Identifying transcript-level differential expression in primary human immune cells.

BACKGROUND: Multipotential hematopoietic stem cells differentiate into a wide variety of immune cells with a diversity of functions, including the ability to respond to a variety of stimuli. Importantly, numerous studies have demonstrated the importance of gene transcription in defining cell identity and functions. While these studies have primarily been performed at the level of the gene, it is known that key immune genes such as CD44 and CD45 generate multiple different transcripts that are differentially expressed across different immune cells, and that encode proteins with different sequences and functions. Prior genomic surveys have shown that the mechanisms for generating diversity in expressed transcripts (alternate splicing, alternate transcription start sites, etc.) are very active in immune cells, but have been lacking in terms of identifying genes with multiple transcripts, that are differentially expressed, and likely to affect cell functions. METHODS: We first identified the set of genes that had at least two transcripts expressed in our RNA sequencing dataset generated from purified populations of neutrophils, monocytes and five lymphocyte populations (B, NK, γδ T, CD4 + T and CD8 + T) from twelve healthy donors. Next, we developed a heuristic approach to identify genes where two or more transcripts have distinct expression patterns across lymphoid and/or myeloid populations. We then focused our annotation and interpretation on differentially expressed transcripts that affect the coding sequence. This process was repeated to identify transcripts that were differentially expressed between monocytes and populations of macrophages and LPS-stimulated macrophages derived from these monocytes in vitro. RESULTS: We found that over 55 % of genes had two or more expressed transcripts, with an average ∼3 transcripts per gene, and that 70 % of these had at least two of the transcripts that encoded proteins with different sequences. As expected, we identified a complex pattern of differential expression for multiple transcripts encoding the CD45 transmembrane protein, but we also found similar evidence for ten other genes (CD300A, FYB1, GPI, LITAF, PSMA1, PTMA, RPL32, SEPTIN9, SH3BP2, SH3KBP1) when comparing the expression patterns of transcripts within myeloid and lymphoid cells. We also identified five genes with differentially expressed transcripts associated with the transition from monocytes to macrophages (FNBP1, KLF6, and SEPTIN9) or between macrophages and LPS-stimulated macrophages (CD44, OAZ2, and SEPTIN9). For the most part, we found that the different transcripts of these genes are expected to impact specific biological functions, for example the different transcripts of SEPTIN9 likely regulate the cytoskeleton in immune cells via their interactions with actins filaments and microtubules. CONCLUSIONS: This analytic approach successfully identified multi-transcript genes that are differentially expressed across immune cells and could be applied to other transcriptomic data. DATA AVAILABILITY STATEMENT: Researchers can request access to the individual-level data from the current study by contacting the Montreal Heart Institute ethics committee at the following institutional email address: cer.icm@icm-mhi.org.

Prostaglandins and calprotectin are genetically and functionally linked to the Inflammatory Bowel Diseases.

BACKGROUND: Genome wide association studies (GWAS) have identified and validated more than 200 genomic loci associated with the inflammatory bowel disease (IBD), although for most the causal gene remains unknown. Given the importance of myeloid cells in IBD pathogenesis, the current study aimed to uncover the role of genes within IBD genetic loci that are endogenously expressed in this cell lineage. METHODS: The open reading frames (ORF) of 42 genes from IBD-associated loci were expressed via lentiviral transfer in the THP-1 model of human monocytes and the impact of each of these on the cell's transcriptome was analyzed using a RNA sequencing-based approach. We used a combination of genetic and pharmacologic approaches to validate our findings in the THP-1 line with further validation in human induced pluripotent stem cell (hiPSC)-derived-monocytes. RESULTS: This functional genomics screen provided evidence that genes in four IBD GWAS loci (PTGIR, ZBTB40, SLC39A11 and NFKB1) are involved in controlling S100A8 and S100A9 gene expression, which encode the two subunits of calprotectin (CP). We demonstrated that increasing PTGIR expression and/or stimulating PTGIR signaling resulted in increased CP expression in THP-1. This was further validated in hiPSC-derived monocytes. Conversely, knocking-down PTGIR endogenous expression and/or inhibiting PTGIR signaling led to decreased CP expression. These analyses were extended to the known IBD gene PTGER4, whereby its specific agonist also led to increased CP expression. Furthermore, we demonstrated that the PTGIR and PTGER4 mediated control of CP expression was dependent on signaling via adenylate cyclase and STAT3. Finally, we demonstrated that LPS-mediated increases in CP expression could be potentiated by agonists of PTGIR and PTGER4, and diminished by their antagonists. CONCLUSION: Our results support a causal role for the PTGIR, PTGER4, ZBTB40, SLC39A11 and NFKB1 genes in IBD, with all five genes regulating the expression of CP in myeloid cells, as well as potential roles for the prostacyclin/prostaglandin biogenesis and signaling pathways in IBD susceptibility and pathogenesis.

