Protocol for the induction of innate immune memory in human smooth muscle cells and endothelial cells in vitro.

Non-immune cells, like innate immune cells, can develop a memory-like phenotype in response to priming with microbial compounds or certain metabolites, which enables an enhanced response to a secondary unspecific stimulus. This paper describes a step-by-step protocol for the induction and analysis of trained immunity in human endothelial and smooth muscle cells. We then describe steps for cell culture with cryopreserved vascular cells, subcultivation, and induction of trained immunity. We then provide detailed procedures for downstream analysis using ELISA and qPCR. For complete details on the use and execution of this protocol, please refer to Sohrabi et al. (2020)1 and Shcnack et al.2.

LXRα Regulates oxLDL-Induced Trained Immunity in Macrophages.

Reprogramming of metabolic pathways in monocytes and macrophages can induce a proatherosclerotic inflammatory memory called trained innate immunity. Here, we have analyzed the role of the Liver X receptor (LXR), a crucial regulator of metabolism and inflammation, in oxidized low-density lipoprotein (oxLDL)-induced trained innate immunity. Human monocytes were incubated with LXR agonists, antagonists, and oxLDL for 24 h. After five days of resting time, cells were restimulated with the TLR-2 agonist Pam3cys. OxLDL priming induced the expression of LXRα but not LXRß. Pharmacologic LXR activation was enhanced, while LXR inhibition prevented the oxLDL-induced inflammatory response. Furthermore, LXR inhibition blocked the metabolic changes necessary for epigenetic reprogramming associated with trained immunity. In fact, enrichment of activating histone marks at the IL-6 and TNFα promotor was reduced following LXR inhibition. Based on the differential expression of the LXR isoforms, we inhibited LXRα and LXRß genes using siRNA in THP1 cells. As expected, siRNA-mediated knock-down of LXRα blocked the oxLDL-induced inflammatory response, while knock-down of LXRß had no effect. We demonstrate a specific and novel role of the LXRα isoform in the regulation of oxLDL-induced trained immunity. Our data reveal important aspects of LXR signaling in innate immunity with relevance to atherosclerosis formation.

Targeting inflammation with collagen.

Tissue damage caused by an infection oran autoimmune disease triggers degradation of collagen in the extracellular matrix (ECM), which further enhances inflammation. Therefore, improving ECM in aninflamed tissue can be exploited as a potential therapeutic target. A recentstudy emphasised an innovative approach against COVID-19 using polymerised type I collagen (PTIC) that improves disease severity through a hitherto unknownmechanism. In this paper, we provide an overview of potential mechanism thatmay explain the anti-inflammatory effect of collagen peptides. In addition,the paper includes a brief summary of possible side effect of collagendeposition in inflammatory diseases. Altogether, current knowledge suggeststhat collagen may potentially reduce the residual risk in inflammatorydiseases; however, the detailed mechanism remains elusive.

LDL-C augments whereas HDL-C prevents inflammatory innate immune memory.

Recent studies emphasize the importance of low-density lipoprotein cholesterol (LDL-C) in altering the hematopoietic cell compartment of bone marrow and of high-density lipoprotein cholesterol (HDL-C) in inhibiting metabolic endotoxemia-induced inflammation. The data suggest that these lipoproteins may exert their inflammatory or anti-inflammatory roles by modulating innate immune memory. Targeting specific LDL-C and HDL-C subfractions could therefore potentially reduce the residual risk in hepatic and cardiometabolic disease.

Synthesis and in vitro evaluation of cyclodextrin hyaluronic acid conjugates as a new candidate for intestinal drug carrier for steroid hormones.

