Analysis of Map3k-dependent molecular mechanisms regulating signaling in iNKT and B cells

Abstract

Abstract

Our immune system consists of two types of lymphocytes; B lymphocytes which produce antibodies in response to an antigen, and T lymphocytes which differentiate into particular effector T lymphocytes such as a cytotoxic, helper or regulatory T cell in response to an antigen. Type 1 invariant natural killer T cells (iNKT cells) are a unique population of T cells whose phenotypical, developmental and functional characteristics differ from conventional T cells (Novak and Lehuen, 2011). Mitogen-Activated Protein Kinase (MAPK) signaling is critical for T lymphocyte development and effector responses. We define the roles of Map3k1 and Map3k7 genes in Natural Killer T (NKT) cells. We identify an elevated expression of the cell cycle inhibitor Cdkn1b during the Map3k1ΔKD iNKT cell clonal burst. We find aberrant Interleukin (IL) -17 production by Map3k1ΔKD iNKT cells and altered expression of Rorc, Mmp9 and Il17rb in Map3k1ΔKD iNKT cells. The liver damage seen in Map3k1ΔKD mice could be the cause of upregulation of Il-17 and IL-1β cytokines, and IL-1β related genes (Il1r2 and Il1f9). Additionally we find that Map3k1+/ΔKD mice injected with keyhole limpet hemocyanin (KLH) display a larger population of germinal center B cells compared to Map3k1ΔKD mice suggesting an importance of Map3k1 in germinal center formation.

Abstract

Popular science summary:

Analysis of Map3k-dependent molecular mechanisms regulating signaling in lymphocytes

My research involves investigating the roles of Map3k1 and Map3k7 genes in relation to invariant natural killer T (iNKT) cells. iNKT cells are a unique population of T cells whose phenotypical, developmental and functional characteristics differ from conventional T cells. iNKT cells express a highly restricted T cell receptor (TCR), a majority of iNKT cells express the Vα14Jα18 chains with a restricted set of β chains Vβ8.2, Vβ7 and Vβ2 in mice. iNKT cells are restricted by CD1d, a family of major histocompatibility complex (MHC) like molecules that specialize in presenting lipid antigens to T cells. iNKT cells can be detected with α-galactosylceramide (α-GalCer) loaded CD1d tetramers. Mitogen-Activated Protein Kinase (MAPK) signaling is critical for T lymphocyte development and effector responses. MAPKs are major components of pathways controlling embyogenesis, cell differentiation, cell proliferation and cell death. Four main groups of MAPKs have been found: extracellular signal regulated kinases (ERKs) 1 and 2 (ERK1/2), c-Jun amino terminal kinases or stress activated protein kinases (JNK/SAPK) 1-3, p38MAPKα, β, γ and δ and ERK5. Map3k1 is involved in the ERK and JNK kinase pathways and Map3k7 is involved in JNK and p38 kinase pathways.

For this research study Map3k1ΔKD (kinase domain knocked out, inactivating the gene making it mutant), Map3k1+/ΔKD (1 allele expresses the gene and the other has been knocked out making it wildtype), Lck-Cre+Map3k7f/f (entire gene knocked out making it mutant) and Lck-Cre+Map3k7+/f (1 allele expresses the gene and the other has been knocked out making it wildtype) mice have been generated.

Mice were injected with KRN7000 an analogue of α-GalCer to stimulate splenic iNKT cells over a 6 day period to establish whether there is a role for Map3k1 kinase signaling in effector iNKT cell responses to glycolipid antigens. A peak response of iNKT cells was observed on day 3 followed by a steep decline on day 6. There were a reduced number of iNKT cells in Map3k1ΔKD mice on day 3 suggesting there was a defect in iNKT expansion.

To assess the molecular mechanisms during this effect, day 3 isolated iNKT cell mRNA from Map3k1ΔKD and Map3k1+/ΔKD mice was processed and hybridized into Affymetrix Mouse Gene 1.0 ST arrays to compare the gene expression profiles between the two groups. Significant microarray hits with a gene expression change of higher than 2 fold between the two genetic groups was found. Enhanced expression of Cdkn1b (a cell cycle inhibitor) could explain the hypoproliferation observed in Map3k1ΔKD mice. An upregulation of Rorc and IL-17rb was observed in Map3k1ΔKD mice, Rorc is a transcriptional regulator of Th17 development. This could suggest possible involvement of Map3k1 in the regulation of IL-17 and Th17 cell signaling and a connection between iNKT cells and Th17 cells. iNKT cell expansion was examined in the liver due to the defective iNKT splenic expansion seen in Map3k1ΔKD mice. Liver damage was observed following α-GalCer stimulation and an increase in lymphocyte infiltration. This could be due to increased IL-1β expression which has previously shown to have proinflammatory roles in liver damage.

Further research in the molecular mechanisms regulating signaling in T and iNKT cells could lead to the development of potential therapies in autoimmune diseases.

Supervisor: Dr Ewen Gallagher (Imperial College London)
Master´s degree project 60 credits in Immunology2013
Department of Biology, Lund University

• author: Anwar, Saba
• supervisor: Ewan Gallagher
• organization: Degree Projects in Molecular Biology
• course: MOBT13 20122
• year: 2013
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 4053809
• date added to LUP: 2013-09-18 16:31:05
• date last changed: 2013-09-18 16:31:05