Student projects
Epithelial cells alarming the immune system in COPD
Keywords:
COPD, cell death, cigarette smoke, autoimmunity, innate immune response, adaptive immune response, danger signals, DAMPs
Names:
Type of project:
Stage Wetenschap / Research project
Type of research:
Experimental research, cell biology, cellular and molecular immunology
Background/introduction:
The pathogenesis of chronic obstructive pulmonary disease (COPD) is thought to be mediated, at least in part, by an aberrant immune- and inflammatory reaction in response to cigarette smoking, the most important risk factor for the development of COPD. However, the initiation of this aberrant immune reaction is still largely unknown.
Recently, a three-step process leading to COPD has been proposed (1). Cigarette smoke (CS) induced injury to epithelial cells, which release “danger signals” was proposed to be the first step in this process. In patients with COPD, airway epithelial injury and cell death have been related to the presence of both increased apoptosis and necrosis in the lung (2;5). In line with this, we have observed that cigarette smoke exposure of human airway epithelial cells (cell-lines and primary cells) disturbs mitochondrial function leading to a shift from apoptosis to necrosis (5) . It is well-known that necrotic cell-death, in contrast to apoptotic cell death, leads to the release of “damage associated molecular patterns” (DAMPs) and alarmins that trigger the innate- and adaptive immune response (4). In preliminary experiments, we found that CS-induced necrotic epithelial cells leads to the release of ATP, dsDNA and HSP70 which are all considered DAMPs. Moreover, incubation of epithelial cells with supernatants of these CS-induced necrotic cells, but not from control cells, induced the release op pro-inflammatory interleukin-6 (IL-6) and IL-8. These cytokine responses induced by epithelial cells alarm the immune system by promoting the recruitment and activation of host leukocytes e.g. neutrophilic granulocytes that are characteristic for COPD (4).
We propose that the presence of DAMPs derived from airway epithelial injury/cell death serve to induce the maturation of dendritic cells at the site of injury. Mature dendritic cells phagocytose dying cells and are able to migrate back to the lymphoid tissues were they can present auto-antigens from death cells to T cells thereby initiating an adaptive immune response to auto-antigens (1). Overall, this research will evaluate if DAMPs released from cigarette-smoke induced epithelial cell necrosis plays an important role in the maturation of dendritic cells and in the activation of innate- and adaptive immune reactions, including auto-immunity, observed in COPD.
Aim/purpose of the research:
Aim 1: To characterize the profile of DAMPs induced by cigarette smoke exposed lung epithelial cells in vitro and its relation with the type of epithelial cell death (apoptosis, necrosis, parthanatos).
Aim 2: To investigate the profile of DAMPs in the bronchoalveolar lavage fluid of smoking- or non-smoking COPD patients and healthy controls and of control and cigarette-smoke exposed BALB/c mice.
Aim 3: To investigate the effect of different DAMPs on the induction of a proinflammatory phenotype (cytokine production) of lung epithelial cells in vitro.
Aim 4: To investigate if DAMPs released by cigarette-smoke exposed epithelial cells induce maturation of dendritic cells and the production of T-cell skewing cytokines
Working programme:
The student will set out to study the effect of cigarette smoke on DAMP release in both Beas-2b lung epithelial cells and primary bronchial epithelial cells from patients and healthy controls. Cell damage will be assessed by measuring apoptosis/necrosis by Annexin V/PI (Flow cytometry) and LDH levels. Release of DAMPs will be assessed by measuring a.o. HMGB1, ATP, IL-33 and HSP70 levels by ELISA. The student will also use these epithelial cells to for aim 3. For the assessment of the pro-inflammatory phenotype of epithelial cells in response to DAMPs, the release of inflammatory mediators like IL-1β, IL-6, IL-8, TNF a , MCP-1 and MIP-1 a will be measured by ELISA. Furthermore, the student will generate immature monocyte-derived dendritic cells using blood monocytes cultured with IL-4 and GM-CSF. The effects of DAMPs on dendritic cell maturation will be determined by flow cytometry (MHC-II, CD80, CD86) and by cytokine production (IL-6, IL-10, IL-12, TGF b ) as measured by ELISA.
Reference List
1. Cosio, M. G., Saetta, M., and Agusti, A. (2009) Immunologic aspects of chronic obstructive pulmonary disease. N. Engl. J. Med. 360, 2445-2454
2. Hodge, S., Hodge, G., Holmes, M., and Reynolds, P. N. (2005) Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation. Eur. Respir. J. 25, 447-454
3. Makris, D., Vrekoussis, T., Izoldi, M., Alexandra, K., Katerina, D., Dimitris, T., Michalis, A., Tzortzaki, E., Siafakas, N. M., and Tzanakis, N. (2009) Increased apoptosis of neutrophils in induced sputum of COPD patients. Respir. Med. 103, 1130-1135
4. Oppenheim, J. J., Tewary, P., de la, R. G., and Yang, D. (2007) Alarmins initiate host defense. Adv. Exp. Med. Biol. 601, 185-194
5. van der Toorn, M., Slebos, D. J., de Bruin, H. G., Leuvenink, H. G., Bakker, S. J., Gans, R. O., Koeter, G. H., van Oosterhout, A. J., and Kauffman, H. F. (2007) Cigarette smoke-induced blockade of the mitochondrial respiratory chain switches lung epithelial cell apoptosis into necrosis. Am. J. Physiol Lung Cell Mol. Physiol 292, L1211-L1218
| Laatst gewijzigd: | 28 januari 2014 15:38 |