Primary Human Monocyte-derived Dendritic Cells Research Articles

A comparative proteomics analysis of four contact allergens in THP-1 cells shows distinct alterations in key metabolic pathways

Allergic contact dermatitis (ACD) is the predominant form of immunotoxicity in humans. The sensitizing potential of chemicals can be assessed in vitro. However, a better mechanistic understanding could improve the current OECD-validated test battery. The aim of this study was to get insights into toxicity mechanisms of four contact allergens, p-benzoquinone (BQ), 2,4-dinitrochlorobenzene (DNCB), p-nitrobenzyl bromide (NBB) and NiSO4, by analyzing differential proteome alterations in THP-1 cells using two common proteomics workflows, stable isotope labeling by amino acids in cell culture (SILAC) and label-free quantification (LFQ). Here, SILAC was found to deliver more robust results. Overall, the four allergens induced similar responses in THP-1 cells, which underwent profound metabolic reprogramming, including a striking upregulation of the TCA cycle accompanied by pronounced induction of the Nrf2 oxidative stress response pathway. The magnitude of induction varied between the allergens with DNCB and NBB being most potent. A considerable overlap between transcriptome-based signatures of the GARD assay and the proteins identified in our study was found. When comparing the results of this study to a previous proteomics study in human primary monocyte-derived dendritic cells, we found a rather low share in regulated proteins. However, on pathway level, the overlap was high, indicating that affected pathways rather than single proteins are more eligible to investigate proteomic changes induced by contact allergens. Overall, this study confirms the potential of proteomics to obtain a profound mechanistic understanding, which may help improving existing in vitro assays for skin sensitization.

The Autophagy Nucleation Factor ATG9 Forms Nanoclusters with the HIV-1 Receptor DC-SIGN and Regulates Early Antiviral Autophagy in Human Dendritic Cells.

Dendritic cells (DC) are critical cellular mediators of host immunity, notably by expressing a broad panel of pattern recognition receptors. One of those receptors, the C-type lectin receptor DC-SIGN, was previously reported as a regulator of endo/lysosomal targeting through functional connections with the autophagy pathway. Here, we confirmed that DC-SIGN internalization intersects with LC3+ autophagy structures in primary human monocyte-derived dendritic cells (MoDC). DC-SIGN engagement promoted autophagy flux which coincided with the recruitment of ATG-related factors. As such, the autophagy initiation factor ATG9 was found to be associated with DC-SIGN very early upon receptor engagement and required for an optimal DC-SIGN-mediated autophagy flux. The autophagy flux activation upon DC-SIGN engagement was recapitulated using engineered DC-SIGN-expressing epithelial cells in which ATG9 association with the receptor was also confirmed. Finally, Stimulated emission depletion (STED) microscopy performed in primary human MoDC revealed DC-SIGN-dependent submembrane nanoclusters formed with ATG9, which was required to degrade incoming viruses and further limit DC-mediated transmission of HIV-1 infection to CD4+ T lymphocytes. Our study unveils a physical association between the Pattern Recognition Receptor DC-SIGN and essential components of the autophagy pathway contributing to early endocytic events and the host's antiviral immune response.

Rescue and in vitro characterization of a divergent TBEV-Eu strain from the Netherlands

Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland.

NCoR1 controls immune tolerance in conventional dendritic cells by fine-tuning glycolysis and fatty acid oxidation

