Induction Of Phagocytosis Research Articles

Targeting CD47 and TNFAIP8 By Mir-155 Overcomes Drug Resistance and Inhibits Tumor Growth through Induction of Phagocytosis and Apoptosis in Multiple Myeloma

CD47, an integrin-associated receptor, is highly expressed in multiple myeloma (MM). CD47 on the surface of MM cells interacts with signal regulatory protein-a (SIRPa) on macrophages and activates “don't eat me” signaling pathway inhibiting the phagocytosis of CD47-expressing MM cells by macrophages. We have shown that overexpression of CD47 is associated with shorter progression free and overall survival in patients with MM. However, the molecular mechanisms underlying CD47 overexpression and its role in drug resistance of MM remain undefined.To explore the role of CD47 in drug resistance of MM, we evaluated its expression level in drug resistant MM cells (8226-R5 and MM.1R) and identified that CD47 mRNA and protein were significantly upregulated in drug resistant MM cells compared with their parental lines (8226 and MM.1S, respectively). Analysis of the MM datasets also revealed that CD47 expression was significantly upregulated in relapsed MM compared with newly diagnosed MM and normal donors. Using miRNA target scan algorithm, we identified miR-155 as a regulator of CD47. miR-155 was downregulated in drug resistant MM cells compared with their parental lines and its downregulation was associated with advanced stages of MM disease. In addition, datasets analysis indicated a significant negative correlation between CD47 and miR-155 level in MM patients. We further demonstrated that miR-155 directly targeted CD47-3'UTR using luciferase assay. Moreover, overexpression of miR-155 markedly decreased CD47 protein level in MM resistant cells. FACS analysis of CD47 stained MM cells also revealed that cell surface expression of CD47 was decreased after miR-155 overexpression.Furthermore, we showed that targeting of CD47 by miR-155 abrogated the protection against phagocytosis and promoted the phagocytosis of MM cells by macrophages resulting in inhibition of MM cells growth. To explore whether CD47 overexpression could attenuate the enhancing effect of miR-155 on phagocytosis of MM cells, we performed functional rescue assay by overexpressing CD47 in 8226-R5 and MM.1R cells and co-transfecting with miR-155 mimics or scrambled miRNA. CD47 overexpression abolished the phagocytosis of MM cells by macrophages confirming that miR-155 activated “eat me” signal in MM cells by targeting CD47.On the other hand, we demonstrated that beside promoting phagocytosis, miR-155 overexpression induced apoptosis through targeting of TNFAIP8, a negative mediator of apoptosis, and re-sensitized the resistant MM cells to bortezomib (BTZ). Analysis of the MM patient's pair dataset revealed that TNFAIP8 was significantly upregulated in relapse vs newly diagnosed stages of the disease. We also identified a significant negative correlation between TNFAIP8 and miR-155 expression level in MM patients. Moreover, TNFAIP8 level was remarkably upregulated in drug resistant cells compared to their parental cells. The luciferase assay using wild-type and mutant 3'UTR of TNFAIP8 confirmed that miR-155 directly targeted TNFAIP8. Transfection of MM resistant cells with miR-155 mimics could also reduce the expression level of TNFAIP8 and induce apoptosis in drug resistant MM cells.In order to translate our findings to a therapeutic model, we next stablished a xenograft model and investigated the effect of miR-155 on tumorigenesis of MM resistant cells in vivo. Administration of miR-155 mimics significantly suppressed the tumor growth in combination with BTZ. The miR-155 or BTZ alone moderately prolonged survival compared to control treatment. However, the combination treatment of miR-155 mimics and BTZ significantly extended the overall survival. In addition, TNFAIP8 and CD47 levels were decreased in miR-155 overexpressing groups compared with controls, indicating in vivo targeting of these genes by miR-155. IHC analysis of xenograft tumor sections showed that combination of miR-155 mimics and BTZ treatment resulted in a decrease in the proliferation index (Ki67) and an increase in the apoptotic index (Tunnel), compared to either BTZ or miR-155 mimics alone. Collectively, these results support that regulatory interaction between miR-155 and its targets, CD47 and TNFIP8, provides the rationale that restoration of miR-155 may serve as a promising therapeutic approach by promoting of phagocytosis and inducing of apoptosis at the same time in patients with refractory/relapsed MM. DisclosuresNo relevant conflicts of interest to declare.

