Serum Opsonins Research Articles

Peptidoglycan from Bacillus anthracis Inhibits Human Macrophage Efferocytosis in Part by Reducing Cell Surface Expression of MERTK and TIM-3.

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVβ5, CD36, and TIM-3, whereas TIM-1, αVβ3, CD300b, CD300f, STABILIN-1, and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant, suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.

Targeting Ligand Independent Tropism of siRNA-LNP by Small Molecules for Directed Therapy of Liver or Myeloid Immune Cells.

Hepatic clearance of lipid nanoparticles (LNP) with encapsulated nucleic acids restricts their therapeutic applicability. Therefore, tools for regulating hepatic clearance are of high interest for nucleic acid delivery. To this end, this work employs wild-type (WT) and low-density lipoprotein receptor (Ldlr)-/- mice pretreated with either a leukotriene B4 receptor inhibitor (BLT1i) or a high-density lipoprotein receptor inhibitor (HDLRi) prior to the injection of siRNA-LNP. This work is able to demonstrate significantly increased hepatic uptake of siRNA-LNP by the BLT1i in Ldlr-/- mice by in vivo imaging and discover an induction of specific uptake-related proteins. Irrespective of the inhibitors and Ldlr deficiency, the siRNA-LNP induced RNA-binding and transport-related proteins in liver, including haptoglobin (HP) that is also identified as most upregulated serum protein. This work observes a downregulation of proteins functioning in hepatic detoxification and of serum opsonins. Most strikingly, the HDLRi reduces hepatic uptake and increases siRNA accumulation in spleen and myeloid immune cells of blood and liver. RNA sequencing demonstrates leukocyte recruitment by the siRNA-LNP and the HDLRi through induction of chemokine ligands in liver tissue. The data provide insights into key mechanisms of siRNA-LNP biodistribution and indicate that the HDLRi has potential for extrahepatic and leukocyte targeting.

Shape and Phase Transitions in a PEGylated Phospholipid System.

Poly(ethylene glycol) (PEG) polymers and PEG-conjugated lipids are widely used in bioengineering and drug transport applications. A PEG layer in a drug carrier increases hydrophilic repulsion, inhibits membrane fusion and serum opsonin interactions, and prolongs the storage and circulation time. It can also change the carrier shape and have an influence on many properties related to the content release of the carrier. In this paper, we focus on the physicochemical effects of PEGylation in the lipid bilayer. We introduce laurdanC as a fluorophore for shape recognition and phase transition detection. Together with laurdanC, cryogenic transmission electron microscopy, differential scanning calorimetry, molecular dynamics simulations, and small-angle X-ray scattering/wide-angle X-ray scattering, we acquire information of the particle/bilayer morphology and phase behavior in systems containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG(2000) with different fractions. We find that PEGylation leads to two important and potentially usable features of the system. (1) Spherical vesicles present a window of elevated chain-melting temperatures and (2) lipid packing shape-controlled liposome-to-bicelle transition. The first finding is significant for targets requiring multiple release sequences and the second enables tuning the release by composition and the PEG polymer length. Besides drug delivery systems, the findings can be used in other smart soft materials with trigger-polymers as well.

Kingella kingae Surface Polysaccharides Promote Resistance to Human Serum and Virulence in a Juvenile Rat Model.

Kingella kingae is a Gram-negative coccobacillus that is increasingly being recognized as an important cause of invasive disease in young children. The pathogenesis of K. kingae disease begins with colonization of the oropharynx, followed by invasion of the bloodstream, survival in the intravascular space, and dissemination to distant sites. Recent studies have revealed that K. kingae produces a number of surface factors that may contribute to the pathogenic process, including a polysaccharide capsule and an exopolysaccharide. In this study, we observed that K. kingae was highly resistant to the bactericidal effects of human serum complement. Using mutant strains deficient in expression of capsule, exopolysaccharide, or both in assays with human serum, we found that elimination of both capsule and exopolysaccharide was required for efficient binding of IgG, IgM, C4b, and C3b to the bacterial surface and for complement-mediated killing. Abrogation of the classical complement pathway using EGTA-treated human serum restored survival to wild-type levels by the mutant lacking both capsule and exopolysaccharide, demonstrating that capsule and exopolysaccharide promote resistance to the classical complement pathway. Consistent with these results, loss of both capsule and exopolysaccharide eliminated invasive disease in juvenile rats with an intact complement system but not in rats lacking complement. Based on these observations, we conclude that the capsule and the exopolysaccharide have important redundant roles in promoting survival of K. kingae in human serum. Each of these surface factors is sufficient by itself to fully prevent serum opsonin deposition and complement-mediated killing of K. kingae, ultimately facilitating intravascular survival and promoting K. kingae invasive disease.