Dendritic cells (DCs) undergo rapid metabolic reprogramming to generate signal-specific immune responses. The fine control of cellular metabolism underlying DC immune tolerance remains elusive. We have recently reported that NCoR1 ablation generates immune-tolerant DCs through enhanced IL-10, IL-27 and SOCS3 expression. In this study, we did comprehensive metabolic profiling of these tolerogenic DCs and identified that they meet their energy requirements through enhanced glycolysis and oxidative phosphorylation (OXPHOS), supported by fatty acid oxidation-driven oxygen consumption. In addition, the reduced pyruvate and glutamine oxidation with a broken TCA cycle maintains the tolerogenic state of the cells. Mechanistically, the AKT-mTOR–HIF–1α-axis mediated glycolysis and CPT1a-driven β-oxidation were enhanced in these tolerogenic DCs. To confirm these observations, we used synthetic metabolic inhibitors and found that the combined inhibition of HIF-1α and CPT1a using KC7F2 and etomoxir, respectively, compromised the overall transcriptional signature of immunological tolerance including the regulatory cytokines IL-10 and IL-27. Functionally, treatment of tolerogenic DCs with dual KC7F2 and etomoxir treatment perturbed the polarization of co-cultured naïve CD4+ T helper (Th) cells towards Th1 than Tregs, ex vivo and in vivo. Physiologically, the Mycobacterium tuberculosis (Mtb) infection model depicted significantly reduced bacterial burden in BMcDC1 ex vivo and in CD103+ lung DCs in Mtb infected NCoR1DC−/−mice. The spleen of these infected animals also showed increased Th1-mediated responses in the inhibitor-treated group. These findings suggested strong involvement of NCoR1 in immune tolerance. Our validation in primary human monocyte-derived DCs (moDCs) showed diminished NCOR1 expression in dexamethasone-derived tolerogenic moDCs along with suppression of CD4+T cell proliferation and Th1 polarization. Furthermore, the combined KC7F2 and etomoxir treatment rescued the decreased T cell proliferative capacity and the Th1 phenotype. Overall, for the first time, we demonstrated here that NCoR1 mediated control of glycolysis and fatty acid oxidation fine-tunes immune tolerance versus inflammation balance in murine and human DCs.

HLAII peptide presentation of infliximab increases when complexed with TNF.

CD4+ T-cell activation through recognition of Human Leukocyte Antigen II (HLAII)-presented peptides is a key step in the development of unwanted immune response against biotherapeutics, such as the generation of anti-drug antibodies (ADA). Therefore, the identification of HLAII-presented peptides derived from biotherapeutics is a crucial part of immunogenicity risk assessment and mitigation strategies during drug development. To date, numerous CD4+ T-cell epitopes have been identified by HLAII immunopeptidomics in antibody-based biotherapeutics using either their native or aggregated form. Antibody-target immune complexes have been detected in patients with ADA and are thought to play a role in ADA development by enhancing the presentation of CD4+ T-cell epitopes at the surface of antigen presenting cells (APCs). The aim of this study was to investigate the effect of biotherapeutic antibody-target immune complexes on the HLAII peptide presentation of biotherapeutics in human primary monocyte-derived dendritic cells (DCs). The trimeric tumor necrosis factor (TNF) and its biotherapeutic antagonists infliximab (INFL), adalimumab (ADAL), and a single armed Fab’ were used as a model system. The HLAII immunopeptidome of DCs loaded with antagonists or their immune complexes with TNF was analyzed by trapped ion mobility time-of-flight mass spectrometry (timsTOF MS) leading to the identification of ~ 12,000 unique HLAII-associated peptides per preparation. Anti-TNF sequences were detected at a median of 0.3% of the total immunopeptidome, against a majority background of peptides from endogenous and media-derived proteins. TNF antagonist presentation spanned the variable and constant regions in a widespread manner in both light and heavy chains, consistent with previously discovered HLAII peptides. This investigation extends the collection of observed HLAII peptides from anti-TNF biotherapeutics to include sequences that at least partially span the complementary determining regions (CDRs), such as the LCDR1 for both INFL and ADAL. Although antagonist presentation varied significantly across donors, peptides from both bivalent antagonists INFL and ADAL were more highly presented relative to the Fab’. While TNF immune complexes did not alter overall HLAII presentation, a moderate increase in presentation of a subset of peptide clusters was observed in the case of INFL-TNF, which included HCDR2, HCDR3 and LCDR2 sequences.

Haptoglobin Induces a Specific Proteomic Profile and a Mature-Associated Phenotype on Primary Human Monocyte-Derived Dendritic Cells.

