Blood-brain barrier permeability towards small and large tracers in a mouse model of osmotic demyelination syndrome.

During osmotic demyelination syndrome (ODS), myelin and oligodendrocyte are lost according to specific patterns in centro- or extra-pontine regions. In both experimental model of ODS and human cases, brain lesions are locally correlated with the disruption of the blood brain-barrier (BBB). The initiation, the degree and the duration of blood-brain barrier (BBB) opening as well as its contribution to brain damages are still a matter of debate. Using a panel of intravascular tracers from low- to high- molecular weight (from 0.45 kDa 150 kDa), we have assessed the BBB permeability at different timings of ODS induced experimentally in mice. ODS was mimicked according to a protocol of rapid correction of a chronic hyponatremia. We demonstrated that BBB leakage towards smallest tracers Lucifer Yellow (0.45 kDa) and Texas Red-dextran (3 kDa) was delayed by 36 h compared to the first clues of oligodendrocyte loss (occurring 12 h post-correction of hyponatremia). At 48 h post-correction and concomitantly to myelin loss, BBB was massively disrupted as attested by accumulation of Evans Blue (69 kDa) and IgG (150 kDa) in brain parenchyma. Analysis of BBB ultrastructure verified that brain endothelial cells had minimal alterations during chronic hyponatremia and at 12 h post-correction of hyponatremia. However, brain endothelium yielded worsened alterations at 48 h, such as enlarged vesicular to tubular-like cytoplasmic profiles of pinocytosis and/or transcytosis, local basal laminae abnormalities and sub-endothelial cavities. The protein expressions of occludin and claudin-1, involved in inter-endothelial tight junctions, were also downregulated at 48 h post-correction of hyponatremia. Our results revealed that functional BBB opening occured late in pre-established ODS lesions, and therefore was not a primary event initiating oligodendrocyte damages in the mouse model of ODS.

Characterization of CYP26B1-Selective Inhibitor, DX314, as a Potential Therapeutic for Keratinization Disorders.

Inhibition of CYP450-mediated retinoic acid (RA) metabolism by RA metabolism blocking agents increases endogenous retinoids and is an alternative to retinoid therapy. Currently available RA metabolism blocking agents (i.e., liarozole and talarozole) tend to have fewer adverse effects than traditional retinoids but lack target specificity. Substrate-based inhibitor DX314 has enhanced selectivity for RA-metabolizing enzyme CYP26B1 and may offer an improved treatment option for keratinization disorders such as congenital ichthyosis and Darier disease. In this study, we used RT-qPCR, RNA sequencing, pathway, upstream regulator, and histological analyses to demonstrate that DX314 can potentiate the effects of all-trans-RA in healthy and diseased reconstructed human epidermis. We unexpectedly discovered that DX314, but not all-trans-RA or previous RA metabolism blocking agents, appears to protect epidermal barrier integrity. In addition, DX314-induced keratinization and epidermal proliferation effects are observed in a rhino mice model. Altogether, the results indicate that DX314 inhibits all-trans-RA metabolism with minimal off-target activity and shows therapeutic similarity to topical retinoids in vitro and in vivo. Findings of a barrier-protecting effect require further mechanistic study but may lead to a unique strategy in barrier-reinforcing therapies. DX314 is a promising candidate compound for further study and development in the context of keratinization disorders.

Regional oligodendrocytopathy and astrocytopathy precede myelin loss and blood-brain barrier disruption in a murine model of osmotic demyelination syndrome.

