Human Phagocytic Cells Research Articles

Sac1 links phosphoinositide turnover to cryptococcal virulence.

Cryptococcus neoformans is an environmentally acquired fungal pathogen that causes over 140,000 deaths per year. Cryptococcal infection occurs when infectious particles are deposited into the lung, where they encounter host phagocytic cells. C. neoformans may be engulfed by these phagocytes, an important step of infection that leads to outcomes ranging from termination of infection to cryptococcal dissemination. To study this critical process, we screened approximately 4,700 cryptococcal gene deletion mutants for altered uptake, using primary mouse and human phagocytic cells. Among the hits of these two screens, we identified 93 mutants with perturbed uptake in both systems, as well as others with differences in uptake by only one cell type. We further screened the hits for changes in thickness of the capsule, a protective polysaccharide layer around the cell which is an important cryptococcal virulence factor. The combination of our three screens yielded 45 mutants, including one lacking the phosphatidylinositol-4-phosphate phosphatase Sac1. In this work, we implicate Sac1 in both host cell uptake and capsule production. We found that sac1 mutants exhibit lipid trafficking defects, reductions in secretory system function, and changes in capsule size and composition. Many of these changes occur specifically in tissue culture media, highlighting the role of Sac1 phosphatase activity in responding to the stress of host-like conditions. Overall, these findings show how genome-scale screening can identify cellular factors that contribute to our understanding of cryptococcal biology and demonstrate the role of Sac1 in determining fungal virulence.IMPORTANCECryptococcus neoformans is a fungal pathogen with significant impact on global health. Cryptococcal cells inhaled from the environment are deposited into the lungs, where they first contact the human immune system. The interaction between C. neoformans and host cells is critical because this step of infection can determine whether the fungal cells die or proliferate within the human host. Despite the importance of this stage of infection, we have limited knowledge of cryptococcal factors that influence its outcome. In this study, we identify cryptococcal genes that affect uptake by both human and mouse cells. We also identify mutants with altered capsule, a protective coating that surrounds the cells to shield them from the host immune system. Finally, we characterize the role of one gene, SAC1, in these processes. Overall, this study contributes to our understanding of how C. neoformans interacts with and protects itself from host cells.

Systematic Analysis of Composition, Interfacial Performance and Effects of Pulmonary Surfactant Preparations on Cellular Uptake and Cytotoxicity of Aerosolized Nanomaterials

The interplay of particles with pulmonary surfactant, a lipid‐protein material pivotal for lung function, is hypothesized as a key factor that has not been routinely considered in the current in vitro models when determining the fate of inhaled nanomaterials. To explain its influence on cellular uptake and protective effects, nanoparticles are studied on two models of alveolar cells, in the absence or presence of pulmonary surfactant. Composition and interfacial performance of native human and porcine surfactants, a commercially available bovine surfactant (Alveofact), and an artificial lung lining fluid are characterized using shotgun lipidomics and biophysical approaches (i.e., Langmuir surface balances and captive bubble surfactometry). Plain and aminofunctionalized silica nanoparticles and a novel antimycobacterial nanoformulated benzothiazinone (BTZ043) are selected as examples of neutral, positively charged and therapeutically relevant nanoparticles, respectively. They are deposited onto monocultures of human alveolar epithelial and phagocytic cell lines in the presence or absence of the surfactant preparations, modeling the alveolar milieu. Only surfactant preparations with high interfacial activity and distinctive composition mitigated the toxicity of aerosolized particles, along with a tendency of aerosolized particles to aggregate. Key requirements of surfactant preparations needed when studying interactions of nanomaterials with the pulmonary air‐blood barrier in vitro are identified.

