Soft extracellular matrix drives endoplasmic reticulum stress-dependent S quiescence underlying molecular traits of pulmonary basal cells.

Cell culture on soft matrix, either in 2D and 3D, preserves the characteristics of progenitors. However, the mechanism by which the mechanical microenvironment determines progenitor phenotype, and its relevance to human biology, remains poorly described. Here we designed multi-well hydrogel plates with a high degree of physico-chemical uniformity to reliably address the molecular mechanism underlying cell state modification driven by physiological stiffness. Cell cycle, differentiation and metabolic activity could be studied in parallel assays, showing that the soft environment promotes an atypical S-phase quiescence and prevents cell drift, while preserving the differentiation capacities of human bronchoepithelial cells. These softness-sensitive responses are associated with calcium leakage from the endoplasmic reticulum (ER) and defects in proteostasis and enhanced basal ER stress. The analysis of available single cell data of the human lung also showed that this non-conventional state coming from the soft extracellular environment is indeed consistent with molecular feature of pulmonary basal cells. Overall, this study demonstrates that mechanical mimicry in 2D culture supports allows to maintain progenitor cells in a state of high physiological relevance for characterizing the molecular events that govern progenitor biology in human tissues. STATEMENT OF SIGNIFICANCE: This study focuses on the molecular mechanism behind the progenitor state induced by a soft environment. Using innovative hydrogel supports mimicking normal human lung stiffness, the data presented demonstrate that lung mechanics prevent drift while preserving the differentiation capabilities of lung epithelial cells. Furthermore, we show that the cells are positioned in a quiescent state in the atypical S phase. Mechanistically, we demonstrate that this quiescence: i) is driven by calcium leakage from the endoplasmic reticulum (ER) and basal activation of the PERK branch of ER stress signalling, and ii) protects cells from lethal ER stress caused by metabolic stress. Finally, we validate using human single-cell data that these molecular features identified on the soft matrix are found in basal lung cells. Our results reveal original and relevant molecular mechanisms orchestrating cell fate in a soft environment and resistance to exogenous stresses, thus providing new fundamental and clinical insights into basal cell biology.

Poly-l-lysine/Laminin Surface Coating Reverses Glial Cell Mechanosensitivity on Stiffness-Patterned Hydrogels.

Brain tissues demonstrate heterogeneous mechanical properties, which evolve with aging and pathologies. The observation in these tissues of smooth to sharp rigidity gradients raises the question of brain cell responses to both different values of rigidity and their spatial variations, in dependence on the surface chemistry they are exposed to. Here, we used recent techniques of hydrogel photopolymerization to achieve stiffness texturing down to micrometer resolution in polyacrylamide hydrogels. We investigated primary neuron adhesion and orientation as well as glial cell proliferative properties on these rigidity-textured hydrogels for two adhesive coatings: fibronectin or poly-l-lysine/laminin. Our main observation is that glial cell adhesion and proliferation is favored on the stiffer regions when the adhesive coating is fibronectin and on the softer ones when it consists of poly-l-lysine/laminin. This behavior was unchanged by the presence or the absence of neuronal cells. In addition, glial cells were not confined by sharp, micron-scaled gradients of rigidity. Our observations suggest that rigidity sensing could involve adhesion-related pathways that profoundly depend on surface chemistry.

Cells on Hydrogels with Micron-Scaled Stiffness Patterns Demonstrate Local Stiffness Sensing.

Cell rigidity sensing-a basic cellular process allowing cells to adapt to mechanical cues-involves cell capabilities exerting force on the extracellular environment. In vivo, cells are exposed to multi-scaled heterogeneities in the mechanical properties of the surroundings. Here, we investigate whether cells are able to sense micron-scaled stiffness textures by measuring the forces they transmit to the extracellular matrix. To this end, we propose an efficient photochemistry of polyacrylamide hydrogels to design micron-scale stiffness patterns with kPa/µm gradients. Additionally, we propose an original protocol for the surface coating of adhesion proteins, which allows tuning the surface density from fully coupled to fully independent of the stiffness pattern. This evidences that cells pull on their surroundings by adjusting the level of stress to the micron-scaled stiffness. This conclusion was achieved through improvements in the traction force microscopy technique, e.g., adapting to substrates with a non-uniform stiffness and achieving a submicron resolution thanks to the implementation of a pyramidal optical flow algorithm. These developments provide tools for enhancing the current understanding of the contribution of stiffness alterations in many pathologies, including cancer.

