Sharissa L. Latham

Microparticles from Mycobacteria-Infected Macrophages Promote Inflammation and Cellular Migration

Mycobacterium tuberculosis infection is characterized by a strong inflammatory response whereby a few infected macrophages within the granuloma induce sustained cellular accumulation. The mechanisms coordinating this response are poorly characterized. We hypothesized that microparticles (MPs), which are submicron, plasma membrane-derived vesicles released by cells under both physiological and pathological conditions, are involved in this process. Aerosol infection of mice with M. tuberculosis increased CD45+ MPs in the blood after 4 wk of infection, and in vitro infection of human and murine macrophages with mycobacteria enhanced MP release. MPs derived from mycobacteria-infected macrophages were proinflammatory, and when injected into uninfected mice they induced significant neutrophil, macrophage, and dendritic cell recruitment to the injection site. When incubated with naive macrophages, these MPs enhanced proinflammatory cytokine and chemokine release, and they aided in the disruption of the integrity of a respiratory epithelial cell monolayer, providing a mechanism for the egress of cells to the site of M. tuberculosis infection in the lung. In addition, MPs colocalized with the endocytic recycling marker Rab11a within macrophages, and this association increased when the MPs were isolated from mycobacteria-infected cells. M. tuberculosis–derived MPs also carried mycobacterial Ag and were able to activate M. tuberculosis–specific CD4+ T cells in vivo and in vitro in a dendritic cell–dependent manner. Collectively, these data identify an unrecognized role for MPs in host response against M. tuberculosis by promoting inflammation, intercellular communication, and cell migration.

Endothelial Microparticles Interact with and Support the Proliferation of T Cells

Endothelial cells closely interact with circulating lymphocytes. Aggression or activation of the endothelium leads to an increased shedding of endothelial cell microparticles (MP). Endothelial MP (EMP) are found in high plasma levels in numerous immunoinflammatory diseases, such as atherosclerosis, sepsis, multiple sclerosis, and cerebral malaria, supporting their role as effectors and markers of vascular dysfunction. Given our recently described role for human brain microvascular endothelial cells (HBEC) in modulating immune responses, we investigated how HBEC-derived MP could interact with and support the proliferation of T cells. Like their mother cells, EMP expressed molecules important for Ag presentation and T cell costimulation, that is, β2-microglobulin, MHC II, CD40, and ICOSL. HBEC were able to take up fluorescently labeled Ags with EMP also containing fluorescent Ags, suggestive of Ag carryover from HBEC to EMP. In cocultures, fluorescently labeled EMP from resting or cytokine-stimulated HBEC formed conjugates with both CD4+ and CD8+ subsets, with higher proportions of T cells binding EMP from cytokine-stimulated cells. The increased binding of EMP from cytokinestimulated HBEC to T cells was VCAM-1 and ICAM-1 dependent. Finally, in CFSE T cell proliferation assays using anti-CD3 mAb or T cell mitogens, EMP promoted the proliferation of CD4+ T cells and that of CD8+ T cells in the absence of exogenous stimuli and in the T cell mitogenic stimulation. Our findings provide novel evidence that EMP can enhance T cell activation and potentially ensuing Ag presentation, thereby pointing toward a novel role for MP in neuroimmunological complications of infectious diseases.

Cuticle Thickening in a Pyrethroid-Resistant Strain of the Common Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae).

Thickening of the integument as a mechanism of resistance to insecticides is a well recognised phenomenon in the insect world and, in recent times, has been found in insects exhibiting pyrethroid-resistance. Resistance to pyrethroid insecticides in the common bed bug, Cimex lectularius L., is widespread and has been frequently inferred as a reason for the pest’s resurgence. Overexpression of cuticle depositing proteins has been demonstrated in pyrethroid-resistant bed bugs although, to date, no morphological analysis of the cuticle has been undertaken in order to confirm a phenotypic link. This paper describes examination of the cuticle thickness of a highly pyrethroid-resistant field strain collected in Sydney, Australia, in response to time-to-knockdown upon forced exposure to a pyrethroid insecticide. Mean cuticle thickness was positively correlated to time-to-knockdown, with significant differences observed between bugs knocked-down at 2 hours, 4 hours, and those still unaffected at 24 hours. Further analysis also demonstrated that the 24 hours survivors possessed a statistically significantly thicker cuticle when compared to a pyrethroid-susceptible strain of C. lectularius. This study demonstrates that cuticle thickening is present within a pyrethroid-resistant strain of C. lectularius and that, even within a stable resistant strain, cuticle thickness will vary according to time-to-knockdown upon exposure to an insecticide. This response should thus be considered in future studies on the cuticle of insecticide-resistant bed bugs and, potentially, other insects.

