Hiding in Plain Site: The Immune System, Tuberculosis, and Antibiotic Resistance

1. Hiding in Plain Site: The Immune System, Tuberculosis, and Antibiotic Resistance  Mark Stephansky,1; Jillian Richmond,2 and William Cruikshank,21Whitman-Hanson Regional High School, Whitman, MA 02382, 2 Pulmonary Center, Boston University School of Medicine, Boston, MA 02118 MATERIALS & METHODS A Sampling of Laboratory Experiences Encountered During Summer Research Program in Immunology Human Jurkat T-lymphocyte cell line and Human Primary T-lymphocytes Human Jurkat T-lymphocyte cells lines were obtained from ATCC and were cultured in RPMI complete medium supplemented with 25mM HEPES, 10% fetal bovine serum, and 1% Penicillin/Streptomycin. Primary T lymphocytes were isolated from whole blood via density gradient centrifugation and were cultured in RPMI complete medium. Both primary cells and cell lines were incubated at 37ºC, 5% CO2. Cell lines were kept in a logarithmic growth phase for the entirety of experiments. CURRICULUM OVERVIEW This high school biology unit is designed to present students in a typical high school setting with the following aspects of Cell Biology and Immunology:  • Students will be able to list and describe the two main defensive strategies of the immune system: innate immunity and acquired immunity. • Students will be able to distinguish between antibody-mediated immunity and cell-mediated immunity. • Students will be able to compare and contrast the cell components of the immune system. • Students will be able to distinguish between antigens and antibodies. • Students will be able to list and draw the different classes of immunoglobulins. • Students will be able to describe the basics of an ELISA assay and practical uses of this type of lab assay. • Students will research and describe the disease tuberculosis and how it interacts with the human immune system. • Students will perform a bacterial transformation. • Students will relate the bacterial transformation lab to evolution in general and to the evolution of bacterial resistance in particular. • Students will communicate their knowledge of the immune system with two narrative essays. This unit may be incorporated into several units of study (biochemistry, disease and immunity, evolution, microbiology, biotechnology) of a typical biology, advanced biology or anatomy and physiology class. It is meant to complement and extend existing curricula rather than exist as a stand-alone unit. BACTERIAL TRANSFORMATION A major objective of this curriculum unit is for students to forge a link between the inability of a compromised immune system to completely eradicate a pathogen borne threat with the ability of bacteria to evolve resistance to antibiotic drugs designed to aid the immune system. Students research tuberculosis and the stages of disease. They learn about bacterial transformation and how bacteria receive resistance genes to antibiotics, Fig 5. Students then conduct their own bacterial transformation lab Fig 6. ABSTRACT # 818999 This past summer I worked in the Cruikshank lab at the Boston University School of Medicine. The Cruikshank research team determined that mannose lipoarabinomannan, (ManLAM), a cell wall component of M. tuberculosis can decrease S1P sensitivity of activated T cells as well as to alter production of selected cytokines. These alterations likely affect the normal immune response by changing T cell homing patterns and decreasing production of response cytokines thereby increasing the mTB’s chances of survival and optimal infection. This research along with the history of tuberculosis inspired me to create a high school biology unit. This unit is designed to link the study of the immune system with evolution through a closer examination of mTB, particularly, its two main forms: latent TB infection and active TB disease. The unit includes teacher presentations and demonstrations, an internet activity entitled TB in the Media, and laboratory activities. Students are first introduced to the basic functions of the immune system through its defense against Mycobacterium tuberculosis infection. Then, students work through a simulated ELISA (Enzyme-linked immunosorbent assay) to see its importance in diagnosing disease. Finally, we turn to the emergence of multi-drug resistant mTB as students carry out a laboratory exercise on bacterial transformation. Students present a summary of their learning through the creation of a pamphlet/poster where they investigate a specific disease and describe its symptoms, treatments, suggested causes and immune system interactions. Isolation of mouse peripheral T-lymphocytes T-lymphocytes were obtained from 6 week old C57BL/6J mice by removing the axillary, cervical, inguinal, and mediastinal nodes and suspending in RPMI complete media. Cells were dissociated from lymph node stroma and filtered to remove excess tissue. In addition, the spleen was perfused with media and the supernatant collected. A red blood cell lysis was performed, then cells were washed, and resuspended in media. Isolated T-lymphocytes were resuspended in RPMI. The Boston University Institutional Animal Care and Use Committee Approved all experiments. Jurkat cell stimulation and Arabinogalactan pretreatment 2.5x105 Jurkat cells were isolated and allowed to incubate at 37ºC, 5% CO2 for 24 hours prior to PHA stimulation. Following incubation cells were pretreated with 10ng/mL Arabinogalactan (AG provided by Colorado State University TBVRTM Contract) for 2 hours. Cell stimulation was performed using Phaseolusvulgaris agglutinin (PHA, Sigma-Aldrich) at 100μg/mL and Ionomycin calcium salt (Sigma-Aldrich) at 2μM. Stimulated cell suspensions were allowed to incubate for 72 hours prior to supernatant collection and cytokine analysis by ELISA. BACKGROUND Tuberculosis, second only to HIV as the worldwide cause of death from infection, has haunted humanity for the past 500,000 years. Currently one-third of the world’s population is infected with the TB