Biofilm-stimulated epithelium modulates the inflammatory responses in co-cultured immune cells

Abstract The gingival epithelium is a physical and immunological barrier to the microbiota of the oral cavity, which interact through soluble mediators with the immune cells that patrol the tissue at the gingival epithelium. We sought to develop a three-dimensional gingivae-biofilm interface model using a commercially available gingival epithelium to study the tissue inflammatory response to oral biofilms associated with “health”, “gingivitis” and “periodontitis”. These biofilms were developed by sequential addition of microorganisms to mimic the formation of supra- and sub-gingival plaque in vivo. Secondly, to mimic the interactions between gingival epithelium and immune cells in vivo, we integrated peripheral blood mononuclear cells and CD14+ monocytes into our three-dimensional model and were able to assess the inflammatory response in the immune cells cultured with and without gingival epithelium. We describe a differential inflammatory response in immune cells cultured with epithelial tissue, and more so following incubation with epithelium stimulated by “gingivitis-associated” biofilm. These results suggest that gingival epithelium-derived soluble mediators may control the inflammatory status of immune cells in vitro, and therefore targeting of the epithelial response may offer novel therapies. This multi-cellular interface model, both of microbial and host origin, offers a robust in vitro platform to investigate host-pathogens at the epithelial surface.

Download Full-text

A 3D In Vitro Model for Burn Wounds: Monitoring of Regeneration on the Epidermal Level

Biomedicines ◽

10.3390/biomedicines9091153 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1153

Author(s):

Verena Schneider ◽

Daniel Kruse ◽

Ives Bernardelli de Mattos ◽

Saskia Zöphel ◽

Kendra-Kathrin Tiltmann ◽

...

Keyword(s):

Wound Healing ◽

Inflammatory Response ◽

Healing Process ◽

Three Dimensional ◽

Source Model ◽

Burn Wound ◽

Thermal Burn ◽

Burn Wounds

Burns affect millions every year and a model to mimic the pathophysiology of such injuries in detail is required to better understand regeneration. The current gold standard for studying burn wounds are animal models, which are under criticism due to ethical considerations and a limited predictiveness. Here, we present a three-dimensional burn model, based on an open-source model, to monitor wound healing on the epidermal level. Skin equivalents were burned, using a preheated metal cylinder. The healing process was monitored regarding histomorphology, metabolic changes, inflammatory response and reepithelialization for 14 days. During this time, the wound size decreased from 25% to 5% of the model area and the inflammatory response (IL-1β, IL-6 and IL-8) showed a comparable course to wounding and healing in vivo. Additionally, the topical application of 5% dexpanthenol enhanced tissue morphology and the number of proliferative keratinocytes in the newly formed epidermis, but did not influence the overall reepithelialization rate. In summary, the model showed a comparable healing process to in vivo, and thus, offers the opportunity to better understand the physiology of thermal burn wound healing on the keratinocyte level.

Download Full-text

Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

Infection and Immunity ◽

10.1128/iai.00293-20 ◽

2020 ◽

Vol 88 (11) ◽

Author(s):

Marloes I. Hofstee ◽

Martijn Riool ◽

Igors Terjajevs ◽

Keith Thompson ◽

Martin J. Stoddart ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Immune Cells ◽

Early Stage ◽

Three Dimensional ◽

Collagen Gel ◽

Maximum Size ◽

Human Neutrophils ◽

Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

Download Full-text

Mesenchymal-like glioma cells are enriched in the gelatin methacrylate hydrogels

10.1101/2021.03.24.436751 ◽

2021 ◽

Author(s):

Nameeta Shah ◽

Pavan M. Hallur ◽

Raksha A. Ganesh ◽

Pranali Sonpatki ◽

Divya Naik ◽

...

