Comparison of the Regulation of β-Catenin Signaling by Type I,Type II and Type III Interferons in Hepatocellular Carcinoma Cells

Background/Objective

IFNs are a group of cytokines that possess potent antiviral and antitumor activities, while β-catenin pathway is a proliferative pathway involved in carcinogenesis. Interaction between these two pathways has not been well elaborated in hepatocellular carcinoma (HCC).

Methods

HCC cell lines, HepG2 and Huh7, were used in this study. β-catenin protein levels and corresponding signaling activities were observed by flow cytometry and luciferase assay, respectively. Cell proliferation was quantified by counting viable cells under microscope, and apoptosis by TUNEL assay. DKK1 and GSK3β levels were determined by flow cytometry. Secreted DKK1 was tested by ELISA. FLUD, S3I and aDKK1 were used to inhibit STAT1, STAT3 and DKK1 activities, respectively.

Results

Our findings show that all three types of IFNs, IFNα, IFNγ and IFNλ, are capable of inhibiting β-catenin signaling activity in HepG2 and Huh7 cells, where IFNγ was the strongest (p<0.05). They expressed suppression of cellular proliferation and induced apoptosis. IFNγ expressed greater induction ability when compared to IFNα and IFNλ (p<0.05). All tested IFNs could induce DKK1 activation but not GSK3β in HepG2 and Huh7 cells. IFNs induced STAT1 and STAT3 activation but by using specific inhibitors, we found that only STAT3 is vital for IFN-induced DKK1 activation and apoptosis. In addition, DKK1 inhibitor blocked IFN-induced apoptosis. The pattern of STAT3 activation by different IFNs is found consistent with the levels of apoptosis with the corresponding IFNs (p<0.05).

Conclusions

In hepatocellular carcinoma, all three types of IFNs are found to induce apoptosis by inhibiting β-catenin signaling pathway via a STAT3- and DKK1-dependent pathway. This finding points to a cross-talk between different IFN types and β-catenin signaling pathways which might be carrying a biological effect not only on HCC, but also on processes where the two pathways bridge.     

Increased Peripheral Blood Pro-Inflammatory/Cytotoxic Lymphocytes in Children with Bronchiectasis

Objective

Bronchiectasis (BE) in children is common in some communities including Indigenous children in Australia. Relatively little is known about the nature of systemic inflammation in these children, especially the contribution of specific pro-inflammatory and cytotoxic lymphocyte subsets: T-cells, natural killer (NK) cells and NKT-like cells. We have shown that these cells produce increased cytotoxic (granzyme b and perforin) and inflammatory (IFNγ and TNFα) mediators in several adult chronic lung diseases and hypothesised that similar changes would be evident in children with BE.

Methods

Intracellular cytotoxic mediators perforin and granzyme b and pro-inflammatory cytokines were measured in T cell subsets, NKT-like and NK cells from blood and bronchoalveolar samples from 12 children with BE and 10 aged-matched control children using flow cytometry.

Results

There was a significant increase in the percentage of CD8+ T cells and T and NKT-like subsets expressing perforin/granzyme and IFNγ and TNFα in blood in BE compared with controls. There was a further increase in the percentage of pro-inflammatory cytotoxic T cells in Indigenous compared with non-Indigenous children. There was no change in any of these mediators in BAL.

Conclusions

Childhood bronchiectasis is associated with increased systemic pro-inflammatory/cytotoxic lymphocytes in the peripheral blood. Future studies need to examine the extent to which elevated levels of pro-inflammatory cytotoxic cells predict future co-morbidities.     

Epstein-Barr Virus Interferes with the Amplification of IFNα Secretion by Activating Suppressor of Cytokine Signaling 3 in Primary Human Monocytes

Background

Epstein-Barr virus is recognized to cause lymphoproliferative disorders and is also associated with cancer. Evidence suggests that monocytes are likely to be involved in EBV pathogenesis, especially due to a number of cellular functions altered in EBV-infected monocytes, a process that may affect efficient host defense. Because type I interferons (IFNs) are crucial mediators of host defense against viruses, we investigated the effect of EBV infection on the IFNα pathway in primary human monocytes.

