Complement C5a induces the formation of neutrophil extracellular traps by myeloid-derived suppressor cells to promote metastasis.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.

Development of Potent and Selective Agonists for Complement C5a Receptor 1 with In Vivo Activity.

The anaphylatoxin C5a is a complement peptide associated with immune-related disorders. C5a binds with equal potency to two GPCRs, C5aR1 and C5aR2. Multiple C5a peptide agonists have been developed to interrogate the C5a receptor function but none show selectivity for C5aR1. To address these limitations, we developed potent and stable peptide C5aR1 agonists that display no C5aR2 activity and over 1000-fold selectivity for C5aR1 over C3aR. This includes BM213, which induces C5aR1-mediated calcium mobilization and pERK1/2 signaling but not ß-arrestin recruitment, and BM221, which exhibits no signaling bias. Both ligands are functionally similar to C5a in human macrophage cytokine release assays and in a murine in vivo neutrophil mobilization assay. BM213 showed antitumor activity in a mouse model of mammary carcinoma. We anticipate that these C5aR1-selective agonists will be useful research tools to investigate C5aR1 function.

An Immunoregulatory Role for Complement Receptors in Murine Models of Breast Cancer.

The complement system has demonstrated roles in regulating tumor growth, although these may differ between tumor types. The current study used two murine breast cancer models (EMT6 and 4T1) to investigate whether pharmacological targeting of receptors for complement proteins C3a (C3aR) and C5a (C5aR1) is protective in murine breast cancer models. In contrast to prior studies in other tumor models, treatment with the selective C5aR1 antagonist PMX53 had no effect on tumor growth. However, treatment of mice with a dual C3aR/C5aR1 agonist (YSFKPMPLaR) significantly slowed mammary tumor development and progression. Examination of receptor expression by quantitative polymerase chain reaction (qPCR) analysis showed very low levels of mRNA expression for either C3aR or C5aR1 by EMT6 or 4T1 mammary carcinoma cell lines compared with the J774 macrophage line or bone marrow-derived macrophages. Moreover, flow cytometric analysis found no evidence of C3aR or C5aR1 protein expression by either EMT6 or 4T1 cells, leading us to hypothesize that the tumor inhibitory effects of the dual agonist are indirect, possibly via regulation of the anti-tumor immune response. This hypothesis was supported by flow cytometric analysis of tumor infiltrating leukocyte populations, which demonstrated a significant increase in T lymphocytes in mice treated with the C3aR/C5aR1 agonist. These results support an immunoregulatory role for complement receptors in primary murine mammary carcinoma models. They also suggest that complement activation peptides can influence the anti-tumor response in different ways depending on the cancer type, the host immune response to the tumor and levels of endogenous complement activation within the tumor microenvironment.

C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

The canonical complement component 5a (C5a) receptor (C5aR) 1 has well-described roles in tumorigenesis but the contribution of the second receptor, C5aR2, is unclear. The present study demonstrates that B16.F0 melanoma cells express mRNA for both C5aR1 and C5aR2 and signal through ERK and p38 MAPKs in response to C5a. Despite this, C5a had no impact on melanoma cell proliferation or migration in vitro. In vivo studies demonstrated that the growth of B16.F0 melanoma tumors was increased in C5aR2-/- mice but reduced in C5aR1-/- mice and wild-type mice treated with a C5aR1 antagonist. Analysis of tumor-infiltrating leukocyte populations showed no significant differences between wild-type and C5aR2-/- mice. Conversely, percentages of myeloid-derived suppressor cells, macrophages, and regulatory T lymphocytes were lower in tumors from C5aR1-/- mice, whereas total (CD3+) T lymphocytes and CD4+ subsets were higher. Analysis of cytokine and chemokine levels also showed plasma IFN-γ was higher and tumor C-C motif chemokine ligand 2 was lower in the absence of C5aR1. The results suggest that C5aR1 signaling supports melanoma growth by promoting infiltration of immunosuppressive leukocyte populations into the tumor microenvironment, whereas C5aR2 has a more restricted but beneficial role in limiting tumor growth. Overall, these data support the potential of C5aR1-inhibitory therapies for melanoma.-Nabizadeh, J. A., Manthey, H. D., Panagides, N., Steyn, F. J., Lee, J. D., Li, X. X., Akhir, F. N. M., Chen, W., Boyle, G. M., Taylor, S. M., Woodruff, T. M., Rolfe, B. E. C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

