Elevated microRNA-21 Is a Brake of Inflammation Involved in the Development of Nasal Polyps

BackgroundCRSwNP is an inflammatory disease but the mechanism is not yet fully understood. MiR-21, a member of miRNAs, has been reported to play roles in mediating inflammation. However, the expression of miR-21 and its role in patients with CRSwNP remain elusive.MethodsTurbinates from control subjects, uncinate processes from CRSsNP, polyp tissues from CRSwNP, and nasal epithelial cells brushed from nasal mucosa were collected. The expression of miR-21 and cytokines in nasal tissues and epithelial cells were detected by qPCR. The localization of miR-21 was detected by ISH, and its target was identified by bioinformation analysis, qPCR, IHC, WB, and luciferase reporter system. The protein and mRNA of PDCD4 and NF-κB P65 were determined by WB and qPCR after miR-21 transfection in HNEpC. The role of miR-21 on cytokines was analyzed in HNEpC and nasal polyp explants.ResultsMiR-21 was upregulated in CRSwNP relative to control subjects by qPCR, which was determined mainly in nasal epithelial cells of CRSwNP by ISH. Both pro-inflammation cytokines (IL-1β, IL-6, IL-8, IL-25, and TSLP) and a suppressive cytokine (IL-10) were overexpressed in the epithelial cells of CRSwNP. The expression of miR-21 was positively correlated with IL-10 and negatively correlated with IL-6, IL-8, IL-33, and TSLP in the epithelial cells of CRSwNP. As a potential target of miR-21, the expression of PDCD4 was negatively correlated with miR-21 in CRSwNP. In HNEpC, miR-21 could reduce the expression of PDCD4 at both mRNA and protein levels, and bioinformation analysis and luciferase reporter system confirmed PDCD4 as one target of miR-21. Furthermore, miR-21 could decrease the activation of NF-κB and increase IL-10 mRNA. Both SEB and LPS could elevate miR-21, with IL-25, IL-33, TSLP induced by SEB and IL-1β, IL-6, IL-8 induced by LPS, while the miR-21 could regulate the expression of IL-33, TSLP, IL-1β, IL- 6 and IL-8 in vitro and ex vivo. Clinically, miR-21 expression was inversely correlated with the Lund-Mackay CT scores and the Lund-Kennedy scores in CRSwNP.ConclusionMiR-21 could be a prominent negative feedback factor in the inflammation process to attenuate the expression of pro-inflammatory cytokines, thereby playing an anti-inflammation role in CRSwNP.

p53-R273H Sustains ROS, Pro-Inflammatory Cytokine Release and mTOR Activation While Reducing Autophagy, Mitophagy and UCP2 Expression, Effects Prevented by wtp53

p53 is the most frequently mutated or inactivated gene in cancer, as its activity is not reconcilable with tumor onset and progression. Moreover, mutations in the p53 gene give rise to mutant proteins such as p53-R273H that, besides losing the wild type p53 (wtp53) capacity to safeguard genome integrity, may promote carcinogenesis, mainly due to its crosstalk with pro-oncogenic pathways. Interestingly, the activation of oncogenic pathways is interconnected with reactive oxygen species (ROS) and the release of pro-inflammatory cytokines that contribute to create an inflammatory/pro-tumorigenic milieu. In this study, based on experiments involving p53-R273H silencing and transfection, we showed that this mutant p53 (mutp53) promoted cancer cell survival by increasing intracellular ROS level and pro-inflammatory/immune suppressive cytokine release, activating mTOR, reducing autophagy and mitophagy and downregulating uncoupling protein 2 (UCP2). Interestingly, p53-R273H transfection into cancer cells carrying wtp53 induced none of these effects and resulted in p21 upregulation. This suggests that wtp53 may counteract several pro-tumorigenic activities of p53-R273H and this could explain the lower aggressiveness of cancers carrying heterozygous mutp53 in comparison to those harboring homozygous mutp53.