Epigenetic reader SP140 loss of function drives Crohn's disease due to uncontrolled macrophage topoisomerases.

How mis-regulated chromatin directly impacts human immune disorders is poorly understood. Speckled Protein 140 (SP140) is an immune-restricted PHD and bromodomain-containing epigenetic "reader," and SP140 loss-of-function mutations associate with Crohn's disease (CD), multiple sclerosis (MS), and chronic lymphocytic leukemia (CLL). However, the relevance of these mutations and mechanisms underlying SP140-driven pathogenicity remains unexplored. Using a global proteomic strategy, we identified SP140 as a repressor of topoisomerases (TOPs) that maintains heterochromatin and macrophage fate. In humans and mice, SP140 loss resulted in unleashed TOP activity, de-repression of developmentally silenced genes, and ultimately defective microbe-inducible macrophage transcriptional programs and bacterial killing that drive intestinal pathology. Pharmacological inhibition of TOP1/2 rescued these defects. Furthermore, exacerbated colitis was restored with TOP1/2 inhibitors in Sp140-/- mice, but not wild-type mice, in vivo. Collectively, we identify SP140 as a TOP repressor and reveal repurposing of TOP inhibition to reverse immune diseases driven by SP140 loss.

IBD-associated G protein-coupled receptor 65 variant compromises signalling and impairs key functions involved in inflammation.

BACKGROUND AND AIMS: Inflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. METHODS: We used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. RESULTS: We confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1ß secretion in response to an inflammatory stimulus. CONCLUSIONS: We demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.

Functional screen of inflammatory bowel disease genes reveals key epithelial functions.

BACKGROUND: Genetic studies have been tremendously successful in identifying genomic regions associated with a wide variety of phenotypes, although the success of these studies in identifying causal genes, their variants, and their functional impacts has been more limited. METHODS: We identified 145 genes from IBD-associated genomic loci having endogenous expression within the intestinal epithelial cell compartment. We evaluated the impact of lentiviral transfer of the open reading frame (ORF) of these IBD genes into the HT-29 intestinal epithelial cell line via transcriptomic analyses. By comparing the genes in which expression was modulated by each ORF, as well as the functions enriched within these gene lists, we identified ORFs with shared impacts and their putative disease-relevant biological functions. RESULTS: Analysis of the transcriptomic data for cell lines expressing the ORFs for known causal genes such as HNF4a, IFIH1, and SMAD3 identified functions consistent with what is already known for these genes. These analyses also identified two major clusters of genes: Cluster 1 contained the known IBD causal genes IFIH1, SBNO2, NFKB1, and NOD2, as well as genes from other IBD loci (ZFP36L1, IRF1, GIGYF1, OTUD3, AIRE and PITX1), whereas Cluster 2 contained the known causal gene KSR1 and implicated DUSP16 from another IBD locus. Our analyses highlight how multiple IBD gene candidates can impact on epithelial structure and function, including the protection of the mucosa from intestinal microbiota, and demonstrate that DUSP16 acts a regulator of MAPK activity and contributes to mucosal defense, in part via its regulation of the polymeric immunoglobulin receptor, involved in the protection of the intestinal mucosa from enteric microbiota. CONCLUSIONS: This functional screen, based on expressing IBD genes within an appropriate cellular context, in this instance intestinal epithelial cells, resulted in changes to the cell's transcriptome that are relevant to their endogenous biological function(s). This not only helped in identifying likely causal genes within genetic loci but also provided insight into their biological functions. Furthermore, this work has highlighted the central role of intestinal epithelial cells in IBD pathophysiology, providing a scientific rationale for a drug development strategy that targets epithelial functions in addition to the current therapies targeting immune functions.

