Gut cytokines modulate olfaction through metabolic reprogramming of glia.

Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival1,2. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms3-5. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. Here we show that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. Our findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies.

Anti-VEGF Treatment Enhances CD8+ T-cell Antitumor Activity by Amplifying Hypoxia.

Antiangiogenic therapies that target the VEGF pathway have been used clinically to combat cancer for over a decade. Beyond having a direct impact on blood vessel development and tumor perfusion, accumulating evidence indicates that these agents also affect antitumor immune responses. Numerous clinical trials combining antiangiogenic drugs with immunotherapies for the treatment of cancer are ongoing, but a mechanistic understanding of how disruption of tumor angiogenesis may impact immunity is not fully discerned. Here, we reveal that blockade of VEGF-A with a mAb to VEGF augments activation of CD8+ T cells within tumors and potentiates their capacity to produce cytokines. We demonstrate that this phenomenon relies on the disruption of VEGFR2 signaling in the tumor microenvironment but does not affect CD8+ T cells directly. Instead, the augmented functional capacity of CD8+ T cells stems from increased tumor hypoxia that initiates a hypoxia-inducible factor-1α program within CD8+ T cells that directly enhances cytokine production. Finally, combinatorial administration of anti-VEGF with an immunotherapeutic antibody, anti-OX40, improved antitumor activity over single-agent treatments. Our findings illustrate that anti-VEGF treatment enhances CD8+ T-cell effector function and provides a mechanistic rationale for combining antiangiogenic and immunotherapeutic drugs for cancer treatment.

Untangling the brain's neuroinflammatory and neurodegenerative transcriptional responses.

A common approach to understanding neurodegenerative disease is comparing gene expression in diseased versus healthy tissues. We illustrate that expression profiles derived from whole tissue RNA highly reflect the degenerating tissues' altered cellular composition, not necessarily transcriptional regulation. To accurately understand transcriptional changes that accompany neuropathology, we acutely purify neurons, astrocytes and microglia from single adult mouse brains and analyse their transcriptomes by RNA sequencing. Using peripheral endotoxemia to establish the method, we reveal highly specific transcriptional responses and altered RNA processing in each cell type, with Tnfr1 required for the astrocytic response. Extending the method to an Alzheimer's disease model, we confirm that transcriptomic changes observed in whole tissue are driven primarily by cell type composition, not transcriptional regulation, and identify hundreds of cell type-specific changes undetected in whole tissue RNA. Applying similar methods to additional models and patient tissues will transform our understanding of aberrant gene expression in neurological disease.

Balanced interactions between Lyn, the p85alpha regulatory subunit of class I(A) phosphatidylinositol-3-kinase, and SHIP are essential for mast cell growth and maturation.

The growth and maturation of bone marrow-derived mast cells (BMMCs) from precursors are regulated by coordinated signals from multiple cytokine receptors, including KIT. While studies conducted using mutant forms of these receptors lacking the binding sites for Src family kinases (SFKs) and phosphatidylinositol-3-kinase (PI3K) suggest a role for these signaling molecules in regulating growth and survival, how complete loss of these molecules in early BMMC progenitors (MCps) impacts maturation and growth during all phases of mast cell development is not fully understood. We show that the Lyn SFK and the p85α subunit of class I(A) PI3K play opposing roles in regulating the growth and maturation of BMMCs in part by regulating the level of PI3K. Loss of Lyn in BMMCs results in elevated PI3K activity and hyperactivation of AKT, which accelerates the rate of BMMC maturation due in part to impaired binding and phosphorylation of SHIP via Lyn's unique domain. In the absence of Lyn's unique domain, BMMCs behave in a manner similar to that of Lyn- or SHIP-deficient BMMCs. Importantly, loss of p85α in Lyn-deficient BMMCs not only represses the hyperproliferation associated with the loss of Lyn but also represses their accelerated maturation. The accelerated maturation of BMMCs due to loss of Lyn is associated with increased expression of microphthalmia-associated transcription factor (Mitf), which is repressed in MCps deficient in the expression of both Lyn and p85α relative to controls. Our results demonstrate a crucial interplay of Lyn, SHIP, and p85α in regulating the normal growth and maturation of BMMCs, in part by regulating the activation of AKT and the expression of Mitf.

