Monday, December 9, 2013

Autoimmunity, Rheumatoid Arthritis, and Abatacept

                        Figure 1: Example of rheumatoid arthritis effects.

You’ve likely heard of rheumatoid arthritis (RA), an autoimmune disease that results in a chronic, systemic inflammatory disorder. More than 2 million adults in the United States suffer from this disease, with women being two to three times more likely to develop it than men (UCSF Medical Center 2013; http://www.ucsfhealth.org/conditions/rheumatoid_arthritis/). The disease can occur at any age, although it typically affects those over 40 years old (Mayo Clinic 2013; http://www.mayoclinic.com/health/rheumatoid-arthritis/DS00020). RA is caused by an autoimmune attack on antigens expressed in the synovial tissue and cartilage of joints. Wrists, fingers, knees, feet, and ankles are most commonly affected. Early phase symptoms are characterized by morning stiffness in the affected joints. Over time, inflammation, cartilage destruction, and bone erosion may lead to deformations and crippling. The general explanation for these effects is as follows. The immune system attacks the synovium, which is the lining of the membrane that surrounds joints. Inflammation ensues which results in the thickening of the synovium and can eventually lead to the destruction of the cartilage and bone within the joint. The tendons and ligaments that hold the joint together also weaken and stretch, and can suffer degradation as a result of proteases (enzymes that break down proteins) secreted by activated macrophages (phagocyte meaning that they engulf solid particles). In this fashion, the joint gradually loses its shape and alignment, leading to deformities and crippling.

                            Figure 2: RA commonly affected joints and impact on joints.

So, more specifically, how does this all happen? Activated macrophages and DCs extravasate (essentially escape from a blood vessel into tissues) into the joint and produce large amounts of pro-inflammatory cytokines (intercellular mediators), especially TNF. The blood vessels in the inflamed joint take on the characteristics of high endothelial venules (HEVs), which specializes them for lymphocyte (small white blood cell) extravasation (Villani 2012; http://www.ipbs.fr/?High-endothelial-venules-HEVs). The inflammation is perpetuated by the eventual infiltration of the joint by CD4+ Th effectors (white blood cells that assist other immune cells) and CD8+ CTLs (white blood cells responsible for causing cell death of infected/damaged cells), which produce cytokines like TNF and IL-17. RA synovial tissues also contain “ectopic” germinal centers (sites where mature B lymphocytes proliferate, differentiate, and mutate and switch the class of their antibodies), meaning that these structures have developed in the wrong tissue. Plasma cells in these abnormal germinal centers produce autoantibodies (an antibody formed in response to and reacting against an antigenic constituent of its own tissues) directed against antigens (substance that induces an immune antibody response) in the synovial membrane and cartilage. Common markers of RA include the presence of rheumatoid factor and anti-citrullinated protein antibodies (ACPA) in the serum. These markers represent autoantibodies that have significant diagnostic values (da Mota et al. 2012; http://www.ncbi.nlm.nih.gov/pubmed/22187055).

 Figure 3: General immune overview of RA joint. Retrieved from Nutrition Remarks (2013) (http://www.nutritionremarks.com/2013/03/09/fish-oil-can-reduce-rheumatoid-arthritis-flu/)

ACPAs are autoantibodies present in the majority of patients with RA. They have proven to be useful biomarkers and allow for the diagnoses of RA at an early stage (da Mota et al. 2012; http://www.ncbi.nlm.nih.gov/pubmed/22187055). During inflammation, in a process known as citrullination, arginine residues in proteins can be converted to citrulline ones (Suurmond et al. 2011; http://www.ncbi.nlm.nih.gov/pubmed/21339220). If this change significantly alters the shape of the proteins, they may be seen as antigens and an immune response will be generated. Autoantibodies are generated against these citrullinated proteins (including fibrinogen or vimentin for example), forming the basis of an autoimmune disease. It is important to note that rheumatoid arthritis patients can be either ACPA-positive or ACPA-negative, and this status can have a significant influence on the intensity and therapy of RA. In fact, ACPA-positive and ACPA-negative RA have been recognized as distinct disease sub-entities, which demonstrate significant differences with regards to HLA-association, genetic and environmental risk factors, disease phenotype, and treatment response. 
Commonly used biological therapies for RA target mainly cytokine pathways. However, abatacept is a chimerical CTLA4 (protein receptor on the surface of T cells which acts as an off switch for T cell attack) and IgG Fc (antibody isotype) fusion protein modulating T cell activation. Abatacept is thought to work by blocking CD28 costimulation, and consequently interfering with T cell-APC interaction and limiting T cell activation. CD28 is a molecule expressed on T cells that provides co-stimulatory signals by interacting with B7 (peripheral membrane protein key for costimulation signals) molecules CD80 and CD86 on antigen presenting cells (APCs). CD28 is thus required for T cell activation. This abatacept mediated blockade could potentially change the activity and lifespan of APCs and limit the activation of CD4+ T cells. Using abatacept has been associated with reduced joint inflammation and pain and joint damage in patients with active RA (Maxwell & Singh 2009; http://www.ncbi.nlm.nih.gov/pubmed/19821401).

