Mitochondrial complex II in intestinal epithelial cells regulates T cell-mediated immunopathology.

Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.

Selective Inhibition of mTORC1 Signaling Supports the Development and Maintenance of Pluripotency.

Insight into the molecular mechanisms governing the development and maintenance of pluripotency is important for understanding early development and the use of stem cells in regenerative medicine. We demonstrate the selective inhibition of mTORC1 signaling is important for developing the inner cell mass (ICM) and the self-renewal of human embryonic stem cells. S6K suppressed the expression and function of pluripotency-related transcription factors (PTFs) OCT4, SOX2, and KLF4 through phosphorylation and ubiquitin proteasome-mediated protein degradation, indicating that S6K inhibition is required for pluripotency. PTFs inhibited mTOR signaling. The phosphorylation of S6 was decreased in PTF-positive cells of the ICM in embryos. Activation of mTORC1 signaling blocked ICM formation and the selective inhibition of S6K by rapamycin increased the ICM size in mouse blastocysts. Thus, selective inhibition of mTORC1 signaling supports the development and maintenance of pluripotency.

Development of a major histocompatibility complex class II conditional knockout mouse to study cell-specific and time-dependent adaptive immune responses in peripheral nerves.

INTRODUCTION/AIMS: The precise relationship between molecular mimicry and tissue-specific autoimmunity is unknown. Major histocompatibility complex (MHC) class II antigen presenting cell-CD4+ T-cell receptor complex interactions are necessary for adaptive immunity. This study aimed to determine the role of endoneurial endothelial cell MHC class II in autoimmune polyneuropathy. METHODS: Cryopreserved Guillain-Barré syndrome (GBS) patient sural nerve biopsies and sciatic nerves from the severe murine experimental autoimmune neuritis (sm-EAN) GBS model were studied. Cultured conditional ready MHC Class II antigen A-alpha chain (H2-Aa) embryonic stem cells were used to generate H2-Aaflox/+ C57BL/6 mice. Mice were backcrossed and intercrossed to the SJL background to generate H2-Aaflox/flox SJL mice, bred with hemizygous Tamoxifen-inducible von Willebrand factor Cre recombinase (vWF-iCre/+) SJL mice to generate H2-Aaflox/flox; vWF-iCre/+ mice to study microvascular endothelial cell adaptive immune responses. Sm-EAN was induced in Tamoxifen-treated H2-Aaflox/flox; vWF-iCre/+, H2-Aaflox/flox; +/+, H2-Aa+/+; vWF-iCre/+ and untreated H2-Aaflox/flox; vWF-iCre/+ adult female SJL mice. Neurobehavioral, electrophysiological and histopathological assessments were performed at predefined time points. RESULTS: Endoneurial endothelial cell MHC class II expression was observed in normal and inflamed human and mouse peripheral nerves. Tamoxifen-treated H2-Aaflox/flox; vWF-iCre/+ mice were resistant to sm-EAN despite extensive MHC class II expression in lymphoid and non-lymphoid tissues. DISCUSSION: A conditional MHC class II knockout mouse to study cell- and time-dependent adaptive immune responses in vivo was developed. Initial studies show microvascular endothelial cell MHC class II expression is necessary for peripheral nerve specific autoimmunity, as advocated by human in vitro adaptive immunity and ex vivo transplant rejection studies.

Resting zone of the growth plate houses a unique class of skeletal stem cells.