Deficiency of Src family kinases compromises the repopulating ability of hematopoietic stem cells.

OBJECTIVE: Src family kinases (SFK) have been implicated in regulating growth factor and integrin-induced proliferation, migration, and gene expression in multiple cell types. However, little is known about the role of these kinases in the growth, homing, and engraftment potential of hematopoietic stem and progenitor cells. RESULTS: Here we show that loss of hematopoietic-specific SFKs Hck, Fgr, and Lyn results in increased number of Sca-1(+)Lin(-) cells in the bone marrow, which respond differentially to cytokine-induced growth in vitro and manifest a significant defect in the long-term repopulating potential in vivo. Interestingly, a significant increase in expression of adhesion molecules, known to coincide with the homing potential of wild-type bone marrow cells is also observed on the surface of SFK(-/-) cells, although, this increase did not affect the homing potential of more primitive Lin(-)Sca-1(+) SFK(-/-) cells. The stem cell-repopulating defect observed in mice transplanted with SFK(-/-) bone marrow cells is due to the loss of Lyn Src kinase, because deficiency of Lyn, but not Hck or Fgr, recapitulated the long-term stem cell defect observed in mice transplanted with SFK(-/-) bone marrow cells. CONCLUSIONS: Taken together, our results demonstrate an essential role for Lyn kinase in positively regulating the long-term and multilineage engraftment of stem cells, which is distinct from its role in mature B cells and myeloid cells.

Src family kinase-mediated negative regulation of hematopoietic stem cell mobilization involves both intrinsic and microenvironmental factors.

OBJECTIVE: The intracellular signals that contribute to granulocyte colony-stimulating factor (G-CSF) receptor induced stem cell mobilization are poorly characterized. METHODS: We show enhanced G-CSF induced mobilization of stem cells in mice deficient in expression of Src family kinases (SFK-/-), which is associated with hypersensitivity of SFK-/- bone marrow cells to G-CSF as well as sustained activation of signal transducer and activator of transcription-3. RESULTS: A proteome map of the bone marrow fluid derived from wild-type and SFK-/- mice revealed a significant global reduction in the number of proteins in SFK-/- mice compared to controls, which was associated with elevated matrix metalloproteinase-9 levels, reduced stromal-derived factor-1 expression, and enhanced breakdown of vascular cell adhesion molecule-1. Transplantation of wild-type or SFK-/- stem cells into wild-type mice and treatment with G-CSF recapitulated the G-CSF-induced increase in stem cell mobilization noted in SFK-/- nontransplanted mice; however, the increase was significantly less. G-CSF treatment of SFK-/- mice engrafted with wild-type stem cells also demonstrated a modest increase in stem cell mobilization compared to controls, however, the observed increase was greatest in mice completely devoid of SFKs. CONCLUSIONS: These data suggest an involvement of both hematopoietic intrinsic and microenvironmental factors in Src kinase-mediated mobilization of stem cells and identify Src kinases as potential targets for modulating stem cell mobilization.

Genetic and pharmacologic evidence implicating the p85 alpha, but not p85 beta, regulatory subunit of PI3K and Rac2 GTPase in regulating oncogenic KIT-induced transformation in acute myeloid leukemia and systemic mastocytosis.