Damage-associated molecular patterns (DAMPs) play a critical role in dendritic cells (DCs) ability to trigger a specific and efficient adaptive immune response for different physiological and pathological scenarios. We have previously identified constitutive DAMPs (HMGB1 and Calreticulin) as well as new putative inducible DAMPs such as Haptoglobin (HP), from a therapeutically used heat shock-conditioned melanoma cell lysate (called TRIMEL). Remarkably, HP was shown to be the most abundant protein in the proteomic profile of heat shock-conditioned TRIMEL samples. However, its relative contribution to the observed DCs phenotype has not been fully elucidated. Human DCs were generated from monocytes isolated from PBMC of melanoma patients and healthy donors. DC lineage was induced with rhIL-4 and rhGM-CSF. After additional stimulation with HP, the proteome of these HP-stimulated cells was characterized. In addition, DCs were phenotypically characterized by flow cytometry for canonical maturation markers and cytokine production. Finally, in vitro transmigration capacity was assessed using Transwell plates. Our results showed that the stimulation with HP was associated with the presence of exclusive and higher relative abundance of specific immune-; energy production-; lipid biosynthesis-; and DAMPs-related proteins. Importantly, HP stimulation enhanced the expression of specific DC maturation markers and pro-inflammatory and Th1-associated cytokines, and an in vitro transmigration of primary human DCs. Taken together, these data suggest that HP can be considered as a new inducible DAMP with an important role in in vitro DC activation for cancer immunotherapy.

Primary Human Dendritic Cells and Whole-Blood Based Assays to Evaluate Immuno-Modulatory Properties of Heat-Killed Commensal Bacteria.

There is mounting evidence that the microbiome plays a critical role in training and maturation of the host immune system. Pre-clinical and clinical studies have shown that microbiome perturbation is correlated with sub-optimal host responses to vaccines and cancer immunotherapy. As such, identifying species of commensal bacteria capable of modulating immunological outcomes is of considerable interest. Currently, the lack of reliable primary immune cell-based assays capable of differentiating immuno-modulatory properties of various commensal bacteria is a major limitation. Here, we demonstrate that primary human monocyte-derived dendritic cells (MoDC) are capable of stratifying different strains of live and heat-killed commensal bacteria in an in vitro culture system. Specifically, heat-killed bacterial strains were able to differentially modulate co-stimulation/maturation markers CD80, CD83, and HLA-DR, as well as cytokine/chemokine signatures, such as IL-1b, MIP-1a, and TNFa in primary human MoDC. We further validated our observations using the TruCulture® (Myriad RBM, Inc., Austin, TX, USA) whole-blood ex vivo culture system. Using this ex vivo system allowed us to measure immune-altering effects of commensal bacteria in primary human whole-blood. As such, we report that both these primary in vitro and ex vivo systems are robust and enable identification, stratification, and differentiation of various commensal bacteria as potential modulators of host immunity.

HAMLET a human milk protein‐lipid complex induces a pro‐inflammatory phenotype of myeloid cells

HAMLET is a protein‐lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune‐stimulatory effects on primary human monocyte‐derived dendritic cells and macrophages (Mo‐DC and Mo‐M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha‐lactalbumin or oleic acid, induces mature CD14low/–CD83+ Mo‐DC and M1‐like CD14+CD86++ Mo‐M surface phenotypes. Concomitantly, inflammatory mediators, including IL‐2, IL‐6, IL‐10, IL‐12 and MIP‐1α, were released in the supernatant of HAMLET‐stimulated cells, indicating a mainly pro‐inflammatory phenotype. The HAMLET‐induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo‐DCs and calcium, NFκB and ERK signaling in Mo‐M as inhibitors of these pathways almost completely blocked the induction of mature Mo‐DCs and M1‐like Mo‐M. Compared to unstimulated Mo‐DCs, HAMLET‐stimulated Mo‐DCs were more potent in inducing T cell proliferation and HAMLET‐stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET‐stimulated DCs and macrophages. Combined, we propose that HAMLET has a two‐fold anti‐bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.

Macrolets: Outsized Extracellular Vesicles Released from Lipopolysaccharide-Stimulated Macrophages that Trap and Kill Escherichia coli.