Targeted Inhibition of CD47-Sirp Alpha Requires Fc-Fc Gamma Receptor Interactions to Maximize Phagocytosis in T-Cell Lymphomas

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Monoclonal antibodies (mAbs) that inhibit engagement of the “don't eat me” signal CD47 with SIRPα on myeloid cells may induce phagocytosis and have exhibited promise in multiple cancers. We dissected the role of CD47 blockade in opsonization and phagocytosis-induction through Fc-Fc gamma receptor (FcγR) interactions. CD47 was expressed at higher levels on 22 TCL cell lines by flow cytometry compared to quiescent T cells from 10 healthy donors (mean fluorescent intensity, 176.9 versus 69.85; Welch t-test p=0.002). We determined tumor cell-specific protein expression of CD47 by calculation of H scores in a tissue microarray (TMA) of 68 primary TCLs. The common subtypes (PTCL-NOS (n=16), AITL (n=19), and ALCL (n=12)) each had heterogeneous CD47 expression that was not associated with progression-free or overall survival. We utilized B6H12, an anti-CD47 mAb to assess apoptosis, antibody-dependent cell mediated-cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) of TCL cells (HuT-78, HH, Myla, SU-PM2, KIJK, MAC-2A, SMZ1 and K-299) and no increase was observed except for ADCC by murine FcγRIII-expressing engineered Jurkat cells. Upon addition of B6H12 or SRF231, a fully human IgG4 mAb against CD47 that is currently in a Phase I clinical trial (NCT03512340), phagocytosis of CFSE+ TCL cells with human monocyte-derived macrophages (hMDMs) and murine bone-marrow derived macrophages (mBMDMs) was increased. In contrast, induction of phagocytosis of resting T cells from healthy donors was tested using B6H12 and no increase was observed (22.7 and 19.5%). A flow cytometry-based receptor occupancy assay demonstrated a linear correlation between receptor binding and phagocytosis induction in Jurkat T-cell lymphoblastic leukemia cells. hMDMs failed to induce phagocytosis of TCL cells with F(ab')2 portion of B6H12 suggesting complete dependence on Fc receptors. In contrast, mBMDM-mediated phagocytosis of TCL cells with the F(ab')2 portion of B6H12 (mean, 21.5%) was increased compared to negative control but reduced compared to full length (mean, 33.4%), suggesting both Fc-dependent and Fc-independent mechanisms. mBMDMs from FcγR knockout mice demonstrated a statistically significant decrease in phagocytosis (compared to wild-type mBMDMs) of Jurkat, HuT-78, MAC2A and K-299 cells with B6H12, confirming a role for Fc-FcγR engagement. Phagocytosis of tumor cells during CD47 blockade may depend on the ability of SLAMF7 to interact with the integrin Mac-1 (Chen J, Nature 2017). Phagocytosis assays of TCL cells with mBMDMs from Mac-1 knockout mice with full length and F(ab')2 B6H12 demonstrated a reduction in phagocytosis using Mac-1 knockout macrophages (compared to wild-type) only with full-length B6H12. Thus, the Mac-1 contribution to phagocytosis induced by B6H12 in this context is Fc-dependent. We tested SRF231 in vivo using patient-derived xenograft (PDX) models of systemic hepatosplenic (HSTL), transformed cutaneous, and NKTCL. After 10 days, SRF231-treated mice had remarkable reductions in tumor burden compared to untreated mice, across multiple compartments. SRF231 treatment resulted in prolonged survival despite a short course of therapy (Figure 1). Bone marrow from xenografted mice, collected at day 11 post-treatment with SRF231, had significantly increased cytokines known to promote monocyte recruitment. Ex-vivo phagocytosis assays using mBMDMs and hMDMs, enhanced engulfment of HSTL, T-PLL and anaplastic large cell-lymphoma PDX harvested cells upon exposure to SRF231. Finally, we observed that in our HSTL PDX model, depletion of macrophages with clodronate completely abrogated the antitumor efficacy of SRF231 while depletion of neutrophils with anti-Ly6G Ab had no effect, highlighting macrophages as the essential effectors of phagocytosis in this context. In summary, monoclonal antibodies that disrupt CD47-SIRPα have striking in vitro and in vivo preclinical efficacy but require interactions with human FcγR on macrophages to maximize efficacy. [Display omitted] DisclosuresPeluso:Surface Oncology: Employment, Equity Ownership. O'Connor:Surface Oncology: Employment, Equity Ownership. Paterson:Surface Oncology: Employment, Equity Ownership. Holland:Surface Oncology: Employment, Equity Ownership. Weinstock:Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties; Travera: Equity Ownership; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Genentech/Roche, Monsanto: Consultancy; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy.

