Targeting Macrophage Polarization for Reinstating Homeostasis following Tissue Damage.

Tissue regeneration and remodeling involve many complex stages. Macrophages are critical in maintaining micro-environmental homeostasis by regulating inflammation and orchestrating wound healing. They display high plasticity in response to various stimuli, showing a spectrum of functional phenotypes that vary from M1 (pro-inflammatory) to M2 (anti-inflammatory) macrophages. While transient inflammation is an essential trigger for tissue healing following an injury, sustained inflammation (e.g., in foreign body response to implants, diabetes or inflammatory diseases) can hinder tissue healing and cause tissue damage. Modulating macrophage polarization has emerged as an effective strategy for enhancing immune-mediated tissue regeneration and promoting better integration of implantable materials in the host. This article provides an overview of macrophages' functional properties followed by discussing different strategies for modulating macrophage polarization. Advances in the use of synthetic and natural biomaterials to fabricate immune-modulatory materials are highlighted. This reveals that the development and clinical application of more effective immunomodulatory systems targeting macrophage polarization under pathological conditions will be driven by a detailed understanding of the factors that regulate macrophage polarization and biological function in order to optimize existing methods and generate novel strategies to control cell phenotype.

Modelled-Microgravity Reduces Virulence Factor Production in Staphylococcus aureus through Downregulation of agr-Dependent Quorum Sensing.

Bacterial contamination during space missions is problematic for human health and damages filters and other vital support systems. Staphylococcus aureus is both a human commensal and an opportunistic pathogen that colonizes human tissues and causes acute and chronic infections. Virulence and colonization factors are positively and negatively regulated, respectively, by bacterial cell-to-cell communication (quorum sensing) via the agr (accessory gene regulator) system. When cultured under low-shear modelled microgravity conditions (LSMMG), S. aureus has been reported to maintain a colonization rather than a pathogenic phenotype. Here, we show that the modulation of agr expression via reduced production of autoinducing peptide (AIP) signal molecules was responsible for this behavior. In an LSMMG environment, the S. aureus strains JE2 (methicillin-resistant) and SH1000 (methicillin-sensitive) both exhibited reduced cytotoxicity towards the human leukemia monocytic cell line (THP-1) and increased fibronectin binding. Using S. aureus agrP3::lux reporter gene fusions and mass spectrometry to quantify the AIP concentrations, the activation of agr, which depends on the binding of AIP to the transcriptional regulator AgrC, was delayed in the strains with an intact autoinducible agr system. This was because AIP production was reduced under these growth conditions compared with the ground controls. Under LSMMG, S. aureus agrP3::lux reporter strains that cannot produce endogenous AIPs still responded to exogenous AIPs. Provision of exogenous AIPs to S. aureus USA300 during microgravity culture restored the cytotoxicity of culture supernatants for the THP-1 cells. These data suggest that microgravity does not affect AgrC-AIP interactions but more likely the generation of AIPs.

Differential patterns of cross-reactive antibody response against SARS-CoV-2 spike protein detected for chronically ill and healthy COVID-19 naïve individuals.

Immunity to previously encountered viruses can alter response to unrelated pathogens. We reasoned that similar mechanism may also involve SARS-CoV-2 and thereby affect the specificity and the quality of the immune response against the virus. Here, we employed high-throughput next generation phage display method to explore the link between antibody immune response to previously encountered antigens and spike (S) glycoprotein. By profiling the antibody response in COVID-19 naïve individuals with a diverse clinical history (including cardiovascular, neurological, or oncological diseases), we identified 15 highly antigenic epitopes on spike protein that showed cross-reactivity with antigens of seasonal, persistent, latent or chronic infections from common human viruses. We observed varying degrees of cross-reactivity of different viral antigens with S in an epitope-specific manner. The data show that pre-existing SARS-CoV-2 S1 and S2 cross-reactive serum antibody is readily detectable in pre-pandemic cohort. In the severe COVID-19 cases, we found differential antibody response to the 15 defined antigenic and cross-reactive epitopes on spike. We also noted that despite the high mutation rates of Omicron (B.1.1.529) variants of SARS-CoV-2, some of the epitopes overlapped with the described mutations. Finally, we propose that the resolved epitopes on spike if targeted by re-called antibody response from SARS-CoV-2 infections or vaccinations can function in chronically ill COVID-19 naïve/unvaccinated individuals as immunogenic targets to boost antibodies augmenting the chronic conditions. Understanding the relationships between prior antigen exposure at the antibody epitope level and the immune response to subsequent infections with viruses from a different strain is paramount to guiding strategies to exit the COVID-19 pandemic.

