IL-1α Treatment Research Articles

Interleukin-1 increases SERPINE1 expression in human granulosa-lutein cell via P50/P52 signaling pathways

It has been reported that immune factors are associated with the occurrence of polycystic ovary syndrome (PCOS). Interleukin-1 (IL-1) is a member of the interleukin family that widely participates in the regulation of the inflammatory response in the immune system. In addition, it has been reported that aberrant IL-1 accumulation in serum is associated with the occurrence of PCOS. However, little is known about how IL-1 participates in the pathogenesis of PCOS. In the present study, we demonstrated that the immune microenvironment was altered in follicular fluid from PCOS patients and that the expression levels of two IL-1 cytokines, IL-1α and IL-1β were increased. Transcriptome analysis revealed that IL-1α and IL-1β treatment induced primary human granulosa-lutein (hGL) cell inflammatory response and increased the expression of serpin family E member 1 (SERPINE1). Mechanistically, we demonstrated that IL-1α and IL-1β upregulated SERPINE1 expression through IL-1R1-mediated activation of downstream P50 and P52 signaling pathways in human granulosa cells. Our study highlighted the role of immune state changes in the occurrence of PCOS and provided new insight into the treatment of patients with IL-1-induced ovarian function disorders.

Melatonin Ameliorates Axonal Hypomyelination of Periventricular White Matter by Transforming A1 to A2 Astrocyte via JAK2/STAT3 Pathway in Septic Neonatal Rats.

BackgroundAstrocyte A1/A2 phenotypes may play differential role in the pathogenesis of periventricular white matter (PWM) damage in septic postnatal rats. This study aimed to determine whether melatonin (MEL) would improve the axonal hypomyelination through shifting A1 astrocytes towards A2.MethodsOne-day-old Sprague–Dawley rats were divided into control, LPS, and LPS+MEL groups. Immunofluorescence was performed to detect C1q, IL-1α, TNF-α, IBA1, GFAP, MAG, C3 and S100A10 immunoreactivity in the PWM of neonatal rats. Electron microscopy was conducted to observe alterations of axonal myelin sheath in the PWM; moreover, myelin protein expression was assessed using in situ hybridization. The effects of MEL on neurological function were evaluated by behavioral tests. In vitro, A1 astrocytes were induced by IL-1α, C1q and TNF-α, and following which the effect of MEL on C3 and S100A10 expression was determined by Western blot and immunofluorescence.ResultsAt 1 and 3 days after LPS injection, IBA1+ microglia in the PWM were significantly increased in cell numbers which generated excess amounts of IL-1α, TNF-α, and C1q. The number of A1 astrocytes was significantly increased at 7–28d after LPS injection. In rats given MEL treatment, the number of A1 astrocytes was significantly decreased, but that of A2 astrocytes, PLP+, MBP+ and MAG+ cells was increased. By electron microscopy, ultrastructural features of axonal hypomyelination were attenuated by MEL. Furthermore, MEL improved neurological dysfunction as evaluated by different neurological tests. In vitro, MEL decreased the C3 significantly, and upregulated expression of S100A10 in primary astrocytes subjected to IL-1α, TNF-α and C1q treatment. Importantly, JAK2/STAT3 signaling pathway was found to be involved in modulation of A1/A2 phenotype transformation.ConclusionMEL effectively alleviates PWMD of septic neonatal rats, which is most likely through modulating astrocyte phenotypic transformation from A1 to A2 via the MT1/JAK2/STAT3 pathway.

Pulsed Electromagnetic Fields Reduce Interleukin-6 Expression in Intervertebral Disc Cells Via Nuclear Factor-κβ and Mitogen-Activated Protein Kinase p38 Pathways.

This is an in vitro study of bovine disc cells exposed to pulsed electromagnetic fields. The purpose of the present study was to investigate whether pulsed electromagnetic fields (PEMF) effects on the expression of interleukin-6 (IL-6) expression is mediated by two known inflammation regulators, nuclear factor-κB (NF-κβ) and phosphorylated mitogen-activated protein kinase p38 (p38-MAPK) signaling pathways SUMMARY OF BACKGROUND DATA.: Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Increasing evidence showed that PEMF, a noninvasive biophysical stimulation, can have physiologically beneficial effects on inflammation and tissue repair. Our previous research shows that PEMF treatment can reduce IL-6 expression by intervertebral disc cells. However, the underlying mechanisms of PEMF action are yet to be uncovered. Intervertebral disc nuclear pulposus cells were challenged with interleukin-1α (IL-1α) (for mimicking inflammatory microenvironment) and treated with PEMF simultaneously up to 4 hours. Cells were then collected for NF-κβ and phosphorylated p38-MAPK protein detection with Western blot. Additionally, the RelA (p65) subunit of NF-κβ was examined with immunostaining for assessment of NF-κβ activation. As expected, Western blot results showed that both NF-κβ and phosphorylated p38 expression were significantly increased by IL-1α treatment. This induction was significantly inhibited to control condition levels by PEMF treatment. Immunostaining demonstrated similar trends, that PEMF treatment reduced the NF-κβ activation induced by IL-1α exposure. Our data indicate that the previously-reported inhibitory effect of PEMF treatment on disc inflammation is mediated by NF-κβ and phosphorylated p38-MAPK signaling pathways. These results further establish PEMFs anti-inflammatory activity, and may inform potential future clinical uses for management of inflammation associated with disc degeneration. N/A.