Phosphatidylinositol-(3,4,5)-Trisphosphate Induces Phagocytosis of Nonmotile Pseudomonas aeruginosa.

Pathogenic bacteria that establish chronic infections in immunocompromised patients frequently undergo adaptation or selection for traits that are advantageous for their growth and survival. Clinical isolates of Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterial pathogen, exhibit a temporal transition from a motile to a nonmotile phenotype through loss of flagellar motility during the course of chronic infection. This progressive loss of motility is associated with increased resistance to both antibiotic and immune clearance. We have previously shown that loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and in vivo and fails to activate the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phagocytic pathway. Therefore, we tested the hypothesis that clearance of phagocytosis-resistant bacteria could be induced by exogenously pretreating innate immune cells with the Akt-activating molecule phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). Here, we demonstrate that PIP3 induces the uptake of nonmotile P. aeruginosa by primary human neutrophils >25-fold, and this effect is phenocopied with the use of murine phagocytes. However, surprisingly, mechanistic studies revealed that the induction of phagocytosis by PIP3 occurs because polyphosphoinositides promote bacterial binding by the phagocytes rather than bypassing the requirement for PI3K. Moreover, this induction was selective since the uptake of other nonmotile Gram-negative, but not Gram-positive, bacteria can also be induced by PIP3 Since there is currently no treatment that effectively eradicates chronic P. aeruginosa infections, these findings provide novel insights into a potential methodology by which to induce clearance of nonmotile pathogenic bacteria and into the endogenous determinants of phagocytic recognition of P. aeruginosa.

Oligosaccharide ligand tuning in design of third generation carbohydrate pneumococcal vaccines

Abstract Streptococcus pneumoniae can cause many types of dangerous infectious diseases such as otitis media, pneumonia, meningitis and others that are more common in the very young and very old age. Available to date commercial vaccines based on capsular polysaccharides of S. pneumoniae of clinically important strains (first generation carbohydrate vaccines) and conjugated vaccines based on these polysaccharides (second generation carbohydrate vaccines) have certain limitations in protective efficiency. However, the efficiency of vaccines can be increased by the use of third generation vaccines based on synthetic oligosaccharide ligands representing in their structures the protective epitopes of capsular polysaccharides. The proper choice of an optimal oligosaccharide ligand is the most important step in the design of third generation carbohydrate vaccines. Herein we overview our works on the synthesis of three oligosaccharides corresponding to one, “one and a half” and two repeating units of S. pneumoniae type 14 capsular polysaccharide, immunogenic conjugates thereof and comparative immunological study of their conjugates with bovine serum albumin, which was used as a model protein carrier. The ability of obtained products to raise antibodies specific to capsular polysaccharide and homologous oligosaccharides, the induction of phagocytosis by immune antisera and active protection of immunized animals from S. pneumoniae type 14 infection were evaluated. On the basis of the results obtained tetrasaccharide comprising the repeating unit of S. pneumoniae type 14 capsular polysaccharide is an optimal carbohydrate ligand to be used as a part of the third generation carbohydrate pneumococcal vaccine.