Skeletal stem cells regulate bone growth and homeostasis by generating diverse cell types, including chondrocytes, osteoblasts and marrow stromal cells. The emerging concept postulates that there exists a distinct type of skeletal stem cell that is closely associated with the growth plate1-4, which is a type of cartilaginous tissue that has critical roles in bone elongation5. The resting zone maintains the growth plate by expressing parathyroid hormone-related protein (PTHrP), which interacts with Indian hedgehog (Ihh) that is released from the hypertrophic zone6-10, and provides a source of other chondrocytes11. However, the identity of skeletal stem cells and how they are maintained in the growth plate are unknown. Here we show, in a mouse model, that skeletal stem cells are formed among PTHrP-positive chondrocytes within the resting zone of the postnatal growth plate. PTHrP-positive chondrocytes expressed a panel of markers for skeletal stem and progenitor cells, and uniquely possessed the properties of skeletal stem cells in cultured conditions. Cell-lineage analysis revealed that PTHrP-positive chondrocytes in the resting zone continued to form columnar chondrocytes in the long term; these chondrocytes underwent hypertrophy, and became osteoblasts and marrow stromal cells beneath the growth plate. Transit-amplifying chondrocytes in the proliferating zone-which was concertedly maintained by a forward signal from undifferentiated cells (PTHrP) and a reverse signal from hypertrophic cells (Ihh)-provided instructive cues to maintain the cell fates of PTHrP-positive chondrocytes in the resting zone. Our findings unravel a type of somatic stem cell that is initially unipotent and acquires multipotency at the post-mitotic stage, underscoring the malleable nature of the skeletal cell lineage. This system provides a model in which functionally dedicated stem cells and their niches are specified postnatally, and maintained throughout tissue growth by a tight feedback regulation system.

Slow oscillations persist in pancreatic beta cells lacking phosphofructokinase M.

Pulsatile insulin secretion by pancreatic beta cells is necessary for tight glucose control in the body. Glycolytic oscillations have been proposed as the mechanism for generating the electrical oscillations underlying pulsatile insulin secretion. The glycolytic enzyme 6-phosphofructokinase-1 (PFK) synthesizes fructose-1,6-bisphosphate (FBP) from fructose-6-phosphate. It has been proposed that the slow electrical and Ca2+ oscillations (periods of 3-5 min) observed in islets result from allosteric feedback activation of PFKM by FBP. Pancreatic beta cells express three PFK isozymes: PFKL, PFKM, and PFKP. A prior study of mice that were engineered to lack PFKM using a gene-trap strategy to delete Pfkm produced a mosaic reduction in global Pfkm expression, but the islets isolated from the mice still exhibited slow Ca2+ oscillations. However, these islets still expressed residual PFKM protein. Thus, to more fully test the hypothesis that beta cell PFKM is responsible for slow islet oscillations, we made a beta-cell-specific knockout mouse that completely lacked PFKM. While PFKM deletion resulted in subtle metabolic changes in vivo, islets that were isolated from these mice continued to exhibit slow oscillations in electrical activity, beta cell Ca2+ concentrations, and glycolysis, as measured using PKAR, an FBP reporter/biosensor. Furthermore, simulations obtained with a mathematical model of beta cell activity shows that slow oscillations can persist despite PFKM loss provided that one of the other PFK isoforms, such as PFKP, is present, even if its level of expression is unchanged. Thus, while we believe that PFKM may be the main regulator of slow oscillations in wild-type islets, PFKP can provide functional redundancy. Our model also suggests that PFKM likely dominates, in vivo, because it outcompetes PFKP with its higher FBP affinity and lower ATP affinity. We thus propose that isoform redundancy may rescue key physiological processes of the beta cell in the absence of certain critical genes.

Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse.

Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice ('rescues') using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing, Arl6ip5 was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced) de novo mutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p = 0.003) in the corresponding pedigree. Thrombosis suppression by heterozygous Plcb4 loss of function was confirmed through analysis of an independent, CRISPR/Cas9-induced Plcb4 mutation (p = 0.01).

Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo.

Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.

Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a painful and debilitating side effect of cancer chemotherapy with an unclear pathogenesis. Consequently, the available therapies for this neuropathic pain syndrome are inadequate, leading to a significantly reduced quality of life in many patients. Complement, a key component of the innate immune system, has been associated with neuroinflammation, a potentially important trigger of some types of neuropathic pain. However, the role of complement in CIPN remains unclear. To address this issue, we developed a C3 knockout (KO) rat model and induced CIPN in these KO rats and wild-type littermates via the i.p. administration of paclitaxel, a chemotherapeutic agent associated with CIPN. We then compared the severity of mechanical allodynia, complement activation, and intradermal nerve fiber loss between the groups. We found that 1) i.p. paclitaxel administration activated complement in wild-type rats, 2) paclitaxel-induced mechanical allodynia was significantly reduced in C3 KO rats, and 3) the paclitaxel-induced loss of intradermal nerve fibers was markedly attenuated in C3 KO rats. In in vitro studies, we found that paclitaxel-treated rat neuronal cells activated complement, leading to cellular injury. Our findings demonstrate a previously unknown but pivotal role of complement in CIPN and suggest that complement may be a new target for the development of novel therapeutics to manage this painful disease.

