Morphological analysis of the acute effects of cigarette smoke on normal human oral mucosa explants

Cigarette smoke is one of the five leading risk factors for mortality in the world and smoking related lung cancer is the main cause of deaths from cancer in both sexes in the United States. This habit is particularly popular in Western low- and middle- income countries, with about one billion males and 250 million females smoking in the world. Human oral mucosa is the combustion chamber of cigarette, but scanty evidence is available about the early smoke effects. The present work aimed at evaluating from a morphological point of view whole smoke early effects on epithelial intercellular adhesion and keratinocyte terminal differentiation in a three-dimensional model of human oral mucosa. Biopsies of keratinized oral mucosa of healthy non smoking women (n = 5) were collected. After culturing in a Transwell system, one fragment of each biopsy was exposed to the smoke of one single cigarette; the remnant represented the internal control. The distribution of epithelial differentiation markers (keratin-10, K10, and keratin-14, K14, for suprabasal and basal cells respectively), desmosomes (desmoglein-1, desmoglein-3), tight junctions (occludin), adherens junctions (E-cadherin, β-catenin), and apoptotic cells (p53, caspase-3) were evaluated by immunofluorescence. Quantitative analysis of K14 immunolabeling revealed an over expression in the suprabasal layers as early as 3 hours after smoke exposure, without impairment of the epithelial junctional apparatus and apoptosis induction. The present study showed that the early response of the normal human oral mucosa to acute exposure to the smoke of one single cigarette induced K14 expression in suprabasal oral keratinocytes, without impairment of the epithelial junctional apparatus and apoptosis induction, suggesting that the first significant response to cigarette smoke arise from the basal and suprabasal layers of the human oral epithelium. The novelty of the present work consists in the combination of human oral explants with a non hermetic smoke exposure system. The main improvement offered by this setting is the ability of reproducing the cyclic condition by which cigarette smoke normally comes in contact with the oral mucosa. Furthermore, it allows administering simultaneously both gaseous phase and particulate matter of cigarette smoke

Desmocollin 1 and desmoglein 1 expression in human epidermis and keratinizing oral mucosa: a comparative immunohistochemical and molecular study

Epidermis and keratinizing oral mucosa (KOM) are effective barriers against a wide spectrum of insults. The optimal form of protection provided by each epithelium is determined also by the molecular composition of desmosomes. Up to now, the expression of the "skin type" desmosomal cadherins, i.e. desmocollin 1 (Dsc1) and desmoglein 1 (Dsg1), was correlated with the morphological features of keratinocyte terminal differentiation in epidermis, but not in KOM. The aim of the present study was to investigate Dsc1 and Dsg1 expression in adult human KOM compared to epidermis. Biopsies of epidermis and KOM were obtained from young healthy adults (n=6) and simultaneously processed for immunofluorescence analysis, post-embedding immunogold electron microscopy (immunogold EM), and RT-PCR analysis. For molecular biology analysis, as a negative control, we considered human fibroblasts. By immunofluorescence and immunogold EM, Dsc1 labeling was not detected in any suprabasal layer of KOM, but it was present in the upper spinous/granular layers of epidermis. Immunofluorescence and transmission electron microscopy analysis showed that (i) Dsg1 expression was evident in the spinous, granular, and horny layer of the oral epithelium and (ii) Dsg1 immunoreactivity was always lower in desmosomes between oral keratinocytes than in all epidermal junctions. RT-PCR analysis confirmed that in KOM Dsc1 gene expression was undetectable. On the whole, these observations suggest a weakened adhesion in KOM, allowing oral keratinocytes to undergo a faster transition throughout the living layers of the epithelium. The intrinsic and specific regulation of the molecular composition of desmosomes can contribute in defining a specific keratinocyte phenotype in KOM and in epidermis

Chronic alendronate therapy impairs epithelial morphology and homeostasis in the human oral mucosa

