Bilal Osta

Classical and Paradoxical Effects of TNF-α on Bone Homeostasis

Tumor necrosis factor-α (TNF-α) plays an essential role in the regulation of bone homeostasis in several chronic immune and inflammatory joint diseases, where inhibition of TNF has led to significant clinical improvement. However, TNF-activated pathways and mechanisms involved in bone remodeling remain unclear. So far, TNF-α was known as an inhibitor of osteoblast differentiation and an activator of osteoclastogenesis. Recent contradictory findings indicated that TNF-α can also activate osteoblastogenesis. The paradoxical role of TNF-α in bone homeostasis seems to depend on the concentration and the differentiation state of the cell type used as well as on the exposure time. This review aims to summarize the recent contradictory findings on the regulation of bone homeostasis by TNF-α at the isolated cell, whole bone, and whole body levels. In addition, the involvement of TNF-α in the bone remodeling imbalance is observed in inflammatory joint diseases including rheumatoid arthritis and ankylosing spondylitis, which are associated with bone destruction and ectopic calcified matrix formation, respectively. Both diseases are associated with systemic/vertebral osteoporosis.

Bone marrow–derived and synovium-derived mesenchymal cells promote Th17 cell expansion and activation through caspase 1 activation: Contribution to the chronicity of rheumatoid arthritis†

OBJECTIVE Th17 cells have been implicated in rheumatoid arthritis (RA). We hypothesized that the interaction of T cells with bone marrow-derived mesenchymal stem cells (BM-MSCs) or with fibroblast- like synoviocytes (FLS) might, with the help of T cell-secreted inflammatory cytokines (i.e., interleukin-17A [IL-17A], tumor necrosis factor α [TNFα], and/or interferon-γ [IFNγ]), promote Th17 cell expansion and activation. METHODS Peripheral blood mononuclear cells (PBMCs) from healthy blood donors were cocultured with BM-MSCs or FLS from RA patients or osteoarthritis (OA) patients. Cocultures were exposed to phytohemagglutinin with or without IL-17A, TNFα, or IFNγ. Quantitative reverse transcription-polymerase chain reaction analysis, enzyme-linked immunosorbent assay, and cytofluorometry were used to measure IL-17A production. RESULTS Interaction of PBMCs with BM-MSCs inhibited Th1 and Th2 responses, but promoted Th17 cell expansion, as early as 24 hours, as demonstrated by increases in retinoic acid receptor-related orphan nuclear receptor γ or IL-17A messenger RNA (mRNA) levels, IL-17A secretion levels, and IL-17A-secreting cell frequency, as well as by T cell switching to the Th17 pathway after 2 rounds of stimulation with MSCs. IL-17A production was also increased in PBMCs stimulated with anti-CD3 plus anti-CD28 or in isolated CD3+ or CD45RO+ T cells, thus demonstrating the role of T cell activation. Levels of mRNA for IL-6, IL-8, and IL-1β were further amplified when T cell-secreted inflammatory cytokines were added. Interestingly, OA FLS or RA FLS also enhanced IL-17A and IL-6 production, but only RA FLS enhanced IFNγ and IL-1β production. We further demonstrated that MSC-mediated Th17 promotion requires caspase 1 activation by using an inhibitory peptide and measuring its activity. CONCLUSION We found that the interaction of MSCs or FLS with T cells promotes the activation and expansion of Th17 cells through caspase 1 activation. Since proinflammatory and T cell-secreted inflammatory cytokines are also amplified, this mechanism may participate in the chronicity of RA.

