivation of Dicer in the T cell lineage didn’t lead to lethality. But it has been shown to be associated with impaired T cell development, aberrant T helper cell differentiation and cytokine production. These observations indicate the importance of miRNAs in the immune system via regulating, at least in part, the development, differentiation and function of T cells.

MicroRNAs are involved in the regulation of both innate and adaptive immune responses. For example the Toll-like receptors, which recognize the bacterial constituents and viral nucleic acids has been reported to induce expression of some miRNAs, which in turn regulate expression of proteins involved in the innate immune responses.

According to a latest report published in Nature Cell Biology 12 (2010), some miRNAs can regulate innate antiviral immunity by inhibiting expression of the p300 co-activator, which interact with transcription factors and contributes to gene expression. Thus, miRNAs might have a broad role in the regulation of antiviral immunity.

MicroRNAs in diseases
In addition to their roles in the regulation of immune responses, miRNAs has also been shown to be involved in the pathogenesis of different types of cancers including leukemias and lymphomas. This is due to the regulatory roles of miRNAs in physiologic processes including cell proliferation and cell death (apoptosis). Different studies have also elucidated the role of miRNAs in immune tolerance and autoimmunity. For example, selective miRNA disruption in T-regulatory cells has been reported to cause uncontrolled autoimmunity. In some recent studies, a functional connection between miRNA expression and allergic diseases such as asthma has been reported. Thus, targeting some miRNAs has been suggested as a method for the treatment of allergic airway inflammation. Elucidation of the roles of few hundred human miRNAs in health and diseases, in combination with many virally-encoded miRNAs, which regulate expression of viral as well as cellular genes, has been a great challenge for the researchers, especially for therapeutic purposes.

Rheumatoid arthritis (RA)

Rheumatic diseases

Rheumatic diseases are different painful medical conditions, which primarily affect joints, tendons, ligaments, bones, and muscles. Rheumatic diseases can also affect internal organs, skin and blood vessels. Common symptoms are pain, swelling, and stiffness. Rheumatic diseases are characterized by loss of functions of connective or supportive tissues. There are many different types of rheumatic diseases, arthritis, and related conditions. The picture, from medicalgeek.com, demonstrates the arthritic and normal joints.

Rheumatoid arthritis (RA)
RA is an autoimmune rheumatic disease affecting millions of people, and is an important cause of disability and even mortality across the world. Arthritis means joint inflammation and is just a part of the rheumatic diseases. RA most often affects the joints of the hands and feet, which are usually inflamed in a symmetrical pattern so that both sides of the body are affected. RA is most likely triggered by a combination of factors such as genetic susceptibility, abnormal autoimmune responses and even environmental or biologic factors, such as infection.

Autoantigens
Autoantigens are normal tissue constituents, which can become the target of a humoral or cell-mediated immune response initiating autoimmune diseases. Determining autoantigens targeted by the autoimmune response in RA has been a priority and an essential step for understanding the molecular mechanisms involved in pathenogenesis of RA and also for the immunotherapy of patients. So far, several autoantogens such as cartilage antigens, heat shock proteins, viral/bacterial antigens and several other autoantigens have been shown to be asscociated with RA. It should be noted that RA is a systemic disease. Thus, the candidate autoantigens may not necessarily be restricted to the joint components.

Genetic factors
Genetic factors play key roles in RA either by increasing susceptibility to the disease or by worsening the disease process. The association of RA with the human leukocyte antigens (HLA) was discovered when the frequency of HLA haplotypes, such as HLA-Dw4, HLA-B27 and HLA-DR4, were found to be increased in RA patients. The HLA system or the human major histocompatibility complex (MHC) are composed of cell surface proteins, which are encoded by the genes located on chromosome 6. HLA play an essential role in normal  immune responses by presenting both non-self (microorganisms) and self antigens to T cell receptors (TCR) on T cells. This presentation may also lead to development of autoimmunity. Genetic mutation, for example, in type II collagen, has also been shown to be associated with some form of arthritis.

Immunology of RA
The immune responses in RA involve numerous different immune cell types, including B cells, T cells, neutrophils, professional antigen presenting cells such as DCs and macrophages. They also involve molecules such as antibodies and components of the complement system, which can cause further recruitment and activation of the macrophages and neutrophils to the site of inflammation. Cytokines that regulate a broad range of inflammatory responses are also involved in the pathogenesis of RA. Besides, chemokines, which are involved in recruitment of leukocytes to the site of inflammation have also been shown to play a role in the pathogenesis of RA by recruiting leukocytes to synovial tissues.

