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. Crohn's 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.

Distinct mechanisms govern populations of myeloid-derived suppressor cells in chronic viral infection and cancer.

Myeloid-derived suppressor cells (MDSCs) are major negative regulators of immune responses in cancer and chronic infections. It remains unclear if regulation of MDSC activity in different conditions is controlled by similar mechanisms. We compared MDSCs in mice with cancer and lymphocytic choriomeningitis virus (LCMV) infection. Chronic LCMV infection caused the development of monocytic MDSCs (M-MDSCs) but did not induce polymorphonuclear MDSCs (PMN-MDSCs). In contrast, both MDSC populations were present in cancer models. An acquisition of immune-suppressive activity by PMN-MDSCs in cancer was controlled by IRE1α and ATF6 pathways of the endoplasmic reticulum (ER) stress response. Abrogation of PMN-MDSC activity by blockade of the ER stress response resulted in an increase in tumor-specific immune response and reduced tumor progression. In contrast, the ER stress response was dispensable for suppressive activity of M-MDSCs in cancer and LCMV infection. Acquisition of immune-suppressive activity by M-MDSCs in spleens was mediated by IFN-γ signaling. However, it was dispensable for suppressive activity of M-MDSCs in tumor tissues. Suppressive activity of M-MDSCs in tumors was retained due to the effect of IL-6 present at high concentrations in the tumor site. These results demonstrate disease- and population-specific mechanisms of MDSC accumulation and the need for targeting different pathways to achieve inactivation of these cells.

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-kappaB 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-alpha alpha 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-alpha alpha 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 (CD62L(low)/CD44(high)) 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.

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.

Ca2+/calmodulin-dependent protein kinase II is a modulator of CARMA1-mediated NF-kappaB activation.

CARMA1 is a central regulator of NF-kappaB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-kappaB 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 Ca(2+)/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-kappaB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-kappaB 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-kappaB activation.

Nore1B regulates TCR signaling via Ras and Carma1.

Nore1A was originally identified as a potential Ras effector, and Nore1B is an alternatively spliced isoform. Both share a Ras/Rap association domain (RA domain) but only Nore1A contains sequence motifs that predict SH3 domain binding and diacylglycerol/phorbol ester binding in the amino-terminal region. Here we report that Carma1 binds to Nore1A and Nore1B through the RA domain and that Carma1 interacts with active Ras in the presence of Nore1B. RNA interference against Nore1B attenuates NF-kappaB activation induced by T cell receptor (TCR) ligation, but not NF-kappaB activation induced by TNFalpha or lipoteichoic acid. In addition, Nore1B is also required for KiRas GV12-mediated ERK1 activation and Elk1 reporter activity in T cells. We also provide evidence that knockdown of Nore1B also impairs polarized redistribution of Ras at the B cell-T cell immune interface. Together, these findings suggest that endogenous Nore1B recruits active Ras to the APC-T cell interface and mediates the interaction between Ras and Carma1.

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.

Erythropoietin regulates endothelial progenitor cells.

Circulating bone marrow-derived endothelial progenitor cells (EPCs) promote vascular reparative processes and neoangiogenesis, and their number in peripheral blood correlates with endothelial function and cardiovascular risk. We tested the hypothesis that the cytokine erythropoietin (EPO) stimulates EPCs in humans. We studied 11 patients with renal anemia and 4 healthy subjects who received standard doses of recombinant human EPO (rhEPO). Treatment with rhEPO caused a significant mobilization of CD34(+)/CD45(+) circulating progenitor cells in peripheral blood (measured by flow cytometry), and increased the number of functionally active EPCs (measured by in vitro assay) in patients (week 2, 312% +/- 31%; week 8, 308% +/- 40%; both P <.01 versus baseline) as well as in healthy subjects (week 8, 194% +/- 15%; P <.05 versus baseline). The effect on EPCs was already observed with an rhEPO dose of about 30 IU/kg per week. Administration of rhEPO increased the number of functionally active EPCs by differentiation in vitro in a dose-dependent manner, assessed in cell culture and by tube formation assay. Furthermore, rhEPO activates the Akt protein kinase pathway in EPCs. Erythropoietin increases the number of functionally active EPCs in humans. Administration of rhEPO or EPO analogs may open new therapeutic strategies in regenerative cardiovascular medicine.