Polyacrylamide Hydrogels with Rigidity-Independent Surface Chemistry Show Limited Long-Term Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells on Soft Substrates.

In general, cells are cultured and adapted to the in vitro rigidities of plastic or glass ranging between 1 and 10 GPa, which is very far from physiological values that are mostly in the kilopascal range. Stem cells however show a high sensitivity to the rigidity of their culture environment, which impacts their differentiation program. Here, we address the impact of rigidity on the long-term maintenance of pluripotency in human induced pluripotent stem cells (hiPSCs) to determine whether soft substrates could provide a new standard for hiPSC expansion and maintenance. To do this, we set up a fabrication process of polyacrylamide-based culture supports with a rigidity-decoupled surface chemistry. Soft elastic substrates with uniform and reproducible physicochemical properties were designed. The maintenance of pluripotency of two hiPSCs lines on substrates with stiffnesses ranging from 3 to 25 kPa was studied with an identical chemical coating consisting of a truncated recombinant vitronectin with defined surface density. Based on the analysis of cellular adhesion, survival, growth kinetics, three-dimensional distribution, and gene and protein expressions, we demonstrate that below 25 kPa hiPSCs do not maintain pluripotency on long-term culture, while pluripotency and self-renewal capacities are maintained above 25 kPa. In contrast to previous studies, no drift toward a specific germ line lineage was revealed. On soft substrates, cell colonies started to grow in three-dimensional (3D), suggesting that softness allows cells to limit contact with the synthetic matrix and to build their own microenvironment. These observations drastically limit the benefit of using standardized soft substrates to expand hiPSCs, at least with the current culture conditions. The development of a robust technology for the design of soft substrates nevertheless opens up perspectives to fine-tune physicochemical properties of the culture environment in addition to or in replacement of soluble growth factors to finely direct cell fate.

Reactivity of chemical sensitizers toward amino acids in cellulo plays a role in the activation of the Nrf2-ARE pathway in human monocyte dendritic cells and the THP-1 cell line.

Allergic contact dermatitis resulting from skin sensitization is an inflammatory skin disease linked to the use of chemicals termed haptens. Chemical reactivity is necessary for a chemical to be a sensitizer, allowing both covalent binding to proteins and maturation of dendritic cells (DCs) by mimicking "danger signals." The aim of this study was to evaluate how the reactivity of chemical sensitizers toward amino acids translates into a biological response using the activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway, which was assessed by the induction of three Nrf2 target genes (ho-1, nqo1, and il-8) and Nrf2 protein accumulation. Nrf2 activation is known to play a role in numerous detoxification mechanisms that could regulate danger signal outcomes in myeloid cells. Monocyte-derived DCs and THP-1 cells were exposed to (a) haptens with cysteine, lysine, or cysteine/lysine reactivity, (b) pro-/prehaptens, and (c) nonsensitizing molecules with reducing or oxidative properties (17 molecules in total). Chemicals were classified as "Nrf2 pathway activators" when at least two Nrf2 target genes associated with Nrf2 protein expression were induced. Results showed that most chemical sensitizers having cysteine and cysteine/lysine affinities were inducers of the Nrf2 pathway in both cell models, whereas lysine-reactive chemicals were less efficient. In THP-1 cells, the Nrf2 pathway was also activated by pro-/prehaptens. Regression analysis revealed that ho-1 and nqo1 expressions were found to be associated with chemical sensitizer reactivity to cysteine, providing evidence of the importance of chemical reactivity, as a part of danger signals, in DC biology.

