Improved tropoelastin synthesis in the skin by codon optimization and nucleotide modification of tropoelastin-encoding synthetic mRNA.

Loss of elastin due to aging, disease, or injury can lead to impaired tissue function. In this study, de novo tropoelastin (TE) synthesis is investigated in vitro and in vivo using different TE-encoding synthetic mRNA variants after codon optimization and nucleotide modification. Codon optimization shows a strong effect on protein synthesis without affecting cell viability in vitro, whereas nucleotide modifications strongly modulate translation and reduce cell toxicity. Selected TE mRNA variants (3, 10, and 30 µg) are then analyzed in vivo in porcine skin after intradermal application. Administration of 30 µg of native TE mRNA with a me1 Ψ modification or 10 and 30 µg of unmodified codon-optimized TE mRNA is required to increase TE protein expression in vivo. In contrast, just 3 µg of a codon-optimized TE mRNA variant with the me1 Ψ modification is able to increase protein expression. Furthermore, skin toxicity is investigated in vitro by injecting 30 µg of mRNA of selected TE mRNA variants into a human full-thickness skin model, and no toxic effects are observed. Thereby, for the first time, an increased dermal TE synthesis by exogenous administration of synthetic mRNA is demonstrated in vivo. Codon optimization of a synthetic mRNA can significantly increase protein expression and therapeutic outcome.

Acute and chronic inflammation alter immunometabolism in a cutaneous delayed-type hypersensitivity reaction (DTHR) mouse model.

T-cell-driven immune responses are responsible for several autoimmune disorders, such as psoriasis vulgaris and rheumatoid arthritis. Identification of metabolic signatures in inflamed tissues is needed to facilitate novel and individualised therapeutic developments. Here we show the temporal metabolic dynamics of T-cell-driven inflammation characterised by nuclear magnetic resonance spectroscopy-based metabolomics, histopathology and immunohistochemistry in acute and chronic cutaneous delayed-type hypersensitivity reaction (DTHR). During acute DTHR, an increase in glutathione and glutathione disulfide is consistent with the ear swelling response and degree of neutrophilic infiltration, while taurine and ascorbate dominate the chronic phase, suggesting a switch in redox metabolism. Lowered amino acids, an increase in cell membrane repair-related metabolites and infiltration of T cells and macrophages further characterise chronic DTHR. Acute and chronic cutaneous DTHR can be distinguished by characteristic metabolic patterns associated with individual inflammatory pathways providing knowledge that will aid target discovery of specialised therapeutics.

Immunomodulatory role of reactive oxygen species and nitrogen species during T cell-driven neutrophil-enriched acute and chronic cutaneous delayed-type hypersensitivity reactions.

Rationale: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important regulators of inflammation. The exact impact of ROS/RNS on cutaneous delayed-type hypersensitivity reaction (DTHR) is controversial. The aim of our study was to identify the dominant sources of ROS/RNS during acute and chronic trinitrochlorobenzene (TNCB)-induced cutaneous DTHR in mice with differently impaired ROS/RNS production. Methods: TNCB-sensitized wild-type, NADPH oxidase 2 (NOX2)- deficient (gp91phox-/-), myeloperoxidase-deficient (MPO-/-), and inducible nitric oxide synthase-deficient (iNOS-/-) mice were challenged with TNCB on the right ear once to elicit acute DTHR and repetitively up to five times to induce chronic DTHR. We measured ear swelling responses and noninvasively assessed ROS/RNS production in vivo by employing the chemiluminescence optical imaging (OI) probe L-012. Additionally, we conducted extensive ex vivo analyses of inflamed ears focusing on ROS/RNS production and the biochemical and morphological consequences. Results: The in vivo L-012 OI of acute and chronic DTHR revealed completely abrogated ROS/RNS production in the ears of gp91phox-/- mice, up to 90 % decreased ROS/RNS production in the ears of MPO-/- mice and unaffected ROS/RNS production in the ears of iNOS-/- mice. The DHR flow cytometry analysis of leukocytes derived from the ears with acute DTHR confirmed our in vivo L-012 OI results. Nevertheless, we observed no significant differences in the ear swelling responses among all the experimental groups. The histopathological analysis of the ears of gp91phox-/- mice with acute DTHRs revealed slightly enhanced inflammation. In contrast, we observed a moderately reduced inflammatory immune response in the ears of gp91phox-/- mice with chronic DTHR, while the inflamed ears of MPO-/- mice exhibited the strongest inflammation. Analyses of lipid peroxidation, 8-hydroxy-2'deoxyguanosine levels, redox related metabolites and genomic expression of antioxidant proteins revealed similar oxidative stress in all experimental groups. Furthermore, inflamed ears of wild-type and gp91phox-/- mice displayed neutrophil extracellular trap (NET) formation exclusively in acute but not chronic DTHR. Conclusions: MPO and NOX2 are the dominant sources of ROS/RNS in acute and chronic DTHR. Nevertheless, depletion of one primary source of ROS/RNS exhibited only marginal but conflicting impact on acute and chronic cutaneous DTHR. Thus, ROS/RNS are not a single entity, and each species has different properties at certain stages of the disease, resulting in different outcomes.