Cooperation between β- and γ-cytoplasmic actins in the mechanical regulation of endothelial microparticle formation

Elevated endothelial microparticle (MP) levels are observed in numerous diseases, increasingly supporting roles as effectors and valuable markers of vascular dysfunction. While a contractile role for the actin cytoskeleton has been implicated in vesiculation, i.e., MP production, the precise interactions and mechanisms of its constituents, β‐ and γ‐cytoplasmic actins, is unknown. Human cerebral microvascular endothelial cells were stimulated with known agonists, and vesiculation development was monitored by scanning electron microscopy (SEM) and flow cytometry. These data in combination provide new insight into the kinetics, patterns of vesiculating cell recruitment, and degrees of response specific to stimuli. Reorganization of β‐and γ‐actins, F‐actin, vinculin, and talin accompanied significant MP release. β‐Actin redistribution into basal stress fibers following stimulation was associated with increased apically situated actin‐rich particulate structures, which in turn directly correlated with electron‐lucent membrane protrusions observed by SEM. Y‐27632 Rho‐kinase inhibition abolished basal β‐actin fiber formation, minimizing apically associated actin‐rich structures, significantly reducing membrane protrusions and MP release to near basal levels. Cytoskeletal protein expression and distribution varied between MPs and mother cells, as determined by Western blot. These data strongly suggest that β‐actin plays an active facilitative role in agonist‐induced protuberance formation, through mechanical interactions with newly described actin‐rich structures.—Latham, S. L., Chaponnier, C., Dugina, V., Couraud, P.‐O., Grau, G. E. R., Combes, V. Cooperation between β‐ and γ‐cytoplasmic actins in the mechanical regulation of endothelial microparticle formation. FASEB J. 27, 672–683 (2013). www.fasebj.org

Immuno-analysis of microparticles: probing at the limits of detection.

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30–40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data.

Exploring experimental cerebral malaria pathogenesis through the characterisation of host-derived plasma microparticle protein content

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection responsible for thousands of deaths in children in sub-Saharan Africa. CM pathogenesis remains incompletely understood but a number of effectors have been proposed, including plasma microparticles (MP). MP numbers are increased in CM patients’ circulation and, in the mouse model, they can be localised within inflamed vessels, suggesting their involvement in vascular damage. In the present work we define, for the first time, the protein cargo of MP during experimental cerebral malaria (ECM) with the overarching hypothesis that this characterisation could help understand CM pathogenesis. Using qualitative and quantitative high-throughput proteomics we compared MP proteins from non-infected and P. berghei ANKA-infected mice. More than 360 proteins were identified, 60 of which were differentially abundant, as determined by quantitative comparison using TMTTM isobaric labelling. Network analyses showed that ECM MP carry proteins implicated in molecular mechanisms relevant to CM pathogenesis, including endothelial activation. Among these proteins, the strict association of carbonic anhydrase I and S100A8 with ECM was verified by western blot on MP from DBA/1 and C57BL/6 mice. These results demonstrate that MP protein cargo represents a novel ECM pathogenic trait to consider in the understanding of CM pathogenesis.

Plasma levels of endothelial and B-cell-derived microparticles are restored by fingolimod treatment in multiple sclerosis patients

Background: No molecular marker can monitor disease progression and treatment efficacy in multiple sclerosis (MS). Circulating microparticles represent a potential snapshot of disease activity at the blood brain barrier. Objectives and methods: To profile plasma microparticles by flow cytometry in MS and determine how fingolimod could impact endothelial microparticles production. Results: In non-treated MS patients compared to healthy and fingolimod-treated patients, endothelial microparticles were higher, while B-cell-microparticle numbers were lower. Fingolimod dramatically reduced tumour necrosis factor (TNF)-induced endothelial microparticle release in vitro. Conclusion: Fingolimod restored dysregulated endothelial and B-cell-microparticle numbers, which could serve as a biomarker in MS.