The effects of the dietary compound L-sulforaphane against respiratory pathogens

L-sulforaphane (LSF) is an isothiocyanate derived from cruciferous vegetables that has long been known for its anticarcinogenic, antioxidant and anti-inflammatory effects. LSF also possesses antimicrobial properties, although the evidence for this is limited. Respiratory pathogens, such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes and respiratory syncytial virus (RSV), are leading global causes of illness and death among children aged under five years, particularly in resource-poor countries where access to vaccines are limited or, in the case of S. pyogenes and RSV, vaccines have not been licensed for use in humans. Therefore, alternative strategies to prevent and/or treat these common infectious diseases are urgently needed. This study was conducted to investigate the antimicrobial effects of LSF against common respiratory pathogens, S. pneumoniae (serotypes 1 and 6B), H. influenzae type B (HiB), non-typeable H. influenzae (NTHi), S. pyogenes and RSV in relevant human cell-based models. LSF significantly inhibited the growth of H. influenzae, but not S. pneumoniae or S. pyogenes. LSF did not improve opsonophagocytic capacity or killing by human phagocytic cell lines (HL-60s and THP-1 macrophages) for S. pneumoniae yet showed some improved killing for H. influenzae species in THP-1 macrophages. However, LSF significantly reduced RSV infection in human lung epithelial cells, associated with increased expression of cyclin D1 (CCND1) gene as well as the antioxidant genes, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HMOX-1). Overall, LSF represents an exciting avenue for further antimicrobial research, particularly as a novel therapy against H. influenzae species and RSV.

Coronin-1 is phosphorylated at Thr-412 by protein kinase Cα in human phagocytic cells

Coronin-1, a hematopoietic cell-specific actin-binding protein, is thought to be involved in the phagocytic process through its interaction with actin filaments. The dissociation of coronin-1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin-1 is phosphorylated by protein kinase C (PKC), 2) coronin-1 has two phosphorylation sites, Ser-2 and Thr-412, and 3) Thr-412 of coronin-1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoform is responsible for the phosphorylation of coronin-1 at Thr-412 by using isotype-specific PKC inhibitors and small interfering RNAs (siRNAs). Thr-412 phosphorylation of coronin-1 was suppressed by Gö6976, an inhibitor of PKCα and PKCβI. This phosphorylation was attenuated by siRNA for PKCα, but not by siRNA for PKCβ. Furthermore, Thr-412 of coronin-1 was phosphorylated by recombinant PKCα in vitro, but not by recombinant PKCβ. We next examined the effects of Gö6976 on the intracellular distribution of coronin-1 in HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin-1 was not dissociated from phagosomes in Gö6976-treated cells. These results indicate that phosphorylation of coronin-1 at Thr-412 by PKCα regulates intracellular distribution during phagocytosis.

Contribution of Heptose Metabolites and the cag Pathogenicity Island to the Activation of Monocytes/Macrophages by Helicobacter pylori.

The human gastric pathogen Helicobacter pylori activates human epithelial cells by a particular combination of mechanisms, including NOD1 and ALPK1-TIFA activation. These mechanisms are characterized by a strong participation of the bacterial cag pathogenicity island, which forms a type IV secretion system (CagT4SS) that enables the bacteria to transport proteins and diverse bacterial metabolites, including DNA, glycans, and cell wall components, into human host cells. Building on previous findings, we sought to determine the contribution of lipopolysaccharide inner core heptose metabolites (ADP-heptose) in the activation of human phagocytic cells by H. pylori. Using human monocyte/macrophage-like Thp-1 cells and human primary monocytes and macrophages, we were able to determine that a substantial part of early phagocytic cell activation, including NF-κB activation and IL-8 production, by live H. pylori is triggered by bacterial heptose metabolites. This effect was very pronounced in Thp-1 cells exposed to bacterial purified lysates or pure ADP-heptose, in the absence of other bacterial MAMPs, and was significantly reduced upon TIFA knock-down. Pure ADP-heptose on its own was able to strongly activate Thp-1 cells and human primary monocytes/macrophages. Comprehensive transcriptome analysis of Thp-1 cells co-incubated with live H. pylori or pure ADP-heptose confirmed a signature of ADP-heptose-dependent transcript activation in monocyte/macrophages. Bacterial enzyme-treated lysates (ETL) and pure ADP-heptose–dependent activation differentiated monocytes into macrophages of predominantly M1 type. In Thp-1 cells, the active CagT4SS was less required for the heptose-induced proinflammatory response than in epithelial cells, while active heptose biosynthesis or pure ADP-heptose was required and sufficient for their early innate response and NF-κB activation. The present data suggest that early activation and maturation of incoming and resident phagocytic cells (monocytes, macrophages) in the H. pylori–colonized stomach strongly depend on bacterial LPS inner core heptose metabolites, also with a significant contribution of an active CagT4SS.