Interleukin-1α leads to growth hormone deficiency in adamantinomatous craniopharyngioma by targeting pericytes: implication in pituitary fibrosis

BackgroundThe incidence of growth hormone deficiency (GHD) in adamantinomatous craniopharyngioma (aCP) is significantly higher than in other sellar region tumors, but the possible mechanism is still elusive. A high level of inflammatory responses is another feature of aCP. We investigated the internal connection between interleukin-1α (IL-1α) and GHD, while focusing on its biological activities in pituitary fibrosis. Materials and methodsTo diagnosis of GHD, the Body Mass Index (BMI), Insulin Like Growth Factor-1(IGF-1) and peak growth hormone (GH) values after insulin stimulation test of 15 aCP patients were recorded. Histological staining was performed on the aCP samples. Levels of 9 proinflammatory cytokines in tumor tissue and cell supernatant were detected using Millipore bead arrays. The effect of IL-1α on GH secretion was evaluated in vivo and in vitro. Western blot, qRT-PCR and cell functional assays were used to explore the potential mechanism through which IL-1α acts on GH secretion. The stereotactic ALZET osmotic pump technique was used to simulate aCP secretion of proinflammatory cytokines in rats. Recombinant IL-1α (rrIL-1α) and conditioned media (CM) prepared from the supernatant of aCP cells was infused directly into the intra-sellar at a rate of 1 μl/h over 28 days, and then the effects of IL-1α treatment on pathological changes of pituitary gland and GH secretion were measured. To further confirm whether IL-1α affects GH secretion through IL-1R1, an IL-1R1 blocker (IL-1R1a, 10 mg/kg body weight, once daily) was administered subcutaneously from the first day until day 28. ResultsThere was a significant positive correlation between pituitary fibrosis and GHD (rS = 0.756, P = 0.001). A number of cytokines, in particular IL-1α, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1), were elevated in tumor tissue and cell supernatant. Only IL-1α showed a significant difference between the GHD group and the No-GHD group (P < 0.001, F = 6.251 in tumor tissue; P = 0.003, F = 1.529 in cell supernatant). IL-1α significantly reduced GH secretion in coculture of GH3 and pericytes. The activation of pericytes induced by IL-1α was mediated by the IL-1R1 signaling pathway. In vivo, IL-1α induces pituitary fibrosis, further leading to a decreased level of GH. This pathological change was antagonized by IL-1R1a. ConclusionThis study found that the cross talk between aCP cells and stroma cells in the pituitary, i.e. pericytes, is an essential factor in the formation of GHD, and we propose that neutralization of IL-1α signaling might be a potential therapy for GHD in aCP.

Abstract 5734: Interleukin-1 signaling is required for HNSCC tumor response to cetuximab

Abstract Despite the incorporation of the epidermal growth factor receptor (EGFR) inhibitor cetuximab into the clinical management of head and neck squamous cell carcinoma (HNSCC), limited to no long-term changes in overall survival are observed in HNSCC patients even though EGFR is expressed at high levels in these tumors. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that cetuximab activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the implications of activating this pathway are unclear. The IL-1 pathway plays a central role in immune response and displays both pro-tumor and anti-tumor activities. IL-1 may promote tumor growth by upregulating the secretion of pro-inflammatory mediators involved in angiogenesis and metastasis. On the other hand, IL-1 signaling may promote antitumor immunity specifically via natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC), which is also an important mechanism of action of cetuximab. The goal of our work is to determine how modulation of the IL-1 pathway affects HNSCC tumor response to cetuximab. We found that blockade of IL-1 signaling using an IL-1-receptor antagonist (IL-1RA, anakinra), neutralizing IL-1α/IL-1β antibodies, and genetic knockdown of the IL-1R all suppressed the anti-tumor efficacy of cetuximab, while IL-1α overexpression and treatment with recombinant IL-1α enhanced HNSCC tumor response to cetuximab in immunodeficient and immunocompetent HNSCC mouse models. Mechanistically, these results appear to be due to modulation of ADCC, as we found that IL-1 blockade significantly reduced cetuximab-mediated ADCC in vitro. Additionally, we found that HNSCC patients with high baseline circulating levels of IL-1 ligands (IL-1α, IL-1β) were significantly more likely to respond favorably to cetuximab monotherapy compared to patients with low or no baseline circulating IL-1 ligands. Altogether, these results suggest that IL-1 signaling is necessary for HNSCC tumor response to cetuximab. Therefore, IL-1α warrants further study as a novel therapeutic to enhance response to cetuximab and as an immunologic biomarker for cetuximab response. Citation Format: Madelyn M. Espinosa-Cotton, Rachel A. Dahl, Elana Fertig, Isaac Jensen, Ayana J. McLaren, Kenley Miller, Samuel N. Rodman, Sandra Schmitz, Andrean L. Simons. Interleukin-1 signaling is required for HNSCC tumor response to cetuximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5734.

