Optimization of Interleukin-10 incorporation for dendritic cells embedded in Poly(ethylene glycol) hydrogels.

Translational research in biomaterials and immunoengineering is leading to the development of novel advanced therapeutics to treat diseases such as cancer, autoimmunity, and viral infections. Dendritic cells (DCs) are at the center of these therapeutics given that they bridge innate and adaptive immunity. The biomaterial system developed herein uses a hydrogel carrier to deliver immunomodulatory DCs for amelioration of autoimmunity. This biomaterial vehicle is comprised of a poly (ethylene glycol)-4 arm maleimide (PEG-4MAL) hydrogels, conjugated with the immunosuppressive cytokine, interleukin-10, IL-10, and cross-linked with a collagenase-degradable peptide sequence for the injectable delivery of immunosuppressive DCs to an anatomical disease-relevant site of the cervical lymph nodes, for intended application to treat multiple sclerosis. The amount of IL-10 incorporated in the hydrogel was optimized to be 500 ng in vitro, based on immunological endpoints. At this concentration, DCs exhibited the best viability, most immunosuppressive phenotype, and protection against proinflammatory insult as compared with hydrogel-incorporated DCs with lower IL-10 loading amounts. Additionally, the effect of the degradability of the PEG-4MAL hydrogel on the release rate of incorporated IL-10 was assessed by varying the ratio of degradable peptides: VPM (degradable) and DTT (nondegradable) and measuring the IL-10 release rates. This IL-10-conjugated hydrogel delivery system for immunosuppressive DCs is set to be assessed for in vivo functionality as the immunosuppressive cytokine provides a tolerogenic environment that keeps DCs in their immature phenotype, which consequently enhances cell viability and optimizes the system's immunomodulatory functionality.

Brief exposure to hyperglycemia activates dendritic cells in vitro and in vivo.

Dendritic cells are key players in regulating immunity. These cells both activate and inhibit the immune response depending on their cellular environment. Their response to hyperglycemia, a condition common amongst diabetics wherein glucose is abnormally elevated, remains to be elucidated. In this study, the phenotype and immune response of dendritic cells exposed to hyperglycemia were characterized in vitro and in vivo using the streptozotocin-induced diabetes model. Dendritic cells were shown to be sensitive to hyperglycemia both during and after differentiation from bone marrow precursor cells. Dendritic cell behavior under hyperglycemic conditions was found to vary by phenotype, among which, tolerogenic dendritic cells were particularly sensitive. Expression of the costimulatory molecule CD86 was found to reliably increase when dendritic cells were exposed to hyperglycemia. Additionally, hydrogel-based delivery of the anti-inflammatory molecule interleukin-10 was shown to partially inhibit these effects in vivo.

Biomaterial Strategies for Immunomodulation.

Strategies to enhance, suppress, or qualitatively shape the immune response are of importance for diverse biomedical applications, such as the development of new vaccines, treatments for autoimmune diseases and allergies, strategies for regenerative medicine, and immunotherapies for cancer. However, the intricate cellular and molecular signals regulating the immune system are major hurdles to predictably manipulating the immune response and developing safe and effective therapies. To meet this challenge, biomaterials are being developed that control how, where, and when immune cells are stimulated in vivo, and that can finely control their differentiation in vitro. We review recent advances in the field of biomaterials for immunomodulation, focusing particularly on designing biomaterials to provide controlled immunostimulation, targeting drugs and vaccines to lymphoid organs, and serving as scaffolds to organize immune cells and emulate lymphoid tissues. These ongoing efforts highlight the many ways in which biomaterials can be brought to bear to engineer the immune system.

Differential functional effects of biomaterials on dendritic cell maturation.

