The enzyme HPGD is critical for regulatory T cell function in adipose tissue

Regulatory T cells (Treg cells) are essential for maintaining immune homeostasis. However, how Treg cells exert their function in tissue specific environments is often unknown. We have found hydroxyprostaglandin dehydrogenase (Hpgd), the major Prostaglandin E2 (PGE2) metabolizing enzyme, to be significantly upregulated in Treg cells compared to conventional T cells (Tconv). In the murine system, this upregulation is especially pronounced in the visceral adipose tissue (VAT), a prostaglandin-rich environment. Furthermore, we could show that through the metabolism of PGE2 into 15-keto-PGE2 Hpgd enhances the suppressive capabilities of Treg cells in an, at least partially, Pparγ-dependent manner. In vivo, we found that Hpgd-deficient Treg cells were less efficient in preventing the onset of both DSS-induced and adoptive transfer colitis, further indicating that Hpgd plays a role in the suppressive capacity of Treg cells. However, analysis of the transcriptome of these Hpgd-deficient Treg cells did not differ significantly from Hpgd-competent Treg cells, indicating that the observed changes are due to the extrinsic effect caused by the loss of the enzymatic function of Hpgd. When analyzing the VAT of aged animals with Hpgd-deficient Treg cells, we could detect an influx of non-functional Treg cells as well as an accumulation of pro-inflammatory macrophages and an increase in adipocyte size. Furthermore, while we could neither detect a change in body or organ weight of these animals, nor a change in motility, food and water intake, or respiration, we could observe impaired metabolic signaling. Aged animals with Hpgd-deficient Treg cells respond less to insulin and glucose challenge and show a reduction in insulin signaling. When subjecting animals with Hpgd-deficient Treg cells to a high fat diet (HFD), we could not detect a difference in weight gain when compared to wildtype littermate control animals. Even though we could detect a slight decrease in insulin responsiveness in animals on a HFD with Hpgd-deficient Treg cells, no difference in the VAT-resident immune cell population or in any other metabolic parameters could be observed. Additionally, in peripheral blood from human type II diabetes (T2D) patients we observed a dysregulation of the Treg cell population as well as a decrease in HPGD expression in these cells compared to healthy, age-matched controls. Taken together, these data indicate that both in humans and in the murine system, HPGD expression in Treg cells might be involved in metabolic regulation. Finally, we analyzed the role of the Treg cell specific transcription factor mesenchyme homeobox 1 (MEOX1) for HPGD expression. We found that MEOX1 is highly upregulated in human Treg cells, especially after stimulation with interleukin (IL) 2. Furthermore, we could show that while MEOX1 expression, like HPGD, is regulated by FOXP3, a loss of MEOX1 does not affect HPGD expression, thus disproving our hypothesis that HPGD may be regulated by the transcription factor MEOX1. Taken together, we could describe that HPGD is an important mediator of Treg cell suppression, independently of MEOX1. We found that a Treg cell specific deletion of Hpgd in the mouse leads to a dysregulation of the metabolism, and that HPGD levels are significantly decreased in Treg cells isolated from the peripheral blood of T2D patients compared to Treg cells isolated from healthy subjects

Stereolithography

The stereolithography (SLA) process and its methods are introduced in this chapter. After establishing SLA as pertaining to the high-resolution but also high-cost spectrum of the 3D printing technologies, different classifications of SLA processes are presented. Laser-based SLA and digital light processing (DLP), as well as their specialized techniques such as two-photon polymerization (TPP) or continuous liquid interface production (CLIP) are discussed and analyzed for their advantages and shortcomings. Prerequisites of SLA resins and the most common resin compositions are discussed. Furthermore, printable materials and their applications are briefly reviewed, and insight into commercially available SLA systems is given. Finally, an outlook highlighting challenges within the SLA process and propositions to resolve these are offered

Topology Optimization and 3D printing of Large Deformation Compliant Mechanisms for Straining Biological Tissues

This paper presents a synthesis approach in a density-based topology optimization setting to design large deformation compliant mechanisms for inducing desired strains in biological tissues. The modelling is based on geometrical nonlinearity together with a suitably chosen hypereleastic material model, wherein the mechanical equilibrium equations are solved using the total Lagrangian finite element formulation. An objective based on least-square error with respect to target strains is formulated and minimized with the given set of constraints and the appropriate surroundings of the tissues. To circumvent numerical instabilities arising due to large deformation in low stiffness design regions during topology optimization, a strain-energy based interpolation scheme is employed. The approach uses an extended robust formulation i.e. the eroded, intermediate and dilated projections for the design description as well as variation in tissue stiffness. Efficacy of the synthesis approach is demonstrated by designing various compliant mechanisms for providing different target strains in biological tissue constructs. Optimized compliant mechanisms are 3D-printed and their performances are recorded in a simplified experiment and compared with simulation results obtained by a commercial software.Comment: 23 pages, 14 figure

