Susan N. Christo

Innate Immunity and Biomaterials at the Nexus: Friends or Foes

Biomaterial implants are an established part of medical practice, encompassing a broad range of devices that widely differ in function and structural composition. However, one common property amongst biomaterials is the induction of the foreign body response: an acute sterile inflammatory reaction which overlaps with tissue vascularisation and remodelling and ultimately fibrotic encapsulation of the biomaterial to prevent further interaction with host tissue. Severity and clinical manifestation of the biomaterial-induced foreign body response are different for each biomaterial, with cases of incompatibility often associated with loss of function. However, unravelling the mechanisms that progress to the formation of the fibrotic capsule highlights the tightly intertwined nature of immunological responses to a seemingly noncanonical “antigen.” In this review, we detail the pathways associated with the foreign body response and describe possible mechanisms of immune involvement that can be targeted. We also discuss methods of modulating the immune response by altering the physiochemical surface properties of the biomaterial prior to implantation. Developments in these areas are reliant on reproducible and effective animal models and may allow a “combined” immunomodulatory approach of adapting surface properties of biomaterials, as well as treating key immune pathways to ultimately reduce the negative consequences of biomaterial implantation.

The Role of Surface Nanotopography and Chemistry on Primary Neutrophil and Macrophage Cellular Responses

Synthetic materials employed for enhancing, replacing, or restoring biological functionality may be compromised by the host immune responses that they evoke. Surface modification has attracted substantial attention as a tool to modulate the host response to synthetic materials; however, how surface nanotopography combined with chemistry affects immune effector cell responses is still poorly understood. To address this open question, a unique set of model surfaces with controlled surface nanotopography in the range of 16, 38, and 68 nm has been generated. Tailored outermost surface chemistry that was amine, carboxyl, or methyl group rich has been provided. The combinations of these properties yield 12 surface types that are subject to functional assays assessing key immune effector cells, namely, primary neutrophil and macrophage responses in vitro. The data demonstrate that surface nanotopography leads to enhanced matrix metalloproteinase-9 production from primary neutrophils, and a decrease in pro-inflammatory cytokine secretion from primary macrophages. Together, these results are the first to directly compare the immunomodulatory effects of the cooperative interplay between surface nanotopography and chemistry.

Solid-state capture and real-time analysis of individual T cell activation via self-assembly of binding multimeric proteins on functionalized materials surfaces

Polyfunctional T cell responses are increasingly underpinning new and improved vaccination regimens. Studies of the nature and extent of these T cell responses may be facilitated if specific T cell populations can be assessed from mixed populations by ligand-mediated capture in a solid-state assay format. Accordingly, we report here the development of a novel strategy for the solid-state capture and real-time activation analyses of individual cognate T cells which utilizes a spontaneous self-assembly process for generating multimers of biotinylated class I major histocompatibility-peptide complex (MHCp) directly on the solid-state assay surface while also ensuring stability by covalent interfacial binding. The capture surface was constructed by the fabrication of multilayer coatings onto standard slides. The first layer was a thin polymer coating with surface aldehyde groups, onto which streptavidin was covalently immobilized, followed by the docking of multimers of biotinylated MHCp or biotinylated anti-CD45.1 monoclonal antibody. The high binding strength at each step of this immobilization sequence aims to ensure that artefacts such as (partial) detachment, or displacement by proteins from solution, would not interfere with the intended biological assays. The multilayer coating steps were monitored by X-ray photoelectron spectroscopy; data indicated that the MHCp proteins self-assembled in a multimeric form onto the streptavidin surface. Immobilized multimeric MHCp demonstrated the capacity to bind and retain antigen-specific T cells from mixed populations of cells onto the solid carrier. Furthermore, real-time confocal microscopic detection and quantification of subsequent calcium flux using paired fluorescent ratiometric probes facilitated the analysis of individual T cell response profiles, as well as population analyses using a combination of individual T cell events.

The contribution of inflammasome components on macrophage response to surface nanotopography and chemistry.

Implantable devices have become an established part of medical practice. However, often a negative inflammatory host response can impede the integration and functionality of the device. In this paper, we interrogate the role of surface nanotopography and chemistry on the potential molecular role of the inflammasome in controlling macrophage responses. To achieve this goal we engineered model substrata having precisely controlled nanotopography of predetermined height and tailored outermost surface chemistry. Bone marrow derived macrophages (BMDM) were harvested from genetically engineered mice deficient in the inflammasome components ASC, NLRP3 and AIM2. These cells were then cultured on these nanoengineered substrata and assessed for their capacity to attach and express pro-inflammatory cytokines. Our data provide evidence that the inflammasome components ASC, NLRP3 and AIM2 play a role in regulating macrophage adhesion and activation in response to surface nanotopography and chemistry. The findings of this paper are important for understanding the inflammatory consequences caused by biomaterials and pave the way to the rational design of future implantable devices having controlled and predictable inflammatory outcomes.

Scrutinizing calcium flux oscillations in T lymphocytes to deduce the strength of stimulus.

The capture and activation of individual T cells on functionalised surfaces enables real-time analyses of the magnitude and rhythm of intracellular calcium release. Application of Haarlet transformations generate a calcium flux ‘threshold’, with the frequency of the ‘threshold crossings’ correlating with the strength of the original T cell stimulus. These findings represent a new method to evaluate graduations in T cell activation in real time, and at a single-cell level.

