Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling.

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.

Yolk Sac Macrophages, Fetal Liver, and Adult Monocytes Can Colonize an Empty Niche and Develop into Functional Tissue-Resident Macrophages.

Tissue-resident macrophages can derive from yolk sac macrophages (YS-Macs), fetal liver monocytes (FL-MOs), or adult bone-marrow monocytes (BM-MOs). The relative capacity of these precursors to colonize a niche, self-maintain, and perform tissue-specific functions is unknown. We simultaneously transferred traceable YS-Macs, FL-MOs, and BM-MOs into the empty alveolar macrophage (AM) niche of neonatal Csf2rb(-/-) mice. All subsets produced AMs, but in competition preferential outgrowth of FL-MOs was observed, correlating with their superior granulocyte macrophage-colony stimulating factor (GM-CSF) reactivity and proliferation capacity. When transferred separately, however, all precursors efficiently colonized the alveolar niche and generated AMs that were transcriptionally almost identical, self-maintained, and durably prevented alveolar proteinosis. Mature liver, peritoneal, or colon macrophages could not efficiently colonize the empty AM niche, whereas mature AMs could. Thus, precursor origin does not affect the development of functional self-maintaining tissue-resident macrophages and the plasticity of the mononuclear phagocyte system is largest at the precursor stage.

Granulocyte Macrophage Colony-Stimulating Factor-Activated Eosinophils Promote Interleukin-23 Driven Chronic Colitis.

The role of intestinal eosinophils in immune homeostasis is enigmatic and the molecular signals that drive them from protective to tissue damaging are unknown. Most commonly associated with Th2 cell-mediated diseases, we describe a role for eosinophils as crucial effectors of the interleukin-23 (IL-23)-granulocyte macrophage colony-stimulating factor (GM-CSF) axis in colitis. Chronic intestinal inflammation was characterized by increased bone marrow eosinopoiesis and accumulation of activated intestinal eosinophils. IL-5 blockade or eosinophil depletion ameliorated colitis, implicating eosinophils in disease pathogenesis. GM-CSF was a potent activator of eosinophil effector functions and intestinal accumulation, and GM-CSF blockade inhibited chronic colitis. By contrast neutrophil accumulation was GM-CSF independent and dispensable for colitis. In addition to TNF secretion, release of eosinophil peroxidase promoted colitis identifying direct tissue-toxic mechanisms. Thus, eosinophils are key perpetrators of chronic inflammation and tissue damage in IL-23-mediated immune diseases and it suggests the GM-CSF-eosinophil axis as an attractive therapeutic target.

The Cytokine GM-CSF Drives the Inflammatory Signature of CCR2+ Monocytes and Licenses Autoimmunity.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2(+)Ly6C(hi) monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2(+)Ly6C(hi) monocytes and their progeny, which was essential for tissue damage.

A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common ß chain (CSF2RB) protein stability.

The IL-3, IL-5, and GM-CSF family of cytokines play an essential role in the growth, differentiation, and effector functions of multiple hematopoietic cell types. Receptors in this family are composed of cytokine-specific α chains and a common ß chain (CSF2RB), responsible for the majority of downstream signaling. CSF2RB abundance and stability influence the magnitude of the cellular response to cytokine stimulation, but the exact mechanisms of regulation are not well understood. Here, we use genetic screens in multiple cellular contexts and cytokine conditions to identify STUB1, an E3 ubiquitin ligase, and CHIC2 as regulators of CSF2RB ubiquitination and protein stability. We demonstrate that Stub1 and Chic2 form a complex that binds Csf2rb and that genetic inactivation of either Stub1 or Chic2 leads to reduced ubiquitination of Csf2rb. The effects of Stub1 and Chic2 on Csf2rb were greatest at reduced cytokine concentrations, suggesting that Stub1/Chic2-mediated regulation of Csf2rb is a mechanism of reducing cell surface accumulation when cytokine levels are low. Our study uncovers a mechanism of CSF2RB regulation through ubiquitination and lysosomal degradation and describes a role for CHIC2 in the regulation of a cytokine receptor.

BHLHE40 Promotes TH2 Cell-Mediated Antihelminth Immunity and Reveals Cooperative CSF2RB Family Cytokines.

