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SummaryBy mirroring their function as tissue repair organizers in normal tissues, immune cells regulate tumor growth. To understand the different facets of immune-tumor collaboration through genetics, spatial transcriptomics, and immunological manipulation with non-invasive, longitudinal imaging, we generated a penetrant double oncogene-driven autochthonous model of neuroblastoma. Using spatial transcriptomic analysis, we co-localized CD4+and myeloid populations within the tumor parenchyma, while CD8+T cells and B cells were peripherally dispersed. Depletion of CD4+T cells or CCR2+macrophages, but not B cells, CD8+, or NK cells, prevented tumor formation. Tumor CD4+T cells displayed unconventional phenotypes, were clonotypically diverse, and antigen-independent. Within the myeloid fraction, tumor growth required myeloid cells expressing Arginase-1. Overall, our results suggest that arginine-metabolizing myeloid cells conspire with pathogenic CD4+T cells to create permissive conditions for tumor formation, and therefore suggest that these pro-tumorigenic pathways can be disabled by targeting myeloid amino acid metabolism.

Immune cells regulate tumor growth by mirroring their function as tissue repair organizers in normal tissues. To understand the different facets of immune–tumor collaboration through genetics, spatial transcriptomics, and immunologic manipulation with noninvasive, longitudinal imaging, we generated a penetrant double oncogene–driven autochthonous model of neuroblastoma. Spatial transcriptomic analysis showed that CD4+ and myeloid populations colocalized within the tumor parenchyma, while CD8+ T cells and B cells were peripherally dispersed. Depletion of CD4+ T cells or CCR2+ macrophages, but not B cells, CD8+ T cells, or natural killer (NK) cells, prevented tumor formation. Tumor CD4+ T cells displayed unconventional phenotypes and were clonotypically diverse and antigen independent. Within the myeloid fraction, tumor growth required myeloid cells expressing arginase-1. Overall, these results demonstrate how arginine-metabolizing myeloid cells conspire with pathogenic CD4+ T cells to create permissive conditions for tumor formation, suggesting that these protumorigenic pathways could be disabled by targeting myeloid arginine metabolism. Significance: A new model of human neuroblastoma provides ways to track tumor formation and expansion in living animals, allowing identification of CD4+ T-cell and macrophage functions required for oncogenesis.

The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer1–5. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1β and IL-18 and drives cell fate towards pyroptosis—a form of programmed inflammatory cell death that has major roles in health and disease6–12. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions. The RNA helicase DDX3X has a critical role in regulating both the induction of stress granules and the activation of the NLRP3 inflammasome in cells under stress conditions.

Summary Regulatory T (Treg) cells are critical mediators of immune tolerance whose activity depends upon T cell receptor (TCR) and mTORC1 kinase signaling, but the mechanisms that dictate functional activation of these pathways are incompletely understood. Here, we showed that amino acids license Treg cell function by priming and sustaining TCR-induced mTORC1 activity. mTORC1 activation was induced by amino acids, especially arginine and leucine, accompanied by the dynamic lysosomal localization of the mTOR and Tsc complexes. Rag and Rheb GTPases were central regulators of amino acid-dependent mTORC1 activation in effector Treg (eTreg) cells. Mice bearing RagA-RagB- or Rheb1-Rheb2-deficient Treg cells developed a fatal autoimmune disease and had reduced eTreg cell accumulation and function. RagA-RagB regulated mitochondrial and lysosomal fitness, while Rheb1-Rheb2 enforced eTreg cell suppressive gene signature. Together, these findings reveal a crucial requirement of amino acid signaling for licensing and sustaining mTORC1 activation and functional programming of Treg cells.

Caspase-1, also known as interleukin-1β (IL-1β)-converting enzyme (ICE), regulates antimicrobial host defense, tissue repair, tumorigenesis, metabolism and membrane biogenesis. On activation within an inflammasome complex, caspase-1 induces pyroptosis and converts pro-IL-1β and pro-IL-18 into their biologically active forms. “ICE−/−” or “Casp1−/−” mice generated using 129 embryonic stem cells carry a 129-associated inactivating passenger mutation on the caspase-11 locus, essentially making them deficient in both caspase-1 and caspase-11. The overlapping and unique functions of caspase-1 and caspase-11 are difficult to unravel without additional genetic tools. Here, we generated caspase-1–deficient mouse (Casp1Null) on the C57BL/6 J background that expressed caspase-11. Casp1Null cells did not release IL-1β and IL-18 in response to NLRC4 activators Salmonella Typhimurium and flagellin, canonical or non-canonical NLRP3 activators LPS and ATP, Escherichia coli, Citrobacter rodentium and transfection of LPS, AIM2 activators Francisella novicida, mouse cytomegalovirus and DNA, and the infectious agents Listeria monocytogenes and Aspergillus fumigatus. We further demonstrated that caspase-1 and caspase-11 differentially contributed to the host defense against A. fumigatus infection and to endotoxemia.