Complement C3 as a Prompt for Human Macrophage Death during Infection with Francisella tularensis Strain SCHU S4.

Tularemia is caused by the Gram-negative bacterial pathogen Francisella tularensis Infection of macrophages and their subsequent death are believed to play important roles in the progression of disease. Because complement is a particularly effective opsonin for Francisella, we asked whether complement-dependent uptake of F. tularensis strain SCHU S4 affects the survival of primary human macrophages during infection. Complement component C3 was found to be an essential opsonin in human serum not only for greatly increased uptake of SCHU S4 but also for the induction of macrophage death. Single-cell analysis also revealed that macrophage death did not require a high intracellular bacterial burden. In the presence of C3, macrophage death was observed at 24 h postinfection in a quarter of the macrophages that contained only 1 to 5 bacterial cells. Macrophages infected in the absence of C3 rarely underwent cell death, even when they contained large numbers of bacteria. The need for C3, but not extensive replication of the pathogen, was confirmed by infections with SCHU S4 ΔpurMCD, a mutant capable of phagosome escape but of only limited cytosolic replication. C3-dependent Francisella uptake alone was insufficient to induce macrophage death, as evidenced by the failure of the phagosome escape-deficient mutant SCHU S4 ΔfevR to induce cell death despite opsonization with C3. Together, these findings indicate that recognition of C3-opsonized F. tularensis, but not extensive cytosolic replication, plays an important role in regulating macrophage viability during intracellular infections with type A F. tularensis.

Hemocytes and hemocytic responses in the mole crab Emerita emeritus (Linnaeus 1767).

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).

Assessment of the cytotoxic and genotoxic effects of TiO2 nanoparticles in A549 cells

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).

Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer

Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes. Over the past decades, the molecular and cell biological events associated with efferocytosis have been rigorously studied, and many eat-me signals and receptors have been identified. The externalization of phosphatidylserine (PS) is arguably the most emblematic eat-me signal that is in turn bound by a large number of serum proteins and opsonins that facilitate efferocytosis. Under physiological conditions, externalized PS functions as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. Pathologically, the innate immunosuppressive effect of externalized PS has been hijacked by numerous viruses, microorganisms, and parasites to facilitate infection, and in many cases, establish infection latency. PS is also profoundly dysregulated in the tumor microenvironment and antagonizes the development of tumor immunity. In this review, we discuss the biology of PS with respect to its role as a global immunosuppressive signal and how PS is exploited to drive diverse pathological processes such as infection and cancer. Finally, we outline the rationale that agents targeting PS could have significant value in cancer and infectious disease therapeutics.

Exploring zwitterionic siRNA polyplex coronas as an alternative to traditional PEGylated architectures for improved intravenous pharmacokinetics