Effect of Cytochalasin B on Human Monocyte Binding and Sphering of IgG-coated Human Erythrocytes

The ability of human mononuclear phagocytic cells to bind IgG-coated human erythrocytes (EA) and to cause bound EA to become osmotically fragile (sphered) was investigated in the presence of cytochalasin B, a known inhibitor of phagocytosis. Cytochalasin B inhibited the binding of EA to mononuclear cells in a dose-dependent fashion; 80% inhibition of binding was observed at a concentration of 5 mug/ml. This profound effect on EA binding together with presently available data suggested a role for IgG receptor mobility in the macrophage binding of IgG-coated erythrocytes. Cytochalasin B, however, had a minimal effect on the capacity of mononuclear cells to sphere adherent EA, suggesting that the processes involved in macrophage-induced spherocytosis may differ from those operable in phagocytosis.

Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

Existing antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune response to mycobacterial infection. We used high-throughput CRISPR knockout and CRISPR interference (CRISPRi) screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Guérin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus, high-throughput functional genomic screens, by elucidating highly complex host-pathogen interactions, can serve to identify HDTs to potentially improve TB treatment.

Infection and nuclear interaction in mammalian cells by \u2018Candidatus Berkiella cookevillensis\u2019, a novel bacterium isolated from amoebae

Background‘Candidatus Berkiella cookevillensis’ and ‘Ca. Berkiella aquae’ have previously been described as intranuclear bacteria of amoebae. Both bacteria were isolated from amoebae and were described as appearing within the nuclei of Acanthamoeba polyphaga and ultimately lysing their host cells within 4 days. Both bacteria are Gammaproteobacteria in the order Legionellales with the greatest similarity to Coxiella burnetii. Neither bacterium grows axenically in artificial culture media. In this study, we further characterized ‘Ca. B. cookevillensis’ by demonstrating association with nuclei of human phagocytic and nonphagocytic cell lines.ResultsTransmission electron microscopy (TEM) and confocal microscopy were used to confirm nuclear co-localization of ‘Ca. B. cookevillensis’ in the amoeba host A. polyphaga with 100% of cells having bacteria co-localized with host nuclei by 48 h. TEM and confocal microscopy demonstrated that the bacterium was also observed to be closely associated with nuclei of human U937 and THP-1 differentiated macrophage cell lines and nonphagocytic HeLa human epithelial-like cells. Immunofluorescent staining revealed that the bacteria-containing vacuole invaginates the nuclear membranes and appears to cross from the cytoplasm into the nucleus as an intact vacuole.ConclusionResults of this study indicate that a novel coccoid bacterium isolated from amoebae can infect human cell lines by associating with the host cell nuclei, either by crossing the nuclear membranes or by deeply invaginating the nuclear membranes. When associated with the nuclei, the bacteria appear to be bound within a vacuole and replicate to high numbers by 48 h. We believe this is the first report of such a process involving bacteria and human cell lines.