Single-Cell Metabolic Profiling of Macrophages Using 3D OrbiSIMS: Correlations with Phenotype.

Macrophages are important immune cells that respond to environmental cues acquiring a range of activation statuses represented by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at each end of their spectrum. Characterizing the metabolic signature (metabolic profiling) of different macrophage subsets is a powerful tool to understand the response of the human immune system to different stimuli. Here, the recently developed 3D OrbiSIMS instrument is applied to yield useful insight into the metabolome from individual cells after in vitro differentiation of macrophages into naïve, M1, and M2 phenotypes using different cytokines. This analysis strategy not only requires more than 6 orders of magnitude less sample than traditional mass spectrometry approaches but also allows the study of cell-to-cell variance. Characteristic metabolites in macrophage subsets are identified using a targeted lipid and data-driven multivariate approach highlighting amino acids and other small molecules. The diamino acids alanylasparagine and lipid sphingomyelin SM(d18/16:0) are uniquely found in M1 macrophages, while pyridine and pyrimidine are observed at increased intensity in M2 macrophages, findings which link to known biological pathways. The first demonstration of this capability illustrates the great potential of direct cell analysis for in situ metabolite profiling with the 3D OrbiSIMS to probe functional phenotype at the single-cell level using molecular signatures and to understand the response of the human body to implanted devices and immune diseases.

Melanoma-specific antigen-associated antitumor antibody reactivity as an immune-related biomarker for targeted immunotherapies.

Background: Immunotherapies, including cancer vaccines and immune checkpoint inhibitors have transformed the management of many cancers. However, a large number of patients show resistance to these immunotherapies and current research has provided limited findings for predicting response to precision immunotherapy treatments. Methods: Here, we applied the next generation phage display mimotope variation analysis (MVA) to profile antibody response and dissect the role of humoral immunity in targeted cancer therapies, namely anti-tumor dendritic cell vaccine (MelCancerVac®) and immunotherapy with anti-PD-1 monoclonal antibodies (pembrolizumab). Results: Analysis of the antibody immune response led to the characterization of epitopes that were linked to melanoma-associated and cancer-testis antigens (CTA) whose antibody response was induced upon MelCancerVac® treatments of lung cancer. Several of these epitopes aligned to antigens with strong immune response in patients with unresectable metastatic melanoma receiving anti-PD-1 therapy. Conclusions: This study provides insights into the differences and similarities in tumor-specific immunogenicity related to targeted immune treatments. The antibody epitopes as biomarkers reflect melanoma-associated features of immune response, and also provide insights into the molecular pathways contributing to the pathogenesis of cancer. Concluding, antibody epitope response can be useful in predicting anti-cancer immunity elicited by immunotherapy.

Immune-instructive materials as new tools for immunotherapy.

Immune instructive materials, are materials with the ability to modulate or mimic the function of immune cells, provide exciting opportunities for developing new therapies in many areas including medical devices, chronic inflammation, cancer, and autoimmune diseases. In this review we highlight some of the latest research involving material-based strategies for modulating macrophage phenotype and dendritic cell function, as well as a brief description on biomaterial use in T cell and natural killer cell engineering. We highlight studies on material topography, size, shape and surface chemistry to reduce inflammation, along with scaffold and hydrogel delivery systems that are used for modulating DC phenotype and influencing T cell polarization. Artificial antigen presenting cells are also reviewed as a promising approach to cancer immunotherapy.

