Foundational Article: Mechnikov I, 1909: Intestinal Bacteriotherapy.

In 1908, Ilya Metchnikov, then Assistant director of the Pasteur Institute, writes about the potential of bacterial cultures to remedy a range of intestinal ailments. Translated from French.

The gram-negative sensing receptor PGRP-LC contributes to grooming induction in Drosophila.

Behavioral resistance protects insects from microbial infection. However, signals inducing insect hygiene behavior are still relatively unexplored. Our previous study demonstrated that olfactory signals from microbes enhance insect hygiene behavior, and gustatory signals even induce the behavior. In this paper, we postulated a cross-talk between behavioral resistance and innate immunity. To examine this hypothesis, we employed a previously validated behavioral test to examine the function of taste signals in inducing a grooming reflex in decapitated flies. Microbes, which activate different pattern recognition systems upstream of immune pathways, were applied to see if there was any correlation between microbial perception and grooming reflex. To narrow down candidate elicitors, the grooming induction tests were conducted with highly purified bacterial components. Lastly, the role of DAP-type peptidoglycan in grooming induction was confirmed. Our results demonstrate that cleaning behavior can be triggered through recognition of DAP-type PGN by its receptor PGRP-LC.

The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors.

The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. Here we describe rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. We find that rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. We propose that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat.

ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors.

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin ß receptor (LTßR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTßR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

Sensing Gram-negative bacteria: a phylogenetic perspective.

Gram-negative bacteria represent a major group of pathogens that infect all eukaryotes from plants to mammals. Gram-negative microbe-associated molecular patterns include lipopolysaccharides and peptidoglycans, major immunostimulatory determinants across phyla. Recent advances have furthered our understanding of Gram-negative detection beyond the well-defined pattern recognition receptors such as TLR4. A B-type lectin receptor for LPS and Lysine-motif containing receptors for peptidoglycans were recently added to the plant arsenal. Caspases join the ranks of mammalian cytosolic immune detectors by binding LPS, and make TLR4 redundant for septic shock. Fascinating bacterial evasion mechanisms lure the host into tolerance or promote inter-bacterial competition. Our review aims to cover recent advances on bacterial messages and host decoding systems across phyla, and highlight evolutionarily recurrent strategies.

Different flavors of Toll guide olfaction.

Toll-like receptors are historically linked to immunity across animal phyla, but accumulating evidence suggests they play additional roles in neuronal networks and in cell-cell interactions. Ward and colleagues now identify Toll-6 and Toll-7 as instructive guidance cues during Drosophila olfactory development.

The Black cells phenotype is caused by a point mutation in the Drosophila pro-phenoloxidase 1 gene that triggers melanization and hematopoietic defects.

Melanization contributes to arthropod-specific innate immunity through deposition of melanin at wound sites or around parasites, with concomitant release of microbicidal reactive oxygen species. Melanization requires sequential activation of proteolytic enzymes in the hemolymph, including the final enzyme pro-phenoloxidase. Black cells (Bc) is a mutation causing spontaneous melanization of Drosophila crystal cells, a hemocyte cell type producing phenoloxidases. Bc individuals exhibit circulating black spots but fail to melanize upon injury. Although Bc is widely used as a loss-of-function mutant of phenoloxidases, the mutation causing Bc remained unknown. Here, we identified a single point mutation in the pro-phenoloxidase 1 (PPO1) gene of Bc flies causing an Alanine to Valine change in the C-terminal domain of PPO1, predicted to affect the conformation of the N-terminal pro-domain cleavage site at a distance and causing uncontrolled catalytic activity. Genomic insertion of a PPO1(A480V) transgene phenocopies Black cells, proving that A480V is indeed the causal mutation of the historical Bc phenotype.

Prophenoloxidase activation is required for survival to microbial infections in Drosophila.

