IL-8 Secretion In Cells Research Articles

Thermoresponsive nanosponges: Efficient antigen delivery carriers and adjuvants for in vivo vaccination

BackgroundVaccination is an effective disease prevention strategy involving immune cell stimulation and formation in response to foreign substances. However, the poor immunogenicity and deliverability of certain antigens necessitate novel methods to establish continuous immunity. Therefore, for efficient antigen delivering and simultaneously performing as a nanoadjuvant, herein, we prepared core (polylactic acid)-shell (poloxamer) thermoresponsive nanosponges (TNSs). MethodsBy simple nanoprecipitation, we prepared the thermoresponsive nanosponges (TNSs) with core-to-shell ratios of 1:2 and 1:20. Thereafter, antigens (ovalbumin, OVA) were loaded into TNSs by swelling behavior. ResultsThe TNSs were prepared with different sizes using differing core-to-shell ratios: TNS1:2 (160 nm) and TNS1:20 (67 nm). When loaded with the model antigen ovalbumin (OVA), TNS1:2 (289 nm) showed a seven-fold higher IL-2 secretion response cross-presentation ability than TNS1:20 (74 nm) and was used in subsequent experiments. TNS1:2 promoted dendritic cell maturation and exhibited high OVA encapsulation (optimal loading: 50 wt%), sufficient long-term stability, and controlled OVA release under physiological conditions. It was not toxic to dendritic cells even at high concentrations. Compared to free OVA, 50 wt% OVA loaded TNS1:2 exhibited better cellular uptake and a superior ability to elicit immune cell IL-2 secretion. The OVA antigen-specific activation of immune cells in mice was examined by monitoring serum IgG levels, cross-presentation to splenocyte CD8+ T-cells, and IFN-γ cytokine levels. ConclusionsThe results highlight TNSs as promising nanoadjuvants and carriers for antigen delivery in vaccine applications.

Mucosal Administration of Lactobacillus casei Surface-Displayed HA1 Induces Protective Immune Responses against Avian Influenza A Virus in Mice.

Avian influenza is a serious threat to both public health and the poultry industry worldwide. This respiratory virus can be combated by eliciting robust immune responses at the site of infection through mucosal immunization. Recombinant probiotics, specifically lactic acid bacteria, are safe and effective carriers for mucosal vaccines. In this study, we engineered recombinant fusion protein by fusing the hemagglutinin 1 (HA1) subunit of the A/Aquatic bird/Korea/W81/2005 (H5N2) with the Bacillus subtilis poly γ-glutamic acid synthetase A (pgsA) at the surface of Lactobacillus casei (pgsA-HA1/L. casei). Using subcellular fractionation and flow cytometry we confirmed the surface localization of this fusion protein. Mucosal administration of pgsA-HA1/L. casei in mice resulted in significant levels of HA1-specific serum IgG, mucosal IgA and neutralizing antibodies against the H5N2 virus. Additionally, pgsA-HA1/L. casei-induced systemic and local cell-mediated immune responses specific to HA1, as evidenced by an increased number of IFN-γ and IL-4 secreting cells in the spleens and higher levels of IL-4 in the local lymphocyte supernatants. Finally, mice inoculated with pgsA-HA1/L. casei were protected against a 10LD50 dose of the homologous mouse-adapted H5N2 virus. These results suggest that mucosal immunization with L. casei displaying HA1 on its surface could be a potential strategy for developing a mucosal vaccine against other H5 subtype viruses.

CpG 1018 Is an Effective Adjuvant for Influenza Nucleoprotein.

Current influenza vaccines mainly induce neutralizing antibodies against the highly variable surface antigen hemagglutinin and require annual manufacturing and immunization. Different from surface antigens, intracellular nucleoprotein (NP) is highly conserved and has been an attractive target to develop universal T cell vaccines against influenza. Yet, influenza NP protein mainly induces humoral immune responses and lacks the ability to induce potent cytotoxic T lymphocyte (CTL) responses, key for the success of universal T cell vaccines. This study compared CpG 1018 and AddaVax to enhance recombinant NP-induced CTL responses and protection in murine models. CpG 1018 was explored to boost intradermal NP immunization, while AddaVax was explored to boost intramuscular NP immunization due to the high risk of AddaVax adjuvant to induce significant local reactions following intradermal delivery. We found CpG 1018 was highly effective to enhance NP-induced humoral and cellular immune responses superior to AddaVax adjuvant. Furthermore, CpG 1018 potentiated Th1-biased antibody responses, while AddaVax enhanced Th1/Th2-balanced antibody responses. CpG 1018 significantly enhanced IFNγ-secreting Th1 cells, while AddaVax adjuvant significantly increased IL4-secreting Th2 cells. Influenza NP immunization in the presence of CpG 1018 induced significant protection against lethal viral challenges, while influenza NP immunization in the presence of AddaVax failed to elicit significant protection. Our data validated CpG 1018 as an effective adjuvant to enhance influenza NP-induced CTL responses and protection.

