Jérémy Malaisse

Knockdown of Filaggrin in a Three-Dimensional Reconstructed Human Epidermis Impairs Keratinocyte Differentiation

Atopic dermatitis is a chronic inflammatory skin disorder characterized by defects in the epidermal barrier and keratinocyte differentiation. The expression of filaggrin, a protein thought to have a major role in the function of the epidermis, is downregulated. However, the impact of this deficiency on keratinocytes is not really known. This was investigated using lentivirus-mediated small-hairpin RNA interference in a three-dimensional reconstructed human epidermis (RHE) model, in the absence of other cell types than keratinocytes. Similar to what is known for atopic skin, the experimental filaggrin downregulation resulted in hypogranulosis, a disturbed corneocyte intracellular matrix, reduced amounts of natural moisturizing factor components, increased permeability and UV-B sensitivity of the RHE, and impaired keratinocyte differentiation at the messenger RNA and protein levels. In particular, the amounts of two filaggrin-related proteins and one protease involved in the degradation of filaggrin, bleomycin hydrolase, were lower. In addition, caspase-14 activation was reduced. These results demonstrate the importance of filaggrin for the stratum corneum properties/functions. They indicate that filaggrin downregulation in the epidermis of atopic patients, either acquired or innate, may be directly responsible for some of the disease-related alterations in the epidermal differentiation program and epidermal barrier function.

Epidermal morphogenesis during progressive in vitro 3D reconstruction at the air-liquid interface.

Keratinocyte monolayers, cultured in immersed conditions, constitute a frequently used in vitro model system to study keratinocytes behaviour in response to environmental assaults. However, monolayers lack the keratinocyte terminal differentiation and the organization of the epidermal tissue, which are observed in vivo. Advancements of in vitro techniques were used to reconstruct three‐dimensional equivalents that mimic human epidermis in terms of layering, differentiation and barrier function. Here, we update a published method and illustrate the progressive morphogenesis responsible for in vitro reconstruction. The analysis of cell proliferation, expression of differentiation markers and barrier efficacy demonstrate the excellent similarity of the reconstructed tissue with normal human epidermis. Availability of epidermal tissue during its reconstruction phase in culture appears crucial for studies intending to challenge the barrier function.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Lack of hyaluronidases exacerbates renal post-ischemic injury, inflammation, and fibrosis.

Renal ischemia-reperfusion injury (IRI) is a pathological process that may lead to acute renal failure and chronic dysfunction in renal allografts. During IRI, hyaluronan (HA) accumulates in the kidney, but suppression of HA accumulation during IRI protects the kidney from ischemic insults. Here we tested whether Hyal1-/- and Hyal2-/- mice display exacerbated renal damage following unilateral IRI due to a higher HA accumulation in the post-ischemic kidney compared with that in the kidney of wild-type mice. Two days after IRI in male mice there was accumulation of HA and CD44 in the kidney, marked tubular damage, infiltration, and increase creatininemia in wild-type mice. Knockout mice exhibited higher amounts of HA and higher creatininemia. Seven days after injury, wild-type mice had a significant decrease in renal damage, but knockout mice still displayed exacerbated inflammation. HA and CD44 together with α-smooth muscle actin and collagen types I and III expression were increased in knockout compared with wild-type mice 30 days after IRI. Thus, both HA-degrading enzymes seem to be protective against IRI most likely by reducing HA accumulation in the post-ischemic kidney and decreasing the inflammatory processes. Deficiency in either HYAL1 or HYAL2 leads to enhanced HA accumulation in the post-ischemic kidney and consequently worsened inflammatory response, increased tubular damage, and fibrosis.

