IL-6 mRNA Stability Research Articles

APOBEC-1 Complementation Factor: From RNA Binding to Cancer.

APOBEC-1 complementation factor (A1CF) and Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1 (APOBEC-1) constitute the minimal proteins necessary for the editing of apolipoprotein B (apoB) mRNA in vitro. Unlike APOBEC-1 and apoB mRNA, the ubiquitous expression of A1CF in human tissues suggests its unique biological significance, with various factors such as protein kinase C, thyroid hormones, and insulin regulating the activity and expression of A1CF. Nevertheless, few studies have provided an overview of this topic. We conducted a literature review to describe the molecular mechanisms of A1CF and its relevance to human diseases. In the PubMed database, we used the keywords 'A1CF' and 'APOBEC-1 complementation factor' to collect peer-reviewed articles published in English from 2000 to 2023. Two authors independently reviewed the articles and reached the consensus. After reviewing 127 articles, a total of 61 articles that met the inclusion criteria were included in the present review. Studies revealed that A1CF is involved in epigenetic regulation of reproductive cells affecting embryonic development, and that it is closely associated with the occurrence of gout due to its editing properties on apoB. A1CF can also affect the process of epithelial-mesenchymal transition in renal tubular epithelial cells and promote liver regeneration by controlling the stability of IL-6 mRNA, but no influence on cardiac function was found. Furthermore, increasing evidence suggests that A1CF may promote the occurrence and development of breast cancer, lung cancer, renal cell carcinoma, hepatocellular carcinoma, endometrial cancer, and glioma. This review clarifies the association between A1CF and other complementary factors and their impact on the development of human diseases, aiming to provide guidance for further research on A1CF, which can help treat human diseases and promote health.

Stimulation of ectopically expressed muscarinic receptors induces IFN-γ but suppresses IL-2 production by inhibiting activation of pAKT pathways in primary T cells

T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCβ1 is coexpressed. Resting peripheral hM3Dq+PLCβ1 (hM3Dq/β1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCβ1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/β1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/β1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/β1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

CircPTPN12 promotes the progression and sunitinib resistance of renal cancer via hnRNPM/IL-6/STAT3 pathway

Renal cell carcinoma (RCC) is characterized by the difficulties in early diagnosis and the propensity to metastases. For advanced RCC, sunitinib targeted therapy is the clinically recommended first-line drug and the major challenge of sunitinib treatment is adaptive resistance. Therefore, it is imperative to research the mechanisms underlying sunitinib resistance. In this study, we discovered that circPTPN12 was highly expressed in RCC tissues and was associated with poorer clinical outcomes. circPTPN12 could promote the proliferation, migration, invasion, and sunitinib resistance of RCC cells. Mechanistically, circPTPN12 was found to form a complex with hnRNPM, which was involved in the regulation of mRNA processing. The combination with circPTPN12 enhanced the ability of hnRNPM to maintain the stability of IL-6 mRNA and further activated the STAT3 signaling pathway. The study revealed that circPTPN12/hnRNPM/IL-6/STAT3 axis promoted RCC progression and sunitinib resistance, which might be a promising therapeutic target for relieving sunitinib resistance in RCC.

Dexamethasone, Interleukin 6, and the Adrenal Gland are the Real Battles for COVID-19: Our Molecular Docking, Physiological, and Immunological Explanations of Why the Clinical Benefit of Dexamethasone is More Evident in Males

