Selective neuronal lineages derived from Dll4‐expressing progenitors/precursors in the retina and spinal cord

Results: We generated a Dll4‐Cre BAC transgenic mouse line that drives Cre recombinase expression mimicking that of the endogenous Dll4 in the developing retina and spinal cord. By fate‐mapping analysis, we found that Dll4‐expressing progenitors/precursors give rise to essentially all cone, amacrine and horizontal cells, a large portion of rod and ganglion cells, but only few bipolar and Müller cells. In the spinal cord, Dll4‐expressing progenitors/precursors generate almost all V2a and V2c cells while producing only a fraction of the cells for other interneuron and motor neuron subtypes along the dorsoventral axis. 相似文献   

IFNγ‐responsiveness of endothelial cells leads to efficient angiostasis in tumours involving down‐regulation of Dll4

Although IFNγ is regarded as a key cytokine in angiostatic response, our poor understanding of its effective cellular target drastically limits its clinical trials against angiogenesis‐related disorders. Here, we investigated the effect of IFNγ on endothelial cells (ECs) and possible molecular mechanisms in angiostasis. By employing Tie2^IFNγR mice, in which IFNγR expression was reconstituted under the control of Tie2 promoter in IFNγR‐deficient mice, we found that the response of ECs to IFNγ was highly effective in inhibiting blood supply and retarding tumour growth. Interestingly, the expression of IFNγR on Tie2^? cells did not inhibit, but promoted tumour growth in control wild‐type mice. Mechanism studies showed that IFNγ reacting on ECs down‐regulated the delta‐like ligand 4 (Dll4)/Notch signalling pathway. Accordingly, overexpression of Dll4 in human ECs diminished the effect of IFNγ on ECs. This study demonstrates that the action of IFNγ on ECs, but not other cells, is highly effective for tumour angiostasis, which involves down‐regulating Dll4. It provides insights for EC‐targeted angiostatic therapy in treating angiogenesis‐associated disorders in the clinic. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Plasma membrane Toll‐like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon‐β

Lactobacillus acidophilus induces a potent interferon‐β (IFN‐β) response in dendritic cells (DCs) by a Toll‐like receptor 2 (TLR2) ‐dependent mechanism, in turn leading to strong interleukin‐12 (IL‐12) production. In the present study, we investigated the involvement of different types of endocytosis in the L. acidophilus‐induced IFN‐β and IL‐12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN‐β and IL‐12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place in the immature cells as well as by spleen tyrosine kinase (Syk) ‐dependent phagocytosis but without involvement of plasma membrane TLR2. Stimulation with TLR2 or TLR4 ligands increased macropinocytosis in a Syk‐independent manner. As a consequence, incubation of DCs with TLR ligands before incubation with L. acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN‐β and IL‐12 was strongly inhibited. As L. acidophilus‐induced IFN‐β depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading to increased macropinocytosis abrogates IFN‐β induction we conclude that plasma membrane TLR stimulation leading to increased macropinocytosis decreases endosomal induction of IFN‐β and speculate that this is due to competition between compartments for molecules involved in the signal pathways. In summary, endosomal signalling by L. acidophilus that leads to IFN‐β and IL‐12 production is inhibited by TLR stimulation from the plasma membrane.     

IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

Dll3 is expressed in developing hair cells in the mammalian cochlea.

Notch mediates the process of lateral inhibition that controls the production of hair cells in the inner ear. Hair cells are known to express Notch ligands Dll1 and Jag2, which signal through Notch1 in adjacent supporting cells. However, recent genetic and pharmacological studies indicate that the level of Notch-mediated lateral inhibition is greater than can be accounted for by Dll1 and Jag2. Here, we report that another Notch ligand, Dll3, is expressed in developing hair cells, in a pattern that overlaps that of Dll1 and Jag2. We analyzed the cochleae of Dll3(pu) mutant mice, but did not detect any abnormalities. However, earlier studies have demonstrated that there is functional redundancy among Notch ligands in cochlear development and loss of one ligand can be at least partially compensated for by another. Thus Dll3 may play a role in lateral inhibition similar to that of Dll1 and Jag2.     

Dosage-sensitive requirement for mouse Dll4 in artery development

Involvement of the Notch signaling pathway in vascular development has been demonstrated by both gain- and loss-of-function mutations in humans, mice, and zebrafish. In zebrafish, Notch signaling is required for arterial identity by suppressing the venous fate in developing artery cells. In mice, the Notch4 receptor and the Delta-like 4 (Dll4) ligand are specifically expressed in arterial endothelial cells, suggesting a similar role. Here we show that the Dll4 ligand alone is required in a dosage-sensitive manner for normal arterial patterning in development. This implicates Dll4 as the specific mammalian endothelial ligand for autocrine endothelial Notch signaling, and suggests that Dll4 may be a suitable target for intervention in arterial angiogenesis.     