T cells
The strong association of RA with specific HLA molecules and the observations that in experimental animal models of RA the disease can be transferred by isolated T cells, are among the most acceptable arguments in favor of an essential role for T cells in RA. Among the T helper cell populations, Th1 and Th17 cells have been reported to play a pathogenic role in RA. Several reports support a role for IL-17 in promoting rheumatoid arthritis. However, careful investigations are required to further identify the importance of Th17 cells in RA. Regulatory T cells (Treg) can play a critical role in preventing autoimmune diseases. However, the importance of Treg cells in RA and whether or not defect in these cells can contribute to the pathogenesis RA is not completely known. It has been shown that patients with RA exhibited substantial increased frequency of Epstein Barr Virus (EBV)-specific effector memory CD8+ T cells, indicating a role for these cells in RA. Note that EBV can trigger RA.

B cells
B cells most likely play several roles in the development of RA via presentation of antigens, activation of T cells, secretion of antibodies and proinflammatory cytokines such as TNF and IL-6. Several autoantibodies, such as rheumatoid factor (RF), have been shown to be associated with RA. RF is an autoantibody produced against the Fc portion of IgG, which form immune complexes and contribute to RA. Conversely, a negative regulatory role for IL-10- and TGF beta-producing B cell population in animal model of RA has been reported.

Macrophages and Dendritic cells (DCs)
These professional antigen-presenting cells (APC) are very crucial for immune responces especially for activation of T cells via processing and presenting antigens bound to their MHC class II molecules. Macrophages are involved in leukocyte migration to the site of inflammation, matrix degradation and angiogenesis. These cells contribute significantly to the pathology of many chronic inflammatory diseases, including RA. The cytokine produced by these cells, such as TNF, IL-6 and IL-1 has been targeted by different biological therapies. DC also play a central role in immune and inflammatory responses and contribute significantly to the maintenance and progression of RA. Interestingly, a group of DC, plasmacytoid DC, have been shown to mediate immune tolerance. These cells play a regulatory role in RA via induction of IL-10- and TGF beta-producing Treg cells.

For further information, I encourage you to refer to the recently published review articles in the journal "Nature Reviews Rheumatology"  at: http://www.nature.com/nrrheum/index.html

Glucocorticoid hormones and immune system

In 1950, the Nobel Prize for Physiology or Medicine was awarded to three scientists for the discovery of glucocorticoid hormones (GC), and their therapeutic values in the treatment of rheumatoid arthritis.

Synthesis
GC are a group of steroid hormones and are synthesized from cholesterol in the cortex of the adrenal gland. We and others have previously shown that GC can also be synthesized in the thymus with some paracrine effects on the development of T lymphocytes. Synthesis of GC is under the control of corticotropin-releasing hormone (CRH) and adrenocorticotropin hormone (ACTH) produced by hypothalamus and pituitary gland respectively. On the other hand, high blood concentration of GC inhibits ACTH and CRH secretion, which leads to inhibition of GC production. Stressful conditions leads to elevation of blood GC concentrations. (Picture from thebrain.mcgill.ca).

Mechanism of action
GC diffuse through the cell membrane and binds to the glucocorticoid receptor (GR), which is a transcription factor expressed in almost all cell types. GR is sequestered in the cytoplasm. However, after binding to the GC, the hormone-receptor complex will translocate into the nucleus, where GR binds to its DNA response elements in the promoter region of target genes and by which modulates the gene transcription.

Immunological importance
GCs have wide spectrum of physiological effects involved in, for example, development, glucose metabolism and immune system. GC are well-known for their anti-inflammatory and immunosuppressive properties and thus have been widely used for the treatment of various autoimmune diseases, allergic reaction and also in transplantation. This is particularly the case when they are used at pharmacological doses. However, we and others have shown that GC at the physiological concentrations can also play important roles in the development and functions of immune system.

Glucocorticoids can inhibit expression of several genes involved in both cell-mediated and humoral immunity such as interleukin (IL)-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-12, IL-17, IFN gamma, FC receptors etc. These immunosuppressive actions of GC is known to be mainly via cross-talk between GR and other transcription factors such as nuclear factor-kappa B (NF-kappaB), which is a major transcription factor required for the expression of many molecules involved in different types of cell-mediated and humoral immunity. GC may also prevent the transcriptional activities of NF-kappaB by inducing the expression of I-KappaB alpha, which is the natural inhibitor of NF-kappaB.

Other important functions of GC are induction of apoptosis and inhibition of immune cell proliferation. This is particularly the case for T and B lymphocytes. These are the key mechanisms mediating the therapeutic effects of GC in the treatment of leukemias and lymphomas.

