Michael J. Williams

Comparative genomic analysis of the Tribolium immune system.

BackgroundTribolium castaneum is a species of Coleoptera, the largest and most diverse order of all eukaryotes. Components of the innate immune system are hardly known in this insect, which is in a key phylogenetic position to inform us about genetic innovations accompanying the evolution of holometabolous insects. We have annotated immunity-related genes and compared them with homologous molecules from other species.ResultsAround 300 candidate defense proteins are identified based on sequence similarity to homologs known to participate in immune responses. In most cases, paralog counts are lower than those of Drosophila melanogaster or Anopheles gambiae but are substantially higher than those of Apis mellifera. The genome contains probable orthologs for nearly all members of the Toll, IMD, and JAK/STAT pathways. While total numbers of the clip-domain serine proteinases are approximately equal in the fly (29), mosquito (32) and beetle (30), lineage-specific expansion of the family is discovered in all three species. Sixteen of the thirty-one serpin genes form a large cluster in a 50 kb region that resulted from extensive gene duplications. Among the nine Toll-like proteins, four are orthologous to Drosophila Toll. The presence of scavenger receptors and other related proteins indicates a role of cellular responses in the entire system. The structures of some antimicrobial peptides drastically differ from those in other orders of insects.ConclusionA framework of information on Tribolium immunity is established, which may serve as a stepping stone for future genetic analyses of defense responses in a nondrosophiline genetic model insect.

Drosophila Hemopoiesis and Cellular Immunity

In Drosophila melanogaster larvae, three classes of circulating cellular immune surveillance cells (hemocytes) can be identified: plasmatocytes, crystal cells, and lamellocytes. Plasmatocytes are professional phagocytes most similar to the mammalian monocyte/macrophage lineage and make up ∼95% of circulating hemocytes. The other ∼5% of circulating hemocytes consists of crystal cells, which secrete components necessary for the melanization of invading organisms, as well as for wound repair. A third cell type known as lamellocytes are rarely seen in healthy larvae and are involved in the encapsulation of invading pathogens. There are no obvious mammalian counterparts for crystal cells or lamellocytes, and there is no equivalent to the lymphoid lineage in insects. In this review, I will discuss what is currently known about Drosophila hemopoiesis and the cellular immune response and where possible compare it to vertebrate mechanisms.

Rac1 signalling in the Drosophila larval cellular immune response.

The Drosophila larval cellular immune response involves cells (hemocytes) that can be recruited from a hematopoietic organ located behind the brain, as well as a sessile population of cells found just underneath the larval cuticle arranged in a segmental pattern. By using two Rac1 GTPase effector-loop mutants together with epistasis studies, we show that Rac1 requires the Drosophila melanogaster Jun N-terminal kinase Basket (Bsk), as well as stable actin formation to recruit the sessile hemocyte population. We show that actin stabilization is necessary for Rac1-induced hemocyte activation by lowering cofilin (encoded by the twinstar gene tsr) expression in blood cells. Removing Bsk by RNAi suppressed Rac1-induced release of sessile hemocytes. RNAi against Bsk also suppressed Rac1 induction of lamellocytes, a specialized population of hemocytes necessary for the encapsulation of invading pathogens. Furthermore, Rac1 and Bsk are involved in regulating the formation of actin- and focal adhesion kinase (FAK)-rich placodes in hemocytes. Lastly, Rac1 and Bsk are both required for the proper encapsulation of eggs from the parasitoid wasp Leptipolina boulardi. From these data we conclude that Rac1 induces Bsk activity and stable actin formation for cellular immune activation, leading to sessile hemocyte release and an increase in the number of circulating hemocytes.

Drosophila melanogaster Rac2 is necessary for a proper cellular immune response.

It has been reported that during Drosophila embryonic development, and in cell culture, that the Rac GTPases are redundant. To better elucidate Rac function in Drosophila, we decided to study the role of Rac2 in larval cellular defense reactions against the parasitiod Leptopilina boulardi. Here we show a dramatic effect in the context of cellular immunity where, unlike embryonic development, Rac2 appears to have a non‐redundant function. When an invading parasitoid is recognized as foreign, circulating hemocytes (blood cells) should recognize and attach to the egg chorion. After attachment the hemocytes should then spread to form a multilayered capsule surrounding the invader. In Rac2 mutants this process is disrupted. Immune surveillance cells, known as plasmatocytes, adhere to the parasitoid egg but fail to spread, and septate junctions do not assemble, possibly due to mislocalization of the Protein 4.1 homolog Coracle. Finally, larger cells known as lamellocytes attach to the capsule but also fail to spread, and there is a lack of melanization. From these results it appears that Rac2 is necessary for the larval cellular immune response.