The immunological outcome of dendritic cell (DC) treatment with different biomaterials was assessed to demonstrate the range of DC phenotypes induced by biomaterials commonly used in combination products. Immature DCs (iDCs) were derived from human peripheral blood monocytes, and treated with different biomaterial films of alginate, agarose, chitosan, hyaluronic acid (HA), or 75:25 poly(lactic-co-glycolic acid) (PLGA) and a comprehensive battery of phenotypic functional outcomes was assessed. Different levels of functional changes in DC phenotype were observed depending on the type of biomaterial films used to treat the DCs. Treatment of DCs with PLGA or chitosan films supported DC maturation, with higher levels of DC allostimulatory capacity, pro-inflammatory cytokine release, and expression of CD80, CD86, CD83, HLA-DQ and CD44 compared with iDCs, and lower endocytic ability compared with iDCs. Alginate film induced pro-inflammatory cytokine release from DCs at levels higher than from iDCs. Dendritic cells treated with HA film expressed lower levels of CD40, CD80, CD86 and HLA-DR compared with iDCs. They also exhibited lower endocytic ability and CD44 expression than iDCs, possibly due to an insolubilized (cross-linked) form of high molecular weight HA. Interestingly, treatment of DCs with agarose film maintained the DC functional phenotype at levels similar to iDCs except for CD44 expression, which was lower than that of iDCs. Taken together, these results can provide selection criteria for biomaterials to be used in immunomodulating applications and can inform potential outcomes of biomaterials within combination products on associated immune responses as desired by the application.

Macrophage and dendritic cell phenotypic diversity in the context of biomaterials.

Macrophages (Mϕ) and dendritic cells (DCs) are critical antigen presenting cells that play pivotal roles in host responses to biomaterial implants. Although Mϕs have been widely studied for their roles in the inflammatory responses against biomaterials, the roles that DCs play in the host responses toward implanted materials have only recently been explored. DCs are of significant research interest because of the emergence of a large number of combination products that cross-traditional medical device boundaries. These products combine biomaterials with biologics, including cells, nucleic acids, and/or proteins. The biomaterial component may evoke an inflammatory response, primarily mediated by neutrophils and Mϕs, whereas the biologic component may elicit an immunogenic immune response, initiated by DCs involving lymphocyte activation. Control of Mϕ phenotypic balance from proinflammatory M1 to reparative M2 is a goal of investigators to optimize the host response to biomaterials. Similarly, control of DC phenotype from proinflammatory to toleragenic is of interest in vaccine delivery and tissue engineering/transplantation situations, respectively. This review discusses the interconnection between innate and adaptive immunity, the comparative and contrasting phenotypes and roles of Mϕs and DCs in immunity, their responses to biomaterials and the strategies to modulate their phenotype for applications in tissue engineering and vaccine delivery. Furthermore, the collaboration between and unique roles of DCs and Mϕs needs to be addressed in future studies to gain a more complete picture of host responses toward combination products.

Altered adherent leukocyte profile on biomaterials in Toll-like receptor 4 deficient mice.

The host response to a biomaterial is characterized by both acute recruitment and attachment of cells as well as chronic encapsulating tissue reaction. The implantation procedure induces production of damage-associated molecular patterns (DAMPs) which may contribute to host recognition of the material. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that bind not only pathogen-associated molecular patterns (PAMPs) but also DAMPs. We sought to investigate whether TLR4/DAMP interactions were involved in the acute and chronic inflammatory response to an implanted biomaterial. When PET discs were implanted intraperitoneally for 16h, no differences were found in the number of leukocytes recruited between TLR4(+) (C57BL/10J) and TLR4(-) (C57BL/10ScNJ) mice. However, a significant shift in the leukocyte profile on the biomaterial surface was observed for TLR4(-) mice. While the total number of adherent cells was the same in both strains, TLR4(+) mice had a profile with equivalent neutrophil and monocyte/macrophage presence on the material surface, and TLR4(-) mice had a profile of predominantly neutrophils with fewer monocyte/macrophages. When implants were placed subcutaneously for 2 weeks, the fibrous capsule thicknesses were not different between TLR4(+) and TLR4(-) mouse strains. These findings illustrate that TLR4 may play a role in the initial recognition of a biomaterial by directing the adhesive cellular profile.