A transcriptome-based approach to identify functional modules within and across primary human immune cells.

Genome-wide transcriptomic analyses have provided valuable insight into fundamental biology and disease pathophysiology. Many studies have taken advantage of the correlation in the expression patterns of the transcriptome to infer a potential biologic function of uncharacterized genes, and multiple groups have examined the relationship between co-expression, co-regulation, and gene function on a broader scale. Given the unique characteristics of immune cells circulating in the blood, we were interested in determining whether it was possible to identify functional co-expression modules in human immune cells. Specifically, we sequenced the transcriptome of nine immune cell types from peripheral blood cells of healthy donors and, using a combination of global and targeted analyses of genes within co-expression modules, we were able to determine functions for these modules that were cell lineage-specific or shared among multiple cell lineages. In addition, our analyses identified transcription factors likely important for immune cell lineage commitment and/or maintenance.

Lrrk2 alleles modulate inflammation during microbial infection of mice in a sex-dependent manner.

Variants in the leucine-rich repeat kinase-2 (LRRK2) gene are associated with Parkinson's disease, leprosy, and Crohn's disease, three disorders with inflammation as an important component. Because of its high expression in granulocytes and CD68-positive cells, LRRK2 may have a function in innate immunity. We tested this hypothesis in two ways. First, adult mice were intravenously inoculated with Salmonella typhimurium, resulting in sepsis. Second, newborn mouse pups were intranasally infected with reovirus (serotype 3 Dearing), which induced encephalitis. In both mouse models, wild-type Lrrk2 expression was protective and showed a sex effect, with female Lrrk2-deficient animals not controlling infection as well as males. Mice expressing Lrrk2 carrying the Parkinson's disease-linked p.G2019S mutation controlled infection better, with reduced bacterial growth and longer animal survival during sepsis. This gain-of-function effect conferred by the p.G2019S mutation was mediated by myeloid cells and was abolished in animals expressing a kinase-dead Lrrk2 variant, p.D1994S. Mouse pups with reovirus-induced encephalitis that expressed the p.G2019S Lrrk2 mutation showed increased mortality despite lower viral titers. The p.G2019S mutant Lrrk2 augmented immune cell chemotaxis and generated more reactive oxygen species during virulent infection. Reovirus-infected brains from mice expressing the p.G2019S mutant Lrrk2 contained higher concentrations of α-synuclein. Animals expressing one or two p.D1994S Lrrk2 alleles showed lower mortality from reovirus-induced encephalitis. Thus, Lrrk2 alleles may alter the course of microbial infections by modulating inflammation, and this may be dependent on the sex and genotype of the host as well as the type of pathogen.

Characterization of a Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for the Study of Variant Pathogenicity: Validation of a KCNJ2 Mutation.