SummaryMacrophages release a variety of extracellular vesicles (EVs). Here we describe a previously unreported class of EVs that are released from macrophages in response to Escherichia coli endotoxin, lipopolysaccharide (LPS), that we have named "macrolets" since they are extruded as large "droplets" released from macrophages. Morphologically, macrolets are anuclear, bounded by a single lipid membrane and structurally dependent on an actin cytoskeleton. Macrolets are enriched in tetraspanins and separable on this basis from their parent macrophages. Macrolets are distinguished from classic exosomes by their larger size (10–30 μm), discoid shape, and the presence of organelles. Macrolets are rich in both interleukin 6 (IL-6) and interleukin 6 receptor (IL-6R),and are capable of trapping and killing E. coli in association with production of reactive oxygen species. Our observations offer insights into the mechanisms by which macrophage activities may be amplified in sites of infection, inflammation, and healing.

Abstract PR08: Novel microbiome-derived peptides modulate immune cell activity and the tumor microenvironment

Abstract The gut microbiota has emerged as an important player in cancer pathology, and increasing evidence supports its role in clinical response to immune checkpoint inhibitor (ICI) therapy. However, the specific microbiome-derived factors responsible for the improved response to ICI therapy remain unknown. Second Genome has developed a unique discovery platform to identify, screen, and validate microbiome-derived peptides that promote response to cancer immunotherapy. Using our multitechnology meta-analysis of published datasets and characterizing the baseline microbiome of melanoma patients treated with anti-PD-1, we have identified gut microbiome strains differentially abundant in responders versus nonresponders that are concordant across multiple cohorts. Next, peptides from strains associated with responder signatures were predicted from their genome sequences. In addition, we predicted peptides from assembled metagenomes that were associated with responders. The predicted peptides were screened using phage display technology to identify binders to immune cells known to play a role in the tumor microenvironment (TME). Peptides that bound to specific immune cells were then evaluated for activity in cell-based assays using isolated primary human T cells, dendritic cells (DCs), and macrophages. We have demonstrated that several microbiome-derived peptides induce secretion of proinflammatory cytokines and chemokines such as CXCL10 and TNF-α by primary human monocyte-derived dendritic cells (moDCs), as well as secretion of effector cytokines such as IFNγ and IL-2 by primary human T cells. We have also identified microbiome-derived peptides with the capacity to inhibit an M2-like phenotype in macrophages (decreased LPS-induced IL-10 secretion). These effects were dose dependent and evident across immune cells derived from multiple human blood donors. In a coculture assay using allogeneic moDCs and T cells from human donors, combination of our DC-activating peptides with CD40 agonistic antibody and/or anti-PD-L1 induced secretion of proinflammatory cytokines such as IFNγ and TNF-α. In vivo, peritumoral administration of a candidate DC-activating peptide into RENCA tumor-bearing mice led to a significant reduction in tumor volume as compared to the control-treated mice. Collectively, these data demonstrate the potential of the microbiome-derived peptides identified by Second Genome’s discovery platform to modulate immune-cell effector functions in vitro and promote antitumor immunity in vivo. These results validate the unique approach of Second Genome’s discovery platform to identify novel microbiome-derived agents with potential for use as therapeutics in cancer immunotherapy. This abstract is also being presented as Poster B19. Citation Format: Dhwani D. Haria, Jayamary Divya Ravichandar, Lynn Yamamoto, Bernat Baeza-Raja, Ashil Bans, Cheryl-Emiliane Chow, Jill Desnoyer, Joanna Dreux, Shoko Iwai, Sabina Lau, Jina Lee, Michelle Lin, Paul Loriaux, Nicole Narayan, Eskedar Nigatu, Erica Rutherford, Michi Wilcoxon, Yonggan Wu, Todd DeSantis, Toshihiko Takeuchi, Karim Dabbagh, Helena Kiefel. Novel microbiome-derived peptides modulate immune cell activity and the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR08.

The citrus flavonoid naringenin impairs the in vitro infection of human cells by Zika virus

The Zika virus (ZIKV) is an arthropod-borne virus that belongs to the Flaviviridae family. The ZIKV infection is usually asymptomatic or is associated with mild clinical manifestations; however, increased numbers of cases of microcephaly and birth defects have been recently reported. To date, neither a vaccine nor an antiviral treatment has become available to control ZIKV replication. Among the natural compounds recognized for their medical properties, flavonoids, which can be found in fruits and vegetables, have been found to possess biological activity against a variety of viruses. Here, we demonstrate that the citrus flavanone naringenin (NAR) prevented ZIKV infection in human A549 cells in a concentration-dependent and ZIKV-lineage independent manner. NAR antiviral activity was also observed when primary human monocyte-derived dendritic cells were infected by ZIKV. NAR displayed its antiviral activity when the cells were treated after infection, suggesting that NAR acts on the viral replication or assembly of viral particles. Moreover, a molecular docking analysis suggests a potential interaction between NAR and the protease domain of the NS2B-NS3 protein of ZIKV which could explain the anti-ZIKV activity of NAR. Finally, the results support the potential of NAR as a suitable candidate molecule for developing anti-ZIKV treatments.