Comparative characterization of cultures of primary human macrophages or dendritic cells relevant to biomaterial studies.

Macrophages are central mediators of biomaterial-associated wound healing. Dendritic cells (DCs) link innate and adaptive immunity and are important in the context of the host response to combination products. Starting with human peripheral blood mononuclear cells (PBMCs), DCs were derived from monocytes upon culture with granulocyte macrophage colony-stimulating factor and interleukin-4; macrophages were derived from monocytes upon culture without cytokines. Macrophage or DC cultures were characterized at relevant timepoints in both adherent and nonadherent fractions on control Primaria surfaces to characterize and define these inflammatory/immune cells as a prequel to their use in in vitro test biomaterial-host response studies. At day 10 (typical time for harvesting macrophages for subsequent treatment with test biomaterials), macrophages were CD11c+, macrophage mannose receptor (MMR)+, CD14+, and CD64+. At day 6 (typical time for harvesting of DCs after 24-h treatment with test biomaterials), DCs were CD1c+, CD11c+, CD123+, MMR+, CD14+, and CD64-. Furthermore, CD3+ and CD4+ T lymphocytes and CD19+ and CD24+ B lymphocytes were present in both cultures at all timepoints, although to different extents. Immature DCs (approximately 15 microm), were rounded but presented extensive dendritic processes upon maturation with lipopolysaccharide. Alternatively, adherent macrophages (approximately 15-20 microm) displayed internalized lipids and exhibited few membrane processes. The characterization and comparison of existing techniques to establish reliable, reproducible primary cultures of DCs or macrophages provides an important basis for examining and interpreting complex macrophage/DC-lymphocyte-orchestrated host responses in future studies with equivalent cell populations on test biomaterials.

Dendritic cell responses to self-assembled monolayers of defined chemistries.

Biomaterial contact triggers dendritic cell (DC) maturation, to an extent depending on the biomaterial, ultimately enhancing an immune response toward associated antigens, implying a role for biomaterials as adjuvants. Self-assembled monolayers (SAM) of alkanethiols on titanium/gold-coated surfaces presenting different chemistries were used to study effects of biomaterial surface chemistry on DC maturation. Although DCs treated with OH, COOH, or NH(2) SAMs showed modest maturation, those treated with CH(3) SAMs were least mature, all based on cytospins, allostimulatory capacity, or maturation marker expression. Surprisingly, DCs treated with CH(3) SAMs secreted highest levels of proinflammatory tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6) but were least mature. Secretion of anti-inflammatory mediators by DCs treated with CH(3) SAMs was not responsible for mitigating DC maturation under these conditions. Interestingly, elevated levels of apoptotic markers were measured associated with DCs and T cells upon CH(3) SAMs contact. Since phagocytosis of apoptotic DCs has strong immunosuppressive effects on DCs, more apoptotic DCs on CH(3) SAMs may account for lower DC maturation. Finally, higher expression of cytotoxic T lymphocyte associated antigen receptor-4 (CTLA-4) on T cells may imply a mechanism of T cell inhibition on CH(3) SAMs.

Reduced acute inflammatory responses to microgel conformal coatings.

Implantation of synthetic materials into the body elicits inflammatory host responses that limit medical device integration and biological performance. This inflammatory cascade involves protein adsorption, leukocyte recruitment and activation, cytokine release, and fibrous encapsulation of the implant. We present a coating strategy based on thin films of poly(N-isopropylacrylamide) hydrogel microparticles (i.e. microgels) cross-linked with poly(ethylene glycol) diacrylate. These particles were grafted onto a clinically relevant polymeric material to generate conformal coatings that significantly reduced in vitro fibrinogen adsorption and primary human monocyte/macrophage adhesion and spreading. These microgel coatings also reduced leukocyte adhesion and expression of pro-inflammatory cytokines (TNF-alpha, IL-1beta, MCP-1) in response to materials implanted acutely in the murine intraperitoneal space. These microgel coatings can be applied to biomedical implants as a protective coating to attenuate biofouling, leukocyte adhesion and activation, and adverse host responses for biomedical and biotechnological applications.

