Natália G. Sampaio

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Phosphorylation of CSF-1R Y721 mediates its association with PI3K to regulate macrophage motility and enhancement of tumor cell invasion

Colony stimulating factor-1 (CSF-1) regulates macrophage morphology and motility, as well as mononuclear phagocytic cell proliferation and differentiation. The CSF-1 receptor (CSF-1R) transduces these pleiotropic signals through autophosphorylation of eight intracellular tyrosine residues. We have used a novel bone-marrow-derived macrophage cell line system to examine specific signaling pathways activated by tyrosine-phosphorylated CSF-1R in macrophages. Screening of macrophages expressing a single species of CSF-1R with individual tyrosine-to-phenylalanine residue mutations revealed striking morphological alterations upon mutation of Y721. M−/−.Y721F cells were apolar and ruffled poorly in response to CSF-1. Y721-P-mediated CSF-1R signaling regulated adhesion and actin polymerization to control macrophage spreading and motility. Moreover, the reduced motility of M−/− .Y721F macrophages was associated with their reduced capacity to enhance carcinoma cell invasion. Y721 phosphorylation mediated the direct association of the p85 subunit of phosphoinositide 3-kinase (PI3K) with the CSF-1R, but not that of phospholipase C (PLC) γ2, and induced polarized PtdIns(3,4,5)P3 production at the putative leading edge, implicating PI3K as a major regulator of CSF-1-induced macrophage motility. The Y721-P-motif-based motility signaling was at least partially independent of both Akt and increased Rac and Cdc42 activation but mediated the rapid and transient association of an unidentified ~170 kDa phosphorylated protein with either Rac-GTP or Cdc42-GTP. These studies identify CSF-1R-Y721-P–PI3K signaling as a major pathway in CSF-1-regulated macrophage motility and provide a starting point for the discovery of the immediate downstream signaling events.

Agonist-Independent Interactions between β-Arrestins and Mutant Vasopressin Type II Receptors Associated with Nephrogenic Syndrome of Inappropriate Antidiuresis

Nephrogenic syndrome of inappropriate antidiuresis is a recently identified genetic disease first described in two unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. It was found that each infant had a different mutation of the vasopressin type II receptor (V2R) at codon 137 where arginine was converted to cysteine or leucine (R137C or R137L), resulting in constitutive signaling. Interestingly, a missense mutation at the same codon, converting arginine to histidine (R137H), leads to the opposite disease phenotype with a loss of the kidneys ability to concentrate urine resulting in nephrogenic diabetes insipidus. This mutation is associated with impaired signaling, although whether this is predominantly due to impaired trafficking to the plasma membrane, agonist-independent internalization, or G protein uncoupling is currently unclear. Using bioluminescence resonance energy transfer and confocal microscopy, we demonstrate that both V2R-R137C and V2R-R137L mutants interact with beta-arrestins in an agonist-independent manner resulting in dynamin-dependent internalization. This phenotype is similar to that observed for V2R-R137H, which is intriguing considering that it is accompanied by constitutive rather than impaired signaling. Consequently, it would seem that agonist-independent internalization per se is unlikely to be the major determinant of impaired V2R-R137H signaling. Our findings indicate that the V2R-R137C and V2R-R137L mutants traffic considerably more efficiently to the plasma membrane than V2R-R137H, identifying this as a potentially important mutation-dependent difference affecting V2R function.

Specific inhibition of PI3K p110δ inhibits CSF‐1‐induced macrophage spreading and invasive capacity

Colony stimulating factor‐1 (CSF‐1) stimulates mononuclear phagocytic cell survival, growth and differentiation into macrophages through activation and autophosphorylation of the CSF‐1 receptor (CSF‐1R). We have previously demonstrated that CSF‐1‐induced phosphorylation of Y721 (pY721) in the receptor kinase insert triggers its association with the p85 regulatory subunit of phosphoinositide 3′‐kinase (PI3K). Binding of p85 PI3K to the CSF‐1R pY721 motif activates the associated p110 PI3K catalytic subunit and stimulates spreading and motility in macrophages and enhancement of tumor cell invasion. Here we show that pY721‐based signaling is necessary for CSF‐1‐stimulated PtdIns(3,4,5)P production. While primary bone marrow‐derived macrophages and the immortalized bone marrow‐derived macrophage cell line M−/−.WT express all three class IA PI3K isoforms, p110δ predominates in the cell line. Treatment with p110δ‐specific inhibitors demonstrates that the hematopoietically enriched isoform, p110δ, mediates CSF‐1‐regulated spreading and invasion in macrophages. Thus GS‐1101, a potent and selective p110δ inhibitor, may have therapeutic potential by targeting the infiltrative capacity of tumor‐associated macrophages that is critical for their enhancement of tumor invasion and metastasis.

Merozoite Antigens of Plasmodium falciparum Elicit Strain-Transcending Opsonizing Immunity

ABSTRACT It is unclear whether naturally acquired immunity to Plasmodium falciparum results from the acquisition of antibodies to multiple, diverse antigens or to fewer, highly conserved antigens. Moreover, the specific antibody functions required for malaria immunity are unknown, and hence informative immunological assays are urgently needed to address these knowledge gaps and guide vaccine development. In this study, we investigated whether merozoite-opsonizing antibodies are associated with protection from malaria in a strain-specific or strain-transcending manner by using a novel field isolate and an immune plasma-matched cohort from Papua New Guinea with our validated assay of merozoite phagocytosis. Highly correlated opsonization responses were observed across the 15 parasite strains tested, as were strong associations with protection (composite phagocytosis score across all strains in children uninfected at baseline: hazard ratio of 0.15, 95% confidence interval of 0.04 to 0.63). Opsonizing antibodies had a strong strain-transcending component, and the opsonization of transgenic parasites deficient for MSP3, MSP6, MSPDBL1, or P. falciparum MSP1-19 (PfMSP1-19) was similar to that of wild-type parasites. We have provided the first evidence that merozoite opsonization is predominantly strain transcending, and the highly consistent associations with protection against diverse parasite strains strongly supports the use of merozoite opsonization as a correlate of immunity for field studies and vaccine trials. These results demonstrate that conserved domains within merozoite antigens targeted by opsonization generate strain-transcending immune responses and represent promising vaccine candidates.

Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors

STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.STING is an intracellular DNA sensor that can alter response to infection, but in the case of malaria it is unclear how parasite DNA in red blood cells (RBCs) reaches DNA sensors in immune cells. Here the authors show that STING in human monocytes can sense P. falciparum nucleic acids transported from infected RBCs via parasite extracellular vesicles.

Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.

A major role of colony‐stimulating factor‐1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony‐stimulating factor‐1 receptor transduces colony‐stimulating factor‐1 signaling, and we have shown previously that phosphatidylinositol 3‐kinase p110δ is a critical mediator of colony‐stimulating factor‐1–stimulated motility through the colony‐stimulating factor‐1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony‐stimulating factor‐1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony‐stimulating factor‐1–induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony‐stimulating factor‐1–induced macrophage differentiation. Hck associated with the activated colony‐stimulating factor‐1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony‐stimulating factor‐1 receptor activation and colony‐stimulating factor‐1–induced macrophage spreading, motility, and invasion. Distinct colony‐stimulating factor‐1–induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony‐stimulating factor‐1.

The role of extracellular vesicles in malaria biology and pathogenesis

In the past decade, research on the functions of extracellular vesicles in malaria has expanded dramatically. Investigations into the various vesicle types, from both host and parasite origin, has revealed important roles for extracellular vesicles in disease pathogenesis and susceptibility, as well as cell–cell communication and immune responses. Here, work relating to extracellular vesicles in malaria is reviewed, and the areas that remain unknown and require further investigations are highlighted.

The occurrence and severity of grass toxicoses in Australian alpaca (Vicugna pacos) herds

A survey of 108 alpaca producers registered with the Australian Alpaca Association examined the occurrence and severity of ‘staggers’ (a colloquial term embracing various diseases of the nervous system in animals, characterised by neck tremors and head nodding in the milder alpaca cases and a lack of coordination in moving, a staggering gait and frequent falling in severe cases) in Australian alpaca and the presence of four pasture grasses, during three production seasons in 2004–2006. There have been few studies on the susceptibility of alpaca to staggers and its effect on productivity and animal welfare. The survey found that 23% of alpaca producers had observed staggering animals, with Victoria and South Australia being the most severely affected states. Clinical signs of staggers were most frequent in January–March, with a mean duration of 3 months. Some animals showed clinical signs lasting up to 12 months. A strong correlation was found between the presence of perennial ryegrass (P < 0.001) and phalaris (P < 0.003) and the occurrence of staggers. Based on grass presence and the timing when staggers was observed, it was concluded that perennial ryegrass toxicosis was the main cause of staggers in alpaca. About 12% and 9% of alpaca grazing pasture containing perennial ryegrass exhibited staggers in the two full seasons for which data were collected. Herds with staggering animals were correlated with those exhibiting possible subclinical effects – heat sensitivity and ill-thrift (P < 0.01) and reduced fertility (P < 0.05) – of perennial ryegrass endophyte toxins. Results indicate that weanlings and cria are more likely to stagger than adults. Thirteen of the 15 mixed farms with staggering alpaca did not observe staggers in other livestock, suggesting that alpaca may be more sensitive to the causal toxins than sheep and cattle.

Investigation of interactions between TLR2, MyD88 and TIRAP by bioluminescence resonance energy transfer is hampered by artefacts of protein overexpression

Toll like receptors (TLRs) are important pattern recognition receptors that can detect pathogen and danger associated molecular patterns to initiate an innate immune response. TLR1 and 2 heterodimerize at the plasma membrane upon binding to triacylated lipopeptides from bacterial cell walls, or to the synthetic ligand Pam3CSK4. TLR1/2 dimers interact with adaptor molecules TIRAP and MyD88 to initiate a signalling cascade that leads to activation of key transcription factors, including NF-kB. Despite TLRs being extensively studied over the last two decades, the real-time kinetics of ligand binding and receptor activation remains largely unexplored. We aimed to study the kinetics of TLR activation and recruitment of adaptors, using TLR1/2 dimer interactions with adaptors MyD88 and TIRAP. Bioluminescence resonance energy transfer (BRET) allows detection of real-time protein-protein interactions in living cells, and was applied to study adaptor recruitment to TLRs. Energy transfer showed interactions between TLR2 and TIRAP, and between TLR2 and MyD88 only in the presence of TIRAP. Quantitative BRET and confocal microscopy confirmed that TIRAP is necessary for MyD88 interaction with TLR2. Furthermore, constitutive proximity between the proteins in the absence of Pam3CSK4 stimulation was observed with BRET, and was not abrogated with lowered protein expression, changes in protein tagging strategies, or use of the brighter NanoLuc luciferase. However, co-immunoprecipitation studies did not demonstrate constitutive interaction between these proteins, suggesting that the interaction observed with BRET likely represents artefacts of protein overexpression. Thus, caution should be taken when utilizing protein overexpression in BRET studies and in investigations of the TLR pathway.