The transcription factor BHLHE40 is an emerging regulator of the immune system. Recent studies suggest that BHLHE40 regulates type 2 immunity, but this has not been demonstrated in vivo. We found that BHLHE40 is required in T cells for a protective TH2 cell response in mice infected with the helminth Heligmosomoides polygyrus bakeri H. polygyrus elicited changes in gene and cytokine expression by lamina propria CD4+ T cells, many of which were BHLHE40 dependent, including production of the common ß (CSF2RB) chain family cytokines GM-CSF and IL-5. In contrast to deficiency in GM-CSF or IL-5 alone, loss of both GM-CSF and IL-5 signaling impaired protection against H. polygyrus Overall, we show that BHLHE40 regulates the TH2 cell transcriptional program during helminth infection to support normal expression of Csf2, Il5, and other genes required for protection and reveal unexpected redundancy of common ß chain-dependent cytokines previously thought to possess substantially divergent functions.

GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells.

GM-CSF (Csf-2) is a critical cytokine for the in vitro generation of dendritic cells (DCs) and is thought to control the development of inflammatory DCs and resident CD103(+) DCs in some tissues. Here we showed that in contrast to the current understanding, Csf-2 receptor acts in the steady state to promote the survival and homeostasis of nonlymphoid tissue-resident CD103(+) and CD11b(+) DCs. Absence of Csf-2 receptor on lung DCs abrogated the induction of CD8(+) T cell immunity after immunization with particulate antigens. In contrast, Csf-2 receptor was dispensable for the differentiation and innate function of inflammatory DCs during acute injuries. Instead, inflammatory DCs required Csf-1 receptor for their development. Thus, Csf-2 is important in vaccine-induced CD8(+) T cell immunity through the regulation of nonlymphoid tissue DC homeostasis rather than control of inflammatory DCs in vivo.

Pulmonary macrophage transplantation therapy.

Bone-marrow transplantation is an effective cell therapy but requires myeloablation, which increases infection risk and mortality. Recent lineage-tracing studies documenting that resident macrophage populations self-maintain independently of haematological progenitors prompted us to consider organ-targeted, cell-specific therapy. Here, using granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor-ß-deficient (Csf2rb(-/-)) mice that develop a myeloid cell disorder identical to hereditary pulmonary alveolar proteinosis (hPAP) in children with CSF2RA or CSF2RB mutations, we show that pulmonary macrophage transplantation (PMT) of either wild-type or Csf2rb-gene-corrected macrophages without myeloablation was safe and well-tolerated and that one administration corrected the lung disease, secondary systemic manifestations and normalized disease-related biomarkers, and prevented disease-specific mortality. PMT-derived alveolar macrophages persisted for at least one year as did therapeutic effects. Our findings identify mechanisms regulating alveolar macrophage population size in health and disease, indicate that GM-CSF is required for phenotypic determination of alveolar macrophages, and support translation of PMT as the first specific therapy for children with hPAP.

Negative Regulation of the Differentiation of Flk2- CD34- LSK Hematopoietic Stem Cells by EKLF/KLF1.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2- CD34- Lin- Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor ß subunit (CSF2RB). As a result, Flk2- CD34- LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2- CD34- LSK-HSCs.

Evidence for an association of interferon gene variants with sudden infant death syndrome.

