Richa Mishra
Senior Thesis | 2024
Activation of the Complement System in Systemic Lupus Erythematosus
Richa Mishra
Prospectus:
The immune system is a vast and complicated network affecting all mechanisms of the body. Immunology remains a growing field as demands for its critical research increase with vaccination and immunotherapy becoming more popular health services. This research focuses on the complement system, a component of one’s immune system that acts as a surveillance system and defense mechanism against invading organisms. The complement system uses around 50 plasma proteins that interact with each other through a series of events to maintain homeostasis. Depending on how intensely the complement system is activated, the proteins’ interactions will follow the classical pathway, alternative pathway, or lectin pathway. This experiment examines the relationship between the complement system and Systemic Lupus Erythematosus (SLE), an autoimmune disease that is continuously affecting more people each year, especially women of reproductive age. Autoimmune diseases occur when someone’s immune system attacks
healthy cells and SLE is one where the body attacks its own tissues. Like most autoimmune infections, the cause of SLE is unknown. However, recent studies present evidence that deficiency or overactivation of the complement system contribute to the pathogenesis of SLE and then eventually lupus nephritis (LN), the last stage of SLE which causes severe inflammation of the kidney. 8 mice that had developed SLE on their own were the subjects of this study. SLE in mice and SLE in humans resemble each other in processing and symptoms. A C3 inhibitor was inserted into the mice with a goal to suppress the mouse’s complement system by stopping its central protein C3 and hopefully prevent kidney damage.
Introduction:
The complement system is the immune system's surveillance system and acts as the first line of defense against invading organisms [1]. It uses nearly 50 distinct plasma proteins that are expressed as intracellular proteins or exist in the circulation [2]. They interact with each other and bound antibodies through a series of events to maintain homeostasis by preventing
infection and removing dead or injured cells. The liver is the greater producer of plasma proteins but more recent research has revealed the spinal cord and neurons to also secrete plasma proteins. The primary stage which typifies tissue injury is characterized by complement activation and recruitment and activation of neutrophils and other inflammatory cells. The complement system is vital in the recognition and removal of selfthreats, including apoptotic cells, and the initiation of a broad inflammatory response and sustenance of the innate immune responses [1,3,4]. The central complement proteins are labeled as C1, C2, C3, C4, C5, C6, C7, C8, and C9 based on the order of their discovery [5]. The complex nomenclature of the complement proteins and the effect of their activation in many diseases is still not well understood. Understanding the intricacies of the structure and functioning of proteins is crucial for researchers to apply the molecular mechanisms of the complement system effectively in areas such as clinical diagnosis, pathophysiology, and treatment
strategies. This research paper investigates the relationship between the complement system and the pathology of Systemic Lupus Erythematosus and how it can be utilized for diagnosis and treatment.
Immune mechanisms are usually differentiated as either part of the innate immune system or the adaptive immune system. The innate immune system can absorb and destroy pathogens, disease causing microorganisms, without any prior exposure to it. Due to the lack of requirements, innate immune mechanisms have the same response to any invading organism, which is why innate immunity is often referred to as the nonspecific immune system. The adaptive immune system produces antibodies to fight pathogens by binding to the antigens on the pathogen’s surface. For this antibody antigen complex to form, the antibodies have to be very specific to the target pathogen and can only be produced after exposure to the pathogen. Therefore, the adaptive immune mechanisms are slower and production of an antibody requires five
to seven days. However, the specificity between antibodies and pathogens allows for a more effective response to infection than a nonspecific response [6]. These two systems do not operate separately. When it fails to stop infection, innate immunity will trigger the adaptive immunity, resulting in a powerful defense mechanism remembered by the organism's immunological memory for future encounters with the same pathogen [7].
Complement Activation:
The close interrelationship between the innate and adaptive immune system is demonstrated by the complement system. Depending on how the complement system is activated, the proteins’ cascade of reactions occurs through the classical, alternate, or lectin pathway. The various pathways display how the complement system can serve as both an innate and adaptive immune mechanism, making it a critical component for maintaining homeostasis. The classical pathway is an instrument of the adaptive immune system because it is dependent upon an
antibody while the lectin and alternative pathways are both innate procedures because they are antibody independent [8].
