Development of Imiquimod-induced HaCaT-THP-1 co-culture for modeling of psoriasis.

Psoriasis is one of the most prevalent and chronic inflammatory disease of the skin, associated with disrupted barrier function. Currently, a widely accepted, generally usable cell culture model has not been developed yet. In the present work, we aimed to establish a co-culture model with human keratinocyte (HaCaT) and human monocyte cells (THP-1) induced by Imiquimod (IMQ), which acts on the TLR7 receptor. The role of TLR7 expressed on THP-1 cells was confirmed by immunofluorescence staining of NF-κB activation. Chloroquine (CH) was used as a receptor inhibitor, in the presence or absence of which the NF-κB pathway was activated. We determined the most effective proliferation-stimulating IMQ concentration by RTCA method and the hyperproliferative effect was investigated by wound-healing test. The effect of IMQ was compared with the effects of the anthocyanin (AC) components from the anti-inflammatory sour cherry extract that we have already studied. We found that IMQ significantly increased the migration rate however, the combined treatment resulted in a decreased migration rate compared to the IMQ treatment alone. Inflammatory cytokines were measured from the supernatant of co-culture by ELISA. During the development of the co-culture intended to model psoriasis, we confirmed the induction effect of IMQ and in the case of AC treatment, we supported the stabilizing effect of the barrier.

Effectiveness of Anthocyanin-Rich Sour Cherry Extract on Gliadin-Induced Caco-2 Barrier Damage.

Several types of gluten-related disorders are known, in which the common starting point is gluten-induced zonulin release. Zonulin results in varying degrees of increased permeability in certain gluten-related disorders but is largely responsible for the development of further pathogenic processes and symptoms. Therefore, it is important to know the barrier-modulating role of individual nutritional components and to what extent the antioxidant substance supports the protection of gliadin-induced membrane damage with its radical scavenging capacity. We investigated the pH dependence of the gliadin-anthocyanin interaction using UV photometry, during which a concentration-dependent interaction was observed at pH 6.8. The barrier modulatory effect of the anthocyanin-rich sour cherry extract (AC) was analyzed on Caco-2 cell culture with pepsin-trypsin-resistant gliadin (PT-gliadin) exposure by TEER measurement, zonula occludens-1 (ZO-1), and Occludin immunohistochemistry. In addition to the TEER-reducing and TJ-rearranging effects of PT-gliadin, NF-κB activation, an increase in cytokine (TNF-α, IFN-γ, and IL-8) release, and mitochondrial ROS levels were observed. We confirmed the anti-inflammatory, stabilizing, and restoring roles of AC extract during gliadin treatment on the Caco-2 monolayer. The extract was able to significantly reduce cytokine and ROS levels despite the known interaction of the main components of the extract with PT-gliadin.

A vapBC-type toxin-antitoxin module of Sinorhizobium meliloti influences symbiotic efficiency and nodule senescence of Medicago sativa.

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti carries a large number of toxin-antitoxin (TA) modules both on the chromosome and megaplasmids. One of them, the vapBC-5 module that belongs to the type II systems was characterized here. It encodes an active toxin vapC-5, and was shown to be controlled negatively by the complex of its own proteins. Different mutants of the vapBC-5 genes exhibited diverse effects on symbiotic efficiency during interaction with the host plant Medicago sativa. The absence of the entire vapBC-5 region had no influence on nodule formation and nitrogen fixation properties. The strain carrying an insertion in the antitoxin gene showed a reduced nitrogen fixation capacity resulting in a lower plant yield. In contrast, when the toxin gene was mutated, the strain developed more efficient symbiosis with the host plant. The nitrogen fixing root nodules had a delayed senescent phenotype and contained elevated level of plant-derived molecules characteristic of later steps of nodule development. The longer bacteroid viability and abundance of active nitrogen fixing zone resulted in increased production of plant material. These data indicate that modification of the toxin/antitoxin production may influence bacteroid metabolism and may have an impact on the adaptation to changing environmental conditions.

LIN, a novel type of U-box/WD40 protein, controls early infection by rhizobia in legumes.

The formation of a nitrogen-fixing nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. Based on its mutant phenotype, lumpy infections (lin), LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. We show that spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase is independent of LIN; thus, LIN is not necessary for nodule organogenesis. From this, we infer that LIN predominantly functions during rhizobial colonization and that the abortion of this process in lin mutants leads to a suppression of nodule development. Here, we identify the LIN gene in Medicago truncatula and Lotus japonicus, showing that it codes for a predicted E3 ubiquitin ligase containing a highly conserved U-box and WD40 repeat domains. Ubiquitin-mediated protein degradation is a universal mechanism to regulate many biological processes by eliminating rate-limiting enzymes and key components such as transcription factors. We propose that LIN is a regulator of the component(s) of the nodulation factor signal transduction pathway and that its function is required for correct temporal and spatial activity of the target protein(s).

Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/DMI2 signalling pathway.

Legumes form two different types of intracellular root symbioses, with fungi and bacteria, resulting in arbuscular mycorrhiza and nitrogen-fixing nodules, respectively. Rhizobial signalling molecules, called Nod factors, play a key role in establishing the rhizobium-legume association and genes have been identified in Medicago truncatula that control a Nod factor signalling pathway leading to nodulation. Three of these genes, the so-called DMI1, DMI2 and DMI3 genes, are also required for formation of mycorrhiza, indicating that the symbiotic pathways activated by both the bacterial and the fungal symbionts share common steps. To analyse possible cross-talk between these pathways we have studied the effect of treatment with Nod factors on mycorrhization in M. truncatula. We show that Nod factors increase mycorrhizal colonization and stimulate lateral root formation. The stimulation of lateral root formation by Nod factors requires both the same structural features of Nod factors and the same plant genes (NFP, DMI1, DMI2, DMI3 and NSP1) that are required for other Nod factor-induced symbiotic responses such as early nodulin gene induction and cortical cell division. A diffusible factor from arbuscular mycorrhizal fungi was also found to stimulate lateral root formation, while three root pathogens did not have the same effect. Lateral root formation induced by fungal signal(s) was found to require the DMI1 and DMI2 genes, but not DMI3. The idea that this diffusible fungal factor might correspond to a previously hypothesized mycorrhizal signal, the 'Myc factor', is discussed.