More than two years have passed since the pandemic and despite all the efforts of researchers, the pathogenesis of the SARS-CoV-2 has not yet been resolved. The SARS-CoV-2 coronavirus that causes COVID-19 has high ability of mutation resulting in different variants of the virus. This mutation helps the virus bypass the body's immune defenses such as innate and adaptive immunity. Interleukin (Il-6), neutrophils, high ferritin, and high D dimer are crucial markers for all sorts of cells on the lengthy path to treatment of COVID-19. IL-6 is one of the key mediators of inflammation and viral cytokine storm in COVID-19 patients. In humans, interleukin (IL)-6 is a strong stimulator of the hypothalamic-pituitary-adrenal (HPA) axis at all levels, and it appears to have a pathogenic role in chronic stress and physiological aging. The adrenal glands have earned further recognition as key modulators of immune function, because secretory products of the adrenal glands. Dexamethasone inhibits TNF-α–induced IL-6 mRNA expression and protein secretion by reducing IL-6 mRNA stability. Although Dexamethasone provides benefits in patients with coronavirus disease but there is sex linked difference between male and female at response to this drug. It was showed that this drug might indeed have an effect in treating covid-19 but the clinical benefit of dexamethasone is more evident in males. Here we propose a testable hypothesis that dexamethasone may be effective in men better than female owing to two factors the first is the ability of males to induce high secretion of adrenal hormone under stress vs female. In males both stressors induced a significant increase, whereas in female adrenal e excretion remained on the same level under the two stress conditions. Both Adrenal e hormones and dexamethasone are hypothesized to promote blood neutrophil levels by increasing neutrophil exit from bone marrow, delaying neutrophil outflow from circulation, and lengthening the half-life of circulating neutrophils. Neutrophils a type of white blood cells that can downregulate interferon-stimulated genes, i.e., suppress their activity. When male patients received the steroid treatment, the dysregulated interferon signals went away quickly. But in female patients, the proportions of neutrophils were not as high and they did not react to the steroids in the same way. The second factor is that ACTH response to il-6 is stronger in males than females and ACTH was found to induce il-6. Therefore, male interact with dexamethasone better than females. Additionally, dexamethasone does not affect Il-6 secreted from the adrenal cortex, which allows Il-6 to be produced during stress and increase neutrophil ratio leading to stronger response in males. We need a new classification of IL-6 at physiological and pathological aspects, as the stimulatory factors are different. it was found that ACTH is h Endocrine Il-6 is stimulated by ACTH, but immune Il-6 is stimulated by il-1β and angiotensin II. Of note, dexamethasone critically saves COVID-19 patients but has no effect on Il-6 of endocrine (ACTH stimulated) origin, either basal or stimulated. We studied the molecular docking of dexamethasone with interleukin-6 and showed that the binding interaction of dexamethasone with interleukin-6 was with high binding affinity (-6.7 Kcal/mol)

POS-486 SECONDARY POLYCYTHEMIA IN NEPHROTIC SYNDROME – AN UNCOMMON ASSOCIATION

Polycythaemia is defined as haemoglobin (Hb) of over16.5 g/dL in men and 16 g/dL in women or haematocrit of over 49% in men and 48% in women (WHO 2017). Primary polycythaemiais caused by erythropoietin (EPO) receptor mutations or Janus Kinase 2 (JAK2) mutations. Secondary polycythaemia is occasionally seen with renal diseases such as cystic kidney disease, tumours, renal artery stenosis and after renal transplantation. Polycythaemia associated with Nephrotic Syndrome (NS) is very rare. This association was first described by Emmanuel DA and Wenzel FJ in 1962. Here, we describe four patients with NS who had polycythaemia at presentation.Heavy protienuria leading to renal interstitial oedema causing hypoxia and increased EPO production and high serum IL8 and increased stability of IL8 mRNA(in minimal change disease) are thought to cause secondary polycythaemia in NS. The resolution of polycythaemia following reduction in proteinuria further strengthens the causation.The exact pathogenesis remains to be elucidated. This is a retrospective observational study conducted in NU Hospitals, Bengaluru, India.There were four patients with NS and polycythaemia, between February 2015 to May 2021. All patients with NSwere subjected to biochemical investigations and ultrasonography (USG) of the abdomen after a detailed history and physical examination as per our hospital protocol. Coagulation profile and blood grouping were done prior to renal biopsy. Percutaneous renal biopsy was done under real-time USG guidance. The tissue obtained was sent for Light microscopic examination, Immunofluorescence microscopy and Electron microscopy. In view of the polycythaemia, all these patients underwent a Chest X Ray, arterial blood gas (ABG) analysis, serum EPO and serum JAK2 mutation studies. All four patients in our study were men. Mean age was 32.25 ± 11.70 years. Mean Hb was 18.75 ± 1.22 g/dL. Mean hematocrit was 54.67 ± 6.5 %. Mean proteinuria at onset was 7.62 ± 7.45 grams/day. Mean serum creatinine at the onset was 1.30 ± 0.58 mg/dL. None had splenomegaly. All had normal total white cell and platelet count. None were smokers. ABG was normal in all. All patients had normal serum EPO levels (mean 3.4±1.64 mU/ml) and negative JAK2 mutation analysis. The etiology of NS was minimal change disease (MCD), primary membranous nephropathy (MGN), primary focal segmental glomerulosclerosis (FSGS) and IgA nephropathy in each one of them. PLA2R antibodies were positive in the serum and renal tissue of the patient with MGN.Immunosuppression was initiated in all patients except the patient with IgA nephropathy who had significant chronicity. Three out of four patients had resolution of polycythemia in parallel with remission of NS.The patient with MCD was detected to have polycythaemia during a relapse. This study highlights polycythaemia as an unusual complication of NS.Most cases of NS and polycythemia documented in the literature correspond to FSGS, mainly associated with polycythaemia vera. There are case reports of secondary polycythaemia associated with primary FSGS, MGN and MCD. We didn't do IL8 and IL11 assay in our patients. To the best of our knowledge ours is probably the largest case series of polycythaemia associated with NS.

