RESUMO
Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn's disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-ß strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn's disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn's disease patients and may play a role in the pathology of the disease.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Linfócitos/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Perforina/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Células Cultivadas , Doença de Crohn/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunidade Inata/genética , Imunidade Inata/fisiologia , Inflamação/imunologia , Inflamação/metabolismo , Linfócitos/imunologia , Perforina/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: As fluorescent microscopy has developed, significant insights have been gained into the establishment of immune response within secondary lymphoid organs, particularly in draining lymph nodes. While established techniques such as confocal imaging and intravital multi-photon microscopy have proven invaluable, they provide limited insight into the architectural and structural context in which these responses occur. To interrogate the role of the lymph node environment in immune response effectively, a new set of imaging tools taking into account broader architectural context must be implemented into emerging immunological questions. METHODS AND RESULTS: Using two different methods of whole-organ imaging, optical clearing and three-dimensional reconstruction of serially sectioned lymph nodes, fluorescent representations of whole lymph nodes can be acquired at cellular resolution. Using freely available post-processing tools, images of unlimited size and depth can be assembled into cohesive, contextual snapshots of immunological response. Through the implementation of robust iterative analysis techniques, these highly complex three-dimensional images can be objectified into sortable object data sets. These data can then be used to interrogate complex questions at the cellular level within the broader context of lymph node biology. CONCLUSIONS: By combining existing imaging technology with complex methods of sample preparation and capture, we have developed efficient systems for contextualizing immunological phenomena within lymphatic architecture. In combination with robust approaches to image analysis, these advances provide a path to integrating scientific understanding of basic lymphatic biology into the complex nature of immunological response.