Deregulated phenotype of autoreactive Th17 and Treg clone cells in pemphigus vulgaris after in-vitro treatment with desmoglein antigen (Dsg-3)

The loss of balance between regulatory T (Treg) and T helper 17 (Th17) causes loss of tolerance against desmoglein (Dsg)-3 leading to pemphigus vulgaris (PV), an autoimmune bullous skin disorder associated with autoantibodies against Dsg-3. We aimed to elucidate the complex relationship of Th17 and Treg cells, their molecules, and the underlying mechanism in the development of PV disease. Using cytokine secretion assays, Th17 and Treg cells were sorted by FACS Aria-III within Dsg-3-responsive PBMC population and homogeneous T cell clones were generated in-vitro. Different cell surface molecules like CD25, GITR, CD122, CD152, CD45RO, IL-23R, STAT3, STAT5, CD127, HLA-DR, CCR4, CCR5, CCR6 and CCR7 were studied. The functional response of Th17 and Treg cells were elucidated by measuring the levels of various cytokines released by IL-10 and IL-17 T cells. The mRNA expression of transcription factors (FoxP3 and RORγt) was also analyzed. IL-17 secreting (Th17) cells with phenotype CD4+IL-17+ were greatly increased and IL-10 secreting (Treg) cells with phenotype CD4+IL-10+ were reduced in PV cases than healthy controls. The qPCR analysis showing high expression of retinoic acid receptor-related orphan receptor gamma (RORγt) mRNA in comparison to forkhead box P3 (FoxP3) mRNA confirmed the development of pro-inflammatory Th17 response in PV. Further, the cytokine profile of pro-inflammatory and anti-inflammatory cytokines suggested defective suppressive functions in Treg cells with high inflammatory response. Our findings indicate that autoantigen Dsg-3 specifically allows the proliferation of IL-17 secreting T cells though has a negative effect on IL-10 secreting T cells leading to dysregulation of immunity in PV patients. This antagonistic relationship between Dsg-3-specific Th17 and Treg cells may be critical for the onset and persistence of inflammation in PV cases.

CGAMP-adjuvanted multivalent influenza mRNA vaccines induce broadly protective immunity through cutaneous vaccination in mice

Increasing preclinical and clinical results have demonstrated that mRNA vaccines efficiently prevent infectious diseases and are safe in animal models and humans. In this study, we fabricated a multivalent influenza mRNA lipid nanoparticle (LNP) vaccine with mRNAs of hemagglutinins from influenza H1N1 and H3N2 viruses, matrix protein 1, and nucleoprotein. We found that cutaneous immunization with mRNA LNPs induced strong Th1 and Th2 cellular immunity with robust antigen-specific antibody titers and increased cytokine-secreting splenocytes and antibody-secreting cells. The supplement of cGAMP improved the immunogenicity of mRNA LNPs. Compared with αGC or cGAMP/αGC adjuvanted mRNA LNP formulations in our study, cGAMP mRNA LNPs induced more robust antibody responses. Enhanced cellular immunity with more IL-4 and IFN-γ secreting cells and effector memory Tcell populations in spleens, as well as increased CD4+ resident memory (TRM) Tcells in lungs were observed in cGAMP mRNA LNPs immunized group. These results demonstrated that cGAMP is an effective adjuvant for cutaneous vaccination of multivalent mRNA LNP vaccines in mice to induce stronger immune responses in the spleen and lung, and the cGAMP-adjuvanted mRNA LNPs protected against homologous and heterologous viral infection.