Eosinophils, a Jack of All Trades in Immunity: Therapeutic Approaches for Correcting Their Functional Disorders

Background: Eosinophils are primitive myeloid cells derived from bonemarrow precursors and require the intervention of interleukin (IL)-5 for their survival and persistence in blood and tissues. Under steady-state conditions, they contribute to immune regulation and homeostasis. Under pathological circumstances, eosinophils are involved in host protection against parasites and participate in allergy and inflammation. Discussion: Mostly, in asthma, eosinophils provoke airway damage via the release of granule contents and IL-13 with mucus hypersecretion and differentiation of goblet cells. Then, tissue remodeling follows with the secretion of transforming growth factor-β. Eosinophils are able to kill helminth larvae acting as antigen-presenting cells with the involvement of T helper (h)-2 cells and subsequent antibody response. However, they also exert pro-worm activity with the production of suppressive cytokine (IL- 10 and IL-4) and inhibition of nitric oxide. Eosinophils may play a pathogenic role in the course of chronic and autoimmune disease, e.g., inflammatory bowel disease and eosinophilic gastroenteritis, regulating Th2 responses and promoting a profibrotic effect. In atopic dermatitis, eosinophils are commonly detected and may be associated with disease severity. In cutaneous spontaneous urticaria, eosinophils participate in the formation of wheals, tissue remodeling and modifications of vascular permeability. With regard to tumor growth, it seems that IgE can exert anti-neoplastic surveillance via mast cell and eosinophil-mediated cytotoxicity, the so-called allergo-oncology. From a therapeutic point of view, monoclonal antibodies directed against IL-5 or the IL-5 receptors have been shown to be very effective in patients with severe asthma. Finally, as an alternative treatment, polyphenols for their anti-inflammatory and anti-allergic activities seem to be effective in reducing serum IgE and eosinophil count in bronchoalveolar lavage in murine asthma. Conclusion: Eosinophils are cells endowed with multiple functions and their modulation with monoclonal antibodies and nutraceuticals may be effective in the treatment of chronic disease.

High Smad7 in the early post-operative recurrence of Crohn’s disease

Background: In Crohn’s disease (CD), one of the major inflammatory bowel disease (IBD) in human beings, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine TGF-β1. The aim of this study was to assess whether Smad7 over-expression occurs in the early and/or late phases of CD. Methods: Mucosal samples were taken from the neo-terminal ileum of CD patients undergoing ileocolonic resection, with or without (early CD) post-operative endoscopic recurrence, and terminal ileum of CD patients with long-standing disease undergoing intestinal resection (late CD). Smad7 was examined by immunohistochemistry and cytokine expression was analysed by flow-cytometry. Results: Before the appearance of endoscopic lesions, the mucosa of the neo-terminal ileum contained high number of Smad7-expressing cells in both the epithelial and lamina propria compartments. Transition from this stage to endoscopic recurrence was marked by persistence of high number of Smad7-positive cells, which reduced significantly in the late stages of the disease, where Smad7 expression remained, however, greater than that seen in normal controls. In samples with early lesions, Smad7 expression positively correlated with the number of interferon-g-secreting cells. Conclusions: Smad7 induction is an early event in the inflammatory sequence occurring in CD, thus suggesting that knockdown of Smad7 can help prevent post-operative recurrence.

High Smad7 in the early post-operative recurrence of Crohn’s disease

Background: In Crohn’s disease (CD), one of the major inflammatory bowel disease (IBD) in human beings, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine TGF-β1. The aim of this study was to assess whether Smad7 over-expression occurs in the early and/or late phases of CD. Methods: Mucosal samples were taken from the neo-terminal ileum of CD patients undergoing ileocolonic resection, with or without (early CD) post-operative endoscopic recurrence, and terminal ileum of CD patients with long-standing disease undergoing intestinal resection (late CD). Smad7 was examined by immunohistochemistry and cytokine expression was analysed by flow-cytometry. Results: Before the appearance of endoscopic lesions, the mucosa of the neo-terminal ileum contained high number of Smad7-expressing cells in both the epithelial and lamina propria compartments. Transition from this stage to endoscopic recurrence was marked by persistence of high number of Smad7-positive cells, which reduced significantly in the late stages of the disease, where Smad7 expression remained, however, greater than that seen in normal controls. In samples with early lesions, Smad7 expression positively correlated with the number of interferon-g-secreting cells. Conclusions: Smad7 induction is an early event in the inflammatory sequence occurring in CD, thus suggesting that knockdown of Smad7 can help prevent post-operative recurrence.