The osmotic demyelination syndrome (ODS) is a non-primary inflammatory disorder of the central nervous system myelin that is often associated with a precipitous rise of serum sodium concentration. To investigate the physiopathology of ODS in vivo, we generated a novel murine model based on the abrupt correction of chronic hyponatremia. Accordingly, ODS mice developed impairments in brainstem auditory evoked potentials and in grip strength. At 24 hr post-correction, oligodendrocyte markers (APC and Cx47) were downregulated, prior to any detectable demyelination. Oligodendrocytopathy was temporally and spatially correlated with the loss of astrocyte markers (ALDH1L1 and Cx43), and both with the brain areas that will develop demyelination. Oligodendrocytopathy and astrocytopathy were confirmed at the ultrastructural level and culminated with necroptotic cell death, as demonstrated by pMLKL immunoreactivity. At 48 hr post-correction, ODS brains contained pathognomonic demyelinating lesions in the pons, mesencephalon, thalamus and cortical regions. These damages were accompanied by blood-brain barrier (BBB) leakages. Expression levels of IL-1ß, FasL, TNFRSF6 and LIF factors were significantly upregulated in the ODS lesions. Quiescent microglial cells type A acquired an activated type B morphology within 24 hr post-correction, and reached type D at 48 hr. In conclusion, this murine model of ODS reproduces the CNS demyelination observed in human pathology and indicates ambiguous causes that is regional vulnerability of oligodendrocytes and astrocytes, while it discards BBB disruption as a primary cause of demyelination. This study also raises new queries about the glial heterogeneity in susceptible brain regions as well as about the early microglial activation associated with ODS.

Phagocyte-specific S100A8/A9 is upregulated in primary Sjögren's syndrome and triggers the secretion of pro-inflammatory cytokines in vitro.

OBJECTIVES: To determine the role of S100A8/A9 in the pathogenesis of primary Sjögren's syndrome (pSS). METHODS: The serum levels of S100A8/A9 were determined in pSS patients and healthy controls by ELISA. The expression of S100A8/A9 in salivary glands was assessed by immunohistochemistry. The phenotype of S100A8+ and S100A9+ cells was identified using double immunofluorescence. The effects of S100A8/A9 on cytokine production by peripheral blood mononuclear cells (PBMCs) from pSS patients were determined in vitro by flow cytometry. The effects of pro-inflammatory cytokines on S100A8/A9 secretion were additionally investigated in vitro by ELISA in PBMCs from pSS patients and control subjects. RESULTS: Serum levels of S100A8/A9 were significantly increased in pSS patients compared to healthy controls. The tissular expression of S100A8 and S100A9, identified in professional phagocytes (neutrophils, monocytes and plasmacytoid dendritic cells), was increased in the salivary glands of pSS patients and correlated with focus score. In vitro, recombinant S100A8/A9 increased the production of IL-1ß, IL-6, TNF-α, IFN-γ, IL-10, IL-17A and IL-22 by PBMCs. The S100A8/A9-induced increase in TNF-α production in pSS patients was significant relative to controls. Furthermore, IL-1ß, TNF-α, IL-6, and IL-17A stimulated release of S100A8/A9 from PBMCs in pSS patients. CONCLUSIONS: S100A8/A9 is increased in pSS patients contributing to the in vitro increased production of pro-inflammatory cytokines. As such, S100A8/A9 in concert with other cytokines might contribute to the pathogenesis of pSS.

TMEM45A Is Dispensable for Epidermal Morphogenesis, Keratinization and Barrier Formation.

TMEM45A gene encodes an initially uncharacterized predicted transmembrane protein. We previously showed that this gene is highly expressed in keratinocytes where its expression correlates with keratinization, suggesting a role in normal epidermal physiology. To test this hypothesis, we generated TMEM45A knockout mice and found that these mice develop without any evident phenotype. The morphology of the epidermis assessed by histology and by labelling differentiation markers in immunofluorescence was not altered. Toluidine blue permeability assay showed that the epidermal barrier develops normally during embryonic development. We also showed that depletion of TMEM45A in human keratinocytes does not alter their potential to form in vitro 3D-reconstructed epidermis. Indeed, epidermis with normal morphogenesis were generated from TMEM45A-silenced keratinocytes. Their expression of differentiation markers quantified by RT-qPCR and evidenced by immunofluorescence labelling as well as their barrier function estimated by Lucifer yellow permeability were similar to the control epidermis. In summary, TMEM45A gene expression is dispensable for epidermal morphogenesis, keratinization and barrier formation. If this protein plays a role in the epidermis, its experimental depletion can possibly be compensated by other proteins in the two experimental models analyzed in this study.