BACKGROUND: There is evidence that inflammation plays a role in the etiology of sudden infant death syndrome (SIDS). Immune system dysregulation seems to be the background of higher infection susceptibility in SIDS infants. This phenotype is possibly determined by genetic factors. METHODS: Twenty-three single nucleotide polymorphisms (SNPs) in the following 13 candidate genes governing the immune system were successfully genotyped in 251 Caucasian SIDS cases and 336 controls from Germany: ADAR1, CSF2RB, DDX58, IFNA1, IFNA21, IFNA8, IFNAR2, IFNG, IL6, MX2, OAS1, OAS3, and TNFA. Associations between genotypes and SIDS were then statistically evaluated using logistic regression analyses. RESULTS: Overall analysis revealed statistically significant results for two variants in interferon gamma (IFNG) (rs2069705: OR 1.40 (1.07; 1.83), p = 0.01; and rs2069727: OR 0.75 (0.59; 0.96), p = 0.02) and for one variant in interferon alpha 8 (IFNA8) (rs1330321: OR 1.85 (1.06; 3.21), p = 0.03). Haplotype analyses identified a three-marker risk IFNG haplotype rs2069727-rs2069718-rs2069705 associated with SIDS (OR = 1.62, 95% CI 1.23-2.13; p = 0.0003). Subgroup associations were found for variants in adenosine deaminase acting on RNA1 (ADAR1), 2',5'-oligoadenylate synthetase-1 (OAS1) and colony stimulating factor 2 receptor beta common subunit (CSF2RB). CONCLUSION: In summary, this large study of 251 SIDS cases for common variants in 13 candidate genes governing the immune system has provided first evidence for a role of IFNG in the etiology of SIDS and should stimulate further research into the clinicopathological relevance of immunomodulatory genes for this fatal syndrome.

Multiple QTL underlie milk phenotypes at the CSF2RB locus.

BACKGROUND: Over many years, artificial selection has substantially improved milk production by cows. However, the genes that underlie milk production quantitative trait loci (QTL) remain relatively poorly characterised. Here, we investigate a previously reported QTL located at the CSF2RB locus on chromosome 5, for several milk production phenotypes, to better understand its underlying genetic and molecular causes. RESULTS: Using a population of 29,350 taurine dairy cows, we conducted association analyses for milk yield and composition traits, and identified highly significant QTL for milk yield, milk fat concentration, and milk protein concentration. Strikingly, protein concentration and milk yield appear to show co-located yet genetically distinct QTL. To attempt to understand the molecular mechanisms that might be mediating these effects, gene expression data were used to investigate eQTL for 11 genes in the broader interval. This analysis highlighted genetic impacts on CSF2RB and NCF4 expression that share similar association signatures to those observed for lactation QTL, strongly implicating one or both of these genes as responsible for these effects. Using the same gene expression dataset representing 357 lactating cows, we also identified 38 novel RNA editing sites in the 3' UTR of CSF2RB transcripts. The extent to which two of these sites were edited also appears to be genetically co-regulated with lactation QTL, highlighting a further layer of regulatory complexity that involves the CSF2RB gene. CONCLUSIONS: This locus presents a diversity of molecular and lactation QTL, likely representing multiple overlapping effects that, at a minimum, highlight the CSF2RB gene as having a causal role in these processes.

Dysregulated genes and miRNAs in the apoptosis pathway in colorectal cancer patients.

Apoptosis is genetically regulated and involves intrinsic and extrinsic pathways. We examined 133 genes within these pathways to identify whether they are expressed differently in colorectal carcinoma (CRC) and normal tissue (N = 217) and if they are associated with similar differential miRNA expression. Gene expression data (RNA-Seq) and miRNA expression data (Agilent Human miRNA Microarray V19.0) were generated. We focused on dysregulated genes with a fold change (FC) of > 1.50 or < 0.67, that were significant after adjustment for multiple comparisons. miRNA:mRNA seed-region matches were determined. Twenty-three genes were significantly downregulated (FC < 0.67) and 18 were significantly upregulated (FC > 1.50). Of these 41 genes, 11 were significantly associated with miRNA differential expression. BIRC5 had the greatest number of miRNA associations (14) and the most miRNAs with a seed-region match (10). Four of these matches, miR-145-5p, miR-150-5p, miR-195-5p, and miR-650, had a negative beta coefficient. CSF2RB was associated with ten total miRNAs (five with a seed-region match, and one miRNA, miR-92a-3p, with a negative beta coefficient). Of the three miRNAs associated with CTSS, miR-20b-5p, and miR-501-3p, had a seed-region match and a negative beta coefficient between miRNA:mRNA pairs. Several miRNAs that were associated with dysregulated gene expression, seed-region matches, and negative beta coefficients also were associated with CRC-specific survival. Our data suggest that miRNAs could influence several apoptosis-related genes. BIRC5, CTSS, and CSF2R all had seed-region matches with miRNAs that would favor apoptosis. Our study identifies several miRNA associated with apoptosis-related genes, that if validated, could be important therapeutic targets.