The RNA binding protein neuro‐oncological ventral antigen 1 (NOVA1) regulates IL-6 mRNA stability to enhance JAK2-STAT3 signaling in CRC

The molecular mechanisms governing the metastasis of colorectal cancer (CRC) are incompletely understood. In the present study, we found NOVA1 to be expressed at higher levels in CRC cell lines and tissue samples, and this upregulation was positively correlated with TNM stage (p = 0.034), poor differentiation (p = 0.001), and lymph node metastasis (p = 0.008). Both overall survival (OS) and relapse-free survival (RFS) were both significantly decreased in patients with high NOVA1 expression relative to those with low expression. Through a multivariate analysis, we determined that NOVA1 independently predicted poor outcomes in those with CRC. In further functional studies, we found that NOVA1 expression controlled the proliferation and invasive characteristics of CRC cells via a mechanism wherein NOVA1 bound and stabilized the IL6 mRNA, enhancing IL-6/JAK2/STAT3 signaling to in turn upregulate matrix metalloproteinases (MMPs) 2, 7, and 9. NOVA1 therefore plays key functional roles in regulating CRC progression, and our results further indicate that it serve as a valuable prognostic biomarker and potentially a target for therapeutic treatment in individuals with CRC.

Synergistic effect of interleukin-17 and tumour necrosis factor-α on inflammatory response in hepatocytes through interleukin-6-dependent and independent pathways.

The proinflammatory cytokines interleukin (IL)-17 and tumour necrosis factor (TNF)-α are targets for treatment in many chronic inflammatory diseases. Here, we examined their role in liver inflammatory response compared to that of IL-6. Human hepatoma cells (HepaRG, Huh7.5 and HepG2 cells) and primary human hepatocytes (PHH) were cultured with IL-6, IL-17 and/or TNF-α. To determine the contribution of the IL-6 pathway in the IL-17/TNF-α-mediated effect, an anti-IL-6 receptor antibody was used. IL-17 and TNF-α increased in synergy IL-6 secretion by HepaRG cells and PHH but not by Huh7.5 and HepG2 cells. This IL-17/TNF-α synergistic cooperation enhanced the levels of C-reactive protein (CRP) and aspartate aminotransferase (ASAT) in HepaRG cell and PHH cultures through the induction of IL-6. IL-17/TNF-α also up-regulated IL-8, monocyte chemoattractant protein (MCP)-1 and chemokine (C-C motif) ligand 20 (CCL20) chemokines in synergy through an IL-6-independent pathway. Interestingly, first exposure to IL-17, but not to TNF-α, was crucial for the initiation of the IL-17/TNF-α synergistic effect on IL-6 and IL-8 production. In HepaRG cells, IL-17 enhanced IL-6 mRNA stability resulting in increased IL-6 protein levels. The IL-17A/TNF-α synergistic effect on IL-6 and IL-8 induction was mediated through the activation of extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase, nuclear factor-κB and/or protein kinase B (Akt)-phosphatidylinositol 3-kinase signalling pathways. Therefore, the IL-17/TNF-α synergistic interaction mediates systemic inflammation and cell damage in hepatocytes mainly through IL-6 for CRP and ASAT induction. Independently of IL-6, the IL-17A/TNF-α combination may also induce immune cell recruitment by chemokine up-regulation. IL-17 and/or TNF-α neutralization can be a promising therapeutic strategy to control both systemic inflammation and liver cell attraction.

ZCCHC6 (TUT7) deletion inhibit the expression of IL-6 and MMP-13 and reduces the severity of post-traumatic osteoarthritis in a murine model