Wound healing factor IL1α: a plausible trigger for zinc-mediated cartilage erosion?

Purpose: Chronic low-grade inflammation is a hallmark of osteoarthritis (OA) in patient subgroups but the cytokine signalling networks that influence the chondrocyte catabolic phenotype are incompletely understood. Zinc homeostasis is governed by importers (ZIPs), exporters (ZnTs) and the metallothionein (MT) storage genes and ZIP8 has been shown to be upregulated by IL1β in murine chondrocytes. ZIP8 overexpression in murine chondrocytes leads to severe cartilage erosion via ZIP8-mediated zinc entry, MTF1 nuclear localisation and enhanced transcription of ADAMTS5 and MMP13. Correlations between promoter hypomethylation, increased inflammatory gene expression and zinc importers ZIP8 and ZIP14 were observed in a subset of OA hip patients stratified by their DNA methylome. It is unknown if IL1α, a cytokine implicated in wound healing following necrosis, disrupts zinc homeostasis in human cartilage. We hypothesize that ILα signalling increases locally within the join as a result of injury, leads to disruption of zinc homeostasis and increases inflammatory gene expression contributing to detrimental effects upon cartilage tissue. Methods: Human articular chondrocytes (HACs) from 8 knee arthroplasty patients (ages 61-81) were extracted from cartilage post-surgery, cultured for one passage then treated ± 0.1ng/ml IL1α for P2 and cell pellets were collected. The rib chondrocyte TC28a2 and osteosarcoma SW1353 cell lines (n=3-5) were cultured ± 0.25ng/ml IL1α for a total of three passages over 9 days, then allowed to recover from stimulation three days (passage 4). RNA was extracted, reverse transcribed into cDNA and gene expression determined by quantitative real-time PCR (qPCR). DNA was extracted for future methylation studies. A panel of 35 genes involved in inflammation (5), zinc transportation (24), proliferation (3) and senescence (3) were assessed. The HAC qPCR data was analysed with a Wilcoxon t-test for P2 and the cell line data with a one-way ANOVA with Sidak’s test for P1. Results: IL-6, IL-8 and CCL2 inflammatory genes were upregulated in IL1α-treated HACs and these results were recapitulated in the cell lines. The extracellular-matrix degrading gene ADAMTS5 was upregulated 1.3 fold (p=0.0078) in IL1α-treated HACs, though the same observation was not made in TC28a2 or SW1353 cells. The zinc importers ZIP8 and ZIP14 were upregulated in HACs (4.3 fold, p=0.0078 and 2.6 fold, p=0.0078 respectively). Both genes were significantly upregulated after 1 passage of IL1α treatment in SW1353s (2.4 fold, (p=0.002) and 2.8 fold (p=0.0001) for ZIP8 and ZIP14 respectively), with ZIP14 also upregulated in TC28a2 cells. All MT genes tested (MT1A, MT1E, MT1F, MT1G, MT1H, MT1X and MT2A) were significantly upregulated by IL1α in HACs. IL1α-treated HACs had reduced cell numbers at the end of P2 than untreated cells (p=0.000529) from the same donor and this was associated with decreased expression of the proliferation markers MKI67 (0.4 fold, p=0.0078), TOP2A (0.7 fold, p=0.0078) and TPX2 (0.6 fold, p=0.0078). No effect of IL1α treatment on cell number was observed in SW1353 or TC28a2 cells. Conclusions: IL1α treatment of HACs increased inflammatory gene expression and specifically the zinc importers ZIP8 and ZIP14. This result was mimicked in chondrocyte cell lines, supporting the observations made in the primary cells. MTs and ADAMTS5, both target genes of the zinc sensing transcription factor, are upregulated after IL1α treatment and future experiments will investigate the role of IL1α on MTF1 nuclear translocation and activity.