Development and in vitro assay of oxidative stress modifying formulations for wound healing promotion.

Often presented as metabolism byproducts, reactive oxygen species are linked to detrimental effects such as chronic wound, mutagenesis, cancer and skin ageing. However, recent in vitro and in vivo observations suggest that ROS, and mainly hydrogen peroxide, interfere with cell signaling acting like second messenger and inducing adaptive responses. This is particularly observed in skin wound healing where cells are exposed to H2O2 following injury. In this study, we developed and characterized an innovative formulation producing H2O2 at low concentrations, in order to mimic physiological inflammation phase. Then, this pro-oxidative formulation (CAM-GOx) was assayed in vitro on keratinocytes cell culture, compared to the blank formulation (CAM) and the anti-oxidative formulation (CAM-CAT) to assess whether oxidative stress was implied or not in cellular responses.

[Reactive oxygen species and oxidative stress]. / Espèces réactives de l'oxygène et stress oxydant.

Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) and the antioxidant capacity of the cell. For long, ROS have been considered as harmful by-products of the normal aerobic metabolism process of the mitochondria, implicated in a large variety of diseases. But there are now growing evidences that controlled ROS production also play physiological roles especially in regulating cell redox homeostasis and cell signaling. Biological ROS effects are now well documented. Data show that living organisms have not only adapted themselves to coexist with free radicals but have also developed mechanisms to use them advantageously. However their main sources and mechanisms of action remain poorly described. This review focuses on the main properties of ROS and their paradoxical effects.

Internalisation of hybrid titanium dioxide/para-amino benzoic acid nanoparticles in human dendritic cells did not induce toxicity and changes in their functions.

Nanoparticles (NPs) have been reported to penetrate into human skin through lesional skin or follicular structures. Therefore, their ability to interact with dendritic cell (DC) was investigated using DCs generated from monocytes (mono-DCs). Hybrid titanium dioxide/para-amino benzoic acid (TiO(2)/PABA) NPs did not induce any cell toxicity. NPs were internalised into DCs through macropinocytosis and not by a receptor-mediated mechanism. Confocal microscopy showed that NPs were not detected in the nucleus. These data are confirmed by electronic microscopy which demonstrated that hybrid NPs were rapidly in contact with cellular membrane and localised into cytoplasmic vesicles without colocalisation with clathrin-coated vesicles. Hybrid NPs did not induce CD86 or HLA-DR overexpression or cytokine secretion (IL-8 and TNF-α) indicating no DC activation. Internalisation of hybrid NPs did not modify DC response towards sensitisers such as nickel and thimerosal or LPS used as positive controls. Moreover, hybrid NPs did not induce any oxidative stress implicated in DC activation process. After mono-DC irradiation by ultraviolet A (UVA), hybrid NP-treated cells did not produce UVA-induced reactive oxygen species (ROS) and exhibited a better cell viability compared with UVA-irradiated control cells, suggesting a protecting effect of hybrid TiO(2)/PABA NPs against UVA-induced ROS.

Sensitization effect of thimerosal is mediated in vitro via reactive oxygen species and calcium signaling.