For decades, genome engineering relied on techniques that took years to master, required the generation of large and often complex DNA constructs containing selection markers, and could be applied to only a few organisms. However, the ease and efficiency of current technologies to edit genomes are unprecedented. With the advent of targetable nucleases, most notably the CRISPR-Cas9 technology, genomes of all species are now easily accessible to modifications. This advance has provided countless opportunities not only to further our understanding of gene functions and disease mechanisms but also to correct disease-causing mutations, modify crops and livestock, and perhaps modify our environment. This chapter discusses the advances in genome-editing technologies and their current and future applications.

The effects of cyclic AMP-related compounds and beta adrenoceptor agonists on the basal and lipopolysaccharide (LPS)-stimulated release of endothelin-1 (ET-1) from guinea-pig tracheal epithelial cells (GPTEpCs) in culture were studied. Forskolin (a potent activator of adenylyl cyclase), 8-bromo-cyclic AMP (a cyclic AMP analogue), salbutamol and salmeterol (two beta 2-adrenoceptor agonists), were used to increase cyclic AMP levels. Cultured GPTEpCs released ET-1 continuously over a 24 h incubation period. The values reached 1,938 +/- 122 pg/mg of total cell proteins after 24 h. LPS (10 microg/ml) significantly stimulated the release of ET-1 by 1.6- to 1.8-fold, up to 1,262 +/- 56 pg/mg total cell proteins after an 8 h incubation period. Compound 8-bromo-cyclic AMP (10(-5), 10(-4) and 10(-3) M) reduced the basal release of ET-1 from GPTEpCs by up to 31% (P0.01) and the LPS stimulated release by up to 42% (P0.05), after an 8 h incubation period. Forskolin (10(-6), 10(-5) and 10(-4) M) also inhibited the basal release of ET-1 by up to 28% (P0.05) and LPS-stimulated release of ET-1 by up to 50% (P0.05), after an 8 h incubation period. At the concentration of 10(-5) M, forskolin increased cyclic AMP levels in GPTEpCs by 17-fold (P0.001) in the medium, 15 min after the beginning of the incubation. Salbutamol (10(-8) to 10(-6) M) had no effect on the basal production and release of ET-1 after 8 h. Conversely, this short acting beta 2-adrenoceptor agonist significantly reduced LPS-mediated increase of ET-1 production by up to 55% (P0.05) after an 8 h incubation period. Salmeterol (10(-9) M to 10(-5) M) inhibited basal and LPS-stimulated production and release of ET-1 after an 8 h incubation period (between 44 and 51%, P0.01). Both salbutamol and salmeterol (10(-6) M) increase cyclic AMP levels by five- and twofold, respectively (P0.05). In summary, these observations indicate that beta 2-adrenoceptor agonists or cyclic AMP enhancers can modulate both basal and more markedly, the enhanced production of ET-1 from LPS-activated guinea pig airway EpCs. In addition, these compounds increase cyclic AMP levels in the cells. It is suggested that there is a correlation between cyclic AMP increase and inhibition of ET-1 release by guinea pig airway EpCs. Since ET-1 production was shown to be elevated in asthmatic subjects and in patients suffering from other inflammatory lung disorders, the inhibition of its production by beta adrenoceptor agonists, such as salbutamol and salmeterol, could be added to their therapeutical benefits.

LC3-associated phagocytosis (LAP) is a process wherein elements of canonical autophagy conjugate LC3 to the membranes of phagosomes, facilitating maturation upon fusion to lysosomes. Here, we characterize the molecular requirements for LAP, and identify a protein, Rubicon, required for LAP but not canonical autophagy. During LAP, Rubicon is recruited to the LAP-engaged phagosome (“LAPosome”) and is required for the activity of a Class III PI3K complex containing Beclin-1, UVRAG, and VPS34, but lacking the canonical autophagy components ATG14 and Ambra1. This allows for the sustained localization of PI(3)P, which is critical for the recruitment of downstream autophagic proteins and the stabilization of the NOX2 complex to produce ROS. Both PI(3)P and ROS, as well as ATG7, ATG3, ATG5, ATG12, and ATG16L, are required for the conjugation of LC3 to the LAPosome and subsequent association with LAMP1+ lysosomes. LAP is also induced by engulfment of Aspergillus fumigatus, a fungal pathogen that commonly afflicts immunocompromised hosts, and is required for its optimal clearance in vivo. Therefore, we have identified molecules that distinguish LAP from canonical autophagy, thereby elucidating the importance of LAP in the response to Aspergillus fumigatus infection.