Abstract TP102: Delayed Interleukin-1 alpha Treatment is Profoundly Neuroreparative in Experimental Ischemic Stroke

Despite recent advancements in blood vessel recanalization, ischemic stroke remains a leading cause of significant morbidity. Therefore, new stroke therapies are urgently needed, but have thus far failed in clinical trials despite promising animal data. Studies in our lab have suggested that proteolytic fragments of the extracellular matrix heparan sulfate proteoglycan perlecan are neuroprotective and neuroreparative in experimental ischemic stroke. Further studies have suggested that one such fragment, perlecan LG3, is generated by multiple cells of the neurovascular unit in response to the pro-inflammatory cytokine, interleukin-1alpha (IL-1α). Long thought to be deleterious to the brain after stroke, we now provide data that suggests that IL-1α is neuroreparative upon delayed intravenous administration in experimental ischemic stroke. Coupled with other work in our lab that suggests that acute post-stroke IL-1α administration is neuroprotective, we here aim to show that IL-1α is also an attractive therapeutic target in later phases of stroke. 3-month-old C57Bl6 mice underwent transient middle cerebral artery occlusion for one hour and received either vehicle or IL-1α treatment (1ng/mouse) intravenously 3 days following experimental stroke. For up to 14 days after stroke, all groups were evaluated on an 11 point neuroscore. Mice were sacrificed 7 or 14 days post stroke and their brains analyzed for cellular dysmorphia, endothelial cell activation, overall cerebral vascularization, and evidence of neurogenesis at the subventricular zone (SVZ). IL-1α treated mice have less brain dysmorphia (p &gt; 0.0001), greater overall vascularization (p &gt; 0.05), a trending increase in neurogenesis at the SVZ, and significantly improved behavioral outcomes. Collectively, our results suggest that IL-1α could represent an unexpected and novel therapeutic approach for ischemic stroke with a broad therapeutic window.

CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues.

Proinflammatory cytokines such as interleukin-1 (IL-1) are found in elevated levels in diseased or injured tissues and promote rapid tissue degradation while preventing stem cell differentiation. This study was undertaken to engineer inflammation-resistant murine induced pluripotent stem cells (iPSCs) through deletion of the IL-1 signaling pathway and to demonstrate the utility of these cells for engineering replacements for diseased or damaged tissues. Targeted deletion of the IL-1 receptor type I (IL-1RI) gene in murine iPSCs was achieved using the RNA-guided, site-specific clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome engineering system. Clonal cell populations with homozygous and heterozygous deletions were isolated, and loss of receptor expression and cytokine signaling was confirmed by flow cytometry and transcriptional reporter assays, respectively. Cartilage was engineered from edited iPSCs and tested for its ability to resist IL-1-mediated degradation in gene expression, histologic, and biomechanical assays after a 3-day treatment with 1 ng/ml of IL-1α. Three of 41 clones isolated possessed the IL-1RI+/- genotype. Four clones possessed the IL-1RI-/- genotype, and flow cytometry confirmed loss of IL-1RI on the surface of these cells, which led to an absence of NF-κB transcription activation after IL-1α treatment. Cartilage engineered from homozygous null clones was resistant to cytokine-mediated tissue degradation. In contrast, cartilage derived from wild-type and heterozygous clones exhibited significant degradative responses, highlighting the need for complete IL-1 blockade. This work demonstrates proof-of-concept of the ability to engineer custom-designed stem cells that are immune to proinflammatory cytokines (i.e., IL-1) as a potential cell source for cartilage tissue engineering.

IL-1α Up-Regulates IL-6 Expression in Bovine Granulosa Cells via MAPKs and NF-κB Signaling Pathways

Background/Aims: IL-6 is one of the main cytokines in regulating ovarian follicular development and ovulation. However, the factors that regulate IL-6 expression in follicles are still unclear. The aim of this study was to elucidate the mechanisms underlying the effect of IL-1α on IL-6 expression in granulosa cells. Methods: IL-6 expression after IL-1α with/without inhibitors treatment was analyzed by RT-qPCR and ELISA. The phosphorylation of proteins induced by IL-1α was analyzed by western blot. The intracellular cAMP level was assayed by immunoassay kit. Results: IL-1α has a dose-dependent effect on IL-6 expression in granulosa cells. This promoting effect can be significantly attenuated by Erk, c-Jun, p38 and IκB proteins inhibitors, respectively. Moreover, the phosphorylation levels of Erk, c-Jun, p38 and IκBα proteins were significantly increased after IL-1α treatment. In addition, we also found that IL-1α not only reversed the cAMP attenuated IL-6 expression， but also increased IL-1α mRNA expression in granulosa cells. Conclusion: The regulation of IL-1α on IL-6 expression is mediated by activation of MAPKs and NF-κB signaling pathways. Moreover，IL-1α may regulate the ovulation-related genes expression in granulosa cells by an autocrine and/or paracrine manner.