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

Factor V Leiden (F5L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/- ). F8 deficiency enhanced the survival of F5L/LTfpi+/- mice, demonstrating that F5L/LTfpi+/- lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+Tfpi+/- females were crossed to generate 6,729 progeny, with 98 F5L/LTfpi+/- offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (MF5L1-16)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/- ) suppressed F5L/LTfpi+/- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/LTfpi+/- lethality (P = 1.7 × 10-6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.

An upstream enhancer regulates Gpihbp1 expression in a tissue-specific manner.

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1Enh/Enh). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia-even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1Enh/- mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.

Functional roles of MMP14 and MMP15 in early postnatal mammary gland development.

During late embryogenesis, mammary epithelial cells initiate migration programs that drive ductal invasion into the surrounding adipose-rich mesenchyme. Currently, branching morphogenesis is thought to depend on the mobilization of the membrane-anchored matrix metalloproteinases MMP14 (MT1-MMP) and MMP15 (MT2-MMP), which drive epithelial cell invasion by remodeling the extracellular matrix and triggering associated signaling cascades. However, the roles that these proteinases play during mammary gland development in vivo remain undefined. Here, we characterize the impact of global Mmp14 and Mmp15 targeting on early postnatal mammary gland development in mice. Unexpectedly, both Mmp14-/- and Mmp15-/- mammary glands retain the ability to generate intact ductal networks. Although neither proteinase is required for branching morphogenesis, transcriptome profiling reveals a key role for MMP14 and MMP15 in regulating mammary gland adipocyte differentiation. Whereas MMP14 promotes the generation of white fat depots crucial for energy storage, MMP15 differentially controls the formation of thermogenic brown fat. Taken together, these data not only indicate that current paradigms relevant to proteinase-dependent morphogenesis need be revisited, but also identify new roles for the enzymes in regulating adipocyte fate determination in the developing mammary gland.

Mutations in RELT cause autosomal recessive amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt-/- mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt-/- enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations.

The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension.

Measures of the adipokine chemerin are elevated in multiple cardiovascular diseases, including hypertension, but little mechanistic work has been done to implicate chemerin as being causative in such diseases. The chemerin knockout (KO) rat was created to test the hypothesis that removal of chemerin would reduce pressure in the normal and hypertensive state. Western analyses confirmed loss of chemerin in the plasma and tissues of the KO vs. wild-type (WT) rats. Chemerin concentration in plasma and tissues was lower in WT females than in WT males, as determined by Western analysis. Conscious male and female KO rats had modest differences in baseline measures vs. the WT that included systolic, diastolic, mean arterial and pulse pressures, and heart rate, all measured telemetrically. The mineralocorticoid deoxycorticosterone acetate (DOCA) and salt water, combined with uninephrectomy as a hypertensive stimulus, elevated mean and systolic blood pressures of the male KO higher than the male WT. By contrast, all pressures in the female KO were lower than their WT throughout DOCA-salt treatment. These results revealed an unexpected sex difference in chemerin expression and the ability of chemerin to modify blood pressure in response to a hypertensive challenge.-Watts, S. W., Darios, E. S., Mullick, A. E., Garver, H., Saunders, T. L., Hughes, E. D., Filipiak, W. E., Zeidler, M. G., McMullen, N., Sinal, C. J., Kumar, R. K., Ferland, D. J., Fink, G. D. The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension.

Endothelial and perivascular cells maintain haematopoietic stem cells.

