A pathologically increased activation of complement appears in autoimmune diseases which might enhance the inflammatory reaction. Disruption or exhaustion of complement-mediated clearance of immune complexes and apoptotic cells, and of its bridging to adaptive immunity, are contributing factors of autoimmune diseases. Additionally, the endocrine system is a target for autoimmune diseases where local effects of sex hormones seems to consist mainly in modulation of cytokine production.
The goal of this proposal is to evaluate the role of sex hormones in complement-mediated therapy as a contributor factor in autoimmune diseases.
In particular, the objectives of this project are:
- to determine if manipulated levels of estrogen contribute to complement-related therapies of autoimmune disorders
- to study the signaling crosstalk of TLRs and sex hormones receptors in innate immunity response.
- and measurement of inflammatory cytokines.
According to the National Institute of Allergy and Infectious Diseases, the function of immune system is to prevent or limit infection. This system divide into innate and adaptive immunity. The first line defense of innate immunity system is called complement and consists of small plasma proteins, around 30 and synthesized by the liver. The complement proteins circulate in an inactive form. However, when a pathogen enters in our body the complement protects it against infection .
Therefore, three main pathways involved classical (CP), alternative (AP) and lectin (LP). First, it generates large numbers of activated complement proteins that bind covalently to pathogens and phagocytes opsonizing them. Second, the small fragments of some complement proteins enhance inflammation. Third, the terminal complement components damage certain bacteriaby creating pores on the surface of the pathogen called membrane attack complex (MAC) .
Inaccurate activation and regulation of the complement may contribute to complement-related diseases such as inflammatory, autoimmune, neurodegenerative and infectious diseases . Consequently, inhibition or modulation of complement activity used as a promising therapeutic strategy .
Specifically, complement appears to contribute to a number of autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) or anti-phospholipid antibody syndrome (APS) . Autoimmune diseases are the result of specific immune responses directed against structures of the self. Complement involvement is usually complex and may include an inappropriate initiation of the cascade and deficiencies in specific components . For example, deposition of IgG immune complexes during systemic lupus erythematosus initiates the classical pathway by binding of C1q to these complexes . The Toll-like receptors (TLRs) and complement are key innate defense systems that are rapidly triggered upon infection .In different autoimmune diseases TLR-mediated activation mechanisms leading to the induction of Th17 T cell differentiation .
Noteworthy, American Autoimmune Related Diseases Association mention that autoimmune diseases strike women three times more than men. The sexual steroid hormones estrogens have been recently recognized to influence numerous autoimmune and inflammatory responses. In vivo study indicate that 17b-Estradiol promotes TLR4-triggered pro-inflammatory through estrogen receptor α in macrophages .
Materials and Methods
Evaluate the sex hormones levels and their association with complement components will be studied. It is known that, autoimmune diseases can be influenced by autoantibodies and different types of disorders activate different pathway, for example in RA the alternative pathway is involved but in systemic lupus erythematosus SLE deficiencies participate the classical pathway. On the other hand, sex hormones, includingestrogens, influence the immune system in a complex manner, playing an important role in the pathophysiology ofautoimmune diseases and statistical analyses shows that women affected more than men. The therapy for autoimmune diseases depends on the initial target of altered the immune regulation. Biologic therapies of autoimmune disease focuses on anti-cytokines include anti-tumor necrosis factor (TNF-α), anti-interleukin 1(IL-1), and anti-interleukin 6(IL-6) molecules.
Depend on the autoimmune disease model that it is studied, complement inhibitors for C3 and C5 will be used in male and female mouse.
Mice will be treated (injection) with anti-C3 (compstatin), anti-C5 (eculizumab or natural inhibitors from Staphylococcus aureus like SSL7) combination with anti-TNFa mAb will be benefit with this disorders. As a control mice carrying the disorder without any treatments will be used.
Five different mice groups will be used for this study, one for control and four treatments.
- Group 1: control, no treatments on this group
- Group 2: treatments with anti- C3 (compstatin)
- Group 3: treatments with anti-C5 (eculizumab or natural inhibitors from S. aureus like SSL7)
- Group 4: treatments with anti- C3 (compstatin) combined with anti- TNF-α
- Group 5: treatments with anti- C5 (eculizumab or natural inhibitors from S. aureus like SSL7) combined with anti- TNF-α
The above groups are consist of both female and male mice.
ELISA (Enzyme-Linked Immunosorbent Assay) for measuring the levels of cytokines (IL-1, IL-6, TNF-α) and estrogen in plasma.
Immunohistochemistry staining with H&E in different tissues (kidney, bone, liver) or
Immunofluorescence with specific antibodies that indicates inflammation.
Signaling with Western blot (homogenized tissue- protein extraction and purification – test for specific molecules an the signaling pathway)
Real-time PCR amplification using specific primers for genes related to autoimmune and inflammatory response
The innate immune system is critically involved in the defense against pathogens and depending in the cells differentiation promote development of autoimmune diseases. Autoimmune disease is one of the top 10 leading causes of death in female children and women in all age groups up to 64 years of age and there is no cure. Mostly, treatment comes in the form of relief the symptoms and commonly used immunosuppressant treatments lead to devastating long-term side effects. Complement targeted drugs with estrogen therapies for autoimmune diseases may be contribute to a better understanding. Furthermore, the findings will allow the development of therapeutic strategies to combat these disorders in patients associated with gender.
Finding the association between complement inhibition and autoimmune diseases may be lead to a tool to mitigate the symptoms and decrease the risk of complications in patients who already suffer from this condition, understanding how to modulate immune system activity will benefit transplant recipients, cancer patients, AIDS patients and infectious disease patients. Finally, this proposal focuses on gender separation, in male and female groups as autoimmune disorders associated with sex hormones, as mentioned above.