Caco-2 세포에서 커큐민 처리에 의한 IL-1α로 유도된 소장 상피세포의 tight junction 투과성 저해

The intestinal tight junction (TJ) plays an important role as a paracellular barrier. Impaired TJ permeability and enhanced proinflammatory cytokine production are crucial pathophysiological mechanisms in inflammatory bowel diseases (IBDs). Although proinflammatory cytokines, tumor necrosis factor-alpha and interluekin-1 beta, which are markedly increased in IBD patients, have been reported to increase intestinal TJ permeability, the role of interleukin-1 alpha (IL-1α) in the TJ has not been studied. Phytochemicals could prevent proinflammatory cytokine-caused TJ alteration. Curcumin (CCM), a biologically active component of turmeric, has a strong anti-inflammatory activity. The purpose of this study was to elucidate the effect of IL-1α on intestinal epithelial TJ permeability and the role of CCM in IL-1α’s action on TJ in an in vitro intestinal epithelial system, Caco-2 monolayers. The TJ integrity of Caco-2 monolayers was estimated by measuring the flux of FITC-labeled dextran and transepithelial electrical resistance (TEER). Apical IL-1α (100 ng/ml) treatment elevated TJ permeability and suppressed TEER of Caco-2 monolayers. Pretreatment with CCM (20 μM) for 30 min significantly inhibited IL-1α-induced TJ alterations, such as increased TJ permeability and decreased in TEER values. These results demonstrated that IL-1α-induced increases in Caco-2 TJ permeability and CCM blocked the action of IL-1α in the TJ.

IL-1α induces apoptosis and inhibits the osteoblast differentiation of MC3T3-E1 cells through the JNK and p38 MAPK pathways.

Interleukin(IL)-1 is a proinflammatory cytokine that plays important roles in inflammation and host responses to infection. The present study aimed to evaluate the effects of IL-1α on the apoptosis and differentiation of osteoblasts, and to elucidate the mechanism responsible for these effects in the osteoblast‑like cell line MC3T3-E1. The MC3T3-E1 cells were non-treated or treated with IL-1α. Following treatment, cell viability, alkaline phosphatase(ALP) activity and caspase-3 activity were evaluated. The expression of osteoblast-specific genes as well as Bax, Bcl-2 and caspase-3 were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein levels of Bax, Bcl-2, caspase-3 and the phosphorylation of mitogen-activated protein kinases(MAPKs, also known as MAP kinases) were evaluated using western blot analysis. The MAPK signaling pathway was blocked by pre-treatment with MAPK inhibitors SB203580, PD98059 and SP600125. IL-1α treatment induced a significant decrease in cell viability and ALP activity in the MC3T3-E1 cells. IL-1α also significantly decreased the mRNA expression and protein levels of osteoblast-related genes in the MC3T3-E1 cells. On the other hand, IL-1α significantly upregulated the mRNA expression and protein levels of Bax and caspase-3 as well as caspase-3 activity, whereas Bcl-2 expression was decreased in the MC3T3-E1 cells. Furthermore, IL-1α activated the apoptotic signaling pathway by increasing the phosphorylation of c-JunN-terminal kinase(JNK) and p38-MAPK, whereas it inhibited the phosphorylation of extracellular signal-regulated kinase1/2(ERK1/2). Moreover, pre-treatment with MAPK inhibitors attenuated the phosphorylation of JNK, p38 and Bax expression enhanced by IL-1α. However, MAPK inhibitors markedly increased the protein expression of osteoblast-related genes and Bcl-xL in the MC3T3-E1 cells downregulated by IL-1α. Taken together, these findings suggest that IL-1α induces the apoptosis of osteoblasts and inhibits osteoblast differentiation by activating the JNK and the p38MAPK pathways.

Inflammatory Cytokine Il-1α Up-Regulates Piezo1 and Hyper-Sensitizes Chondrocytes to Compression

In diarthrodial joints, chondrocyte mechanotransduction play a critical role in the maintenance of cartilage and in the pathogenesis of osteoarthritis (OA). The mechanistic understanding of chondrocyte mechanotransduction both in health and disease is essential for the rational prevention and treatment of joint diseases. We recently identified that injurious mechanical stress is transduced by the mechano-sensitive ion channels Piezo1 and Piezo2 in chondrocytes; blocking Piezo1/2 protected chondrocytes from injurious mechanical stress, suggesting a possible therapeutic benefit of this approach in cartilage injury or OA. Yet, little is known about how the OA joint environment may influence Piezo1/2-mediated mechanotransduction. We hypothesize that mechanotransduction of chondrocytes is altered by OA-related inflammatory stress, and that Piezo1/2 gene expression and function is altered in the context of OA.Here, we present new data in support of increased expression of Piezo1 in chondrocytes under inflammatory conditions, associated with sensitization to mechanical stimulation. We found that in the presence of pathophysiologically relevant concentrations of IL-1α, (1) the basal Ca2+ concentration of articular chondrocytes was increased (∼90nM), (2) mechanically activated Ca2+ influx was increased (∼50 nM by a compression force of 300nN), (3) Piezo1 mRNA levels increased (∼2 fold). These findings suggest sensitization of chondrocytes to mechanical cues via enhanced expression of Piezo1 channels. Also IL-1α treatment resulted in increased cell strain (∼4%) at the same compressive load and attenuated cortical actin labeling, indicative of increased actin destabilization, whereas β-tubulin remained unaffected. These data indicate that IL-1α-exposed chondrocytes (and potentially their membranes) can deform more readily, thereby elevating gating probabilities of mechanosensitive ion channels.Our study shows that the OA-associated inflammatory cytokine IL-1α alters chondrocyte mechanotransduction, suggesting that chondrocytes’ mechanical hypersensitivity is due to upregulated Piezo1 mRNA expression caused by IL-1α-signaling.