Classical Pathway:
The classical pathway of the complement system is an adaptive mechanism because it requires an antigen antibody complex for activation. The first protein used, C1, is composed of three proteins called C1q, C1r, and Cls [5]. The antibodies attached to the pathogen must bind C1q at C1q binding sites. These locations first have to be revealed by a specific class of antibodies called IgG or IgM antibodies. At least one IgM molecule or multiple adjacent IgG molecules are needed to bind with the antigens on the pathogen’s surface to reveal C1 binding sites. C1q binds to the IgM or multiple IgG molecules to activate the C1r which then activates the C1s. IgM is a planar polymeric molecule that hides the C1 binding sites until attaching to the antigens and undergoing conformational change. IgG molecules are monomers with exposed C1 binding sites from the start but have a weaker
bond to antigens than IgM does. The intensity of the classical pathway is altered by the number of engaged IgG molecules and distribution of antigens [1].
Once C1s is activated, it will interact with C4 and cause it to split into two parts called C4a and C4b. When complement proteins of the classical pathway split in two, each half is labeled as part a and part b, where part b is always bigger. C4b will covalently bind to C2, allowing it to also be split by Cls into C2a and C2b. C4b and C2b join together to form and provide enzymatic activity for the C3 convertase. This complex is essential to execute any complement mechanism . It is very important that C4b is covalently bound to the pathogen surface. Its highly reactive thioester bond that occurred from the cleavage of C4 allows C4b to bind to close molecules of adjacent proteins. This ensures that the C3 convertase is strongly attached to the pathogen and will not carry out its functions on the host cells. The C3 convertase will split into C3a and C3b. C4b and C3b are
structurally similar, meaning that C3b can also covalently bind to molecules on the surface of the pathogen. While there are many effects of complement activation, the most important outcome is to produce large amounts of C3b on the initiating pathogen. C3b will then bind to C5 and cause it to split into C5a and C5b. C5b will interact with C6, C7, C8, and C9, also known as the terminal complement proteins. These binds will result in many conformational changes that create the membrane attack complex (MAC). The membrane attack complex has a hydrophobic membrane which allows it to make holes in the lipid bilayer membrane of a pathogen which can cause the pathogen to burst from ruining the proton gradient across its membrane [5].
Besides pathogen elimination through the membrane attack complex, the complement system also has other actions for host defense against infections. C3a, C4a, and C5a are anaphylatoxins or peptide mediators which induce inflammatory responses and recruit more phagocytes for assistance. Although the membrane
attack complex has the most dramatic effect, deficiencies in the terminal complement proteins have been associated with susceptibility to very few diseases compared to the amount associated with anaphylatoxins. Therefore, the smaller fragments of the complement proteins that are produced in earlier stages of the cascade are more important for immunity [5].
Lectin pathway:
The lectin pathway is one of the three primary pathways that trigger the activation of the complement system. Alongside the classical and alternative pathways, the lectin pathway provides a specialized mechanism for recognizing and responding to microbial invaders. The initiation of the lectin pathway begins with the recognition of specific carbohydrate patterns on the surface of pathogens by pattern recognition molecules. Mannose-binding lectin (MBL), ficolins, and collectins are examples of these pattern recognition molecules. MBL and ficolins are known to recognize structures such as mannose, N-acetylglucosamine, and acetyl
groups, while collectins, such as surfactant proteins A and D, recognize carbohydrates and lipid moieties on the surface of pathogens. Upon binding to these pathogen-associated molecular patterns (PAMPs), the recognition molecules form complexes with MBL-associated serine proteases (MASPs), specifically MASP-1, MASP-2, and MASP-3. Among these, MASP-2 plays a central role in the lectin pathway. Activation of MASP-2 leads to the cleavage of complement factors C4 and C2. This cleavage generates the C3 convertase, known as C4b2a, which is a pivotal enzyme in the complement cascade. The C3 convertase triggers a series of downstream events very similar to the ones of the classical pathway, including the cleavage of complement factor C3 into C3a and C3 and formation of the membrane attack complex. The lectin pathway of complement activation provides a specialized immune response mechanism by recognizing specific carbohydrate patterns on pathogens, ultimately leading to opsonization, phagocytosis, and lysis for effective elimination of the invaders [9,10,11].