Cord Blood Regulatory T Cells for Prevention of Graft Versus Host Disease

Background: Regulatory T-cells (Tregs) are a T-cell subset capable of suppressing effector T-cells (Teffs) and, when given at the time of allogeneic hematopoietic stem cell transplantation (Allo-SCT), have been shown to prevent graft v. host disease (GVHD). We previously demonstrated that cord blood (CB) Tregs can be ex vivo expanded to clinically meaningful doses and prevent GVHD in a xenogenic mouse model. The efficacy of the ex vivo expanded CB Tregs in preventing GVHD was markedly improved with fucosylation, which adds fucose resulting in creation of a siayl-Lewis X moiety found on P-selectin ligand, thereby facilitating trafficking through the endothelium into inflammatory sites. We hypothesized that adoptive therapy with fucosylated Tregs could prevent GVHD even when given at suboptimal Tregs:Teffs ratios. We conducted a Phase I clinical trial to evaluate the clinical safety of fucosylated CB Treg infusion for GVHD prophylaxis (ww.clinicaltrials.gov NCT02423915). We now report on 5 patients who received GVHD prophylaxis with 3rdparty, ex-vivo expanded CB Tregs at a dose level of 1.0 x10e6 cells/ kg (2 un-fucosylated and 3 fucosylated CB Treg products).Patients and methods: A 3rd party CB unit with at least 3/6 HLA matching to the recipient was selected. Treg isolation and expansion was performed as shown previously. When indicated, expanded CB Tregs were incubated with substrate (GDP β-fucose) and fucosyltrasferase-VI (FT6) enzyme (TZ101, Targazyme, Carlsbad, CA) for 30 minutes, washed and infused on day -1. The first 2 patients underwent double cord transplant with Flu/Cy/TBI reduced intensity conditioning and received unfucosylated CB Tregs at a dose of 1 x 10e6/kg. The subsequent 3 patients underwent peripheral blood (PB) matched unrelated donor (MUD) transplant with Fludarabine/ Melphalan myeloablative conditioining and received fucosylated CB Tregs at a dose of 1 x 10e6/kg. All patients received additional pharmacologic GVHD prophylaxis with sirolimus and mycophenolate mofetil. All patients received their designated CB Treg dose. The median proportion of CD4+25+CD127- in the infused Treg product was 96% (94-98%). Expanded CB Treg product showed suppression of T cell proliferation using CFSE assay. For the first 2 patients, the infused graft Teffs dose was at least 12 times more than the CB Tregs and for the subsequent 3 patients, the dose of infused Teffs dose was at least 132 times more than that of the infused fucosylated CB Tregs. No infusion toxicities were observed.Results: All patients receiving fucosylated CB Tregs followed by PB MUD transplants exhibited non-infectious high fevers up to 40οC accompanied by an erythematous rash starting on post-transplant day +5 that lasted for 5-7 days and resolved after steroid administration (IV methylprednisolone 1mg/kg) for 24-96 hours. Skin biopsies were consistent with engraftment syndrome and not GVHD. All 5 patients engrafted at a median of 13 days. The three patients who received fucosylated CB Tregs and one of two patients receiving untreated CB Tregs developed ≥ grade II acute GVHD at a median of day +22 which had completely resolved by day 130 post-transplant. One patient had late onset aGVHD. No cGVHD was documented in any patient. One recipient of non-fucosylated Tregs died on day +40 due to unrelated causes (intracranial bleeding). No GVHD was observed in this patient. PB Flow cytometric analysis revealed an increase in inflammatory T-cell subsets in the first week post-transplant with a concurrent bimodal increase in the Treg populations on day -1 and day +7 (figure 1A). NK and DC but not B cell subsets were significantly increased. A consistent increase in the cell populations secreting IFNү, IL-4 and IL-17 was observed in the post-transplant period and a bi-modal increase in the IL-10 secreting cells was observed on Day -1 and Day +7 consistent with the circulating Treg cells (figure 1B).Conclusions: Our group is the first to show safety of infusing 3rd party, ex-vivo expanded CB Tregs in a PB MUD transplant setting. At a dose level of 1.0 x 10e6 /kg, 3rd party CB Tregs were administered without acute infusional toxicity and no negative impact on engraftment. The cause for the high fevers observed in the fucosylated CB Treg recipients is unclear at this time. We are now enrolling additional patients with unfucosylated Tregs in a PB MUD transplant setting to investigate the mechanisms underlying this phenomenon. [Display omitted] DisclosuresKhoury:Kiromics: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding; Pfizer: Research Funding.

Adiponectin/AdipoR1 Axis Promotes IL-10 Release by Human Regulatory T Cells.

BackgroundAdiponectin is an important immunomodulatory mediator in inflammatory conditions. While we previously showed that adiponectin receptor 1 (AdipoR1) is expressed in murine regulatory T cells (Tregs), its expression in human Tregs remain unknown. Here, we examined the expression of AdipoR1 in human Tregs and whether its ligand, globular adiponectin (gAd) affects the Treg ability to secrete IL-10 and the role of Type 2 (T2) inflammation in such process.MethodsHuman Tregs from peripheral blood were analyzed by flow cytometry for AdipoR1, Helios and IL-10 expression. CD4+ T cells enriched from peripheral blood mononuclear cells (PBMCs) were cultured in the presence or the absence of gAd or the chemical adiponectin receptor agonist, AdipoRon, or in a T2 cytokine milieu. Flow cytometry was then used to assess intracellular IL-10, IL-10 secreting cells, FOXP3 and Helios expression, and phosphorylated p38 MAP kinase (MAPK). IL-10 levels in CD4+ T cell supernatants were quantified by ELISA.ResultsWe found that a subset of human Tregs expressed AdipoR1. Importantly, more Helios- cells expressed AdipoR1 than Helios+ cells. Likewise, there was a higher frequency of IL-10+ cells within Helios- AdipoR1+ Tregs compared to Helios+ AdipoR1+ Tregs. In contrast, the IL-10 mean fluorescence intensity (MFI) was higher in Helios+ AdipoR1+ Tregs compared to Helios-AdipoR1+ Tregs. When human CD4+ T cells were treated with gAd or AdipoRon, a significant increase in IL-10 secretion, FOXP3 expression, and p38 MAPK phosphorylation was observed in Helios- AdipoR1+ Tregs. Interestingly, gAd under T2 cytokine milieu significantly increased the intracellular levels of IL-10, mainly in Helios+ AdipoR1+ Tregs, and IL-10 levels in supernatants of CD4+ T cells.ConclusionsCollectively, our findings suggest that adiponectin/AdipoR1 axis promotes IL-10 release by Tregs, mainly in Helios- Tregs, and the effect was amplified by T2 inflammation in Helios+ Tregs.

