Human Innate Immune Response Research Articles

Natural molecules induce and synergize to boost expression of the human antimicrobial peptide β-defensin-3

Antimicrobial peptides (AMPs) are mucosal defense effectors of the human innate immune response. In the intestine, AMPs are produced and secreted by epithelial cells to protect the host against pathogens and to support homeostasis with commensals. The inducible nature of AMPs suggests that potent inducers could be used to increase their endogenous expression for the prevention or treatment of diseases. Here we aimed at identifying molecules from the natural pharmacopoeia that induce expression of human β-defensin-3 (HBD3), one of the most efficient AMPs, without modifying the production of proinflammatory cytokines. By screening, we identified three molecules isolated from medicinal plants, andrographolide, oridonin, and isoliquiritigenin, which induced HBD3 production in human colonic epithelial cells. This effect was observed without activation of the NF-κB pathway or the expression of associated proinflammatory cytokines. We identified the EGF receptor as the target of these compounds and characterized the downstream-activated MAPK pathways. At the chromatin level, molecules increased phosphorylation of histone H3 on serine S10 and recruitment of the c-Fos, c-Jun, and Elk1 or c-Myc transcription factors at the HBD3 promoter. Interestingly, stimulating cells with a combination of andrographolide and isoliquiritigenin synergistically enhanced HBD3 induction 10-fold more than observed with each molecule alone. Finally, we investigated the molecular basis governing the synergistic effect, confirmed our findings in human colonic primary cells, and demonstrated that synergism increased cellular antimicrobial activity. This work shows the capability of small molecules to achieve induction of epithelial antimicrobial defenses while simultaneously avoiding the deleterious risks of an inflammatory response.

Backbone 1H, 13C, and 15N resonance assignments of deubiquitinase A in non-phosphorylated and phosphorylated forms.

Ubiquitination is one of the most prevalent forms of post-translational modifications that are important for regulating many cellular processes in eukaryotes. Deubiquitinases are proteases that hydrolyze the isopeptide or peptide bonds formed between ubiquitin and the target proteins or within a polyubiquitin chain. Deubiquitinase A (DUBA) is a deubiquitinase known to be a negative regulator of innate immune responses in humans by suppressing production of type I interferons (INF-I). Excess INF-I production has been associated with autoimmune diseases. Phosphorylation of a single serine residue at position 177 is essential for the protease activity of DUBA. The structural and mechanistic basis of DUBA activation by phosphorylation and substrate specificity is not well understood. Here, we report the backbone resonance assignments of the isoform 2 of DUBA in both non-phosphorylated and phosphorylated forms. The reported assignments form the basis for future NMR studies on the structural and dynamical properties of both active and inactive forms of DUBA.

Studies on the interactions of neutral Galleria mellonella cecropin D with living bacterial cells

Cecropins constitute an important family of insect antimicrobial peptides involved in humoral innate immune response. In comparison with the highly basic cecropins A and B, cecropins D are less cationic and more hydrophobic. Interestingly, cecropins D were described only in lepidopteran insects, e.g., the greater wax moth Galleria mellonella. In the present study, interactions of neutral cecropin D (pI 6.47) purified from hemolymph of G. mellonella with living Escherichia coli cells were investigated. Fluorescence lifetime imaging microscopy using fluorescein isothiocyanate-labeled cecropin D revealed very fast binding of the peptide to E. coli cells. Fourier transform infrared spectroscopy analyses showed that G. mellonella cecropin D interacted especially with E. coli LPS and probably other lipid components of the bacterial cell envelope and exhibited an ordering effect with regard to lipid chains. This effect is consistent with the peptide binding mechanism based upon its incorporation into the lipid phase of the cell membrane. The interaction resulted in permeabilization of the bacterial cell membrane. Upon cecropin D binding, the cells lost characteristic surface topography, which was accompanied by altered nanomechanical properties, as revealed by atomic force microscopy. The interaction of the peptide with the bacterial cells also led to intracellular damage, i.e., loss of the cell envelope multilayer structure, formation of membrane vesicles, and enlargement of periplasmic space, which eventually caused death of the bacteria. In summary, it can be concluded that amphipathic character of α-helices, exposure of small positively charged patches on their polar surfaces and hydrophobic interactions are important physicochemical characteristics related to effective binding to E. coli cells and antibacterial activity of neutral G. mellonella cecropin D.