Engulfment, persistence and fate of Bdellovibrio bacteriovorus predators inside human phagocytic cells informs their future therapeutic potential

In assessing the potential of predatory bacteria, such as Bdellovibrio bacteriovorus, to become live therapeutic agents against bacterial infections, it is crucial to understand and quantify Bdellovibrio host cell interactions at a molecular level. Here, we quantify the interactions of live B. bacteriovorus with human phagocytic cells, determining the uptake mechanisms, persistence, associated cytokine responses and intracellular trafficking of the non-growing B. bacteriovorus in PMA-differentiated U937 cells. B. bacteriovorus are engulfed by U937 cells and persist for 24 h without affecting host cell viability and can be observed microscopically and recovered and cultured post-uptake. The uptake of predators is passive and depends on the dynamics of the host cell cytoskeleton; the engulfed predators are eventually trafficked through the phagolysosomal pathway of degradation. We have also studied the prevalence of B. bacteriovorus specific antibodies in the general human population. Together, these results quantify a period of viable persistence and the ultimate fate of B. bacteriovorus inside phagocytic cells. They provide new knowledge on predator availability inside hosts, plus potential longevity and therefore potential efficacy as a treatment in humans and open up future fields of work testing if predators can prey on host-engulfed pathogenic bacteria.

Hemolysins of <i>Staphylococcus aureus</i>—An Update on Their Biology, Role in Pathogenesis and as Targets for Anti-Virulence Therapy

Staphylococcus aureus is a dangerous gram positive bacterial pathogen which, not only evades the host’s immune system but also can destroy the leucocytes especially neutrophils. It has an embodiment of virulence factors most of which are secreted. Staphylococcus aureus secretes a number of toxins which cause tissue damage and facilitate spreading and nutrients uptake. Among the toxins, hemolysins α, β, γ, δ and Panton Valentine Leukocidin (PVL) are unique that they drill pores in the membrane, leading to the efflux of vital molecules and metabolites. Hemolysins also help in the scavenging of iron, although many of them also have leucolytic properties. α-hemolysin, also known as α-toxin, is the most prominent cytotoxin which damages a wide range of host cells including epithelial cells, endothelial cells, erythrocytes, monocytes, keratinocytes and it damages cell membrane and induces apoptosis. β-Hemolysin significantly affects human immune cell function. It has Mg2+ dependent sphingomyelinase activity and degrades sphingomyelin of plasma membrane into phosphorylcholine and ceramides. The bi-component leukocidins, which include γ-hemolysin and PVL, attack human phagocytic cells and greatly contribute to immune evasion. Delta toxin is a low molecular weight exotoxin with a broad cytolytic activity. Virulence determinants, quorum sensing and biofilm synthesis provide some attractive targets for design and development of a new group of antimicrobial compounds. This review provides an update on the structure, biological functions of hemolysins and their role in quorum sensing/biofilm synthesis (if any) and as effective therapeutic targets for anti-virulence drug development. We have tried to bring together information available on various aspects of hemolysins and highlighted their distribution among all species of Staphylococcus and other bacteria. We have updated the status of development of candidate drugs targeting the hemolysins for anti-virulence therapy as it offers an additional strategy to reduce the severity of infection and which would, through quorum quenching, delay the development biofilms leading to drug resistance.

Human phagocytic cell response to histamine derived from potential probiotic strains of Lactobacillus reuteri