T lymphocyte family at a glance

T lymphocytes constitute the most important cells of the immune system. The ”T” stands for the thymus, where these cells are developed in. The vast majority of these cells express T cell receptor (TCR) molecules, which are required for recognition of antigenic peptides presented by the major histocompatibility complex (MHC) molecules expressed on different cell types. Recognition of antigens is followed by activation and proliferation of these cells, which is required for different types of immune responses. Below, different groups of T cells are described.

CD8+ T cells
These cells are also called cytotoxic T cells (CTL), and are characterized by expression of CD8 molecules. CTL recognize their targets by binding to antigenic peptide presented by MHC-I molecules expressed by almost all cells. CTL can recognize and destroy virally infected cells and tumor cells by inducing apoptosis in these cells. This is done by releasing the cytotoxic molecules such as perforin and granzymes. Perforin forms pores in the target cell's membrane. This is followed by activation of caspases by granzymes, which leads to  cell death by a programed cell death (apoptosis). CTL are also involved in graft rejection.

CD4+ T cells
These cells constitute a big group of T cells, which play a central and key role in the immune responses. CD4+ T cells recognize their targets by binding to antigenic peptides presented by MHC-II molecules expressed by antigen presenting cells sush as macrophages or dendritic cells.

Naive CD4+ T cells can differentiate into 4 distinct T cell populations including T helper 1 (Th1), Th2, regulatory T cells (Treg) and Th17 cells depending on what kind of cytokines or growth factors are present in cells' environment. The presence of interleukin (IL)-12 skews towards Th1, IL-4 towards Th2, transforming growth factor beta (TGF beta)  towards Treg and finally a combination of IL-6 and TGF-beta stimulates differentiastion of naive CD4+  T cells towards Th17 cells. The differentiated cells are characterized by expression of different transcription factors such as T-bet, GATA-3, FoxP3 and  RORγ for Th1, Th2, Tregs and Th17 cells respectively.

CD4+ T cells have different characteristics and are involved in different types of immune responses. Th1 cells secret IFN gamma and TNF beta and are mainly involved in cellular immune responses via stimulating macrophages, dendritic cells and CD8+ cytotoxic T cells. They can also stimulate B cells to generate antibodies involved in the antibody-dependent cell-mediated cytotoxicity. Th2 cells produce IL-4, IL-5,.IL-10, IL-13 and are mainly involved in humoral immune response via stimulating B cells to proliferate and generate antibodies. Interestingly, Th1 and Th2 cells can regulate each others differentiation or activation. For example, Th1-derived IFN gamma prevents IL-4 production. On the other hand, Th2-derived IL-10 can prevent IFN gamma  and L-12 production. Treg cells secert IL-10 and TGF beta, and are known to play a suppressive or regulatory roles in immune responses. Absence of these cells has been reported to be associated with the development of autoimmunity. Unlike Treg cells, which have anti-inflammatory properties, the Th17 cells are involved in induction of local inflammation and autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, allergic reactions and in mediating allograft rejection via secretion of proinflammatory cytokines IL-21, IL-22 and IL-17, which may further stimulate secretion of other proinflammatory cytokines.

Natural killer T (NKT) cells 
NKT cells are small group of T cells, which co-express some of the NK cells' and T cells' markers such as CD16 and TCR. After activation, they can produce cytokines or cytolytic molecules. Thus, they have both T cell and NK cell activities as well. These cells normally recognize glycolipid antigens presented by CD1d molecules. They are also able to recognize and eliminate some tumor cells and cells infected with some viruses. They can also be involved in autoimmunity, allergic inflammation or transplant immunity.

γδ T cells
These cells are present mainly within the intraepithelial lymphocytes in the gut. The nature of antigens and the mechanism by which these cells recognize them is not fully understood. However, unlike the majority of T cells, the γδ T cells are not MHC restricted. They have also been shown to have some phagocytic activities. These cells can apparently participate in both innate and adaptive immune responses.

Programmed Cell Death (apoptosis)

Programmed cell death, which is also called apoptosis involves a series of biochemical events making some morphological changes in the cells leading to cell death.   In fact, this is a mechanism by which a cell, under certain conditions, commits a kind of suicide. 

The cell shrinkage and nuclear/DNA fragmentation are among the most recognized morphological changes associated with apoptosis. The apoptotic cells can finally break into several sealed membrane vesicles called apoptotic bodies. Apototic cells/bodies are removed by the neighboring phagocytic cells such as macrophages (Picture from Genetics Home Reference shows engulfment of apoptotic bodies by a macrophage). 