Interactive effects of dietary vegetable oil and carbohydrate incorporation on the innate immune response of European seabass (Dicentrarchus labrax) juveniles subjected to acute stress

This study aims to understand the interactive effects of high dietary vegetable oils (VO) and carbohydrate (CH) content in European seabass cellular, humoral, and molecular innate immune responses to an acute stress. For that purpose, European sea bass juveniles (74.0 ± 1.5 g) were fed four diets differing in lipid source (fish oil (FO) or a blend of vegetable oils (VO)) and carbohydrate content (0% (CH–) or 20% (CH+) gelatinized starch). Nine fish per dietary treatment were sampled after 73 days of feeding and used as control whereas the remaining fish were subjected to an acute stress (netting, 1 min air exposure, and transfer to smaller tanks). Those fish were then sampled after 1 h and considered the stressed group. In the present study, dietary VO incorporation affected fish humoral immune parameters with a decrease of haematocrit, plasma peroxidase activity, and head-kidney mc2r and gr1 mRNA expression levels. Regardless of dietary treatment, higher cortisol levels were accompanied by an increase of several haematological, cellular, and humoral parameters in response to an acute stress. Dietary VO incorporation led to lower neutrophil numbers, plasma antiproteases activity, and head-kidney cox2 expression level in stressed fish. Fish fed CH+ diets showed a reduction of head-kidney mc2r expression levels in response to stress, while plasma cortisol remained unchanged, plasma NO decreases and antiproteases activity increased. In fish fed the VO diets, CH+ led to a decrease of plasma bactericidal activity compared to that of group CH-, while the opposite pattern was observed for plasma peroxidase. In conclusion, dietary substitution of FO by VO in CH– diet negatively affected the immune response in both undisturbed and stressed fish, while the effect of dietary CH incorporation in VO diet was not clear.

Mosquito Innate Immunity

Mosquitoes live under the endless threat of infections from different kinds of pathogens such as bacteria, parasites, and viruses. The mosquito defends itself by employing both physical and physiological barriers that resist the entry of the pathogen and the subsequent establishment of the pathogen within the mosquito. However, if the pathogen does gain entry into the insect, the insect mounts a vigorous innate cellular and humoral immune response against the pathogen, thereby limiting the pathogen’s propagation to nonpathogenic levels. This happens through three major mechanisms: phagocytosis, melanization, and lysis. During these processes, various signaling pathways that engage intense mosquito–pathogen interactions are activated. A critical overview of the mosquito immune system and latest information about the interaction between mosquitoes and pathogens are provided in this review. The conserved, innate immune pathways and specific anti-pathogenic strategies in mosquito midgut, hemolymph, salivary gland, and neural tissues for the control of pathogen propagation are discussed in detail.

Dibutyl phthalate modulates phenotype of granulocytes in human blood in response to inflammatory stimuli

BackgroundPhthalates are plasticizers used in many common commercial products. They are ubiquitous environmental contaminants and epidemiological studies suggest that phthalate exposure is associated with development or worsening of airway diseases. Dibutyl phthalate (DBP) is a type of phthalate, found in high concentrations in indoor air, which appears to have a high inflammatory potential. In vitro studies on innate immune cells like macrophages have shown a reduction in phagocytic and antigen-presenting capacity and decreased production of stimuli-induced cytokines after DBP exposure. ObjectiveWe aimed to assess how DBP may alter the in vitro cellular and humoral innate immune response to inflammatory stimuli in blood innate immune cells. MethodsHuman whole blood was stimulated with inflammatory stimuli (lipopolysaccharide (LPS), resiquimod (R848) and phorbol 12-myristate 13-acetate (PMA)) in the presence or absence of DBP. The expression of surface markers CD16, CD24, CD69 and CD14 on granulocytes and monocytes was quantified by flow cytometry analysis. The release of TNFα, IFNγ, IL8 and IL10 cytokines was measured by ELISA. ResultsThe presence of DBP reduced the inflammatory stimuli-induced expression of CD24 on neutrophils and eosinophils and CD69 on activated eosinophils, whereas expression of CD16 on neutrophils was increased. DBP also had a dampening effect on the release of pro-inflammatory mediators TNFα and IFNγ in response to the inflammatory stimuli. ConclusionsThese responses may reflect an immunosuppressive effect of DBP through impairment of immune cell function.