Human Lymphoid Stromal Cells Contribute to Polarization of Follicular T Cells Into IL-4 Secreting Cells.

Fibroblastic reticular cells (FRCs) are the specialized lymphoid stromal cells initially identified as triggering T-cell recruitment and dynamic motion in secondary lymphoid organs. Interestingly, FRCs also display antigen presentation capacities and support lymphocyte survival. CXCR5+CD4+ follicular T cells are important players of B-cell maturation and antibody response. Our study reported that in vitro-differentiated FRC-like cells enhanced the growth of the whole CXCR5+CD4+ T-cell compartment, while enhancing IL-4 secretion specifically by the PD1dimCXCR5+CD4+ cell subset, in a Notch- and ICAM1/LFA1-dependent manner. In addition, we revealed that in follicular lymphoma (FL) tissues, previously identified as enriched for PD1hiCXCR5hiCD4+ mature follicular helper T cells, PD1dimCXCR5+CD4+ T cells displayed an enrichment for Notch and integrin gene signatures, and a Notch and ICAM-1-dependent overexpression of IL-4 compared to their non-malignant counterparts. These findings suggest that the crosstalk between FRCs and CXCR5+PD1dimCD4+ T cells may contribute to the FL IL-4 rich environment, thus providing new insights in FL lymphomagenesis.

M3 muscarinic acetylcholine receptor-reactive Th17 cells in primary Sjögren's syndrome.

M3 muscarinic acetylcholine receptor (M3R) is one of the autoantigens associated with Sjögren's syndrome (SS) and is localized in exocrine glands where disease-specific inflammation occurs. The inflammatory lesion is characterized by infiltration of CD4+ T cells, including clonally expanded Th17 cells. We undertook this study to identify circulating M3R-specific Th17 cells and to determine functional properties of those cells. Using the enzyme-linked immunospot assay (ELISpot) method, we identified M3R-reactive Th17 cells in the peripheral blood of patients with primary SS (pSS). Among 10 examined pSS patients, 10 healthy subjects (HS), and 5 IgG4-related disease (IgG4-RD) patients, M3R-reactive IL-17 secreting cells were significantly increased in 5 pSS patients specifically. The most common T cell epitope, which was analyzed and confirmed by coculture of isolated CD4+ T cells with antigen presenting cells plus M3R peptides in vitro, was peptide 83-95 of M3R. Peptide recognition was partly in an HLA-DR-restricted manner, confirmed by blocking assay. M3R-reactive Th17 cells positivity correlated with higher titers of anti-M3R antibodies, whose systemic disease activity score tended to be higher. Our studies highlight the role of tissue-specific autoantigen-derived circulating Th17 cells in pSS, for which further work might lead to antigen-specific targeted therapy.

Discriminative expression of CD39 and CD73 in Cerebrospinal fluid of patients with Multiple Sclerosis and Neuro-Behçet’s disease

Treg‐mediated immune suppression involves many molecular mechanisms including the cleavage of inflammatory extracellular ATP to adenosine by CD39 ectoenzyme. In the peripheral blood of Multiple Sclerosis (MS) patients, it has been suggested that CD39+ Treg cells have the potential to suppress pro-inflammatory IL-17 secreting cells. Herein, we studied cellular phenotype and mRNA expression of CD39 and CD73 ectoenzymes in the Cerebrospinal fluid (CSF) of MS patients and another neuro-inflammatory disease: the Neuro-behçet’s disease (NBD).Using qRT-PCR, we assessed mRNA expression of CD39 and CD73 as well as anti-inflammatory (IL-10) and pro-inflammatory (IL-6, TNF-α, IL-1β) cytokines in patients Peripheral blood mononuclear cells (PBMCs) and CSF of 28 relapsing-remitting multiple sclerosis (RRMS), 20 NBD and 22 controls with non inflammatory neurological disorders (NIND). The most substantial result in the CSF was the higher expression of CD39 in both RRMS and NBD patients compared to NIND. While, the expression of CD73 in CSF samples of NBD was low. In RRMS samples, we detected a significant positive correlation of both CD39 and CD73 with IL-10 expression. Moreover, results by flow cytometry revealed a high percentage of CD39 Treg cells in RRMS CSF. CD39 was preferentially expressed on B cells of NBD. Regarding inflammatory response, we showed a significant increase of IL-6 mRNA expression in NBD patients CSF while in RRMS this increase concerned TNF-α. These results bring evidence that CD39 correlates positively with an anti-inflammatory IL-10 response in RRMS. In contrast, no such association was observed in CSF of NBD patients and CD39 was preferentially expressed on B cells.