Interleukin 1-α: A Modulator of Melanocyte Homeostasis in Vitiligo

Background: Vitiligo is a hypomelanotic autoimmune skin disorder arising from a breakdown in immunological self-tolerance, which leads to aberrant immune responses against melanocytes leading to selective destruction of melanocytes. High levels of Interleukin 1(IL1) have been reported in various autoimmune disorders including vitiligo. The aim of present study was to explore the effect of IL1-α on melanocyte biology by monitoring the melanocyte viability, IL1R1 membrane expression, melanogenesis (TYR, TYRP1 and MITF-M) and other immuno modulatory molecules (IL1A, IL1B, IL1RN,IL1R1 IL8, TNFA, IL6, and ICAM1) upon exogenous stimulation of IL1- α on primary cultured normal human melanocytes (NHM). Materials and methods: Melanocytes were isolated from normal c skin biopsies and cultured in vitro. The NHMs were treated with IL1-α (0-100 ng/ml) and cell viability was monitored by MTT assay, IL1R1 membrane expression was monitored using flow cytometry and relative gene expression was performed using semi-quantitative RT-PCR. Results: The dose dependent effect of IL1-α on melanocytes showed ~12% melanocyte death and ~22% increase in IL1R1 membrane expression upon 100 ng/ml IL1-α treatment for 48 hrs. Further, IL1RN, IL1A, IL1B, IL6, TNFA, ICAM1 showed significantly increased expression (p<0.05) and MITF-M showed significantly decreased expression (p<0.05) upon IL1-α (10 and 100 ng/ml, 48 hrs) stimulation on NHM; while TYR, TYRP1, IL8 and IL1R1 showed no difference. Conclusion: Overall, the present study suggests the crucial role of IL1-α in melanocyte destruction in vitiligo by regulating MITF-M and other immunomodulatory molecules.

Interleukin-1α deficiency attenuates endoplasmic reticulum stress-induced liver damage and CHOP expression in mice

Background & AimsER stress promotes liver fat accumulation and induction of inflammatory cytokines, which contribute to the development of steatohepatitis. Unresolved ER stress upregulates the pro-apoptotic CHOP. IL-1α is localized to the nucleus in apoptotic cells, but is released when these cells become necrotic and induce sterile inflammation. We investigated whether IL-1α is involved in ER stress-induced apoptosis and steatohepatitis. MethodsWe employed WT and IL-1α-deficient mice to study the role of IL-1α in ER stress-induced steatohepatitis. ResultsLiver CHOP mRNA was induced in a time dependent fashion in the atherogenic diet-induced steatohepatitis model, and was twofold lower in IL-1α deficient compared to WT mice. In the ER stress-driven steatohepatitis model, IL-1α deficiency decreased the elevation in serum ALT levels, the number of apoptotic cells (measured as caspase-3-positive hepatocytes), and the expression of IL-1β, IL-6, TNFα, and CHOP, with no effect on the degree of fatty liver formation. IL-1α was upregulated in ER-stressed-macrophages and the protein was localized to the nucleus. IL-1β mRNA and CHOP mRNA and protein levels were lower in ER-stressed-macrophages from IL-1α deficient compared to WT mice. ER stress induced the expression of IL-1α and IL-1β also in mouse primary hepatocytes. Recombinant IL-1α treatment in hepatocytes did not affect CHOP expression but upregulated both IL-1α and IL-1β mRNA levels. ConclusionWe show that IL-1α is upregulated in response to ER stress and IL-1α deficiency reduces ER stress-induced CHOP expression, apoptosis and steatohepatitis. As a dual function cytokine, IL-1α may contribute to the induction of CHOP intracellularly, while IL-1α released from necrotic cells accelerates steatohepatitis via induction of inflammatory cytokines by neighboring cells.