Reversed-engineered human alveolar lung-on-a-chip model.

Here, we present a physiologically relevant model of the human pulmonary alveoli. This alveolar lung-on-a-chip platform is composed of a three-dimensional porous hydrogel made of gelatin methacryloyl with an inverse opal structure, bonded to a compartmentalized polydimethylsiloxane chip. The inverse opal hydrogel structure features well-defined, interconnected pores with high similarity to human alveolar sacs. By populating the sacs with primary human alveolar epithelial cells, functional epithelial monolayers are readily formed. Cyclic strain is integrated into the device to allow biomimetic breathing events of the alveolar lung, which, in addition, makes it possible to investigate pathological effects such as those incurred by cigarette smoking and severe acute respiratory syndrome coronavirus 2 pseudoviral infection. Our study demonstrates a unique method for reconstitution of the functional human pulmonary alveoli in vitro, which is anticipated to pave the way for investigating relevant physiological and pathological events in the human distal lung.

Discovery of synergistic material-topography combinations to achieve immunomodulatory osteoinductive biomaterials using a novel in vitro screening method: The ChemoTopoChip.

Human mesenchymal stem cells (hMSCs) are widely represented in regenerative medicine clinical strategies due to their compatibility with autologous implantation. Effective bone regeneration involves crosstalk between macrophages and hMSCs, with macrophages playing a key role in the recruitment and differentiation of hMSCs. However, engineered biomaterials able to simultaneously direct hMSC fate and modulate macrophage phenotype have not yet been identified. A novel combinatorial chemistry-topography screening platform, the ChemoTopoChip, is used here to identify materials suitable for bone regeneration by screening 1008 combinations in each experiment for human immortalized mesenchymal stem cell (hiMSCs) and human macrophage response. The osteoinduction achieved in hiMSCs cultured on the "hit" materials in basal media is comparable to that seen when cells are cultured in osteogenic media, illustrating that these materials offer a materials-induced alternative to osteo-inductive supplements in bone-regeneration. Some of these same chemistry-microtopography combinations also exhibit immunomodulatory stimuli, polarizing macrophages towards a pro-healing phenotype. Maximum control of cell response is achieved when both chemistry and topography are recruited to instruct the required cell phenotype, combining synergistically. The large combinatorial library allows us for the first time to probe the relative cell-instructive roles of microtopography and material chemistry which we find to provide similar ranges of cell modulation for both cues. Machine learning is used to generate structure-activity relationships that identify key chemical and topographical features enhancing the response of both cell types, providing a basis for a better understanding of cell response to micro topographically patterned polymers.

Corrigendum to 'LC-MS metabolomics comparisons of cancer cell and macrophage responses to methotrexate and polymer-encapsulated methotrexate' [International Journal of Pharmaceutics: X Volume 1 (2019) 100036].

[This corrects the article DOI: 10.1016/j.ijpx.2019.100036.].

Role of exosomes in malignant glioma: microRNAs and proteins in pathogenesis and diagnosis.