The melanization reaction is a major immune response in Arthropods and involves the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones, which subsequently polymerize into melanin. The Drosophila genome encodes three POs, which are primarily produced as zymogens or prophenoloxidases (PPO). Two of them, PPO1 and PPO2, are produced by crystal cells. Here we have generated flies carrying deletions in PPO1 and PPO2. By analyzing these mutations alone and in combination, we clarify the functions of both PPOs in humoral melanization. Our study shows that PPO1 and PPO2 are responsible for all the PO activity in the hemolymph. While PPO1 is involved in the rapid early delivery of PO activity, PPO2 is accumulated in the crystals of crystal cells and provides a storage form that can be deployed in a later phase. Our study also reveals an important role for PPO1 and PPO2 in the survival to infection with Gram-positive bacteria and fungi, underlining the importance of melanization in insect host defense.

Methods to study Drosophila immunity.

Innate immune mechanisms are well conserved throughout evolution, and many theoretical concepts, molecular pathways and gene networks are applicable to invertebrate model organisms as much as vertebrate ones. Drosophila immunity research benefits from an easily manipulated genome, a fantastic international resource of transgenic tools and over a quarter century of accumulated techniques and approaches to study innate immunity. Here we present a short collection of ways to challenge the fruit fly immune system with various pathogens and parasites, as well as read-outs to assess its functions, including cellular and humoral immune responses. Our review covers techniques for assessing the kinetics and efficiency of immune responses quantitatively and qualitatively, such as survival analysis, bacterial persistence, antimicrobial peptide gene expression, phagocytosis and melanisation assays. Finally, we offer a toolkit of Drosophila strains available to the research community for current and future research.

An apolipoprotein A-I mimetic targets scavenger receptor A on tumor-associated macrophages: A prospective anticancer treatment?

Tumor-associated macrophages polarize toward an M2 phenotype and express scavenger receptor A (SRA), hence promoting tumor progression. We demonstrated that SRA can be therapeutically targeted in vivo with the small peptide inhibitor 4F to prevent metastatic spread. Beyond our study, 4F is emerging as a promising anticancer agent.

dRYBP contributes to the negative regulation of the Drosophila Imd pathway.

The Drosophila humoral innate immune response fights infection by producing antimicrobial peptides (AMPs) through the microbe-specific activation of the Toll or the Imd signaling pathway. Upon systemic infection, the production of AMPs is both positively and negatively regulated to reach a balanced immune response required for survival. Here, we report the function of the dRYBP (drosophila Ring and YY1 Binding Protein) protein, which contains a ubiquitin-binding domain, in the Imd pathway. We have found that dRYBP contributes to the negative regulation of AMP production: upon systemic infection with Gram-negative bacteria, Diptericin expression is up-regulated in the absence of dRYBP and down-regulated in the presence of high levels of dRYBP. Epistatic analyses using gain and loss of function alleles of imd, Relish, or skpA and dRYBP suggest that dRYBP functions upstream or together with SKPA, a member of the SCF-E3-ubiquitin ligase complex, to repress the Imd signaling cascade. We propose that the role of dRYBP in the regulation of the Imd signaling pathway is to function as a ubiquitin adaptor protein together with SKPA to promote SCF-dependent proteasomal degradation of Relish. Beyond the identification of dRYBP as a novel component of Imd pathway regulation, our results also suggest that the evolutionarily conserved RYBP protein may be involved in the human innate immune response.

Macrophage scavenger receptor a promotes tumor progression in murine models of ovarian and pancreatic cancer.