A lipid A-based TLR4 mimetic effectively adjuvants a Yersinia pestis rF-V1 subunit vaccine in a murine challenge model

Vaccination can significantly reduce worldwide morbidity and mortality to infectious diseases, thereby reducing the health burden as a result of microbial infections. Effective vaccines contain three components: a delivery system, an antigenic component of the pathogen, and an adjuvant. With the growing use of purely recombinant or synthetic antigens, there is a need to develop novel adjuvants that enhance the protective efficacy of a vaccine against infection. Using a structure–activity relationship (SAR) model, we describe here the synthesis of a novel TLR4 ligand adjuvant compound, BECC438, by bacterial enzymatic combinatorial chemistry (BECC). This compound was identified using an in vitro screening pipeline consisting of (i) NFκB activation and cytokine production by immortalized cell lines, (ii) cytokine production by primary human PBMCs, and (iii) upregulation of surface costimulatory markers by primary human monocyte-derived dendritic cells. Using this SAR screening regimen, BECC438 was shown to produce an innate immune activation profile comparable to the well-characterized TLR4 agonist adjuvant compound, phosphorylated hexa-acyl disaccharide (PHAD). To evaluate the in vivo adjuvant activity of BECC438, we used the known protective Yersinia pestis (Yp) antigen, rF1-V, in a murine prime-boost vaccination schedule followed by lethal challenge. In addition to providing protection from lethal challenge, BECC438 stimulated production of higher levels of rF1-V-specific total IgG as compared to PHAD after both prime and boost vaccinations. Similar to PHAD, BECC438 elicited a balanced IgG1/IgG2c response, indicative of active TH2/TH1-driven immunity. These data demonstrate that the novel BECC-derived TLR4L adjuvant, BECC438, elicits cytokine profiles in vitro similar to PHAD, induces high antigen-specific immune titers and a TH1-associated IgG2c immune titer skew, and protects mice against a lethal Yp challenge.

Feasibility and biological rationale of repurposing sunitinib and erlotinib for dengue treatment

There is an urgent need for strategies to combat dengue virus (DENV) infection; a major global threat. We reported that the cellular kinases AAK1 and GAK regulate intracellular trafficking of multiple viruses and that sunitinib and erlotinib, approved anticancer drugs with potent activity against these kinases, protect DENV-infected mice from mortality. Nevertheless, further characterization of the therapeutic potential and underlying mechanism of this approach is required prior to clinical evaluation. Here, we demonstrate that sunitinib/erlotinib combination achieves sustained suppression of systemic infection at approved dose in DENV-infected IFN-α/β and IFN-γ receptor–deficient mice. Nevertheless, treatment with these blood-brain barrier impermeable drugs delays, yet does not prevent, late-onset paralysis; a common manifestation in this immunodeficient mouse model but not in humans. Sunitinib and erlotinib treatment also demonstrates efficacy in human primary monocyte-derived dendritic cells. Additionally, DENV infection induces expression of AAK1 transcripts, but not GAK, via single-cell transcriptomics, and these kinases are important molecular targets underlying the anti-DENV effect of sunitinib and erlotinib. Lastly, sunitinib/erlotinib combination alters inflammatory cytokine responses in DENV-infected mice. These findings support feasibility of repurposing sunitinib/erlotinib combination as a host-targeted antiviral approach and contribute to understanding its mechanism of antiviral action.

TSPAN7, effector of actin nucleation required for dendritic cell-mediated transfer of HIV-1 to T cells.

