Aimee Landry

Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease

Following recent success in genome-wide association studies, a critical focus of human genetics is to understand how genetic variation at implicated loci influences cellular and disease processes. Crohns disease (CD) is associated with SNPs around IRGM, but coding-sequence variation has been excluded as a source of this association. We identified a common, 20-kb deletion polymorphism, immediately upstream of IRGM and in perfect linkage disequilibrium (r2 = 1.0) with the most strongly CD-associated SNP, that causes IRGM to segregate in the population with two distinct upstream sequences. The deletion (CD risk) and reference (CD protective) haplotypes of IRGM showed distinct expression patterns. Manipulation of IRGM expression levels modulated cellular autophagy of internalized bacteria, a process implicated in CD. These results suggest that the CD association at IRGM arises from an alteration in IRGM regulation that affects the efficacy of autophagy and identify a common deletion polymorphism as a likely causal variant.

The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naïve T cells

The Mst1 and Mst2 protein kinases are the mammalian homologs of hippo, a major inhibitor of cell proliferation in Drosophila. Mst1 is most abundant in lymphoid tissues. Mice lacking Mst1 exhibit markedly reduced levels of the Mst1 regulatory protein Nore1B/RAPL in lymphoid cells, whereas Mst2 abundance is unaltered. Mst1-null mice exhibit normal T cell development but low numbers of mature naïve T cells with relatively normal numbers of effector/memory T cells. In vitro, the Mst1-deficient naïve T cells exhibit markedly greater proliferation in response to stimulation of the T cell receptor whereas the proliferative responses of the Mst1-null effector/memory T cell cohort is similar to wild type. Thus, elimination of Mst1 removes a barrier to the activation and proliferative response of naïve T cells. The levels of Mst1 and Nore1B/RAPL in wild-type effector/memory T cells are approximately 10% those seen in wild-type naïve T cells, which may contribute to the enhanced proliferative responses of the former. Freshly isolated Mst1-null T cells exhibit high rates of ongoing apoptosis, a likely basis for their low numbers in vivo; they also exhibit defective clustering of LFA-1, as previously observed for Nore1B/RAPL-deficient T cells. Among known Mst1 substrates, only the phosphorylation of the cell cycle inhibitory proteins MOBKL1A/B is lost entirely in TCR-stimulated, Mst1-deficient T cells. Mst1/2-catalyzed MOBKL1A/B phosphorylation slows proliferation and is therefore a likely contributor to the anti-proliferative action of Mst1 in naïve T cells. The Nore1B/RAPL-Mst1 complex is a negative regulator of naïve T cell proliferation.

Ca2+/Calmodulin-Dependent Protein Kinase II Is a Modulator of CARMA1-Mediated NF-κB Activation

ABSTRACT CARMA1 is a central regulator of NF-κB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-κB signaling downstream of the T-cell receptor (TCR). Computational analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca2+/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-κB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-κB activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF-κB activation.

Differential requirement for CARMA1 in agonist-selected T-cell development

Caspase recruitment domain‐containing membrane‐associated guanylate kinase protein‐1 (CARMA1) is a critical component of the NF‐κB signaling cascade mediated by TCR engagement. In addition to activation of naïve T cells, TCR signaling is important for the development of agonist‐selected T‐cell subsets such as Treg, NKT cells, and CD8‐αα T cells. However, little is known about the role of CARMA1 in the development of these lineages. Here we show that CARMA1‐deficient mice (CARMA1−/−) have altered populations of specific subsets of agonist‐selected T cells. Specifically, CARMA1−/− mice have impaired natural and adaptive Treg development, whereas NKT cell numbers are normal compared with wild‐type mice. Interestingly, CD8‐αα T cells, which may also be able to develop through an extrathymic selection pathway, are enriched in the gut of CARMA1−/− mice, whereas memory‐phenotype CD4+ T cells (CD62Llow/CD44high) are present at reduced numbers in the periphery. These results indicate that CARMA1 is essential for Treg development, but is not necessary for the development of other agonist‐selected T‐cell subsets. Overall, these data reveal an important but differential role for CARMA1‐mediated TCR signaling in T‐cell development.

DLGH1 is a negative regulator of T-lymphocyte proliferation

ABSTRACT Discs large homolog 1 (DLGH1), a founding member of the membrane-associated guanylate kinase family of proteins containing PostSynaptic Density-95/Discs large/Zona Occludens-1 domains, is an ortholog of the Drosophila tumor suppressor gene Discs large. In the mammalian embryo, DLGH1 is essential for normal urogenital morphogenesis and the development of skeletal and epithelial structures. Recent reports also indicate that DLGH1 may be a critical mediator of signals triggered by the antigen receptor complex in T lymphocytes by functioning as a scaffold coordinating the activities of T-cell receptor (TCR) signaling proteins at the immune synapse. However, it remains unclear if DLGH1 functions to enhance or attenuate signals emanating from the TCR. Here, we used Dlgh1 gene-targeted mice to determine the requirement for DLGH1 in T-cell development and activation. Strikingly, while all major subsets of T cells appear to undergo normal thymic development in the absence of DLGH1, peripheral lymph node Dlgh1−/− T cells show a hyper-proliferative response to TCR-induced stimulation. These data indicate that, consistent with the known function of Discs large proteins as tumor suppressors and attenuators of cell division, in T lymphocytes, DLGH1 functions as a negative regulator of TCR-induced proliferative responses.