Event Abstract Back to Event Exploring zwitterionic siRNA polyplex coronas as an alternative to traditional PEGylated architectures for improved intravenous pharmacokinetics Meredith Jackson1, Thomas A. Werfel1, Zoe E. Johnson1, Todd D. Giorgio1 and Craig L. Duvall1 1 Vanderbilt University, Biomedical Engineering, United States Introduction: Despite the use of PEGylation to improve in vivo stability of traditional polymeric siRNA delivery vehicles, circulation half-lives remain short (~20 min) [1]. Serum opsonins and complement proteins adsorb onto these particles, decreasing stability and increasing recognition and uptake by the mononuclear phagocyte system (MPS)[2]-[4]. To improve intravenous polyplex delivery of RNAi therapeutics, it is imperative to design nanocarriers with improved resistance to the challenges of a complex protein environment. The current work compares zwitterionic corona chemistry to traditional linear and branched PEG-based architectures for predictors of best in vivo function. To our knowledge, this work describes and provides initial characterization of the first endosomolytic zwitterion-based polymeric siRNA delivery system. Materials and Methods: Poly[(ethylene glycol)-b-[(2-dimethylamino)ethyl methacrylate) (DMAEMA)-co-(butyl methacrylate) (BMA)] (PEG-DB), poly[(ethyl ether methacrylate) (POEGMA)-b-p(DMAEMA-co-BMA)] (POEGMA-DB), and poly(methacryloyloxyethylphosphorylcholine (PMPC)-b-p(DMAEMA-co-BMA)] (PMPC-DB), were RAFT polymerized with an optimized 50:50 ratio of DMAEMA and BMA[5]. Polymers were characterized with GPC and 1H-NMR. Polyplexes formulated from these polymers and siRNA were characterized with DLS and TEM. These polyplexes were assessed for uptake and in vitro knockdown of the model gene luciferase in breast cancer cells (MDA-MB-231), as well as for endosomal escape function and cytotoxicity. Finally, isothermal titration calorimetry was used to probe adsorption of bovine serum albumin. Results and Discussion: Overall, zwitterionic PMPC-DB notably outperformed PEG-DB and POEGMA-DB in terms of increased cell uptake (Fig 1C) and reduced protein adsorption (Fig 2). Each polymer formed siRNA polyplexes of similar size (~150 nm) and had neutral zeta potential with low polydispersities. All polyplexes exhibit similar knockdown of the model gene luciferase (Fig 1B) and equally low levels of toxicity. Based on isothermal titration calorimetry (Fig 2), zwitterionic PMPC-DB was least favorable to protein adsorption compared to PEG-DB, POEGMA-DB, and a control formulation with a purely cationic DMAEMA corona (D-DPB), as evidenced by its large change in Gibbs free energy (ΔG). However, PMPC-DB had the greatest increase in enthalpy, suggesting that more energy is required to displace the hydration shell of zwitterionic polyplex coatings than for traditional PEG coatings. While the displacement of cage-like hydration shells resulted in favorable entropy changes for PMPC-DB, this effect was outweighed by the enthalpy required to displace them, resulting in higher ΔG values. Conclusion: In conclusion, novel zwitterionic siRNA polyplex coronas show promise for improved in vivo pharmacokinetics relative to conventional PEG based on increased uptake and reduced adsorption of serum proteins. This is a significant finding as there is a known direct correlation between level of opsonization and rapidity of hepatic uptake of IV-injected nanocarriers [4]. In vivo pharmacokinetic effects of the PMPC corona will be compared to PEG in ongoing studies. This work was supported in part by National Science Foundation Graduate Research Fellowship Grant No. 1445197.; We would like to thank members of the Duvall and Giorgio labs for scientific discussions and support regarding this work.; We would like to thank Dr. Joel Tellinghuisen for his guidance regarding isothermal titration calorimetry methods.; This work was supported in part by the NIH through the grant 1R01EB019409.

Shielding Therapeutic Drug Carriers from the Mononuclear Phagocyte System: A Review.

The mononuclear phagocyte system (MPS) defends the body against the invasion of microorganisms by phagocytosis. In the presence of opsonins, the invading matter is readily recognized by phagocytes because of the interaction between receptors on the phagocytic cell surfaces and the modified conformation of opsonins. The particulate carriers, which are otherwise capable of optimizing drug delivery, are subjected to opsonization and phagocytosis by the MPS immediately following intravenous administration. These drug carriers should remain in the bloodstream in order to spatially locate the drug to the target site and temporally control the drug's release from there on; however, they are devastated by opsonization by serum proteins. Therefore, to restrict opsonization, which is critical for recognition of particulate carriers by the MPS, stealth devices have been developed by engineering the carriers' surface characteristics. Physicochemical properties that influence protein immunogenicity include particle size, surface charge, and surface hydrophobicity. Steric stabilization using polyethylene glycol (PEG) and polyethylene oxide (PEO) chains attached to the particle surface is principally effective in preventing the adsorption of serum opsonins. This article reviews the literature on the MPS and its development and functions, as well as approaches for designing long-circulating carrier particles. It also comprehensively reviews parameters affecting the steric characteristics of drug carriers, such as particle size, shape, surface charge, and surface affinity, including PEGylation of carriers.