Application of high resolution DLP stereolithography for fabrication of tricalcium phosphate scaffolds for bone regeneration

Bone regeneration requires porous and mechanically stable scaffolds to support tissue integration and angiogenesis, which is essential for bone tissue regeneration. With the advent of additive manufacturing process, production of complex porous architecture has become feasible. However, a balance has to be sorted between porous architecture and mechanical stability which facilitates bone regeneration for load bearing applications. 
 Current study evaluates used of high resolution digital light processing (DLP) -based additive manufacturing to produce complex but mechanical stable scaffolds based on β-tricalcium phosphate (β-TCP) for bone regeneration. 
 Four different geometries, a rectilinear Grid, hexagonal Kagome, schwart primitive and hollow Schwarz are designed with 400 µm pores and 75 or 50 vol.% porosity. However after initial screening for design stability and mechanical properties, only a rectilinear Grid structure, a hexagonal Kagome structure are found to be reproducible and showed higher mechanical properties. 
 Micro computed tomography (µ-CT) analysis shows < 2 vol.% error in porosity and < 6 % relative deviation of average pore sizes for the Grid structures. At 50 vol.% porosity, this architecture also has the highest compressive strength of 44.7 MPa (Weibull modulus is 5.28), while bulk specimens reach 235 ± 37 MPa. 
 To evaluate suitability of 3D scaffolds produce by DLP methods for bone regeneration, scaffolds were cultured with murine preosteoblastic MC3T3-E1 cells. Short term study showed cells growth over 14 days, with more than two-fold increase of alkaline phosphatase (ALP) activity compared to cells on 2D tissue culture plastic. Collagen deposition was increased by a factor of 1.5 – 2 when compared to the 2D controls. This confirm retention of biocompatible and osteo-inductive properties of β-TCP following DLP process. 
 This study has implications for designing of the high resolution porous scaffolds for bone regenerative applications and contributes to understanding of DLP based additive manufacturing process for medical applications

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2.

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies

MCT4 blockade increases the efficacy of immune checkpoint blockade

Background & Aims Intratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be prognostic for the survival of patients with rectal cancer, indicating that single MCT4 blockade might be a promising strategy to overcome glycolysis-related therapy resistance. Methods To determine whether blockade of MCT4 alone is sufficient to improve the efficacy of immune checkpoint blockade (ICB) therapy, we examined the effects of the selective MCT1 inhibitor AZD3965 and a novel MCT4 inhibitor in a colorectal carcinoma (CRC) tumor spheroid model co-cultured with blood leukocytes in vitro and the MC38 murine CRC model in vivo in combination with an antibody against programmed cell death ligand-1(PD-L1). Results Inhibition of MCT4 was sufficient to reduce lactate efflux in three-dimensional (3D) CRC spheroids but not in two-dimensional cell-cultures. Co-administration of the MCT4 inhibitor and ICB augmented immune cell infiltration, T-cell function and decreased CRC spheroid viability in a 3D co-culture model of human CRC spheroids with blood leukocytes. Accordingly, combination of MCT4 and ICB increased intratumoral pH, improved leukocyte infiltration and T-cell activation, delayed tumor growth, and prolonged survival in vivo. MCT1 inhibition exerted no further beneficial impact. Conclusions These findings demonstrate that single MCT4 inhibition represents a novel therapeutic approach to reverse lactic-acid driven immunosuppression and might be suitable to improve ICB efficacy

Transcriptome-Based Network Analysis Reveals a Spectrum Model of Human Macrophage Activation

SummaryMacrophage activation is associated with profound transcriptional reprogramming. Although much progress has been made in the understanding of macrophage activation, polarization, and function, the transcriptional programs regulating these processes remain poorly characterized. We stimulated human macrophages with diverse activation signals, acquiring a data set of 299 macrophage transcriptomes. Analysis of this data set revealed a spectrum of macrophage activation states extending the current M1 versus M2-polarization model. Network analyses identified central transcriptional regulators associated with all macrophage activation complemented by regulators related to stimulus-specific programs. Applying these transcriptional programs to human alveolar macrophages from smokers and patients with chronic obstructive pulmonary disease (COPD) revealed an unexpected loss of inflammatory signatures in COPD patients. Finally, by integrating murine data from the ImmGen project we propose a refined, activation-independent core signature for human and murine macrophages. This resource serves as a framework for future research into regulation of macrophage activation in health and disease