Methodology/Principal Findings

Infection of monocytes with EBV induced IFNα secretion but inhibited the positive feedback loop for the amplification of IFNα. We showed that EBV infection induced the expression of suppressor of cytokine signaling 3 (SOCS3) and, to a lesser extent, SOCS1, two proteins known to interfere with the amplification of IFNα secretion mediated by the JAK/STAT signal transduction pathway. EBV infection correlated with a blockage in the activation of JAK/STAT pathway members and affected the level of phosphorylated IFN regulatory factor 7 (IRF7). Depletion of SOCS3, but not SOCS1, by small interfering RNA (siRNA) abrogated the inhibitory effect of EBV on JAK/STAT pathway activation and significantly restored IFNα secretion. Finally, transfection of monocytes with the viral protein Zta caused the upregulation of SOCS3, an event that could not be recapitulated with mutated Zta.

Conclusions/Significance

We propose that EBV protein Zta activates SOCS3 protein as an immune escape mechanism that both suppresses optimal IFNα secretion by human monocytes and favors a state of type I IFN irresponsiveness in these cells. This immunomodulatory effect is important to better understand the aspects of the immune response to EBV.     

Examination of a Viral Infection Mimetic Model in Human iPS Cell-Derived Insulin-Producing Cells and the Anti-Apoptotic Effect of GLP-1 Analogue

Aims

Viral infection is associated with pancreatic beta cell destruction in fulminant type 1 diabetes mellitus. The aim of this study was to investigate the acceleration and protective mechanisms of beta cell destruction by establishing a model of viral infection in pancreatic beta cells.

Methods

Polyinosinic:polycytidylic acid was transfected into MIN6 cells and insulin-producing cells differentiated from human induced pluripotent stem cells via small molecule applications. Gene expression was analyzed by real-time PCR, and apoptosis was evaluated by caspase-3 activity and TUNEL staining. The anti-apoptotic effect of Exendin-4 was also evaluated.

Results

Polyinosinic:polycytidylic acid transfection led to elevated expression of the genes encoding IFNα, IFNβ, CXCL10, Fas, viral receptors, and IFN-inducible antiviral effectors in MIN6 cells. Exendin-4 treatment suppressed the elevated gene expression levels and reduced polyinosinic:polycytidylic acid-induced apoptosis both in MIN6 cells and in insulin-producing cells from human induced pluripotent stem cells. Glucagon-like peptide-1 receptor, protein kinase A, and phosphatidylinositol-3 kinase inhibitors counteracted the anti-apoptotic effect of Exendin-4.

Conclusions

Polyinosinic:polycytidylic acid transfection can mimic viral infection, and Exendin-4 exerted an anti-apoptotic effect both in MIN6 and insulin-producing cells from human induced pluripotent stem cells.     

Differential Expression and Release of Activin A and Follistatin in Chronic Rhinosinusitis with and without Nasal Polyps

Background

Chronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP) should be regarded as distinct clinical entities based on differential inflammatory mediator and remodeling profiles. Activin A, a member of the TGF-β superfamily, plays an important role in inflammation and remodeling in the lower airways, although its expression and release in the upper airways remain undescribed.

Objective

To investigate the expression of activin A and its inhibitor follistatin in nasal tissue samples from CRSsNP and CRSwNP patients, and to monitor the spontaneous release of these molecules in a human mucosal model.

Methods

Protein levels were determined using ELISA for activin A, follistatin, TGF-β1 and indicator proteins (IL-5, ECP, IFNγ) in 13 CRSsNP, 23 CRSwNP, and 10 control samples. The spontaneous release rate and the release ratios of activin A, follistatin and TGF-β1 were determined in 9 CRSsNP and 7 CRSwNP tissue fragments cultured ex-vivo. The induction of activin A and TGF-β1 by one another was studied in 7 CRSsNP tissue fragments cultured ex-vivo.

Results

Significantly higher concentrations of activin A, follistatin, TGF-β1, and IFNγ were observed in CRSsNP compared with CRSwNP samples, whereas the concentrations of IL-5 and ECP were significantly lower. Follistatin was positively and linearly correlated with activin A in CRSsNP and CRSwNP. Activin A, follistatin and TGF-β1 were all spontaneously released by the samples, although the relative ratios released by tissue fragments from CRSsNP and CRSwNP samples were significantly different, with a higher follistatin/activin A-ratio and a follistatin/TGFß1-ratio (with less overall TGF-β1) in CRSwNP than in CRSsNP. Furthermore, TGF-β1 enhanced activin A secretion in CRSsNP tissue fragments cultured ex-vivo.