Activation of the EPOR-ß common receptor complex by cibinetide ameliorates impaired wound healing in mice with genetic diabetes.

Diabetes is characterized by poor wound healing which currently lacks an efficacious treatment. The innate repair receptor (IRR) is a master regulator of tissue protection and repair which is expressed as a response injury or metabolic stress, including in diabetes. Activation of the IRR might provide benefit for diabetic wound healing. A specific IRR agonist cibinetide was administered in an incisional wound healing model performed mice with genetic diabetes (db+/db+) and compared to the normal wild-type. Animals were treated daily with cibinetide (30µg/kg/s.c.) or vehicle and euthanized 3, 7, and 14days after the injury to quantitate vascular endothelial growth factor (VEGF), malondialdehyde (MAL), phospho-Akt (pAkt), phospho e-NOS (p-eNOS), and nitrite/nitrate content within the wound. Additional evaluations included quantification of skin histological change, angiogenesis, scar strength, and time to complete wound closure. Throughout the wound healing process diabetic animals treated with vehicle exhibited increased wound MAL with reduced VEGF, pAkt, peNOS and nitrite/nitrate, all associated with poor re-epitheliziation, angiogenesis, and wound breaking strength. Cibenitide administration significantly improved these abnormalities. The results suggest that cibinetide-mediated IRR activation may represent an interesting strategy to treat diabetes-associated wound healing.

Characterisation of the porcine cytokines which activate the CD131ßc common sub-unit, for potential immune-augmentation.

Early acting cytokines and growth factors such as those of the CD131 ßc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.

Expansion of EPOR-negative macrophages besides erythroblasts by elevated EPOR signaling in erythrocytosis mouse models.

Activated erythropoietin (EPO) receptor (EPOR) signaling causes erythrocytosis. The important role of macrophages for the erythroid expansion and differentiation process has been reported, both in baseline and stress erythropoiesis. However, the significance of EPOR signaling for regulation of macrophages contributing to erythropoiesis has not been fully understood. Here we show that EPOR signaling activation quickly expands both erythrocytes and macrophages in vivo in mouse models of primary and secondary erythrocytosis. To mimic the chimeric condition and expansion of the disease clone in the polycythemia vera patients, we combined Cre-inducible Jak2V617F/+ allele with LysM-Cre allele which expresses in mature myeloid cells and some of the HSC/Ps (LysM-Cre;Jak2V617F/+ mice). We also generated inducible EPO-mediated secondary erythrocytosis models using Alb-Cre, Rosa26-loxP-stop-loxP-rtTA, and doxycycline inducible EPAS1-double point mutant (DPM) alleles (Alb-Cre;DPM mice). Both models developed a similar degree of erythrocytosis. Macrophages were also increased in both models without increase of major inflammatory cytokines and chemokines. EPO administration also quickly induced these macrophages in wild-type mice before observable erythrocytosis. These findings suggest that EPOR signaling activation could induce not only erythroid cell expansion, but also macrophages. Surprisingly, an in vivo genetic approach indicated that most of those macrophages do not express EPOR, but erythroid cells and macrophages contacted tightly with each other. Given the importance of the central macrophages as a niche for erythropoiesis, further elucidation of the EPOR signaling mediated-regulatory mechanisms underlying macrophage induction might reveal a potential therapeutic target for erythrocytosis.

Interleukin-3 stimulates matrix metalloproteinase 12 production from macrophages promoting thoracic aortic aneurysm/dissection.