Purpose: Approximately 20-50% of patients diagnosed with osteoarthritis (OA) develop the disease in joints that previously sustained trauma. RNA terminal uridyltransferases (TUTs), including Zcchc6 (zinc-finger CCHC-domain containing protein 6/TUT7), have been implicated in the post-transcriptional regulation of inflammatory mediators. However, role of Zcchc6 (TUT7) in the pathogenesis of OA or post-traumatic OA has not been explored. Methods: All murine studies were approved by the IACUC and the studies with deidentified, discarded human cartilage were approved by the IRB. Zcchc6 knockout mice were generated by gene targeting (KOMP, UC-Davis). Human or murine chondrocytes were prepared by the enzymatic digestion of the OA cartilage or knee cartilage respective and treated with recombinant human or mouse IL-1β (5ng/ml, R&D Systems, #: 201-LB-025 and #:401-ML-005 respectively) for in vitro studies. IL-6 and MMP13 gene and protein expression in chondrocytes was analyzed by qPCR and immunoblotting respectively. IL-6 mRNA stability was determined by Actinomycin-D chase experiments. For in vivo studies, post-traumatic OA in the knee joints of Zcchc6 deficient male mice and wild type littermates was induced by the surgical destabilization of medial meniscus (DMM) and mice were sacrificed after eight weeks of DMM and the severity of OA was determined per OARSI guidelines for murine OA. Contra lateral knee joints with sham surgery served as controls. Expression of MMP13 (Santa Cruz Biotechnology, #sc-30071), Type-II Collagen cleavage (IBEX Pharmaceuticals, #C1,2C) and Zcchc6 (Santa Cruz Biotechnology, #sc-137947) in vivo were analyzed by immunohistochemistry. Fluorescence in situ hybridization (FISH) was performed using the RNAScope technology. RNA immunoprecipitation (RIP) was performed using validated antibodies. siRNAs were used to deplete the expression of ZCCHC6. Results: Knee cartilage chondrocytes prepared from Zcchc6 KO mice showed increased levels of IL-6 and MMP13 mRNA expression compared to WT chondrocytes in response to IL-1β stimulation. Of interest was the observation that the protein levels of IL-6 and MMP13 were significantly downregulated in IL-1β stimulated Zcchc6 KO chondrocytes lysate and culture supernatants in comparison to WT chondrocytes. FISH and RIP analysis revealed that ZCCHC6 protein binds to IL-6 mRNAs in primary human OA chondrocytes under pathological conditions. Furthermore, IL-6 mRNA half-life was reduced by 50% in Zcchc6 KO murine chondrocytes and ZCCHC6 depleted human primary chondrocytes. The expression of Zcchc6 in the knee joints of wild type mice with DMM surgery was highly upregulated in comparison to control joints. Zcchc6 KO mice expressed low levels of MMP13 and showed lesser matrix degradation in the joints with DMM surgery compared to joints of the WT mice and the control contralateral joints and developed less severe OA as determined by Safranin-O-Fast green staining followed by OARSI scoring. Synovitis was also found to be decreased in the KO mice DMM joints in comparison to WT DMM joints. Conclusions: Our data demonstrate that Zcchc6 is upregulated in DMM joints and functions as a positive regulator of IL-6 and MMP13 expression in murine chondrocytes and cartilage in vivo. Severity of the posttraumatic OA and cartilage degradation was significantly low in Zcchc6 KO mice and correlated with reduced expression of MMP-13. These results identify ZCCHC6 as a potential therapeutic target for the treatment of OA.

CRABP-II enhances pancreatic cancer cell migration and invasion by stabilizing interleukin 8 expression.

Our previous study shows that cellular retinoic acid binding protein II (CRABP-II) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and pre-cancerous lesions, but not detected in normal pancreatic tissues. In this study, we show that deletion of CRABP-II in PDAC cells by CRISPR/Cas9 does not affect cancer cell proliferation, but decreases cell migration and invasion. Gene expression microarray analysis reveals that IL-8 is one of the top genes whose expression is down-regulated upon CRABP-II deletion, while expression of MMP-2 and MMP-14, two targets of IL-8 are also significantly down-regulated. Moreover, we found that CRABP-II is able to form a complex with HuR, which binds to the 3′UTR of IL-8 messenger RNA (mRNA) and enhances IL-8 mRNA stability. Ectopic expression of flag-CRABP-II in CRABP-II knockout cells is able to rescue the expression of IL-8, MMP-2/MMP-14 and recovers cell migration. Using the orthotopic xenograft model, we further demonstrate that CRABP-II deletion impairs tumor metastasis to nearby lymph nodes. Taken together, our results reveal a novel pathway linking CRABP-II expression to enhanced PDAC metastasis, and hence we propose CRABP-II may serve as a new PDAC therapeutic target.

