Alternative activation of macrophages by filarial nematodes is MyD88-independent

AbstractAlternative macrophage activation is largely defined by IL-4Rα stimulation but the contribution of Toll-like receptor (TLR) signaling to this phenotype is not currently known. We have investigated macrophage activation status under Th2 conditions in the absence of the core TLR adaptor molecule, MyD88. No impairment was observed in the ability of MyD88-deficient bone marrow derived macrophages to produce or express alternative activation markers, including arginase, RELM-α or Ym1, in response to IL-4 treatment in vitro. Further, we observed no difference in the ability of peritoneal exudate cells from nematode implanted wild type (WT) or MyD88-deficient mice to produce arginase or express the alternative activation markers RELM-α or Ym1. Therefore, MyD88 is not a fundamental requirement for Th2-driven macrophage alternative activation, either in vitro or in vivo

Synthesis, G-Quadruplex Stabilisation, Docking Studies, and Effect on Cancer Cells of Indolo[3,2-b]quinolines with One, Two, or Three Basic Side Chains.

G-quadruplex (G4) DNA structures in telomeres and oncogenic promoter regions are potential targets for cancer therapy, and G4 ligands have been shown to modulate telomerase activity and oncogene transcription. Herein we report the synthesis and G4 thermal stabilisation effects, determined by FRET melting assays, of 20 indolo[3,2-b]quinolines mono-, di-, and trisubstituted with basic side chains. Molecular modelling studies were also performed in an attempt to rationalise the ligands' binding poses with G4. Overall, the results suggest that ligand binding and G4 DNA thermal stabilisation increase with an N5-methyl or a 7-carboxylate group and propylamine side chains, whereas selectivity between G4 and duplex DNA appears to be modulated by the number and relative position of basic side chains. From all the indoloquinoline derivatives studied, the novel trisubstituted compounds 3 d and 4 d, bearing a 7-(aminoalkyl)carboxylate side chain, stand out as the most promising compounds; they show high G4 thermal stabilisation (ΔTm values between 17 and 8 °C) with an inter-G4 ΔTm trend of Hsp90A>KRas21R≈F21T>c-Kit2, 10-fold selectivity for G4 over duplex DNA, and 100-fold selectivity for the HCT116 cancer cell line (IC50 and IC90 : <10 μM) over primary rat hepatocytes. Compounds 3 d and 4 d also decreased protein expression levels of Hsp90 and KRas in HCT116 cancer cells

MHCII-mediated dialog between group 2 innate lymphoid cells and CD4+ T cells potentiates type 2 immunity and promotes parasitic helminth expulsion

Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity

Discovery of new G-quadruplex binding chemotypes.

We report here on the discovery and preliminary evaluation of a novel non-macrocyclic low molecular weight quadruplex-stabilizing chemotype. The lead compounds, based on a furan core, show high G-quadruplex stabilisation and selectivity as well as potent in vitro anti-proliferative activity

DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential.

Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy.The Balasubramanian laboratory is core-funded by Cancer Research UK (C14303/A17197) and further supported by a Cancer Research UK programme grant (C9681/A18618). S.B. is a Wellcome Trust Senior Investigator (099232/Z/12/Z)

CD11c depletion severely disrupts Th2 induction and development in vivo

Although dendritic cells (DCs) are adept initiators of CD4+ T cell responses, their fundamental importance in this regard in Th2 settings remains to be demonstrated. We have used CD11c–diphtheria toxin (DTx) receptor mice to deplete CD11c+ cells during the priming stage of the CD4+ Th2 response against the parasitic helminth Schistosoma mansoni. DTx treatment significantly depleted CD11c+ DCs from all tissues tested, with 70–80% efficacy. Even this incomplete depletion resulted in dramatically impaired CD4+ T cell production of Th2 cytokines, altering the balance of the immune response and causing a shift toward IFN-γ production. In contrast, basophil depletion using Mar-1 antibody had no measurable effect on Th2 induction in this system. These data underline the vital role that CD11c+ antigen-presenting cells can play in orchestrating Th2 development against helminth infection in vivo, a response that is ordinarily balanced so as to prevent the potentially damaging production of inflammatory cytokines

Murine but Not Human Basophil Undergoes Cell-Specific Proteolysis of a Major Endoplasmic Reticulum Chaperone

Basophil has been implicated in anti-parasite defense, allergy and in polarizing T(H)2 response. Mouse model has been commonly used to study basophil function although the difference between human and mouse basophils is underappreciated. As an essential chaperone for multiple Toll-like receptors and integrins in the endoplasmic reticulum, gp96 also participates in general protein homeostasis and in the ER unfolded protein response to ensure cell survival during stress. The roles of gp96 in basophil development are unknown.We genetically delete gp96 in mice and examined the expression of gp96 in basophils by Western blot and flow cytometry. We compared the expression pattern of gp96 between human and mouse basophils.We found that gp96 was dispensable for murine basophil development. Moreover, gp96 was cleaved by serine protease(s) in murine but not human basophils leading to accumulation of a nun-functional N-terminal ∼50 kDa fragment and striking induction of the unfolded protein response. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. We also demonstrated that the ectopic expression of a mouse-specific tryptase mMCP11 does not lead to gp96 cleavage in human basophils.Our study revealed a remarkable biochemical event of gp96 silencing in murine but not human basophils, highlighting the need for caution in using mouse models to infer the function of basophils in human immune response. Our study also reveals a novel mechanism of shutting down gp96 post-translationally in regulating its function

The impact of negative selection on thymocyte migration in the medulla

Developing thymocytes are screened for self-reactivity before they exit the thymus, but how thymocytes scan the medulla for self antigens is unclear. Using two-photon microscopy, we observed that medullary thymocytes migrated rapidly and made frequent, transient contacts with dendritic cells. In the presence of a negative selecting ligand, thymocytes slowed, became confined to areas of approximately 30 mum in diameter and had increased contact with dendritic cells surrounding confinement zones. One third of polyclonal medullary thymocytes also showed confined, slower migration and may correspond to autoreactive thymocytes. Our data suggest that many autoreactive thymocytes do not undergo immediate arrest and death after encountering a negative selecting ligand but instead adopt an altered migration program while remaining in the medullary microenvironment

Helminth Genomics: The Implications for Human Health

More than two billion people (one-third of humanity) are infected with parasitic roundworms or flatworms, collectively known as helminth parasites. These infections cause diseases that are responsible for enormous levels of morbidity and mortality, delays in the physical development of children, loss of productivity among the workforce, and maintenance of poverty. Genomes of the major helminth species that affect humans, and many others of agricultural and veterinary significance, are now the subject of intensive genome sequencing and annotation. Draft genome sequences of the filarial worm Brugia malayi and two of the human schistosomes, Schistosoma japonicum and S. mansoni, are now available, among others. These genome data will provide the basis for a comprehensive understanding of the molecular mechanisms involved in helminth nutrition and metabolism, host-dependent development and maturation, immune evasion, and evolution. They are likely also to predict new potential vaccine candidates and drug targets. In this review, we present an overview of these efforts and emphasize the potential impact and importance of these new findings