COMP negatively influences keratinocyte proliferation via α5β1-integrin: Potential relevance of altered COMP expression in psoriasis

In psoriasis, non-lesional skin shows alterations at the dermal–epidermal junction (DEJ) compared to healthy skin. Cartilage oligomeric matrix protein (COMP) is part of the papillary dermis of healthy skin, and its expression has not yet been studied in psoriatic skin. In this study, we found that COMP localization extended deeper into the dermis and formed a more continuous layer in psoriatic non-lesional skin compared to healthy skin, while in psoriatic lesions, COMP showed a partially discontinuous deposition at the DEJ. COMP and β1-integrin showed strong co-localization in non-lesional skin, where the laminin layer within the basement membrane (BM) is discontinuous. In in vitro models, the presence of exogenous COMP decreased the proliferation rate of keratinocytes and this proliferation-suppressing effect was diminished by blocking α5β1-integrin. Our results suggest that COMP can interact with α5β1-integrin of basal keratinocytes through the disrupted BM, and this interaction might stabilize the epidermis in the non-lesional state by contributing to the suppression of keratinocyte proliferation. The antiproliferative effect of COMP is likely to be relevant to other skin diseases in which chronic non-healing wounds are coupled with massive COMP accumulation

Abnormal basement membrane results in increased keratinocyte-derived periostin expression in psoriasis similar to wound healing

The psoriatic skin resembles wound healing, and it shows abnormalities at the basement membrane (BM), also in the non-lesional skin. Fibroblast-derived dermal periostin has well-known functions in wound healing and Th2-mediated diseases, such as atopic dermatitis. Here we show that serum periostin level was elevated in psoriatic patients, remarkably in the systemically treated ones. Obvious periostin positivity was detected in basal keratinocytes of the non-lesional, lesional, and previously-lesional psoriatic vs. healthy skin. Ex vivo skin models were generated to examine how different skin injuries affect periostin expression during wound healing. Our newly developed cultured salt-split model demonstrated that BM-injury induced periostin expression in basal keratinocytes, and periostin levels in the supernatant were also increased upon healing. In wound healing models, β1-integrin expression was similarly induced. β1-integrin blocking caused reduced periostin expression in in vitro scratch assay, indicating that β1-integrin can mediate periostin production. In contrast to atopic dermatitis, psoriatic basal keratinocytes are in an activated state and show a stable wound healing-like phenotype with the overexpression of periostin. This abnormal BM-induced wound healing as a potential compensatory mechanism can be initiated already in the non-lesional skin present in the lesion and keratinocytes can remain activated in the healed skin

Psoriatic Resolved Skin Epidermal Keratinocytes Retain Disease-Residual Transcriptomic and Epigenomic Profiles

The disease-residual transcriptomic profile (DRTP) within psoriatic healed/resolved skin and epidermal tissue-resident memory T (TRM) cells have been proposed to be crucial for the recurrence of old lesions. However, it is unclear whether epidermal keratinocytes are involved in disease recurrence. There is increasing evidence regarding the importance of epigenetic mechanisms in the pathogenesis of psoriasis. Nonetheless, the epigenetic changes that contribute to the recurrence of psoriasis remain unknown. The aim of this study was to elucidate the role of keratinocytes in psoriasis relapse. The epigenetic marks 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) were visualized using immunofluorescence staining, and RNA sequencing was performed on paired never-lesional and resolved epidermal and dermal compartments of skin from psoriasis patients. We observed diminished 5-mC and 5-hmC amounts and decreased mRNA expression of the ten-eleven translocation (TET) 3 enzyme in the resolved epidermis. SAMHD1, C10orf99, and AKR1B10: the highly dysregulated genes in resolved epidermis are known to be associated with pathogenesis of psoriasis, and the DRTP was enriched in WNT, TNF, and mTOR signaling pathways. Our results suggest that epigenetic changes detected in epidermal keratinocytes of resolved skin may be responsible for the DRTP in the same regions. Thus, the DRTP of keratinocytes may contribute to site-specific local relapse

Comprehensive Proteomic Analysis Reveals Intermediate Stage of Non-Lesional Psoriatic Skin and Points out the Importance of Proteins Outside this Trend

To better understand the pathomechanism of psoriasis, a comparative proteomic analysis was performed with non-lesional and lesional skin from psoriasis patients and skin from healthy individuals. Strikingly, 79.9% of the proteins that were diferentially expressed in lesional and healthy skin exhibited expression levels in non-lesional skin that were within twofold of the levels observed in healthy and lesional skin, suggesting that non-lesional skin represents an intermediate stage. Proteins outside this trend were categorized into three groups: I. proteins in non-lesional skin exhibiting expression similar to lesional skin, which might be predisposing factors (i.e., CSE1L, GART, MYO18A and UGDH); II. proteins that were diferentially expressed in non-lesional and lesional skin but not in healthy and lesional skin, which might be non-lesional characteristic alteration (i.e., CHCHD6, CHMP5, FLOT2, ITGA7, LEMD2, NOP56, PLVAP and RRAS); and III. proteins with contrasting diferential expression in non-lesional and lesional skin compared to healthy skin, which might contribute to maintaining the non-lesional state (i.e., ITGA7, ITGA8, PLVAP, PSAPL1, SMARCA5 and XP32). Finally, proteins diferentially expressed in lesions may indicate increased sensitivity to stimuli, peripheral nervous system alterations, furthermore MYBBP1A and PRKDC were identifed as potential regulators of key pathomechanisms, including stress and immune response, proliferation and diferentiation