Clay nanoparticles co-deliver three antigens to promote potent immune responses against pathogenic Escherichia coli.

Currently, there are few strategies for controlling pathogenic bacteria, especially the pathotypes of Escherichia coli which are an emerging threat to public health worldwide. Here, multivalent vaccine formulations are reported for control of pathogenic E. coli. The formulations utilised clay nanoparticles, either layered double hydroxides (LDH) or hectorite (HEC), to complex with a cocktail of three recombinant antigens, intimin ß (IB), proprietary antigen 1 (PAg1) and proprietary antigen 2 (PAg2). Acting as nano-adjuvants, LDH and HEC were able to stimulate strong, durable and balanced immune responses in mice. Moreover, LDH-IB-PAg1-PAg2 and HEC-IB-PAg1-PAg2 immunised mice developed potent mucosal immune responses and efficiently prevented adherence of enterohemorrhagic E. coli serotype O26 to mammalian cells. Notably, the multi-faceted immune responses elicited by the clay nanoparticle formulations were significantly higher than those induced by a QuilA formulation, without antigenic competition observed for the first time. The results of this study suggest that LDH and HEC offer considerable promise as effective multivalent vaccine carriers against important pathogens such as enteropathogenic E. coli.

The Pathways for Layered Double Hydroxide Nanoparticles to Enhance Antigen (Cross)-Presentation on Immune Cells as Adjuvants for Protein Vaccines.

Nanoparticles (NPs) are intensively investigated as adjuvants in new generation vaccines, while how these NPs promote the immune responses has not been well understood. In this research, we have tried to elucidate the possible pathways for layered double hydroxide (LDH) NPs to provoke immune responses. As previously reported, LDH NPs efficiently deliver antigens to antigen presenting cells (APCs). In this research, we have found that these internalized LDH NPs are not released by these APCs within 8 h. We have for the first time found that macrophage cells exchange the internalized LDH NPs with other surrounding ones, which may promote immune responses in an additional way. Moreover, the internalized LDH-antigen NPs significantly facilitate the maturation of immature DCs and enhance cross-presentation of epitope/MHC class I complexes on the DC surface. This research would help understand the NP adjuvant mechanism and further assist the design of new specific NPs as more efficient nano-adjuvants.

High F-Content Perfluoropolyether-Based Nanoparticles for Targeted Detection of Breast Cancer by 19F Magnetic Resonance and Optical Imaging.

Two important challenges in the field of 19F magnetic resonance imaging (MRI) are the maintenance of high fluorine content without compromising imaging performance, and effective targeting of small particles to diseased tissue. To address these challenges, we have developed a series of perfluoropolyether (PFPE)-based hyperbranched (HBPFPE) nanoparticles with attached peptide aptamer as targeting ligands for specific in vivo detection of breast cancer with high 19F MRI sensitivity. A detailed comparison of the HBPFPE nanoparticles (NPs) with the previously reported trifluoroethyl acrylate (TFEA)-based polymers demonstrates that the mobility of fluorinated segments of the HBPFPE nanoparticles is significantly enhanced (19F T2 > 80 ms vs 31 ms), resulting in superior MR imaging sensitivity. Selective targeting was confirmed by auto- and pair correlation analysis of fluorescence microscopy data, in vitro immunofluorescence, in vivo 19F MRI, ex vivo fluorescence and 19F NMR. The results highlight the high efficiency of aptamers for targeting and the excellent sensitivity of the PFPE moieties for 19F MRI. Of relevance to in vivo applications, the PFPE-based polymers exhibit much faster clearance from the body than the previously introduced perfluorocarbon emulsions ( t1/2 ∼ 20 h vs up to months). Moreover, the aptamer-conjugated NPs show significantly higher tumor-penetration, demonstrating the potential of these imaging agents for therapeutic applications. This report of the synthesis of polymeric aptamer-conjugated PFPE-based 19F MRI CAs with high fluorine content (∼10 wt %) demonstrates that these NPs are exciting candidates for detecting diseases with high imaging sensitivity.