Malignant gliomas are the most common and deadly type of central nervous system tumors. Despite some advances in treatment, the mean survival time remains only about 1.25 years. Even after surgery, radiotherapy and chemotherapy, gliomas still have a poor prognosis. Exosomes are the most common type of extracellular vesicles with a size range of 30 to 100 nm, and can act as carriers of proteins, RNAs, and other bioactive molecules. Exosomes play a key role in tumorigenesis and resistance to chemotherapy or radiation. Recent evidence has shown that exosomal microRNAs (miRNAs) can be detected in the extracellular microenvironment, and can also be transferred from cell to cell via exosome secretion and uptake. Therefore, many recent studies have focused on exosomal miRNAs as important cellular regulators in various physiological and pathological conditions. A variety of exosomal miRNAs have been implicated in the initiation and progression of gliomas, by activating and/or inhibiting different signaling pathways. Exosomal miRNAs could be used as therapeutic agents to modulate different biological processes in gliomas. Exosomal miRNAs derived from mesenchymal stem cells could also be used for glioma treatment. The present review summarizes the exosomal miRNAs that have been implicated in the pathogenesis, diagnosis and treatment of gliomas. Moreover, exosomal proteins could also be involved in glioma pathogenesis. Exosomal miRNAs and proteins could also serve as non-invasive biomarkers for prognosis and disease monitoring. Video Abstract.

Metabolic characterisation of THP-1 macrophage polarisation using LC-MS-based metabolite profiling.

INTRODUCTION: Macrophages constitute a heterogeneous population of functionally distinct cells involved in several physiological and pathological processes. They display remarkable plasticity by changing their phenotype and function in response to environmental cues representing a spectrum of different functional phenotypes. The so-called M1 and M2 macrophages are often considered as representative of pro- and anti-inflammatory ends of such spectrum. Metabolomics approach is a powerful tool providing important chemical information about the cellular phenotype of living systems, and the changes in their metabolic pathways in response to various perturbations. OBJECTIVES: This study aimed to characterise M1 and M2 phenotypes in THP-1 macrophages in order to identify characteristic metabolites of each polarisation state. METHODS: Herein, untargeted liquid chromatography (LC)-mass spectrometry (MS)-based metabolite profiling was applied to characterise the metabolic profile of M1-like and M2-like THP-1 macrophages. RESULTS: The results showed that M1 and M2 macrophages have distinct metabolic profiles. Sphingolipid and pyrimidine metabolism was significantly changed in M1 macrophages whereas arginine, proline, alanine, aspartate and glutamate metabolism was significantly altered in M2 macrophages. CONCLUSION: This study represents successful application of LC-MS metabolomics approach to characterise M1 and M2 macrophages providing functional readouts that show unique metabolic signature for each phenotype. These data could contribute to a better understanding of M1 and M2 functional properties and could pave the way for developing new therapeutics targeting different immune diseases.

A composite Gelatin/hyaluronic acid hydrogel as an ECM mimic for developing mesenchymal stem cell-derived epithelial tissue patches.

Here we report fabrication of Gelatin-based biocomposite films and their application in developing epithelial patches. The films were loaded with an epithelial cell growth factor cocktail and used as an extracellular matrix mimic for in vitro regeneration of organized respiratory epithelium using Calu-3 cell line and mesenchymal stem cells (MSCs). Our data show differentiation of Calu-3 cells on composite films as evidenced by tight junction protein expression and barrier formation. The films also supported attachment, migration, and proliferation of alveolar basal epithelial cell line A549. We also show the suitability of the composite films as a biomimetic scaffold and growth factor delivery platform for differentiation of human MSCs to epithelial cells. MSCs differentiation to the epithelial lineage was confirmed by staining for epithelial and stem cell specific markers. Our data show that the MSCs acquire the epithelial characteristics after 2 weeks with significant reduction in vimentin, increase in pan cytokeratin expression, and morphological changes. However, despite the expression of epithelial lineage markers, these cells did not form fully functional tight junctions as evidenced by low expression of junctional protein ZO1. Further optimisation of culture conditions and growth factor cocktail is required to enhance tight junction formation in MSCs-derived epithelial cells on the composite hydrogels. Nevertheless, our data clearly highlight the possibility of using MSCs in epithelial tissue engineering and the applicability of the composite hydrogels as transferrable extracellular matrix mimics and delivery platforms with potential applications in regenerative medicine and in vitro modelling of barrier tissues.