Arid5a exacerbates IFN-γ–mediated septic shock by stabilizing T-bet mRNA

Adenine-thymine (AT)-rich interactive domain containing protein 5a (Arid5a) is an RNA-binding protein that has been shown to play an important immune regulatory function via the stabilization of IL-6 and STAT3 mRNA. However, the role of Arid5a in the overwhelming and uncontrolled immune response that leads to septic shock is unknown. Here, we report that Arid5a-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxic shock and secrete lower levels of major proinflammatory cytokines, including IFN-γ, IL-6, and TNF-α, than WT mice in response to LPS. Arid5a deficiency resulted in decreased levels of IFN-γ under Th1 cell conditions, in which T-box expressed in T cells (T-bet) mRNA expression was inhibited. Arid5a bound to the conserved stem loop structure of the 3'UTR of T-bet and stabilized its mRNA. Arid5a-deficient mice were also resistant to Propionibacterium acnes-primed LPS injection, which is considered to be a T-cell-mediated IFN-γ dependent endotoxic shock mouse model. Thus, regulation of IFN-γ by Arid5a via the stabilization of T-bet mRNA in Th1 cells contributes to the development of septic shock in mice. In addition, our previous study suggests that Arid5a control the IL-6 level in vivo in response to LPS by stabilization of IL-6 mRNA. We also observed that neutralization of IFN-γ and IL-6 significantly recovered the mice from endotoxic shock. Taken together, we conclude that Arid5a regulates the augmentation of IL-6 and IFN-γ in response to LPS, which possibly works synergistically for amplification of various other cytokines that ultimately cause the development of septic shock in mice.

Transmembrane TNF–TNFR2 Antagonizes TH17 Differentiation by Promoting Il2 Expression

Abstract While IL-17 is critical for host defense, its overabundance promotes autoimmunity. IL-2 represses TH17 differentiation, but a role for tumor necrosis factor (TNF) in this process is not well defined. TNF binds TNF receptor 1 (TNFR1) and TNFR2 to stimulate opposing signaling cascades. Whereas sTNF signals through TNFR1, tmTNF preferentially activates TNFR2. We have previously demonstrated reduced IL-2 production in TNFR1−/− TNFR2−/− double knockout CD4+ T cells. To further explore the mechanism by which TNF regulates IL-2 production, we generated TNFR1−/−, TNFR2−/−, and TNFR1−/− TNFR2−/− 5C.C7 TCR Il2-GFP mice to study Il2 transcription at the single cell level. Our findings indicate that CD4+ T cell–intrinsic tmTNF/TNFR2 stimulates Il2 promoter activity and Il2 mRNA stability. Pharmacological blockade of TNF in wild-type C57BL/6 mice and tmTNF Foxp3-GFP mice also demonstrated tmTNF/TNFR2-mediated augmentation of Il2 expression. We further report elevated expression of TNFR2 on the surface of TH17-polarized CD4+ T cells and show that tmTNF/TNFR2, but not sTNF/TNFR1, inhibits TH17 differentiation. Under TH17-polarizing conditions, elevated IL-17 production by TNFR2-knockout CD4+ T cells correlated with increased STAT3 activity and was prevented by exogenous IL-2. We conclude that increased IL-2 production in response to CD4+ T cell–intrinsic tmTNF/TNFR2 signaling is sufficient to inhibit TH17 differentiation in a Foxp3-independent manner.

NF-κB and MAPK signaling pathways regulate the IL6 mRNA stability under TLR4 by regulating the expression and degradation of Arid5a

Abstract AT-rich interactive domain (ARID) 5a controls the IL-6 mRNA stability by binding to its 3′UTR region and preventing it from degradation by Regnase-1. This mechanism was shown to contribute to the elevation of IL-6 levels, which causes the development of autoimmune diseases. However, almost nothing is known about the regulatory mechanisms of Arid5a mRNA and protein expression. In the present study, we have investigated the regulatory mechanisms of Arid5a at transcriptional, post-transcriptional and post-translational levels under TLR4 signaling. We found that in response to LPS stimulation NF-κB binds to the promoter region of Arid5a and induce its gene expression. After expression of Arid5a mRNA, MKP-1 signaling induces AUF-1 to translocate from the nucleus to the cytoplasm where it binds to the 3′UTR region of Arid5a and resulting in its destabilization. Inhibition of MKP-1 and AUF-1 results in the stabilization of Arid5a mRNA, which leads to higher expression of IL6, suggesting that this pathway has a significant role in regulating IL6 mRNA through Arid5a. We also show that during the late phase of LPS stimulation, p38 MAPK phosphorylates the Arid5a protein at Ser 253, 433 and 458 positions that lead to its ubiquitination at lysine 80 and 93 positions resulting in degradation. Inhibition of Arid5a phosphorylation and degradation causes the over-production IL6 mRNA further supports the importance of Arid5a in the regulation of IL6. Our data demonstrate that LPS signaling controls IL6 mRNA stability; therefore, its regulation through up-regulation of Arid5a by NF-κB at the early phase, and down-regulation of Arid5a at the mRNA and protein level by MKP-1-mediated AUF1 and P38 MAPK signaling, respectively at the later phase.