BACKGROUND: Long-QT syndrome is a potentially fatal condition for which 30% of patients are without a genetically confirmed diagnosis. Rapid identification of causal mutations is thus a priority to avoid at-risk situations that can lead to fatal cardiac events. Massively parallel sequencing technologies are useful for the identification of sequence variants; however, electrophysiological testing of newly identified variants is crucial to demonstrate causality. Long-QT syndrome could, therefore, benefit from having a standardized platform for functional characterization of candidate variants in the physiological context of human cardiomyocytes. METHODS AND RESULTS: Using a variant in Kir2.1 (Gly52Val) revealed by whole-exome sequencing in a patient presenting with symptoms of long-QT syndrome as a proof of principle, we demonstrated that commercially available human induced pluripotent stem cell-derived cardiomyocytes are a powerful model for screening variants involved in genetic cardiac diseases. Immunohistochemistry experiments and whole-cell current recordings in human embryonic kidney cells expressing the wild-type or the mutant Kir2.1 demonstrated that Kir2.1-52V alters channel cellular trafficking and fails to form a functional channel. Using human induced pluripotent stem cell-derived cardiomyocytes, we not only confirmed these results but also further demonstrated that Kir2.1-52V is associated with a dramatic prolongation of action potential duration with evidence of arrhythmic activity, parameters which could not have been studied using human embryonic kidney cells. CONCLUSIONS: Our study confirms the pathogenicity of Kir2.1-52V in 1 patient with long-QT syndrome and also supports the use of isogenic human induced pluripotent stem cell-derived cardiomyocytes as a physiologically relevant model for the screening of variants of unknown function.

Loss of hepatic LRPPRC alters mitochondrial bioenergetics, regulation of permeability transition and trans-membrane ROS diffusion.

The French-Canadian variant of Leigh Syndrome (LSFC) is an autosomal recessive oxidative phosphorylation (OXPHOS) disorder caused by a mutation in LRPPRC, coding for a protein involved in the stability of mitochondrially-encoded mRNAs. Low levels of LRPPRC are present in all patient tissues, but result in a disproportionately severe OXPHOS defect in the brain and liver, leading to unpredictable subacute metabolic crises. To investigate the impact of the OXPHOS defect in the liver, we analyzed the mitochondrial phenotype in mice harboring an hepatocyte-specific inactivation of Lrpprc. Loss of LRPPRC in the liver caused a generalized growth delay, and typical histological features of mitochondrial hepatopathy. At the molecular level, LRPPRC deficiency caused destabilization of polyadenylated mitochondrial mRNAs, altered mitochondrial ultrastructure, and a severe complex IV (CIV) and ATP synthase (CV) assembly defect. The impact of LRPPRC deficiency was not limited to OXPHOS, but also included impairment of long-chain fatty acid oxidation, a striking dysregulation of the mitochondrial permeability transition pore, and an unsuspected alteration of trans-membrane H2O2 diffusion, which was traced to the ATP synthase assembly defect, and to changes in the lipid composition of mitochondrial membranes. This study underscores the value of mitochondria phenotyping to uncover complex and unexpected mechanisms contributing to the pathophysiology of mitochondrial disorders.

IL23R (Interleukin 23 Receptor) Variants Protective against Inflammatory Bowel Diseases (IBD) Display Loss of Function due to Impaired Protein Stability and Intracellular Trafficking.

Genome-wide association studies as well as murine models have shown that the interleukin 23 receptor (IL23R) pathway plays a pivotal role in chronic inflammatory diseases such as Crohn disease (CD), ulcerative colitis, psoriasis, and type 1 diabetes. Genome-wide association studies and targeted re-sequencing studies have revealed the presence of multiple potentially causal variants of the IL23R. Specifically the G149R, V362I, and R381Q IL23Rα chain variants are linked to protection against the development of Crohn disease and ulcerative colitis in humans. Moreover, the exact mechanism of action of these receptor variants has not been elucidated. We show that all three of these IL23Rα variants cause a reduction in IL23 receptor activation-mediated phosphorylation of the signal-transducing activator of transcription 3 (STAT3) and phosphorylation of signal transducing activator of transcription 4 (STAT4). The reduction in signaling is due to lower levels of cell surface receptor expression. For G149R, the receptor retention in the endoplasmic reticulum is due to an impairment of receptor maturation, whereas the R381Q and V362I variants have reduced protein stability. Finally, we demonstrate that the endogenous expression of IL23Rα protein from V362I and R381Q variants in human lymphoblastoid cell lines exhibited lower expression levels relative to susceptibility alleles. Our results suggest a convergent cause of IL23Rα variant protection against chronic inflammatory disease.

