We have identified two redundant GTPase activating proteins (GAPs) -RGA-3 and RGA-4 -that regulate Rho GTPase function at the plasma membrane in early Caenorhabditis elegans embryos. Knockdown of both RhoGAPs resulted in extensive membrane ruffling, furrowing and pronounced pseudo-cleavages. In addition, the non-muscle myosin NMY-2 and RHO-1 accumulated on the cortex at sites of ruffling. RGA-3 and RGA-4 are GAPs for RHO-1, but most probably not for CDC-42, because only RHO-1 was epistatic to the two GAPs, and the GAPs had no obvious influence on CDC-42 function. Furthermore, knockdown of either the RHO-1 effector, LET-502, or the exchange factor for RHO-1, ECT-2, alleviated the membrane-ruffling phenotype caused by simultaneous knockdown of both RGA-3 and RGA-4 [rga-3/4 (RNAi)]. GFP::PAR-6 and GFP::PAR-2 were localized at the anterior and posterior part of the early C. elegans embryo, respectively showing that rga-3/4 (RNAi) did not interfere with polarity establishment. Most importantly, upon simultaneous knockdown of RGA-3, RGA-4 and the third RhoGAP present in the early embryo, CYK-4, NMY-2 spread over the entire cortex and GFP::PAR-2 localization at the posterior cortex was greatly diminished. These results indicate that the functions of CYK-4 are temporally and spatially distinct from RGA-3 and RGA-4 (RGA-3/4). RGA-3/4 and CYK-4 also play different roles in controlling LET-502 activation in the germ line, because rga-3/4 (RNAi), but not cyk-4 (RNAi), aggravated the let-502(sb106) phenotype. We propose that RGA-3/4 and CYK-4 control with which effector molecules RHO-1 interacts at particular sites at the cortex in the zygote and in the germ line.

Alternariol (AOH) is a secondary metabolite formed by black mold of the genus Alternaria alternata. Due to limited hazard and occurrence data, AOH is still considered as an “emerging mycotoxin” and, as such, not monitored and regulated yet. Recent studies indicate immunosuppressive effects in vitro by altering the expression of CD molecules and proinflammatory cytokines, which are indispensable in mounting an innate immune response. However, the mode of action by which AOH exerts its immunosuppressive effects has not been unraveled yet. The present study aimed to characterise the impact of AOH on the nuclear factor kappa B (NF-κB) pathway, the expression of NF-κB target cytokines and involved regulatory microRNAs (miRNAs). In THP-1 derived macrophages, AOH (1–20 µM) was found to suppress lipopolysaccharide (LPS)-induced NF-κB pathway activation, decrease secretion of the proinflammatory cytokines IL-8, IL-6, TNF-α and to induce secretion of the anti-inflammatory IL-10. Thereby, a distinct pattern of cytokine mRNA levels was monitored, varying between short- and long-term exposure. Concomitantly, AOH (2–20 µM) affected the transcription levels of miR-146a and miR-155 in LPS-stimulated THP-1 derived macrophages dose-dependently by down- and upregulation, respectively. In contrast, transcription of miR-16 and miR-125b, two other immune-related miRNAs, was not modulated. In the absence of a LPS stimulus, AOH (20 µM) did not affect basal NF-κB activity, but increased IL-10 transcription. Collectively, our results indicate, that AOH itself does not induce a proinflammatory immune response in human macrophages; however, in an inflamed environment it possesses the ability to repress inflammation by targeting the NF-κB signalling pathway and regulatory miRNAs.

ScopeAlternariol (AOH), a toxic secondary metabolite of Alternaria spp., may contaminate a broad spectrum of food and feed. Besides its cytotoxic, genotoxic, and estrogenic properties, several studies report the potential of AOH to suppress the rich network of immune responses. The specific effect of AOH on inflammation‐related signaling in non‐immune cells of the intestinal epithelial layer has, however, not been investigated yet. Since intestinal epithelial cells (IECs) are, compared to underlying cells, exposed to higher concentrations of the ingested mycotoxin, the question is addressed whether immunomodulation by AOH at the gastrointestinal barrier must be considered.Methods and resultsThe impact of AOH (0.02–40 µm) on inflammatory signaling in either IL‐1β‐stimulated or non‐stimulated differentiated Caco‐2 cells is determined. AOH significantly reduces IL‐1β transcription after 5 h but shows an increasing tendency on IL‐8 transcript levels after long‐term exposure (20 h). In IL‐1β‐stimulated cells, AOH (20–40 µm) augments TNF‐α transcripts while repressing IL‐8, IL‐6, and IL‐1β transcription as well as IL‐8 secretion. Furthermore, inflammation‐related microRNAs miR‐16, miR‐146a, miR‐125b, and miR‐155 are altered in response to AOH.ConclusionThe obtained data indicate that AOH represses immune responses in an inflamed environment, possibly leading to higher susceptibility to diseases.

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An anthocyanin-rich diet is considered to protect against chronic inflammatory processes although the bioavailability of anthocyanins is regarded as rather low. Moreover, the immunomodulatory role of anthocyanins is not fully understood yet. In the present study, fractions of blackberry (Rubus fruticosus) juice were investigated in plasma-relevant concentrations with respect to their immunomodulatory properties in lipopolysaccharide (LPS)-challenged THP-1-derived macrophages. The complex blackberry extract acted ineffective as well as potential degradation products. Cyanidin-3O-glucoside (Cy3glc), the main constituent of blackberry anthocyanins, diminished TNF-α levels at a concentration of 0.02 µg/mL, indicating protective effects as measured with quantitative RT-PCR and multiplex cytokine assays. LPS-boosted activity of transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) of differentiated THP-1 reporter gene cells was marginally inhibited by Cy3glc. LPS-induced microRNA-155 was further increased, supporting the evidence of protection. Of note, fractions obtained from blackberry juice, in particular cyanidin-3O-(6″-dioxalylglucoside), were displaying potential pro-inflammatory properties as these elevated IL-6 and TNF-α levels. In conclusion, highly purified anthocyanin fractions of blackberry juice display both anti- and pro-inflammatory properties at plasma-relevant concentrations depending on their structure and substitution pattern.