Dietary administration of eryngii mushroom (Pleurotus eryngii) powder on haemato-immunological responses, bactericidal activity of skin mucus and growth performance of koi carp fingerlings (Cyprinus carpio koi)

The aim of present study was to evaluate the effects of edible eryngii mushroom powder, Pleurotus eryngii (PE), for 63 days on haematological parameters, the serum immune responses, skin mucus, bactericidal activity, stress resistance, growth performance and digestive enzyme activities of Koi carp fingerlings (Cyprinus carpio koi). Fish were divided into five groups and each group was fed with dietary PE with five graded levels (0, 0.5, 1, 1.5 and 2%). The results showed a significant dose-dependent increase of Ht, Hb, MCV and MCH levels in fish fed dietary PE (P < 0.05). The highest levels of WBCs, lymphocytes and monocytes were measured in fish fed 1.5% and 2% of dietary PE (P < 0.05). The activities of total IG, lysozyme, Alternative haemolytic complement activity in serum of fish fed with 2% of dietary PE for 63 days as well as 5-min air exposure challenge test were significantly higher than other groups (P < 0.05). The most bactericidal activity was observed in skin mucus of fish fed with 1.5% of dietary PE against Streptococcus iniae (P < 0.05). The highest ratio of the lactobacillus count to the total viable count was observed in fish fed 2% of dietary PE. The α-amylase activity of fish fed with dietary PE (1, 1.5 and 2%) were significantly higher than control group (P < 0.05). Feeding fish 2% of dietary PE increased the trypsin and lipase activity compared to others groups (P < 0.05). The growth performance of fish fed 1.5% of dietary PE improved compared to control group (P < 0.05). The results revealed that feeding koi fish with dietary supplementation of PE (1.5 and 2%) improved the selected humoral innate immune responses, bactericidal activity of skin mucus and growth performance of koi fish.

Assessing the cellular and humoral immune response in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) infected with Leishmania infantum (Nicolle, 1908)

The aim of this study was to evaluate aspects of the innate cellular and humoral immune response by evaluating hemocyte dynamics, phagocytosis, phenoloxidase (PO) activity and nitric oxide (NO) production in Rhipicephalus sanguineus sensu lato (s.l.) (Acari: Ixodidae) infected with Leishmania infantum and to assess the persistence of parasites at time 0 and 1, 2, 5, and 7 days post-infection (dpi). The total and differential count of the five types of hemocytes circulating in the hemolymph of R. sanguineus s.l. females showed the average total number of hemocytes in the group infected with L. infantum to be significantly higher (p < 0.05) on the 1st and 2nd dpi compared to the control group. The hemocyte differential count showed that the average number of plasmatocytes and granulocytes increased significantly on the 1st, 2nd, and 5th dpi with L. infantum compared to the control group (p < 0.001). Phagocytosis assays revealed that plasmatocytes and granulocytes were able to perform phagocytosis of latex beads and L. infantum on the 1st and 2nd dpi, respectively. NO production was significantly increased (p < 0.001) on the 1st, 2nd, and 5th dpi with L. infantum and PO activity increased significantly (p < 0.05) only on the 5th dpi. L. infantum DNA was significantly increased (p < 0.001) on the 5th and 7th dpi compared to time 0. Although there are no studies describing the response of R. sanguineus s.l. to an infection with L. infantum, these results suggest that R. sanguineus s.l. activates the cellular and humoral immune response after infection with L. infantum. Further studies are however, needed to assess the impact of such a response on fighting infection.

Immune response to recombinant Burkholderia pseudomallei FliC.

Burkholderia pseudomallei is a flagellated Gram-negative bacterium which is the causative agent of melioidosis. The disease poses a major public health problem in tropical regions and diabetes is a major risk factor. The high mortality rate of melioidosis is associated with severe sepsis which involves the overwhelming production of pro-inflammatory cytokines. Bacterial flagellar protein (flagellin) activates Toll-like receptor 5 (TLR5)-mediated innate immune signaling pathways and induces adaptive immune response. However, previous studies of TLR5 signaling in melioidosis have been performed using recombinant flagellin from Salmonella Typhimurium instead of B. pseudomallei. This study aimed to investigate human innate immune response and antibody response against a recombinant B. pseudomallei flagellin (rFliC). We prepared B. pseudomallei rFliC and used it to stimulate HEK-BlueTM-hTLR5 and THP1-DualTM cells to assess TLR5 activation. Subsequently, whole blood stimulation assays with rFliC were performed ex vivo. TLR5-flagellin interactions trigger activation of transcription factor NF-κB in HEK-BlueTM-hTLR5 cells. Pro-inflammatory cytokine (IL-1β, IL-6, and TNF-α) productions from whole blood in response to rFliC differed between fourteen healthy individuals. The levels of these cytokines changed in a dose and time-dependent manner. ELISA was used to determine rFliC-specific antibodies in serum samples from different groups of melioidosis patients and healthy subjects. IgG antibody to rFliC in melioidosis patients with diabetes were higher compared with non-diabetic patients. Our results show that B. pseudomallei flagellin is a potent immune stimulator and that the immune responses to rFliC are different among individuals. This may provide valuable insights toward the potential use of rFliC in vaccine development.