DIANNEXIN DOWN-MODULATES TNF-INDUCED ENDOTHELIAL MICROPARTICLE RELEASE BY BLOCKING MEMBRANE BUDDING PROCESS

BACKGROUND Microparticles are now recognised as true biological effectors with a role in immunopathology through their ability to disseminate functional properties. Diannexin, a homodimer of annexin V, binds to PS with a higher affinity and longer blood half-life than the monomer, inhibits prothrombinase complex activity thereby diminishing coagulation and reperfusion injury mediators and prevent microvesicle-mediated material transfer. Our aim was to determine if Diannexin could modulate microparticle production by endothelial cells by interacting with the phosphatidylserine exposure occurring during the release of these vesicles. RESULTS In this study we showed that fluorescently labelled Diannexin binds to calcimycin-activated endothelial cells but not to resting cells. After overnight incubation, Diannexin enters cells and their released MP carry Diannexin. Some Diannexin seems to be processed via early endosomes and later is found in lysosomes. Both unlabelled Diannexin and fluorescent Diannexin inhibit MP release from TNF-activated endothelial cells. However, Diannexin treatment does not prevent endothelial activation by TNF. In addition, the inhibitory effect of Diannexin on MP release could be observed when cells were pre-, concomitantly or post-treated with cytokines. Scanning electron microscopy showed differences in the numbers and types of protuberances at the cell surface when cells were treated or not with Diannexin. Finally, there is no apparent congruency between fluorescent Diannexin labelling and surface protuberances as shown by correlative microscopy. CONCLUSIONS Altogether these data suggest that Diannexin can inhibit endothelial vesiculation by binding PS present either at the cell surface or at the level of the inner leaflet of the plasma membrane.

Divergent roles of β- and γ-actin isoforms during spread of vaccinia virus

Actin is a major component of the cytoskeleton and is present as two isoforms in non‐muscle cells: β‐ and γ‐cytoplasmic actin. These isoforms are strikingly conserved, differing by only four N‐terminal amino acids. During spread from infected cells, vaccinia virus (VACV) particles induce localized actin nucleation that propel virus to surrounding cells and facilitate cell‐to‐cell spread of infection. Here we show that virus‐tipped actin comets are composed of β‐ and γ‐actin. We employed isoform‐specific siRNA knockdown to examine the role of the two isoforms in VACV‐induced actin comets. Despite the high level of similarity between the actin isoforms, and their colocalization, VACV‐induced actin nucleation was dependent exclusively on β‐actin. Knockdown of β‐actin led to a reduction in the release of virus from infected cells, a phenotype dependent on virus‐induced Arp2/3 complex activity. We suggest that local concentrations of actin isoforms may regulate the activity of cellular actin nucleator complexes.

Thrombocytopenia Microcephaly Syndrome - a novel phenotype associated with ACTB mutations

Until recently missense germ-line mutations in ACTB, encoding the ubiquitously expressed β-cytoplasmic actin (CYA), were exclusively associated with Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF), a complex developmental disorder1,2. Here, we report six patients with previously undescribed heterozygous variants clustered in the 3’-coding region of ACTB. These patients present with clinical features different from BWCFF, including thrombocytopenia, microcephaly, and mild developmental disability. Patient derived cells are morphologically and functionally distinct from controls. Assessment of cytoskeletal constituents identified a discrete filament population altered in these cells, which comprises force generating and transmitting actin binding proteins (ABP) known to be associated with thrombocytopenia3–8. In silico modelling and molecular dynamics (MD)-simulations support altered interactions between these ABP and mutant β-CYA. Our results describe a new clinical syndrome associated with ACTB mutations with a distinct genotype-phenotype correlation, identify a cytoskeletal protein interaction network crucial for thrombopoiesis, and provide support for the hypomorphic nature of these actinopathy mutations.