Clay Nanoparticles Elicit Long-Term Immune Responses by Forming Biodegradable Depots for Sustained Antigen Stimulation.

Nanomaterials have been widely tested as new generation vaccine adjuvants, but few evoke efficient immunoreactions. Clay nanoparticles, for example, layered double hydroxide (LDH) and hectorite (HEC) nanoparticles, have shown their potent adjuvanticity in generating effective and durable immune responses. However, the mechanism by which clay nanoadjuvants stimulate the immune system is not well understood. Here, it is demonstrated that LDH and HEC-antigen complexes form loose agglomerates in culture medium/serum. They also form nodules with loose structures in tissue after subcutaneous injection, where they act as a depot for up to 35 d. More importantly, clay nanoparticles actively and continuously recruit immune cells into the depot for up to one month, and stimulate stronger immune responses than FDA-approved adjuvants, Alum and QuilA. Sustained antigen release is also observed in clay nanoparticle depots, with 50-60% antigen released after 35 d. In contrast, Alum-antigen complexes show minimal antigen release from the depot. Importantly, LDH and HEC are more effective than QuilA and Alum in promoting memory T-cell proliferation. These findings suggest that both clay nanoadjuvants can serve as active vaccine platforms for sustained and potent immune responses.

Differential immunological profiles herald magnetic resonance imaging-defined perioperative cerebral infarction.

BACKGROUND: The perioperative period is associated with a high risk for human ischaemic stroke. Although inflammatory mechanisms are known to have an important role in cerebral infarction in the nonoperative setting, their role in modulating perioperative risk remains unclear. METHODS: In this prospective case-control study, we compared 10 patients (cases) who developed magnetic resonance imaging (MRI) evidence of cerebral infarction following transcatheter aortic valve implantation with 10 patients (controls) who underwent the same procedure without neurological complication. Blood sampling was performed preoperatively (baseline) and at 24 h, 48 h and 72 h postoperatively and analysed for specific cytokines, chemokines and complement factors. RESULTS: Baseline serum assessments identified significant differences between the two cohorts for levels of complement C3, complement C4b, granulocyte-macrophage colony-stimulating factor, interleukin-15 and macrophage inflammatory protein-1ß. Longitudinal regression analysis and best-fit polynomial curves of postoperative analyte profiles identified significantly higher levels of complement C3 and matrix metalloproteinase-9, and lower levels of interferon-γ and macrophage inflammatory protein-1ß levels in cases versus controls. CONCLUSIONS: These results support a potentially important role for inflammatory mechanisms in MRI-defined perioperative stroke and reveal a potentially important role for complement components in this process.

Influence of Charge on Hemocompatibility and Immunoreactivity of Polymeric Nanoparticles.