Effects of Interleukin-17A on Osteogenic Differentiation of Isolated Human Mesenchymal Stem Cells

Objectives: Rheumatoid arthritis (RA) is characterized by defective bone repair and excessive destruction and ankylosing spondylitis (AS) by increased ectopic bone formation with syndesmophytes. Since TNF-α and IL-17A are involved in both diseases, this study investigated their effects on the osteogenic differentiation of isolated human bone marrow-derived mesenchymal stem cells (hMSCs). Methods: Differentiation of hMSCs into osteoblasts was induced in the presence or absence of IL-17A and/or TNF-α. Matrix mineralization (MM) was evaluated by alizarin red staining and alkaline phosphatase (ALP) activity. mRNA expression was measured by qRT-PCR for bone morphogenetic protein (BMP)-2 and Runx2, genes associated with osteogenesis, DKK-1, a negative regulator of osteogenesis, Schnurri-3 and receptor activator of nuclear factor kappa B ligand (RANKL), associated with the cross talk with osteoclasts, and TNF-α receptor type I and TNF-α receptor type II (TNFRII). Results: TNF-α alone increased both MM and ALP activity. IL-17A alone increased ALP but not MM. Their combination was more potent. TNF-α alone increased BMP2 mRNA expression at 6 and 12 h. These levels decreased in combination with IL-17A at 6 h only. DKK-1 mRNA expression was inhibited by TNF-α and IL-17A either alone or combined. Supporting an imbalance toward osteoblastogenesis, RANKL expression was inhibited by TNF-α and IL-17A. However, TNF-α but not IL-17 alone decreased Runx2 mRNA expression at 6 h. In parallel, TNF-α but not IL-17 alone increased Schnurri-3 expression with a synergistic effect with their combination. This may be related to an increase of TNFRII overexpression. Conclusion: IL-17 increased the effects of TNF-α on bone matrix formation by hMSCs. However, IL-17 decreased the TNF-α-induced BMP2 inhibition. Synergistic interactions between TNF-α and IL-17 were seen for RANKL inhibition and Schnurri-3 induction. Such increase of Schnurri-3 may in turn activate osteoclasts leading to bone destruction as in RA. Conversely, in the absence of osteoclasts, this could promote ectopic bone formation as in AS.

The lipid alterations in the stratum corneum of dogs with atopic dermatitis are alleviated by topical application of a sphingolipid-containing emulsion

Background.  Atopic dermatitis (AD) results from an altered skin barrier associated with defects in the lipid composition of the skin. Dogs with AD present similar clinical symptoms to humans, and may be a useful model for investigations into AD.

Increased sensitivity of rheumatoid synoviocytes to Schnurri-3 expression in TNF-α and IL-17A induced osteoblastic differentiation

OBJECTIVE To compare the effects of TNF-α and IL-17A on osteogenic differentiation of isolated fibroblast-like synoviocytes (FLS) from healthy donors, osteoarthritis (OA) and rheumatoid arthritis (RA) patients. METHODS FLS were cultured in osteogenic medium, with and without TNF-α and/or IL-17A. Extracellular matrix mineralization was evaluated by alizarin red staining and alkaline phosphatase activity (ALP) measurement. mRNA expression was analyzed by qRT-PCR for Wnt5a, BMP2 and Runx2, genes associated with osteogenesis, for DKK1 and RANKL, genes associated with osteogenesis inhibition and Schnurri-3, a new critical gene in the cross talk with osteoclasts. IL-6 and IL-8 production was measured by ELISA. RESULTS In osteogenic medium, matrix mineralization and increased ALP activity indicated that FLS can undergo osteogenic differentiation, which was increased with TNF-α and IL-17A. The expression of osteogenesis activators (BMP2 and Wnt5a) was increased with cytokines and that of the osteogenesis inhibitor DKK1 was decreased. There was no difference between all three cell types. In contrast, RA FLS were particularly sensitive to the synergistic increase of Shn3 with TNF-α and IL-17A. Levels of IL-6 and IL-8 were also higher for RA-FLS, compared to healthy and OA FLS. CONCLUSION IL-17A and/or TNF-α treatment favor an osteogenesis induction in isolated FLS, independent of their origin. RA-FLS were more sensitive to the synergistic increase of Schnurri-3 expression. Combined with the higher levels of inflammation, this may in turn activate osteoclastogenesis, leading to increased bone destruction seen in destructive arthritis.