Alternatively activated macrophages express the pattern recognition receptor scavenger receptor A (SR-A). We demonstrated previously that coculture of macrophages with tumor cells upregulates macrophage SR-A expression. We show in this study that macrophage SR-A deficiency inhibits tumor cell migration in a coculture assay. We further demonstrate that coculture of tumor-associated macrophages and tumor cells induces secretion of factors that are recognized by SR-A on tumor-associated macrophages. We tentatively identified several potential ligands for the SR-A receptor in tumor cell-macrophage cocultures by mass spectrometry. Competing with the coculture-induced ligand in our invasion assay recapitulates SR-A deficiency and leads to similar inhibition of tumor cell invasion. In line with our in vitro findings, tumor progression and metastasis are inhibited in SR-A(-/-) mice in two in vivo models of ovarian and pancreatic cancer. Finally, treatment of tumor-bearing mice with 4F, a small peptide SR-A ligand able to compete with physiological SR-A ligands in vitro, recapitulates the inhibition of tumor progression and metastasis observed in SR-A(-/-) mice. Our observations suggest that SR-A may be a potential drug target in the prevention of metastatic cancer progression.

Tissue- and ligand-specific sensing of gram-negative infection in drosophila by PGRP-LC isoforms and PGRP-LE.

The Drosophila antimicrobial response is one of the best characterized systems of pattern recognition receptor-mediated defense in metazoans. Drosophila senses Gram-negative bacteria via two peptidoglycan recognition proteins (PGRPs), membrane-bound PGRP-LC and secreted/cytosolic PGRP-LE, which relay diaminopimelic acid (DAP)-type peptidoglycan sensing to the Imd signaling pathway. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their peptidoglycan binding specificities. In this study, we used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of PGRP-LC and assessed the tissue-specific roles of PGRP-LC isoforms and PGRP-LE in the antibacterial response. Our in vivo studies demonstrate the key role of PGRP-LCx in sensing DAP-type peptidoglycan-containing Gram-negative bacteria or Gram-positive bacilli during systemic infection. We also highlight the contribution of PGRP-LCa/x heterodimers to the systemic immune response to Gram-negative bacteria through sensing of tracheal cytotoxin (TCT), whereas PGRP-LCy may have a minor role in antagonizing the immune response. Our results reveal that both PGRP-LC and PGRP-LE contribute to the intestinal immune response, with a predominant role of cytosolic PGRP-LE in the midgut, the central section of endodermal origin where PGRP-LE is enriched. Our in vivo model also definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a loss-of-tolerance model. In conclusion, our study delineates how a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila.

Role of macrophage scavenger receptors in atherosclerosis.

Accumulating evidence indicates that atherosclerosis is a chronic inflammatory disease. The key innate immune cells that are involved in the pathogenesis of atherosclerosis are circulating monocytes and plaque macrophages. Complex interplay between immune and metabolic processes results in pathological activity of these cells. The best understood pathological process mediated by macrophages is their inability to process modified lipoproteins properly resulting in the formation of foamy cells, which are a dangerous component of atherosclerotic plaques. Key molecules involved in the recognition and processing of modified lipoproteins are scavenger receptors (SR). This is a large family of surface expressed structurally heterogeneous receptors with a broad spectrum of endogenous and exogenous ligands. The common functional feature of SR is internalisation of extracellular components and targeting them for lysosomal degradation. However, these relatively simple functions can have complex consequences, since they are linked to diverse specific signalling pathways and to other membrane transport pathways. Moreover, scavenger receptors can co-operate with other types of receptors increasing the variability of the macrophage response to multiple extracellular ligands. At least some SRs respond to modified lipoproteins by amplification of inflammation and accumulation of macrophages in the plaque, while some SRs may support tolerogenic reactions. Outcome of different SR activities will be the decision of monocytes and macrophage to guard homeostatic balance, support atherosclerosis progression and plaque instability by inflammatory reactions, or support rapid fibrotic processes in the plaque that stabilise it. Despite the accumulating knowledge about the molecular mechanisms of scavenger receptor action, their role in the progression of atherosclerosis remains controversial. The activities of scavenger receptors that can contribute to each of these processes are a subject of current review.

Identification of scavenger receptor ligands.