Specific targeting of the IL-23 receptor, using a novel small peptide noncompetitive antagonist, decreases the inflammatory response.

IL-23 is part of the IL-12 family of cytokines and is composed of the p19 subunit specific to IL-23 and the p40 subunit shared with IL-12. IL-23 specifically contributes to the inflammatory process of multiple chronic inflammatory autoimmune disorders, including psoriasis, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. So far, one antibody targeting the shared p40 subunit of IL-12 and IL-23, Ustekinumab, is approved clinically to treat psoriasis. However, there are no treatments inhibiting specifically the IL-23 proinflammatory response. We have developed small IL-23R-specific antagonists by designing all D-peptides arising from flexible regions of IL-23R. Of these peptides, we selected 2305 (teeeqqly), since in addition to its soluble properties, it inhibited IL-23-induced STAT3 phosphorylation in spleen cells. Peptide 2305 specifically binds to IL-23R/IL-12Rß1-expressing HEK-293 cells and not to cells devoid of the receptor. Peptide 2305 showed functional selectivity by modulating IL-23-induced gene expression in IL-23R/IL-12Rß1-expressing cells and in Jurkat cells; 2305 does not inhibit IL-12-induced cytokine expression in IL-12Rß-IL-12Rß2-HEK-293 cells. Finally, compared with anti-p40 treatment, 2305 effectively and selectively inhibits IL-23-induced inflammation in three in vivo mouse models: IL-23-induced ear inflammation, anti-CD40-induced systemic inflammatory response, and collagen-induced arthritis. We, hereby, describe the discovery and characterization of a potent IL-23R small-peptide modulator, 2305 (teeeqqly), that is effective in vivo. 2305 may be more convenient, less cumbersome, less costly, and most importantly, more specific than current biologics for the treatment of inflammatory conditions, and conceivably complement the actual therapies for these chronic and debilitating inflammatory diseases.

The dichotomous pattern of IL-12r and IL-23R expression elucidates the role of IL-12 and IL-23 in inflammation.

IL-12 and IL-23 cytokines respectively drive Th1 and Th17 type responses. Yet, little is known regarding the biology of these receptors. As the IL-12 and IL-23 receptors share a common subunit, it has been assumed that these receptors are co-expressed. Surprisingly, we find that the expression of each of these receptors is restricted to specific cell types, in both mouse and human. Indeed, although IL-12Rß2 is expressed by NK cells and a subset of γδ T cells, the expression of IL-23R is restricted to specific T cell subsets, a small number of B cells and innate lymphoid cells. By exploiting an IL-12- and IL-23-dependent mouse model of innate inflammation, we demonstrate an intricate interplay between IL-12Rß2 NK cells and IL-23R innate lymphoid cells with respectively dominant roles in the regulation of systemic versus local inflammatory responses. Together, these findings support an unforeseen lineage-specific dichotomy in the in vivo role of both the IL-12 and IL-23 pathways in pathological inflammatory states, which may allow more accurate dissection of the roles of these receptors in chronic inflammatory diseases in humans.

The non-classical MAP kinase ERK3 controls T cell activation.

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.

Unusual selection and peripheral homeostasis for immunoregulatory CD4(-) CD8(-) T cells.

Immunoregulatory CD4(-)  CD8(-) (double-negative; DN) T cells exhibit a unique antigen-specific mode of suppression, yet the ontogeny of DN T cells remains enigmatic. We have recently shown that 3A9 T-cell receptor (TCR) transgenic mice bear a high proportion of immunoregulatory 3A9 DN T cells, facilitating their study. The 3A9 TCR is positively selected on the H2(k) MHC haplotype, is negatively selected in mice bearing the cognate antigen, namely hen egg lysozyme, and there is absence of positive selection on the H2(b) MHC haplotype. Herein, we take advantage of this well-defined 3A9 TCR transgenic model to assess the thymic differentiation of DN T cells and its impact on determining the proportion of these cells in secondary lymphoid organs. We find that the proportion of DN T cells in the thymus is not dictated by the nature of the MHC-selecting haplotype. By defining DN T-cell differentiation in 3A9 TCR transgenic CD47-deficient mice as well as in mice bearing the NOD.H2(k) genetic background, we further demonstrate that the proportion of 3A9 DN T cells in the spleen is independent of the MHC selecting haplotype. Together, our findings suggest that immunoregulatory DN T cells are subject to rules distinct from those imposed upon CD4 T cells.