146. Regulation of Inflammation in Cystic Fibrosis Lung Epithelial Cells by miR-155

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation, F508del, is associated with failure of the mutant CFTR to traffic to the plasma membrane, with concomitant loss of cAMP-activated chloride conductance, and hypersecretion of pro-inflammatory IL-8. Our data indicate that the mis-expression of microRNAs (miRNAs, miRs) in CF cells, both in culture and in ex vivo bronchial biopsies, contribute to the inflammatory disease phenotype of CF. Our studies have demonstrated that the aberrant up-regulation of miR-155, in CF cells compared to CFTR-repaired control cells, induces hyper-expression of IL-8 through reduction in AKT1 activation. Our data indicate that, in addition to regulation of IL-8 mRNA stability by targeting SHIP1 gene, miR-155 also promotes increased translation of IL-8 mRNA. Therefore, we have analyzed the role of pro-inflammatory RNA-binding proteins (RBPs) and RNAs in miR-155-mediated regulation of IL-8 expression in CF lung epithelial cells. We have employed an in vivo cross-linking and affinity purification strategy to isolate and identify proteins and RNA in the miR-155-induced IL-8-mRNP complexes that regulate IL-8 expression in CF cells. We have thus identified the antagonistic role of HuR and miR-16 in the regulation of IL-8 expression in CF. Additionally, we have also identified CF disease-specific inflammatory target genes of miR-155, viz. RPTOR, SIRT1 and NR2F2, in CF lung epithelial cells that regulate the CF disease phenotype. Understanding these mechanisms will ultimately lead to the development of novel miR-based anti-inflammatory therapeutics for CF and other pulmonary disorders.

Transmembrane TNF-TNFR2 Impairs Th17 Differentiation by Promoting Il2 Expression.

The double-edged sword nature by which IL-2 regulates autoimmunity and the unpredictable outcomes of anti-TNF therapy in autoimmunity highlight the importance for understanding how TNF regulates IL-2. Transmembrane TNF (tmTNF) preferentially binds TNFR2, whereas soluble TNF (sTNF) binds TNFR1. We previously showed reduced IL-2 production in TNFR1(-/-) TNFR2(-/-) CD4(+) T cells. In this study, we generated TNFR1(-/-), TNFR2(-/-), or TNFR1(-/-) TNFR2(-/-) 5C.C7 TCR Il2-GFP mice and report that CD4(+) T cell-intrinsic tmTNF/TNFR2 stimulates Il2 promoter activity and Il2 mRNA stability. We further used tmTNF Foxp3 gfp reporter mice and pharmacological TNF blockade in wild-type mice to report a tmTNF/TNFR2 interaction for Il2 expression. IL-17 is critical for host defense, but its overabundance promotes autoimmunity. IL-2 represses Th17 differentiation, but the role for TNFR2 in this process is not well understood. We report elevated expression of TNFR2 under Th17-polarization conditions. Genetic loss-of-function experimental models, as well as selective TNF blockade by etanercept and XPro1595 in wild-type mice, demonstrate that impaired tmTNF/TNFR2, but not sTNF/TNFR1, promotes Th17 differentiation in vivo and in vitro. Under Th17-polarizing conditions, elevated IL-17 production by TNFR2-knockout CD4(+) T cells was associated with increased STAT3 activity and decreased STAT5 activity. Increased IL-17 production in TNFR2-knockout T cells was prevented by adding exogenous IL-2. We conclude that CD4(+) T cell-intrinsic tmTNF/TNFR2 promotes IL-2 production that inhibits the generation of Th17 cells in a Foxp3-independent manner. Moreover, under Th17-polarizing conditions, selective blockade of CD4(+) T cell-intrinsic TNFR2 appears to be sufficient to promote Th17 differentiation.

Olive oil phenolic extract regulates interleukin-8 expression by transcriptional and posttranscriptional mechanisms in Caco-2 cells.