An in vitro/ex vivo human lung infection model for studying the macrophage innate immune response to Ebola virus Kikwit

Abstract Ebola virus causes a severe and often fatal hemorrhagic fever in people, for which no approved vaccines or therapeutics are available. Disease pathogenesis and countermeasure studies are typically performed using animal models; however, ex vivo models recapitulating human immune responses may provide valuable information. We optimized engineered lung tissue constructs comprised of human lung epithelial cells and human endothelial cells that mimic the vascularized lower airway alveolar tissue micro-environment to study the human innate immune response to Ebola virus Kikwit (EBOV/Kik). Human lung tissue macrophages are difficult to obtain due to practical and ethical considerations, so macrophages were chemically derived from human monocytes. We inoculated the constructs, in the presence and absence of monocyte derived macrophages (MDM), and quantified cytokines/chemokines secreted on days 0, 3, 6, 9, and 15 post-infection using 29-plex and 1-plex human cytokine/chemokine kits. Prior to infection, MDM were secreting IL-13, MCP-1, MIP-1a, MIP-1b; however, these levels rapidly declined by day 3 post-infection (pi). The MDM secreted peak levels of IL-4, IFN-a2, Eotaxin, IL-12P40, and IFNg at day 6pi, and IL-1ra peaked at day 9 pi. In response to EBOV/Kik, the human lung epithelial cells in the engineered lung tissue construct secreted G-CSF, IL-1ra, IL-8, IP-10, and PDGF-AA/BB. G-CSF and IL-8, cytokines known to mobilize hematopoietic progenitors from bone marrow to blood and recruit neutrophils to sites of infection, were abundantly secreted by both endothelial cells and macrophages in response to infection. This model system may be a valuable resource to the field to study human immune responses during acute infections.

C-Reactive Protein in Atherothrombosis and Angiogenesis.

C-reactive protein (CRP) is a short pentraxin mainly found as a pentamer in the circulation, or as non-soluble monomers CRP (mCRP) in tissues, exerting different functions. This review is focused on discussing the role of CRP in cardiovascular disease, including recent advances on the implication of CRP and its forms specifically on the pathogenesis of atherothrombosis and angiogenesis. Besides its role in the humoral innate immune response, CRP contributes to cardiovascular disease progression by recognizing and binding multiple intrinsic ligands. mCRP is not present in the healthy vessel wall but it becomes detectable in the early stages of atherogenesis and accumulates during the progression of atherosclerosis. CRP inhibits endothelial nitric oxide production and contributes to plaque instability by increasing endothelial cell adhesion molecules expression, by promoting monocyte recruitment into the atheromatous plaque and by enzymatically binding to modified low-density lipoprotein. CRP also contributes to thrombosis, but depending on its form it elicits different actions. Pentameric CRP has no involvement in thrombogenesis, whereas mCRP induces platelet activation and thrombus growth. In addition, mCRP has apparently contradictory pro-angiogenic and anti-angiogenic effects determining tissue remodeling in the atherosclerotic plaque and in infarcted tissues. Overall, CRP contributes to cardiovascular disease by several mechanisms that deserve an in-depth analysis.

Time-dependent distinct roles of Toll-like receptor 4 in a house dust mite-induced asthma mouse model.

House dust mites (HDMs) are a common source of allergens that trigger both allergen-specific and innate immune responses in humans. Here, we examined the effect of allergen concentration and the involvement of Toll-like receptor 4 (TLR4) in the process of sensitization to house dust mite allergens in an HDM extract-induced asthma mouse model. Intranasal administration of HDM extract induced an immunoglobulin E response and eosinophilic inflammation in a dose-dependent manner from 2.5 to 30μg/dose. In TLR4-knockout mice, the infiltration of eosinophils and neutrophils into the lung was decreased compared with that in wild-type mice in the early phase of inflammation (total of three doses). However, in the late phase of inflammation (total of seven doses), eosinophil infiltration was significantly greater in TLR4-knockout mice than in wild-type mice. This suggests that the roles of TLR4 signaling are different between the early phase and the later phase of HDM allergen-induced inflammation. Thus, innate immune response through TLR4 regulated the response to HDM allergens, and the regulation was altered during the phase of inflammation.