Investigating the intracellular effects of hyperbranched polycation-DNA complexes on lung cancer cells using LC-MS-based metabolite profiling.

Cationic polymers have emerged as a promising alternative to viral vectors in gene therapy. They are cheap to scale up, easy to functionalise and are potentially safer than viral vectors, however many are cytotoxic. The large number of polycations, designed to address the toxicity problem, raises a practical need to develop a fast and reliable method for assessing the safety of these materials. In this regard, metabolomics provides a detailed and comprehensive method that can assess the potential toxicity at the cellular and molecular level. Here, we applied metabolomics to investigate the impact of hyperbranched polylysine, hyperbranched polylysine-co-histidine and branched polyethyleneimine polyplexes at sub-toxic concentrations on the metabolic pathways of A459 and H1299 lung carcinoma cell lines. The study revealed that the polyplexes downregulated metabolites associated with glycolysis and the TCA cycle, and induced oxidative stress in both cell lines. The relative changes of the metabolites indicated that the polyplexes of polyethyleneimine and hyperbranched polylysine affected the metabolism much more than the polyplexes of hyperbranched polylysine-co-histidine. This was in line with transfection results, suggesting a correlation between the toxicity and transfection efficiency of these polyplexes. Our work highlights the importance of the metabolomics approach not just to assess the potential toxicity of polyplexes but also to understand the molecular mechanisms underlying any adverse effects, which could help in designing more efficient vectors.

LC-MS metabolomics comparisons of cancer cell and macrophage responses to methotrexate and polymer-encapsulated methotrexate.

Methotrexate (MTX) is a folate analogue antimetabolite widely used for the treatment of rheumatoid arthritis and cancer. A number of studies have shown that MTX delivered via nanoparticle carriers is more potent against cancer cells than free MTX, a phenomenon attributed to higher cellular uptake of the particles compared to the saturable folate receptor pathway. In this study, a cell-based global metabolic profiling approach was applied to study the effects of MTX in both free drug form and when encapsulated in -poly(lactide-co-glycolide) (PLGA) nanoparticles on a cancer cell line, A549, and also on human-like THP-1 macrophages. The results showed that MTX loaded nanoparticles had less impact on the macrophages than free MTX, and the effects on macrophages were limited to changes in nucleotide metabolism and suppression of the tricarboxylic acid cycle, whereas free MTX also led to a drop in glycolytic activity and impairment in redox homeostasis. In contrast, MTX loaded nanoparticles showed a greater impact on A549 cells than the free drug, which was in accord with studies in other cell lines in prior literature with MTX-carrier nanoparticles.

Photocrosslinkable Gelatin Hydrogels Modulate the Production of the Major Pro-inflammatory Cytokine, TNF-α, by Human Mononuclear Cells.

Hydrogels are an attractive class of biomaterials in tissue engineering due to their inherently compatible properties for cell culture. Gelatin methacryloyl (GelMA) has shown significant promise in the fields of tissue engineering and drug delivery, as its physical properties can be precisely tuned depending on the specific application. There is a growing appreciation for the interaction between biomaterials and cells of the immune system with the increasing usage of biomaterials for in vivo applications. Here, we addressed the current lack of information regarding the immune-modulatory properties of photocrosslinked GelMA. We investigated the ability of human mononuclear cells to mount inflammatory responses in the context of a GelMA hydrogel platform. Using lipopolysaccharide to stimulate a pro-inflammatory immune response, we found tumor necrosis factor-α (TNF-α) expression was suppressed in GelMA culture conditions. Our findings have important implications on the future use of GelMA, and potentially similar hydrogels, and highlight the significance of investigating the potential immune-modulatory properties of biomaterials.

Incorporation of resident macrophages in engineered tissues: Multiple cell type response to microenvironment controlled macrophage-laden gelatine hydrogels.