Figure 4: Abatacept functional mechanism. Retrieved from Rheumatologist (2011) (http://www.rheumatologysa.com/biologics.html)

In their August 2013 paper (http://www.biomedcentral.com/1471-2172/14/34), Pieper et al. examined T cell functionality in the context of ACPA status in RA patients with regards to abatacept therapy. The authors recognized that RA has a strong MHC class II (molecules on APCs and B cell lymphocytes) component, indicative of a HLA (human leukocyte antigen; the genes encoding the MHC in humans) associated autoimmune disease, which implies that CD4+ T cells are important. This is also supported by the presence of ACPAs in patients and the fact that CD4+ T cells are abundant in synovial tissue and fluid. The role of CD4+ T cells in RA may be mediated through Th1 effector functions (enhanced cytotoxic mechanisms), such as IFN-γ secretion, Th17 activity, or induction of ACPA. It has also been suggested that regulatory T cell function is impaired in RA. Tregs are important as they serve as T cells that modulate the immune system. In this study, the authors wanted to look at the effect of abatacept therapy on T cell subsets and their associated cytokines in ACPA-positive versus negative patients. They accomplished this by performing an anti-CCP assay to determine anti-CCP levels in the patients. They also performed intracellular cytokine staining and luminex analysis of cell culture supernatants to examine cytokine levels. Treg levels were analyzed and phenotypically characterized in peripheral blood mononuclear cells by flow cytometry. Finally, synovial fluid mononuclear cells were examined in vitro in the presence or absence of abatacept.

  Figure 5: T cell subsets. Retrieved from Peterson (2012) (http://livingwellnessblog.wordpress.com/2012/10/12/am-i-th1-or-th2-or-th17/)

This study examined 33 patients starting on abatacept therapy, 23 of whom were ACPA-positive and 10 of whom were ACPA-negative. Peripheral blood was collected from these patients at baseline, after 3 months, and after 6 months. The authors noted diminished T cell effector functions in patients treated with abatacept. Specifically, the authors began by investigating IFN-γ, TNF, and IL-17 as these cytokines are the most relevant in the context of RA, are implicated in disease pathogenesis, and are central in Th1 and Th17 function. T cells of the Th1 subset were found to be affected by abatacept, as both TNF and IFN-γ production by CD4+ T cells decreased. IL-17A production exhibited a decreasing trend. In contrast, in ACPA-negative patients, increases in TNF, IFN-γ, and IL-17A production were noted (reduced cytokine output).
Abatacept limits the immune response by binding to CD80 and CD86 (B7 proteins) on APCs. Consequently, the authors further examined the effect of abatacept on key cytokines influencing Th1, Th2, and Th17 subsets. The general trend was a decrease in cytokine levels (most notably IL-3, IL-13, IL-23) in ACPA-positive patients, while an increase in cytokine levels (most notably IFN-γ) was observed in ACPA-negative patients. The impact of abatacept on regulatory T cell frequencies in vivo was also examined. This was accomplished by investigated FOXP3, Helios, CD39, CTLA4, and CD45RA as these proteins have all been implicated in Treg function in RA. All Treg subset frequencies were considerably reduced. To assess the impact of abatacept on T cells and Tregs in the synovial fluid of affected joints, the authors examined the affect of in vitro added abatacept on both synovial T effector and Treg function. Ultimately, the authors found that abatacept reduces T cell proliferation. A general reduction in Treg frequency in the periphery was also noted.
This study demonstrated a significant down-regulation of all key T cell effector subsets by abatacept, but only in APCA-positive patients. A general decrease in Treg cell frequency was also observed. Ultimately, the study sought to tease out the impact of the abatacept costimulation blockade on cell population level. The authors accomplished this using both peripheral blood and synovial fluid cells. Most strikingly, a reduction in representative Th1, Th2, and Th17 cytokines was observed in ACPA-positive patients, but not in negative ones. Overall, abatacept was found to have a significant impact on T effector functions of the Th1, Th2, and Th17 subsets and the effects were predominantly observed in ACPA-positive patients. These findings demonstrate that RA is a disease with several-different sub-entities, and support the notion that ACPA-positive and ACPA-negative patients represent immunologically distinct disease phenotypes and require tailored treatment strategies.
This paper is highly relevant because of the significant impact of RA in the world today. Many of us know someone who suffers from this immune disease and have seen how debilitating it can be. For that reason, it is important that we continue to investigate potential treatments for this disease and analyze the mechanisms fueling these treatments. This study provided insight into how the abatacept costimulation blockade can modulate T cell effector functions. The better we understand the mechanism of action and effects of a treatment, the more effectively we will be able to determine what treatments will work best. This study also points out the complexity of RA in that it can be characterized by immunologically distinct disease phenotypes. This can have repercussions for which treatment strategies should be employed, as certain treatment methods may work better with a certain disease phenotype, as this study demonstrated. Further studies should work to expand upon the direction of this study and examine the mechanistic effects of other RA therapies and how effective the therapy is in relation to different RA disease phenotypes. In this way, the most effective therapy can be matched to different RA disease phenotypes. Although this study indicates that abatacept therapy effectively modulates T cell effector functions in ACPA-positive patients, further studies should seek to identify a more effective treatment for ACPA-negative patients.  