The process of apoptosis is under the control of different extrinsic and intrinsic factors, such as cytokines, hormones, toxins, radiation, which damage the DNA, viral infections and cell membrane/intracellular proteins. However, the executive elements involved in inducing this form of cell death are already inside the cell. It is in fact the cell itself, which utilizes those elements such as pro-apoptotic proteins or a group of proteases called caspases to kill itself.  Mitochondria can play an important role in the regulation of apoptosis. Pro-apoptotic proteins such as those in the bcl-2 family, can induce the permeability transition pores in the mitochondrial membrane. This leads to mitochondrial swelling and release of factors such as cytochrome C, which activates caspases leading to programmed cell death or apoptosis.

Apoptosis plays an essential role in homeostasis to keep the number of cells relatively constant. It is also involved in the development of cells and tissues. Billions of cells die each day due to apoptosis. Defective apoptotic processes have been reported in variety of diseases such as cancer or autoimmunity. On the other hand, excessive apoptosis can cause tissue damage, immune deficiency or diseases such as Alzheimer. Billions of immune cells undergo apoptosis during thier development or after activation. In the immune system, apoptosis is a mechanism by which useless or auto-reactive immune cells such as T and B cells are removed. On the other hand, immune system can induce apoptosis in virally-infected cells. This is done via activation of cytotoxic lymphocytes, which make pores in the target cell's membrane by releasing chemicals such as perforin, which leads to activation of caspases and programmed cell death in the target cells. Sometimes viruses can prevent apoptosis and this can lead to formation of tumors. 

Diabetes

There are two different types of diabetes. In type 1 diabetes the body's insulin production is highly impaired. The body's immune system for some reason, attacks and destroys the insulin-producing cells in the pancreas, which can ultimately lead to complete insulin deficiency. Why the immune system, which is responsible to defend the body against infection, attacks and destroys its own insulin-producing cells is not known. Many researchers believe that a combination of both genetic and environmental factor such as viruses or chemicals can cause immune system's auto-reactivities. Type 1 diabetes diagnosed normally in children and young people. Most of the patients, who suffer from type-1 diabetes do not have a close relative with type 1 diabetes. Patients are normally treated with insulin with some form of injections. (Picture from medical-look)

 The first signs are usually large amounts of urine, increased thirst, unusual tiredness and sometimes weight loss. The large volume of urine is because of high glucose (sugar) levels in the blood, which makes the kidneys to extract the glucose in the urine and the sugar draws water with it, which cause increased thirst. Fatigue and weight loss can also be other symptoms, which is due to serious disruptions in metabolism due to insulin deficiency. Unfortunately, the symptoms appear only when most of the insulin-producing cells are destroyed.

Type 2 diabetes is the most common form of diabetes (>80 %), in which the body either doesn't produce enough insulin or the cells don't respond to the insulin. Many people with type 2 diabetes are overweight and may produce much more insulin than normal level. The exposure of cells to high concentrations of insulin may lead to insulin resistance and thus diabetes. Both types of diabetes can have similar symptoms. Unlike type-1 diabetes, the type-2 diabetes is not an autoimmune disease and is diagnosed normally in people above 40 years of age.

Atherosclerosis

Atherosclerosis is an inflammatory disease in which plaque is made inside the arteries. These blood vessels are responsible for carrying oxygen-rich blood to the vital organs such as heart and other parts of the body. Gradually, growing and hardening of the plaque results in narrowing arteries, which in turn limits the flow of blood to the organs or tissues (Picture from Ohiohealth). Any artery can be affected by atherosclerosis. This can lead to different diseases such as heart attack or stroke causing serious problems and even death. Lack of physical activity, smoking and an unhealthy diet are among most important causes of atherosclerosis.

Immunology of atherosclerosis

It has now become clear that atherosclerosis is an immune system-mediated inflammatory disorder involving antibodies, components of the complement system, immune cells such as macrophages and lymphocytes infiltrating the walls of the arteries, by which participating in the formation of the plaques. Cytokines that regulate a broad range of inflammatory responses are also involved in the pathogenesis of atherosclerosis. In addition, chemokines, which are involved in recruitment of leukocytes to the site of inflammation have also been reported to play a role in atherosclerosis. It is believed that the inflammatory reactions are triggered by the damaged artery, caused by the oxidized low-density lipoprotein molecules. These molecules carry cholesterol throughout the body.

Psoriasis

Psoriasis and atopic eczema are the most common chronic inflammatory skin diseases affecting a large number of patients worldwide. Both diseases have a substantial negative impact on the patients' quality of life.

