Human Properdin Opsonizes Nanoparticles and Triggers a Potent Pro-inflammatory Response by Macrophages without Involving Complement Activation.

Development of nanoparticles as tissue-specific drug delivery platforms can be considerably influenced by the complement system because of their inherent pro-inflammatory and tumorigenic consequences. The complement activation pathways, and its recognition subcomponents, can modulate clearance of the nanoparticles and subsequent inflammatory response and thus alter the intended translational applications. Here, we report, for the first time, that human properdin, an upregulator of the complement alternative pathway, can opsonize functionalized carbon nanotubes (CNTs) via its thrombospondin type I repeat (TSR) 4 and 5. Binding of properdin and TSR4+5 is likely to involve charge pattern/polarity recognition of the CNT surface since both carboxymethyl cellulose-coated carbon nanotubes (CMC-CNT) and oxidized (Ox-CNT) bound these proteins well. Properdin enhanced the uptake of CMC-CNTs by a macrophage cell line, THP-1, mounting a robust pro-inflammatory immune response, as revealed by qRT-PCR, multiplex cytokine array, and NF-κB nuclear translocation analyses. Properdin can be locally synthesized by immune cells in an inflammatory microenvironment, and thus, its interaction with nanoparticles is of considerable importance. In addition, recombinant TSR4+5 coated on the CMC-CNTs inhibited complement consumption by CMC-CNTs, suggesting that nanoparticle decoration with TSR4+5, can be potentially used as a complement inhibitor in a number of pathological contexts arising due to exaggerated complement activation.

Pulmonary surfactant protein SP-D opsonises carbon nanotubes and augments their phagocytosis and subsequent pro-inflammatory immune response.

Carbon nanotubes (CNTs) are increasingly being developed for use in biomedical applications, including drug delivery. One of the most promising applications under evaluation is in treating pulmonary diseases such as tuberculosis. Once inhaled or administered, the nanoparticles are likely to be recognised by innate immune molecules in the lungs such as hydrophilic pulmonary surfactant proteins. Here, we set out to examine the interaction between surfactant protein D (SP-D), a key lung pattern recognition molecule and CNTs, and possible downstream effects on the immune response via macrophages. We show here that a recombinant form of human SP-D (rhSP-D) bound to oxidised and carboxymethyl cellulose (CMC) coated CNTs via its C-type lectin domain and enhanced phagocytosis by U937 and THP-1 macrophages/monocytic cell lines, together with an increased pro-inflammatory response, suggesting that sequestration of SP-D by CNTs in the lungs can trigger an unwanted and damaging immune response. We also observed that functionalised CNTs, opsonised with rhSP-D, continued to activate complement via the classical pathway, suggesting that C1q, which is the recognition sub-component of the classical pathway, and SP-D have distinct pattern recognition sites on the CNTs. Consistent with our earlier reports, complement deposition on the rhSP-D opsonised CNTs led to dampening of the pro-inflammatory immune response by THP-1 macrophages, as evident from qPCR, cytokine array and NF-κB nuclear translocation analyses. This study highlights the importance of understanding the interplay between innate immune humoral factors including complement in devising nanoparticle based drug delivery strategies.

The progressive ankylosis protein ANK facilitates clathrin- and adaptor-mediated membrane traffic at the trans-Golgi network-to-endosome interface.

Dominant or recessive mutations in the progressive ankylosis gene ANKH have been linked to familial chondrocalcinosis (CCAL2), craniometaphyseal dysplasia (CMD), mental retardation, deafness and ankylosis syndrome (MRDA). The function of the encoded membrane protein ANK in cellular compartments other than the plasma membrane is unknown. Here, we show that ANK localizes to the trans-Golgi network (TGN), clathrin-coated vesicles and the plasma membrane. ANK functionally interacts with clathrin and clathrin associated adaptor protein (AP) complexes as loss of either protein causes ANK dispersion from the TGN to cytoplasmic endosome-like puncta. Consistent with its subcellular localization, loss of ANK results in reduced formation of tubular membrane carriers from the TGN, perinuclear accumulation of early endosomes and impaired transferrin endocytosis. Our data indicate that clathrin/AP-mediated cycling of ANK between the TGN, endosomes, and the cell surface regulates membrane traffic at the TGN/endosomal interface. These findings suggest that dysfunction of Golgi-endosomal membrane traffic may contribute to ANKH-associated pathologies.

Complement Deposition on Nanoparticles Can Modulate Immune Responses by Macrophage, B and T Cells.

Nanoparticles are attractive drug delivery vehicles for targeted organ-specific as well as systemic therapy. However, their interaction with the immune system offers an intriguing challenge to the success of nanotherapeutics in vivo. Recently, we showed that pristine and derivatised carbon nanotubes (CNT) can activate complement mainly via the classical pathway leading to enhanced uptake by phagocytic cells, and transcriptional down-regulation of pro-inflammatory cytokines. Here, we report the interaction of complement-activating CC-CNT and RNA-CNT, and non-complement-activating gold-nickel (Au-Ni) nanowires with cell lines representing macrophage, B and T cells. Complement deposition considerably enhanced uptake of CNTs by immune cells known to overexpress complement receptors. Real-Time qPCR and multiplex array analyses showed complement-dependent down-regulation of TNF-α and IL-1ß and up-regulation of IL-12 by CMC- and RNA-CNTs, in addition to revealing IL-10 as a crucial regulator during nanoparticle-immune cell interaction. It appears that complement system can recognize molecular patterns differentially displayed by nanoparticles and thus, modulate subsequent processing of nanoparticles by antigen capturing and antigen presenting cells, which can shape innate and adaptive immune axes.