The benefits of nanomedicine may be restricted by hemocompatibility and immunoreactivity problems arising from administration of exogenous materials into the bloodstream. To understand how surface charge influences the interaction of polymeric nanoparticles with blood components, we synthesized three well-defined, charge-varied hyperbranched polymers (HBPs) of similar size and analyzed both hemocompatibility and immunoreactivity of these methacrylate-based HBPs ex vivo using primary human blood cell assays and image analyses following intravenous injection into mice. The results show that, regardless of charge, endotoxin-free HBPs had minimal effects on coagulation, platelet, complement, or T cell activation. However, high concentrations (100 µg mL-1) of cationic HBPs led to significant dendritic cell activation, suggesting the potential application of these nanoparticles as vaccine adjuvants to aid efficient antigen presentation. Biodistribution studies showed that intravenously administered charge-neutral HBPs had a longer retention time in the circulation than cationic or anionic HBPs; whereas these neutral HBPs were eventually cleared in the urine, charged HBPs mainly accumulated in liver and spleen. Overall, these results demonstrate that, regardless of surface charge, HBPs display a high level of hemocompatibility. In contrast, immunoreactivity and biodistribution are significantly influenced by charge. Manipulation of surface charge may thus be a useful method by which nanomaterials such as HBPs can be tailored to different clinical applications.

High adjuvant activity of layered double hydroxide nanoparticles and nanosheets in anti-tumour vaccine formulations.

Effective adjuvants in anti-tumour vaccine formulations are very important in the development of new-generation vaccines. In this study, two layered double hydroxide (LDH) nanomaterial forms, i.e. nanoparticles (NPs) and nanosheets (NSs), were synthesised and examined as adjuvants to provoke the immune responses for anti-tumour purpose. Immunogen ovalbumin (OVA) delivered by both nanomaterials induced much stronger humoral and cell-medicated immune responses, together with an immune stimulant (TLR9 ligand CpG), as evidenced by higher levels of IgG1, IgG2a and interferon-γ. By comparison, LDH NSs showed higher activity to promote specific antibody responses than LDH NPs but with a similar cell-mediated immune response. The mice immunised with OVA-CpG vaccines formulated with both nanomaterials showed stronger inhibition of the inoculated tumour growth and had a longer survival. Altogether, these data indicate that LDH NPs and NSs can be used as potential nanoadjuvants for efficient protein-based anti-tumour vaccines.

Layered double hydroxide nanoparticles: Impact on vascular cells, blood cells and the complement system.

The mounting interest in layered double hydroxide (LDH) nanoparticles as drug carriers and bio-imaging contrast agents makes biosafety evaluation of LDH essential. Considering the important role of blood circulation in bio-distribution of nanoparticles, the present work evaluated the impact of MgAl-LDHs on key components of the circulatory system, including vascular cells (vascular smooth muscle cells (SMCs) and endothelial cells (HUVECs)), red blood cells (RBCs), and complement activation. The results showed that LDH had no effects on SMCs and HUVECs at concentrations up to 500 and 10 µg/mL respectively, in terms of cell proliferation and viability. LDH (10 µg/mL) did not change either the migration distance or the number of migrating SMCs in culture. Moreover, LDH (400 µg/mL) had a negligible effect on RBCs' lysis, and there was no significant increase in levels of complement activation product, C5a, in the presence of LDH (20 or 200 µg/mL). The low toxicity for vascular cells and blood cells combined with low immunogenicity sheds a light on the biosafety of LDH nanoparticles, and encourages further studies into their biomedical applications.

Effects of Surface Charge of Hyperbranched Polymers on Cytotoxicity, Dynamic Cellular Uptake and Localization, Hemotoxicity, and Pharmacokinetics in Mice.