CARMA3 Mediates Lysophosphatidic Acid–Stimulated Cytokine Secretion by Bronchial Epithelial Cells

NF-kappaB activation in bronchial epithelial cells is important for the development of allergic airway inflammation, and may control the expression of critical mediators of allergic inflammation such as thymic stromal lymphopoietin (TSLP) and the chemokine CCL20. Members of the caspase recruitment domain (CARD) family of proteins are differentially expressed in tissue and help mediate NF-kappaB activity in response to numerous stimuli. Here we demonstrate that CARMA3 (CARD10) is specifically expressed in human airway epithelial cells, and that expression of CARMA3 in these cells leads to activation of NF-kappaB. CARMA3 has recently been shown to mediate NF-kappaB activation in embryonic fibroblasts after stimulation with lysophosphatidic acid (LPA), a bioactive lipid-mediator that is elevated in the lungs of individuals with asthma. Consistent with this, we demonstrate that stimulation of airway epithelial cells with LPA leads to increased expression of TSLP and CCL20. We then show that inhibition of CARMA3 activity in airway epithelial cells reduces LPA-mediated NF-kappaB activity and the production of TSLP and CCL20. In conclusion, these data demonstrate that LPA stimulates TSLP and CCL20 expression in bronchial epithelial cells via CARMA3-mediated NF-kappaB activation.

Selective activation of nuclear factor kappa B in the cochlea by sensory and inflammatory stress.

Damage response pathways triggered by mechanical stress might reasonably be expected to be conserved throughout evolution. However, using a nuclear factor kappa B (NF-kappaB) reporter mouse we show here that this phylogenetically recent transcription factor plays a major role in the response to mechanosensory stress in the mammalian inner ear. The protective action of NF-kappaB is exerted in neither sensory nor non-sensory epithelial cells, but rather in connective tissue cells within the spiral ligament and spiral limbus. In the spiral ligament, predominantly type I fibrocytes are activated following noise exposure, whereas type II fibrocytes are activated following systemic inflammatory stress. Immune-mediated and acoustic trauma-mediated hearing loss syndromes in humans may in part result from the vulnerability of type II and type I fibrocytes to systemic inflammatory stress and acoustic trauma, respectively. Unexpected cell-specific and stress-specific NF-kappaB activation found in the inner ear in this in vivo study suggest that this approach may have wide applications in demonstrating similar specializations of stress responses in other tissues, including the brain.

MAST3: a Novel IBD Risk Factor that Modulates TLR4 Signaling

Inflammatory bowel disease (IBD) is a chronic disorder caused by multiple factors in a genetically susceptible host. Significant advances in the study of genetic susceptibility have highlighted the importance of the innate immune system in this disease. We previously completed a genome-wide linkage study and found a significant locus (IBD6) on chromosome 19p. We were interested in identifying the causal variant in IBD6. We performed a two-stage association mapping study. In stage 1, 1530 single-nucleotide polymorphisms (SNPs) were selected from the HapMap database and genotyped in 761 patients with IBD. Among the SNPs that passed the threshold for replication, 26 were successfully genotyped in 754 additional patients (stage 2). One intronic variant, rs273506, located in the microtubule-associated serine/threonine-protein kinase gene-3 (MAST3), was found to be associated in both stages (pooled P=1.8 × 10−4). We identified four MAST3 coding variants, including a non-synonymous SNP rs8108738, correlated to rs273506 and associated with IBD. To test whether MAST3 was expressed in cells of interest, we performed expression assays, which showed abundant expression of MAST3 in antigen-presenting cells and in lymphocytes. The knockdown of MAST3 specifically decreased Toll-like receptor-4-dependent NF-κB activity. Our findings are additional proofs of the pivotal role played by modulators of NF-κB activity in IBD pathogenesis.

Isolation and Analysis of Lipid Rafts in Cell–Cell Interactions

Lipid rafts are dynamic structures made up of proteins and lipids that float freely within the liquid-disordered bilayer of cellular membranes and have the ability to cluster to form larger, more-ordered platforms. These clustered structures have been identified in all cell types and have been shown to play critical roles in signal transduction, cellular transport, and cell-cell communication. Lipid rafts also have been implicated in facilitating bacterial/viral entry into host cells and in human disease, highlighting the significance of understanding the role lipid rafts play in physiological and pathological signaling outcomes. In this chapter, we provide protocols to isolate lipid rafts from polarized and nonpolarized cells and outline novel technologies to analyze signal transduction cascades in vivo.