Molecular Insights Into Regulatory T-Cell Adaptation to Self, Environment, and Host Tissues: Plasticity or Loss of Function in Autoimmune Disease

There has been much interest in the ability of regulatory T cells (Treg) to switch function in vivo, either as a result of genetic risk of disease or in response to environmental and metabolic cues. The relationship between levels of FOXP3 and functional fitness plays a significant part in this plasticity. There is an emerging role for Treg in tissue repair that may be less dependent on FOXP3, and the molecular mechanisms underpinning this are not fully understood. As a result of detailed, high-resolution functional genomics, the gene regulatory networks and key functional mediators of Treg phenotype downstream of FOXP3 have been mapped, enabling a mechanistic insight into Treg function. This transcription factor-driven programming of T-cell function to generate Treg requires the switching on and off of key genes that form part of the Treg gene regulatory network and raises the possibility that this is reversible. It is plausible that subtle shifts in expression levels of specific genes, including transcription factors and non-coding RNAs, change the regulation of the Treg gene network. The subtle skewing of gene expression initiates changes in function, with the potential to promote chronic disease and/or to license appropriate inflammatory responses. In the case of autoimmunity, there is an underlying genetic risk, and the interplay of genetic and environmental cues is complex and impacts gene regulation networks frequently involving promoters and enhancers, the regulatory elements that control gene expression levels and responsiveness. These promoter–enhancer interactions can operate over long distances and are highly cell type specific. In autoimmunity, the genetic risk can result in changes in these enhancer/promoter interactions, and this mainly impacts genes which are expressed in T cells and hence impacts Treg/conventional T-cell (Tconv) function. Genetic risk may cause the subtle alterations to the responsiveness of gene regulatory networks which are controlled by or control FOXP3 and its target genes, and the application of assays of the 3D organization of chromatin, enabling the connection of non-coding regulatory regions to the genes they control, is revealing the direct impact of environmental/metabolic/genetic risk on T-cell function and is providing mechanistic insight into susceptibility to inflammatory and autoimmune conditions.Cheryl Y. Brown, Timothy Sadlon, Christopher M. Hope, Ying Y. Wong, Soon Wong, Ning Liu, Holly Withers, Katherine Brown, Veronika Bandara, Batjargal Gundsambuu, Stephen Pederson, James Breen, Sarah Anne Robertson, Alistair Forrest, Marc Beyer, and Simon Charles Barr

Additive Fertigung von Gerüststrukturen aus Tricalciumphosphat für die Knochenregeneration