Keyword(s):

Therapeutic Response ◽

Immune Cell ◽

Three Dimensional ◽

Glioma Cells ◽

Cellular Phenotype ◽

3D Scaffold ◽

Promising Alternative ◽

Cells Cultured

AbstractGlioblastoma is the most lethal primary malignant brain tumor in adults. Simplified two-dimensional (2D) cell culture and neurospheres in vitro models fail to recapitulate the complexity of the tumor microenvironment, limiting its ability to predict therapeutic response. Three-dimensional (3D) scaffold-based models have emerged as a promising alternative for addressing these concerns. One such 3D system is gelatin methacrylate (GelMA) hydrogels, which can be used for modeling the glioblastoma microenvironment. We characterized the phenotype of patient-derived glioma cells cultured in GelMA hydrogels (3D-GMH) for their tumorigenic properties using invasion and chemoresponse assays. In addition, we used integrated single-cell and spatial transcriptome analysis to compare cells cultured in 3D-GMH to cells in vivo. Finally, we assessed tumor-immune cell interactions with a macrophage infiltration assay and a cytokine array. We show that cells cultured in 3D-GMH develop a mesenchymal-like cellular phenotype found in perivascular and hypoxic regions present in the core of the tumor, and recruit macrophages by secreting cytokines in contrast to the cells grown as neurospheres that match the phenotype of cells of the infiltrative edge of the tumor.

Download Full-text

Multiscale modelling of drug transport and metabolism in liver spheroids

Interface Focus ◽

10.1098/rsfs.2019.0041 ◽

2020 ◽

Vol 10 (2) ◽

pp. 20190041 ◽

Cited By ~ 3

Author(s):

Joseph A. Leedale ◽

Jonathan A. Kyffin ◽

Amy L. Harding ◽

Helen E. Colley ◽

Craig Murdoch ◽

...

Keyword(s):

Drug Transport ◽

Temporal Dynamics ◽

Three Dimensional ◽

Culture Conditions ◽

Isolated Cells ◽

Modelling Framework ◽

The Impact ◽

Cells Cultured

In early preclinical drug development, potential candidates are tested in the laboratory using isolated cells. These in vitro experiments traditionally involve cells cultured in a two-dimensional monolayer environment. However, cells cultured in three-dimensional spheroid systems have been shown to more closely resemble the functionality and morphology of cells in vivo . While the increasing usage of hepatic spheroid cultures allows for more relevant experimentation in a more realistic biological environment, the underlying physical processes of drug transport, uptake and metabolism contributing to the spatial distribution of drugs in these spheroids remain poorly understood. The development of a multiscale mathematical modelling framework describing the spatio-temporal dynamics of drugs in multicellular environments enables mechanistic insight into the behaviour of these systems. Here, our analysis of cell membrane permeation and porosity throughout the spheroid reveals the impact of these properties on drug penetration, with maximal disparity between zonal metabolism rates occurring for drugs of intermediate lipophilicity. Our research shows how mathematical models can be used to simulate the activity and transport of drugs in hepatic spheroids and in principle any organoid, with the ultimate aim of better informing experimentalists on how to regulate dosing and culture conditions to more effectively optimize drug delivery.

Download Full-text

Gelatin methacrylate hydrogels culture model for glioblastoma cells enriches for mesenchymal-like state and models interactions with immune cells

Scientific Reports ◽

10.1038/s41598-021-97059-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nameeta Shah ◽

Pavan M. Hallur ◽

Raksha A. Ganesh ◽

Pranali Sonpatki ◽

Divya Naik ◽

...

Keyword(s):