Histamine derived from lactobacilli isolates is considered to be a potential immunomodulator able to interact with the host immune system. We tested the effect of pure histamine (0.413 mM) together with the effect of cell-culture supernatants (CCS) containing different concentration of histamine produced by two of Lactobacillus reuteri isolates on the activities of antioxidant enzyme, as well as on the phagocytic activity of human leucocytes (HL). Phagocytic activity represents the non-specific immune response of HL homogenate, in vitro. Analysed histamine-producers were represented by a goatling isolate named L. reuteri KO5 and a lamb isolate named L. reuteri E and histamine production was determined using HPLC method connected with UV detection. Concretely, the samples contained the mixture of isolated HL and the addition of lactobacilli CCS at three different final concentrations of histamine ∼ 0.1, 1.8 and 5.4 mM. It was found that pure histamine (0.413 mM) did not significantly influence the oxidant-antioxidant balance in HL demonstrated by unchanged degree of HL lipid peroxidation. However, at the same time, the final activity of catalase and superoxide dismutase were significantly changed (p ≤ 0.001).Moreover, the phagocytic index (p ≤ 0.01), lysozyme (p ≤ 0.05) and peroxidase activity (p ≤ 0.001), and production of IL-1β significantly decreased. CCS containing different concentration of produced histamine were also able to modulate the host non-specific immune response together with the enzymatic activity of SOD and catalase too. However, our findings indicated that the impact of lactobacilli histamine is strictly strain-dependent and concentration dependent. Moreover, it seems that histamine is not the only one lactobacilli metabolite, which may play an important role in overall immunomodulatory and antioxidant potential of tested lactobacilli.

Role of the hepcidin-ferroportin axis in pathogen-mediated intracellular iron sequestration in human phagocytic cells

AbstractUpon infection, pathogen and host compete for the same iron pool, because this trace metal is a crucial micronutrient for all living cells. Iron dysregulation in the host is strongly associated with poor outcomes in several infectious diseases, including tuberculosis, AIDS, and malaria, and inefficient iron scavenging by pathogens severely affects their virulence. Hepcidin is the master regulator of iron homeostasis in vertebrates, responsible for diminishing iron export from macrophages during iron overload or infection. Hepcidin regulation in hepatocytes is well characterized and mostly dependent on interleukin-6 signaling during inflammation, although in myeloid cells, hepcidin induction and the mechanisms leading to intracellular iron regulation remain elusive. Here we show that activation of different Toll-like receptors (TLRs) by their respective ligands leads to increased iron sequestration in macrophages. By measuring the transcriptional levels of iron-related proteins (eg, hepcidin, ferroportin, and ferritin), we observed that TLR signaling can induce intracellular iron sequestration in macrophages through 2 independent but redundant mechanisms. Interestingly, TLR2 ligands or infection with Listeria monocytogenes lead to direct ferroportin transcriptional downregulation, whereas TLR4 ligands, such as lipopolysaccharide, induce hepcidin expression. Infection with Mycobacterium bovis Bacillus Calmette-Guerin promotes intracellular iron sequestration through both hepcidin upregulation and ferroportin downregulation. This is the first study in which TLR1-9–mediated iron homeostasis in human macrophages was evaluated, and the outcome of this study elucidates the mechanism of iron dysregulation in macrophages during infection.

Complement Receptor 3-Mediated Inhibition of Inflammasome Priming by Ras GTPase-Activating Protein During Francisella tularensis Phagocytosis by Human Mononuclear Phagocytes.

Francisella tularensis is a remarkably infectious facultative intracellular bacterium of macrophages that causes tularemia. Early evasion of host immune responses contributes to the success of F. tularensis as a pathogen. F. tularensis entry into human monocytes and macrophages is mediated by the major phagocytic receptor, complement receptor 3 (CR3, CD11b/CD18). We recently determined that despite a significant increase in macrophage uptake following C3 opsonization of the virulent Type A F. tularensis spp. tularensis Schu S4, this phagocytic pathway results in limited pro-inflammatory cytokine production. Notably, MAP kinase/ERK activation is suppressed immediately during C3-opsonized Schu S4-CR3 phagocytosis. A mathematical model of CR3-TLR2 crosstalk predicted early involvement of Ras GTPase-activating protein (RasGAP) in immune suppression by CR3. Here, we link CR3-mediated uptake of opsonized Schu S4 by human monocytes and macrophages with inhibition of early signal 1 inflammasome activation, evidenced by limited caspase-1 cleavage and IL-18 release. This inhibition is due to increased RasGAP activity, leading to a reduction in the Ras-ERK signaling cascade upstream of the early inflammasome activation event. Thus, our data uncover a novel signaling pathway mediated by CR3 following engagement of opsonized virulent F. tularensis to limit inflammasome activation in human phagocytic cells, thereby contributing to evasion of the host innate immune system.