TLR8‐mediated activation of human monocytes inhibits TL1A expression

TLR play important roles in inflammation and innate immune response to pathogens. TLR8 recognizes ssRNA and induces NF‐κB via MyD88 signaling. TL1A is a member of the TNF superfamily that markedly enhances IFN‐γ production by IL‐12/IL‐18‐stimulated peripheral and mucosal CD4⁺ T cells. TL1A expression is increased in the mucosa of patients with inflammatory bowel disease and is considered a key mediator of Crohn's disease (CD). We have previously shown that TL1A is strongly induced by immune complexes (IC) but not TLR ligands in antigen‐presenting cells. However, a potential interaction between these pro‐inflammatory signaling pathways has not been investigated. IC‐induced TL1A expression of monocytes was potently inhibited by a TLR8 or TLR7/8 ligand (R848) in a dose‐dependent manner. Furthermore, when co‐cultured with CD4⁺ T cells, TLR8 ligands inhibited TL1A production, resulting in almost complete inhibition of IFN‐γ production by the CD4⁺ T cells. Furthermore, we demonstrate that IFN‐α is not required for this suppressive effect by TLR8 signaling. Our data demonstrate for the first time a direct interaction between TLR and TL1A signaling pathways. TLR8 activation may be an important, novel pathway for targeted treatment of Th1‐mediated diseases, such as CD.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

Delta‐like 4‐mediated Notch signaling is required for early T‐cell development in a three‐dimensional thymic structure

Delta‐like 4 (Dll4)‐mediated Notch signaling is critical for specifying T‐cell fate, but how Dll4‐mediated Notch signaling actually contributes to T‐cell development in the thymus remains unclear. To explore this mechanism in the thymic three‐dimensional structure, we performed fetal thymus organ culture using Dll4‐deficient mice. DN1a/b+DN2mt cells, which had not yet committed to either the αβ T or γδ T/NK cell lineage, did not differentiate into the αβ T‐cell lineage in Dll4‐deficient thymus despite the lack of cell fate conversion into other lineages. However, DN3 cells efficiently differentiated into a later developmental stage of αβ T cells, the double‐positive (DP) stage, although the proliferation was significantly impaired during the differentiation process. These findings suggest that the requirement for Notch signaling differs between the earliest and pre‐TCR‐bearing precursors and that continued Notch signaling is required for proper differentiation with active proliferation of αβ T lineage cells. Furthermore, we showed that Notch signaling increased the c‐Myc expression in DN3 cells in the thymus and that its overexpression rescued the proliferation and differentiation of DN3 cells in the Dll4‐null thymus. Therefore, c‐Myc plays a central role in the transition from stage DN3 to DP as a downstream target of Notch signaling.     

Expression of vascular Notch ligands Delta-like 4 and Jagged-1 in glioblastoma

Jubb A M, Browning L, Campo L, Turley H, Steers G, Thurston G, Harris A L & Ansorge O
(2012) Histopathology  60, 740–747
Expression of vascular Notch ligands Delta‐like 4 and Jagged‐1 in glioblastoma Aims: The coordinated expression of the Notch ligands Delta‐like 4 (Dll4) and Jagged (Jag)1 is believed to define appropriate endothelial sensitivity to vascular endothelial growth factor (VEGF). Preclinical data suggest that Dll4‐Notch signalling may confer resistance to anti‐VEGF therapy with bevacizumab, and Jag1 may antagonize Dll4–Notch. The aims of this study were to characterize the expression of Dll4 and Jag1 in primary glioblastomas. Methods and results: Immunohistochemistry was performed on 40 glioblastomas and normal brain using validated antibodies against Dll4 and Jag1. In‐situ hybridization for Dll4 was performed on serial sections and compared with protein expression. Dll4 expression was localized to the cytoplasm and membrane of endothelial cells in all glioblastomas; it was weak or absent in normal brain. Jag1 expression was observed in the cytoplasm and membrane of glomeruloid and non‐glomeruloid endothelial cells from 76% and 67% of glioblastomas, respectively. However, endothelial Jag1 expression was less intense and less prevalent than Dll4. There was no association between Dll4 and Jag1 expression. Conclusions: In summary, Dll4 and Jag1 are expressed in glioblastoma vasculature. These data may define subsets of glioblastoma that might be sensitive (Dll4⁺/Jag1⁺) or resistant (Dll4⁺/Jag1^–) to bevacizumab. Our data also suggest that anti‐Dll4 therapy should be evaluated experimentally in glioblastoma.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