Alternative Pathway:
The alternative pathway of the complement system is part of innate immunity because it is always on during normal conditions. It relies on the bioactivated form of the C3 protein instead of a specific antibody. C3 is constantly converted into its bioactive form C3(H2O) through persistent hydrolysis of a thioester bond, allowing the alternative pathway to maintain low levels of activation. This conversion is called tickover and the rate of it can be altered by C3’s interactions. Tickover causes a conformational change in C3 that exposes binding sites for an alternative pathway component called Factor B. When C3(H2O) binds to Factor B, it is cleaved by a plasma serine protease called Factor D, forming C3(H2O)Bb. This fluid phase C3 convertase complex C3(H2O)Bb is the C3 convertase for the alternative pathway because it will interact with the native C3 molecules and cause it to split into C3a and C3b. Similar to C4b, C3b has a reactive thioester bond exposed by the cleavage of C3 that allows
for C3b to covalently bind to any adjacent molecules with hydroxyl groups. However, not all hydroxyl groups attract C3b equally, so the particular positioning and composition of the hydroxyl groups on the pathogen’s surface will affect the successfulness of complement activation. Despite the differences in activation, the classical and alternative pathway execute a similar cascade of events. The covalently bound C3b will initiate the same steps of the classical pathway that occur after the formation and cleaving of the C3 convertase. Therefore, the alternative pathway can amplify the effects of the classical pathway by releasing more C3b molecules on the surface of the pathogen [1].
Outcomes of the Complement System:
The most essential outcome of the complement system is the opsonization of pathogens [5]. Opsonization is an immune process that tags pathogens to be eliminated by phagocytes [12]. Phagocytes are cells that use their plasma membrane to absorb
harmful foreign particles or dead or damaged cells. For opsonization to be carried out by the complement system, certain complement components need to bind to specific complement receptors that are expressed on phagocytes. There are five types of complement receptors with different functions and each are specific for different proteins and cell types. However, most of them are capable of stimulating phagocytosis. Bacteria is more efficiently engulfed by phagocytes when they are also coated with complement components. C3b is an especially powerful opsonin. Although C4b is structurally similar and therefore can also act as an opsonin, a much smaller amount of it is generated compared to C3b [5].
Regulation of the Complement Activation:
Complement activation has to be regulated to protect host cells from the defensive effects of the complement system. In most cases, complement proteins bind directly molecules on the surface of the pathogen that initiated activation. Sometimes these
complement components bind to proteins on host cells which will undergo the elimination process intended for the infecting molecule. Complement regulatory proteins work to protect healthy cells and redirect the complement effect to the surface of pathogens. These cell surface control proteins act on different stages of the complement cascade like the activation of C1, split of the C3 and C5, and the formation of the membrane attack complex. Some of these regulatory proteins have proved their importance by correcting diseases. C1 is controlled by a C1 inhibitor called C1INH which can decrease the amount of time C1s is able to cleave C4 and C2 by a few minutes. Deficiencies in C1NH can cause hereditary angioneurotic edema, a disease where the spontaneous complement activation increases the amount of small complement fragments, leading to swelling and then suffocation. One of the cell surface proteins that regulate the terminal complement proteins is called CD59. By restraining the final assembly of the membrane attack complex, npt blocking, CD59
prevents a disease called paroxysmal nocturnal hemoglobinuria which results from cell lysis of intravascular red blood cells [5].