Signaling pathway for phagocyte priming upon encounter with apoptotic cells

The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin αPS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocyte-specific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signal-transduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells by stimulated phagocytes and is thus considered as a mechanism to prime phagocytes in innate immunity.

Lapatinib induces autophagic cell death and differentiation in acute myeloblastic leukemia.

Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her) superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoblastic leukemia (ALL) cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA) partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of macrophagic differentiation in AML U937 cells by lapatinib. We also noted the synergistic effects of the use of lapatinib and cytotoxic drugs in U937 leukemia cells. These results indicate that lapatinib may have potential for development as a novel antileukemia agent.

The PadR-like transcriptional regulator LftR ensures efficient invasion of Listeria monocytogenes into human host cells.

Invasion of the bacterial pathogen Listeria monocytogenes into human host cells requires specialized surface molecules for attachment and induction of phagocytosis. However, efficient invasion is also dependent on factors with house-keeping functions, such as SecA2-dependent secretion of autolysins for post-divisional segregation of daughter cells. Mutations in this pathway prevent degradation of peptidoglycan cross-walls, so that long cell chains are formed that cannot be phagocytosed. The extreme chaining of such mutants manifests as rough colony phenotype. One rough clone was isolated from a transposon library with a transposon insertion in the uncharacterized lmo0720 gene (lftS) together with a spontaneous point mutation in the secA2 gene. We separated both mutations and demonstrated that this point mutation in the intramolecular regulator 2 domain of SecA2 was sufficient to inactivate the protein. In contrast, lftS deletion did not cause a ΔsecA2-like phenotype. lftS is located in an operon with lftR (lmo0719), encoding a PadR-like transcriptional regulator, and lftR deletion affected growth, invasion and day-light dependent coordination of swarming. Inactivation of lftS partially suppressed these phenotypes, suggesting a functional relationship between LftR and LftS. However, the invasion defect of the ΔlftR mutant was only marginally suppressed by lftS removal. LftR regulates expression of the lmo0979–0980 (lieAB) operon, encoding a putative multidrug resistance transporter and lieAB transcription was strongly upregulated in the absence of LftR. Deletion of lieAB in the ΔlftR background restores wild type-like invasion levels. Hence, we conclude that tight transcriptional repression of the lieAB operon is essential for efficient listerial host cell invasion.

Calreticulin in Myeloproliferative Neoplasms: The Other Side of the Alice Mirror

Calreticulin (CALR), a Ca2+ binding protein mostly localised in the endoplasmic reticulum, regulates Ca2+ homeostasis, chaperones, and other proteins to the nucleus and other cellular compartments. CALR has been implicated in several cellular processes including: signalling, regulation of gene expression, cell adhesion, apoptosis, autoimmunity, and, when expressed on the cell surface, induction of phagocytosis by macrophages. Reports indicating over-expression of CALR in cancer cells suggest that modulation of CALR expression may be exploited to increase their clearance by the immune system. In the hematopoietic system, CALR has been implicated in the activation of the stress pathway as an obligatory partner of the glucocorticoid receptor. More recently, somatic loss-of-function mutations in the CALR gene were discovered in a significant proportion of patients with Philadelphia-negative myeloproliferative neoplasms (MPN) who did not harbour gain-of-function mutations in Janus kinase 2 (JAK2), the first signalling element of cytokine receptors, and myeloproliferative leukaemia virus oncogene (MPL), the thrombopoietin receptor, usually associated with these diseases. This review will summarise current knowledge on the biological activity of CALR and MPL/JAK2 in hematopoiesis, delineate a unifying pathway for the pathogenesis of MPN, and discuss how this pathway may be exploited for therapy.