The success of tissue engineering strategy is strongly related to the inflammatory response, mainly through the activity of macrophages that are key cells in initial immune response to implants. For engineered tissues, the presence of resident macrophages can be beneficial for maintenance of homeostasis and healing. Thus, incorporation of macrophages in engineered tissues can facilitate the integration upon implantation. In this study, an in-vitro model of interaction was developed between encapsulated naive monocytes, macrophages induced with M1/M2 stimulation and incoming cells for immune assisted tissue engineering applications. To mimic the wound healing cascade, naive THP-1 monocytes, endothelial cells and fibroblasts were seeded on the gels as incoming cells. The interaction was first monitored in the absence of the gels. To mimic resident macrophages, THP-1 cells were encapsulated in the presence or absence of IL-4 to control their phenotype and then these hydrogels were seeded with incoming cells. Without encapsulation, activated macrophages induce apoptosis in endothelial cells. Once encapsulated no adverse effects were seen. Macrophage-laden hydrogels attracted more endothelial cells and fibroblasts compared to monocytes-laden hydrogels. The induction (M2 stimulation) of encapsulated macrophages did not change the overall number of attracted cells; but significantly affected their morphology. M1 stimulation by a defined media resulted in more secretion of both pro- and anti-inflammatory cytokines compared to M2 stimulation. It was demonstrated that there is a distinct effect of encapsulated macrophages on the behaviour of the incoming cells; this effect can be harnessed to establish a microenvironment more prone to regeneration upon implantation.

Integrin-Mediated Interactions Control Macrophage Polarization in 3D Hydrogels.

Adverse immune reactions prevent clinical translation of numerous implantable devices and materials. Although inflammation is an essential part of tissue regeneration, chronic inflammation ultimately leads to implant failure. In particular, macrophage polarity steers the microenvironment toward inflammation or wound healing via the induction of M1 and M2 macrophages, respectively. Here, this paper demonstrates that macrophage polarity within biomaterials can be controlled through integrin-mediated interactions between human monocytic THP-1 cells and collagen-derived matrix. Surface marker, gene expression, biochemical, and cytokine profiling consistently indicate that THP-1 cells within a biomaterial lacking cell attachment motifs yield proinflammatory M1 macrophages, whereas biomaterials with attachment sites in the presence of interleukin-4 (IL-4) induce an anti-inflammatory M2-like phenotype and propagate the effect of IL-4 in induction of M2-like macrophages. Importantly, integrin α2ß1 plays a pivotal role as its inhibition blocks the induction of M2 macrophages. The influence of the microenvironment of the biomaterial over macrophage polarity is further confirmed by its ability to modulate the effect of IL-4 and lipopolysaccharide, which are potent inducers of M2 or M1 phenotypes, respectively. Thus, this study represents a novel, versatile, and effective strategy to steer macrophage polarity through integrin-mediated 3D microenvironment for biomaterial-based programming.

Image based Machine Learning for identification of macrophage subsets.

Macrophages play a crucial rule in orchestrating immune responses against pathogens and foreign materials. Macrophages have remarkable plasticity in response to environmental cues and are able to acquire a spectrum of activation status, best exemplified by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at the two ends of the spectrum. Characterisation of M1 and M2 subsets is usually carried out by quantification of multiple cell surface markers, transcription factors and cytokine profiles. These approaches are time-consuming, require large numbers of cells and are resource intensive. In this study, we used machine learning algorithms to develop a simple and fast imaging-based approach that enables automated identification of different macrophage functional phenotypes using their cell size and morphology. Fluorescent microscopy was used to assess cell morphology of different cell types which were stained for nucleus and actin distribution using DAPI and phalloidin respectively. By only analysing their morphology we were able to identify M1 and M2 phenotypes effectively and could distinguish them from naïve macrophages and monocytes with an average accuracy of 90%. Thus we suggest high-content and automated image analysis can be used for fast phenotyping of functionally diverse cell populations with reasonable accuracy and without the need for using multiple markers.