Enhanced IFN-\u03b3, but not IL-2, response to Mycobacterium tuberculosis antigens in HIV/latent TB co-infected patients on long-term HAART

BackgroundHIV-infected individuals with latent TB infection are at increased risk of developing active TB. HAART greatly reduces the incidence rate of TB in HIV-infected patients and reconstitutes Mycobacterium tuberculosis (M. tuberculosis)-specific immune response in the first 12 months of therapy. The durability of the anti-mycobacterial immune restoration after a year of HAART however remains less investigated.MethodA cross-sectional study was conducted to evaluate M. tuberculosis-specific functional immune responses in HIV/latent TB co-infected patients who were on HAART for at least 1.5 up to 9 years as compared to HAART-naïve patients. Three-hundred sixteen HIV-infected patients without active TB were screened by tuberculin skin testing for M. tuberculosis infection and peripheral blood mononuclear cells (PBMCs) were isolated from 61 HIV/latent TB co-infected patients (30 HAART-naïve and 31 HAART-treated). IFN-γ and IL-2 ELISPOT as well as CFSE cell proliferation assays were performed after stimulation with M. tuberculosis antigens PPD and ESAT-6.ResultThe median frequency of PPD and ESAT-6 specific IFN-γ secreting cells was significantly higher in the HAART-treated patients as compared to HAART-naïve patients, p = 0.0021 and p = 0.0081 respectively. However, there was no significant difference in the median frequency of IL-2 secreting cells responding to PPD (p = 0.5981) and ESAT-6 (p = 0.3943) antigens between HAART-naïve and-treated groups. Both IFN-γ and IL-2 responses were independent of CD4+ T cell count regardless of the HAART status. Notably, the frequency of PPD and ESAT-6 specific IL-2 secreting cells was positively associated with CD4+ T cell proliferation while inversely correlated with duration of HAART, raising the possibility that M. tuberculosis-specific IL-2 response that promote the antigen-specific CD4+ T cell proliferation diminish with time on antiretroviral therapy in HIV/latent TB co-infected patients.ConclusionThis study shows an increased M. tuberculosis-specific IFN-γ, but not IL-2, response in HIV/latent TB co-infected patients with long-term HAART, consistent with only partial immune restoration. Future studies should, therefore, be done to prospectively define the rate and extent to which functional immune responses to M. tuberculosis are restored after long-term HAART.

NK Cell IL-10 Production Requires IL-15 and IL-10 Driven STAT3 Activation

Natural killer (NK) cells can produce IFNγ or IL-10 to regulate inflammation and immune responses but the factors driving NK cell IL-10 secretion are poorly-defined. Here, we identified NK cell-intrinsic STAT3 activation as vital for IL-10 production during both systemic Listeria monocytogenes (Lm) infection and following IL-15 cytokine/receptor complex (IL15C) treatment for experimental cerebral malaria (ECM). In both contexts, conditional Stat3 deficiency in NK cells abrogated production of IL-10. Initial NK cell STAT3 phosphorylation was driven by IL-15. During Lm infection, this required capture or presentation of IL-15 by NK cell IL-15Rα. Persistent STAT3 activation was required to drive measurable IL-10 secretion and required NK cell expression of IL-10Rα. Survival-promoting effects of IL-15C treatment in ECM were dependent on NK cell Stat3 while NK cell-intrinsic deficiency for Stat3, Il15ra, or Il10ra abrogated NK cell IL-10 production and increased resistance against Lm. NK cell Stat3 deficiency did not impact production of IFNγ, indicating the STAT3 activation initiated by IL-15 and amplified by IL-10 selectively drives the production of anti-inflammatory IL-10 by responding NK cells.