Phase I study of oncolytic virus (OV) MG1 maraba/MAGE-A3 (MG1MA3), with and without transgenic MAGE-A3 adenovirus vaccine (AdMA3) in incurable advanced/metastatic MAGE-A3-expressing solid tumours: CCTG IND.214.

e14637 Background: OVs display oncolytic activity and boost adaptive cell immunity. MG1MA3 is a Maraba virus modified to express tumour antigen MAGE-A3. MG1MA3, both alone and after immune priming with a MAGE-A3 modified adenovirus (AdMA3) may trigger anti-tumour T-cell responses. Methods: N = 41 patients (pts) with MAGE-A3 expressing solid tumours were evaluated in 3 groups (A) Dose escalation of MG1MA3 iv d1+4, q8w (n = 9); (B) Single fixed dose AdMA3 1e10 pfu IM d1 (n = 6); (C) AdMA3 priming d(-14) followed by dose escalated MG1MA3 (d1+4, q8w) (n = 25). Arm A and C had a 3+3 design. Pre + post treatment blood and tissue biopsies were evaluated for viral and immune markers. Endpoints included MTD/MFD, RP2DL, safety, tolerability, pharmacokinetics, viral delivery and replication. Results: Dose limiting toxicities (hypoxia/dyspnea, vomiting, headache) occurred in 4 pts (2 each Arm A +C). RP2DL for arm C was AdMA3 1e10 pfu IM d(-14) then MG1MA3 1e11 pfu iv d1+4. Common treatment related toxicities on Arm C occurring hours to a few days after MG1MA3 included diarrhea, nausea, vomiting, anorexia, chills, fatigue, fever, flu-like symptoms, hypophosphatemia, headache, and hypotension. Preliminary tumour gene expression results reveal induction of pro-inflammatory genes, including chemokines (CCL2, CCL5, CX3CL1, CXCL10), acute phase response proteins (IL-6, TNF), antigen presenting cell (APC) activation markers (CD80, HLA-A, HLA-B), markers of APC and Natural Killer cell infiltration (CD56, CD68, TLR3), as well as a co-incident decrease in the suppressive cytokine TGF-β. MG1MA3 replication was observed in some pts, inferred by detection of circulating genomes on days 4, 8 and 15 after clearance of the input dose. Induction of anti-tumour immune responses (CD8 T cells and antibodies vs MAGE-A3) was demonstrated in 3 of 6 Arm C pts evaluated to date. In one patient, over 1% of circulating CD8 T cells were directed against MAGE-A3. Conclusions: AdMA3 prime followed by MG1MA3 OV boost is feasible with a defined RP2DL, and capable of inducing potent anti-tumour immune response. Alternate schedules will be evaluated. Clinical trial information: NCT02285816.

Schistosoma mansoni Larvae Do Not Expand or Activate Foxp3+Regulatory T Cells during Their Migratory Phase

Foxp3+regulatory T (Treg) cells play a key role in suppression of immune responses during parasitic helminth infection, both by controlling damaging immunopathology and by inhibiting protective immunity. During the patent phase ofSchistosoma mansoniinfection, Foxp3+Treg cells are activated and suppress egg-elicited Th2 responses, but little is known of their induction and role during the early prepatent larval stage of infection. We quantified Foxp3+Treg cell responses during the first 3 weeks of murineS. mansoniinfection in C57BL/6 mice, a time when larval parasites migrate from the skin and transit the lungs en route to the hepatic and mesenteric vasculature. In contrast to other helminth infections,S. mansonidid not elicit a Foxp3+Treg cell response during this early phase of infection. We found that the numbers and proportions of Foxp3+Treg cells remained unchanged in the lungs, draining lymph nodes, and spleens of infected mice. There was no increase in the activation status of Foxp3+Treg cells upon infection as assessed by their expression of CD25, Foxp3, and Helios. Furthermore, infection failed to induce Foxp3+Treg cells to produce the suppressive cytokine interleukin 10 (IL-10). Instead, only CD4+Foxp3−IL-4+Th2 cells showed increased IL-10 production upon infection. These data indicate that Foxp3+Treg cells do not play a prominent role in regulating immunity toS. mansonilarvae and that the character of the initial immune response invoked byS. mansoniparasites contrasts with the responses to other parasitic helminth infections that promote rapid Foxp3+Treg cell responses.