Complimentary endothelial cell/smooth muscle cell co-culture systems with alternate smooth muscle cell phenotypes.

Development of in vitro models of native and injured vasculature is crucial for better understanding altered wound healing in disease, device implantation, or tissue engineering. Conditions were optimized using polyethyleneteraphalate transwell filters for human aortic endothelial cell (HAEC)/smooth muscle cell (HASMC) co-cultures with divergent HASMC phenotypes ('more or less secretory') while maintaining quiescent HAECs. Resulting HASMC phenotype was studied at 48 and 72 h following co-culture initiation, and compared to serum and growth factor starved monocultured 'forced contractile' HASMCs. Forced contractile HASMCs demonstrated organized alpha-smooth muscle actin filaments, minimal interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion, and low intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and tissue factor expression. Organization of alpha-smooth muscle actin was lost in 'more secretory' HASMCs in co-culture with HAECs, and IL-8 and MCP-1 secretion, as well as ICAM-1, VCAM-1, and tissue factor expression were significantly upregulated at both time points. Alternately, 'less secretory' HASMCs in co-culture with HAECs showed similar characteristics to forced contractile HASMCs at the 48 h time point, while by the 72 h time point they behaved similarly to 'more secretory' HASMCs. These co-culture systems could be useful in better understanding vascular healing, however there remain time constraint considerations for maintaining culture integrity/cell phenotype.

Effect of poly(lactic-co-glycolic acid) contact on maturation of murine bone marrow-derived dendritic cells.

Understanding of biomaterial adjuvant effect and its mechanisms is essential for the effective design and selection of appropriate materials for specific applications. We have previously shown that poly(lactic-co-glycolic acid) (PLGA), one of the most commonly studied polymers in tissue engineering, supports an adjuvant effect as measured by enhanced immune response against a co-delivered model antigen, which was dependent on the form of the biomaterial. Furthermore, we have shown that PLGA induces the maturation of human peripheral blood mononuclear cell-derived dendritic cells (DCs) in vitro. In this study, the effect of PLGA contact on the maturation of murine bone marrow-derived DCs was investigated in part to explain the biomaterial adjuvant effect observed. Treatment of bone marrow-derived DCs from C57BL6 mice with PLGA microparticles or films lead to maturation of these cells as exemplified by increased expression of co-stimulatory molecules CD80 and CD86 and production of proinflammatory cytokines TNF-alpha and IL-6. These results suggest that PLGA contact induces maturation of murine DCs, supporting our observations with human DCs. With the techniques developed in this study and given the results, our future goal is to utilize transgenic murine models to delineate the mechanisms of biomaterial-induced DC maturation.

Molecular aspects of microparticle phagocytosis by dendritic cells.

The ability of immature dendritic cells (iDCs) derived from human peripheral blood mononuclear cells to phagocytose poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) as compared to polystyrene MPs and the molecular aspects of this phagocytosis were investigated. Treating iDCs with PLGA or polystyrene fluorospheres of approximately 3 microm in diameter resulted in the internalization of the particles as evidenced by confocal laser scanning micrographs. This uptake of fluorospheres by DCs was decreased by pretreatment of cells with cytochalasin D or by incubation with the fluorospheres at 4 degrees C, and was sensitive to EDTA and trypsin pretreatments in a dose-dependent manner. In agreement with our previous studies, treatment of iDCs with PLGA MPs, but not with polystyrene MPs, led to DC maturation, as measured by increase in release of the autocrine maturation cytokine, tumor necrosis factor-alpha, which was dependent on ratio of PLGA MPs to DCs. Taken together, this work begins to address the role of phagocytosis on PLGA MP-induced DC maturation and the molecular mechanisms involved.

Differential effects of agarose and poly(lactic-co-glycolic acid) on dendritic cell maturation.