1728-P: Inhibition of PKC-Theta in Prediabetic NOD Mice Prevents Diabetes Onset and Increases Regulatory CD4 T Cells in a Subset of Animals

The novel PKC isoform, PKC-theta, is particularly enriched in T lymphocyte populations, serving a critical role in signal transduction after CD3/CD28 T cell receptor stimulation. PKC-theta is known to be involved in promoting T cell mediated immunity and has been implicated in other autoimmune diseases. We hypothesized that inhibition of this isoform would increase regulatory T cell phenotypes, promote an anti-inflammatory environment, and lead to delayed onset of type 1 diabetes in the NOD mouse model. A cell permeable pseudosubstrate peptide inhibitor was identified with potent suppression of IL-2 secretion at concentrations ≤15 μg in T cell enriched populations stimulated in vitro with CD3 and CD28 antibodies and a significant reduction in stimulated IL-2 secretion after 7 days of intraperitoneal treatment with 50 μg of pseudosubstrate inhibitor. Five-week-old prediabetic NOD mice were either treated daily with 50 μg of inhibitor or mock treated with saline for 4 weeks, then allowed to progress until 17 weeks of age. An 80% conversion to diabetes, based on IP glucose tolerance tests, was observed in saline mice compared to 30% conversion in inhibitor-treated mice, which correlated with a greater frequency of inflammation-free islets and islets with periinsulitis only in inhibitor-treated mice. Inhibitor-treated mice had increased frequencies of IL-10 secreting cells by ELISPOT, and increased Tregs in spleen at 17 weeks of age while reduced frequencies of IL-17+ CD4+ T cells. This study was repeated with a scrambled peptide control and later endpoint to determine if T1D would be prevented entirely. At 25 weeks of age a 66% conversion to diabetes was observed in scrambled peptide treated mice, while only 33% converted in the peptide treated. Our data demonstrate that PKC theta activation contributes to the progression of beta cell destruction in NOD mice, where inhibition of this isoform may confer an increase in protective regulatory T cell populations. Disclosure J.E. DiLisio: None. N. Mishkin: None. B. Podell: None. Funding National Institutes of Health (5K01OD016997 to B.P.); National Center for Advancing Translational Sciences (UL1TR001082 to B.P.)

Preconditioned or IL4-Secreting Mesenchymal Stem Cells Enhanced Osteogenesis at Different Stages

Pathogen-associated molecular patterns, damage-associated molecular patterns, and other noxious stimuli activate macrophages to induce the proinflammatory responses. Modulation of inflammatory macrophages (M1) into an anti-inflammatory tissue repair macrophage (M2) phenotype at the appropriate time optimizes bone remodeling and regeneration. Simulating the proinflammatory stimuli by using preconditioned mesenchymal stem cells (MSCs) at an earlier stage, and alleviate the inflammation by using IL4-secreting MSCs at a later stage could further optimize bone regeneration in chronic inflammatory conditions, including periprosthetic osteolysis.

PO-021 Merkel cell polyomavirus-encoded miRNA subverts the host cell immune response

IntroductionVirus-encoded miRNAs are emerging as key regulators of persistent infection and host-cell immune evasion. Merkel cell polyomavirus (MCPyV), the predominant aetiological agent of the rare but deadly skin cancer, Merkel cell carcinoma (MCC), encodes a single miRNA, MCV-miR-M1, which targets the oncogenic MCPyV large T antigen (LT). MCV-miR-M1 has previously been shown to play an important role in establishment of long-term infection, however, the underlying mechanism is not fully understood. A key unanswered question is whether, in addition to auto-regulating LT, MCV-miR-M1 also targets cellular transcripts to orchestrate an environment conducive for persistent infection, which is a prerequisite for oncovirus-mediated transformation.Material and methodsWe adopted an unbiased RNA-Seq-based approach to identify differentially-expressed (DE) targets of MCV-miR-M1 in cell expressing either the 5 p or 3 p arm of the miRNA. Bioinformatic analysis of DE transcripts was carried out using DAVID. DE targets identified by RNA-seq were confirmed via RT-qPCR in a variety of cell lines expressing either MCV-miR-M1 mimics or an MCPyV replication system based on synthetic MCPyV genomes. MCV-miR-M1 interaction with full length 3’UTR regions of DE genes was analysed via dual-luciferase assay to identify direct MCV-miR-M1 targets. IL-8 secretion was determined by ELISA and neutrophil chemotaxis assessed via transwell migration assays.Results and discussionsBioinformatic analysis of DE transcripts revealed numerous genes implicated in immune evasion. Subsequent target validation led to the identification of the innate immunity protein, SP100, as a direct target of MCV-miR-M1. Intriguingly, MCV-miR-M1-mediated modulation of SP100 was associated with a significant decrease in IL-8 secretion in cells harbouring synthetic MCPyV genomes. Moreover, the observed MCV-miR-M1-mediated reduction in IL-8 secretion was significantly increased by ectopic expression of MCV-miR-M1-resistant SP100. The attenuation of IL-8 secretion resulted in a significant reduction in neutrophil chemotaxis towards cells harbouring replicative MCPyV.ConclusionThese data represent the fist global analysis of MCPyV miRNA targets. Based on our observations, we propose that MCV-miR-M1 targets key immune response regulators in the host cell to help facilitate persistent infection, in part by attenuating neutrophil chemotaxis towards infected cells.