TLR2 mediates phagocytosis and autophagy through JNK signaling pathway in Staphylococcus aureus-stimulated RAW264.7 cells

Toll-like receptor 2 (TLR2) is involved in phagocytosis and autophagy to enhance host innate immune response to bacterial infection. TLR2 has been reported to participate in the recognition of Staphylococcus aureus (S. aureus). However, the role of TLR2 in phagocytosis and autophagy in S. aureus-stimulated macrophages and the underlying mechanisms as yet remain unclear. In the present study, stimulation of mouse macrophage cell line RAW264.7 with S. aureus activated multiple signaling pathways including mitogen-activated protein kinases (MAPKs), myeloid differentiation factor 88 (MyD88), phosphatidylinositide 3-kinase (PI3K) and Rac1 and triggered autophagy process. Knockdown of TLR2 by siRNA significantly reduced phagocytosis and autophagy of macrophages upon S. aureus infection. Interestingly, TLR2 siRNA markedly attenuated S. aureus-induced phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 or extracellular regulated protein kinase (ERK) in macrophages. Similarly, SP600125, a JNK inhibitor, also down-regulated phagocytosis and autophagy in S. aureus-stimulated macrophages. Furthermore, TLR2 siRNA and SP600125 simultaneous treatment showed similar phagocytosis and autophagy compared to that in TLR2 siRNA treatment alone. Collectively, our results indicate that TLR2 may be critical for phagocytosis and autophagy through JNK signaling pathway, and provide an underlying mechanistic link between innate immune receptor and induction of phagocytosis and autophagy in S. aureus-stimulated macrophages.

Induction of Phagocytosis and Intracellular Signaling by an Inhibitory Channel Catfish Leukocyte Immune-Type Receptor: Evidence for Immunoregulatory Receptor Functional Plasticity in Teleosts

Immunoregulatory receptors are categorized as stimulatory or inhibitory based on their engagement of unique intracellular signaling networks. These proteins also display functional plasticity, which adds versatility to the control of innate immunity. Here we demonstrate that an inhibitory catfish leukocyte immune-type receptor (IpLITR) also displays stimulatory capabilities in a representative myeloid cell model. Previously, the receptor IpLITR 1.1b was shown to inhibit natural killer cell-mediated cytotoxicity. Here we expressed IpLITR 1.1b in rat basophilic leukemia-2H3 cells and monitored intracellular signaling and functional responses. Although IpLITR 1.1b did not stimulate cytokine secretion, activation of this receptor unexpectedly induced phagocytosis as well as extracellular signal-related kinase 1/2- and protein kinase B (Akt)-dependent signal transduction. This novel IpLITR 1.1b-mediated response was independent of an association with the FcRγ chain and was likely due to phosphotyrosine-dependent adaptors associating with prototypical signaling motifs within the distal region of its cytoplasmic tail. Furthermore, compared to a stimulatory IpLITR, IpLITR 1.1b displayed temporal differences in the induction of intracellular signaling, and IpLITR 1.1b-mediated phagocytosis had reduced sensitivity to EDTA and cytochalasin D. Overall, this is the first demonstration of functional plasticity for teleost LITRs, a process likely important for the fine-tuning of conserved innate defenses.

Effects of microparticle size and Fc density on macrophage phagocytosis.

Controlled induction of phagocytosis in macrophages offers the ability to therapeutically regulate the immune system as well as improve delivery of chemicals or biologicals for immune processing. Maximizing particle uptake by macrophages through Fc receptor-mediated phagocytosis could lead to new delivery mechanisms in drug or vaccine development. Fc ligand density and particle size were examined independently and in combination in order to optimize and tune the phagocytosis of opsonized microparticles. We show the internalization efficiency of small polystyrene particles (0.5 µm to 2 µm) is significantly affected by changes in Fc ligand density, while particles greater than 2 µm show little correlation between internalization and Fc density. We found that while macrophages can efficiently phagocytose a large number of smaller particles, the total volume of phagocytosed particles is maximized through the non-specific uptake of larger microparticles. Therefore, larger microparticles may be more efficient at delivering a greater therapeutic payload to macrophages, but smaller opsonized microparticles can deliver bio-active substances to a greater percentage of the macrophage population. This study is the first to treat as independent variables the physical and biological properties of Fc density and microparticle size that initiate macrophage phagocytosis. Defining the physical and biological parameters that affect phagocytosis efficiency will lead to improved methods of microparticle delivery to macrophages.