Conclusion

The differences in tissue concentrations and spontaneous release rates for activin A and follistatin in different CRS samples support the hypothesis that CRSsNP and CRSwNP are two distinct disease entities with respect to remodeling patterns.     

Skewed Differentiation of Circulating Vγ9Vδ2 T Lymphocytes in Melanoma and Impact on Clinical Outcome

Objective

The aim of this study was to evaluate over time circulating γδ T lymphocytes in melanoma patients in terms of frequency, effector functions, and relationship with clinical stage and evolution, by comparing preoperative values to those obtained at a mean follow-up of 36 months or in the event of recurrence or disease progression, and to those of healthy controls. Also, we correlated the presence of tumor-infiltrating γδ T lymphocytes with clinical evolution of melanoma.

Results

Mean frequencies of circulating γδ T cells before and after melanoma removal were very similar and comparable to healthy subjects, but patients who progressed to stage III or IV showed a significantly decreased frequency of circulating Vγ9Vδ2 T cells. The distribution of Vγ9Vδ2 memory and effector subsets was similar in healthy subjects and melanoma patients at diagnosis, but circulating γδ T cells of patients after melanoma removal had a skewed terminally-differentiated effector memory phenotype. Highly suggestive of progressive differentiation toward a cytotoxic phenotype, Vγ9Vδ2T cells from patients at follow up had increased cytotoxic potential and limited cytokine production capability, while the opposite pattern was detected in Vγ9Vδ2T cells from patients before melanoma removal.

Conclusions

Follow-up data also showed that tumor infiltrating γδ T cells were significantly associated with lower mortality and relapse rates, suggesting that they may serve as a prognostic biomarker, for human melanoma.     

Preserved Antigen-Specific Immune Response in Patients with Multiple Sclerosis Responding to IFNβ-Therapy

Background

Interferon-beta (IFNβ) regulates the expression of a complex set of pro- as well as anti-inflammatory genes. In cohorts of MS patients unstratified for therapeutic response to IFNβ, normal vaccine-specific immune responses have been observed. Data capturing antigen-specific immune responses in cohorts of subjects defined by response to IFNβ-therapy are not available.

Objective

To assess antigen-specific immune responses in a cohort of MS patients responding clinically and radiologically to IFNβ.

Methods

In 26 MS patients, clinical and MRI disease activity were assessed before and under treatment with IFNβ. Humoral and cellular immune response to influenza vaccine was prospectively characterized in these individuals, and 33 healthy controls by influenza-specific Enzyme-Linked Immunosorbent Assay (ELISA) and Enzyme Linked Immuno Spot Technique (ELISPOT).

Results

Related to pre-treatment disease activity, IFNβ reduced clinical and radiological MS disease-activity. Following influenza vaccination, frequencies of influenza-specific T cells and concentrations of anti-influenza A and B IgM and IgG increased comparably in MS-patients and in healthy controls.

Conclusions

By showing in a cohort of MS-patients responding to IFNβ vaccine-specific immune responses comparable to controls, this study indicates that antigen-specific immune responses can be preserved under successful IFNβ-therapy.     

Effect of Different Interferonα2 Preparations on IP10 and ET-1 Release from Human Lung Cells

Background

Alfa-interferons (IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b) are effective treatments for chronic hepatitis C infection. However, their usage has been associated with a variety of adverse events, including interstitial pneumonitis and pulmonary arterial hypertension. Although rare, these adverse events can be severe and potentially life-threatening, emphasizing the need for simple biomarkers of IFN-induced lung toxicity.

Methods

Human lung microvascular endothelial cells (HLMVEC), human pulmonary artery smooth muscle (HPASM) cells and A549 cells were grown under standard conditions and plated into 96- or 6-well plates. Cells were stimulated with various concentrations of different IFNs in hydrocortisone-free medium. After 24 and 48 hours, IP10 and ET-1 were measured by ELISA in conditioned medium. In a second set of experiments, cells were pre-treated with tumour necrosis factor-α (TNF-α) (10 ng/mL).