Psoriasis is a noncontagious, lifelong skin disease. According to the National Institutes of Health, as many as 7.5 million Americans have psoriasis. In Sweden around 3% of people suffer from psoriasis. It usually causes red scaly patches on the skin. The scaly patches caused by psoriasis, called psoriatic plaques, which are areas of inflammation and excessive skin production. Skin rapidly accumulates at the sites and has a silvery-white appearance. Plaques mainly occur on the skin of the elbows and knees, but can affect any area such as scalp and genitals.

Psoriasis can also cause inflammation of the joints, which is known as psoriatic arthritis. Up to 30 percent of people with psoriasis also develop psoriatic arthritis, which causes pain and swelling in and around the joints. Early diagnosis and treatment of psoriatic arthritis can considerably relieve pain and inflammation.

Immune system and psoriasis

In psoriasis immune cells antigen presenting cells such as DCs and also some T cells produce the key inflammatory molecules such as tumor necrosis factor (TNF), which plays a role in almost all psoriasis symptoms such as inflammation, redness, pain, and itching in the plaques. It can make blood vessels multiply, and white cells move from the blood vessels into the skin. This may explain why patients bleed so easily when they scratch the plaques. DCs can also produce and release the cytokine IL-23, which stimulates a group of CD4+ Th cells, Th17 cells, to produce the key pathogenic cytokines, IL-17 and IL-22, in the psoriatic lesions. In addition to the Th17 cells, antigen presenting cells-derived IL-12 can also mediate differentiation of CD4+ Th cells towards Th1 cells, which can produce TNF and IFN-gamma. All the mentioned cytokines have been shown to be involved in the pathogenesis of psoriasis. IL-17, IL-23 and IFN-gamma can activate the keratinocytes, and these cells in turn will release proinflammatory cytokines such as IL-8 and other factors, which play a role in inflammatory responses.

Conditional inactivation of a gene using a Cre/loxP inducible system

Creating KO mice is one of the best and advanced methods for studying the function of a gene in vivo. However, in a considerable number of cases, it has turned out to be problematic, as it can cause early embryonic lethality. Even though this is a clear indication for the importance of the candidate gene in the embryonic development, the real function of the gene in adults remains unanswered. Apart from this, inactivation of a gene in all tissues will make interpretation of the results very complicated, as it can not easily be concluded whether the phynotpe in a tissue is the consequence of gene inactivation in that particular tissue or it is due to inactivation of that gene in other tissues. To address these issues, a more advanced system has been created, by which a gene can conditionally be inactivated either in a particular cell types or in a specific time.

The Cre-loxP system has been widely used for inactivation of a gene in a particular tissue. In this system, the Cre recombinase mediates excision, recombination between loxP sites and consequently inactivation of a floxed gene or in other word a target gene flanked by two loxP sites. For creation of this system in mice two different lines of mice are required. First, a conventional transgenic mouse line expressing Cre recombinase protein in a specific tissue or cell type, and secondly a mouse line that carries a floxed gene. Crossing these two mouse lines creates a double transgenic line carrying the floxed gene and expressing the Cre protein. Expression of the cre is under the control a promoter, which directs the expression of this protein to a particular tissue or cell type. Thus, inactivation of the target gene occurs only in those cells expressing Cre. The main advantage of using this system is inactivation of a gene in a cell type-specific manner. However, similar to the conventional methods, the embryonic lethality can still be a problem.

The Cre/loxp inducible system is currently one of the most advanced tools for not only cell type-specific but also time-specific inactivation of a target gene. In this system Cre protein is fused to a mutated form of estrogen receptor (ER). In unstimulated cells Cre-ER is sequestered in the cytoplasm but after addition of the the drug Tamoxifen, which is a ligand for the mutated ER, the ER and Cre translocate to the nucleus, where Cre can excise and inactivate the gene in a particular time. See the attached film for more details.

The Age-Associated Thymic Involution

The thymus gland is the heart of the immune system, especially during its development. It is the main site for the development of T lymphocytes. These cells are more often called T cells. T cells play a central and crucial role in the immune system. Thymus is the site where bone marrow derived very immature T cells are developed, so that mature and competent T cells can be produced and exported via circulation to the peripheral lymphoid tissues such as lymph nodes or spleen to play their active role in the immune system. (Picture from the National Cancer Institute)

The thymus shrinks gradually by age, a process called the “age-associated thymic involution”. This process starts normally after puberty. Thymic involution results is a decreased number of thymocytes or in other word immature T cells in the thymus. This leads to production of less mature T cells. Thymic involution may also involved in Immunosenescence, which refers to the gradual deterioration of the immune system especially in aged people. This might contribute to higher susceptibility of aged people to cancer and infection.