PO-024 SETD1A regulates cell proliferation by modulating miR205 in triple negative breast cancers (TNBCS)

IntroductionVirus-encoded miRNAs are emerging as key regulators of persistent infection and host-cell immune evasion. Merkel cell polyomavirus (MCPyV), the predominant aetiological agent of the rare but deadly skin cancer, Merkel cell carcinoma (MCC), encodes a single miRNA, MCV-miR-M1, which targets the oncogenic MCPyV large T antigen (LT). MCV-miR-M1 has previously been shown to play an important role in establishment of long-term infection, however, the underlying mechanism is not fully understood. A key unanswered question is whether, in addition to auto-regulating LT, MCV-miR-M1 also targets cellular transcripts to orchestrate an environment conducive for persistent infection, which is a prerequisite for oncovirus-mediated transformation.Material and methodsWe adopted an unbiased RNA-Seq-based approach to identify differentially-expressed (DE) targets of MCV-miR-M1 in cell expressing either the 5 p or 3 p arm of the miRNA. Bioinformatic analysis of DE transcripts was carried out using DAVID. DE targets identified by RNA-seq were confirmed via RT-qPCR in a variety of cell lines expressing either MCV-miR-M1 mimics or an MCPyV replication system based on synthetic MCPyV genomes. MCV-miR-M1 interaction with full length 3’UTR regions of DE genes was analysed via dual-luciferase assay to identify direct MCV-miR-M1 targets. IL-8 secretion was determined by ELISA and neutrophil chemotaxis assessed via transwell migration assays.Results and discussionsBioinformatic analysis of DE transcripts revealed numerous genes implicated in immune evasion. Subsequent target validation led to the identification of the innate immunity protein, SP100, as a direct target of MCV-miR-M1. Intriguingly, MCV-miR-M1-mediated modulation of SP100 was associated with a significant decrease in IL-8 secretion in cells harbouring synthetic MCPyV genomes. Moreover, the observed MCV-miR-M1-mediated reduction in IL-8 secretion was significantly increased by ectopic expression of MCV-miR-M1-resistant SP100. The attenuation of IL-8 secretion resulted in a significant reduction in neutrophil chemotaxis towards cells harbouring replicative MCPyV.ConclusionThese data represent the fist global analysis of MCPyV miRNA targets. Based on our observations, we propose that MCV-miR-M1 targets key immune response regulators in the host cell to help facilitate persistent infection, in part by attenuating neutrophil chemotaxis towards infected cells.

BAY-11-7082 induces apoptosis of multiple myeloma U266 cells through inhibiting NF-κB pathway.

To study the effects of BAY-11-7082 on proliferation and apoptosis of U266 cells and its mechanism of action. Multiple myelomas U266 cells were cultured and divided into control group and gradient-concentration BAY-11-7082 groups (1 μmol/L, 2 μmol/L, 4 μmol/L and 8 μmol/L). Cells in BAY-11-7082 groups were treated with drugs in different concentrations for 4 h, while those in control group were added with an equal volume of solvent. The cell viability was detected via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and lactate dehydrogenase (LDH) release assay was used to detect the cytotoxicity. Furthermore, cells in low-concentration and high-concentration BAY-11-7082 groups were compared with those in control group. The cell proliferation level was evaluated via cell cycle assay, the interleukin-6 (IL-6) level in cells was detected via enzyme-linked immunosorbent assay (ELISA), and the β-catenin protein expression was detected via Western blotting. Moreover, flow cytometry and Hoechst staining were performed to detect the number of apoptotic cells, and the apoptosis level was detected via caspase3 activity and apoptosis-related protein expression. Finally, the levels of p65, p50 and inhibitor kappa B kinase β (IKKβ) were detected via polymerase chain reaction (PCR), and the expressions and changes of phosphorylated (p)-p65 and p-IKKβ were detected via Western blotting. BAY-11-7082 could reduce the U266 cell viability and increase the cytotoxic effect. Based on gradient concentration, 2 μmol/L was selected as the low concentration, while 4 μmol/L was selected as the high concentration. Compared with those in control group, the number of cells in the S and G2/M phases in drug administration groups was significantly decreased, but that in the G0/G1 phase was significantly increased. Besides, the secretion of IL-6 in cells in drug administration groups was significantly decreased compared with that in control group. The β-catenin protein expression was decreased in drug administration groups, and there was also a difference between high concentration group and low concentration group. Flow cytometry and Hoechst staining showed that the proportion of apoptotic cells in drug administration groups was significantly increased. Western blotting and detection of caspase3 activity revealed that the expression and activation of apoptosis-related protein were increased in drug administration groups. It was found in the detection of nuclear factor-κB (NF-κB) pathway that the NF-κB pathway was inhibited in drug administration groups, and there was also a statistically significant difference between high concentration group and low concentration group. BAY-11-7082 inhibits the proliferation and induces the apoptosis of U266 cells through inhibiting NF-κB pathway.