Hydrojet-based delivery of footprint-free iPSC-derived cardiomyocytes into porcine myocardium.

The reprogramming of patient´s somatic cells into induced pluripotent stem cells (iPSCs) and the consecutive differentiation into cardiomyocytes enables new options for the treatment of infarcted myocardium. In this study, the applicability of a hydrojet-based method to deliver footprint-free iPSC-derived cardiomyocytes into the myocardium was analyzed. A new hydrojet system enabling a rapid and accurate change between high tissue penetration pressures and low cell injection pressures was developed. Iron oxide-coated microparticles were ex vivo injected into porcine hearts to establish the application parameters and the distribution was analyzed using magnetic resonance imaging. The influence of different hydrojet pressure settings on the viability of cardiomyocytes was analyzed. Subsequently, cardiomyocytes were delivered into the porcine myocardium and analyzed by an in vivo imaging system. The delivery of microparticles or cardiomyocytes into porcine myocardium resulted in a widespread three-dimensional distribution. In vitro, 7 days post-injection, only cardiomyocytes applied with a hydrojet pressure setting of E20 (79.57 ± 1.44%) showed a significantly reduced cell viability in comparison to the cells applied with 27G needle (98.35 ± 5.15%). Furthermore, significantly less undesired distribution of the cells via blood vessels was detected compared to 27G needle injection. This study demonstrated the applicability of the hydrojet-based method for the intramyocardial delivery of iPSC-derived cardiomyocytes. The efficient delivery of cardiomyocytes into infarcted myocardium could significantly improve the regeneration.

Temporal Dynamics of Reactive Oxygen and Nitrogen Species and NF-κB Activation During Acute and Chronic T Cell-Driven Inflammation.

PURPOSE: Reactive oxygen and nitrogen species (ROS/RNS) production and the NF-κB activation are critically involved in inflammatory responses, but knowledge about the temporal dynamics during acute and chronic inflammation is limited. Here, we present a comparative longitudinal in vivo study of both parameters in an experimental model of acute and chronic T cell-driven delayed-type hypersensitivity reaction (DTHR) using noninvasive optical imaging. PROCEDURES: Trinitrochlorobenzene (TNCB)-sensitized NF-κB-luciferase-reporter and wild-type mice were TNCB challenged on the right ear to elicit acute DTHR and then repetitively challenged (up to five times) to induce chronic DTHR. Mice were treated with the ROS-scavenging and NF-κB inhibiting molecule N-acetylcysteine (NAC) or underwent sham treatment. ROS/RNS production was noninvasively analyzed in vivo using the ROS-/RNS-sensitive chemiluminescent probe L-012, and NF-κB activation was measured using NF-κB-luciferase-reporter mice. H&E staining, CD3 and myeloperoxidase (MPO) immunohistochemistry (IHC), and quantitative PCR (qPCR) analyses were employed to investigate immune cell infiltration and expression of NF-κB- and ROS-/RNS-driven genes. RESULTS: In acute DTHR, we found strongly elevated ROS/RNS production and NF-κB activation 12 h after the 1st TNCB ear challenge, peaking at 24 h after the challenge. In chronic DTHR, ROS production peaked as early as 4 h after the 5th TNCB challenge, whereas NF-κB activity peaked after 12 h. The increase in ROS/RNS production in acute DTHR was higher than the increase in NF-κB activity but the relationship was inverse in chronic DTHR. Treatment with the ROS scavenger NAC had differential effects on ROS/RNS production and NF-κB activation during acute and chronic DTHR. Ex vivo cross-validation by histopathology and qPCR analysis correlated closely with the in vivo imaging results. CONCLUSIONS: Noninvasive in vivo imaging is capable of assessing the temporal dynamics of ROS/RNS production and NF-κB activation during progression from acute to chronic DTHR and enables monitoring of anti-inflammatory treatment responses.