Innate immune humoral factors, C1q and factor H, with differential pattern recognition properties, alter macrophage response to carbon nanotubes.

Interaction between the complement system and carbon nanotubes (CNTs) can modify their intended biomedical applications. Pristine and derivatised CNTs can activate complement primarily via the classical pathway which enhances uptake of CNTs and suppresses pro-inflammatory response by immune cells. Here, we report that the interaction of C1q, the classical pathway recognition molecule, with CNTs involves charge pattern and classical pathway activation that is partly inhibited by factor H, a complement regulator. C1q and its globular modules, but not factor H, enhanced uptake of CNTs by macrophages and modulated the pro-inflammatory immune response. Thus, soluble complement factors can interact differentially with CNTs and alter the immune response even without complement activation. Coating CNTs with recombinant C1q globular heads offers a novel way of controlling classical pathway activation in nanotherapeutics. Surprisingly, the globular heads also enhance clearance by phagocytes and down-regulate inflammation, suggesting unexpected complexity in receptor interaction. FROM THE CLINICAL EDITOR: Carbon nanotubes (CNTs) maybe useful in the clinical setting as targeting drug carriers. However, it is also well known that they can interact and activate the complement system, which may have a negative impact on the applicability of CNTs. In this study, the authors functionalized multi-walled CNT (MWNT), and investigated the interaction with the complement pathway. These studies are important so as to gain further understanding of the underlying mechanism in preparation for future use of CNTs in the clinical setting.

Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells.

The directed differentiation of human pluripotent stem cells offers the unique opportunity to generate a broad spectrum of human cell types and tissues for transplantation, drug discovery, and studying disease mechanisms. Here, we report the stepwise generation of bone-resorbing osteoclasts from human embryonic and induced pluripotent stem cells. Generation of a primitive streak-like population in embryoid bodies, followed by specification to hematopoiesis and myelopoiesis by vascular endothelial growth factor and hematopoietic cytokines in serum-free media, yielded a precursor population enriched for cells expressing the monocyte-macrophage lineage markers CD14, CD18, CD11b, and CD115. When plated in monolayer culture in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-kappaB ligand (RANKL), these precursors formed large, multinucleated osteoclasts that expressed tartrate-resistant acid phosphatase and were capable of resorption. No tartrate-resistant acid phosphatase-positive multinucleated cells or resorption pits were observed in the absence of RANKL. Molecular analyses confirmed the expression of the osteoclast marker genes NFATc1, cathepsin K, and calcitonin receptor in a RANKL-dependent manner, and confocal microscopy demonstrated the coexpression of the alphavbeta3 integrin, cathepsin K and F-actin rings characteristic of active osteoclasts. Generating hematopoietic and osteoclast populations from human embryonic and induced pluripotent stem cells will be invaluable for understanding embryonic bone development and postnatal bone disease.

Endocytic trafficking in actively resorbing osteoclasts.

Endocytosis and the subsequent intracellular trafficking of the endocytosed material are important determinants of cellular function. Osteoclasts, cells of the monocyte/macrophage family, are specialized for the internalization and processing of bone matrix. Transcytosis of endocytosed material has been observed in osteoclasts but the precise mechanism controlling this process is unclear. Here, we investigate the regulation of these trafficking events. To establish the directionality and kinetics of trafficking events in resorbing osteoclasts, we devised a system using fluorescent low-molecular-weight markers as probes to follow the route taken by the digested bone matrix. We demonstrate that this route is largely distinct from the pathway followed by proteins taken up by receptor-mediated endocytosis at the basolateral plasma membrane. Endocytosis and transcytosis from the ruffled border are fast processes, with a half-life of the endocytosed material inside the cells of 22 minutes. We demonstrate the crucial role of the microtubule network in transport from the ruffled-border area and provide evidence for a role of the cytoskeleton in the overall efficacy of trafficking. Moreover, we analyse the effect of the V-ATPase inhibitor bafilomycin A1 on endocytic uptake, which gives insight into the pH-dependent regulation of membrane trafficking and resorption in osteoclasts.

Formation and function of the ruffled border in osteoclasts.

Osteoclasts are multinucleated hematopoietic cells specialised for bone resorption. Dissolution of the inorganic fraction of the bone matrix is mediated by acidification of the bone surface in contact with the osteoclast whereas secreted lysosomal enzymes digest organic components. Through massive exocytosis, the plasma membrane in contact with the bone surface enlarges into the ruffled border, which has unusual features more similar to endosomal/lysosomal membranes. Maintenance of the ruffled border during resorption is achieved through a balance between exocytosis and endocytosis. Inactivation of proteins necessary for the extracellular acidification or of the proteases involved in matrix degradation leads to osteopetrosis; a disease characterised by dense bones.