Dendritic cells (DCs) have essential roles in early detection of pathogens and activation of both innate and adaptive immune responses. Whereas human DCs are resistant to productive HIV-1 replication, they have a unique ability to take up virus and transmit it efficiently to T lymphocytes. By doing that, HIV-1 may evade, at least in part, the first line of defense of the immune system, exploiting DCs instead to facilitate rapid infection of a large pool of immune cells. While performing an shRNA screen in human primary monocyte-derived DCs, to gain insights into this cell biological process, we discovered the role played by tetraspanin-7 (TSPAN7). This member of the tetraspanin family appears to be a positive regulator of actin nucleation and stabilization, through the ARP2/3 complex. By doing so, TSPAN7 limits HIV-1 endocytosis and maintains viral particles on actin-rich dendrites for an efficient transfer toward T lymphocytes. While studying the function of TSPAN7 in the control of actin nucleation, we also discovered the existence in DCs of two opposing forces at the plasma membrane: actin nucleation, a protrusive force which seems to counterbalance actomyosin contraction.

Characterization of the Kynurenine Pathway in CD8+ Human Primary Monocyte-Derived Dendritic Cells.

The kynurenine (KYN) pathway (KP) is a major degradative pathway of the amino acid, L-tryptophan (TRP), that ultimately leads to the anabolism of the essential pyridine nucleotide, nicotinamide adenine dinucleotide. TRP catabolism results in the production of several important metabolites, including the major immune tolerance-inducing metabolite KYN, and the neurotoxin and excitotoxin quinolinic acid. Dendritic cells (DCs) have been shown to mediate immunoregulatory roles that mediated by TRP catabolism. However, characterization of the KP in human DCs has so far only been partly delineated. It is critical to understand which KP enzymes are expressed and which KP metabolites are produced to be able to understand their regulatory effects on the immune response. In this study, we characterized the KP in human monocyte-derived DCs (MDDCs) in comparison with the human primary macrophages using RT-PCR, high-pressure gas chromatography, mass spectrometry, and immunocytochemistry. Our results show that the KP is entirely expressed in human MDDC. Following activation of the KP using interferon gamma, MDDCs can mediate apoptosis of T h cells in vitro. Understanding the molecular mechanisms regulating KP metabolism in MDDCs may provide renewed insight for the development of novel therapeutics aimed at modulating immunological effects and peripheral tolerance.

Profile of Class I Histone Deacetylases (HDAC) by Human Dendritic Cells after Alcohol Consumption and In Vitro Alcohol Treatment and Their Implication in Oxidative Stress: Role of HDAC Inhibitors Trichostatin A and Mocetinostat.

Epigenetic mechanisms have been shown to play a role in alcohol use disorders (AUDs) and may prove to be valuable therapeutic targets. However, the involvement of histone deacetylases (HDACs) on alcohol-induced oxidative stress of human primary monocyte-derived dendritic cells (MDDCs) has not been elucidated. In the current study, we took a novel approach combining ex vivo, in vitro and in silico analyses to elucidate the mechanisms of alcohol-induced oxidative stress and role of HDACs in the periphery. ex vivo and in vitro analyses of alcohol-modulation of class I HDACs and activity by MDDCs from self-reported alcohol users and non-alcohol users was performed. Additionally, MDDCs treated with alcohol were assessed using qRT-PCR, western blot, and fluorometric assay. The functional effects of alcohol-induce oxidative stress were measured in vitro using PCR array and in silico using gene expression network analysis. Our findings show, for the first time, that MDDCs from self-reported alcohol users have higher levels of class I HDACs compare to controls and alcohol treatment in vitro differentially modulates HDACs expression. Further, HDAC inhibitors (HDACi) blocked alcohol-induction of class I HDACs and modulated alcohol-induced oxidative stress related genes expressed by MDDCs. In silico analysis revealed new target genes and pathways on the mode of action of alcohol and HDACi. Findings elucidating the ability of alcohol to modulate class I HDACs may be useful for the treatment of alcohol-induced oxidative damage and may delineate new potential immune-modulatory mechanisms.