Oncogenic activation loop KIT mutations are observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants, the activation loop mutants are insensitive to imatinib mesylate. Furthermore, as prior studies primarily used heterologous cell lines, the molecular mechanism(s) underlying oncogenic KIT-induced transformation in primary cells is poorly understood. We demonstrate that expression of KITD814V in primary hematopoietic stem/progenitor cells (HSC/Ps) and mast cell progenitors (MCps) induces constitutive KIT autophosphorylation, supports ligand-independent hyperproliferation, and promotes promiscuous cooperation with multiple cytokines. Genetic disruption of p85 alpha, the regulatory subunit of class IA lipid kinase phosphoinositol-3-kinase (PI3K), but not of p85 beta, or genetic disruption of the hematopoietic cell-specific Rho GTPase, Rac2, normalizes KITD814V-induced ligand-independent hyperproliferation. Additionally, deficiency of p85 alpha or Rac2 corrects the promiscuous hyperproliferation observed in response to multiple cytokines in both KITD814V-expressing HSC/Ps and MCps. Treatment of KITD814V-expressing HSC/Ps with a Rac inhibitor (NC23766) or with rapamycin showed a dose-dependent suppression in ligand-independent growth. Taken together, our results identify p85 alpha and Rac2 as potential novel therapeutic targets for the treatment of KITD814V-bearing AML and SM.

p85alpha subunit of class IA PI-3 kinase is crucial for macrophage growth and migration.

Macrophages play an essential role in defending against invading pathogens by migrating to the sites of infection, removing apoptotic cells, and secreting inflammatory cytokines. The molecular mechanisms whereby macrophages regulate these processes are poorly understood. Using bone marrow-derived macrophages (BMMs) deficient in the expression of p85alpha-subunit of class IA phosphatidylinositol 3 (PI-3) kinase, we demonstrate 50% reduction in proliferation in response to macrophage-colony-stimulating factor (M-CSF) as well as granulocyte macrophage-colony-stimulating factor (GM-CSF) compared with wild-type controls. Furthermore, p85alpha-/- BMMs demonstrate a significant reduction in migration in a wound-healing assay compared with wild-type controls. The reduction in migration due to p85alpha deficiency in BMMs is associated with reduced adhesion and directed migration on fibronectin and vascular cell adhesion molecule-1. In addition, deficiency of p85alpha in BMMs also results in defective phagocytosis of sheep red blood cells. Biochemically, loss of p85alpha in BMMs results in reduced activation of Akt and Rac, but not Erk (extracellular signal-related kinase) mitogen-activated protein (MAP) kinase. Taken together, our results provide genetic evidence for the importance of p85alpha in regulating both actin- and growth-based functions in macrophages, and provide a potential therapeutic target for the treatment of diseases involving macrophages, including inflammation.

Genetic evidence for convergence of c-Kit- and alpha4 integrin-mediated signals on class IA PI-3kinase and the Rac pathway in regulating integrin-directed migration in mast cells.

Mast cells play a critical role in host defense against a number of pathogens. Increased mast cell infiltration has been described in allergic asthma, in rheumatoid arthritis, and during helminthes infection. Despite the importance of mast cells in allergic disease and defense against infection, little is known about the mechanisms by which mast cells migrate to various tissues under steady state conditions or during infection or inflammation. Here, we show that activation of c-Kit by its ligand, stem cell factor (SCF), cooperates with alpha4 integrin in inducing directed migration of mast cells on fibronectin. A reduction in migration and activation of a small G protein, Rac, was observed in mast cells derived from class IA phosphoinositide-3 kinase (PI-3kinase)-deficient mice in response to SCF stimulation and in mast cells expressing the dominant-negative Rac (RacN17), as well as in mast cells deficient in the hematopoietic-specific small G protein, Rac2. In addition, a PI-3kinase inhibitor inhibited alpha4- as well as SCF-induced migration in a dose-dependent fashion. In contrast, a mitogen-activated protein kinase (MAPK) inhibitor had little effect. Consistent with the pharmacologic results, abrogating the binding of the p85alpha subunit of class IA PI-3kinase to c-Kit also resulted in inhibition of SCF-induced migration on fibronectin. These genetic and biochemical data demonstrate that both c-Kit and alpha4 integrin signaling are linked to class IA PI-3kinase and Rac pathways and regulate integrin-directed (haptotactic) migration in mast cells.