Therapeutic effects of probiotics on Multiple Sclerosis (MS) via TH17 cells

MS-specific disease progression and emerging symptoms are heterogeneous, it is difficult to diagnose and find treatment. The abundance of immune cells, such as products of T lymphocytes and MS patients with CNS lesions, supports the view that MS is an immunomodulatory disorder. Th17 cells appear to play an important role in the immunopathogenesis of EAE and MS. Th17 cells are a subset of cells independent of Th1 and Th2. Although the Th17 cells in steady state are important for host defense, pathogenic Th17 cells play a role in the development of many diseases. Th17 cells are known to secrete IL-17 cytokines. IL-17 secreting cells have been identified in MS and many other diseases. In addition, IL-17 levels were found to be high in cerebrospinal fluid from MS patients. Probiotic bacteria are known to be important in terms of human health and have therapeutic properties. Many studies have suggested that probiotics have the ability to inhibit proinflammatory IL-17 production and activation in Th17-related diseases.

The Significance of Discordant Serology in Chagas Disease: Enhanced T-Cell Immunity to Trypanosoma cruzi in Serodiscordant Subjects.

Subjects are considered infected with Trypanosoma cruzi when tested positive by at least two out of three serological tests, whereas a positive result in only one of up to three tests is termed "serodiscordant" (SD). Assessment of parasite-specific T-cell responses may help discriminate the uninfected from infected individuals among SD subjects. Peripheral blood mononuclear cells from SD and seropositive (SP) subjects, who were born in areas endemic for T. cruzi infection but living in Buenos Aires city, Argentina, at the time of the study, and seronegative unexposed subjects were included for analysis. The function and phenotype of T cells were assessed by interferon-γ (IFN-γ) and interleukin (IL)-2 enzyme-linked immunospot assay and multiparameter flow cytometry. T. cruzi-specific antibodies were quantified by conventional serology and a multiplex assay format. SD subjects exhibited immunity cell responses to T. cruzi but in contrast to SP subjects, T cells in SD subjects more often display the simultaneous production of IFN-γ and IL-2 in response to T. cruzi antigens and have a resting phenotype. SD individuals also have higher IFN-γ spot counts, polyfunctional CD4+ T-cells enriched in IL-2 secreting cells and low levels of antibodies specific for a set of T. cruzi-derived recombinant proteins compared with the SP group. Long-term follow-up of SD individuals confirmed that humoral and T-cell responses fluctuate but are sustained over time in these subjects. T cells in SD subjects for T. cruzi infection did not recognize Leishmania antigens. Both T-cell and humoral responses in most subjects assessed by conventional tests as SD for T. cruzi infection indicate prior exposure to infection and the establishment of immunological memory suggestive of a resolved infection.

Suppression of IL-8 Release by Sweet Olive Ethanolic Extract and Compounds in WiDr Colon Adenocarcinoma Cells.

Oxidative stress can stimulate the secretion of pro-inflammatory cytokines. Interleukin-8 (IL-8) has been implicated in the pathogenesis of inflammatory bowel disease and the metastatic spread of colorectal cancer. The flowers of Osmanthus fragrans (sweet olive) are used to alleviate dysentery with blood in the bowel, as well as stomach ache and diarrhea. However, the evidence of their therapeutic effects on these symptoms remains unclear. In the present study, the protective effects of sweet olive flower ethanolic extract (OFE) against oxidative stress in WiDr cells was assessed by evaluating its 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. In addition, cellular IL-8 secretion was evaluated. Notably, high-performance liquid chromatography showed verbascoside to be the primary constituent in OFE; it exhibited a DPPH scavenging activity with an IC50 of 8.23 μg/mL. Moreover, OFE (1 to 100 μg/mL) showed a potent, dose-dependent inhibitory effect on H2 O2 -induced IL-8 secretion in WiDr cells. Nine compounds were isolated from OFE based on a protective effect-guided purification process. Of these compounds, 5 phenolic compounds-verbascoside, phillygenin, tyrosol, methyl 4-hydroxycinnamate, and eutigoside A-reduced IL-8 secretion at 10 μg/mL treatment concentrations. Further analysis showed that the anti-inflammatory effects of OFE likely occurred via nuclear factor-κB pathway inhibition, which attenuates IL-8 secretion in cells. Collectively, these data suggest that OFE could be developed as an agent that suppresses IL-8 secretion to treat chronic inflammatory diseases.