Immune Cell ◽

Three Dimensional ◽

Glioma Cells ◽

Glioblastoma Cells ◽

Treatment Resistant ◽

3D Scaffold ◽

Promising Alternative ◽

Cells Cultured

AbstractGlioblastoma is the most lethal primary malignant brain tumor in adults. Simplified two-dimensional (2D) cell culture and neurospheres in vitro models fail to recapitulate the complexity of the tumor microenvironment, limiting its ability to predict therapeutic response. Three-dimensional (3D) scaffold-based models have emerged as a promising alternative for addressing these concerns. One such 3D system is gelatin methacrylate (GelMA) hydrogels, and we aimed to understand the suitability of using this system to mimic treatment-resistant glioblastoma cells that reside in specific niches. We characterized the phenotype of patient-derived glioma cells cultured in GelMA hydrogels (3D-GMH) for their tumorigenic properties using invasion and chemoresponse assays. In addition, we used integrated single-cell and spatial transcriptome analysis to compare cells cultured in 3D-GMH to neoplastic cells in vivo. Finally, we assessed tumor-immune cell interactions with a macrophage infiltration assay and a cytokine array. We show that the 3D-GMH system enriches treatment-resistant mesenchymal cells that are not represented in neurosphere cultures. Cells cultured in 3D-GMH resemble a mesenchymal-like cellular phenotype found in perivascular and hypoxic regions and recruit macrophages by secreting cytokines, a hallmark of the mesenchymal phenotype. Our 3D-GMH model effectively mimics the phenotype of glioma cells that are found in the perivascular and hypoxic niches of the glioblastoma core in situ, in contrast to the neurosphere cultures that enrich cells of the infiltrative edge of the tumor. This contrast highlights the need for due diligence in selecting an appropriate model when designing a study‘s objectives.

Download Full-text

Preparation of Gelatin and Gelatin/Hyaluronic Acid Cryogel Scaffolds for the 3D Culture of Mesothelial Cells and Mesothelium Tissue Regeneration

International Journal of Molecular Sciences ◽

10.3390/ijms20184527 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4527 ◽

Cited By ~ 1

Author(s):

Hao-Hsi Kao ◽

Chang-Yi Kuo ◽

Kuo-Su Chen ◽

Jyh-Ping Chen

Keyword(s):

Hyaluronic Acid ◽

Three Dimensional ◽

3D Culture ◽

Mesothelial Cells ◽

In Vivo Studies ◽

3D Scaffold ◽

Scaffold Degradation ◽

Cells Cultured

Mesothelial cells are specific epithelial cells that are lined in the serosal cavity and internal organs. Nonetheless, few studies have explored the possibility to culture mesothelial cells in a three-dimensional (3D) scaffold for tissue engineering applications. Towards this end, we fabricated macroporous scaffolds from gelatin and gelatin/hyaluronic acid (HA) by cryogelation, and elucidated the influence of HA on cryogel properties and the cellular phenotype of mesothelial cells cultured within the 3D scaffolds. The incorporation of HA was found not to significantly change the pore size, porosity, water uptake kinetics, and swelling ratios of the cryogel scaffolds, but led to a faster scaffold degradation in the collagenase solution. Adding 5% HA in the composite cryogels also decreased the ultimate compressive stress (strain) and toughness of the scaffold, but enhanced the elastic modulus. From the in vitro cell culture, rat mesothelial cells showed quantitative cell viability in gelatin (G) and gelatin/HA (GH) cryogels. Nonetheless, mesothelial cells cultured in GH cryogels showed a change in the cell morphology and cytoskeleton arrangement, reduced cell proliferation rate, and downregulation of the mesothelium specific maker gene expression. The production of key mesothelium proteins E-cadherin and calretinin were also reduced in the GH cryogels. Choosing the best G cryogels for in vivo studies, the cell/cryogel construct was used for the transplantation of allograft mesothelial cells for mesothelium reconstruction in rats. A mesothelium layer similar to the native mesothelium tissue could be obtained 21 days post-implantation, based on hematoxylin and eosin (H&E) and immunohistochemical staining.

Download Full-text

miR-21-3p/IL-22 Axes Are Major Drivers of Psoriasis Pathogenesis by Modulating Keratinocytes Proliferation-Survival Balance and Inflammatory Response

Cells ◽

10.3390/cells10102547 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2547

Author(s):

Florence Abdallah ◽

Elodie Henriet ◽

Amandine Suet ◽

Ali Arar ◽

Rudy Clemençon ◽

...