Cysteine-type cathepsins promote the effector phase of acute cutaneous delayed-type hypersensitivity reactions.

Cysteine-type cathepsins such as cathepsin B are involved in various steps of inflammatory processes such as antigen processing and angiogenesis. Here, we uncovered the role of cysteine-type cathepsins in the effector phase of T cell-driven cutaneous delayed-type hypersensitivity reactions (DTHR) and the implication of this role on therapeutic cathepsin B-specific inhibition. Methods: Wild-type, cathepsin B-deficient (Ctsb-/-) and cathepsin Z-deficient (Ctsz-/-) mice were sensitized with 2,4,6-trinitrochlorobenzene (TNCB) on the abdomen and challenged with TNCB on the right ear to induce acute and chronic cutaneous DTHR. The severity of cutaneous DTHR was assessed by evaluating ear swelling responses and histopathology. We performed fluorescence microscopy on tissue from inflamed ears and lymph nodes of wild-type mice, as well as on biopsies from psoriasis patients, focusing on cathepsin B expression by T cells, B cells, macrophages, dendritic cells and NK cells. Cathepsin activity was determined noninvasively by optical imaging employing protease-activated substrate-like probes. Cathepsin expression and activity were validated ex vivo by covalent active site labeling of proteases and Western blotting. Results: Noninvasive in vivo optical imaging revealed strong cysteine-type cathepsin activity in inflamed ears and draining lymph nodes in acute and chronic cutaneous DTHR. In inflamed ears and draining lymph nodes, cathepsin B was expressed by neutrophils, dendritic cells, macrophages, B, T and natural killer (NK) cells. Similar expression patterns were found in psoriatic plaques of patients. The biochemical methods confirmed active cathepsin B in tissues of mice with cutaneous DTHR. Topically applied cathepsin B inhibitors significantly reduced ear swelling in acute but not chronic DTHR. Compared with wild-type mice, Ctsb-/- mice exhibited an enhanced ear swelling response during acute DTHR despite a lack of cathepsin B expression. Cathepsin Z, a protease closely related to cathepsin B, revealed compensatory expression in inflamed ears of Ctsb-/- mice, while cathepsin B expression was reciprocally elevated in Ctsz-/- mice. Conclusion: Cathepsin B is actively involved in the effector phase of acute cutaneous DTHR. Thus, topically applied cathepsin B inhibitors might effectively limit DTHR such as contact dermatitis or psoriasis. However, the cathepsin B and Z knockout mouse experiments suggested a complementary role for these two cysteine-type proteases.

Staphylococcal Enterotoxins Dose-Dependently Modulate the Generation of Myeloid-Derived Suppressor Cells.

Staphylococcus aureus is one of the major human bacterial pathogens causing a broad spectrum of serious infections. Myeloid-derived suppressor cells (MDSC) represent an innate immune cell subset capable of regulating host-pathogen interactions, yet their role in the pathogenesis of S. aureus infections remains incompletely defined. The aim of this study was to determine the influence of different S. aureus strains and associated virulence factors on human MDSC generation. Using an in vitro MDSC generation assay we demonstrate that low concentrations of supernatants of different S. aureus strains led to an induction of functional MDSC, whereas increased concentrations, conversely, reduced MDSC numbers. The concentration-dependent reduction of MDSC correlated with T cell proliferation and cytotoxicity. Several findings supported a role for staphylococcal enterotoxins in modulating MDSC generation. Staphylococcal enterotoxins recapitulated concentration-dependent MDSC induction and inhibition, T cell proliferation and cytotoxicity, while an enterotoxin-deficient S. aureus strain largely failed to alter MDSC. Taken together, we identified staphylococcal enterotoxins as main modulators of MDSC generation. The inhibition of MDSC generation by staphylococcal enterotoxins might represent a novel therapeutic target in S. aureus infections and beyond in non-infectious conditions, such as cancer.

Myeloid-Derived Suppressor Cells in Bacterial Infections.

Myeloid-derived suppressor cells (MDSCs) comprise monocytic and granulocytic innate immune cells with the capability of suppressing T- and NK-cell responses. While the role of MDSCs has been studied in depth in malignant diseases, the understanding of their regulation and function in infectious disease conditions has just begun to evolve. Here we summarize and discuss the current view how MDSCs participate in bacterial infections and how this knowledge could be exploited for potential future therapeutics.