Serum opsonin ficolin-A enhances host-fungal interactions and modulates cytokine expression from human monocyte-derived macrophages and neutrophils following Aspergillus fumigatus challenge.

Invasive aspergillosis is a devastating invasive fungal disease associated with a high mortality rate in the immunocompromised, such as leukaemia patients, transplant patients and those with HIV/AIDS. The rodent serum orthologue of human L-ficolin, ficolin-A, can bind to and opsonize Aspergillus fumigatus, the pathogen that causes invasive aspergillosis, and may participate in fungal defence. Using human monocyte-derived macrophages and neutrophils isolated from healthy donors, we investigated conidial association and fungal viability by flow cytometry and microscopy. Additionally, cytokine production was measured via cytometric bead arrays. Ficolin-A opsonization was observed to significantly enhance association of conidia, while also inhibiting hyphal growth and contributing to increased fungal killing following incubation with monocyte-derived macrophages and neutrophils. Additionally, ficolin-A opsonization was capable of manifesting a decrease in IL-8, IL-1β, IL-6, IL-10 and TNF-α production from MDM and IL-1β, IL-6 and TNF-α from neutrophils 24h post-infection. In conclusion, rodent ficolin-A is functionally comparable to human L-ficolin and is capable of modulating the innate immune response to A. fumigatus, down-regulating cytokine production and could play an important role in airway immunity.

Susceptibility of Vibrio aestuarianus 01/032 to the antibacterial activity of Mytilus haemolymph: identification of a serum opsonin involved in mannose-sensitive interactions.

The interactions of Vibrio aestuarianus 01/032 with haemolymph of the bivalves Mytilus galloprovincialis and Crassostrea gigas were investigated to understand if haemolymph components (haemocytes and soluble factors) could be involved in the higher resistance to microbial infection shown by mussels in comparison with oysters. Although 01/032 bacteria adhered to haemocytes of both bivalves, they were sensitive to the bactericidal activity of whole haemolymph from mussel, but not from oyster; in addition, adhesion to mussel (but not oyster) haemocytes was affected by D-mannose. Mussel serum opsonins directed towards D-mannose-binding bacterial ligands were purified by affinity chromatography and were shown to mediate 01/032 interactions with M. galloprovincialis haemocytes. Nano-High Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (HPLC-ESI-MS/MS) analysis showed that the purified opsonin matched the protein precursor of Mytilus edulis extrapallial protein (EP). In the presence of M. galloprovincialis EP protein (MgEP), C. gigas haemocytes killed V. aestuarianus 01/032 almost as efficiently as mussel phagocytes. These findings suggest that the different sensitivity of 01/032 strain to the antibacterial activity of oyster and mussel haemolymph might partly depend on the fact that C. gigas serum lacks MgEP-like opsonins. These results represent the basis for understanding the different sensitivity to microbial infections shown by the two bivalve species.

Thermal acclimation in the perch (Perca fluviatilis L.) immunity

Fish immune systems must be able to cope with pathogens over a wide temperature range. Earlier research suggest that fish are more dependent on innate immune responses based on pattern recognition than acquired functions with specific recognition. If this applies to phagocytes, then opsonins (serum factors that augment phagocytosis e.g. immunoglobulins and complement proteins) attached on zymosan (Z) particles should be recognized better at higher temperatures than Z only. Z is recognized by glucan receptor representing pattern recognition. In this study perch were acclimated to 5°C or 16°C for 3–5 weeks. The recognition and activation of respiratory burst reaction of peripheral blood phagocytes was examined at seven different measurement temperatures (5, 10, 16, 20, 24 27, and 30°C) when the cells were stimulated with Z and serum opsonized zymosan (OZ). Respiratory burst was measured as luminol chemiluminescence (CL) from diluted whole blood. OZ-induced CL per volume of blood was on average approximately 4.6 times higher in 16°C acclimated fish than 5°C acclimated perch (P<0.0001). Z-induced CL was approximately 3 times higher at lower temperatures in 16°C acclimated perch than 5°C acclimated fish and 6–9 times higher at 27°C and 30°C (P<0.001), respectively. CL reaction kinetics were faster in perch acclimated to 5°C than 16°C -acclimated fish, especially at low temperatures (P<0.001). Thermal acclimation caused a 3–4°C shift in temperature response curves of CL towards the acclimation temperature (P<0.0001 and P<0.053 in Z and OZ-induced CL, respectively).Serum opsonins activated perch phagocytes substantially better at higher temperatures in both acclimation groups, which is consistent with an earlier study in rainbow trout (O. mykiss). However, opsonin recognition was significantly better in 16°C acclimated perch than 5°C acclimated fish, which was seen as higher CLs for OZ compared to Z, especially at higher temperatures. This is opposite to previously reported results in rainbow trout.Differences between rainbow trout and perch in opsonin recognition by blood phagocytes suggest that the living habits of perch, which prefers approximately a 10°C higher temperature than rainbow trout, may be reflected in immune cell functions. Results of the present examination suggest that also in fish phagocytes pattern recognition is the prevailing system at low temperatures, and specific recognition is more effective at high temperatures.