Abweichender Titel nach Übersetzung der Verfasserin/des VerfassersKnochenbrüche, die durch Traumata oder Krankheiten verursacht werden, können die Lebensqualität erheblich beeinträchtigen und bereiten ein zunehmendes Problem in unserer alternden Gesellschaft [1]. Insbesondere bei großen Defekten kann native Knochenreparatur unzureichend sein, und häufig ist ein chirurgischer Eingriff erforderlich. In diesem Fall hat sich das Tissue-Engineering (TE) als Alternative zu herkömmlich verwendeten Gewebetransplantaten etabliert [2]. In TE werden Scaffolds (Gerüststrukturen) mit Zellen und Wachstumsfaktoren kombiniert, um Bildung von neuem Gewebe zu induzieren. Diese Scaffolds benötigen durchgehend verbundene Porosität, um Zellmigration, Nährstoffund Endotoxintransport sowie Bildung neuer Gefäße zu ermöglichen. Gleichzeitig ist eine ausreichende mechanische Stabilität erforderlich, um die Belastbarkeitdes Knochens zu gewährleisten [3], [4]. Ein Ansatz, um Materialien mit hoher Festigkeit und geringer Dichte herzustellen, ist das Modifizieren der Gittertopologie. In der vorliegenden Arbeit werden durch generative Fertigung die Scaffold-architekturen genau eingestellt, wodurch mechanische Eigenschaften sowie biologische Aktivität optimiert werden können. Tricalciumphosphat (TCP) -Gerüste mit vier unterschiedlichen Porengeometrien werden entworfen. Ein geradliniges Zylindergitter, die Kagome Geometrie mit hexagonaler Einheitszelle, die Schwarz primitive Architektur ‚welche zu den triply periodic minimal surfaces (TPMS) zählt, sowie die Shellular Version dieser TPMS Geometrie werden unter Verwendung von Digital Light Processing (DLP) Stereolitographie (SLA) hergestellt. Die Porengrößen aller Geometrien werden in einem Bereich von 400 bis 600 m bei Porositäten von 25, 50 und 75 % festgelegt. Durch Röngtenphotoelektronen-Spektroskopie (XPS) und Röngtendiffraktion (XRD) kann eine reine -TCP Phase ohne organischen Binderrückständen oder sonstigen Verunreinigungen bestätigt werden. Weiters kann die exakte Geometrie und die homogene Porengrößenverteilung durch mikro-Computer Tomographie (-CT) validiert werden. Nach Einbau experimentel ermittelter Skalierungsfaktoren ist die Zylindergittergeometrie die am besten reproduzierbare Architektur mit < 2 vol.% Fehler in der Porosität und < 6 % relative Abweichung der durchschnittlichen Porengrößen. Diese Struktur hat bei 50 vol.% Porosität auch die höchste Druckfestigkeit von 44.7 MPa, während die Kagome-Architektur eine Stärke von 19.5 MPa aufweist. Porenfreie Proben hingegen haben eine Festigkeit von 235 37 MPa und eine Dichte von 99.50 0.18 % des theoretischen Wertes kann erreicht werden. Nach dem Einlegen in simulierte Körperflüssigkeit (SBF) für 21 Tage zeigen alle untersuchten Scaffolds erhöhte intergranulare Porosität unter dem asterelektronenmikroskop (SEM), aber keine signifikante Änderung in der Masse. Vorläufige Zellkulturstudien mit Maus-Preosteoblasten (MC3T3-E1 Zellen) auf dem Zylindergitter und den Kagome-Scaffolds bestätigen Zellwachstum, und frühe osteogene Marker weisen auf eine Differenzierung zu Osteoblasten hin. Neben diesen vielversprechenden biologischen Eigenschaften sind weiter Voraussetzungen wie die hohe Reinheit des Materials, die ähnlichen mechanischen Eigenschaften zu Knochen und exzellente geometrische Reproduzierbarkeit erfüllt. Somit können sowohl das Zylindergitter, als auch die Kagome-Architekturen zukünftig Anwendung in der regenerativen Medizin als Knochen-TE-Scaffolds finden.Bone fractures caused by trauma or disease can significantly decrease quality of life and pose a growing issue in our aging population. Especially for large defects, native bone repair can be insufficient, and surgical intervention is often required. In that case, bone tissue engineering (TE) has emerged as an alternative to conventionally used tissue grafts. In TE, scaffolds can be combined with cells and growth factors to act as templates for the formation of new tissue. These scaffolds need interconnected porosity to enable cell migration, nutrient and endotoxin transportation, and neo-vascularization. Simultaneously, adequate mechanical integrity is required to replace load bearing purposes of the bone [3], [4]. One approach to attain high strength and low density materials, is by engineering of lattice topology. The present study uses additive manufacturing to precisely define scaffold architectures for tunable mechanical properties and optimized biological activity. Tricalcium phosphate (TCP) scaffolds with four different pore geometries are designed. A rectilinear cylinder grid lattice, a triply periodic minimal surface (TPMS) inspired Schwarz primitive architecture, a shellular type hollow Schwarz primitive scaffold, and finally a Kagome geometry with hexagonal unit cell are created using a digital light processing (DLP) stereolithography (SLA) system. Pore sizes for all geometries are set in a range of 400 - 600 m at porosities of 25, 50, and 75 %. Material characterization by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) confirms presence of the pure -TCP phase without remnants of organic binder or other contaminants. Validation of geometrical accuracy and homogeneous pore distribution throughout the scaffolds is implemented by micro computer tomography (-CT). After incorporation of adequate scaling factors, the cylinder grid geometry is the most reproducible architecture with < 2 % error in porosity and < 6 % deviation of average pore sizes. This structure also has the highest compressive strength of 44.7 MPa at 50 % porosity while the Kagome architecture has 19.5 MPa at the same density. Manufactured bulk specimens reach a density of up to 99.50 0.18 % of crystallographic values and compressive strength of 235 37 MPa. After submersion in simulated body fluid (SBF) for 21 days, all investigated scaffolds show increased intergranular porosity in scanning electron microscopy (SEM) images but no significant change in mass. Preliminary cell culture studies with murine osteoblast-like MC3T3-E1 cells on the cylinder grid and the Kagome scaffolds confirm cell growth and early osteogenic markers indicate differentiation into osteoblasts. With mechanical properties comparable those of bone at similar densities and excellent geometrical reproducibility as well as promising biological attributes, both the cylinder grid and the Kagome architectures could, after further investigation, have future applications in regenerative medicine as bone tissue engineering scaffolds.6