Role of Complement System in Autoimmune Diseases:
Both deficiency and overactivation of the complement system can result in severe and fatal diseases like autoimmune diseases [1]. Autoimmune disorders involve the dysfunction of the immune system where a specific adaptive immune response to a self antigen causes the observed pathology and mistakenly attacks healthy cells. This group of disorders can affect any organ but long term tissue damage, swelling, and joint pain from chronic inflammation are shared symptoms between all autoimmune diseases. Symptoms are usually sustained because the only way to remove the self antigens that caused this autoimmune response is to destroy the cells that produced them. Most treatments focus on suppressing the immune system to decrease inflammatory responses. While treatments to alleviate symptoms have been made, no autoimmune disease is yet curable because the
underlying causes are still unknown. However, it is highly suspected that the triggers are a combination of environmental and genetic factors [5]. Family studies showing higher diagnoses in monozygotic twins than in dizygotic twins suggest that autoimmune diseases are at least partially inheritable [8]. The MHC genotype is the only transparent and consistent genetic sign of susceptibility to autoimmune disorders. This aligns with the nature of autoimmune diseases because they all involve T cells, which rely on the MHC genotype for responding to antigens. Unfortunately, this connection is not yet scientifically proven and anything it implies about susceptibility to autoimmune disorders and potential clinical treatments is hypothetical [5]. Differences in geographical differences have been well documented enough to show that areas with more sunlight have less prevalence of most autoimmune disorders. However, there is no concrete proof that the sun is causative of autoimmune diseases instead of just correlated [8].
Autoimmune diseases can be broadly categorized as either tissue or organ specific as opposed to systemic. Organ or tissue specific means that only one organ or tissue is injured while systemic means many different organs and tissues are affected.
Systemic Lupus Erythematosus (SLE), more commonly known as Lupus, is a systemic autoimmune disorder that primarily affects the kidney, skin, joints, and central nervous system. Symptoms include skin rashes, kidney damage, nerve damage, and arthritis [8]. Like most autoimmune diseases, SLE has no cure and shows a strong sex bias. SLE is most common in women, especially during their prime reproductive years when production of female sex hormones estrogen and progesterone is highest. This hormonal factor is supported by research done on experimental animals that showed female animals are also more susceptible to autoimmune disorders but castration normalizes the difference in incidence between the two sexes.
Role of the Complement System in SLE:
Scientists have been able to define a relationship between SLE and the complement system. SLE is attributed to the formation of immune complexes. Immune complexes are antibodyantigen complexes that are made of many small soluble antigens. They have to be removed from the circulation of blood by complement receptors. Immune complexes can activate the complement system so that C4b and C3b bind which can then bind to complement receptor CR1, which is specific for erythrocytes. The bound erythrocytes and immune complexes are brought to the liver and the spleen where macrophages can remove immune complexes without damaging the erythrocytes and then dissolve the immune complexes. If they are not removed, immune complexes tend to stay in the membranes of blood vessels, particularly in the blood vessels of the glomerulus, the main filtering unit of the kidney. In SLE, the excessive amount of immune complexes results in a huge deposit of antigen, antibodies,
and the complement on podocytes. Podocytes are cells that support the structure and function of the glomerulus. Therefore, when the complement system mistakenly attacks podocytes due to immune complexes, it is harming the glomerulus [5]. Eventually, this can lead to Lupus Nephritis, the last and final stage of SLE where one’s kidney is extremely damaged. The kidney is responsible for filtering blood and removing waste so damage to this area is often fatal. While cyclophosphamide, a chemotherapeutic drug, has been approved for treating severe cases, without a kidney transplantation, there is not much hope for patients who reach this stage of SLE [8].
This research analyzes the mechanisms of the complement system and its relationship with SLE to test a drug designed to inhibit the cleavage of the C3 convertase, the most central complement component. Performing successful complement intervention before these complement proteins leave the liver can
suggest many treatments for SLE and other diseases that affect the kidney.