Nanoscaled polymeric materials are increasingly being investigated as pharmaceutical products, drug/gene delivery vectors, or health-monitoring devices. Surface charge is one of the dominant parameters that regulates nanomaterial behavior in vivo. In this paper, we demonstrated how control over chemical synthesis allowed manipulation of nanoparticle surface charge, which in turn greatly influenced the in vivo behavior. Three methacrylate/methacrylamide-based monomers were used to synthesize well-defined hyperbranched polymers (HBP) by reversible addition-fragmentation chain transfer (RAFT) polymerization. Each HBP had a hydrodynamic diameter of approximately 5 nm as determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Incorporation of a fluorescent moiety within the polymeric nanoparticles allowed determination of how charge affected the in vivo pharmacokinetic behavior of the nanomaterials and the biological response to them. A direct correlation between surface charge, cellular uptake, and cytotoxicity was observed, with cationic HBPs exhibiting higher cellular uptake and cytotoxicity than their neutral and anionic counterparts. Evaluation of the distribution of the differently charged HBPs within macrophages showed that all HBPs accumulated in the cytoplasm, but cationic HBPs also trafficked to, and accumulated within, the nucleus. Although cationic HBPs caused slight hemolysis, this was generally below accepted levels for in vivo safety. Analysis of pharmacokinetic behavior showed that cationic and anionic HBPs had short blood half-lives of 1.82 ± 0.51 and 2.34 ± 0.93 h respectively, compared with 5.99 ± 2.30 h for neutral HBPs. This was attributed to the fact that positively charged surfaces are more readily covered with opsonin proteins and thus more visible to phagocytic cells. This was supported by in vitro flow cytometric and qualitative live cell imaging studies, which showed that cationic HBPs tended to be taken up by macrophages more effectively and rapidly than neutral and anionic particles.

Efficient induction of comprehensive immune responses to control pathogenic E. coli by clay nano-adjuvant with the moderate size and surface charge.

In recent decades, diseases caused by pathogenic Escherichia coli (E. coli), enterohaemorrhagic E. coli (EHEC) O26 have been increasingly reported worldwide, which are as severe as those caused by EHEC strain O157:H7 and require effective intervention strategies. Herein, we report the application of clay nanoparticles, i.e. hectorites as effective nano-adjuvants for vaccination against EHEC O26 colonization. We show that medium size HEC (hectorite, around 73~77 nm diameter) is able to induce efficient humoral and cellular immune responses against EHEC antigen - intimin ß (IB), which are significantly higher than those triggered by commercially used adjuvants - QuilA and Alum. We also demonstrate that mice immunized with IB adjuvanted with HEC nanoparticles elicit sufficient secretion of mucosal IgA, capable of providing effective protection against EHEC O26 binding to ruminant and human cells. In addition, we demonstrate for the first time that hectorites are able to initiate maturation of RAW 264.7 macrophages, inducing expression of co-stimulatory cytokines at a low nanoparticle concentration (10 µg/mL). Together these data strongly suggest that hectorite with optimized size is a highly efficient vaccine nano-adjuvant.

The Complement C3a Receptor Contributes to Melanoma Tumorigenesis by Inhibiting Neutrophil and CD4+ T Cell Responses.

The complement peptide C3a is a key component of the innate immune system and a major fragment produced following complement activation. We used a murine model of melanoma (B16-F0) to identify a hitherto unknown role for C3a-C3aR signaling in promoting tumor growth. The results show that the development and growth of B16-F0 melanomas is retarded in mice lacking C3aR, whereas growth of established melanomas can be arrested by C3aR antagonism. Flow cytometric analysis showed alterations in tumor-infiltrating leukocytes in the absence of C3aR. Specifically, neutrophils and CD4(+) T lymphocyte subpopulations were increased, whereas macrophages were reduced. The central role of neutrophils was confirmed by depletion experiments that reversed the tumor inhibitory effects observed in C3aR-deficient mice and returned tumor-infiltrating CD4(+) T cells to control levels. Analysis of the tumor microenvironment showed upregulation of inflammatory genes that may contribute to the enhanced antitumor response observed in C3aR-deficient mice. C3aR deficiency/inhibition was also protective in murine models of BRAF(V600E) mutant melanoma and colon and breast cancer, suggesting a tumor-promoting role for C3aR signaling in a range of tumor types. We propose that C3aR activation alters the tumor inflammatory milieu, thereby promoting tumor growth. Therapeutic inhibition of C3aR may therefore be an effective means to trigger an antitumor response in melanoma and other cancers.

Efficient and Durable Vaccine against Intimin ß of Diarrheagenic E. Coli Induced by Clay Nanoparticles.