Several cell types have been proposed to create niches for haematopoietic stem cells (HSCs). However, the expression patterns of HSC maintenance factors have not been systematically studied and no such factor has been conditionally deleted from any candidate niche cell. Thus, the cellular sources of these factors are undetermined. Stem cell factor (SCF; also known as KITL) is a key niche component that maintains HSCs. Here, using Scf(gfp) knock-in mice, we found that Scf was primarily expressed by perivascular cells throughout the bone marrow. HSC frequency and function were not affected when Scf was conditionally deleted from haematopoietic cells, osteoblasts, nestin-cre- or nestin-creER-expressing cells. However, HSCs were depleted from bone marrow when Scf was deleted from endothelial cells or leptin receptor (Lepr)-expressing perivascular stromal cells. Most HSCs were lost when Scf was deleted from both endothelial and Lepr-expressing perivascular cells. Thus, HSCs reside in a perivascular niche in which multiple cell types express factors that promote HSC maintenance.

Podocyte-specific JAK2 overexpression worsens diabetic kidney disease in mice.

Activation of JAK-STAT signaling has been implicated in the pathogenesis of diabetic kidney disease. An increased expression of JAK-STAT genes was found in kidney glomerular cells, including podocytes, in patients with early diabetic kidney disease. However, it is not known whether increased expression of JAK or STAT isoforms in glomerular cells can lead to worsening nephropathy in the setting of diabetes. Therefore, we overexpressed JAK2 mRNA specifically in glomerular podocytes of 129S6 mice to determine whether this change alone could worsen diabetic kidney disease. A 2-3 fold increase in glomerular JAK2 expression, an increase similar to that found in humans with early diabetic kidney disease, led to substantial and statistically significant increases in albuminuria, mesangial expansion, glomerulosclerosis, glomerular fibronectin accumulation, and glomerular basement membrane thickening, and a significant reduction in podocyte density in diabetic mice. Treatment with a specific JAK1/2 inhibitor for 2 weeks partly reversed the major phenotypic changes of diabetic kidney disease and specifically normalized expression of a number of downstream STAT3-dependent genes implicated in diabetic kidney disease progression. Thus, moderate increases in podocyte JAK2 expression at levels similar to those in patients with early diabetic kidney disease can lead directly to phenotypic and other alterations of progressive diabetic glomerulopathy. Hence, inhibition of these changes by treatment with a JAK1/2 inhibitor suggests that such treatment may help retard progression of early diabetic kidney disease in patients.

Targeted disruption of Cd40 in a genetically hypertensive rat model attenuates renal fibrosis and proteinuria, independent of blood pressure.

High blood pressure is a common cause of chronic kidney disease. Because CD40, a member of the tumor necrosis factor receptor family, has been linked to the progression of kidney disease in ischemic nephropathy, we studied the role of Cd40 in the development of hypertensive renal disease. The Cd40 gene was mutated in the Dahl S genetically hypertensive rat with renal disease by targeted-gene disruption using zinc-finger nuclease technology. These rats were then given low (0.3%) and high (2%) salt diets and compared. The resultant Cd40 mutants had significantly reduced levels of both urinary protein excretion (41.8 ± 3.1 mg/24 h vs. 103.7 ± 4.3 mg/24 h) and plasma creatinine (0.36 ± 0.05 mg/dl vs. 1.15 ± 0.19 mg/dl), with significantly higher creatinine clearance compared with the control S rats (3.04 ± 0.48 ml/min vs. 0.93 ± 0.15 ml/min), indicating renoprotection was conferred by mutation of the Cd40 locus. Furthermore, the Cd40 mutants had a significant attenuation in renal fibrosis, which persisted on the high salt diet. However, there was no difference in systolic blood pressure between the control and Cd40 mutant rats. Thus, these data serve as the first evidence for a direct link between Cd40 and hypertensive nephropathy. Hence, renal fibrosis is one of the underlying mechanisms by which Cd40 plays a crucial role in the development of hypertensive renal disease.

A knockin mouse model of spinocerebellar ataxia type 3 exhibits prominent aggregate pathology and aberrant splicing of the disease gene transcript.