A meta-analysis of genome-wide association scans identifies IL18RAP, PTPN2, TAGAP, and PUS10 as shared risk loci for Crohn's disease and celiac disease.

Crohn's disease (CD) and celiac disease (CelD) are chronic intestinal inflammatory diseases, involving genetic and environmental factors in their pathogenesis. The two diseases can co-occur within families, and studies suggest that CelD patients have a higher risk to develop CD than the general population. These observations suggest that CD and CelD may share common genetic risk loci. Two such shared loci, IL18RAP and PTPN2, have already been identified independently in these two diseases. The aim of our study was to explicitly identify shared risk loci for these diseases by combining results from genome-wide association study (GWAS) datasets of CD and CelD. Specifically, GWAS results from CelD (768 cases, 1,422 controls) and CD (3,230 cases, 4,829 controls) were combined in a meta-analysis. Nine independent regions had nominal association p-value <1.0 x 10⁻5 in this meta-analysis and showed evidence of association to the individual diseases in the original scans (p-value < 1 x 10⁻² in CelD and < 1 x 10⁻³ in CD). These include the two previously reported shared loci, IL18RAP and PTPN2, with p-values of 3.37 x 10⁻8 and 6.39 x 10⁻9, respectively, in the meta-analysis. The other seven had not been reported as shared loci and thus were tested in additional CelD (3,149 cases and 4,714 controls) and CD (1,835 cases and 1,669 controls) cohorts. Two of these loci, TAGAP and PUS10, showed significant evidence of replication (Bonferroni corrected p-values <0.0071) in the combined CelD and CD replication cohorts and were firmly established as shared risk loci of genome-wide significance, with overall combined p-values of 1.55 x 10⁻¹° and 1.38 x 10⁻¹¹ respectively. Through a meta-analysis of GWAS data from CD and CelD, we have identified four shared risk loci: PTPN2, IL18RAP, TAGAP, and PUS10. The combined analysis of the two datasets provided the power, lacking in the individual GWAS for single diseases, to detect shared loci with a relatively small effect.

Interleukin-10 limits the expansion of immunoregulatory CD4-CD8- T cells in autoimmune-prone non-obese diabetic mice.

Regulatory T cells appear to show great potential for use in cellular therapy. In particular, CD4(-)CD8(-) (double negative (DN)) T cells, which compose 1-3% of the total number of T lymphocytes, exhibit prominent antigen-specific immune tolerance properties and confer immune tolerance in models of allografts and xenografts. We have recently shown that autoimmune-diabetes-prone mice carry fewer DN T cells and that this phenotype contributes to autoimmune-prone diabetes susceptibility, suggesting that increasing DN T-cell number in autoimmune-prone individuals may be of therapeutic interest. To achieve this goal, we must first determine whether the remaining DN T cells in autoimmune-prone mice are functional. In addition, we must identify the parameters that regulate the numbers of DN T cells. Herein, we evaluate the immunoregulatory properties of DN T cells in the autoimmune-prone non-obese diabetic (NOD) genetic background. Using 3A9 TCR transgenic mice, we show that DN T cells from both diabetes-resistant B10.Br and genetically autoimmune-prone NOD.H2(k) mice show an equivalent immunoregulatory potential on a per cell basis. However, upon stimulation, there is a 10-fold increase in the number of 3A9 TCR transgenic DN T cells that produce interleukin 10 (IL-10) from NOD.H2(k) mice in comparison with B10.Br mice. We further showed that IL-10 facilitates DN T-cell apoptosis and thus may regulate the number of DN T cells. Taken together, our results show that, although reduced in number, DN T cells from mice carrying an autoimmune-prone genetic background exhibit a potent cytotoxic potential and that DN T-cell expansion is regulated, at least in part, by IL-10.