Thoracic aortic aneurysm and dissection (TAAD) is due to degeneration of the aorta and causes a high mortality rate, while molecular mechanisms for the development of TAAD are still not completely understood. In the present study, 3-aminopropionitrile (BAPN) treatment was used to induce TAAD mouse model. Through transcriptome analysis, we found the expression levels of genes associated with interleukin-3 (IL-3) signaling pathway were up-regulated during TAAD development in mouse, which were validated by real-time PCR. IL-3 positive cells were increased in TAAD mouse aortas, especially for smooth muscle cells (SMCs). IL-3 deficiency reduced BAPN-induced TAAD formation. We then examined the matrix metalloproteinases (MMPs) expression during TAAD formation in both wild-type and IL-3 deficient mice, showing that MMP12 were significantly down-regulated in IL-3 deficient aortas. Mechanistically, we found recombinant IL-3 could increase MMP12 production and activity from macrophages in vitro Silencing of IL-3 receptor ß, which was mainly expressed in macrophages but not SMCs, diminished the activation of c-Jun N terminal kinase (JNK)/extracellular-regulated protein kinases 1/2 (ERK1/2)/AP-1 signals, and decreased MMP12 expression in IL-3 stimulated macrophages. Moreover, both circulating and aortic inflammation were decreased in IL-3 deficient aortas. Taken together, our results demonstrated that IL-3 stimulated the production of MMP12 from macrophages by a JNK- and ERK1/2-dependent AP-1 pathway, contributing to TAAD formation. Thus, the IL-3/IL-3Rß/MMP12 signals activation may be an important pathological mechanism for progression of TAAD.

Disruption of Glut1 in Hematopoietic Stem Cells Prevents Myelopoiesis and Enhanced Glucose Flux in Atheromatous Plaques of ApoE(-/-) Mice.

RATIONALE: Inflamed atherosclerotic plaques can be visualized by noninvasive positron emission and computed tomographic imaging with (18)F-fluorodeoxyglucose, a glucose analog, but the underlying mechanisms are poorly understood. OBJECTIVE: Here, we directly investigated the role of Glut1-mediated glucose uptake in apolipoprotein E-deficient (ApoE(-/-)) mouse model of atherosclerosis. METHODS AND RESULTS: We first showed that the enhanced glycolytic flux in atheromatous plaques of ApoE(-/-) mice was associated with the enhanced metabolic activity of hematopoietic stem and multipotential progenitor cells and higher Glut1 expression in these cells. Mechanistically, the regulation of Glut1 in ApoE(-/-) hematopoietic stem and multipotential progenitor cells was not because of alterations in hypoxia-inducible factor 1α signaling or the oxygenation status of the bone marrow but was the consequence of the activation of the common ß subunit of the granulocyte-macrophage colony-stimulating factor/interleukin-3 receptor driving glycolytic substrate utilization by mitochondria. By transplanting bone marrow from WT, Glut1(+/-), ApoE(-/-), and ApoE(-/-)Glut1(+/-) mice into hypercholesterolemic ApoE-deficient mice, we found that Glut1 deficiency reversed ApoE(-/-) hematopoietic stem and multipotential progenitor cell proliferation and expansion, which prevented the myelopoiesis and accelerated atherosclerosis of ApoE(-/-) mice transplanted with ApoE(-/-) bone marrow and resulted in reduced glucose uptake in the spleen and aortic arch of these mice. CONCLUSIONS: We identified that Glut1 connects the enhanced glucose uptake in atheromatous plaques of ApoE(-/-) mice with their myelopoiesis through regulation of hematopoietic stem and multipotential progenitor cell maintenance and myelomonocytic fate and suggests Glut1 as potential drug target for atherosclerosis.

Plasma metabolite profiles, cellular cholesterol efflux, and non-traditional cardiovascular risk in patients with CKD.