Method:
All experiments were conducted on MRLlpr and MRLmpj mice, two species that were selectively bred from laboratory mouse to be a model for autoimmune disease research. These mice are useful for demonstrating the etiology and organ pathology of Systemic Lupus Erythematosus. Around 9-12 weeks old, they develop autoimmune diseases due to a mutation that promotes the survival of self reactive lymphocytes. Excessive lymphocytes can attack tissues and organs and lead to autoimmune disorders. While both male and female mice develop these diseases, females tend to have more severe and fatal cases. Both the experimental group and control group were stored in a pathogen free and temperature controlled animal facility at Beth Israel Deaconess Medical Center and followed a 12 to 12 hour light and dark cycle. At 8 to 12 weeks old, the experimental group was intravenously administered
nanoparticles loaded with a pan-cathepsin inhibitor called E64d every week with a final dose of 10 mg/kg. A pan-cathepsin inhibits intracellular complement activation by limiting all relevant cathepsins which are responsible for the cleavage of C3. The control group was intravenously injected with phosphate buffered saline (PBS) or dimethyl sulfoxide (DMSO) under the same conditions.
At 18 weeks both kidneys from each mouse were surgically removed, cut in half, and rinsed with PBS. The kidneys were left in a 10% phosphate buffered formalin solution overnight and then stored in paraffin wax. Hematoxylin and eosin (H&E) staining, a popular and economical method for visualizing cellular and tissue structure, was performed on the sectioned kidneys. Hematoxylin is used to illustrate the nucleus. The actual dye used in H&E staining, hematein, is produced from the oxidation of hematoxylin. Eosin acts as a counterstain to distinguish between the cytoplasm and types of connective tissue fibers. Researchers can control the levels
of hematoxylin and eosin to vary the intensity of the staining, allowing for the removal of certain stains that the viewer does not want.
Immunohistochemistry staining was performed on the formalin fixed paraffin embedded (FFPE) tissue. This technique can show the specific bind between an antibody and antigen.
Endogenous peroxidase activity in the tissue sections, which must be blocked to prevent false positivity, was quenched with 3% hydrogen peroxide. The first step is to retrieve the antigens that are masked by the formalin. Most techniques break protein cross links that are formed from fixation [13]. Antigen retrieval in this experiment was performed using the Retrievagen A system (BD Pharmingen, San Jose, CA) according to the manufacturer’s directions. The tissue sections were then blocked for 60 min at room temperature with 10% BSA/PBS containing the serum from the host species of secondary antibody and incubated overnight at 4°C with primary antibodies. On the following day, the slides were
incubated with a fluorescence conjugated secondary antibody for 60 min at room temperature. Appropriate isotype control was included. The images of the sections were captured using a Nikon eclipse 80i microscope and analyzed using Nikon NIS-Elements software. Intensity measurements were performed in three fields per section of intestinal tissue prepared from two to three mice per experimental group using ImageJ software. The relative image intensity was expressed as mean intensity per area of selected images.
Results and Discussion:
Previous studies have demonstrated positive results when using complement intervention on other diseases affected by the complement system. Complement inhibitors are designed to treat symptoms rather than cure a disease. Atypical Hemolytic Uremic Syndrome is an extremely rare disorder that causes blood clots to form in the blood vessels, blocking blood flow and eventually leading to kidney failure [14]. It is associated with alternative
pathway mutations in the C3 convertase and its regulators.
Screening for these mutations have been an efficient method for diagnosing aHUS. The therapeutic laboratory-produced antibody
Eculizumab was developed to limit the splitting of the C5 convertase and therefore restrain the late effector functions of the complement system. Eculizumab showed significant improvement in clinical outcomes for treating the symptoms of aHUS and is now
FDA approved as a treatment for aHUS and Paroxysmal Nocturnal Hemoglobinuria, a different complement mediated disease.
Although the terminal complement components were restrained, C3 convertases were unaffected by Eculizumab [1].