Alendronate (ALN) is a nitrogen containing bisphosphonate (BP) widely used for the chronic treatment of osteoporotic patients, especially women over 60 years old. The diffusion of BPs in clinical practice has brought attention to one of their most serious side-effects, osteonecrosis of the jaw (ONJ) [1]. Several theories have been proposed to explain its pathogenesis, but the effect of BPs on the oral mucosa is still matter of debate despite its extensive involvement and injury in ONJ. This study aimed at evaluating from a morphological point of view the effects of ALN therapy on the oral epithelium of clinically healthy keratinized oral mucosa. Six women over 60 years old undergoing chronic therapy (2-7 years) with oral ALN after diagnosis of osteoporosis were recruited and compared to a gender and age matched group (n=6). Smoking habit, past history of head and neck cancer treatment, and concomitant assumption of steroidal and antiangiogenic drugs were excluding criteria. Proliferation, apoptosis, intercellular adhesion, and terminal differentiation were investigated by immunofluorescence and transmission electron microscopy (TEM). A significant decrease in keratinocyte proliferation was detected in the oral epithelium of patients undergoing ALN therapy compared to the control group (237.62 BrdU/mm2 ± 92.22 vs 104.16 BrdU/mm2 ± 66.20; p = 0.0002), without any sign of apoptosis induction by light microscopy and TEM. The presence of well established adherens and tight junctions was accompanied by profound alterations in desmosomal ultrastructure and molecular composition in the uppermost layers of the oral epithelium of the ALN group. Proceeding from the lower spinous to the granular layer, TEM analysis showed a progressive reduction in desmosomal thickness paralleled by a lower immunostaining for desmoglein 1 and desmoglein 3 in the suprabasal keratinocytes. In the upper epithelial layers, intermediate filaments gradually aggregated forming electron-dense bundles detached from the desmosomal plaque and a significant decrease in keratin 10 expression was observed. Taken together the reported results suggested a profound impairment in structure and function of the clinically healthy oral epithelium related to chronic ALN assumption. For the first time our results show that epithelial homeostasis in human oral mucosa is profoundly affected by nitrogen containing BPs, confirming previous in vitro studies [2-4] and strongly supporting the need of further investigation on the molecular mechanisms involved in ONJ pathogenesis

Structural and ultrastructural evaluation of the effects induced by IL-22 alone or in combination with psoriatic cytokines in an ex-vivo human skin model

L-22 is a pro-inflammatory cytokine playing a crucial role in the pathogenesis of psoriasis, an autoimmune chronic inflammatory skin disease. The immunological acti- vation during the progression of the psoriatic lesion is driven by IL-22 together with other cytokines, such as (TNF)-alpha and interleukin (IL)-17 [1]. The aim of our study was to evaluate the early, direct, and specific effect of IL-22 alone or in combination with TNF-alpha and IL-17 by immunofluorescence on i) the molecular composition of intercellular junctions (desmocollin (DSC)1, E-cadherin, and occludin), ii) keratin(K) 10 and 17 expression, iii) keratinocyte proliferation, and, by transmission electron micros- copy (TEM), on the ultrastructural morphology of the skin. An innovative model of human skin culture standardized in our laboratory, in which a psoriatic microenviron- ment was reproduced, was used [2]. Skin explants obtained from plastic surgery of healthy 20-40 year-old women (n = 7) after informed consent were cultured overnight in Dulbecco’s modified Eagle’s medium, divided before adding IL-22 or a combination of the three cytokines, and harvested 24, 48, and 72 hours after cytokine incubation.Interestingly, keratinocyte proliferation was inhibited after exposure to the combi- nation of cytokines while was not affected by IL-22 incubation. In both experimental groups, starting from T24, occludin immunostaining was non homogeneously distrib- uted, K10 immunostaining gradually decreased in scattered clusters in the spinous layer, while K17 expression was induced and progressively increased with time in the suprabasal layers of epidermis. By TEM, after IL-22 incubation we observed keratin aggregates in the perinuclear cytoplasm of cells, while the combination of the three cytokines induced an enlargement of intercellular spaces.Altogether, our results suggest that IL-22 mainly affects keratinocyte terminal dif- ferentiation, whereas, for inducing an impairment in cell proliferation, a more com- plex psoriatic-like microenvironment is needed.

Skin morphological analysis upon treatment with anti-TNF-alpha agents in psoriatic patients