The serum opsonin L-ficolin is detected in lungs of human transplant recipients following fungal infections and modulates inflammation and killing of Aspergillus fumigatus.

Invasive aspergillosis (IA) is a life-threatening systemic fungal infection in immunocompromised individuals that is caused by Aspergillus fumigatus. The human serum opsonin, L-ficolin, has been observed to recognize A. fumigatus and could participate in fungal defense. Using lung epithelial cells, primary human monocyte-derived macrophages (MDMs), and neutrophils from healthy donors, we assessed phagocytosis and killing of L-ficolin-opsonized live A. fumigatus conidia by flow cytometry and microscopy. Additionally, cytokines were measured by cytometric bead array, and L-ficolin was measured in bronchoalveolar lavage (BAL) fluid from lung transplant recipients by enzyme-linked immunosorbent assay. L-ficolin opsonization increased conidial uptake and enhanced killing of A. fumigatus by MDMs and neutrophils. Opsonization was also shown to manifest an increase in interleukin 8 release from A549 lung epithelial cells but decreased interleukin 1β, interleukin 6, interleukin 8, interleukin 10, and tumor necrosis factor α release from MDMs and neutrophils 24 hours after infection. The concentration of L-ficolin in BAL fluid from patients with fungal infection was significantly higher than that for control subjects (P = .00087), and receiving operating characteristic curve analysis highlighted the diagnostic potential of L-ficolin for lung infection (area under the curve, 0.842; P < .0001). L-ficolin modulates the immune response to A. fumigatus. Additionally, for the first time, L-ficolin has been demonstrated to be present in human lungs.

Sea bass Dicentrarchus labrax (L.) bacterial infection and confinement stress acts on F-type lectin (DlFBL) serum modulation.

The F-lectin, a fucose-binding protein found from invertebrates to ectothermic vertebrates, is the last lectin family to be discovered. Here, we describe effects of two different types of stressors, bacterial infection and confinement stress, on the modulation of European sea bass Dicentrarchus labrax (L.) F-lectin (DlFBL), a well-characterized serum opsonin, using a specific antibody. The infection of the Vibrio alginolyticus bacterial strain increased the total haemagglutinating activity during the 16-day testing period. The DlFBL value showed an upward regulation on the first, second and last days and underwent a slight downward regulation 4days post-challenge. In contrast, the effect of confinement and density stress showed a decrease in the plasma concentration of lectin, ranging from 50% to 60% compared with the control. The modulation of DlFBL is in line with the hypothesis that humoral lectins could be involved and recruited in the initial recognition step of the inflammation, which leads to agglutination, and the activation of mechanisms responsible for killing of the pathogens.

Delineating the importance of serum opsonins and the bacterial capsule in affecting the uptake and killing of Burkholderia pseudomallei by murine neutrophils and macrophages.