Two X-ray methods combine to map molarity of cellular substructures in a freeze-dried sample

X-ray phase nanotomagraphy maps molarity from X-ray fluorescence data to the subcellular structure of freeze-dried human phagocytic cells with nanometer resolution.

Nanoscale quantification of intracellular element concentration by X-ray fluorescence microscopy combined with X-ray phase contrast nanotomography

We present here a correlative X-ray microscopy approach for quantitative single cell imaging of molar concentrations. By combining the elemental content provided by X-ray fluorescence microscopy and the morphology information extracted from X-ray phase nanotomography, we determine the intracellular molarity distributions. This correlative method was demonstrated on a freeze-dried human phagocytic cell to obtain the absolute elemental concentration maps of K, P, and Fe. The cell morphology results showed a very good agreement with atomic-force microscopy measurements. This work opens the way for non-destructive single cell chemical analysis down to the sub-cellular level using exclusively synchrotron radiation techniques. It will be of high interest in the case where it is difficult to access the morphology using atomic-force microscopy, for example, on frozen-hydrated cells or tissues.

Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model

Mycobacterium avium subsp. hominissuis (MAH) is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS)-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro. Cultures of human peripheral blood mononuclear cells and phagocytic human leukemic cell line THP-1 cells were infected with each MAH isolate and intracellular colony-forming units (CFU) were determined. At 2 h after infection, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types. At 5 days, Mav-A organisms, sharing highly related VNTR-MIRU genotypes, yielded numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types. MIRU-VNTR-based minimum spanning tree analysis of the MAH isolates showed a divergent phylogenetic pathway of Mav-A and Mav-B organisms. Mav-A and Mav-B sequevars might have evolved different pathogenetic properties that might account for their association with different human infections.

Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells.

The human pathogen Burkholderia pseudomallei and the related species Burkholderia thailandensis are facultative intracellular bacteria characterized by the ability to escape into the cytosol of the host cell and to stimulate the formation of multinucleated giant cells (MNGCs). MNGC formation is induced via an unknown mechanism by bacterial type VI secretion system 5 (T6SS-5), which is an essential virulence factor in both species. Despite the vital role of the intracellular life cycle in the pathogenesis of the bacteria, the range of host cell types permissive for initiation and completion of the intracellular cycle is poorly defined. In the present study, we used several different types of human primary cells to evaluate bacterial entry, intracellular survival, and MNGC formation. We report the capacity of B. pseudomallei to enter, efficiently replicate in, and mediate MNGC formation of vein endothelial and bronchial epithelial cells, indicating that the T6SS-5 is important in the host-pathogen interaction in these cells. Furthermore, we show that B. pseudomallei invades fibroblasts and keratinocytes and survives inside these cells as well as in monocyte-derived macrophages and neutrophils for at least 17 h postinfection; however, MNGC formation is not induced in these cells. In contrast, infection of mixed neutrophils and RAW264.7 macrophages with B. thailandensis stimulated the formation of heterotypic MNGCs in a T6SS-5-dependent manner. In summary, the ability of the bacteria to enter and survive as well as induce MNGC formation in certain host cells may contribute to the pathogenesis observed in B. pseudomallei infection.