Psoriasis is the most common immune-mediated skin disease worldwide, with an estimated incidence of about 2%. It is characterized by erythematous scaly plaques and its pathogenesis is mostly due to an interplay among epidermal cells, immunocompetent cells, and pro-inflammatory cytokines. Previous studies on psoriatic skin demonstrated delayed terminal differentiation, keratinocyte hyperproliferation, and abnormal occludin expression in tight junctions. However, evidences about desmosomal cadherin distribution in psoriatic patients are not available so far, and only scattered studies have been carried out on adherens junctions. In psoriasis, Tumor Necrosis Factor- alpha (TNF-alpha) plays a central role, strongly supporting the treatment with anti-TNF-alpha agents, but the effects of these agents on epidermal intercellular adhesion, terminal differentiation, and proliferation have still to be elucidated. In this preliminary study, we investigated by immunofluorescence the expression of transmembrane proteins in tight junctions (occludin), adherens junctions (E-cadherin), and desmosomes (desmocollin-1 and desmoglein-1) in normal (N=5) and psoriatic skin before/after treatment with anti-TNF-alpha agents (N=5). Differentiation biomarkers (keratin-10, keratin-14, and involucrin) and epithelial proliferation were also evaluated. In psoriatic epidermis occludin, keratin-14, and involucrin were expressed also in the spinous layer, differently from controls. Desmoglein-1, desmocollin-1, E-cadherin, and keratin-10 localizations were comparable in psoriatic and healthy subjects. Moreover, in all considered patients the hyperproliferative condition was accompanied by an unusual suprabasal distribution of replicating keratinocytes. Interestingly, the distribution pattern of all considered biomarkers of intercellular adhesion and terminal differentiation was reverted to the physiological condition upon treatment with anti-TNF-alpha agents. What’s more, the proliferative rate registered in anti-TNF-alpha treated patients was reduced and similar to controls, even though scattered suprabasal dividing cells were still present. Our results highlight that anti-TNF-alpha biologicals, next to their proven inhibitory action on the cytokinic pathway, are effective in restoring an efficient junctional apparatus, a more differentiated phenotype, and the typical proliferation rate in the epidermis of psoriatic patients

Mice lacking mitochondrial ferritin are more sensitive to doxorubicin-mediated cardiotoxicity

15noMitochondrial ferritin is a functional ferritin that localizes in themitochondria.Itisexpressedinthetestis, heart,brain,and cells with active respiratory activity. Its overexpression in culturedcellsprotectedagainstoxidativedamageandreduced cytosolic iron availability. However, no overt phenotype was describedinmicewithinactivationoftheFtMtgene.Here,we usedthe doxorubicin model ofcardiac injuryina novel strain of FtMt-null mice to investigate the antioxidant role of FtMt. These mice did not show any evident phenotype, but after acute treatment to doxorubicin, they showed enhanced mortalityandaltered heartmorphologywithfibrildisorganization and severe mitochondrial damage. Signs of mitochondrial damage were present also in mock-treated FtMt−/− mice. The hearts of saline- and doxorubicin-treated FtMt−/− mice had higher thiobarbituric acid reactive substance levels, heme oxygenase 1 expression, and protein oxidation, but did not differ from FtMt+/+ in the cardiac damage marker B-type natriureticpeptide(BNP),ATP levels, and apoptosis.However,the autophagy marker LC3 was activated. The results show that the absence of FtMt, which is highly expressed in the heart, increases the sensitivity of heart mitochondria to the toxicity of doxorubicin. This study represents the first in vivo evidence of the antioxidant role of FtMt.openopenMaccarinelli, Federica; Gammella, Elena; Asperti, Michela; Mariaregon, ; Donetti, Elena; Recalcati, Stefania; Poli, Maura; Finazzi, Dario; Arosio, Paolo; Biasiotto, Giorgio; Emiliaturco, ; Altruda, Fiorella; Lonardi, Silvia; Cornaghi, Laura; Cairo, GaetanoMaccarinelli, Federica; Gammella, Elena; Asperti, Michela; Mariaregon, ; Donetti, Elena; Recalcati, Stefania; Poli, Maura; Finazzi, Dario; Arosio, Paolo; Biasiotto, Giorgio; Emiliaturco, ; Altruda, Fiorella; Lonardi, Silvia; Cornaghi, Laura; Cairo, Gaetan

Langerhans cells and Toll Like Receptors: how do they act and react in an in vitro psoriatic microenvironment?

Tumor Necrosis Factor (TNF)-α, interleukin (IL)-17, IL-22 and IL-23 are involved in the psoriasis pathogenesis and represent a strong proinflammatory stimulus. Both epidermal keratinocytes (KCs) and Langerhans cells (LCs) early respond promoting an early epidermal response [1, 2]. Human skin can count on the cellular response supported by LCs and on innate immunity through the expression of Toll-like Receptors (TLRs) [4]. We aimed at investigate whether the exposure of normal human skin to a combination of TNF-α, IL-17, IL-22, and IL-23 (cytokine mix) affected i) LCs immunophenotype, ii) expression of TLR2 and TLR9 and iii) KC proliferation. Human skin samples were obtained after plastic surgery (n = 5) and exposed to the cytokine mix in a Transwell system at air-liquid interface, with a parallel control group. Samples were harvested 24 and 48 hours after cytokine stimulation, processed in parallel for immunofluorescence or ultrastructural analysis. A decrease of cell proliferation was evident in samples exposed to cytokine mix for 24 hours and this phenomenon was more and more evident later. TLR2 immunopositivity progressively disappeared in the basal layer after cytokine mix exposure compared to the control group, while TLR9 expression was induced in scattered granular keratinocytes. By TEM, LCs showed an activated phenotype. In conclusion, these results suggest that, in a microenvironment mimicking the psoriatic plaque, epidermis early stimulates two important lines of defense, thus proposing that a therapeutic intervention in this direction can interfere with the formation/progression of the psoriatic plaque