This can be very beneficial in some cases because it treats symptoms but still leaves the patient with a somewhat working complement system. Controlling the complement system at an earlier stage might be more effective in alleviating symptoms, especially for diseases that result in kidney damage [15]. The complement system’s cascade of reactions occurs within the
kidney until the split of the C3 convertase. Therefore, all the proteins inhibited by Eculizumab are part of the extracellular complement activation because their reactions occur outside of the kidney. Inhibitors acting on intracellular complement activation have also been developed with the goal of preventing kidney damage. Compstatin is a peptide that binds to C3 and limits its cleavage. It is still undergoing preclinical trials but Compstatin has been observed as restraining the complement system in vitro and in animal models [1]. The E64d pan-cathepsin inhibitor was hypothesized to have the same effect as compstatin.
Figure 1: Blue color staining (DAPI) performed on the kidney of the experimental group to determine the number of nuclei and assess gross cell morphology.
Figure 2: Green color staining performed on the kidney of the experimental group to measure levels of the C3 complement protein.
Figure 3: Orange staining performed on the kidney of the experimental group to visualize the location of the glomerulus.
Figure 4: Blue, green, and orange staining overlapping in one image to measure proportions and physical distances between nuclei, C3 protein, and the glomerulus.
Figures 1 through 4 show the kidneys of the experimental group treated with the E64d inhibitor. The blue stain represents the nucleus, the orange stain represents synaptopodin, and the green stain represents the complement protein C3. Figure 4 shows all three stains in one image. Synaptopodin is a marker for the glomerulus so it is used to locate the glomerulus which can then be further observed for damage. After staining and images of the kidney sections from the control group is performed, a comparison between the control group’s kidney images and the one of the experimental group will show if the E64d inhibitor worked and decreased the amount of C3. Certain software is able to quantify the amount of C3 from these images. C3 is considered a marker for SLE so quantifying the deposition of C3 can be used for diagnosing SLE. Remarkably, mice treated with E64d exhibited a significant reduction in kidney injury compared to mice treated with PBS. The assessment of tissue injury further revealed a notable decrease in injury for E64d- NP treated mice compared to
PBS-treated (control) mice. The resident kidney cells, podocytes, were also observed under in vitro conditions. These findings suggest that complement plays a pivotal role in the kidney injury caused by LN.
Systemic lupus erythematosus stands as a complex autoimmune disease characterized by immune system dysregulation, particularly affecting women of reproductive age and resulting in systemic autoimmunity and organ damage, notably lupus nephritis (LN). Inflammation of the kidney is one of the most severe manifestations of SLE that leads to lupus nephritis. Despite improved understanding and treatment options, LN remains a significant cause of morbidity and mortality in SLE patients [1619]. T and B cells play crucial roles in SLE pathogenesis, with T cells aiding B cells in autoantibody production and contributing to systemic inflammation and organ injury [17-19] . Immune complex deposition and complement system activation are
established processes in LN pathogenesis, leading to tubulointerstitial and glomerular injury.
While the complement system stands as one of the earliest and extensively studied pathways in systemic lupus erythematosus (SLE), the comprehension of the mechanisms underlying complement activation and its role in the pathogenesis of SLE and lupus nephritis (LN) remains incomplete. Excessive complement activation is implicated in promoting tissue inflammation, yet genetic deficiencies in the early complement components invariably lead to autoimmunity. The classical pathway, initiated by the interaction of immune complexes (ICs) with C1q, is recognized as the prevailing route for complement activation in SLE [20]. However, recent studies in LN-prone mice suggest the involvement of factors B and D from the alternative pathway, warranting further validation in human LN [21, 22]. Overall, the extent of complement cascade activation serves as a critical regulator of the innate immune response and, consequently,
clinical outcomes [23]. The comprehensive analysis presented here strengthens the assertion that the complement system plays a significant role in the pathogenesis of SLE. The activation of complement molecules, along with the resulting split products, intricately contributes to complex inflammatory networks.
Importantly, insights from studies on complement activation in LN suggest dual opportunities: the therapeutic targeting of complement components and their utilization as potential biomarkers for disease diagnosis and treatment response monitoring.
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