In this study, we investigated the ability of a phenolic extract from extra virgin olive oil (OPE) to modulate the inflammatory response in intestinal epithelial cells. Undifferentiated and differentiated Caco-2 cells were challenged with LPS (50 μg/mL) or IL-1β (5 ng/mL) to mimic the early and intermediate phase of intestinal inflammation, respectively. The effects of OPE on nuclear factor-κB-driven transcription and IL-8 promoter activity were evaluated in transfection assays, coupled to p65 nuclear translocation. Modulation of IL-8 mRNA levels by OPE was measured by quantitative RT-PCR while effects on protein levels by ELISA. Specific mitogen activated protein kinases inhibitors were used to investigate mRNA stability and the involvement of related signaling pathways. OPE prevented IL-8 expression and secretion in LPS-treated Caco-2 cells. In the presence of IL-1β OPE exhibited opposing effects on IL-8 gene transcription and mRNA/protein levels. While in IL-1β-treated cells IL-8 promoter activity was inhibited by treatment with OPE, IL-8 mRNA stability was strongly enhanced, leading to increased protein expression. Inhibitors of p38 and extracellular signal-regulated kinases partly prevented OPE effect on IL-8 mRNA levels. Intestinal epithelial cells represent a direct target of the action of olive oil phenols where they regulate IL-8 expression by transcriptional and posttranscriptional mechanisms.

Nickel ions selectively inhibit lipopolysaccharide-induced interleukin-6 production by decreasing its mRNA stability.

Nickel (Ni) ions easily elute from many alloys and elicit inflammation and allergies. Previous studies have shown that infections due to the implantation of medical devices cause inflammation and enhance the elution of Ni ions (Ni2+). However, cross-talk between infection- and Ni2+-induced signaling pathways has not yet been elucidated in detail. In the present study, we investigated the effects of Ni2+ on the lipopolysaccharide (LPS)-induced production of cytokines in a LPS-induced air pouch-type inflammation model in BALB/c mice and the murine macrophage cell line RAW264. We demonstrated that Ni2+ inhibited the LPS-induced production of interleukin (IL)-6, but not that of tumor necrosis factor (TNF)-α both in vivo and in vitro. This inhibitory effect was also observed with cobalt ion (Co2+), but not with chloride ion (Cl-), zinc ion (Zn2+), or palladium ion (Pd2+), and was highly selective to the production of IL-6. Ni2+ did not inhibit the activation of ERK1/2, p38 MAPK, or JNK. Although Ni2+ decreased IL-6 mRNA levels, it failed to inhibit the LPS-induced activation of the IL-6 promoter. An experiment using actinomycin D, a transcription inhibitor, revealed that Ni2+ decreased the stability of IL-6 mRNA. Moreover, Ni2+ inhibited the LPS-induced expression of Arid5a, but not regnase-1. These results demonstrated that Ni2+ may have selectively inhibited the LPS-induced production of IL-6 by decreasing the Arid5a-dependent stabilization of IL-6 mRNA.

SIGNR1-mediated phagocytosis, but not SIGNR1-mediated endocytosis or cell adhesion, suppresses LPS-induced secretion of IL-6 from murine macrophages

C-type lectin receptors (CLRs) serve as phagocytosis receptors for pathogens and also function as adhesion molecules and in the recognition and endocytosis of glycosylated self-antigens. In the present study, we demonstrated that phagocytosis mediated by a mouse mannose-binding CLR, SIGNR1 significantly suppressed the LPS-induced secretion of the specific pro-inflammatory cytokines from the resident peritoneal macrophages and the mouse macrophage-like cells that express SIGNR1 (RAW-SIGNR1). LPS-induced secretion of IL-6 from peritoneal macrophages suppressed in response to uptake of oligomannose-coated liposomes (OMLs), and the suppression was partly inhibited by treatment with an anti-SIGNR1 antibody. LPS-induced secretion of IL-6 from RAW-SIGNR1 cells was also clearly inhibited by treatment of the cells with OMLs >0.4μm in diameter, but treatment with OMLs <0.4μm in diameter did not affect the IL-6 secretion. In contrast, LPS-induced TNF-α secretion from the cells was not affected on treatment of the cells with OMLs. Suppression of the IL-6 secretion was not observed following treatment with oligomannose-containing soluble polymers or when cells were bound to an oligomannose-coated solid phase. Phagocytosis of oligomannose-coated liposomes did not interfere with the transcription of IL-6 mRNA, but did affect IL-6 mRNA stability, leading to suppression of IL-6 secretion. Interestingly, treatment of the cells with Ly290042, a PI3 kinase inhibitor, partly blocked the suppression of LPS-induced secretion of IL-6 by OML. Thus, we conclude that SIGNR1-mediated phagocytosis but not SIGNR1-mediated endocytosis and cell adhesion, suppresses the TLR4-mediated production of specific proinflammatory cytokines via PI3 kinase signaling.

Regulation of the innate immune response by fibronectin: synergism between the III-1 and EDA domains.