Genome-wide association identifies multiple ulcerative colitis susceptibility loci.

Ulcerative colitis is a chronic, relapsing inflammatory condition of the gastrointestinal tract with a complex genetic and environmental etiology. In an effort to identify genetic variation underlying ulcerative colitis risk, we present two distinct genome-wide association studies of ulcerative colitis and their joint analysis with a previously published scan, comprising, in aggregate, 2,693 individuals with ulcerative colitis and 6,791 control subjects. Fifty-nine SNPs from 14 independent loci attained an association significance of P < 10(-5). Seven of these loci exceeded genome-wide significance (P < 5 x 10(-8)). After testing an independent cohort of 2,009 cases of ulcerative colitis and 1,580 controls, we identified 13 loci that were significantly associated with ulcerative colitis (P < 5 x 10(-8)), including the immunoglobulin receptor gene FCGR2A, 5p15, 2p16 and ORMDL3 (orosomucoid1-like 3). We confirmed association with 14 previously identified ulcerative colitis susceptibility loci, and an analysis of acknowledged Crohn's disease loci showed that roughly half of the known Crohn's disease associations are shared with ulcerative colitis. These data implicate approximately 30 loci in ulcerative colitis, thereby providing insight into disease pathogenesis.

Implication of the CD47 pathway in autoimmune diabetes.

CD47 and signal regulatory protein (SIRP) interactions have been proposed to take part in autoimmune disease susceptibility. Importantly, a recent genome-wide association study for type 1 diabetes susceptibility highlighted the association of the 20p13 region comprising the SIRP cluster, where some of the SIRP proteins encode functional ligands to CD47. Using a TCR transgenic mouse model at the brink of autoimmune disease, we demonstrate that CD47-deficiency is sufficient to break the immune tolerance and provoke the onset of autoimmune diabetes. Interestingly, CD47-deficient mice show a severe reduction in the number of mature CD4(-)CD8(-) T cells, and passive transfer of these CD4(-)CD8(-) T cells is sufficient to restore immune tolerance and prevent diabetes progression. Together, these findings constitute an in vivo demonstration that CD47 is involved in diabetes susceptibility and controls the homeostatic regulation of CD4(-)CD8(-) T cells.

Absence of CD47 in vivo influences thymic dendritic cell subset proportions but not negative selection of thymocytes.

CD47 is a ubiquitously expressed molecule which has been attributed a role in many cellular processes. Its role in preventing cellular phagocytosis has defined CD47 as an obligatory self-molecule providing a 'don't-eat-me-signal'. Additionally, CD47-CD172a interactions are important for cellular trafficking. Yet, the contribution of CD47 to T cell stimulation remains controversial, acting sometimes as a co-stimulator and sometimes as an inhibitor of TCR signalling or peripheral T cell responses. Most of the experiments leading to this controversy have been carried in in vitro systems. Moreover, the role of CD47 on thymocyte differentiation, which precisely relies on TCR signal strength, has not been evaluated. Here, we examine the in vivo role of CD47 in T cell differentiation using CD47-deficient mice. We find that, in the absence of CD47, thymocyte positive and negative selection processes are not altered. Indeed, our data demonstrate that the absence of CD47 does not influence the strength of TCR signalling in thymocytes. Furthermore, in agreement with a role for CD47-CD172a interactions in CD172a(+) dendritic cell migration, we report a reduced proportion of thymic dendritic cells expressing CD172a in CD47-deficient mice. As the total proportion of dendritic cells is maintained, this creates an imbalance in the proportion of CD172a(+) and CD172a(low) dendritic cells in the thymus. Together, these data indicate that the altered proportion of thymic dendritic cell subsets does not have a primordial influence on thymic selection processes.