SLAM is a microbial sensor which regulates bacterial phagosome functions in macrophages by recruiting a Vps34/beclin 1/UVRAG complex (112.8)

Abstract Phagocytosis is a pivotal process by which macrophages eliminate microorganisms upon recognition by pathogen sensors. Surprisingly, the self-ligand cell surface receptor Slamf1 functions not only as a co-stimulatory molecule but also as a microbial sensor controlling killing of Gram-negative bacteria by macrophages. Slamf1 regulates activity of the Nox2 enzyme and phagolysosomal maturation upon entering the phagosome, by recruiting the enzyme Vps34, which produces PI3P. Here we report that in mouse macrophages, Slamf1 interacted with Vps34, Beclin 1 and UVRAG in E. coli-containing phagosomes. However this interaction was also found without induction of phagocytosis in macrophages, transfectant HEK293 cells and human chronic lymphocytic leukemia cells. Removal of the second ITAM motif in the cytoplasmic tail of Slamf1 seemed to enhance the interaction of Slamf1 with Beclin 1, while Slamf1 tailless totally abolished the interaction. Both the BD and CCD domains of Beclin 1 were required for efficient binding to Slamf1. Slamf1 did not interact with Atg14L or Rubicon, which also complex with Vps34 and Beclin 1 in autophagy. We conclude that Slamf1 recruits a subset of Vps34 associated proteins, which is involved in membrane fusions.

Induction of Strain-Transcending Antibodies Against Group A PfEMP1 Surface Antigens from Virulent Malaria Parasites

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (<10 µg/ml of total IgG) against homologous parasites. Furthermore, the antibodies showed broad cross-reactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four sub-Saharan African countries that showed surface reactivity with either DBLα1.5 antibodies (variant HB3var6) or DBLα1.8 antibodies (variant TM284var1). These data show that parasites with a virulence-associated adhesion phenotype share IE surface epitopes that can be targeted by strain-transcending antibodies to PfEMP1. The existence of shared surface epitopes amongst functionally similar disease-associated P. falciparum parasite isolates suggests that development of therapeutic interventions to prevent severe malaria is a realistic goal.

Proliferating culture of aged microglia for the study of neurodegenerative diseases

Microglial cells’ phenotype and function change with aging. Since microglial cell impairments that are relevant for neurodegenerative diseases appear to be unique to aged individuals, it is important to assess function of aged microglia. However, most studies are done with microglia from neonates, mostly due to lack of reliable protocols to obtain microglia from adult animals. Here, we present a conditioned media-dependent culture system that promotes proliferation of adult microglia. We observed that inflammatory activation was increasingly oxidative in microglia from aged animals. Also, whereas phagocytosis of Aβ by microglia from adult animals was more robust than that of microglia from neonates, the induction of phagocytosis by TGFβ was abolished in aged animals. Our results show the importance of using adult animals cells for the study of neurodegenerative processes or other diseases associated with aging. The proposed culture method is inexpensive and cell yield allows for their assessment by functional bioassays and biochemistry.