Polyglutamine diseases, including spinocerebellar ataxia type 3 (SCA3), are caused by CAG repeat expansions that encode abnormally long glutamine repeats in the respective disease proteins. While the mechanisms underlying neurodegeneration remain uncertain, evidence supports a proteotoxic role for the mutant protein dictated in part by the specific genetic and protein context. To further define pathogenic mechanisms in SCA3, we generated a mouse model in which a CAG expansion of 82 repeats was inserted into the murine locus by homologous recombination. SCA3 knockin mice exhibit region-specific aggregate pathology marked by intranuclear accumulation of the mutant Atxn3 protein, abundant nuclear inclusions and, in select brain regions, extranuclear aggregates localized to neuritic processes. Knockin mice also display altered splicing of the disease gene, promoting expression of an alternative isoform in which the intron immediately downstream of the CAG repeat is retained. In an independent mouse model expressing the full human ATXN3 disease gene, expression of this alternatively spliced transcript is also enhanced. These results, together with recent findings in other polyglutamine diseases, suggest that CAG repeat expansions can promote aberrant splicing to produce potentially more aggregate-prone isoforms of the disease proteins. This report of a SCA3 knockin mouse expands the repertoire of existing models of SCA3, and underscores the potential contribution of alternative splicing to disease pathogenesis in SCA3 and other polyglutamine disorders.

Cutting Edge: IFN-γ Produced by Brain-Resident Cells Is Crucial To Control Cerebral Infection with Toxoplasma gondii.

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations, including LPS. However, the physiological role of IFN-γ production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ-mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b(+) cells suggested that IFN-γ production by microglia, which is the only CD11b(+) cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell-mediated immune responses to control cerebral infection with T. gondii.

Notch pathway targets proangiogenic regulator Sox17 to restrict angiogenesis.

RATIONALE: The Notch pathway stabilizes sprouting angiogenesis by favoring stalk cells over tip cells at the vascular front. Because tip and stalk cells have different properties in morphology and function, their transcriptional regulation remains to be distinguished. Transcription factor Sox17 is specifically expressed in endothelial cells, but its expression and role at the vascular front remain largely unknown. OBJECTIVE: To specify the role of Sox17 and its relationship with the Notch pathway in sprouting angiogenesis. METHODS AND RESULTS: Endothelial-specific Sox17 deletion reduces sprouting angiogenesis in mouse embryonic and postnatal vascular development, whereas Sox17 overexpression increases it. Sox17 promotes endothelial migration by destabilizing endothelial junctions and rearranging cytoskeletal structure and upregulates expression of several genes preferentially expressed in tip cells. Interestingly, Sox17 expression is suppressed in stalk cells in which Notch signaling is relatively high. Notch activation by overexpressing Notch intracellular domain reduces Sox17 expression both in primary endothelial cells and in retinal angiogenesis, whereas Notch inhibition by delta-like ligand 4 (Dll4) blockade increases it. The Notch pathway regulates Sox17 expression mainly at the post-transcriptional level. Furthermore, endothelial Sox17 ablation rescues vascular network from excessive tip cell formation and hyperbranching under Notch inhibition in developmental and tumor angiogenesis. CONCLUSIONS: Our findings demonstrate that the Notch pathway restricts sprouting angiogenesis by reducing the expression of proangiogenic regulator Sox17.

Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells.

Little is known about metabolic regulation in stem cells and how this modulates tissue regeneration or tumour suppression. We studied the Lkb1 tumour suppressor and its substrate AMP-activated protein kinase (AMPK), kinases that coordinate metabolism with cell growth. Deletion of the Lkb1 (also called Stk11) gene in mice caused increased haematopoietic stem cell (HSC) division, rapid HSC depletion and pancytopenia. HSCs depended more acutely on Lkb1 for cell-cycle regulation and survival than many other haematopoietic cells. HSC depletion did not depend on mTOR activation or oxidative stress. Lkb1-deficient HSCs, but not myeloid progenitors, had reduced mitochondrial membrane potential and ATP levels. HSCs deficient for two catalytic α-subunits of AMPK (AMPK-deficient HSCs) showed similar changes in mitochondrial function but remained able to reconstitute irradiated mice. Lkb1-deficient HSCs, but not AMPK-deficient HSCs, exhibited defects in centrosomes and mitotic spindles in culture, and became aneuploid. Lkb1 is therefore required for HSC maintenance through AMPK-dependent and AMPK-independent mechanisms, revealing differences in metabolic and cell-cycle regulation between HSCs and some other haematopoietic progenitors.