Disappearance of centroacinar cells in the Notch ligand‐deficient pancreas

Notch signaling has been shown to contribute to murine pancreatic development at various stages. Delta‐like 1 (Dll1) or Jagged1 (Jag1) are the Notch ligands that solely function to trigger this signaling during the pancreatic bud stage (~e9.5) or after birth, respectively. However, it has not been elucidated whether these Notch ligands are required at the later stage (e10.5–18.5) when the particular pancreas structures form. Here, we detected the dual expression of Dll1 and Jag1 in the epithelium after e10.5, which was restricted to the ductal cell lineage, including centroacinar cells expressing Sox9, CD133 and Hes1 but not the ductal cell markers Hnf1β and DBA, at e18.5. To evaluate the significance of the Notch ligands during this period, we established double‐floxed mice of Dll1 and Jag1 genes with Ptf1a‐Cre knock‐in allele and examined the effects on development. The abrogation of both ligands but not a single one led to the loss of centroacinar cells, which was due to the decrease in cell proliferation and the increase in cell death, as well as to the reduction of Sox9. These results suggested that Dll1 and Jag1 function redundantly and are necessary to maintain the centroacinar cells as an environmental niche in the developing pancreas.     

CD40 ligand and interferon‐γ induce an antimicrobial response against Mycobacterium tuberculosis in human monocytes

The ability of T cells to activate antimicrobial pathways in infected macrophages is essential to host defence against many intracellular pathogens. Here, we compared the ability of two T‐cell‐mediated mechanisms to trigger antimicrobial responses against Mycobacterium tuberculosis in humans, CD40 activation and the release of interferon‐γ (IFN‐γ). Given that IFN‐γ activates a vitamin D‐dependent antimicrobial response, we focused on induction of the key components of this pathway. We show that activation of human monocytes via CD40 ligand (CD40L) and IFN‐γ, alone, and in combination, induces the CYP27b1‐hydroxylase, responsible for the conversion of 25‐hydroxyvitamin D (25D) to the bioactive 1,25‐dihydroxyvitamin D (1,25D), and the vitamin D receptor (VDR). The activation of the vitamin D pathway by CD40L and IFN‐γ results in up‐regulated expression of the antimicrobial peptides, cathelicidin and DEFB4, as well as induction of autophagy. Finally, activation of monocytes via CD40L and IFN‐γ results in an antimicrobial activity against intracellular M. tuberculosis. Our data suggest that at least two parallel T‐cell‐mediated mechanisms, CD40L and IFN‐γ, activate the vitamin D‐dependent antimicrobial pathway and trigger antimicrobial activity against intracellular M. tuberculosis, thereby contributing to human host defence against intracellular infection.     

Caspase‐11 non‐canonical inflammasome: a critical sensor of intracellular lipopolysaccharide in macrophage‐mediated inflammatory responses

Inflammatory responses mediated by macrophages are part of the innate immune system, whose role is to protect against invading pathogens. Lipopolysaccharide (LPS) found in the outer membrane of Gram‐negative bacteria stimulates an inflammatory response by macrophages. During the inflammatory response, extracellular LPS is recognized by Toll‐like receptor 4, one of the pattern recognition receptors that activates inflammatory signalling pathways and leads to the production of inflammatory mediators. The innate immune response is also triggered by intracellular inflammasomes, and inflammasome activation induces pyroptosis and the secretion of pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β) and IL‐18 by macrophages. Cysteine‐aspartic protease (caspase)‐11 and the human orthologues caspase‐4/caspase‐5 were recently identified as components of the ‘non‐canonical inflammasome’ that senses intracellular LPS derived from Gram‐negative bacteria during macrophage‐mediated inflammatory responses. Direct recognition of intracellular LPS facilitates the rapid oligomerization of caspase‐11/4/5, which results in pyroptosis and the secretion of IL‐1β and IL‐18. LPS is released into the cytoplasm from Gram‐negative bacterium‐containing vacuoles by small interferon‐inducible guanylate‐binding proteins encoded on chromosome 3 (GBP^chr3)‐mediated lysis of the vacuoles. In vivo studies have clearly shown that caspase‐11^−/− mice are more resistant to endotoxic septic shock by excessive LPS challenge. Given the evidence, activation of caspase‐11 non‐canonical inflammasomes by intracellular LPS is distinct from canonical inflammasome activation and provides a new paradigm in macrophage‐mediated inflammatory responses.