IL-2, -4, and -15 differentially regulate O-glycan branching and P-selectin ligand formation in activated CD8 T cells

The glycosyltransferase core 2 β1–6 N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) is responsible for formation of branched structures on O-glycans present on cell surface glycoproteins. The O-glycan branch created by C2GnT1 is physiologically important insofar as only this structure can be extended and modified to yield P-selectin ligands that promote initial interactions between extravasating lymphocytes and endothelia. In mature T cells, C2GnT1 activity is thought to be induced as an intrinsic consequence of T cell activation. Through analysis of C2GnT1-dependent epitopes on CD43 and CD45RB we have found that in activated CD8+ T cells expression of C2GnT1 was dependent upon exposure to specific cytokines rather than being induced as a direct consequence of activation. Activated CD8+ cells became receptive to strong induction of C2GnT1 expression and P-selectin ligand expression in response to IL-2, moderate induction by IL-15, and minimal induction in response to IL-4. Our observations clarify the relationship between T cell activation and C2GnT1 expression, demonstrate the differential impact of distinct cytokines on expression of C2GnT1 activity and P-selectin ligand, and reinforce the concept that the cytokine milieu subsequent to activation can influence adhesion systems that dictate lymphocyte homing properties.

Drosophila cellular immunity : a story of migration and adhesion

Research during the past 15 years has led to significant breakthroughs, providing evidence of a high degree of similarity between insect and mammalian innate immune responses, both humoural and cellular, and highlighting Drosophila melanogaster as a model system for studying the evolution of innate immunity. In a manner similar to cells of the mammalian monocyte and macrophage lineage, Drosophila immunosurveillance cells (haemocytes) have a number of roles. For example, they respond to wound signals, are involved in wound healing and contribute to the coagulation response. Moreover, they participate in the phagocytosis and encapsulation of invading pathogens, are involved in the removal of apoptotic bodies and produce components of the extracellular matrix. There are several reasons for using the Drosophila cellular immune response as a model to understand cell signalling during adhesion and migration in vivo: many genes involved in the regulation of Drosophila haematopoiesis and cellular immunity have been maintained across taxonomic groups ranging from flies to humans, many aspects of Drosophila and mammalian innate immunity seem to be conserved, and Drosophila is a simplified and well-studied genetic model system. In the present Commentary, we will discuss what is known about cellular adhesion and migration in the Drosophila cellular immune response, during both embryonic and larval development, and where possible compare it with related mechanisms in vertebrates.

Reciprocal regulation of Rac1 and Rho1 in Drosophila circulating immune surveillance cells.

In many cell types it is evident that the small GTPases Rac and Rho regulate each others activities. What is unclear is exactly how this regulation occurs. To further elucidate this interaction we examined the activities of Rac1 and Rho1 in Drosophila cellular immune surveillance cells. In larvae the cellular immune response involves circulating cells (hemocytes) that can be recruited from a hematopoietic organ located behind the brain, as well as a sessile population found just underneath the larval cuticle. We demonstrate for the first time that Rho-kinase activation requires both Rho1 and the Drosophila c-Jun N-terminal kinase (Basket). We also show that Rac1, via Basket, regulates Rho1 activity, possibly by inhibiting RhoGAPp190. In the reciprocal pathway, co-expression of dominant negative Rho-kinase and constitutive active Rho1 induces a Rac1-like phenotype. This induction requires the formin Diaphanous. Co-expression of dominant negative Rho-kinase and constitutive active Rho1 also induces filopodia formation, with Diaphanous enriched at the tips. The Rac1-like phenotypes, and filopodia formation, could be blocked by co-expression of dominant negative Rac1. Finally, though dominant negative Rac1 is able to block filopodia formation in the overexpression experiments, only Rac2 is necessary for filopodia formed by hemocytes after parasitization.

TM9SF4 is required for Drosophila cellular immunity via cell adhesion and phagocytosis

Nonaspanins are characterised by a large N-terminal extracellular domain and nine putative transmembrane domains. This evolutionarily conserved family comprises three members in Dictyostelium discoideum (Phg1A, Phg1B and Phg1C) and Drosophila melanogaster, and four in mammals (TM9SF1-TM9SF4), the function of which is essentially unknown. Genetic studies in Dictyostelium demonstrated that Phg1A is required for cell adhesion and phagocytosis. We created Phg1A/TM9SF4-null mutant flies and showed that they were sensitive to pathogenic Gram-negative, but not Gram-positive, bacteria. This increased sensitivity was not due to impaired Toll or Imd signalling, but rather to a defective cellular immune response. TM9SF4-null larval macrophages phagocytosed Gram-negative E. coli inefficiently, although Gram-positive S. aureus were phagocytosed normally. Mutant larvae also had a decreased wasp egg encapsulation rate, a process requiring haemocyte-dependent adhesion to parasitoids. Defective cellular immunity was coupled to morphological and adhesion defects in mutant larval haemocytes, which had an abnormal actin cytoskeleton. TM9SF4, and its closest paralogue TM9SF2, were both required for bacterial internalisation in S2 cells, where they displayed partial redundancy. Our study highlights the contribution of phagocytes to host defence in an organism possessing a complex innate immune response and suggests an evolutionarily conserved function of TM9SF4 in eukaryotic phagocytes.

The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells.

Background When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys) is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. Results In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. Significance We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.

The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells.

BackgroundWhen the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes) to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells.ResultsHere evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg) is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1) [1]. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1) fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface.ConclusionThe Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At the cell periphery of haemocytes Neuroglian may be involved in cell-cell interactions, while at the cell centre Neuroglian regulates the localisation of the nucleokinesis complex protein lissencephaly-1.