Results

IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b, but not IFNλ, induced IP10 (CXCL10) release and increased IP10 gene induction in HLMVEC. In addition, all four IFNα preparations induced IP10 release from HPASM cells and A549 cells pre-treated with TNFα. In each of these cell types, 40KDa-PEGIFNα2a was significantly less active than the native forms of IFNα2a, IFNα2b or 12KDa-PEGIFNα2b. Similarly, IFNα2a, IFNα2b and 12KDa-PEGIFNα2b, but not 40KDa-PEGIFNα2a, induced endothelin (ET)-1 release from HPASM cells.

Conclusions

Consistent with other interstitial pulmonary diseases, both IP10 and ET1 may serve as markers to monitor IFN-induced lung toxicity in patients. In addition, both markers may also serve to help characterize the risk associated with IFNα preparations to induce lung toxicity.     

The drug efflux pump Pgp1 in pro-inflammatory lymphocytes is a target for novel treatment strategies in COPD

Background

Pro-inflammatory/cytotoxic T cells (IFNγ, TNFα, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNγ, TNFα and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids.

Methods

Pgp1, granzyme B, IFNγ and TNFα expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (± cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry.

Results

There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNγ, TNFα and granzyme B in COPD patients compared with controls (e.g. %IFNγ/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10^-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all).

Conclusions

Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.     

Coincident Pre-Diabetes Is Associated with Dysregulated Cytokine Responses in Pulmonary Tuberculosis

Background

Cytokines play an important role in the pathogenesis of pulmonary tuberculosis (PTB) - Type 2 diabetes mellitus co-morbidity. However, the cytokine interactions that characterize PTB coincident with pre-diabetes (PDM) are not known.

Methods

To identify the influence of coincident PDM on cytokine levels in PTB, we examined circulating levels of a panel of cytokines in the plasma of individuals with TB-PDM and compared them with those without PDM (TB-NDM).

Results

TB-PDM is characterized by elevated circulating levels of Type 1 (IFNγ, TNFα and IL-2), Type 17 (IL-17A and IL-17F) and other pro-inflammatory (IL-1β, IFNβ and GM-CSF) cytokines. TB-PDM is also characterized by increased systemic levels of Type 2 (IL-5) and regulatory (IL-10 and TGFβ) cytokines. Moreover, TB antigen stimulated whole blood also showed increased levels of pro-inflammatory (IFNγ, TNFα and IL-1β) cytokines as well. However, the cytokines did not exhibit any significant correlation with HbA1C levels or with bacterial burdens.

Conclusion

Our data reveal that pre-diabetes in PTB individuals is characterized by heightened cytokine responsiveness, indicating that a balanced pro and anti - inflammatory cytokine milieu is a feature of pre-diabetes - TB co-morbidity.     

Mefunidone Attenuates Tubulointerstitial Fibrosis in a Rat Model of Unilateral Ureteral Obstruction

Background

Inflammation has a crucial role in renal interstitial fibrosis, which is the common pathway of chronic kidney diseases. Mefunidone (MFD) is a new compound which could effectively inhibit the proliferation of renal fibroblasts in vitro. However, the overall effect of Mefunidone in renal fibrosis remains unknown.

Methods

Sprague-Dawley rats were randomly divided intro 6 groups: sham operation, unilateral ureteral obstruction (UUO), UUO/Mefunidone (25, 50, 100mg/kg/day) and UUO/PFD (500mg/kg/day). The rats were sacrificed respectively on days 3, 7, and 14 after the operation. Tubulointerstitial injury index, interstitial collagen deposition, expression of fibronectin (FN), α-smooth muscle actin (α-SMA), type I and III collagen and the number of CD3+ and CD68+ cells were determined. The expressions of proinflammatory cytokines, p-ERK, p-IκB, and p-STAT3 were measured in human renal proximal tubular epithelial cells of HK-2 or macrophages.

Results

Mefunidone treatment significantly attenuated tubulointerstitial injury, interstitial collagen deposition, expression of FN, α-SMA, type I and III collagen in the obstructive kidneys, which correlated with significantly reduced the number of T cells and macrophages in the obstructive kidneys. Mechanistically, Mefunidone significantly inhibited tumor necrosis factor-α (TNF-α-) or lipopolysaccharide (LPS)-induced production of proinflammatory cytokines. This effect is possibly due to the inhibition of phosphorylation of ERK, IκB, and STAT3.