IRE1α mediates PKR activation in response to Chlamydia trachomatis infection

Protein kinase RNA activated (PKR) is a crucial mediator of anti-viral responses but is reported to be activated by multiple non-viral stimuli. However, mechanisms underlying PKR activation, particularly in response to bacterial infection, remain poorly understood. We have investigated mechanisms of PKR activation in human primary monocyte-derived dendritic cells in response to infection by Chlamydia trachomatis. Infection resulted in potent activation of PKR that was dependent on TLR4 and MyD88 signalling. NADPH oxidase was dispensable for activation of PKR as cells from chronic granulomatous disease (CGD) patients, or mice that lack NADPH oxidase activity, had equivalent or elevated PKR activation. Significantly, stimulation of cells with endoplasmic reticulum (ER) stress-inducing agents resulted in potent activation of PKR that was blocked by an inhibitor of IRE1α RNAse activity. Crucially, infection resulted in robust IRE1α RNAse activity that was dependent on TLR4 signalling and inhibition of IRE1α RNAse activity prevented PKR activation. Finally, we demonstrate that TLR4/IRE1α mediated PKR activation is required for the enhancement of interferon-β production following C. trachomatis infection. Thus, we provide evidence of a novel mechanism of PKR activation requiring ER stress signalling that occurs as a consequence of TLR4 stimulation during bacterial infection and contributes to inflammatory responses.

Abstract A157: Oncolytic poliovirus mediated immune events

Abstract Introduction: Tumor-targeted therapies that efficiently eliminate malignant cells and in the process engage the innate and adaptive immune system are desirable for preventing cancer recurrence. We have pioneered an oncolytic poliovirus therapy, PVSRIPO, which selectively targets and eliminates malignant cells. PVSRIPO is a recombinant polio:rhinovirus chimera, engineered to eliminate neuronal competence and is devoid of neuropathogenicity after intracerebral inoculation in non-human primates and in humans. PVSRIPO has cancer tropism due to widespread ectopic expression of the poliovirus receptor, CD155, in solid cancers. PVSRIPO kills tumor cells with no effect on normal cells. Unlike other oncolytic viruses, PVSRIPO is effective in the presence of neutralizing antibodies and an innate antiviral response. A first-in-human Phase-1 study with PVSRIPO at Duke University has shown remarkable promise in patients with recurrent glioblastoma (GBM), a uniformly lethal disease. PVSRIPO tumor cell killing is associated with the induction of danger- and pathogen-associated molecular patterns (DAMPs and PAMPs) via antiviral type I interferons (IFNs) and simultaneous non-lethal infection of antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs) via CD155. Type I IFNs are known to inhibit tumor proliferation, activate innate immune cells and bridge innate and adaptive immunity among other functions. Methods: To understand immune events associated with poliovirus infection of APCs, we examined the effects of PVSRIPO treatment on the human macrophage cell line (THP1) and primary human monocyte-derived DCs. PVSRIPO-treated DCs were evaluated for expression of maturation/activation markers and compared to untreated immature and mature DCs. To investigate if DCs exposed to PVSRIPO-induced dying tumor cells are capable of presenting tumor antigens to T cells, we performed an in vitro human immunotherapy assay. Human DCs generated from HLA-A2+ donor cells were incubated with PVSRIPO-induced tumor cell lysate and then used to stimulate autologous T cells in vitro followed by a cytotoxic T lymphocyte (CTL) assay. All tumor cell lines used for the human immunotherapy experiments were positive for HLA-A2 expression. The following tumor cell lines were used for this analysis: DM6 (MART+) melanoma cell line, MDA-MB231 (CEA+) triple-negative breast cancer cell line, SUM149 (EGFR+) inflammatory breast cancer cell line and LNCaP (PSA+) prostate cancer cell line. Results: PVSRIPO infection of THP1 macrophages induces Mda5/Stat1 and Stat1 phosphorylation as well as TNFα and IFNβ release. Coculturing of DCs with PVSRIPO-induced tumor cell lysate stimulates DC activation and IL12 production. Human DCs loaded with PVSRIPO-induced tumor cell lysate are capable of stimulating tumor antigen-specific cytotoxic T cells in vitro. Autologous DCs transfected with RNA that encodes for CEA, EGFR, MART or PSA (tumor antigen-expressing cells) and human tumor cell lines were used as targets to assess tumor antigen-specificity of T cells. Conclusion: Our data suggests that along with destruction of the primary tumor, oncolytic poliovirus mediates immune events. Importantly, we demonstrate that human DCs co-incubated with PVSRIPO-induced tumor cell lysate stimulate tumor antigen-specific cytotoxic T cells in an in vitro human immunotherapy assay. In ongoing studies we are analyzing oncolytic poliovirus mediated immune events in syngeneic, immunocompetent murine models of breast cancer and prostate cancer. Citation Format: Michael C. Brown, Eda K. Holl, David Boczkowski, Darell D. Bigner, Matthias Gromeier, Smita K. Nair. Oncolytic poliovirus mediated immune events. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A157.