Steroid hormones became increasingly interesting as active pharmaceutical ingredients for the treatment of endocrine disorders. However, medical applications of many steroidal drugs are inhibited by their very low aqueous solubilities giving rise to low bioavailabilities. Therefore, the prioritized oral administration of steroidal drugs remains problematic. Cyclodextrins are promising candidates for the development of drug delivery systems for oral route applications, since they solubilize hydrophobic steroids and increase their rate of transport in aqueous environments. In this study, the synthesis and characterization of polymeric ß-cyclodextrin derivates is described, which result from the attachment of a hydrophilic ß-CD-thioether to hyaluronic acid. Host-guest complexes of the synthesized ß-cyclodextrin hyaluronic acid conjugates were formed with two poorly soluble model steroids (ß-estradiol, dexamethasone) and compared to monomeric ß-cyclodextrin derivates regarding solubilization and complexation efficiency. The ß-cyclodextrin-drug (host-guest) complexes were evaluated in vitro for their suitability (cytotoxicity and transport rate) as intestinal drug carriers for steroid hormones. In case of ß-estradiol, higher solubilities could be achieved by complexation with both synthesized ß-cyclodextrin derivates, leading to significantly higher intestinal transport rates in vitro. However, this success could not be shown for dexamethasone, which namely solubilized better, but could not enhance the transport rate significantly. Thus, this study demonstrates the biocompatibility of the synthesized and characterized ß-cyclodextrin derivates and shows their potential as new candidate for intestinal drug carrier for steroid hormones like ß-estradiol.

Reversible immobilization of a protein to a gold surface through multiple host-guest interactions.

Monolayers were formed by specific interactions between adamantylated proteins (transferrin, lysozyme) and a ß-cyclodextrin (ß-CD) monolayer on a gold surface. Very high stabilities could be reached by multiple interactions of 3-6 adamantyl moieties linked through triethylene glycol spacers to the protein with ß-CD rings attached to the surface. Furthermore, bound proteins could be completely removed from the surface through competitive binding of an excess of free adamantane. Regenerable protein sensor chips can be constructed by using this supramolecular toolbox. Attached proteins are still recognized by specific antibodies, which was attributed to a loose packing of the protein molecules at the ß-CD monolayer.

Solubilizing steroidal drugs by ß-cyclodextrin derivatives.

Administration of steroidal drugs is hampered by their very low solubilities in water. ß-Cyclodextrin and ß-cyclodextrin derivatives can solubilize steroids and improve bio-availability of these hydrophobic APIs. A systematic overview of the achievable solubility enhancements of various steroids, testosterone, estradiol, progesterone, hydrocortisone, prednisone, dexamethasone, and finasteride, is provided. Beside the spatial fit of the steroid within the cyclodextrin cavity also hydrophilic substituents at the cyclodextrin framework play an important role in the extent of solubilization observed. Uniformly substituted anionic heptakis-6-sulfoethylsulfanyl-6-deoxy-ß-cyclodextrin (HSES) performed best, reaching complexation efficiencies of 60-90mol% for most steroids. Two neutral ß-cyclodextrin thioethers, heptakis-6-methylsulfanyl-6-deoxy-2-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)]-ß-CD (HTMT) and heptakis-6-thioglyceryl-6-deoxy-ß-CD (HTG) showed gender selectivity in binding of hormons: HTMT was selective for testosterone, while HTG was selective for estradiol. Solubilization is mainly due to complexation of the A and B rings as well as C and D rings of the steroid framework as demonstrated by ROESY NMR spectroscopy.

Synthesis of uniform cyclodextrin thioethers to transport hydrophobic drugs.

Methyl and ethyl thioether groups were introduced at all primary positions of α-, ß-, and γ-cyclodextrin by nucleophilic displacement reactions starting from the corresponding per-(6-deoxy-6-bromo)cyclodextrins. Further modification of all 2-OH positions by etherification with iodo terminated triethylene glycol monomethyl ether (and tetraethylene glycol monomethyl ether, respectively) furnished water-soluble hosts. Especially the ß-cyclodextrin derivatives exhibit very high binding potentials towards the anaesthetic drugs sevoflurane and halothane. Since the resulting inclusion compounds are highly soluble in water at temperatures ≤37 °C they are good candidates for new aqueous dosage forms which would avoid inhalation anaesthesia.