Fibronectin is a critical component of the extracellular matrix and alterations to its structure will influence cellular behavior. Matrix fibronectin is subjected to both mechanical and biochemical regulation. The Type III domains of fibronectin can be unfolded in response to increased cellular contractility, included or excluded from the molecule by alternative splicing mechanisms, or released from the matrix by proteolysis. Using Inflammatory Cytokine microarrays we found that the alternatively spliced fibronectin Type III domain, FnEDA, and the partially unfolded III-1 domain, FnIII-1c, induced the expression of a multitude of pro-inflammatory cytokines in human dermal fibroblasts, most notably CXCL1-3, IL-8 and TNF-α. FnIII-1c, a peptide representing an unfolded intermediate structure of the first Type III domain has been shown to initiate the toll-like receptor-4 (TLR4)-NFκB-dependent release of cytokines from human dermal fibroblasts (You, et al., J. Biol. Chem., 2010). Here we demonstrate that FnIII-1c and the alternatively spliced FnEDA domain induce a TLR4 dependent activation of p38 MAP kinase and its downstream effector, MAPKAP Kinase-2 (MK-2), to regulate cytokine expression in fibroblasts. RT-qPCR analysis indicated that the p38-MK-2 pathway regulates IL-8 mRNA stability. Interestingly, addition of FnIII-1c and FnEDA synergistically enhanced TLR4-dependent IL-8 release. These data indicate that Fn contains two Type III domains which can activate TLR signaling to induce an inflammatory response in fibroblasts. Furthermore, our data identifies the NF-κB and p38/MK2 signaling pathways as transducers of signals initiated in response to structural changes in fibronectin.

The RNA-binding protein HuR is required for normal homeostatic control of IL-2 expression in activated CD4 T cells (IRM6P.713)

Abstract Upon T cell activation approximately 50% of genes are regulated at the posttranscriptional level by RNA-binding proteins (RBPs). HuR is a stabilizing RBP which regulates mRNA via interactions with AU-rich elements (ARE) in mRNA 3’UTRs. HuR has been shown to be crucial for T cell development, thymic selection and egression. However, it is unclear how HuR regulates T cell differentiation and cytokine expression. To further investigate these questions, we generated a novel conditional HuR KO mouse, distal-Lck Cre ROSA HuRfl/fl which ablates HuR in mature T cells prior to activation. HuR KO T cells develop and egress from thymus normally; however, they display aberrant cytokine and receptor expression upon activation. Activated HuR KO T cells show striking increases in IL-2 (20-30 fold) and display an inability to turn off IL-2 expression, but significant decreases in IL-4, IL-5 and IL-13 with no change in IFN-γ. Over 90% of HuR KO CD4 cells still make IL-2 five days after activation but display impaired IL-4, IL-5 and IL-13 secretion. Increases in IL-2 in HuR KO cells are due to augmentation of IL-2 transcription and mRNA stability. Moreover, there are significant decreases in proliferation and CD25 (IL-2Rα) expression in HuR KO cells as a result of reductions in CD25 transcription. Thus, HuR is required for cytokine homeostasis in CD4 cells and may well function as a critical off-switch for IL-2 expression during T cell activation.

Sustained stabilization of Interleukin-8 mRNA in human macrophages

The mRNAs of most inflammatory mediators are short-lived due to AU-rich elements (AREs) in their 3′-untranslated regions. AREs ensure a low basal level of expression during homeostasis and a transient nature of expression during the inflammatory response. Here, we report that the mRNA of the pro-inflammatory chemokine IL-8, which contains an archetypal ARE, is unexpectedly constitutively abundant and highly stable in primary human monocytes and macrophages. Using the pre-monocyte-like THP-1 cell line that can differentiate into macrophage-like cells, we show that a low level of unstable IL-8 mRNA in undifferentiated cells (half-life < 30 min) becomes constitutively elevated and the mRNA is dramatically stabilized in differentiated THP-1 cells with a half-life of more than 15 h similar to primary monocytes and macrophages. In contrast, the level and stability of TNF-α mRNA also containing an ARE is only slightly affected by differentiation; it remains low and unstable in primary macrophages and differentiated THP-1 cells with an estimated half-life of less than 20 min. This differentiation-dependent stabilization of IL-8 mRNA is p38 MAPK-independent and is probably coupled with reduced protein translation. Reporter assays in THP-1 cells suggest that the ARE alone is not sufficient for the constitutive stabilization in macrophage-like cells and imply an effect of the natural biogenesis of the transcript on the stabilization of the mature form. We present a novel, cell type-dependent sustained stabilization of an ARE-containing mRNA with similarities to situations found in disease.