Conclusion

Mefunidone treatment attenuated tubulointerstitial fibrosis in a rat model of UUO, at least in part, through inhibition of inflammation.     

Cancer Associated Fibroblasts in Stage I-IIIA NSCLC: Prognostic Impact and Their Correlations with Tumor Molecular Markers

Background

Cancer Associated Fibroblasts (CAFs) are thought to regulate tumor growth and metastasis. Fibroblast Activating Protein 1 (FAP-1) is a marker for fibroblast activation and by many recognized as the main marker of CAFs. Alpha Smooth Muscle Actin (α-SMA) is a general myofibroblast marker, and can be used to identify CAFs. This study investigates the prognostic impact of FAP-1 and α-SMA in non-small cell lung cancer (NSCLC) patients and correlates their expression to 105 proteins investigated in the same cohort.

Methods

Tumor specimens from 536 NSCLC patients were obtained and tissue micro-arrays were constructed. Immunohistochemistry was used to evaluate the expression of FAP-1 and α-SMA and explore their impact on survival and association with other tumor molecular markers in NSCLC patients.

Results

High expression of FAP-1, but not α-SMA, in squamous cell carcinoma (SCC, P = 0.043, HR = 0.63 95% CI 0.40–0.99) was significantly associated with increased disease-specific survival. FAP-1 and α-SMA were not significantly correlated to each other. Analyses of FAP-1 and α-SMA associated with other tumor-related proteins revealed histotype-specific correlation patterns.

Conclusion

The presence of FAP-1 expressing CAFs is an indicator of positive outcome for NSCLC-SCC patients. In addition, correlation analyses suggest FAP-1 and α-SMA to label different subsets of fibroblasts and their associations with other tumor-related proteins diverge according to histological subtype.     

RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

Objective

To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis.

Methods

RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression.

Results

The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01).

Conclusions

RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts.     

Skewed Production of IL-6 and TGFβ by Cultured Salivary Gland Epithelial Cells from Patients with Sj?gren's Syndrome

Objective

To determine the cytokine production profile of cultured salivary gland epithelial (SGE) cells obtained from patients with Sjögren''s syndrome (SS).

Methods

SGE cells obtained from 9 SS patients and 6 normal controls were cultured in the presence of exogenous IFNγ. Cell proliferation and apoptosis in response to IFNγ were determined by WST1 assay and by FACS analysis. The concentrations of IL-6 and TGFβ secreted into culture supernatants were analyzed by ELISA.

Results

IFNγ did not significantly affect the proliferation or apoptosis of SGE cells. However, IL-6 concentrations were higher, and TGFβ concentrations were lower, in culture supernatants of SGE cells from SS patients than from normal controls.

Conclusion

Cytokine production by SGE cells from SS patients showed a skewed balance compared with normal controls, with increased IL-6 and decreased TGFβ secretion. This imbalance may be critical in the regulation of Treg/Th17 cells and may foster a pathogenic milieu that may be causative and predictive in SS.     

Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells

Purpose

To study the role of long non-coding RNA (lncRNA) MALAT1 in transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells.

Methods

ARPE-19 cells were cultured and exposed to TGF-β1. The EMT of APRE-19 cells is confirmed by morphological change, as well as the increased expression of alpha-smooth muscle actin (αSMA) and fibronectin, and the down-regulation of E-cadherin and Zona occludin-1(ZO-1) at both mRNA and protein levels. The expression of lncRNA MALAT1 in RPE cells were detected by quantitative real-time PCR. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA). The effect of inhibition of MALAT1 on EMT, migration, proliferation, and TGFβ signalings were observed. MALAT1 expression was also detected in primary RPE cells incubated with proliferative vitreoretinopathy (PVR) vitreous samples.

Results

The expression of MALAT1 is significantly increased in RPE cells incubated with TGFβ1. MALAT1 silencing attenuates TGFβ1-induced EMT, migration, and proliferation of RPE cells, at least partially through activating Smad2/3 signaling. MALAT1 is also significantly increased in primary RPE cells incubated with PVR vitreous samples.

Conclusion

LncRNA MALAT1 is involved in TGFβ1-induced EMT of human RPE cells and provides new understandings for the pathogenesis of PVR.