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Hyaluronic acid (HA) is a glycosaminoglycan in the extracellular matrix with immunoregulatory properties depending on its molecular weight (MW). However, the impact of matrix-bound HA on dendritic cells (DCs) remains unclear due to varying distribution of HA MW under different physiological conditions. To investigate DCs in defined biosystems, 3D collagen matrices modified with HA of specific MW with similar microstructure and HA levels are used. It is found that HA MW influences cytokine binding to matrix, suggesting modulation of cytokine availability by the different HA MWs. These studies on DC immune potency reveal that low MW HA (8-15 kDa) enhances immature DC differentiation and antigen uptake, while medium (MMW-HA; 500-750 kDa) and high MW HA (HMW-HA; 1250-1500 kDa) increase cytokine secretion in mature DCs. The effect on DC phenotype and cytokine secretion by different MWs of HA is independent of CD44. However, blocking the CD44 receptor reveals its potential role in regulating acute inflammation through increased secretion of CCL2, CXCL8, and IL-6. Additionally, MMW- and HMW-HA matrices reduce migratory capacity of DCs, dependent on CD44. Overall, these findings provide insights into MW-dependent effects of matrix-bound HA on DCs, opening avenues for the design of DC-modulating materials to enhance DC-based therapy.

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Microgravity accelerates the aging of various physiological systems, and it is well acknowledged that aged individuals and astronauts both have increased susceptibility to infections and poor response to vaccination. Immunologically, dendritic cells (DCs) are the key players in linking innate and adaptive immune responses. Their distinct and optimized differentiation and maturation phases play a critical role in presenting antigens and mounting effective lymphocyte responses for long-term immunity. Despite their importance, no studies to date have effectively investigated the effects of microgravity on DCs in their native microenvironment, which is primarily located within tissues. Here, we address a significantly outstanding research gap by examining the effects of simulated microgravity via a random positioning machine on both immature and mature DCs cultured in biomimetic collagen hydrogels, a surrogate for tissue matrices. Furthermore, we explored the effects of loose and dense tissues via differences in collagen concentration. Under these various environmental conditions, the DC phenotype was characterized using surface markers, cytokines, function, and transcriptomic profiles. Our data indicate that aged or loose tissue and exposure to RPM-induced simulated microgravity both independently alter the immunogenicity of immature and mature DCs. Interestingly, cells cultured in denser matrices experience fewer effects of simulated microgravity at the transcriptome level. Our findings are a step forward to better facilitate healthier future space travel and enhance our understanding of the aging immune system on Earth.

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T cells act as the puppeteers in the adaptive immune response, and their dysfunction leads to the initiation and progression of pathological conditions. During their lifetime, T cells experience myriad forces that modulate their effector functions. These forces are imposed by interacting cells, surrounding tissues, and shear forces from fluid movement. In this review, a journey with T cells is made, from their development to their unique characteristics, including the early studies that uncovered their mechanosensitivity. Then the studies pertaining to the responses of T cell activation to changes in antigen-presenting cells' physical properties, to their immediate surrounding extracellular matrix microenvironment, and flow conditions are highlighted. In addition, it is explored how pathological conditions like the tumor microenvironment can hinder T cells and allow cancer cells to escape elimination.

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We have developed a rapid technique for characterizing the biomechanical properties of dendritic cells using dielectrophoretic forces. It is widely recognized that maturing of dendritic cells modulates their stiffness and migration capabilities, which results in T-cell activation triggering the adaptive immune response. Therefore it is important to develop techniques for mechanophenotyping of immature and mature dendritic cells. The technique reported here utilizes nonuniform electric fields to exert a substantial force on the cells to induce cellular elongation for optical measurements. In addition, a large array of interdigitated electrodes allows multiple cells to be stretched simultaneously. Our results indicate a direct correlation between F-actin activity and deformability observed in dendritic cells, determined through mean fluorescence signal intensity of phalloidin.