BACKGROUND: Patients with chronic kidney disease (CKD) experience high rates of atherosclerotic cardiovascular disease and death that are not fully explained by traditional risk factors. In animal studies, defective cellular cholesterol efflux pathways which are mediated by the ATP binding cassette transporters ABCA1 and ABCG1 are associated with accelerated atherosclerosis. We hypothesized that cholesterol efflux in humans would vary in terms of cellular components, with potential implications for cardiovascular disease. METHODS: We recruited 120 CKD patients (eGFR<30mL/min/1.73m2) and 120 control subjects (eGFR ≥60mL/min/1.73m2) in order to measure cholesterol efflux using either patients' HDL and THP-1 macrophages or patients' monocytes and a flow cytometry based cholesterol efflux assay. We also measured cell-surface levels of the common ß subunit of the IL-3/GM-CSF receptor (IL-3Rß) which has been linked to defective cholesterol homeostasis and may promote monocytosis. In addition, we measured plasma inflammatory cytokines and plasma metabolite profiles. RESULTS: There was a strong positive correlation between cell-surface IL-3Rß levels and monocyte counts in CKD (P<0.001). ABCA1 mRNA was reduced in CKD vs. control monocytes (P<0.05), across various etiologies of CKD. Cholesterol efflux to apolipoprotein A1 was impaired in monocytes from CKD patients with diabetic nephropathy (P<0.05), but we found no evidence for a circulating HDL-mediated defect in cholesterol efflux in CKD. Profiling of plasma metabolites showed that medium-chain acylcarnitines were both independently associated with lower levels of cholesterol transporter mRNA in CKD monocytes at baseline (P<0.05), and with cardiovascular events in CKD patients after median 2.6years of follow-up. CONCLUSIONS: Cholesterol efflux in humans varies in terms of cellular components. We report a cellular defect in ABCA1-mediated cholesterol efflux in monocytes from CKD patients with diabetic nephropathy. Unlike several traditional risk factors for atherosclerotic cardiovascular disease, plasma metabolites inversely associated with endogenous cholesterol transporters predicted cardiovascular events in CKD patients. (Funded by the National Institute of Diabetes and Digestive and Kidney DiseasesK23DK097288 and others.).

A Conserved Ectodomain-Transmembrane Domain Linker Motif Tunes the Allosteric Regulation of Cell Surface Receptors.

In many families of cell surface receptors, a single transmembrane (TM) α-helix separates ecto- and cytosolic domains. A defined coupling of ecto- and TM domains must be essential to allosteric receptor regulation but remains little understood. Here, we characterize the linker structure, dynamics, and resulting ecto-TM domain coupling of integrin αIIb in model constructs and relate it to other integrin α subunits by mutagenesis. Cellular integrin activation assays subsequently validate the findings in intact receptors. Our results indicate a flexible yet carefully tuned ecto-TM coupling that modulates the signaling threshold of integrin receptors. Interestingly, a proline at the N-terminal TM helix border, termed NBP, is critical to linker flexibility in integrins. NBP is further predicted in 21% of human single-pass TM proteins and validated in cytokine receptors by the TM domain structure of the cytokine receptor common subunit ß and its P441A-substituted variant. Thus, NBP is a conserved uncoupling motif of the ecto-TM domain transition and the degree of ecto-TM domain coupling represents an important parameter in the allosteric regulation of diverse cell surface receptors.

Genetic Complexity of Crohn's Disease in Two Large Ashkenazi Jewish Families.

BACKGROUND & AIMS: Crohn's disease (CD) is a highly heritable disease that is particularly common in the Ashkenazi Jewish population. We studied 2 large Ashkenazi Jewish families with a high prevalence of CD in an attempt to identify novel genetic risk variants. METHODS: Ashkenazi Jewish patients with CD and a positive family history were recruited from the University College London Hospital. We used genome-wide, single-nucleotide polymorphism data to assess the burden of common CD-associated risk variants and for linkage analysis. Exome sequencing was performed and rare variants that were predicted to be deleterious and were observed at a high frequency in cases were prioritized. We undertook within-family association analysis after imputation and assessed candidate variants for evidence of association with CD in an independent cohort of Ashkenazi Jewish individuals. We examined the effects of a variant in DUOX2 on hydrogen peroxide production in HEK293 cells. RESULTS: We identified 2 families (1 with >800 members and 1 with >200 members) containing 54 and 26 cases of CD or colitis, respectively. Both families had a significant enrichment of previously described common CD-associated risk variants. No genome-wide significant linkage was observed. Exome sequencing identified candidate variants, including a missense mutation in DUOX2 that impaired its function and a frameshift mutation in CSF2RB that was associated with CD in an independent cohort of Ashkenazi Jewish individuals. CONCLUSIONS: In a study of 2 large Ashkenazi Jewish with multiple cases of CD, we found the genetic basis of the disease to be complex, with a role for common and rare genetic variants. We identified a frameshift mutation in CSF2RB that was replicated in an independent cohort. These findings show the value of family studies and the importance of the innate immune system in the pathogenesis of CD.