Differential Effects of IL-17A and TNF-α on Osteoblastic Differentiation of Isolated Synoviocytes and on Bone Explants from Arthritis Patients

Objective TNF-α and IL-17A act on fibroblast-like synoviocytes (FLS) and contribute to cytokine production, inflammation, and tissue destruction in rheumatoid arthritis (RA). The aim of this study was to compare their effects on osteogenic differentiation of isolated FLS and on whole bone explants from RA and osteoarthritis (OA) patients. Methods Fibroblast-like synoviocytes and bone explants were cultured in the presence or absence of TNF-α and/or IL-17A. Mineralization of extracellular matrix of FLS was measured by alizarin red and alkaline phosphatase activity (ALP). mRNA expression was analyzed by qRT-PCR for Wnt5a, BMP2, and RUNX2, key genes associated with osteogenesis. IL-6 and IL-8 levels were measured by enzyme-linked immunosorbent assays. Bone explant structure was quantified by histomorphometry. Results In isolated OA and RA FLS, the combination of TNF-α and IL-17A induced matrix mineralization, increased ALP activity and expression of the osteogenesis-associated genes Wnt5a, BMP2, and Runx2, indicating an osteogenic differentiation. Wnt5a levels increased with TNF-α alone and in combination with IL-17A. BMP2 expression decreased with IL-17A and TNF-α after 12 h with OA FLS and 24 h with RA FLS. Runx2 expression decreased only with combination of TNF-α and IL-17A in OA FLS and with cytokines alone and combined in RA FLS. IL-6 and IL-8 production increased with IL-17A and/or TNF-α in both FLS and bone samples, especially from RA. Treatment of bone explants with cytokine combination increased ALP in OA but not RA samples. A decrease in bone volume was seen with cytokine combination, especially with RA explants. Conclusion Differences were observed for the effects of IL-17A and TNF-α on osteogenic differentiation. In isolated FLS, increased osteoblastogenesis was observed, contrasting with the inhibitory effect in whole bone, specifically in RA. The net effect of IL-17A and TNF-α appears to depend on the disease state and the presence of other cells.

Biomonitoring airborne pollution: a case study of “Urginea maritima” species in Bentael natural reserve – Lebanon

ABSTRACT Biomonitoring airborne pollution has been a widespread practice. Its advantages make it an alternative to costly technological techniques. In this context, a rare species “Urginea maritima” was tested to measure the changes surrounding Bentael natural reserve in Lebanon, triggered by the inauguration of a new road on its south side, and revealed alarming results through two years study (2015–2016). Three environmental indicators were evaluated: Air Pollution Tolerance Index, Total Antioxidant Capacity and leaf Relative Water Content. These factors showed the escalated evolution of the air pollution during the studied months. In addition, six parameters, separated between pollution markers and scavengers, were studied: hydrogen peroxide, pheophytin, proline, ascorbic acid, carotenoids and total phenolic compounds showed a gradual and sharp increase. The passive biomonitoring with plants confirmed to be a reliable method to evaluate airborne pollution effects which will expose new extent to establish a routine monitoring program in Lebanon.

Biochemical profile of non-enzymatic stress markers in the plant species “Urginea maritima” in a Mediterranean natural reserve exposed to oxidative stress