Effect of TNF-alpha and IL-17 on TLR expression and Langerhans cells phenotype in a three-dimensional model of normal human skin: a morphological study

Toll-like receptors (TLRs) are essential for innate immunity and contribute to create the skin barrier. Their abnormal stimulation is involved in the development of several dermatological diseases, among which psoriasis. Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role in the pathogenesis of psoriatic plaques and their proinflammatory activity can affect Langerhans cell (LC) phenotype. In a well characterized three-dimensional model of organotypic cultures of normal human skin [1-3] we evaluated the effect of TNF-alpha and IL-17 on the expression of TLR2 and 9 by immunofluorescence, on the ultrastructural morphology of keratinocytes and LCs by transmission electron microscopy (TEM). Human skin explants (n=7) were cultured at the air-liquid interface overnight in a Transwell system and exposed to 50 ng/ml IL-17 or 100 ng/ml TNF-alpha or a combination of both cytokines. Samples were harvested 24 (T24) and 48h (T48) after cytokines incubation. After incubation with IL-17 and IL-17+TNF-alpha, TLR2 immunostaining was not detectable in the basal layer, differently from controls and TNF-alpha-treated samples. Conversely, TLR9 expression was progressively induced in granular keratinocytes in all cytokine-exposed groups. By TEM, enlargements of intercellular spaces were evident especially and, after IL-17 treatment, LCs showed an activated phenotype. At T24 LCs number increased indicating that TNF-alpha and IL-17+TNF-alpha exert a chemoattractant activity, while at T48 only IL-17+TNF-alpha maintained this effect on trapping LCs in epidermis. TNF-alpha and IL-17 differently affect LCs behaviour and TLR expression, with a specific contribution to the inflammatory loop underlying the lesion formation. These results suggest that the simultaneous inhibition of the effect of different cytokines with a defined role in the pathogenesis of psoriasis could improve psoriasis treatment

A proinflammatory microenvironment induces NFkB activation and beta-defensin expression through specific Toll Like Receptors in a 3D human skin model

Psoriasis is an autoimmune skin disease characterized by the formation and the progression of silvery plaques on the extensory surfaces of our body. Proinflammatory cytokines as Tumor Necrosis Factor (TNF)-alpha, interleukin (IL)-17, IL-22 and IL-23 represent for the normal skin a psoriatic microenvironment. In the 3D human skin model standardized in our lab in the last decade, we were able to dissect the events in which each cytokine exerts a specific effect, e.g. keratinocyte proliferation, Langerhans cell activation, cytoskeleton arrangement, and, more recently, the epidermal expression of Toll like Receptors (TLRs) 2, 7, 9. Several experimental studies reported that in psoriasis TLRs are expressed and their activation triggers i) NFkB translocation from the cytoplasm to the nucleus and ii) the release of beta defensins (HBDs). The present study was aimed at investigating the intracellular NFkB activation and HBD1 and HBD2 expression induced by a cytokine mix (TNF-alpha, IL-17, IL-22, IL-23) by indirect immunofluorescence. Bioptic samples of normal human skin were obtained after aesthetic surgery of young healthy informed women (n=7). After overnight incubation to reduce mechanical and termical stress, skin fragments were incubated in a Transwell system for 5 (T5), 24 (T24), and 48 (T48) hours with the cytokine mix. Parallel control samples were carried out and each patient was represented at all time points. In controls at all time-points NFkB was localized only in the cytoplasm, while, starting from T5, scattered basal nuclei were observed in the cytokine-incubated samples. At later time points, in the upper spinous and granular layers, NFkB nuclear immunostaining was evident. HBD2 expression was affected after cytokine mix exposure, while HBD1 distribution was similar to controls. Thanks to this simple but effective model, a deep knowledge of the early events occurring in the normal epidermis exposed to cytokines can be achieved, excluding the contribution of the blood and lymphatic vessels herein absent. This basic research can thus represent an important tool for targeting and counteracting single phenomenon leading to the formation/progression with the most innovative biological drugs