TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

During inflammation, neutrophils are one of the first responding cells of innate immunity, contributing to a fast clearance of infection and return to homeostasis. However, excessive neutrophil infiltration accelerates unsolicited disproportionate inflammation for instance in autoimmune diseases such as rheumatoid arthritis. The transient-receptor-potential channel-kinase TRPM7 is an essential regulator of immune system homeostasis. Naive murine T cells with genetic inactivation of the TRPM7 enzyme, due to a point mutation at the active site, are unable to differentiate into pro-inflammatory T cells, whereas regulatory T cells develop normally. Moreover, TRPM7 is vital for lipopolysaccharides (LPS)-induced activation of murine macrophages. Within this study, we show that the channel-kinase TRPM7 is functionally expressed in neutrophils and has an important impact on neutrophil recruitment during inflammation. We find that human neutrophils cannot transmigrate along a CXCL8 chemokine gradient or produce reactive oxygen species in response to gram-negative bacterial lipopolysaccharide LPS, if TRPM7 channel or kinase activity are blocked. Using a recently identified TRPM7 kinase inhibitor, TG100-115, as well as murine neutrophils with genetic ablation of the kinase activity, we confirm the importance of both TRPM7 channel and kinase function in murine neutrophil transmigration and unravel that TRPM7 kinase affects Akt1/mTOR signaling thereby regulating neutrophil transmigration and effector function. Hence, TRPM7 represents an interesting potential target to treat unwanted excessive neutrophil invasion

Alkylating sulfur mustard induces migration deficiencies and senescence in human mesenchymal stem cells as possible cause of the wound healing disorder

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The Efficiency of Direct Maturation: the Comparison of Two hiPSC Differentiation Approaches into Motor Neurons

Motor neurons (MNs) derived from human-induced pluripotent stem cells (hiPSC) hold great potential for the treatment of various motor neurodegenerative diseases as transplantations with a low-risk of rejection are made possible. There are many hiPSC differentiation protocols that pursue to imitate the multistep process of motor neurogenesis in vivo. However, these often apply viral vectors, feeder cells, or antibiotics to generate hiPSC and MNs, limiting their translational potential. In this study, a virus-, feeder-, and antibiotic-free method was used for reprogramming hiPSC, which were maintained in culture medium produced under clinical good manufacturing practice. Differentiation into MNs was performed with standardized, chemically defined, and antibiotic-free culture media. The identity of hiPSC, neuronal progenitors, and mature MNs was continuously verified by the detection of specific markers at the genetic and protein level via qRT-PCR, flow cytometry, Western Blot, and immunofluorescence. MNX1- and ChAT-positive motoneuronal progenitor cells were formed after neural induction via dual-SMAD inhibition and expansion. For maturation, an approach aiming to directly mature these progenitors was compared to an approach that included an additional differentiation step for further specification. Although both approaches generated mature MNs expressing characteristic postmitotic markers, the direct maturation approach appeared to be more efficient. These results provide new insights into the suitability of two standardized differentiation approaches for generating mature MNs, which might pave the way for future clinical applications

NAD⁺ Acts as a Protective Factor in Cellular Stress Response to DNA Alkylating Agents

Sulfur mustard (SM) and its derivatives are potent genotoxic agents, which have been shown to trigger the activation of poly (ADP-ribose) polymerases (PARPs) and the depletion of their substrate, nicotinamide adenine dinucleotide (NAD+). NAD+ is an essential molecule involved in numerous cellular pathways, including genome integrity and DNA repair, and thus, NAD+ supplementation might be beneficial for mitigating mustard-induced (geno)toxicity. In this study, the role of NAD+ depletion and elevation in the genotoxic stress response to SM derivatives, i.e., the monofunctional agent 2-chloroethyl-ethyl sulfide (CEES) and the crosslinking agent mechlorethamine (HN2), was investigated with the use of NAD+ booster nicotinamide riboside (NR) and NAD+ synthesis inhibitor FK866. The effects were analyzed in immortalized human keratinocytes (HaCaT) or monocyte-like cell line THP-1. In HaCaT cells, NR supplementation, increased NAD+ levels, and elevated PAR response, however, did not affect ATP levels or DNA damage repair, nor did it attenuate long- and short-term cytotoxicities. On the other hand, the depletion of cellular NAD+ via FK866 sensitized HaCaT cells to genotoxic stress, particularly CEES exposure, whereas NR supplementation, by increasing cellular NAD+ levels, rescued the sensitizing FK866 effect. Intriguingly, in THP-1 cells, the NR-induced elevation of cellular NAD+ levels did attenuate toxicity of the mustard compounds, especially upon CEES exposure. Together, our results reveal that NAD+ is an important molecule in the pathomechanism of SM derivatives, exhibiting compound-specificity. Moreover, the cell line-dependent protective effects of NR are indicative of system-specificity of the application of this NAD+ booster.publishe

Chronic senescent human mesenchymal stem cells as possible contributor to the wound healing disorder after exposure to the alkylating agent sulfur mustard

Wound healing is a complex process, and disturbance of even a single mechanism can result in chronic ulcers developing after exposure to the alkylating agent sulfur mustard (SM). A possible contributor may be SM-induced chronic senescent mesenchymal stem cells (MSCs), unable to fulfil their regenerative role, by persisting over long time periods and creating a proinflammatory microenvironment. Here we show that senescence induction in human bone marrow derived MSCs was time- and concentration-dependent, and chronic senescence could be verified 3 weeks after exposure to between 10 and 40 µM SM. Morphological changes, reduced clonogenic and migration potential, longer scratch closure times, differences in senescence, motility and DNA damage response associated genes as well as increased levels of proinflammatory cytokines were revealed. Selective removal of these cells by senolytic drugs, in which ABT-263 showed initial potential in vitro, opens the possibility for an innovative treatment strategy for chronic wounds, but also tumors and age-related diseases.publishe