Application of biomaterials in combination products in which the biomaterial is presented to the host with a biological component prompts the need for understanding the biomaterial-associated adjuvant effect in the immune response against antigens associated with such a product. We have previously demonstrated that a polymer commonly used in tissue engineering and vaccine delivery, poly(lactic-co-glycolic acid) (PLGA), exerts an adjuvant effect in vivo, which was supported by PLGA-induced dendritic cell (DC) maturation in vitro. In this study, the effects of agarose and PLGA on DC maturation were compared in vitro to establish differential biomaterial effects. Human monocyte-derived DCs were treated with agarose or PLGA microparticles or films, and their maturation effect was measured as expression of costimulatory and MHC class II molecules, allostimulatory capacity, and proinflammatory cytokine secretion. Direct comparison of DC maturation phenotype indicated that PLGA was a stronger stimulus of DC maturation than agarose, and this maturation was not affected by microparticle phagocytosis. However, agarose-treated DCs showed higher activation of nuclear factor kappaB (NFkappaB) 24 h after the initial stimulation of DCs. Taken together, these results demonstrate differential biomaterial effects on DC maturation, substantiating the maturation effect of PLGA, and provide screening methods for biomaterial adjuvant effect for applications in combination products.

Differential levels of dendritic cell maturation on different biomaterials used in combination products.

Immature dendritic cells (iDCs) were derived from human peripheral blood monocytes, and treated with films of biomaterials commonly used in combination products (e.g., tissue engineered constructs or vaccines) to assess the resultant dendritic cell (DC) maturation compared to positive control of lipopolysaccharide (LPS) treatment for DC maturation or negative control of untreated iDCs. The following biomaterials were tested: alginate, agarose, chitosan, hyaluronic acid, 75:25 poly(lactic-co-glycolic acid) (PLGA). The effect of DC culture on these films was undertaken to identify biomaterials which support DC maturation and those biomaterials that did not. Dendritic cells treated with chitosan or PLGA (agarose to a lesser extent) films increased expression levels of CD86, CD40, and HLA-DQ, compared to control iDCs, similar to LPS-matured DCs, whereas DCs treated with alginate or hyaluronic acid films decreased their expression levels of these same molecules. In summary, a differential effect of the biomaterial on which iDCs were cultured was observed as far as the extent of induced DC maturation. The effect of biomaterials on DC maturation, and the associated adjuvant effect, is a novel biocompatibility selection and design criteria for biomaterials to be used in combination products in which immune consequences are potential complications or outcomes.

Procoagulant phenotype of endothelial cells after coculture with biomaterial-treated blood cells.

Understanding endothelial cell (EC)/blood/biomaterial interactions is crucial for the advancement of cardiovascular devices that often fail because of the lack of nonthrombogenic biomaterials. To begin to assess these interactions, a static EC/blood cell/biomaterial model was used. Isolated blood cells were pretreated with model biomaterial beads with different surface chemistries: polystyrene (PS), and PS beads grafted with 3-kDa polyethylene glycol (PEG) with either a hydroxyl (PS-PEG-OH) or amine (PS-PEG-NH(2)) terminal group at 5.4 or 54 x 10(4) beads/mL. Biomaterial-treated monocytes, neutrophils, or platelets were applied to human umbilical vein ECs (HUVECs) for 5 or 24 h of static coculture, and the resultant procoagulant HUVEC phenotype was characterized using several methods. Flow cytometry was used to assess surface expression of tissue factor (TF), adenosine triphosphate diphosphohydrolase, phosphatidylserine, and thrombomodulin, a functional TF assay was used to assess TF activity, and a plasma recalcification assay examined clotting times on HUVECs. Static coculture of HUVEC with biomaterial-treated neutrophils induced a procoagulant phenotype as exemplified by upregulation of TF expression and total functional activity, and downregulation of adenosine triphosphate diphosphohydrolase and thrombomodulin expression. The plasma recalcification assay demonstrated that HUVECs cocultured with biomaterial-treated monocytes significantly shortened clotting times, with some effect of similarly treated neutrophils.