Leptin regulation of ocular immunity

1. Leptin regulation of ocular immunity Leptin regulation of ocular immunity Emma Jakes Manchester-Essex Regional High School, Manchester-by-the-Sea, MA Teacher Dr. Maria Lonnett Burgess, Manchester-Essex High School Mentor, Dr. Andrew Taylor, Boston University School of Medicine Boston University, Boston, MA The immune system is comprised of many molecules that regulate the ability of the body to fight infection. In the eye, the concept of “immune privilege” is defined as the lack of harmful inflammatory response due to the composition of fluids that are present. The eye is vulnerable to a variety of internal and external pathogens that can threaten survival, especially in humans. To avoid this, evolution has provided a spectrum of defense mechanisms that can neutralize pathogens invading the eye’s fluid compartments. Leptin, a hormone that regulates metabolism and other essential body functions, is thought to be involved in systemic inflammatory response. The effect of Leptin on the other regulating factors responsible for ocular immune privilege is described in this study. Another hormone, a-Melanocyte Stimulating (a-MSH) is also suspected to confer this privilege. The goal of this project was to determine if Leptin would promote inflammation in the eye by regulating nitric oxide, or NO. Varying concentrations of Leptin were tested for their ability to affect NO on certain cell lines. Each experiment yielded relatively similar results, showing that different concentrations of Leptin did not affect NO release from the cells. Therefore, our original hypothesis was not confirmed by these experiments. Abstract Objectives Tissue Culture Media DMEM plus 10% Fetal Bovine Serum (FBS) Phenol red-free DMEM plus 10% FBS Serum-free RPMI1640 Phenol red-free Media (SFM) Phenol red-free RPMI1640 plus 10% FBS Procedure Culture J774.2 and RAW cell line (DMEM) Centrifuge to solid pellet for counting Pipette cell concentration into 96 well plates, including duplicates, and incubate Wash cells with new media (SFM) Prepare concentrations of needed solutes, pipette and successively dilute concentrations, incubate for 24 hours Table 1. Dilutions used to test NO levels of LPS stimulated cells. Dilutions used were: a-MSH: 5mg/ml → 1/100 → 50ug/ml → 1/100 → 500ng/ml → 1/42 → 12ng/ml → ⅓ →4ng/ml → 1/3 → 1.2ng/ml → 1/3 → 0.4ng/ml; LPS: 10mg/ml → 1/100 → 100ug/ml → 1/25 → 4ug/ml Pipette water into new well plate Mix sodium nitrite stock for standard curve, add to top wells Mix N-(1-naphthyl)ethylenediaminedihydrochloride (Component A) with Sulfanilic acid (Component B) to create Griess Reagent, add to all wells Pipette samples into new well plate Run NO assay on plate and analyze results Fig 2. Cell media being be transferred to a 96-well plate using a multi-pipette. Methods Leptin does not enhance NO levels produced by RAW and J774 cells in vitro . a-MSH appears to suppress NO at concentrations low as 0.3ng/ml Fig 3. NO levels suppressed by concentrations of a-MSH. Fig 4. NO levels of cells enhanced by Leptin (little variation in results at lower levels compared to Fig 3.) Fig 5. NO levels enhanced by concentrations of Leptin and suppressed simultaneously by single concentration of a-MSH(little variation in results, even with the addition of a-MSH). Results Conclusions Experiments using different concentrations of Leptin were expected to increase NO levels. Experiments using different concentrations of a-MSH were expected to suppress NO levels by suppressing inflammation. NO levels that registered in the assay were between 0 and 400mM in Fig 2, but the sodium nitrate stock-based curve for this experiment was not constant. Neither Leptin or a-MSH enhanced or suppressed NO levels respectively Reasoning: • NO reaction happens faster than can be tested • Cells were old and differentiation could have changed their properties • Leptin may react differently in-vitro References Brzoska T, Böhm M, Lügering A, Loser K, Luger TA. Terminal Signal: Anti-Inflammatory Effects of α-Melanocyte-Stimulating Hormone Related Peptides Beyond the Pharmacophore. Adv Exp Med Biol. 2010;681:107-16. Lam QL, Lu L. Role of leptin in immunity. Cell Mol Immunol. 2007;4(1):1 13.107-16. Lau CH, Taylor AW. The immune privileged retina mediates an alternative activation of J774A.1 cells. OculImmunolInflamm. 2009;17(6):380-9. Otero M, Lago R, Gomez R, Dieguez C, Lago F, Gómez-Reino J, Gualillo O. Towards a pro-inflammatory and immunomodulatory emerging role of leptin. Rheumatology (Oxford). 2006;45(8):944-50. Taylor AW. Ocular immune privilege. Eye (Lond). 2009;23(10):1885-9. Taylor AW, Kitaichi N, Biros D. Melanocortin 5 receptor and ocular immunity. Cell Mol Biol(Noisy-le-grand). 2006;52(2):53-9. Taylor AW, Kitaichi N. The diminishment of experimental autoimmune encephalomyelitis (EAE) by neuropeptide alpha-melanocyte stimulating hormone (a-MSH) therapy. Brain Behav Immun. 2008;22(5):639-46. http://us.123rf.com/400wm/400/400/phakimata/phakimata0806/phakimata080600061/3131934-blue-multi-channel-pipet-used-for-pipetting-a-96-well-plate-with-pink-solution-on-white.jpg 9 www.streilein-foundation.org Acknowledgement Thank you to Dr. Andrew Taylor and David Yee for mentoring me in my research, the BU School of Medicine for allowing me the opportunity to work in their labs and Dr. Burgess for her support and enthusiasm. Contact jakesek@gmail.com for further information. To research Leptin’s role in immune regulation by determining if: Leptin acts as a pro-inflammatory hormone on macrophages in vitro. Elicited increased levels of NO in macrophages are suppressed by a-MSH. Leptin acts as antagonist to anti-inflammatory factors. Fig 1. Diagram of the eye and its major functions. Authentic Science Research Program Manchester-Essex Regional High School, Manchester-by-the-Sea, MA 01944 Emma Jakes