Local estrogen metabolism in epithelial ovarian cancer suggests novel targets for therapy

Epithelial ovarian cancer (EOC) accounts for about 90% of malignant ovarian tumors, and estrogen is often implicated in disease progression. We therefore compared the potential for gating of estrogen action via pre-receptor metabolism in normal human ovarian surface epithelium (OSE), EOC and selected EOC cell lines (SKOV3 and PEO1). Steroid sulphatase (STS), estrogen sulfotransferase (EST), 17β-hydroxysteroid dehydrogenases 2 (17BHSD2) and 5 (17BHSD5) mRNAs, proteins and enzymatic activities were all detectable in primary cell cultures of OSE and EOC, whereas aromatase and 17BHSD1 expression was negligible. qRT-PCR assay on total mRNA revealed significantly higher EST mRNA expression in OSE compared to EOC (P<0.05). Radioenzymatic measurements confirmed reduced sulfoconjugation (neutralization) of free estrogen in EOC relative to OSE. OSE cells were more effective at converting free [3H]-E1 to [3H]-E1S or [3H]-E2S, while EOC cell lines mainly converted [3H]-E1 to [3H]-E2 with minimal formation of [3H]-E1S or [3H]-E2S. IL1α treatment suppressed EST (P<0.01) and 17BHSD2 (P<0.001) mRNA levels in OSE and stimulated STS mRNA levels (P<0.001) in cancer (SKOV3) cells. These results show that estrogen is differentially metabolized in OSE and EOC cells, with E2 ‘activation’ from conjugated estrogen predominating in EOC. Inflammatory cytokines may further augment the local production of E2 by stimulating STS and suppressing EST. We conclude that local estrogen metabolism may be a target for EOC treatment.

Effects of insulin-like growth factor-1 and dexamethasone on cytokine-challenged cartilage: relevance to post-traumatic osteoarthritis

Interleukin-1 is one of the inflammatory cytokines elevated after traumatic joint injury that plays a critical role in mediating cartilage tissue degradation, suppressing matrix biosynthesis, and inducing chondrocyte apoptosis, events associated with progression to post-traumatic osteoarthritis (PTOA). We studied the combined use of insulin-like growth factor-1 (IGF-1) and dexamethasone (Dex) to block these multiple degradative effects of cytokine challenge to articular cartilage. Young bovine and adult human articular cartilage explants were treated with IL-1α in the presence or absence of IGF-1, Dex, or their combination. Loss of sulfated glycosaminoglycans (sGAG) and collagen were evaluated by the DMMB and hydroxyproline assays, respectively. Matrix biosynthesis was measured via radiolabel incorporation, chondrocyte gene expression by qRT-PCR, and cell viability by fluorescence staining. In young bovine cartilage, the combination of IGF-1 and Dex significantly inhibited the loss of sGAG and collagen, rescued the suppression of matrix biosynthesis, and inhibited the loss of chondrocyte viability caused by IL-1α treatment. In adult human cartilage, only IGF-1 rescued matrix biosynthesis and only Dex inhibited sGAG loss and improved cell viability. Thus, the combination of IGF-1 + Dex together showed combined beneficial effects in human cartilage. Our findings suggest that the combination of IGF-1 and Dex has greater beneficial effects than either molecule alone in preventing cytokine-mediated cartilage degradation in adult human and young bovine cartilage. Our results support the use of such a combined approach as a potential treatment relevant to early cartilage degradative changes associated with joint injury.

Use of cartilage derived from murine induced pluripotent stem cells for osteoarthritis drug screening.

The discovery of novel disease-modifying drugs for osteoarthritis (OA) is limited by the lack of adequate genetically defined cartilage tissues for application in high-throughput screening systems. We addressed this need by synthesizing cartilage from induced pluripotent stem cells (iPSCs) to establish and validate an in vitro model of OA. Native or iPSC-derived mouse cartilage samples were treated with the cytokine interleukin-1α (IL-1α) for 3 days to model the inflammatory environment of OA. The biochemical content, mechanical properties, and gene expression of the resulting tissues were assayed. In addition, the inflammatory and catabolic environment of the media was assessed. To establish high-throughput capability, we used a 96-well plate format and conducted a screen of previously identified candidate OA drugs. Glycosaminoglycan (GAG) release into the medium was used as the primary output for screening. Treatment of iPSC-derived or native cartilage with IL-1α induced characteristic features of OA in a rapid and dose-dependent manner. In addition to the loss of GAGs and tissue mechanical properties, IL-1α treatment induced the expression of matrix metalloproteinases and increased the production of the inflammatory mediators nitric oxide and prostaglandin E2 . In the high-throughput screen validation, all candidate OA therapeutic agents provided some benefit, but only the NF-κB inhibitor SC514 effectively reduced cartilage loss in response to IL-1α. This work demonstrates the utility of iPSCs for studying cartilage pathology and provides a platform for identifying novel, patient-specific therapeutic agents that prevent cartilage degradation and modify the course of OA development.

Telomerase Activity in Articular Chondrocytes Is Lost after Puberty.