CD14 and Toll-Like Receptors 2 and 4 Are Required for Fibrillar Aβ-Stimulated Microglial Activation

Microglia are the brain's tissue macrophages and are found in an activated state surrounding beta-amyloid plaques in the Alzheimer's disease brain. Microglia interact with fibrillar beta-amyloid (fAbeta) through an ensemble of surface receptors composed of the alpha(6)beta(1) integrin, CD36, CD47, and the class A scavenger receptor. These receptors act in concert to initiate intracellular signaling cascades and phenotypic activation of these cells. However, it is unclear how engagement of this receptor complex is linked to the induction of an activated microglial phenotype. We report that the response of microglial cells to fibrillar forms of Abeta requires the participation of Toll-like receptors (TLRs) and the coreceptor CD14. The response of microglia to fAbeta is reliant upon CD14, which act together with TLR4 and TLR2 to bind fAbeta and to activate intracellular signaling. We find that cells lacking these receptors could not initiate a Src-Vav-Rac signaling cascade leading to reactive oxygen species production and phagocytosis. The fAbeta-mediated activation of p38 MAPK also required CD14, TLR4, and TLR2. Inhibition of p38 abrogated fAbeta-induced reactive oxygen species production and attenuated the induction of phagocytosis. Microglia lacking CD14, TLR4, and TLR2 showed no induction of phosphorylated IkappaBalpha following fAbeta. These data indicate these innate immune receptors function as members of the microglial fAbeta receptor complex and identify the signaling mechanisms whereby they contribute to microglial activation.

Macrophage fusion leading to foreign body giant cell formation persists under phagocytic stimulation by microspheres in vitro and in vivo in mouse models

The formation of surface-damaging foreign body giant cells (FBGC) from the fusion of macrophages is considered a hallmark of the foreign body response. Experimental evidence indicates that when macrophages are unable to internalize foreign bodies via phagocytosis due to their large size, they acquire a fusogenic phenotype. The mechanism behind this transformation is unclear, and questions, such as which phenotype takes precedence for co-stimulated macrophages engaged in the foreign body response and whether or not such phenotypic alteration is graded, remain unanswered. By recapitulating fusion in vitro using cell lines and primary mouse bone marrow-derived macrophages, we investigated whether concurrent exposure of macrophages to phagocytic and fusogenic stimuli would limit fusion. Induction of phagocytosis by addition of 3.0 mum-diameter polystyrene microspheres to cells under fusogenic conditions, at ratios of 1:10, 1:1, and 10:1 did not prevent fusion. To determine the effect of microsphere phagocytosis on fusion in vivo, we first determined the kinetics of monocyte recruitment, surface adhesion, and fusion following intraperitoneal implantation of a foreign body in a mouse model. Concomitant or subsequent injection of microspheres resulted in their significant accumulation at the biomaterial surface at 2 weeks, but FBGC were still detected. Our findings indicate that despite increasing the abundance of a phagocytic stimulus (microspheres), significant FBGC formation occurs.

Leptin induces phagocytosis of apoptotic bodies by hepatic stellate cells via a Rho guanosine triphosphatase–dependent mechanism

Leptin, a profibrogenic cytokine, plays an important role in the development of non-alcoholic steatohepatitis. Leptin also regulates immune responses, including macrophage phagocytic activity. Stellate cells are key elements in liver fibrogenesis, and previously we have demonstrated that phagocytosis of apoptotic bodies by stellate cells is profibrogenic. To study the effects of leptin on the phagocytic activity of hepatic stellate cells, we exposed both LX-2 cells and primary stellate cells to leptin, and we have observed increased phagocytic activity. In stellate cells isolated from Zucker (fa/fa) rats, the rate of phagocytosis was significantly decreased. To investigate the mechanism by which leptin induces phagocytosis, we focused on the role of Rho-guanosine triphosphate (GTP)-ases. We found that leptin induced the PI3K-dependent activation of Rac1, and that nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase activation was also implicated in this process. Leptin also induced RhoA activation and translocation to the phagosomes. Expression of the constitutive active Rac1 and RhoA both increased the phagocytic rate, whereas inhibition of the Rho-dependent kinase decreased the phagocytic activity. We describe a novel role of leptin in the fibrogenic process, the induction of phagocytosis of apoptotic bodies by hepatic stellate cells. The data provide strong evidence of a Rho-GTPase-mediated regulation of the cytoskeleton during stellate cell phagocytosis. Leptin-mediated phagocytic activity of stellate cells therefore could be an important mechanism responsible for progression of fibrosis in non-alcoholic steatohepatitis.