Bordetella pertussis filamentous hemagglutinin itself does not trigger anti-inflammatory interleukin-10 production by human dendritic cells

Filamentous hemagglutinin (FHA) is an important adhesin of the whooping cough agent Bordetella pertussis and is contained in most acellular pertussis vaccines. Recently, FHA was proposed to exert an immunomodulatory activity through induction of tolerogenic IL-10 secretion from dendritic cells. We have re-evaluated the cytokine-inducing activity of FHA, placing specific emphasis on the role of the residual endotoxin contamination of FHA preparations. We show that endotoxin depletion did not affect the capacity of FHA to bind primary human monocyte-derived dendritic cells, while it abrogated the capacity of FHA to elicit TNF-α and IL-10 secretion and strongly reduced its capacity to trigger IL-6 production. The levels of cytokines induced by the different FHA preparations correlated with their residual contents of B. pertussis endotoxin. Moreover, FHA failed to trigger cytokine secretion in the presence of antibodies that block TLR2 and/or TLR4 signaling. The TLR2 signaling capacity appeared to be linked to the presence of endotoxin-associated components in FHA preparations and not to the FHA protein itself. These results show that the endotoxin-depleted FHA protein does not induce cytokine release from human dendritic cells.

Viral Source-Independent High Susceptibility of Dendritic Cells to Human T-Cell Leukemia Virus Type 1 Infection Compared to That of T Lymphocytes.

Human T-cell leukemia virus type 1 (HTLV-1)-infected CD4(+) T cells and dendritic cells (DCs) are present in peripheral blood from HTLV-1 carriers. While T-cell infection requires cell-cell contact, DCs might be infected with cell-free virus, at least in vitro. However, a thorough comparison of the susceptibilities of the two cell types to HTLV-1 infection using cell-associated and cell-free viral sources has not been performed. We first determined that human primary monocyte-derived dendritic cells (MDDCs) were more susceptible to HTLV-1 infection than their autologous lymphocyte counterparts after contact with chronically infected cells. Next, a comparison of infection efficiency using nonconcentrated or concentrated supernatants from infected cells as well as purified viral biofilm was performed. Integrated provirus was found after exposure of MDDCs or primary lymphocytes to viral biofilm but not to a viral supernatant. Using a large series of primary cell samples (n = 21), we demonstrated a higher proviral load in MDDCs exposed to viral biofilm than in lymphocytes. This higher susceptibility is correlated to a higher expression of neuropilin-1 on MDDCs than on autologous activated T lymphocytes. Moreover, we show that MDDCs infected with viral biofilm can transmit the virus to lymphocytes. In conclusion, MDDCs are more susceptible to HTLV-1 infection than autologous lymphocytes in vitro, supporting a model in which DC infection might represent an important step during primo-infection in vivo. HTLV-1 is able to infect several cell types, but viral DNA is mainly found in T lymphocytes in vivo. This supports a model in which T lymphocytes are the main target of infection. However, during the primo-infection of new individuals, incoming viruses might first encounter dendritic cells (DCs), the specialized immune cells responsible for the antiviral response of the host. HTLV-1 cell-free purified viruses can infect dendritic cells in vitro, while T-cell infection is restricted to cell-to-cell transmission. In order to understand the sequence of HTLV-1 dissemination, we undertook a direct comparison of the susceptibilities of the two cell types using cell-associated and cell-free viral sources. We report here that MDDCs are more susceptible to HTLV-1 infection than autologous lymphocytes in vitro and are able to efficiently transmit the virus to lymphocytes. Our results suggest that DCs may represent a true viral reservoir, as the first cell type to be infected in vivo.