Simulation Model for Dynamics of Dengue with Innate and Humoral Immune Responses.

Dengue virus is a mosquito borne Flavivirus and the most prevalent arbovirus in tropical and subtropical regions around the world. The incidence of dengue has increased drastically over the last few years at an alarming rate. The clinical manifestation of dengue ranges from asymptomatic infection to severe dengue. Even though the viral kinetics of dengue infection is lacking, innate immune response and humoral immune response are thought to play a major role in controlling the virus count. Here, we developed a computer simulation mathematical model including both innate and adaptive immune responses to study the within-host dynamics of dengue virus infection. A sensitivity analysis was carried out to identify key parameters that would contribute towards severe dengue. A detailed stability analysis was carried out to identify relevant range of parameters that contributes to different outcomes of the infection. This study provides a qualitative understanding of the biological factors that can explain the viral kinetics during a dengue infection.

Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity

The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses.

Mycobacterium tuberculosis inhibits human innate immune responses via the production of TLR2 antagonist glycolipids

Mycobacterium tuberculosis is a major human pathogen that is able to survive inside host cells and resist immune clearance. Most particularly, it inhibits several arms of the innate immune response, including phagosome maturation or cytokine production. To better understand the molecular mechanisms by which M. tuberculosis circumvents host immune defenses, we used a transposon mutant library generated in a virulent clinical isolate of M. tuberculosis of the W/Beijing family to infect human macrophages, utilizing a cell line derivative of THP-1 cells expressing a reporter system for activation of the transcription factor NF-κB, a key regulator of innate immunity. We identified several M. tuberculosis mutants inducing a NF-κB activation stronger than that of the wild-type strain. One of these mutants was found to be deficient for the synthesis of cell envelope glycolipids, namely sulfoglycolipids, suggesting that the latter can interfere with innate immune responses. Using natural and synthetic molecular variants, we determined that sulfoglycolipids inhibit NF-κB activation and subsequent cytokine production or costimulatory molecule expression by acting as competitive antagonists of Toll-like receptor 2, thereby inhibiting the recognition of M. tuberculosis by this receptor. Our study reveals that producing glycolipid antagonists of pattern recognition receptors is a strategy used by M. tuberculosis to undermine innate immune defense. Sulfoglycolipids are major and specific lipids of M. tuberculosis, considered for decades as virulence factors of the bacilli. Our study uncovers a mechanism by which they may contribute to M. tuberculosis virulence.

Increased circulating interleukin-8 in patients with resistance to thyroid hormone receptor α.

Innate immune cells have recently been identified as novel thyroid hormone (TH) target cells in which intracellular TH levels appear to play an important functional role. The possible involvement of TH receptor alpha (TRα), which is the predominant TR in these cells, has not been studied to date. Studies in TRα0/0 mice suggest a role for this receptor in innate immune function. The aim of this study was to determine whether TRα affects the human innate immune response. We assessed circulating interleukin-8 concentrations in a cohort of 8 patients with resistance to TH due to a mutation of TRα (RTHα) and compared these results to healthy controls. In addition, we measured neutrophil and macrophage function in one of these RTHα patients (mutation D211G). Circulating interleukin-8 levels were elevated in 7 out of 8 RTHα patients compared to controls. These patients harbor different mutations, suggesting that this is a general feature of the syndrome of RTHα. Neutrophil spontaneous apoptosis, bacterial killing, NAPDH oxidase activity and chemotaxis were unaltered in cells derived from the RTHαD211G patient. RTHα macrophage phagocytosis and cytokine induction after LPS treatment were similar to results from control cells. The D211G mutation did not result in clinically relevant impairment of neutrophil or pro-inflammatory macrophage function. As elevated circulating IL-8 is also observed in hyperthyroidism, this observation could be due to the high-normal to high levels of circulating T3 found in patients with RTHα.

Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system.