Improved strategies are urgently required to control infections with enterohemorrhagic Escherichia coli and enteropathogenic E. coli, two dominant zoonotic enteric pathogens responsible for a wide spectrum of illnesses as well as deaths of human being, with tremendous financial cost worldwide. The present study investigates the capacity of two clay nanoparticles (NPs) with opposite surface charges, namely synthetic layered double hydroxide (LDH) and hectorite (HEC) NPs as adjuvants to promote strong immune responses against the infections. Here both LDH and HEC NPs are showed to be able to carry an appreciable amount of Intimin ß (1.1 and 4.4 mg per mg clay nanomaterials, respectively) and significantly facilitate antigen uptake by antigen-presenting cells. Remarkably, these clay NPs induce strong antibody and cell-mediated immune responses, which are much higher than that by the potent adjuvant, QuilA. Furthermore, these strong immune responses are well maintained for at least four months in the mouse model, during which there are no changes in histopathology of the animal organs. Collectively these data demonstrate the suitability of LDH and HEC NPs as useful adjuvants in new-generation vaccine formulations to control various infectious diseases.

The in vivo fate of nanoparticles and nanoparticle-loaded microcapsules after oral administration in mice: Evaluation of their potential for colon-specific delivery.

Anti-cancer drug loaded-nanoparticles (NPs) or encapsulation of NPs in colon-targeted delivery systems shows potential for increasing the local drug concentration in the colon leading to improved treatment of colorectal cancer. To investigate the potential of the NP-based strategies for colon-specific delivery, two formulations, free Eudragit® NPs and enteric-coated NP-loaded chitosan-hypromellose microcapsules (MCs) were fluorescently-labelled and their tissue distribution in mice after oral administration was monitored by multispectral small animal imaging. The free NPs showed a shorter transit time throughout the mouse digestive tract than the MCs, with extensive excretion of NPs in faeces at 5h. Conversely, the MCs showed complete NP release in the lower region of the mouse small intestine at 8h post-administration. Overall, the encapsulation of NPs in MCs resulted in a higher colonic NP intensity from 8h to 24h post-administration compared to the free NPs, due to a NP 'guarding' effect of MCs during their transit along mouse gastrointestinal tract which decreased NP excretion in faeces. These imaging data revealed that this widely-utilised colon-targeting MC formulation lacked site-precision for releasing its NP load in the colon, but the increased residence time of the NPs in the lower gastrointestinal tract suggests that it is still useful for localised release of chemotherapeutics, compared to NP administration alone. In addition, both formulations resided in the stomach of mice at considerable concentrations over 24h. Thus, adhesion of NP- or MC-based oral delivery systems to gastric mucosa may be problematic for colon-specific delivery of the cargo to the colon and should be carefully investigated for a full evaluation of particulate delivery systems.

Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG.

Modulation of the immune response is an important step in the induction of protective humoral and cellular immunity against pathogens. In this study, we investigated the possibility of using a nanomaterial conjugated with the toll-like receptor (TLR) ligand CpG to modulate the immune response towards the preferred polarity. MgAl-layered double hydroxide (LDH) nanomaterial has a very similar chemical composition to Alum, an FDA approved adjuvant for human vaccination. We used a model antigen, ovalbumin (OVA) to demonstrate that MgAl-LDH had comparable adjuvant activity to Alum, but much weaker inflammation. Conjugation of TLR9 ligand CpG to LDH nanoparticles significantly enhanced the antibody response and promoted a switch from Th2 toward Th1 response, demonstrated by a change in the IgG2a:IgG1 ratio. Moreover, immunization of mice with CpG-OVA-conjugated LDH before challenge with OVA-expressing B16/F10 tumor cells retarded tumor growth. Together, these data indicate that LDH nanomaterial can be used as an immune adjuvant to promote Th1 or Th2 dominant immune responses suitable for vaccination purposes.