Thimerosal, a mercury derivative composed of ethyl mercury chloride (EtHgCl) and thiosalicylic acid (TSA), is widely used as a preservative in vaccines and cosmetic products and causes cutaneous reactions. Since dendritic cells (DCs) play an essential role in the immune response, the sensitization potency of chemicals was studied in vitro using U937, a human promyelomonocytic cell line that is used as a surrogate of monocytic differentiation and activation. Currently, this cell line is under ECVAM (European Center for the Validation of Alternative Methods) validation as an alternative method for discriminating chemicals. Thimerosal and mercury derivatives induced in U937 an overexpression of CD86 and interleukin (IL)-8 secretion similarly to 1-chloro-2,4-dinitrobenzene (DNCB), a sensitizer used as a positive control for DC activation. Non-sensitizers, dichloronitrobenzene (DCNB), TSA and sodium dodecyl sulfate (SDS), an irritant, had no effect. U937 activation was prevented by cell pretreatment with N-acetyl-L-cysteine (NAC) but not with thiol-independent antioxidants except vitamin E which affected CD86 expression by preventing lipid peroxidation of cell membranes. Thimerosal, EtHgCl and DNCB induced glutathione (GSH) depletion and reactive oxygen species (ROS) within 15 min; another peak was detected after 2h for mercury compounds only. MitoSOX, a specific mitochondrial fluorescent probe, confirmed that ROS were essentially produced by mitochondria in correlation with its membrane depolarization. Changes in mitochondrial membrane permeability induced by mercury were reversed by NAC but not by thiol-independent antioxidants. Thimerosal and EtHgCl also induced a calcium (Ca2+) influx with a peak at 3h, suggesting that Ca2+ influx is a secondary event following ROS induction as Ca2+ influx was suppressed after pretreatment with NAC but not with thiol-independent antioxidants. Ca2+ influx was also suppressed when culture medium was deprived of Ca2+ confirming the specificity of the measure. In conclusion, these data suggest that thimerosal induced U937 activation via oxidative stress from mitochondrial stores and mitochondrial membrane depolarization with a primordial effect of thiol groups. A cross-talk between ROS and Ca2+ influx was demonstrated.

Different mechanisms are involved in apoptosis induced by melanoma gangliosides on human monocyte-derived dendritic cells.

Tumor escape is linked to multiple mechanisms, notably the liberation, by tumor cells, of soluble factors that inhibit the function of dendritic cells (DC). We have shown that melanoma gangliosides impair DC differentiation and induce their apoptosis. The present study was aimed to give insight into the mechanisms involved. DC apoptosis was independent of the catabolism of gangliosides since lactosylceramide did not induce cell death. Apoptosis induced by GM3 and GD3 gangliosides was not blocked by inhibitors of de novo ceramide biosynthesis, whereas the acid sphingomyelinase inhibitor desipramine only prevented apoptosis induced by GM3. Furthermore, our results suggest that DC apoptosis was triggered via caspase activation, and it was ROS dependent with GD3 ganglioside, suggesting that GM3 and GD3 induced apoptosis through different mechanisms.

Characterization of early events involved in human dendritic cell maturation induced by sensitizers: cross talk between MAPK signalling pathways.

Dendritic cells (DCs), efficient-antigen presenting cells play an important role in initiating and regulating immune responses. DC maturation following exposure to nickel or DNCB induced an up-regulation of phenotypic markers and inflammatory cytokine secretion. Early intracellular mechanisms involved in DC maturation required to be precise. To address this purpose, DCs derived from human monocytes were treated with sensitizers (nickel, DNCB or thimerosal) in comparison with an irritant (SDS). Our data confirming the up-regulation of CD86, CD54 and cytokine secretion (IL-8 and TNFalpha) induced by sensitizers but not by SDS, signalling transduction involved in DC maturation was investigated using these chemicals. Kinase activity measurement was assessed using two new sensitive procedures (Facetrade mark and CBA) requiring few cells. SDS did not induce changes in signalling pathways whereas NiSO(4), DNCB and thimerosal markedly activated p38 MAPK and JNK, in contrast Erk1/2 phosphorylation was completely inhibited by DNCB or thimerosal and only activated by nickel. A pre-treatment with p38 MAPK inhibitor (SB203580) suppressed Erk1/2 inhibition induced by DNCB or thimerosal demonstrating a direct interaction between p38 MAPK and Erk1/2. A pre-treatment with an antioxidant, N-acetyl-L-cysteine (NAC) markedly reduced Erk1/2 inhibition and p38 MAPK phosphorylation induced by DNCB and thimerosal, suggesting a direct activation of p38 MAPK via an oxidative stress and a regulation of MAPK signalling pathways depending on chemicals. Because of a high sensitivity of kinase activity measurements, these procedures will be suitable for weak or moderate sensitizer screening.