In a recent report in Cell Metabolism, Metghalchi et al. presented evidence suggesting a negative regulatory link between the tryptophan-degrading enzyme IDO1 and IL-10 that was proposed to influence the outcomes of atherosclerosis and colitis (Metghalchi et al., 2015). IDO1 activity was linked to suppression of IL-10 production from activated myeloid cells. Importantly, Metghalchi et al. showed IDO1 expression was localized to macrophages and smooth muscle cells in their model of atherosclerosis (Metghalchi et al., 2015).

International audience; As do cytokine receptors and receptor tyrosine kinases, G protein-coupled receptors (GPCRs) signal to Janus kinases (Jaks) and signal transducers and activators of transcription (STATs). However, the early biochemical events linking GPCRs to this signaling pathway have been unclear. Here we show that GPCR-stimulated Rac activity and the subsequent generation of reactive oxygen species are necessary for activating tyrosine phosphorylation of Jaks and STAT-dependent transcription. The requirement for Rac activity can be overcome by addition of hydrogen peroxide. Expression of activated mutants of Rac1 is sufficient to activate Jak2 and STAT-dependent transcription, and the activation of Jak2 correlates with the ability of Rac1 to bind to NADPH oxidase subunit p67(phox). We further show that GPCR agonists stimulate tyrosine phosphorylation of STAT1 and STAT3 proteins in a Rac-dependent manner. The tyrosine phosphorylation of STAT3 is biphasic; the first peak of phosphorylation is weak and correlates with rapid activation of Jaks by GPCRs, whereas the second peak is stronger and requires the synthesis of an autocrine factor. Rho also plays an essential role in the induction of STAT transcriptional activity. Our results highlight a novel role for Rho GTPases in mediating the regulatory effects of GPCRs on STAT-dependent gene expression.

Prostaglandin E2 (PGE2) increased adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) formation in tracheal epithelial cells and concomitantly decreased the production/secretion of immunoreactive endothelin (irET). Naturally occurring prostanoids and selective and non-selective EP receptor agonists showed the following rank order of potency in stimulating cyclic AMP generation by epithelial cells: PGE2 (EP-selective)>16,16-dimethyl PGE2 (EP-selective)>11-deoxy PGE2 (EP-selective)>>>iloprost (IP/EP1/EP3-selective), butaprost (EP2-selective), PGD2 (DP-selective), PGF2α (FP-selective). The lack of responsiveness of the latter prostanoids indicated that the prostanoid receptor present in these cells is not of the DP, FP, IP, EP1, EP2 or EP3 subtype. Pre-incubating the cells with the selective TP/EP4-receptor antagonists AH23848B and AH22921X antagonized the PGE2-evoked cyclic AMP generation. This suggested that EP4 receptors mediate PGE2 effects. However, in addition to any antagonistic effects at EP4-receptors, both compounds, to a different extent, modified cyclic AMP metabolism. The selective EP1, DP and EP2 receptor antagonist (AH6809) failed to inhibit PGE2-evoked cyclic AMP generation which confirmed that the EP2 receptor subtype did not contribute to the change in cyclic AMP formation in these cells. The PGE2-induced inhibition of irET production by guinea-pig tracheal epithelial cells was due to cyclic AMP generation and activation of the cyclic AMP-dependent protein kinase since this effect was reverted by the cyclic AMP antagonist Rp-cAMPS. These results provide the first evidence supporting the existence of a functional prostaglandin E2 receptor that shares the pharmacological features of the EP4-receptor subtype in guinea-pig tracheal epithelial cells. These receptors modulate cyclic AMP formation as well as ET-1 production/secretion in these cells. British Journal of Pharmacology (2001) 132, 999–1008; doi:10.1038/sj.bjp.0703886