Individual and Population Quantitative Analyses of Calcium Flux in T-Cells Activated on Functionalized Material Surfaces

We have developed a novel method for activating T-cells on material surfaces that enable individual and population-based analyses of intracellular calcium flux, as a quantitative measure of T-cell receptor engagement. Functionalized material surfaces were created using a plasma-polymerized foundation layer to immobilize stimulatory T-cell ligands, which could induce T-cell receptor-dependent calcium flux in naive T-cells. Real-time confocal microscopic detection and quantification of calcium flux using paired fluorescent ratiometric probes facilitated the tracking and analysis of response profiles of individual T-cells, as well as population analyses using a combination of individual T-cell events. This type of combined analysis cannot be achieved using traditional population-based flow cytometric approaches, and thus provides a logical step towards developing the capacity to assess the magnitude and quality of inherently heterogeneous effector T-cell responses to antigenic challenge.

Hybrid core/shell microparticles and their use for understanding biological processes.

Hybrid micro and nanoparticles have become a topic of intense research in recent years. This is due to the special properties of these materials that open new avenues in advanced applications. Herein, we report a novel method for the generation of hybrid particles utilising plasma polymerization. Poly (methyl methacrylate) (PMMA) beads were first coated with a thin allylamine based plasma polymer layer. Gold nanoparticles of engineered size and surface structure were then attached in a controlled manner to the plasma polymer coated beads. To generate uniform chemistry on the outermost surface and to preserve the nanotopography, we deposited a 5-10 nm thin layer of Acpp. We demonstrated that these particles can be utilized in in vivo models to interrogate important biological phenomena. Specifically, we used them in mice to study the inflammatory and foreign body responses to surface nanotopography. The data strongly indicates that surface nanotopography and chemistry can modulate collagen production and the number of adhering immune cells. The method for generating hybrid particles reported here is solvent free and can open new opportunities in fields such as tissue engineering, drug delivery, biosensors, and regenerative medicine.

The functional contribution of calcium ion flux heterogeneity in T cells.

The role of intracellular calcium ion oscillations in T‐cell physiology is being increasingly appreciated by studies that describe how unique temporal and spatial calcium ion signatures can control different signalling pathways. Within this review, we provide detailed mechanisms of calcium ion oscillations, and emphasise the pivotal role that calcium signalling plays in directing crucial events pertaining to T‐cell functionality. We also describe methods of calcium ion quantification, and take the opportunity to discuss how a deeper understanding of calcium signalling combined with new detection and quantification methodologies can be used to better design immunotherapies targeting T‐cell responses.

GM-CSF signalling blockade and chemotherapeutic agents act in concert to inhibit the function of myeloid-derived suppressor cells in vitro

Immune evasion is a recently defined hallmark of cancer, and immunotherapeutic approaches that stimulate an immune response to tumours are gaining recognition. However tumours may evade the immune response and resist immune‐targeted treatment by promoting an immune‐suppressive environment and stimulating the differentiation or recruitment of immunosuppressive cells. Myeloid‐derived suppressor cells (MDSC) have been identified in a range of cancers and are often associated with tumour progression and poor patient outcomes. Pancreatic cancer in particular supports MDSC differentiation via the secretion of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and MDSC are believed to contribute to the profoundly immune‐suppressive microenvironment present in pancreatic tumours. MDSC‐targeted therapies that deplete or inhibit this cell population have been proposed as a way to shift the balance in favour of a tumour‐clearing immune response. In this study, we have modelled MDSC differentiation and function in vitro and this has provided us with the opportunity to test a range of potential MDSC‐targeted therapies to identify candidates for further investigation. Using in vitro modelling we show here that the combination of GM‐CSF‐signalling blockade and gemcitabine suppresses both the MDSC phenotype and the inhibition of T‐cell function by MDSC.

Serrulatane Diterpenoid from Eremophila neglecta Exhibits Bacterial Biofilm Dispersion and Inhibits Release of Pro-inflammatory Cytokines from Activated Macrophages.

The purpose of this study was to assess the biofilm-removing efficacy and inflammatory activity of a serrulatane diterpenoid, 8-hydroxyserrulat-14-en-19-oic acid (1), isolated from the Australian medicinal plant Eremophila neglecta. Biofilm breakup activity of compound 1 on established Staphylococcus epidermidis and Staphylococcus aureus biofilms was compared to the antiseptic chlorhexidine and antibiotic levofloxacin. In a time-course study, 1 was deposited onto polypropylene mesh to mimic a wound dressing and tested for biofilm removal. The ex-vivo cytotoxicity and effect on lipopolysaccharide-induced pro-inflammatory cytokine release were studied in mouse primary bone-marrow-derived macrophage (BMDM) cells. Compound 1 was effective in dispersing 12 h pre-established biofilms with a 7 log10 reduction of viable bacterial cell counts, but was less active against 24 h biofilms (approximately 2 log10 reduction). Compound-loaded mesh showed dosage-dependent biofilm-removing capability. In addition, compound 1 displayed a significant inhibitory effect on tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) secretion from BMDM cells, but interleukin-1 beta (IL-1β) secretion was not significant. The compound was not cytotoxic to BMDM cells at concentrations effective in removing biofilm and lowering cytokine release. These findings highlight the potential of this serrulatane diterpenoid to be further developed for applications in wound management.