Objective:Telomere length and telomerase activity are important indicators of cellular senescence and replicative ability. Loss of telomerase is associated with ageing and the development of osteoarthritis. Implantation of telomerase-positive cells, chondrocytes, or stem cells expressing a normal chondrocyte phenotype is desired for cartilage repair procedures. The objective of this study was to identify at what age chondrocytes and at what passage bone marrow–derived mesenchymal stem cells (MSCs) become senescent based on telomerase activity. The effect of osteogenic protein–1 (OP-1) or interleukin-1α (IL-1α) treatment on telomerase activity in chondrocytes was also measured to determine the response to anabolic or catabolic stimuli.Methods:Articular cartilage was collected from horses (n = 12) aged 1 month to 18 years. Chondrocytes from prepubescent horses (<15 months) were treated with OP-1 or IL-1α. Bone marrow aspirate from adult horses was collected and cultured for up to 10 days to isolate MSCs. Telomerase activity was measured using the TeloTAGGG Telomerase PCR ELISA kit.Results:Chondrocytes from prepubescent horses were positive for telomerase activity. Treatment with IL-1α resulted in a decrease in chondrocyte telomerase activity; however, treatment with OP-1 did not change telomerase activity. One MSC culture sample was positive for telomerase activity on day 2; all samples were negative for telomerase activity on day 10.Conclusions:These results suggest that chondrocytes from prepubescent donors are potentially more suitable for cartilage repair procedures and that telomerase activity is diminished by anabolic and catabolic cytokine stimulation. If MSCs are utilized in cartilage repair, minimal passaging should be performed prior to implantation.

Perivascular leukocyte cluster: an essential structure for efficient effector T cell activation in the skin (IRC5P.467)

Abstract It remains largely unclear how antigen-presenting cells encounter effector/memory T cells efficiently in the periphery. Here we used a murine contact hypersensitivity model to analyze antigen presentation in the skin. By antigen challenge, dendritic cells (DCs) formed clusters with effector T cells in dermal perivascular areas to promote in situ proliferation and activation of effector T cells in antigen- and integrin LFA-1-dependent manners. Intriguingly, DCs were attracted toward perivascular macrophages and DC clustering was abrogated by macrophage-depletion. IL-1α treatment induced CXCL2 production from dermal macrophages, and DC clustering was suppressed with blockade of either IL-1R or CXCR2. These findings suggest that dermal leukocyte cluster is an essential structure for elicitation of the acquired cutaneous immunity.

Bioactivity in an aggrecan 32mer fragment is mediated via toll-like receptors

AIM The aim of this study was to determine whether the naturally-occurring aggrecan 32mer fragment was bioactive, and if so, to elucidate the mechanism. METHODS Native 32mer is derived from the aggrecan interglobular domain following cleavage by both MMP and ADAMTS enzymes. Mouse femoral head cartilage explants, epiphyseal chondrocytes, synovial fibroblasts or peritoneal macrophages were cultured in the presence of synthetic mouse 32mer (FFGVGGEDDITIQTVTWPDLELPLPRNVTEGE; Auspep, Australia) or a scrambled peptide comprising the same amino acids in a random order. Global gene expression in explants treated with IL-1α or 32mer was compared on Illumina mouse W6-6_V2 expression beadchips. Thereafter, changes in gene expression were validated in cell and explant cultures by qPCR and ELISA. RESULTS In the microarray experiment, 32mer-treated cartilage explants down-regulated expression of genes encoding matrix proteins and increased expression of pro-inflammatory/pro-catabolic genes with a striking correlation between genes that were regulated by IL-1α and 32mer treatment. Validation by qPCR confirmed that the 32mer, but not a scrambled 32mer peptide, regulated gene expression in articular chondrocytes, peritoneal macrophages and synovial fibroblasts from wildtype mice. The levels of IL-6 measured by ELISA were also significantly increased in cells treated with 32mer or IL-1α, but not the scrambled peptide. Neither gene expression nor levels of IL-6 cytokine were increased in cells harvested from mice lacking the Toll-like receptor (TLR) adaptor protein, MyD88, thus identifying the 32mer as an activator of TLR signalling. Using complementary hydropathy we derived an antigenic sequence for raising an antibody (αCG11) against a putative 32mer-binding protein, then used αCG11 immunoaffinity chromatography to isolate the putative 32mer-binding protein from 4M GuHCl extracts of human OA and juvenile cartilage. Mass spectrometry analyses identified PRELP as the major protein binding to and eluting from the immunoaffinity column in both samples; this was confirmed by Western blotting with αPRELP antibodies. Decorin and biglycan, the small leucine-rich proteoglycans related to PRELP, are recognised TLR2 and TL4 ligands. Experiments to resolve the biological role of PRELP in 32mer-mediated TLR activation are in progress. CONCLUSION These studies identify the aggrecan 32mer as an endogenous activator of TLR-mediated immunity with the potential to accelerate inflammation and cartilage destruction in vivo.