Protected areas decrease degrading natural ecosystems due to pollution such as air pollution. In 1981, the inhabitants founded Bentael natural reserve in Byblos, Lebanon, to secure their region against urbanization projects, like the recently constructed road that threatens the biodiversity of the reserve. This study was conducted to determine the oxidative stress resulting from this pollution and that menaces 360 floral species among them a rare species “Urginea maritima.” In this research, the biomonitoring approach was experienced to assess the oxidative stress. Biomonitoring possesses has the advantage to be low cost and a constructive method to generate valuable data for further examinations. The studied parameters were air pollutants, ascorbic acid, photosynthetic pigments, leave’s pH, relative water content, proline, carbohydrates, and hydrogen peroxide, in three chosen spots, near the pollution source (P1), opposite the latter spot (P2), and in an area relatively far from the source of contamination and which was chosen as the control site (Ctrl). The results showed in P1 detection of air pollutants higher of about 80% than in Ctrl, modifications in stress markers: increased concentration of the reactive oxygen species “hydrogen peroxide,” rise in the concentration of the osmoregulator amino acid “proline,” and depletion in chlorophyll content, in contrast to an increase in pheophytin. All these findings can be exploited as early diagnosis of air pollution and confirmed the ability to use such biomonitor (“Urginea maritima”) as a way to assess the environmental pollution levels and consequently affirm the danger of such landscape activities on natural reserves.

Can the damage caused by anthropogenic activities on Urginea maritima in Bentael natural reserve be a signal of health problems

In the caza of “Byblos”, Lebanon, a new road has been constructed near the natural reserve of “Bentael”, threatening the fauna and the flora of the reserve. Among the 360 floral species, “Urginea maritima” has been declared rare by the United Nations Environment Program (UNEP)[1]. Concerns on the extinction of some species are getting serious. In order to elucidate the seriousness of these worries, “Urginea maritima” was chosen as a bio indicator to monitor the effects of this pollution on the reserves flora. Ten parameters were studied: pH, total chlorophyll content, pheophytin, carotenoids, ascorbic acid, relative water content, proline, Air Pollution Tolerance Index, total carbohydrates content and hydrogen peroxides. Results show significant correlation between pollution load and the biochemical parameters analyzed from the different sites.

A4.11 Combination of TNF-α and IL-17a promotes osteogenic differentiation, but increases schnurri 3 in isolated human mesenchymal stem cells

Background and Objectives Rheumatoid arthritis (RA) is characterised by defective bone repair and excessive bone destruction, and ankylosing spondylitis (AS) by increased ectopic bone formation. Since TNF-α and IL-17A are involved in the pathogenesis of both diseases, the aim of this study was to investigate their effects on the osteogenic differentiation of isolated human bone marrow derived mesenchymal stem cells (hMSCs). Materials and Methods Differentiation of hMSCs into osteoblasts was induced in the presence or absence of TNF-α and/or IL-17A. Matrix mineralization was evaluated by alizarin red staining and alkaline phosphatase activity measurement. mRNA expression was measured by qRT-PCR for the osteogenesis associated genes BMP2 and RUNX2, for DKK1 the negative regulator of osteogenesis, and for Shn3 and RANKL, genes associated with the cross-talk with osteoclasts. Results Exposure of isolated hMSCs to TNF-α resulted in a significant acceleration of matrix mineralization, which was potentiated by IL-17A (TNF-α, p = 0.03; TNF-α + IL-17A, p = 0.001 vs. induction medium at day 17). This was associated with a significant increase of alkaline phosphatase activity and an acceleration of its kinetics (TNF-α, p = 0.05; IL-17A, p = 0.01; TNF-α + IL-17A 1, p = 0.01 vs. induction). TNF-α but not IL-17A induced significant a early increase of BMP2 mRNA expression (TNF-α, p = 0.007 vs. induction at 6hrs). The combination of TNF-α and IL-17A induced a strong and sustained significant decrease of DKK1 (p = 0.03 vs. induction at 72hr) and RANKL (p = 0.002 vs. induction at 6hr) mRNA expression, but an early significant increase of Schnurri-3 (TNF-α, p = 0.03; TNF-α + IL-17A, p = 0.007 vs. induction at 6hr). On the contrary, Runx2 gene expression was not affected in any conditions. Conclusions The combined action of TNF-α and IL-17A increased hMSCs osteogenesis, via increasing BMP2 and reducing RANKL and DKK1 gene expression. This could be involved in ectopic bone formation as observed in AS, where osteoclasts are not present. Conversely, increase of Schnurri-3 may activate osteoclasts and bone destruction as observed in RA.