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Immune cells undergo metabolic reprogramming upon exposure to inflammatory stimuli. The immuneresponsive gene 1 (Irg1) encodes aconitate decarboxylase (Acod1), an enzyme that generates itaconate from cis-aconitate in the TCA cycle and is one of the most highly induced genes in macrophages during inflammation. Itaconate inhibits succinate dehydrogenase activity leading to the accumulation of succinate. As the adjustment of energy metabolism also plays an important role during the formation of bone-resorbing osteoclasts, we investigated if Irg1 is regulated during osteoclastogenesis. We show that M-CSF/RANKL treatment induces Irg1 at an early time-point in bone marrow-derived macrophages (BMDM) as well as in the RAW264.7 macrophage cell line. Next, we stably overexpressed Acod1 in RAW264.7 cells. The metabolism of these Acod1 cells shifted towards glycolysis, as indicated by an increase in mTOR activation, subsequent 4EB-P1 phosphorylation and reduced ATP levels. When we investigated the ability of Acod1 cells to differentiate into osteoclasts, we observed a remarkable suppression of osteoclast-associated genes and the number of TRAP-positive, multi-nucleated osteoclasts was greatly reduced but not completely abrogated. Surprisingly, NFATc1 was detectable in nuclear extracts in untreated Acod1 cells leading to residual transcriptional NFAT activity in luciferase assays. This is caused by the elevated levels of succinate in Acod1 cells, as succinate can bind extracellularly to its cognate receptor GPR91 leading to Gq-mediated activation of NFATc1. When we investigated the expression of Gpr91, we found RANKL-mediated induction of Gpr91 to be severely reduced in Acod1 cells and we suggest that GPR91 is a target of RANKL-mediated NFATc1 activation. However, on the protein level, the receptor was still expressed at the cell surface. The observed repression of Gpr91 in Acod1 overexpressing cells was also detected by treatment with octyl-itaconate, showing that this is an itaconate-mediated effect. We hypothesize that the itaconate-mediated increase in succinate levels causes activation of NFATc1 signalling, although the transcriptional activity does not lead to osteoclastogenesis. In the presence of RANKL, these pre-activated cells are slow in switching to RANKL-mediated induction of osteoclast genes, which decreases their ability to differentiate into osteoclasts.

Muco-obstructive lung diseases are characterized by airway obstruction and hyperinflation, which can be quantified by imaging. Our aim was to evaluate µCT for longitudinal quantification of muco-obstructive lung disease in β-epithelial Na+ channel overexpressing ( Scnn1b-TG) mice and of the effects of neutrophil elastase (NE) knockout on its progression. Lungs from wild-type (WT), NE−/−, Scnn1b-TG, and Scnn1b-TG/NE−/− mice were scanned with 9-µm resolution at 0, 5, 14, and 60 days of age, and airway and parenchymal disease was quantified. Mucus adhesion lesions (MAL) were persistently increased in Scnn1b-TG compared with WT mice from 0 days (20.25 ± 6.50 vs. 9.60 ± 2.07, P < 0.05), and this effect was attenuated in Scnn1b-TG/NE−/− mice (5.33 ± 3.67, P < 0.001). Airway wall area percentage (WA%) was increased in Scnn1b-TG mice compared with WT from 14 days onward (59.2 ± 6.3% vs. 49.8 ± 9.0%, P < 0.001) but was similar in Scnn1b-TG/NE−/− compared with WT at 60 days (46.4 ± 9.2% vs. 45.4 ± 11.5%, P = 0.97). Air proportion (Air%) and mean linear intercept ( Lm) were persistently increased in Scnn1b-TG compared with WT from 5 days on (53.9 ± 4.5% vs. 30.0 ± 5.5% and 78.82 ± 8.44 µm vs. 65.66 ± 4.15 µm, respectively, P < 0.001), whereas in Scnn1b-TG/NE−/−, Air% and Lm were similar to WT from birth (27.7 ± 5.5% vs. 27.2 ± 5.9%, P = 0.92 and 61.48 ± 9.20 µm vs. 61.70 ± 6.73 µm, P = 0.93, respectively). Our results suggest that µCT is sensitive to detect the onset and progression of muco-obstructive lung disease and effects of genetic deletion of NE on morphology of airways and lung parenchyma in Scnn1b-TG mice, and that it may serve as a sensitive endpoint for preclinical studies of novel therapeutic interventions for muco-obstructive lung diseases.