Keyword(s):

Inflammatory Response ◽

Mouse Model ◽

Immune Cells ◽

Skin Disease ◽

Molecular Targets ◽

Proliferative Potential ◽

Inflammatory Skin Disease ◽

Functional Assays

Psoriasis is a chronic inflammatory skin disease that is mediated by complex crosstalk between immune cells and keratinocytes (KCs). Emerging studies have showed a specific psoriatic microRNAs signature, in which miR-21 is one of the most upregulated and dynamic miRNAs. In this study, we focused our investigations on the passenger miR-21-3p strand, which is poorly studied in skin and in psoriasis pathogenesis. Here, we showed the upregulation of miR-21-3p in an IMQ-induced psoriasiform mouse model. This upregulation was correlated with IL-22 expression and functionality, both in vitro and in vivo, and it occurred via STAT3 and NF-κB signaling. We identified a network of differentially expressed genes involved in abnormal proliferation control and immune regulatory genes implicated in the molecular pathogenesis of psoriasis in response to miR-21-3p overexpression in KCs. These results were confirmed by functional assays that validated the proliferative potential of miR-21-3p. All these findings highlight the importance of miR-21-3p, an underestimated miRNA, in psoriasis and provide novel molecular targets for therapeutic purposes.

Download Full-text

Apolipoprotein B binds to enolase-1 and aggravates inflammation in rheumatoid arthritis

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2018-213444 ◽

2018 ◽

Vol 77 (10) ◽

pp. 1480-1489 ◽

Cited By ~ 8

Author(s):

Joo Youn Lee ◽

Min Jueng Kang ◽

Ji Yong Choi ◽

Ji Soo Park ◽

Jin Kyun Park ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Inflammatory Response ◽

Immune Cells ◽

Knockout Mice ◽

Apolipoprotein B ◽

Arthritis Model ◽

Arthritis Severity ◽

Serum Transfer

ObjectiveImmune cells from patients with rheumatoid arthritis (RA) express more enolase-1 (ENO1) on their surface than those from healthy subjects, and they elicit an enhanced inflammatory response. This study is aimed to identify the ligands of ENO1 that could promote inflammatory loops in vitro and enhance the arthritis severity in vivo.MethodsENO1-binding proteins in RA synovial fluid were identified by mass spectromety, and affinity to ENO1 was evaluated by means of a ligand blotting and binding assay, surface plasmon resonance and confocal microscopy. Proinflammatory response by the interaction between ENO1 and apolipoprotein B (apoB) was tested in vitro and in vivo using peripheral blood mononuclear cells and a K/BxN serum transfer arthritis model and low-density lipoproteins receptor (LDLR) knockout mice.ResultsApoB in the synovid fluid of patients with RA was identified as a specific ligand to ENO1 with a higher affinity than plasminogen, a known ENO1 ligand. ApoB binding to ENO1 on monocytes elicited the production of tumour necrosis factor-α, interleukins (IL)-1β and IL-6 through both p38 mitogen-activated protein kinase and NF-κB pathways. In the K/BxN serum transfer arthritis model, administration of apoB increased the production of proinflammatory cytokines and exaggerated arthritis severity. The severity of K/BxN serum transfer arthritis in LDLR knockout mice was comparable with wild-type mice.ConclusionsA key component of atherogenic lipids, apoB, aggravated arthritis by potentiating the inflammatory response via its interaction with ENO1 expressed on the surface of immune cells. This suggests a novel mechanism by which lipid metabolism regulates chronic inflammation in RA.

Download Full-text

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

1978 ◽

Vol 36 (2) ◽

pp. 434-435

Author(s):

D. Reis ◽

B. Vian ◽

J. C. Roland

Keyword(s):

Cell Wall ◽

Self Assembly ◽

Plant Cell Wall ◽

Higher Plants ◽

Three Dimensional ◽

Cytochemical Study ◽

Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Download Full-text

Inhibitory effects of epimedium herb water extract on the inflammatory response in vitro and in vivo

Planta Medica ◽

10.1055/s-0036-1596615 ◽

2016 ◽

Vol 81 (S 01) ◽

pp. S1-S381

Author(s):

YC Oh ◽

YH Jeong ◽

WK Cho ◽

SJ Lee ◽

JY Ma

Keyword(s):

Inflammatory Response ◽

Water Extract ◽

Inhibitory Effects

Download Full-text