The role of indoleamine 2,3-dioxygenase-aryl hydrocarbon receptor pathway in the TLR4-induced tolerogenic phenotype in human DCs.

A controlled inflammatory response is required for protection against infection, but persistent inflammation causes tissue damage. Dendritic cells (DCs) have a unique capacity to promote both inflammatory and anti-inflammatory processes. One key mechanism involved in DC-mediated immunosuppression is the expression of tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO has been implicated in diverse processes in health and disease but its role in endotoxin tolerance in human DCs is still controversial. Here we investigated the role of IDO in shaping DCs phenotype and function under endotoxin tolerance conditions. Our data show that TLR4 ligation in LPS-primed DCs, induced higher levels of both IDO isoforms together with the transcription factor aryl-hydrocarbon receptor (AhR), compared to unprimed controls. Additionally, LPS conditioning induced an anti-inflammatory phenotype in DCs - with an increase in IL-10 and higher expression of programmed death ligand (PD-L)1 and PD-L2 - which were partially dependent on IDO. Furthermore, we demonstrated that the AhR-IDO pathway was responsible for the preferential activation of non-canonical NF-κB pathway in LPS-conditioned DCs. These data provide new insight into the mechanisms of the TLR4-induced tolerogenic phenotype in human DCs, which can help the better understanding of processes involved in induction and resolution of chronic inflammation and tolerance.

Generation of anti-inflammatory macrophages for implants and regenerative medicine using self-standing release systems with a phenotype-fixing cytokine cocktail formulation.

The immediate tissue microenvironment of implanted biomedical devices and engineered tissues is highly influential on their long term fate and efficacy. The creation of a long-term anti-inflammatory microenvironment around implants and artificial tissues can facilitate their integration. Macrophages are highly plastic cells that define the tissue reactions on the implanted material. Local control of macrophage phenotype by long-term fixation of their healing activities and suppression of inflammatory reactions are required to improve implant acceptance. Herein, we describe the development of a cytokine cocktail (M2Ct) that induces stable M2-like macrophage phenotype with significantly decreased pro-inflammatory cytokine and increased anti-inflammatory cytokine secretion profile. The positive effect of the M2Ct was shown in an in vitro wound healing model; where M2Ct facilitated wound closure by human fibroblasts in co-culture conditions. Using a model for induction of inflammation by LPS we have shown that the M2Ct phenotype is stable for 12days. However, in the absence of M2Ct in the medium macrophages underwent rapid pro-inflammatory re-programming upon IFNg stimulation. Therefore, loading and release of the cytokine cocktail from a self-standing, transferable gelatin/tyraminated hyaluronic acid based release system was developed to stabilize macrophage phenotype for in vivo applications in implantation and tissue engineering. The M2Ct cytokine cocktail retained its anti-inflammatory activity in controlled release conditions. Our data indicate that the direct application of a potent M2 inducing cytokine cocktail in a transferable release system can significantly improve the long term functionality of biomedical devices by decreasing pro-inflammatory cytokine secretion and increasing the rate of wound healing. STATEMENT OF SIGNIFICANCE: Uncontrollable activation of macrophages in the microenvironment of implants and engineered tissues is a significant problem leading to poor integration of implants and artificial tissues. In the current manuscript we demonstrate that self-standing, transferable gelatin/tyraminated hyaluronic acid based thin films are perspective tools for controlled release of anti-inflammatory cytokine combinations and can be used to down-modulate macrophage activation on implant surfaces. We also show that optimized cytokine cocktail consisting of IL4/IL10/TGFß1 (M2Ct) induces long-term anti-inflammatory and pro-healing phenotype in human primary monocyte-derived macrophages. This cocktail formulation could be loaded on gelatin/tyraminated films and promoted favorable M2-like macrophage phenotype with low responsiveness to pro-inflammatory stimuli. Such self-standing release systems can be used for prolonged local control of macrophage phenotype upon implantation.