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Atherosclerosis, the inflammation of artery walls due to the accumulation of lipids, is the most common underlying cause for cardiovascular diseases. Monocytes and macrophages are major cells that contribute to the initiation and progression of atherosclerotic plaques. During this process, an accumulation of LDL-laden macrophages (foam cells) and an alteration in the extracellular matrix (ECM) organization leads to a local vessel stiffening. Current in vitro models are carried out onto two-dimensional tissue culture plastic and cannot replicate the relevant microenvironments. To bridge the gap between in vitro and in vivo conditions, we utilized three-dimensional (3D) collagen matrices that allowed us to mimic the ECM stiffening during atherosclerosis by increasing collagen density. First, human monocytic THP-1 cells were embedded into 3D collagen matrices reconstituted at low and high density. Cells were subsequently differentiated into uncommitted macrophages (M0) and further activated into pro- (M1) and anti-inflammatory (M2) phenotypes. In order to mimic atherosclerotic conditions, cells were cultured in the presence of oxidized LDL (oxLDL) and analyzed in terms of oxLDL uptake capability and relevant receptors along with their cytokine secretomes. Although oxLDL uptake and larger lipid size could be observed in macrophages in a matrix dependent manner, monocytes showed higher numbers of oxLDL uptake cells. By analyzing major oxLDL uptake receptors, both monocytes and macrophages expressed lectin-like oxidized low-density lipoprotein receptor-1 (LOX1), while enhanced expression of scavenger receptor CD36 could be observed only in M2. Notably, by analyzing the secretome of macrophages exposed to oxLDL, we demonstrated that the cells could, in fact, secrete adipokines and growth factors in distinct patterns. Besides, oxLDL appeared to up-regulate MHCII expression in all cells, while an up-regulation of CD68, a pan-macrophage marker, was found only in monocytes, suggesting a possible differentiation of monocytes into a pro-inflammatory macrophage. Overall, our work demonstrated that collagen density in the plaque could be one of the major factors driving atherosclerotic progression via modulation of monocyte and macrophages behaviors.

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Dendritic cells (DCs) are antigen-presenting cells capable of either activating the immune response or inducing and maintaining immune tolerance. Understanding how biophysical properties affect DC behaviors will provide insight into the biology of a DC and its applications. In this work, we studied how cell culture dimensionality (two-dimensional (2D) and three-dimensional (3D)), and matrix density of 3D collagen matrices modulate differentiation and functions of DCs. Besides, we aimed to point out the different conceptual perspectives in modern immunological research, namely tissue-centric and cell-centric perspectives. The tissue-centric perspective intends to reveal how specific microenvironments dictate DC differentiation and in turn modulate DC functionalities, while the cell-centric perspective aims to demonstrate how pre-differentiated DCs behave in specific microenvironments. DC plasticity was characterized in terms of cell surface markers and cytokine secretion profiles. Subsequently, antigen internalization and T cell activation were quantified to demonstrate the cellular functions of immature DCs (iDCs) and mature DCs (mDCs), respectively. In the tissue-centric perspective, we found that expressed surface markers and secreted cytokines of both iDCs and mDCs are generally higher in 2D culture, while they are regulated by matrix density in 3D culture. In contrast, in the cell-centric perspective, we found enhanced expression of cell surface markers as well as distinct cytokine secretion profiles in both iDCs and mDCs. By analyzing cellular functions of cells in the tissue-centric perspective, we found matrix density dependence in antigen uptake by iDCs, as well as on mDC-mediated T cell proliferation in 3D cell culture. On the other hand, in the cell-centric perspective, both iDCs and mDCs appeared to lose their functional potentials to internalization antigen and T cell stimulation. Additionally, mDCs from tissue- and cell-centric perspectives modulated T cell differentiation by their distinct cytokine secretion profiles towards Th1 and Th17, respectively. In sum, our work emphasizes the importance of dimensionality, as well as collagen fibrillar density in the regulation of the immune response of DCs. Besides this, we demonstrated that the conceptual perspective of the experimental design could be an essential key point in research in immune cell-material interactions and biomaterial-based disease models of immunity.