Primary Article:
Pieper, J., Herrath, J., Raghavan, S., Muhammad, K., van Vollenhoben, R., & Malmstrom, V. (2013). CTLA4-Ig (abatacept) therapy modulates T cell effector functions in autoantibody-positive rheumatoid arthritis patients. BMC Immunology, 14(34). http://www.biomedcentral.com/1471-2172/14/34.

Additional Sources:
da Mota, L.M., Dos Santos Neto, L.L., de Carvalho, J.F., Pereira, I.A., Burlingame, R., Menard, H.A., & Laurindo, I.M. (2012). The presence of anti-citrullinated protein antibodies (ACPA) and rheumatoid factor on patients with rheumatoid arthritis (RA) does not interfere with the chance of clinical remission in a follow-up of 3 years. Rheumatol. Int., 32(12): 3807-3812. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/22187055.
Maxwell, L. & Singh, J.A. (2009). Abatacept for rheumatoid arthritis. Cochrane Database Syst Rev., 4: CD007277. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19821401.
Mayo Clinic. (2013). Rheumatoid arthritis. Mayo Foundation for Medical Education and Research. Retrieved from http://www.mayoclinic.com/health/rheumatoid-arthritis/DS00020.

Suurmond, J., Schuerwegh, A.J, & Toes, R.E. (2011). Anti-citrullinated protein antibodies in rheumatoid arthritis: a functional role for mast cells and basophils? Ann. Rheum. Dis., 70: 55-58. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/21339220.

UCSF Medical Center. (2013). Rheumatoid arthritis. The Regents of the University of California. Retrieved from http://www.ucsfhealth.org/conditions/rheumatoid_arthritis/.
Villani, G.G. (2012). High endothelial venules (HEVs), specialized blood vessels for lymphocyte migration. Institute of Pharmacology and Structural Biology. Retrieved from http://www.ipbs.fr/?High-endothelial-venules-HEVs.

Atlantic Apothecary. (2013). Rheumatoid arthritis. Atlantic Apothecary. Retrieved from http://www.atlanticapothecary.com/disease-state-management/arthritis/rheumatoid-arthritis/.
Cedars-Sinai. (2013). Arthritis- Rheumatoid arthritis, osteoarthritis and spinal arthritis. Cedars-Sinai. Retrieved from http://www.cedars-sinai.edu/Patients/Health-Conditions/Arthritis---Rheumatoid-Arthritis-Osteoarthritis-and-Spinal-Arthritis.aspx.
Nutrition Remarks (2013). Fish oil can reduce rheumatoid arthritis. Nutrition Remarks. Retrieved from http://www.nutritionremarks.com/2013/03/09/fish-oil-can-reduce-rheumatoid-arthritis-flu/.
Peterson, D. (2012). Am I Th1 or Th2 or Th17? Living Wellness. Retrieved from http://livingwellnessblog.wordpress.com/2012/10/12/am-i-th1-or-th2-or-th17/.
Rheumatologist. (2011). Biologic drugs. Rheumatologist. Retrieved from http://www.rheumatologysa.com/biologics.html.

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