Human neutrophils phagocytose and kill Acinetobacter baumannii and A. pittii

Acinetobacter baumannii is a common cause of health care associated infections worldwide. A. pittii is an opportunistic pathogen also frequently isolated from Acinetobacter infections other than those from A. baumannii. Knowledge of Acinetobacter virulence factors and their role in pathogenesis is scarce. Also, there are no detailed published reports on the interactions between A. pittii and human phagocytic cells. Using confocal laser and scanning electron microscopy, immunofluorescence, and live-cell imaging, our study shows that immediately after bacteria-cell contact, neutrophils rapidly and continuously engulf and kill bacteria during at least 4 hours of infection in vitro. After 3 h of infection, neutrophils start to release neutrophil extracellular traps (NETs) against Acinetobacter. DNA in NETs colocalizes well with human histone H3 and with the specific neutrophil elastase. We have observed that human neutrophils use large filopodia as cellular tentacles to sense local environment but also to detect and retain bacteria during phagocytosis. Furthermore, co-cultivation of neutrophils with human differentiated macrophages before infections shows that human neutrophils, but not macrophages, are key immune cells to control Acinetobacter. Although macrophages were largely activated by both bacterial species, they lack the phagocytic activity demonstrated by neutrophils.

Camouflaging iRGD-EGFR anchored human cytotoxic T-lymphocyte membranes to the surface of nanoparticles combined with low-dose irradiation: New approach to enhance drug-delivery targeting in gastric cancer.

e14076 Background: We report a biomimetic delivery platform based on human cytotoxic T-lymphocyte membranes. In this platform, T-lymphocyte membranes were camouflaged to the surface of poly-lactic-co-glycolic acid nanoparticles, with local low-dose irradiation (LDI) as a chemoattractant. These carriers were further anchored with the recombinant protein anti-EGFR-iRGD, improving tumor accumulation, facilitating tumor uptake. Methods: The T-lymphocyte membrane coating was verified by dynamic light scattering, transmission electron microscopy and confocal laser scanning microscopy. The particle phagocytosis study was performed using a human phagocytic cell line. In vivo NIR fluorescence imaging was performed to monitor the route of nanoparticles. EGFR expression of tumor cells was tested before and after LDI. Results: This new platform reduced phagocytosis of macrophages by 23.99% (p = 0.002). iRGD-EGFR anchored T-lymphocyte membrane-encapsulated nanoparticles accumulated in tumor site more than unfunctionalized groups, while LDI significantly enhanced the targeting ability. LDI could up-regulate EGFR expression on tumor cells, which was important in the process of localization of iRGD-EGFR anchored T-lymphocyte membrane-encapsulated nanoparticles in tumors. Conclusions: This new platform included both the long circulation time and tumor sites accumulation ability while LDI could significantly enhance the tumor accumulation ability.

The phagosome: Meeting point of the Myddosome, NLRs, and degraded Borrelia burgdorferi

Abstract Phagocytosis of the extracellular bacterium Borrelia burgdorferi (Bb) enhances toll-like receptor, NF-κB mediated cytokine responses in human and murine phagocytic cells. TLR adaptor molecule MyD88 is recruited to Bb containing phagosomes, and the spirochetal cargo does not appear to leak from the phagosome into the cytosol. Transcriptome and Network reconstruction analysis identified several master regulators of diverse cellular and molecular processes, including regulation of the TLR-MyD88 pathway, IRFs, cytoskeletal rearrangement, and resolution of inflammation. Surprisingly, genes that code for inflammasome proteins (NLRP3, NOD2 and caspase-1) were also differentially-expressed. We thereby examined the cellular localization of these molecules in Bb-infected WT BMDMs and observed that NLRP3 and NOD2 were observed to colocalize to phagosomes containing degraded spirochetes. NLRP3 also colocalized with MyD88, but only in phagosomes containing degraded spirochetal cargo. Although caspase-1 also colocalized to the phagosome, infection with Bb did not result in an increase in speck formation or secretion of IL-1β. Our results show that the phagosome is a crucial cellular location not only for degradation of the spirochete, but where components of the Myddosome and inflammasome are recruited. Our findings are in line with recent studies showing that cytosolic receptors can localize to endosomal membranes, and can potentially have an alternate role in regulating an adequate functionality of the phagosome. Our study evidences the complexity of phagosomal signaling in which MyD88 plays a critical role not only in the generation of inflammatory but inducing regulatory signals in murine macrophages upon Bb infection.