TLR ligands (TLR-Ls) represent a class of novel vaccine adjuvants. However, their immunologic effects in humans remain poorly defined in vivo. Using a humanized mouse model with a functional human immune system, we investigated how different TLR-Ls stimulated human innate immune response in vivo and their applications as vaccine adjuvants for enhancing human cellular immune response. We found that splenocytes from humanized mice showed identical responses to various TLR-Ls as human PBMCs in vitro. To our surprise, various TLR-Ls stimulated human cytokines and chemokines differently in vivo compared to that in vitro. For example, CpG-A was most efficient to induce IFN-α production in vitro. In contrast, CpG-B, R848 and Poly I:C stimulated much more IFN-α than CpG-A in vivo. Importantly, the human innate immune response to specific TLR-Ls in humanized mice was different from that reported in C57BL/6 mice, but similar to that reported in nonhuman primates. Furthermore, we found that different TLR-Ls distinctively activated and mobilized human plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs) and monocytes in different organs. Finally, we showed that, as adjuvants, CpG-B, R848 and Poly I:C can all enhance antigen specific CD4+ T cell response, while only R848 and Poly I:C induced CD8+ cytotoxic T cells response to a CD40-targeting HIV vaccine in humanized mice, correlated with their ability to activate human mDCs but not pDCs. We conclude that humanized mice serve as a highly relevant model to evaluate and rank the human immunologic effects of novel adjuvants in vivo prior to testing in humans.

Catalog of Differentially Expressed Long Non-Coding RNA following Activation of Human and Mouse Innate Immune Response.

Despite increasing evidence to indicate that long non-coding RNAs (lncRNAs) are novel regulators of immunity, there has been no systematic attempt to identify and characterize the lncRNAs whose expression is changed following the induction of the innate immune response. To address this issue, we have employed next-generation sequencing data to determine the changes in the lncRNA profile in four human (monocytes, macrophages, epithelium, and chondrocytes) and four mouse cell types (RAW 264.7 macrophages, bone marrow-derived macrophages, peritoneal macrophages, and splenic dendritic cells) following exposure to the pro-inflammatory mediators, lipopolysaccharides (LPS), or interleukin-1β. We show differential expression of 204 human and 210 mouse lncRNAs, with positional analysis demonstrating correlation with immune-related genes. These lncRNAs are predominantly cell-type specific, composed of large regions of repeat sequences, and show poor evolutionary conservation. Comparison within the human and mouse sequences showed less than 1% sequence conservation, although we identified multiple conserved motifs. Of the 204 human lncRNAs, 21 overlapped with syntenic mouse lncRNAs, of which five were differentially expressed in both species. Among these syntenic lncRNA was IL7-AS (antisense), which was induced in multiple cell types and shown to regulate the production of the pro-inflammatory mediator interleukin-6 in both human and mouse cells. In summary, we have identified and characterized those lncRNAs that are differentially expressed following activation of the human and mouse innate immune responses and believe that these catalogs will provide the foundation for the future analysis of the role of lncRNAs in immune and inflammatory responses.

Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells

Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)—an inhibitor of RIP1 kinase activity—dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a “nonadaptive virus.” In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

Abstract 4652: Profiling of immunity biomarkers in whole blood

Abstract Cancer immunotherapies are rapidly changing traditional treatment paradigms and expanding the therapeutic landscape for cancer patients. However, despite the current success of these therapies, significant number of patients do not respond to immunotherapy and responders often experience significant toxicities. Therefore, there is a growing need to identify predictive and prognostic biomarkers that enhance our understanding of dynamic immunity mechanisms involved in cancer development. The genome-wide expression profiling of whole blood is an attractive method for the discovery of biomarkers due to its non-invasiveness, possibility for retrospective simple clinical site processing, and rich biological content. To this end, we have developed GeneNet, a genome-wide targeted RNA expression assay that profiles 19,000 genes based on multiplex RT-PCR followed by NGS in whole blood samples stabilized in PAXgene. The GeneNet 19K assay provides comprehensive and unbiased profiling of immune cell activation and canonical immune pathway genes for both innate adaptive and humoral immune responses. In this study, we present the performance of the assay for immunophenotyping of immune cells in blood samples from TNBC patients and assess the immune responses to complex immunomodulatory stimuli in ex vivo model system. Preliminary studies demonstrate the assay’s unparalleled specificity and sensitivity resulting in better detection of low abundance mRNA transcripts as well as an improved cost-effectiveness for high-throughput clinical applications. Citation Format: Alex Chenchik, Andrei Komarov, Michael Makhanov, Costa Frangou. Profiling of immunity biomarkers in whole blood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4652. doi:10.1158/1538-7445.AM2017-4652