Tumor Inhibition by DepoVax-Based Cancer Vaccine Is Accompanied by Reduced Regulatory/Suppressor Cell Proliferation and Tumor Infiltration

A successful cancer vaccine needs to overcome the effects of immune-suppressor cells such as Treg lymphocytes, suppressive cytokine-secreting Tr1 cells, and myeloid-derived suppressor cells (MDSCs), while enhancing tumor-specific immune responses. Given the relative poor efficacy associated with current cancer vaccines, a novel vaccine platform called DepoVaxTM (DPX) was developed. C3 tumor-challenged mice were immunized with HPV-E7 peptide in DPX- or conventional-emulsion- (CE-) based vaccine. While control mice showed marked increase in Treg/MDSCs in spleen and blood, in mice treated with DPX-E7 the levels remained similar to tumor-free naive mice. Such differences were also seen within the tumor. Antigen-specific IL10-secreting CD4/CD8 T cells and TGF-β+CD8+ T cell frequencies were increased significantly in CE-treated and control mice in contrast to DPX-E7-immunized mice. Analysis of tumor-infiltrating cells revealed higher frequency of suppressor cells in untreated controls than in DPX-E7 group while the converse was true for tumor-infiltrating CD8 T cells. Immunization of tumor-bearing HLA-A2 transgenic mice with human vaccine DPX-0907, a peptide-based vaccine for breast/ovarian/prostate cancers, showed efficient induction of immune response to cancer peptides despite the presence of suppressor cells. Thus, this study provides the rationale for using DPX-based cancer vaccines in immune-suppressed cancer patients, to induce effective anticancer immunity.

Biomarkers of treatment effect in a phase II trial of velimogene aliplasmid in patients with metastatic melanoma.

e19058 Background: Velimogene aliplasmid (Allovectin, A) is a plasmid-based immunotherapeutic that encodes human leukocyte antigen-B7 and β-2 microglobulin. A is designed to induce allogeneic and tumor-antigen-specific T-cell responses against tumor cells. In a phase II trial, 127 patients with stage III or IV recurrent metastatic melanoma were treated in cycles of 6 weekly intralesional injections of 2 mg/dose. Complete (CR) or partial responses (PR) were observed in 15 patients (11.9%, 95% CI: 6.2-17.4), stable disease (SD) in 32 (25.2%), and progressive disease (PD) in 80 (63.0%). Methods: Serum collected before treatment and 3 weeks after each cycle from 29 patients who showed clinical benefit (CB: 15 CR or PR patients and 14 patients with SD for >4 months) and 62 with PD (progressed by the end of the first cycle) were evaluated for 45 inflammation biomarkers in a quantitative multiplex immunoassay. Changes in analyte concentrations relative to baseline were evaluated by paired t-test or Wilcoxon signed-rank test. Results: Overall, 52% of patients had decreases in serum IL-10 after treatment; however, decreases were more prevalent in CB patients (71.4% for CB, 44.4% for PD; p=0.036; Chi-square test). At week 8, mean serum IL-10 concentration within a group was reduced relative to baseline in CB (p=0.008) but not PD patients suggesting greater treatment effect in CB patients. In CB patients, IL-10 remained at lower levels relative to baseline through week 26. CCL-3 decreased in CB patients by week 26 (p=0.021) and von Willebrand factor (a biomarker of vascular endothelium dysfunction) was increased at week 26 (p=0.007). In PD patients, by week 8, several biomarkers associated with metastatic melanoma (TIMP-1, VCAM-1, and TNF-R2) or inflammation (haptoglobin) increased (p=0.001, 0.022, <0.001, 0.004, respectively) consistent with tumor progression, whereas others (IL-8 and stem cell factor) decreased (p=0.007, 0.013, respectively). Conclusions: Consistent with A’s proposed mechanism of action, treatment was associated with a decrease in the production of the T-cell suppressive cytokine, IL-10. Furthermore, biomarker profiles in the two groups were markedly different and analogous with treatment outcomes.