Infection of susceptible hosts by the encapsulated Gram-negative bacterium Burkholderia pseudomallei (Bp) causes melioidosis, with septic patients attaining mortality rates ≥40%. Due to its high infectivity through inhalation and limited effective therapies, Bp is considered a potential bioweapon. Thus, there is great interest in identifying immune effectors that effectively kill Bp. Our goal is to compare the relative abilities of murine macrophages and neutrophils to clear Bp, as well as determine the importance of serum opsonins and bacterial capsule. Our findings indicate that murine macrophages and neutrophils are inherently unable to clear either unopsonized Bp or the relatively-avirulent acapsular bacterium B. thailandensis (Bt). Opsonization of Bp and Bt with complement or pathogen-specific antibodies increases macrophage-uptake, but does not promote clearance, although antibody-binding enhances complement deposition. In contrast, complement opsonization of Bp and Bt causes enhanced uptake and killing by neutrophils, which is linked with rapid ROS induction against bacteria exhibiting a threshold level of complement deposition. Addition of bacteria-specific antibodies enhances complement deposition, but antibody-binding alone cannot elicit neutrophil clearance. Bp capsule provides some resistance to complement deposition, but is not anti-phagocytic or protective against reactive oxygen species (ROS)-killing. Macrophages were observed to efficiently clear Bp only after pre-activation with IFNγ, which is independent of serum- and/or antibody-opsonization. These studies indicate that antibody-enhanced complement activation is sufficient for neutrophil-clearance of Bp, whereas macrophages are ineffective at clearing serum-opsonized Bp unless pre-activated with IFNγ. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis.

Phagocytosis via Complement or Fc-Gamma Receptors Is Compromised in Monocytes from Type 2 Diabetes Patients with Chronic Hyperglycemia

Type 2 diabetes patients (DM2) have a higher risk of tuberculosis (TB) that may be attributed to functional defects in their mononuclear phagocytes given the critical role of these cells in Mycobacterium tuberculosis containment. Our previous findings suggest that monocytes from DM2 have reduced association with serum-opsonized M. tuberculosis. To determine if this alteration is due to defects in phagocytosis via complement or Fc-gamma receptors (FcγRs), in this study we evaluated the uptake of sheep red blood cells coated with IgG or complement, respectively, by monocytes from individuals with and without DM2. We found that chronic hyperglycemia was significantly associated with reduced phagocytosis via either receptor by univariable and multivariable analyses. This defect was independent of host serum opsonins and flow cytometry data indicated this was not attributed to reduced expression of these phagocytic receptors on DM2 monocytes. The positive correlation between both pathways (R = 0.64; p = 0.003) indicate that monocytes from individuals with chronic hyperglycemia have a defect in the two predominant phagocytic pathways of these cells. Given that phagocytosis is linked to activation of effector mechanisms for bacterial killing, it is likely that this defect is one factor contributing to the higher susceptibility of DM2 patients to pathogens like M. tuberculosis.

AB0208 Immune complex formation after exposure of autoantigens on the surface of secondary necrotic cells (SNEC) promotes inflammation in SLE

BackgroundSystemic Lupus Erythematosus (SLE) is a chronic inflammatory disease characterised by the production of autoantibodies, formation of immune complexes (IC), and activation of complement.[1] Accumulation in blood and tissues of...

Immune complex formation after exposure of autoantigens on the surface of secondary necrotic cells (SNEC) promotes inflammation in SLE

Background and objectivesSystemic lupus erythematosus (SLE) is a chronic inflammatory disease characterised by the production of autoantibodies, formation of immune complexes (IC), and activation of complement. Accumulation in blood and...

Pentraxins (CRP, SAP) in the process of complement activation and clearance of apoptotic bodies through Fcγ receptors

Ischemia/reperfusion injury and organ allograft rejection both entail excessive cell and tissue destruction. A number of innate immune proteins, including the pentraxins, participate in the removal of this potentially inflammatory and autoimmunogenic material. The classical pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP) are serum opsonins, which bind to damaged membranes and nuclear autoantigens. Understanding the role of pentraxins in inflammation has been advanced by the recent identification and structural analysis of their receptor interactions. The ligand-binding, complement-activating and opsonic properties of pentraxins have been known for some time. However, the establishment of Fcγ receptors as the primary receptors for pentraxins is a recent finding with important implications for CRP and SAP functions. The crystal structure of SAP in complex with FcγRIIa was recently solved, leading to new insights into function and new opportunities for pentraxin-based therapeutics. In addition, new approaches to inhibit CRP synthesis or binding are being developed based on clinical associations between CRP levels and cardiovascular disease. This review will summarize data on the function of pentraxins in clearance of injured tissue and cells and discuss the implications of this clearance pathway for autoimmunity and ischemia/reperfusion injury.