1. The adenosine receptor subtype mediating adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation and the effect of its activation on endothelin-1 (ET-1) secretion were studied in primary cultures of tracheal epithelial cells. 2. Adenosine analogues showed the following rank order of potency (pD(2) value) and intrinsic activity on the generation of cyclic AMP by tracheal epithelial cells: 5'-N-ethylcarboxyamidoadenosine (NECA, A(1)/A(2A)/A(2B), pD(2): 5.44+/-0.16)>adenosine (ADO, non selective, pD(2): 4.99+/-0. 09; 71+/-9% of NECA response) >/=2-Cl-adenosine (2CADO, non selective, pD(2): 4.72+/-0.14; 65+/-9% of NECA response)>>>CGS21680 (A(2A); inactive at up to 100 microM). 3. Cyclic AMP formation stimulated by NECA in guinea-pig tracheal epithelial cells was inhibited by adenosine receptor antagonist with the following order of apparent affinity (pA(2) value): Xanthine amine congeners (XAC, A(2A)/A(2B), 7.89+/-0.22)>CGS15943 (A(2A)/A(2B), 7.24+/-0. 26)>ZM241385 (A(2A), 6.69+/-0.14)>DPCPX (A(1), 6.51+/-0. 14)>3n-propylxanthine (weak A(2B), 4.30+/-0.10). This rank order of potency is typical for A(2B)-adenosine receptor. 4. Adenosine decreased basal and LPS-stimulated irET production in a concentration-dependent manner. Moreover, NECA but not CGS21680 inhibited LPS-induced irET production. 5. The inhibitory effect of NECA on LPS-induced irET production was reversed by XAC (pA(2)=8.84+/-0. 12) and DPCPX (pA(2)=8.10+/-0.22). 6. These results suggested that adenosine increased cyclic AMP formation and inhibited irET production/secretion by guinea-pig tracheal epithelial cells through the activation of a functional adenosine receptor that is most likely the A(2B) subtype. This adenosine receptor may be involved in the regulation of the level of ET-1 production/secretion by guinea-pig tracheal epithelial cells in physiological as well as in pathophysiological conditions.

The effects of selected bradykinin receptor antagonists on leukocyte infiltration into the lungs were studied in a model of guinea pig lung inflammation induced by the intravenous injection of Sephadex beads. The bradykinin B1 receptor antagonist, [Leu8]desArg9-BK (40 mg kg(-1) 24 h(-1)) and the bradykinin B2 receptor antagonist, DArg[Hyp3,Thi5,DTic7,Oic8]BK (code name HOE 140; 4 mg kg(-1) 24 h(-1)), administered intravenously by osmotic pumps, significantly reduced eosinophil counts by 33% and 42% in bronchoalveolar fluid, respectively. HOE 140 decreased neutrophil counts by 35%. LysLys[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK+ ++ (code name B 9858), a newly described bradykinin B1 receptor antagonist, administered intraperitoneally (1 mg kg(-1)), decreased eosinophil and neutrophil counts by 45% in bronchoalveolar fluid. D-Arg[Hyp3,Igl5,D-Igl7,Oic8]BK (code name B 9430), a non-selective bradykinin B1/B2 receptor antagonist, also administered intraperitoneally (1 mg kg(-1)), decreased eosinophil and macrophage counts by 62% and 80% in bronchoalveolar fluid. These results suggest that bradykinin B1 and B2 receptors are involved in leukocyte recruitment in our model of lung inflammation.

Objective and Design: This study evaluated the complement activation in guinea pigs that were given an intravenous injection of Sephadex and its correlation with markers of the development of inflammation.¶Materials and Methods: Dunkin Hartley guinea pigs (250-300 g) were used. Whole blood was collected by heart puncture in a sodium citrate solution (0.315 g/ml) for complement measurements. Complement activation was measured using a colorimetric haemolytic assay. Bronchoalveolar lavages (BAL) were performed to monitor cell infiltration and inflammation was monitored by measurements of eosinophil peroxidase (EPO), histamine, β-glucuronidase, albumin and total proteins in the BAL fluid.¶Treatment: Guinea pigs were pre-treated with aprotinin (40000 IKU/kg) 30 min before they were given an intravenous injection of Sephadex beads (24 mg/kg). Carboxymethyl (CM)-Sephadex (24 mg/kg) was administered alone.¶Results: Sephadex beads activated the complement system in vitro (14.12 ± 2.29 U/ml) and in vivo (9.95 ± 0.08 U/ml) reaching a peak 6 h after the injection. This activation was accompanied by other characteristic features of inflammation such as leukocyte infiltration and activation. Both CM-Sephadex and aprotinin reduced the blood complement activation and eosinophil infiltration/activation observed.¶Conclusions: Our results strongly suggest that complement is involved in the cascade of events leading to the inflammatory state observed in guinea pig following the intravenous injection of Sephadex beads.

A number of studies using endothelin (ET) receptor antagonists support the participation of ETs in a variety of cardiovascular, renal, and other disorders. It has also been established that a number of cytokines, which are released in such diseases, modulate the expression and production of ETs and thus activate the ET system. This effect may represent one pathway by which these inflammatory mediators operate. By regulating endothelin-converting enzyme (ECE) activities, and thus ET synthesis, one can potentiate or attenuate the production of ETs and the receptor affinity/density in such pathologic conditions. Here, the stimulated (lipopolysaccharide or interleukin-1 beta) production of ET-1 from guinea pig tracheal epithelial cells was abolished by CGS 26303 or CGS 26393, two ECE/neutral endopeptidase (NEP) inhibitors, but was unaffected by CGS 24592, a specific NEP inhibitor. Therefore, such dual, and eventually selective ECE inhibitors are effective agents to prevent the stimulated production of ETs.

The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.