bacterium.1 The unique characterization of the Mycobacterium tuberculosis (mTB) cell wall has been implicated in not only protecting the pathogen but in increasing its adaptive ability. Recent TB research has focused on T-lymphocyte function and migration. Lymphocytes are the key cells of acquired immunity. T-lymphocytes migrate to the thymus to mature, while B-lymphocytes mature in the bone marrow. Naïve T cells constantly circulate between blood and lymph nodes in search of pathogen-derived antigen. The migration into lymph nodes is controlled by the action of adhesion receptors and chemokines, particularly CCL19 and CCL21,2 both of which are produced in the high endothelial venules of lymph nodes, while egress is controlled by sphingosine 1-phosphate (S1P) gradients.3 This is significant in that in order for T cells to carry out their immune functions in peripheral tissue they must first experience physical contact with antigen presenting cells, in particular, dendritic cells within a lymph node. (When dendritic cells bind foreign antigen they become activated and migrate to lymph nodes where antigen sampling by Naïve T cells takes place.) The activated T cells must then egress from lymph nodes to carry out their response. The TB cell wall is unique among prokaryotes and a major contributor to the pathogen’s virulence and survival. Two of the principle components of the cell wall are mannose lipoarabinomannan (ManLAM) and mycolyl arabinogalactans (AG). During TB infection ManLAM portions are thought break off and directly insert into lipid raft microdomains (cholesterol and phosphoinositol rich regions). When primary human T cells were treated with ManLAM, migration to S1P was completely blocked.4 Another cell wall component, Arabinogalactan, is part of the mycolyl arabinogalactan-peptidoglycan (mAGP) complex, a linking of the free lipids and cell wall proteins5 (Fig 1.) that surround the cell membrane. AG is thought to play a role in the regulation of specific cytokines responsible for protecting the TB granuloma that surrounds the bacterium. This past summer the Cruikshank lab determined that ManLAM can decrease S1P sensitivity of activated T cells as well as to alter production of selected cytokines.4 These alterations likely affect the normal immune response by changing T cell homing patterns and decreasing production of response cytokines thereby increasing the mTB’s chances of survival and optimal infection. Figure 5. Representation (left) of how bacteria may receive antibiotic resistance genes from their environment. Scanning electron micrograph (right) showing Multidrug-Resistant Mycobacterium tuberculosis and possible effective treatments. Mag15549X. CDC. CURRICULUM TIME REQUIREMENT Total time for this unit is approximately 7 class periods (7 hours – although the computer exercises may be completed by students at home or at the library). The immune system web tutorial is 1 class period, the antibody investigation is 1 class period, the virtual ELISA is 1 class period, the ELISA Classroom Simulation is 1 class period, the bacterial transformation lab requires 3 – 60 minute lab periods. Figure 6. Schematic representation of bacterial transformation procedure. Figure 2. All cells pretreated with arabinogalactan had no detectable TNF-α or IL-4 concentrations, this may be attributed to concentrations outside the standard range (R2 = 0.98 & 0.934, respectively). IL-4 concentration for the untreated cells was 1.159 ng/mL indicating that AG may inhibit IL-4 production. CONCLUSIONS This has certainly been an enlightening experience. One of my main reasons for applying to the program was to become better informed and more confident in my knowledge of the workings of the immune system. My summer research experience certainly delivered. My experience in the Cruikshank lab at BUMC has bolstered my own knowledge and also provided a number of anecdotes to share with my students not only about immune system research, but life in the lab in general. IVIS Imaging of Qdot labeled NWNATS in Mice Subjected to ManLAM I.T.4 REFERENCES • K. Todar, Mycobacterium tuberculosis and Tuberculosis (2009). Available at http://www.textbookofbacteriology.net/tuberculosis.html (18 February 2010). • Drennan, M., Elewaut, D., Hogquist, K. 2008. Thymic emigration: Sphingosine-1-phosphate receptor-1-dependent models and beyond. Eur J. of Immun.39:4:925-930. • Allende, M.L., Dreier, J.L., Mandala, S., and Proia, R.L. 2004. Expression of the sphingosine-1 phosphate receptor, S1P1, on T-cells controls thymic emigration. J. Biol. Chem. 279, 15396–15401. • J. Richmond & W. Cruikshank, BUMC dept. of Pulmonary, Allergy, and Critical Care Medicine; Unpublished Data • Birch HL, et al. (2008) Biosynthesis of mycobacterial arabinogalactan: Identification of a novel α(1—>3) arabinofuranosyltransferase. Mol Microbiol 69:1191–1206. Fig 1: Mycobacterium Cell Wall • outer lipids 2. mycolic acid • Polysaccharides (arabinogalactan) 4. peptidoglycan 5. plasma membrane 6. lipoarabinomannan (LAM) 7. phosphatidylinositolmannoside 8. cell wall skeleton ACKNOWLEDGEMENTS I would like to sincerely thank the American Association of Immunologists for this opportunity, Dr. William Cruikshank, Jillian Richmond and the entire lab at Boston University Medical School Pulmonary Center for their wisdom, patience, and support of this profession-changing experience. Figure 3. IVIS in vitro imaging was performed on mice subjected to ManLAM I.T. following I.V. injection of Qdot labeled NWNATS. Compared to the PBS control (left) the ManLAM treated mouse (right) showed a twofold increase in T cell concentration within lymphoid tissue. Concentrations are highest in the lymph nodes which drain the lungs in mice. The noninvasive fluorescent IVIS imaging system allowed for live analysis of T cell migration while mice were under sedation. Figure 4. Schematic representation of “sandwich” ELISA (Enzyme-linked immunosorbentassay) screening set. ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.