In a three-dimensional reconstructed human epidermis filaggrin-2 is essential for proper cornification

Atopic dermatitis is a chronic inflammatory skin disease with defects in the epidermal barrier. In a cohort of African-American children, a FLG2 nonsense mutation has been associated with the disease. In the epidermis of European patients, the expression of filaggrin-2, the filaggrin-related protein encoded by FLG2, is decreased. To describe the function of filaggrin-2 and evaluate the impact of its deficiency, its expression was downregulated using lentivirus-mediated shRNA interference in a three-dimensional reconstructed human epidermis (RHE) model. This resulted in parakeratosis and a compact stratum corneum, presence of abnormal vesicles inside the corneocytes, increased pH and reduced amounts of free amino acids at the RHE surface, leading to increased sensitivity to UVB radiations. The expression of differentiation markers was slightly modified. However, we observed reduced proteolytic processing of corneodesmosin, hornerin and filaggrin in parallel with reduced amounts of caspase-14 and bleomycin hydrolase. Our data demonstrated that filaggrin-2 is important for a proper cornification and a functional stratum corneum. Its downregulation in atopic patients may be involved in the disease-associated epidermis impairment.

Hypersensitivity of A8344G MERRF mutated cybrid cells to staurosporine-induced cell death is mediated by calcium-dependent activation of calpains

Mutations in the mitochondrial DNA can lead to the development of mitochondrial diseases such as Myoclonic Epilepsy with Ragged Red Fibers (MERRF) or Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS). We first show that human 143B-derived cybrid cells harboring either the A8344G (MERRF) or the A3243G (MELAS) mutation, are more prone to undergo apoptosis then their wild-type counterpart, when challenged with various apoptotic inducers such as staurosporine, etoposide and TRAIL. In addition, investigating the mechanisms underlying A8344G cybrid cells hypersensitivity to staurosporine-induced cell death, we found that staurosporine treatment activates caspases independently of cytochrome c release in both wild-type and mutated cells. Caspases are activated, at least partly, through the activation of calcium-dependent calpain proteases, a pathway that is more strongly activated in mutated cybrid cells than in wild-type cells exposed to staurosporine. These results suggest that calcium homeostasis perturbation induced by mitochondrial dysfunction could predispose cells to apoptosis, a process that could take part into the progressive cell degeneration observed in MERRF syndrome, and more generally in mitochondrial diseases.

Hyaluronan does not regulate human epidermal keratinocyte proliferation and differentiation

Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal.

Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation.

The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process.

Methyl‐β‐cyclodextrin concurs with interleukin (IL)‐4, IL‐13 and IL‐25 to induce alterations reminiscent of atopic dermatitis in reconstructed human epidermis

Reconstructed human epidermis (RHE) mimic normal human in vivo epidermis in terms of histology, distribution of differentiation markers and barrier functionality.1 A typical transcriptional profile and the activation of signalling pathways reminiscent of atopic dermatitis (AD) lesional skin can be obtained in RHE upon incubation with methylβcyclodextrin (MβCD),2,3 a molecule that extracts cholesterol from plasma membranes, thereby disrupting lipid microdomains. However, barrier function and morphology remain unaltered in those conditions, requiring further refinement of the model.

High TMEM45A expression is correlated to epidermal keratinization.

TMEM45A (DERP7, DNAPTP4 or FLJ10134) gene, belonging to the TMEM family encoding predicted transmembrane proteins, is highly expressed in epidermal keratinocytes. To investigate the potential involvement of TMEM45A during the differentiation and keratinization processes, its expression has been characterized in normal human keratinocytes and the protein subcellular localization has been studied in this cell type, both in vitro and in vivo. TMEM45A expression is upregulated with differentiation, either induced by cultured keratinocyte confluence or enhanced Ca2+ concentration in medium. In vivo, TMEM45A mRNA and protein are mostly found in the granular layer of the epidermis. TMEM45A expression is linked to keratinization, as accumulation of the protein is detected in native and reconstructed epidermis as well as in thymic Hassal bodies, but not in non‐keratinized stratified epithelia. At the subcellular level, co‐detection with ER and Golgi markers reveals that TM protein 45A is associated with the Golgi apparatus and more specifically with the trans‐Golgi/trans‐Golgi network in vitro and in granular layer in vivo. The protein is neither related to lysosomes nor transported within corneodesmosin‐containing lamellar bodies. These data demonstrate a strong correlation between TMEM45A expression and epidermal keratinization, indicating the relevance of this gene in this process.