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T cell activation is triggered by signal molecules on the surface of antigen-presenting cells (APC) and subsequent exertion of cellular forces. Deciphering the biomechanical and biochemical signals in this complex process is of interest and will contribute to an improvement in immunotherapy strategies. To address underlying questions, coculture and biomimetic models are established. Mature dendritic cells (mDC) are first treated with cytochalasin B (CytoB), a cytoskeletal disruption agent known to lower apparent cellular stiffness and reduction in T cell proliferation is observed. It is attempted to mimic mDC and T cell interactions using polyacrylamide (PA) gels with defined stiffness corresponding to mDC (0.2-25 kPa). Different ratios of anti-CD3 (aCD3) and anti-CD28 (aCD28) antibodies are immobilized onto PA gels. The results show T cell proliferation is triggered by both aCD3 and aCD28 in a stiffness-dependent manner. Cells cultured on aCD3 immobilized on gels has significantly enhanced proliferation and IL-2 secretion, compared to aCD28. Furthermore, ZAP70 phosphorylation is enhanced in stiffer substrate a in a aCD3-dependent manner. The biosystem provides an approach to study the reduction of T cell proliferation observed on CytoB-treated mDC. Overall, the biosystem allows distinguishing the impact of biophysical and biochemical signals of APC and T cell interactions in vitro.

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The interaction of immune cells with drugs and/or with other cell types should be mechanistically investigated in order to reduce attrition of new drug development. However, they are currently only limited technologies that address this need. In our work, we developed initial but significant building blocks that enable such immune-drug studies. We developed a novel microfluidic platform replicating the Lymph Node (LN) microenvironment called LN-on-a-chip, starting from design all the way to microfabrication, characterization and validation in terms of architectural features, fluidics, cytocompatibility, and usability. To prove the biomimetics of this microenvironment, we inserted different immune cell types in a microfluidic device, which showed an in-vivo-like spatial distribution. We demonstrated that the developed LN-on-a-chip incorporates key features of the native human LN, namely, (i) similarity in extracellular matrix composition, morphology, porosity, stiffness, and permeability, (ii) compartmentalization of immune cells within distinct structural domains, (iii) replication of the lymphatic fluid flow pattern, (iv) viability of encapsulated cells in collagen over the typical timeframe of immunotoxicity experiments, and (v) interaction among different cell types across chamber boundaries. Further studies with this platform may assess the immune cell function as a step forward to disclose the effects of pharmaceutics to downstream immunology in more physiologically relevant microenvironments.

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Monocytes circulate in the bloodstream, extravasate into the tissue and differentiate into specific macrophage phenotypes to fulfill the immunological needs of tissues. During the tissue repair process, tissue density transits from loose to dense tissue. However, little is known on how changes in tissue density affects macrophage activation and their cellular functions. In this work, monocytic cell line THP-1 cells were embedded in three-dimensional (3D) collagen matrices with different fibril density and were then differentiated into uncommitted macrophages (MPMA) using phorbol-12-myristate-13-acetate (PMA). MPMA macrophages were subsequently activated into pro-inflammatory macrophages (MLPS/IFNγ) and anti-inflammatory macrophages (MIL-4/IL-13) using lipopolysaccharide and interferon-gamma (IFNγ), and interleukin 4 (IL-4) and IL-13, respectively. Although analysis of cell surface markers, on both gene and protein levels, was inconclusive, cytokine secretion profiles, however, demonstrated differences in macrophage phenotype. In the presence of differentiation activators, MLPS/IFNγ secreted high amounts of IL-1ß and tumor necrosis factor alpha (TNFα), while M0PMA secreted similar cytokines to MIL-4/IL-13, but low IL-8. After removing the activators and further culture for 3 days in fresh cell culture media, the secretion of IL-6 was found in high concentrations by MIL-4/IL-13, followed by MLPS/IFNγ and MPMA. Interestingly, the secretion of cytokines is enhanced with an increase of fibril density. Through the investigation of macrophage-associated functions during tissue repair, we demonstrated that M1LPS/IFNγ has the potential to enhance monocyte infiltration into tissue, while MIL-4/IL-13 supported fibroblast differentiation into myofibroblasts via transforming growth factor beta 1 (TGF-ß1) in dependence of fibril density, suggesting a M2a-like phenotype. Overall, our results suggest that collagen fibril density can modulate macrophage response to favor tissue functions. Understanding of immune response in such complex 3D microenvironments will contribute to the novel therapeutic strategies for improving tissue repair, as well as guidance of the design of immune-modulated materials.