Scavenger receptors (SRs) are structurally diverse but functionally related innate immune receptors involved in defence and clearance mechanisms. Their broad specificity relies on evolutionarily conserved pattern recognition domains which interact with a variety of microbial, apoptotic and modified self ligands. Studies of immune functions of SR-expressing cells require the identification of interaction partners for SRs. We have developed an ELISA-based method which allows for large-scale high-throughput screening of complex mixtures. The assay successfully identified bacterial and plasma ligands for macrophage scavenger receptor A and can be adapted to screen for novel exogenous or endogenous ligands for any immune receptor of interest.

Macrophage scavenger receptor A mediates adhesion to apolipoproteins A-I and E.

Macrophage scavenger receptor A (SR-A) is a multifunctional, multiligand pattern recognition receptor with roles in innate immunity, apoptotic cell clearance, and age-related degenerative pathologies, such as atherosclerosis and Alzheimer's disease. Known endogenous SR-A ligands are polyanionic and include modified lipoproteins, advanced glycation end products, and extracellular matrix proteins. No native plasma ligands have been identified, but it is known that SR-A recognition of unidentified serum components mediates integrin-independent macrophage adhesion, which may drive chronic local inflammation. In this study, we used a high-throughput fractionation and screening method to identify novel endogenous SR-A ligands that may mediate macrophage adhesion. SR-A was found to recognize the exchangeable apolipoproteins A-I and E (apo A-I and apo E, respectively) in both lipid-free and lipid-associated form, suggesting the shared amphipathic alpha-helix as a potential recognition motif. Adhesion of RAW 264.7 macrophages to surfaces coated with apo A-I and apo E4 proved to be integrin-independent and could be blocked by anti-SR-A antibodies. The presence of apo A-I and apo E in pathological deposits, such as atherosclerotic lesions and neurotoxic Alzheimer's plaques, suggests a possible contribution of SR-A-dependent adhesion of macrophages to an inflammatory microenvironment.

A sensitive solid-phase assay for identification of class A macrophage scavenger receptor ligands using cell lysate.

In order for macrophages to perform their numerous homeostatic, immunological and tissue remodeling functions they are required to express a broad repertoire of cell-surface receptors. These receptors are particularly important for their host-defense functions in the recognition of foreign pathogens. Delineation of the particular functions of specific receptors requires the identification of ligands recognized by the receptor. We have developed a sensitive, high throughput, solid-phase assay for the detection of ligands for the class A macrophage scavenger receptor (SR-A). Post-nuclear cell lysate from murine bone marrow-derived macrophages is used as a source of receptor and specific ligand binding to SR-A is detected with a monoclonal antibody for SR-A. This assay has been used effectively to identify protein ligands for SR-A on the surface of the bacterium Neisseria meningitidis (Peiser, L. et al. [Peiser, L., Makepeace, K., Pluddemann, A., Savino, S., Wright, J.C., Pizza, M., Rappuoli, R., Moxon, E.R., Gordon, S., 2006. Identification of Neisseria meningitidis nonlipopolysaccharide ligands for class A macrophage scavenger receptor by using a novel assay. Infect. Immun. 74, 5191-5199]). In this paper we describe the method in detail and define the specific variables governing the assay.

Macrophage scavenger receptors and host-derived ligands.

The scavenger receptors are a large family of molecules that are structurally diverse and have been implicated in a range of functions. They are expressed by myeloid cells, selected endothelial cells and some epithelial cells and recognise many different ligands, including microbial pathogens as well as endogenous and modified host-derived molecules. This review will focus on the eight classes of scavenger receptors (class A-H) in terms of their structure, expression and recognition of host-derived ligands. Scavenger receptors have been implicated in a range of physiological and pathological processes, such as atherosclerosis and Alzheimer's disease, and function in adhesion and tissue maintenance. More recently, some of the scavenger receptors have been shown to mediate binding and endocytosis of chaperone proteins, such as the heat shock proteins, thereby playing an important role in antigen cross-presentation.