Separating wounded serosa by physical barriers is the only clinically approved adjunct for postoperative adhesion prevention. Since the optimal adhesion barrier has not been found, it is essential to improve our pathogenic understanding of adhesion formation and to compare the effects of different barrier materials on tissue and cells. Wistar rats underwent standardized peritoneal damage and were treated either with Seprafilm, Adept, Intercoat, Spraygel, SupraSeal or remained untreated as a control. 14 days postoperatively, the lesions were explanted and histomorphologically analyzed using the European ISO score to evaluate material implants. Striking differences between the material groups were present regarding the inflammation, fibrosis, and foreign body reaction. According to the ISO score, Intercoat and Spraygel were considered as nonirritating to tissue. Adept, Seprafilm, and SupraSeal were assessed as mild-irritating materials. Interestingly, the most effective material in adhesion prevention revealed moderate inflammation accompanied by minor fibrosis. The degree of inflammation to barrier materials does not predict the efficacy in the prevention of adhesions. Histopathological investigations are crucial to improve our understanding of the cellular mechanisms during adhesion formation and elucidate the tissue response to material approaches used in adhesion prevention. This will lead to improved antiadhesive strategies and the development of functional barrier biomaterials. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 598-609, 2018.

The axial connective tissue system is a fiber continuum of the lung that maintains alveolar surface area during changes in lung volume. Although the molecular anatomy of the axial system remains undefined, the fiber continuum of the lung is central to contemporary models of lung micromechanics and alveolar regeneration. To provide a detailed molecular structure of the axial connective tissue system, we examined the extracellular matrix of murine lungs. The lungs were decellularized using a 24 hour detergent treatment protocol. Systematic evaluation of the decellularized lungs demonstrated no residual cellular debris; morphometry demonstrated a mean 39±7% reduction in lung dimensions. Scanning electron microscopy (SEM) demonstrated an intact structural hierarchy within the decellularized lung. Light, fluorescence, and scanning electron microscopy of precision-cut lung slices demonstrated that alveolar duct structure was defined by a cable line element encased in basement membrane. The cable line element arose in the distal airways, passed through septal tips and inserted into neighboring blood vessels and visceral pleura. The ropelike appearance, collagenase resistance and anti-elastin immunostaining indicated that the cable was an elastin macromolecule. Our results indicate that the helical line element of the axial connective tissue system is composed of an elastin cable that not only defines the structure of the alveolar duct, but also integrates the axial connective tissue system into visceral pleura and peripheral blood vessels.

Cycloallenes, 9. — 1-Phenyl-1-aza-3,4-cyclohexadiene, the First Isodihydropyridine: Generation and Interception The generation of the title cycloallene 4 from 6,6-dibromo-3-phenyl-3-azabicyclo[3.1.0]hexane (10) with methyllithium according to the Doering-Moore-Skattebol method is reported. In the presence of styrene, 1,3-butadiene, furan, 1,3-cyclohexadiene, and 1,3-cyclopentadiene, 4 is intercepted in [2 + 2] and [4 + 2] cycloadditions with formation of the hexahydrocyclobuta[c]pyridines 6 and 11 and the hexahydroisoquinolines 12–15, respectively. The starting compound 10 is prepared via the cis-disubstituted 1,1-dibromocyclopropanes 8 and 9 from the known dioxepine derivative 7, the synthesis of which has been considerably improved. On treatment with n-butyllithium, the dichlorophenylazabicyclohexane 3 is converted into 4 as well. However, in the competition for 4, styrene is not successful against n-butyllithium, which transforms 4 into the pentadienylamine 5.

The low temperature photooxygenation of the furocoumarins 1a–e, the naphthofurans 1f–h, and the furoquinoline 1i afforded the corresponding dioxetanes 2 in good yields, except the linearly annulated naphthofuran 1f, which led exclusively to the allylic hydroperoxide 5f by ene reaction with singlet oxygen. The dioxetanes 2 were rigorously purified by means of low temperature silica gel chromatography and fully characterized. Thermolysis of the furocoumarin dioxetanes 2a–e led to the expected cleavage products 3a–e (acetyloxy- and acetyl-substituted coumarins), while the naphtho- and quino-linofuran dioxetanes 2g–i gave in addition to the cleavage products 3g–i also the spiroepoxides 4g–i. Intramolecular electron transfer from the aromatic moiety to the dioxetane ring, with subsequent cyclization of the radical-ion species and fragmentation is postulated as mechanism for the formation of this novel rearrangement product of dioxetanes. On heating in chloroform the spiroepoxides 4g,h rearranged into the 1,3-dioxole 6g, but heating of 4g in methanol gave the dioxine 7g by trapping of the dipolar intermediate. — The furocoumarin dioxetanes 2a,c,d showed high mutagenic activity in the Salmonella typhimurium strain TA 100 but not in the strain TA 2638. The naphtho- and quinolinofuran dioxetanes 2h,i and the spiroepoxides 4g,h were nonmutagenic in the TA 100 strain. Epoxide-like DNA damage appears to be responsible for the mutagenicity of the furocoumarin dioxetanes 2a,c,d.