Spinocerebellar Ataxia in a Hungarian Female Patient with a Novel Variant of Unknown Significance in the CCDC88C Gene

Spinocerebellar ataxia (SCA) 40 is an extremely rare subtype of the phenotypically and genetically diverse autosomal dominant ataxias caused by mutations of the CCDC88C gene. Most reported cases of SCA40 are characterized by late-onset cerebellar ataxia and variable extrapyramidal features; however, there is a report of a patient with early-onset spastic paraparesis as well. Here, we describe a novel missense CCDC88C mutation (p.R203W) in the hook domain of the DAPLE protein encoded by the CCDC88C gene that was identified in a female patient who developed late-onset ataxia, dysmetria and intention tremor. To explore the molecular consequences of the newly identified and previously described CCDC88C mutations, we carried out in vitro functional tests. The CCDC88C alleles were expressed in HEK293 cells, and the impact of the mutant DAPLE protein variants on JNK pathway activation and apoptosis was assessed. Our results revealed only a small-scale activation of the JNK pathway by mutant DAPLE proteins; however, increased JNK1 phosphorylation could not be detected. Additionally, none of the examined mutations triggered proapoptotic effect. In conclusion, we identified a novel mutation of the CCDC88C gene from a patient with spinocerebellar ataxia. Our results are not in accord with previous observations and do not support the primary role of the CCDC88C mutations in induction of JNK pathway activation in ataxia. Therefore, we propose that CCDC88C mutations may exert their effects through different and possibly in much broader, yet unexplored, biological processes

Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

The quantitative detection of radiation caused DNA double-strand breaks (DSB) by immunostained γ-H2AX foci using direct stochastic optical reconstruction microscopy (dSTORM) provides a deeper insight into the DNA repair process at nanoscale in a time-dependent manner. Glioblastoma (U251) cells were irradiated with 250 keV X-ray at 0, 2, 5, 8 Gy dose levels. Cell cycle phase distribution and apoptosis of U251 cells upon irradiation was assayed by flow cytometry. We studied the density, topology and volume of the γ-H2AX foci with 3D confocal microscopy and the dSTORM superresolution method. A pronounced increase in γ-H2AX foci and cluster density was detected by 3D confocal microscopy after 2 Gy, at 30 min postirradiation, but both returned to the control level at 24 h. Meanwhile, at 24 h a considerable amount of residual foci could be measured from 5 Gy, which returned to the normal level 48 h later. The dSTORM based γ-H2AX analysis revealed that the micron-sized γ-H2AX foci are composed of distinct smaller units with a few tens of nanometers. The density of these clusters, the epitope number and the dynamics of γ-H2AX foci loss could be analyzed. Our findings suggest a discrete level of repair enzyme capacity and the restart of the repair process for the residual DSBs, even beyond 24 h. The dSTORM superresolution technique provides a higher precision over 3D confocal microscopy to study radiation induced γ-H2AX foci and molecular rearrangements during the repair process, opening a novel perspective for radiation research

Ambroxol Treatment Suppresses the Proliferation of Chlamydia pneumoniae in Murine Lungs

Ambroxol (Ax) is used as a mucolytics in the treatment of respiratory tract infections. Ax, at a general dose for humans, does not alter Chlamydia pneumoniae growth in mice. Therefore, we aimed to investigate the potential anti-chlamydial effect of Ax at a concentration four timed higher than that used in human medicine. Mice were infected with C. pneumoniae and 5-mg/kg Ax was administered orally. The number of recoverable C. pneumoniae inclusion-forming units (IFUs) in Ax-treated mice was significantly lower than that in untreated mice. mRNA expression levels of several cytokines, including interleukin 12 (IL-12), IL-23, IL-17F, interferon gamma (IFN-γ), and surfactant protein (SP)-A, increased in infected mice treated with Ax. The IFN-γ protein expression levels were also significantly higher in infected and Ax-treated mice. Furthermore, the in vitro results suggested that the ERK 1/2 activity was decreased, which is essential for the C. pneumoniae replication. SP-A and SP-D treatments significantly decreased the number of viable C. pneumoniae IFUs and significantly increased the attachment of C. pneumoniae to macrophage cells. Based on our results, a dose of 5 mg/kg of Ax exhibited an anti-chlamydial effect in mice, probably an immunomodulating effect, and may be used as supporting drug in respiratory infections caused by C. pneumoniae