Differential kinase requirements in human and mouse Fc-gamma receptor phagocytosis and endocytosis

Fc gamma receptors (FcgammaRs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (FcgammaR) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src-related tyrosine kinases (SRTKs) and phosphatidyl inositol 3-kinases (PI3K) play important roles in FcgammaR phagocytosis. Herein, we demonstrate that these kinases are not required for FcgammaR endocytosis. Endocytosis of heat-aggregated IgG (HA-IgG) by COS-1 cells stably transfected with FcgammaRIIA or chimeric FcgammaRI-gamma-gamma (EC-TM-CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG-sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow-derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when FcgammaRIIA was cross-linked using HA-IgG or EA. However, inhibition of FcgammaR phosphorylation prevented only FcgammaR phagocytosis. Finally, biochemical analyses of PI3K(p85)-Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during FcgammaR phagocytosis and endocytosis. Overall, our results indicate that FcgammaR endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition-based model in which PI3K and c-Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.

Mechanisms of Statin-mediated Inhibition of Small G-protein Function

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been reported to reduce the risk of Alzheimer disease. We have shown previously that statins inhibit a beta-amyloid (Abeta)-mediated inflammatory response through mechanisms independent of cholesterol reduction. Specifically, statins exert anti-inflammatory actions through their ability to prevent the isoprenylation of members of the Rho family of small G-proteins, resulting in the functional inactivation of these G-proteins. We report that statin treatment of microglia results in perturbation of the cytoskeleton and morphological changes due to alteration in Rho family function. Statins also block Abeta-stimulated phagocytosis through inhibition of Rac action. Paradoxically, the statin-mediated inactivation of G-protein function was associated with increased GTP loading of Rac and RhoA, and this effect was observed in myeloid lineage cells and other cell types. Statin treatment disrupted the interaction of Rac with its negative regulator the Rho guanine nucleotide dissociation inhibitor (RhoGDI), an interaction that is dependent on protein isoprenylation. We propose that lack of negative regulation accounts for the increased GTP loading. Isoprenylation of Rac is also required for efficient interaction with the plasma membrane, and we report that statin treatment dramatically reduces the capacity of Rac to interact with membranes. These results suggest a mechanism by which statins inhibit the actions of Rho GTPases and attenuate Abeta-stimulated inflammation.

Microglial Phagocytosis of Fibrillar β-Amyloid through a β1Integrin-Dependent Mechanism

Microglia are the principle immune effector and phagocytic cells in the CNS. These cells are associated with fibrillar β-amyloid (fAβ)-containing plaques found in the brains of Alzheimer's disease (AD) patients. The plaque-associated microglia undergo a phenotypic conversion into an activated phenotype and are responsible for the development of a focal inflammatory response that exacerbates and accelerates the disease process. Paradoxically, despite the presence of abundant activated microglia in the brain of AD patients, these cells fail to mount a phagocytic response to Aβ deposits but can efficiently phagocytose Aβ fibrils and plaquesin vitro.We report that exposure of microglia to fAβin vitroinduces phagocytosis through mechanisms distinct from those used by the classical phagocytic receptors, the Ig receptors (FcRγI and FcγRIII) or complement receptors. Microglia interact with fAβ through a recently characterized Aβ cell surface receptor complex comprising the B-class scavenger receptor CD36, α6β1integrin, and CD47 (integrin-associated protein). Antagonists specific for each component of the receptor complex blocks fAβ-stimulated phagocytosis. These data demonstrated that engagement of this ensemble of receptors is required for induction of phagocytosis. The phagocytic response stimulated by this receptor complex is driven principally by a β1integrin-linked process that is morphologically and mechanistically distinct from the classical type I and type II phagocytic mechanisms. These data provide evidence for phagocytic uptake of fAβ through a receptor-mediated, nonclassical phagocytic mechanism.